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Volume 25
Issue 22
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Int. J. Mol. Sci., Volume 25, Issue 22 (November-2 2024) – 627 articles

Cover Story (view full-size image): Chronic spontaneous urticaria (CSU) is defined by itchy wheals and/or angioedema lasting at least 6 weeks, driven by prolonged mast cell activation. Platelet-Activating Factor (PAF), a potent lipid mediator, plays a key role in CSU pathogenesis, distinct from IgE-mediated mechanisms. This cover image shows PAF signaling through its receptor (PAFR), activating Gi and Gq proteins, leading to calcium mobilization, protein kinase C (PKC) activation, and β-arrestin-mediated receptor internalization. These pathways drive mast cell degranulation, increased vascular permeability, persistent inflammation, and sensory nerve activation, central to CSU. PAF sustains chronic itch and inflammation through mast cells, basophils, neutrophils, endothelial cells, and sensory neurons. Targeting PAF pathways provides promising strategies for antihistamine-resistant CSU. View this paper
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19 pages, 3619 KiB  
Review
Combating Tuberculosis via Restoring the Host Immune Capacity by Targeting M. tb Kinases and Phosphatases
by Shahinda S. R. Alsayed and Hendra Gunosewoyo
Int. J. Mol. Sci. 2024, 25(22), 12481; https://doi.org/10.3390/ijms252212481 - 20 Nov 2024
Viewed by 1185
Abstract
Mycobacterium tuberculosis (M. tb) is a remarkably versatile pathogen that possesses a unique ability to counteract the host’s defence mechanisms to control the infection. Several mycobacterial protein kinases and phosphatases were found to play a key role in impeding phagosome maturation [...] Read more.
Mycobacterium tuberculosis (M. tb) is a remarkably versatile pathogen that possesses a unique ability to counteract the host’s defence mechanisms to control the infection. Several mycobacterial protein kinases and phosphatases were found to play a key role in impeding phagosome maturation in macrophages and accordingly blocking the phagosome–lysosome fusion, therefore allowing the bacteria to survive. During phagocytosis, both M. tb and the host’s phagocytic cells develop mechanisms to fight each other, resulting in pathogen elimination or survival. In this respect, M. tb uses a phosphorylation-based signal transduction mechanism, whereby it senses extracellular signals from the host and initiates the appropriate adaptation responses. Indeed, the ability of M. tb to exist in different states in the host (persistent quiescent state or actively replicating mode) is mainly mediated through protein phosphorylation/dephosphorylation signalling. The M. tb regulatory and defensive responses coordinate different aspects of the bacilli’s physiology, for instance, cell wall components, metabolic activity, virulence, and growth. Herein, we will discuss the implication of M. tb kinases and phosphatases in hijacking the host immune system, perpetuating the infection. In addition, the role of PknG, MPtpA, MPtpB, and SapM inhibitors in resetting the host immune system will be highlighted. Full article
(This article belongs to the Section Molecular Immunology)
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<p>Overview of the phagocytosis of microbial invaders and phagosome maturation, showing <span class="html-italic">M. tb</span> secreted kinases and phosphatases interfering with the developmental process of phagosomes. Generally, once engulfed, the pathogen remains confined within early phagosomes, which then undergo a maturation process that involves fusion with endosomes and lysosomes to eventually turn into phagolysosomes, the definitive pathogenicidal vacuoles, followed by fission and recycling of the endocytic vesicles. Early phagosomes are marked by the presence of EEA1, PI3<span class="html-italic">P</span>, and Rab5, which contribute to the phagosome–endosome fusion. Upon the procession of phagosomal maturation, V-ATPase accumulate on the phagosomal membrane, lowering the pH of phagosomal lumen. The acidic nature of phagolysosomes constitutes a harsh environment for the microbes and is a prerequisite for the activation of several hydrolytic enzymes. Subsequently, the antigen is degraded and presented, alerting the adaptive immune system. <span class="html-italic">M. tb</span>-secreted virulence factors PknG, MPtpA, MPtpB, and SapM impair the phagolysosome fusion, allowing the bacteria to survive. MPtpA impedes phagosome acidification via hydrolysing VPS33B and inhibiting the trafficking of V-ATPase to late phagosomes. MPtpB hydrolyses PI3<span class="html-italic">P</span> and PI(3,5)<span class="html-italic">P</span><sub>2</sub> that mediate the transition to late phagosomes and phagolysosome, respectively. Similar to MPtpB, SapM blocks the preceding transition events via hydrolysing PI(4,5)<span class="html-italic">P</span><sub>2</sub>, PI3<span class="html-italic">P</span> and binding to Rab7.</p> Full article ">
19 pages, 1623 KiB  
Article
Effect of Pre-Sowing Seed Stimulation on Maize Seedling Vigour
by Paulina Pipiak, Katarzyna Sieczyńska, Dorota Gendaszewska and Monika Skwarek-Fadecka
Int. J. Mol. Sci. 2024, 25(22), 12480; https://doi.org/10.3390/ijms252212480 - 20 Nov 2024
Viewed by 660
Abstract
The aim of this study was to investigate the effects of treating maize (Zea mays L.) seeds with fish collagen hydrolysate (FC) and keratin (KE) derived from animal waste by-products of leather and meat production, as well as poly(hexamethylene biguanide) hydrochloride (P) [...] Read more.
The aim of this study was to investigate the effects of treating maize (Zea mays L.) seeds with fish collagen hydrolysate (FC) and keratin (KE) derived from animal waste by-products of leather and meat production, as well as poly(hexamethylene biguanide) hydrochloride (P) and bentonite (B). This research is in line with the search for new, environmentally friendly methods to increase yields of industrial crops in a way that is compatible with sustainable development. The effect of the binders used was investigated by analysing the grown maize seedlings by determining changes in parameters of chlorophyll fluorescence, photosynthetic pigments, elemental composition and FTIR analysis on maize shoots. The results indicated a slightly higher fresh weight (FW) of shoots in plants treated with fish collagen, PHMB and bentonite (FC+P+B) and FW of roots in plants treated with keratin, PHMB and bentonite (KE+P+B). Unexpectedly, the FW and dry weight (DW) of both roots and shoots of all bentonite-treated plants were significantly higher than the corresponding non-bentonite-treated groups. In addition, changes in chlorophyll-a fluorescence were observed for the keratin, PHMB and bentonite variants. This study showed that the proposed materials could be promising seed pelleting agents to improve seed growth and yield. Full article
(This article belongs to the Section Molecular Plant Sciences)
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<p>Fresh weight (FW; (<b>A</b>)) and dry weight (DW; (<b>B</b>)) of shoots and fresh weight (FW; (<b>D</b>)) and dry weight (DW; (<b>E</b>)) of roots of 21-day-old maize plants (<b>C</b>) subjected to different seed treatments. Values followed by different letters within a given binding agent treatment (NT, FC+P or KE+P) are significantly different (<span class="html-italic">p</span> &lt; 0.05; ANOVA followed by Tukey’s post hoc test; <span class="html-italic">n</span> = 8); values within a given bentonite treatment (H+B, FC+P+B or KE+P+B) are significantly different (* for <span class="html-italic">p</span> &lt; 0.05, ** for <span class="html-italic">p</span> &lt; 0.01; Student’s <span class="html-italic">t</span>-test; <span class="html-italic">n</span> = 8). NT—non-treated seeds, FC—fish collagen-treated seeds, KE—keratin-treated seeds, P-PHMB—poly(hexamethylene biguanide) hydrochloride-treated seeds, H—water-treated seeds, B—bentonite-treated seeds.</p> Full article ">Figure 2
<p>Changes in chlorophyll fluorescence parameters in 21−day−old maize plants exposed to different seed coating treatments. Values followed by different letters within the given binding agent treatment (NT; (<b>A</b>), FC+P; (<b>B</b>) or KE+P; (<b>C</b>)) are significantly different (<span class="html-italic">p</span> &lt; 0.05; ANOVA followed by Tukey’s post hoc test; <span class="html-italic">n</span> = 5). The colour of the letter-based statistical indicators refers to each experimental variant as indicated in the legend. Abbreviations: Fo−basic fluorescence, Fm−maximal fluorescence, Fv−maximal variable fluorescence, Fm′−maximal fluorescence for the light-adapted state, Fv/Fm−maximum photochemical quantum yield of PSII in the dark-adapted state, Fv/Fo−efficiency of the water-splitting complex on the donor side of PSII, Rfd−vitality index, ФPSII—quantum efficiency of PSII, qP−photochemical fluorescence quenching, NPQ−non-photochemical fluorescence quenching.</p> Full article ">Figure 3
<p>Content of chlorophyll <span class="html-italic">a + b</span> (Chl <span class="html-italic">a + b</span>) (<b>A</b>), carotenoids (Cars) (<b>B</b>), ratio of chlorophyll <span class="html-italic">a + b</span> to carotenoids (Chl <span class="html-italic">a + b</span>/Cars) (<b>C</b>), porphyrins: protoporphyrin (Proto) (<b>D</b>), Mg−protoporphyrin (Mg−proto) (<b>E</b>) and protochlorophyllide (Pchlide) (<b>F</b>) in leaf discs of 21−day−old maize plants. Values followed by different letters within a given binding agent treatment (NT, FC+P or KE+P) are significantly different (<span class="html-italic">p</span> &lt; 0.05; ANOVA followed by Duncan’s post hoc test; <span class="html-italic">n</span> = 4). NT−non-treated seeds, FC−fish collagen-treated seeds, KE−keratin-treated seeds, P−PHMB-poly(hexamethylene biguanide) hydrochloride-treated seeds, H−water-treated seeds, B−bentonite-treated seeds.</p> Full article ">Figure 4
<p>FTIR spectra of 21−day−old maize leaves of the tested variants and the control sample: NT (black line), H+B (red line), FC+B (blue line), FC+P+B (pink line), KE+P (green line), KE+P+B (yellow line).</p> Full article ">
17 pages, 312 KiB  
Review
The Role of Alpha-Linolenic Acid and Other Polyunsaturated Fatty Acids in Mental Health: A Narrative Review
by Camilla Bertoni, Cecilia Pini, Alessandra Mazzocchi, Carlo Agostoni and Paolo Brambilla
Int. J. Mol. Sci. 2024, 25(22), 12479; https://doi.org/10.3390/ijms252212479 - 20 Nov 2024
Viewed by 1257
Abstract
The present review investigates the relationship between polyunsaturated fatty acids (PUFAs) and mental health disorders, such as dementia, psychosis, schizophrenia, Alzheimer’s disease, anorexia nervosa, and impairment problems in animals and human models. Data were collected from a variety of studies: randomized intervention trials, [...] Read more.
The present review investigates the relationship between polyunsaturated fatty acids (PUFAs) and mental health disorders, such as dementia, psychosis, schizophrenia, Alzheimer’s disease, anorexia nervosa, and impairment problems in animals and human models. Data were collected from a variety of studies: randomized intervention trials, observational and interventional studies, case reports, and epidemiological studies. The evidence suggests that PUFAs are beneficial for mental health, brain function, and behavior. ALA, EPA, and DHA have very significant neuroprotective properties, particularly in inducing changes to the synaptic membrane and modulating brain cell signaling. In the case of neurodegenerative disorders, PUFAs incorporated into cellular membranes have been shown to protect against cell atrophy and death. The formal analyses of the included studies pointed to a decrease in ALA, EPA, and DHA levels in various populations (e.g., children, adolescents, adults, and seniors) presenting with different types of mental disorders. These results indicate that PUFA supplementation may be considered as an innovative therapeutic strategy to reduce the risk of neuronal degeneration. Full article
(This article belongs to the Section Bioactives and Nutraceuticals)
15 pages, 2491 KiB  
Article
Long Noncoding RNA lncRNA-3 Recruits PRC2 for MyoD1 Silencing to Suppress Muscle Regeneration During Aging
by Zong-Kang Zhang, Daogang Guan, Jintao Xu, Xiaofang Li, Ning Zhang, Shanshan Yao, Ge Zhang and Bao-Ting Zhang
Int. J. Mol. Sci. 2024, 25(22), 12478; https://doi.org/10.3390/ijms252212478 - 20 Nov 2024
Viewed by 633
Abstract
Lowered muscle regenerative capacity in the elderly greatly contributes to the development of multiple diseases. The specific roles of long noncoding RNAs (lncRNAs) in muscle regenerative capacity during aging remain unknown. Here, we identify an elevated lncRNA (lncRNA-3), in association with reduced MyoD [...] Read more.
Lowered muscle regenerative capacity in the elderly greatly contributes to the development of multiple diseases. The specific roles of long noncoding RNAs (lncRNAs) in muscle regenerative capacity during aging remain unknown. Here, we identify an elevated lncRNA (lncRNA-3), in association with reduced MyoD expression and suppressed muscle regenerative capacity, in the skeletal muscle of aged mice. LncRNA-3 could interact with both the MyoD1 promoter and RbAp46/48, a subunit of Polycomb repressive complex 2 (PRC2). LncRNA-3 could recruit PRC2 to the MyoD1 promoter and enhance the MyoD1 silencing, which, in turn, suppressed the muscle regenerative capacity. Muscle-specific lncRNA-3 knockdown could restore the muscle regenerative capacity in the aged mice. Exogenous RbAp46/48 binding motif (Rb-motif-2) treatment in skeletal muscle could compete for the lncRNA-3 binding, and therefore, enhance the muscle regenerative capacity in the aged mice. Taken together, lncRNA-3 requires PRC2 for MyoD1 silencing to suppress muscle regenerative capacity during aging. These findings provide a novel therapeutic target and a new strategy to elevate the muscle regenerative capacity in the aged population. Full article
(This article belongs to the Section Molecular Biology)
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<p>Elevated lncRNA-3 expression level in the skeletal muscle was accompanied by a decreased muscle mass and reduced muscle regenerative capacity in the aged mice. (<b>a</b>–<b>c</b>) Gastrocnemius (GA) muscle-to-body weight ratio (<b>a</b>), H&amp;E staining of the cross-section of GA muscle (<b>b</b>) and the GA muscle specific force (<b>c</b>) between the adult and aged mice. (<b>d</b>,<b>e</b>) Representative images (<b>d</b>) and quantification analysis (<b>e</b>) of the gastrocnemius muscle cross-sections from adult and aged mice on day 0 and day 4 post-muscle injury stained for Pax7 (green) and nuclei (blue). Scale bar: 50 µm. (<b>f</b>) Real-time PCR analysis of top 10 upregulated lncRNA levels in the GA muscle of the adult and aged mice. The most upregulated one was named lncRNA-3. (<b>g</b>) Relative expression levels of lncRNA-3 (green) and the <span class="html-italic">MyoD1</span> mRNA (red) in the GA muscle from the mice at different ages. GAPDH and U6 were used as endogenous controls. <span class="html-italic">n</span> = 10. Data are presented as the mean ± SEM. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 vs. 6 mo. (<b>e</b>) ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 vs. day 0.</p> Full article ">Figure 2
<p>LncRNA-3 inhibited the myogenic differentiation in vitro and muscle regenerative capacity in mice. (<b>a</b>) Expression levels of lncRNA-3 of mouse muscle satellite cells transduced with either lncRNA-3 or shRNA vectors on day 7 of differentiation. (<b>b</b>) Representative images of mouse muscle satellite cells transduced with either lncRNA-3 or shRNA vectors on day 7 of differentiation. Myosin was labeled with green fluorescence, and the nuclei were labeled with DAPI. Scale bar: 50 μm. (<b>c</b>) The fusion index determined by the number of nuclei within the myosin-positive myotubes in each group on day 7 of differentiation. (<b>d</b>,<b>e</b>) Expression levels of the <span class="html-italic">MyoD1</span> mRNA (<b>g</b>) and MyoD protein (<b>h</b>) in the mouse muscle satellite cells in each group on day 7 of differentiation. (<b>f</b>) Real-time PCR analysis of the lncRNA-3 level in the gastrocnemius muscle of empty- or lncRNA-3-vector-infected mice following the CTX treatment. (<b>g</b>–<b>j</b>) Cross-sections from the mid-belly gastrocnemius muscle (<b>g</b>), muscle fiber cross-sectional area (CSA) (<b>h</b>), expression levels of the <span class="html-italic">MyoD1</span> mRNA (<b>i</b>) and MyoD protein (<b>j</b>) in the gastrocnemius muscle of empty- or lncRNA-3-vector-infected mice 28 days after the CTX treatment. Scale bar: 50 µm. Data are presented as the mean ± SEM. <span class="html-italic">n</span> = 3 for in vitro. <span class="html-italic">n</span> = 10 for in vivo. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 vs. baseline. <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 vs. CTX.</p> Full article ">Figure 3
<p>LncRNA-3 interacted with the histone methyltransferase PRC2 and mediated the <span class="html-italic">MyoD1</span> gene silencing in vitro. (<b>a</b>) Schematic of the interaction propensity for the full-length lncRNA-3 with RbAp46/48, as predicted by CatRAPID. (<b>b</b>) Representative Western blot analysis of the tagged-RNA streptavidin pulldown assay. (<b>c</b>) Representative RNA EMSA analysis of the flag-tagged RbAp46/48 pulldown assay. (<b>d</b>) Representative Western blot analysis of the RbAp46/48 co-immunoprecipitation assay using the anti-H3K27me3 antibody. (<b>e</b>) Chromatin immunoprecipitation (ChIP) assay using the anti-H3K27me3 antibody in the lncRNA-3- or empty-vector-transduced mouse muscle satellite cells, where the %input of the <span class="html-italic">MyoD1</span> promoter was detected by real-time PCR. (<b>f</b>) Representative Western blot analysis of the tagged-RNA streptavidin pulldown assay. Full-length or deletion fragments of the <span class="html-italic">MyoD1</span> promoter were transfected into the lncRNA-3-vector-transduced mouse muscle satellite cells and followed with the tagged-DNA streptavidin pulldown assay. (<b>g</b>) ChIP assay using anti-RbAp46/48 antibody in the lncRNA-3- or empty-vector-transduced mouse muscle satellite cells, where the %input of the <span class="html-italic">MyoD1</span> promoter was detected by real-time PCR. (<b>h</b>) Real-time PCR analysis of the <span class="html-italic">MyoD1</span> mRNA level in the mouse muscle satellite cells transfected with the wildtype or mutated RbAp46/48 (Rmut2). (<b>i</b>) Real-time PCR analysis of the <span class="html-italic">MyoD1</span> mRNA level in the mouse muscle satellite cells transduced with lncRNA-3 and treated with peptide with a random sequence (random) or RbAp46/48 motif 2 (Rb-motif-2). (<b>j</b>) Real-time PCR analysis of the <span class="html-italic">MyoD1</span> mRNA level in mouse muscle satellite cells transduced with lncRNA-3 and treated with the <span class="html-italic">MyoD1</span> promoter overexpression vector and empty vector. Data are presented as the mean ± SEM. <span class="html-italic">n</span> = 3. (<b>d</b>) * <span class="html-italic">p</span> &lt; 0.05 vs. Rb-WT. (<b>e</b>,<b>g</b>) * <span class="html-italic">p</span> &lt; 0.05 vs. lncRNA-3 vector. (<b>h</b>) * <span class="html-italic">p</span> &lt; 0.05 vs. wildtype. (<b>i</b>) * <span class="html-italic">p</span> &lt; 0.05 vs. random.</p> Full article ">Figure 4
<p>Skeletal-muscle-specific knockdown of lncRNA-3, the Rb-motif-2 treatment and the <span class="html-italic">MyoD1</span> promoter expression could rescue the muscle regenerative capacity in the muscle-specific lncRNA-3 knockin (Sk-lncRNA-3 KI) mice. (<b>a</b>) Real-time PCR analysis of the lncRNA-3 levels in the gastrocnemius muscle of the wildtype and Sk-lncRNA-3 KI mice. (<b>b</b>) Gastrocnemius muscle-to-body weight ratio in the wildtype and transgenic mice. (<b>c</b>) Cross-sections from the mid-belly gastrocnemius muscle (left) and muscle fiber CSA (right). Scale bar: 50 µm. (<b>d</b>) Western blot analysis of the MyoD protein level in each group. (<b>e</b>–<b>g</b>) Adult Sk-lncRNA-3 KI mice and wildtype mice were intramuscularly injected with CTX to induce muscle injury. The Sk-lncRNA-3 KI mice were followed with an intramuscular administration of lncRNA-3 shRNA, the synthesized peptide Rb-motif-2 and the <span class="html-italic">MyoD1</span>-promoter-overexpression system. (<b>e</b>) Real-time PCR analysis of the lncRNA-3 levels in the gastrocnemius muscle on day 7 post-treatment in each group. (<b>f</b>) Representative images (left) and the intensity (right) of EdU positive staining on day 7 post-treatment in each group. Red: EdU staining, blue: DAPI. (<b>g</b>) Real-time PCR analysis of the <span class="html-italic">MyoD1</span> mRNA levels on day 7 post-treatment in each group. <span class="html-italic">n</span> = 10. GAPDH and U6 small nuclear RNA were used as the endogenous controls of mRNA and lncRNA, respectively. β-actin was used as the endogenous control for MyoD protein. Data are presented as the mean ± SD. (<b>a</b>–<b>d</b>) * <span class="html-italic">p</span> &lt; 0.05 vs. wildtype. (<b>e</b>–<b>g</b>) * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 vs. lncRNA-3 KI.</p> Full article ">Figure 5
<p>Skeletal-muscle-specific lncRNA-3 knockdown and exogenous Rb-motif-2 treatment restored the muscle regenerative capacity in the aged mice. (<b>a</b>) LncRNA-3 level in the gastrocnemius muscle of the aged mice treated with the lncRNA-3 shRNA system or exogenous Rb-motif-2 in the skeletal muscle following a muscle injury. (<b>b</b>) EdU staining (red) and the nuclei (blue) indicating muscle satellite cell proliferation at 7 days post-muscle injury (upper panel) and H&amp;E staining of the cross-sections from the mid-belly gastrocnemius muscle at 28 days post-muscle injury (bottom panel). Scale bar: 50 µm. (<b>c</b>) The intensity of the EdU positive staining in each group at 7 days post-muscle injury. (<b>d</b>) Real-time PCR analysis of the <span class="html-italic">MyoD1</span> mRNA level in the gastrocnemius muscle at 7 days post-muscle injury. <span class="html-italic">n</span> = 10 for each group. Each sample was assessed in triplicate. GAPDH was used as the control for mRNA. Data are presented as the mean ± SEM. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 vs. CTX.</p> Full article ">
24 pages, 6866 KiB  
Article
Novel Exosomal miRNA Expression in Irradiated Human Keratinocytes
by Hebah Almujally, Nizar Abuharfeil and Aseel Sharaireh
Int. J. Mol. Sci. 2024, 25(22), 12477; https://doi.org/10.3390/ijms252212477 - 20 Nov 2024
Viewed by 980
Abstract
The epidermis, the outer layer of the skin, relies on a delicate balance of cell growth and keratinocyte differentiation for its function and renewal. Recent research has shed light on exosomes’ role in facilitating skin communication by transferring molecules like miRNAs, which regulate [...] Read more.
The epidermis, the outer layer of the skin, relies on a delicate balance of cell growth and keratinocyte differentiation for its function and renewal. Recent research has shed light on exosomes’ role in facilitating skin communication by transferring molecules like miRNAs, which regulate gene expression post-transcriptionally. Additionally, these factors lead to skin aging through oxidative stress caused by reactive oxygen species (ROS). In this research project, experiments were conducted to study the impact of Sun2000 solar simulator irradiation on exosomal miRNA profiles in HEKa cells. We hypothesized that acute oxidative stress induced by solar simulator irradiation would alter the expression profile of exosomal miRNAs in HEKa cells. The cells were exposed to different durations of irradiation to induce oxidative stress, and the levels of reactive ROS were measured using the CellROX Deep Red flow cytometry assay kit. Exosomes were isolated from both control and irradiated cells, characterized using DLS and SEM techniques, and their miRNAs were extracted and analyzed using qPCR. Solar simulator irradiation led to a time-dependent increase in intracellular ROS and a decrease in cell viability. Exosomal size increased in irradiated cells. Fifty-nine exosomal miRNAs were differentially expressed in irradiated HEKa cells, including hsa-miR-425-5p, hsa-miR-181b-5p, hsa-miR-196b-5p, hsa-miR-376c-3p, and hsa-miR-15a-5p. This study highlights the significant impact of solar radiation on exosomal miRNA expression in keratinocytes, suggesting their potential role in the cellular response to oxidative stress. Full article
(This article belongs to the Section Biochemistry)
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<p>The true relative ratio of fluorescence intensity (CellROX) of HEKa cells immediately (at selected time intervals (10, 20, 30, 40, 50, 60 min)) and 24 h post-solar simulator irradiation at different exposure times. As shown in the graph, the three biological repeats for both control cells and HEKa irradiated cells were compared immediately after irradiation and at 24 h post-irradiation after the addition of NAC and TBHP as further controls. The average of the true relative ratio of fluorescence intensity of HEKa cells immediately after the solar simulator irradiation was 15.137 ± 2.049. On the other hand, the average of the true relative ratio of fluorescence intensity of HEKa cells 24 h post the solar simulator irradiation was 6.978 ± 1.663. Furthermore, both variables can be assumed normally distributed using Q-Q plots with strong correlation (correlation value = 0.896 and <span class="html-italic">p</span>-value = 0.0156).</p> Full article ">Figure 2
<p>Cell viability of HEKa cells immediately and 24 h (24 h) post-solar simulator irradiation at different exposure times. A comparative analysis between the cell viability of HEKa cells immediately after solar simulator irradiation and 24 h after for each exposure time revealed a reduction in cell viability 24 h post-irradiation measurements compared to the immediate post-irradiation at different exposure times. The average cell viability of HEKa cells immediately after solar simulator irradiation was 72.320 ± 9.239, whereas the average cell viability of HEKa cells 24 h post-solar simulator irradiation was 54.934 ± 11.919. Furthermore, both variables could be assumed as normally distributed using Q-Q plots with strong correlation (correlation value = 0.997 and <span class="html-italic">p</span>-value &lt; 0.0001).</p> Full article ">Figure 3
<p>Quanta FEG-250 scanning electron micrographs of fixed HEKa-derived exosomes.</p> Full article ">Figure 4
<p>Zetasizer 90 data analysis; particle size distribution by intensity analysis of HEKa-derived exosomes. (<b>A</b>): Dynamic light scattering analysis of control samples. (<b>B</b>): Dynamic light scattering analysis of irradiated samples.</p> Full article ">Figure 5
<p>Heatmap with hierarchical clustering. The hierarchical clustering heatmap visualizes the differential expression of exosomal miRNAs in non-irradiated and 40 min irradiated HEKa cells. Each row represents a specific miRNA, and each column represents a biological sample. The color intensity of each cell corresponds to the relative expression level of a particular miRNA in a given sample. Red cells suggest upregulation in the irradiated group, while green cells indicate downregulation. The dendrograms provide insights into the hierarchical relationships among miRNAs and samples, potentially revealing miRNA families or groups with similar functions.</p> Full article ">
14 pages, 2370 KiB  
Article
Effect of Constant Illumination on the Morphofunctional State and Rhythmostasis of Rat Livers at Experimental Toxic Injury
by Sevil A. Grabeklis, Maria A. Kozlova, Lyudmila M. Mikhaleva, Alexander M. Dygai, Rositsa A. Vandysheva, Anna I. Anurkina and David A. Areshidze
Int. J. Mol. Sci. 2024, 25(22), 12476; https://doi.org/10.3390/ijms252212476 - 20 Nov 2024
Viewed by 548
Abstract
The effect of dark deprivation on the morphofunctional state and rhythmostasis of the liver under CCl4 toxic exposure has been studied. The relevance of this study is due to the fact that the hepatotoxic effect of carbon tetrachloride on the liver is [...] Read more.
The effect of dark deprivation on the morphofunctional state and rhythmostasis of the liver under CCl4 toxic exposure has been studied. The relevance of this study is due to the fact that the hepatotoxic effect of carbon tetrachloride on the liver is well studied, but there are very few data on the relationship between CCl4 intoxication and circadian biorhythms, and most of the studies consider the susceptibility of the organism in general and of the liver in particular to the influence of CCl4 in some separate periods of the rhythm, but not the influence of this chemical agent on the structure of the whole rhythm. In addition, earlier studies indicate that light disturbance causes certain changes in the morphofunctional state of the liver and the structure of the circadian rhythm of a number of parameters. As a result of this study, we found that the effect of CCl4 in conditions of prolonged dark deprivation causes more significant structural and functional changes in hepatocytes, as well as leading to significant changes in the circadian rhythms of a number of parameters, which was not observed in the action of CCl4 as a monofactor. We assume that the severity of structural and functional changes is due to the light-induced deficiency of melatonin, which has hepatoprotective properties. Thus, the mechanisms of CCl4 action on CRs under conditions of light regime violations leave a large number of questions requiring further study, including the role of melatonin in these processes. Full article
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
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<p>Liver of rats: (<b>A</b>) control group, hematoxylin and eosin, ×100; (<b>B</b>) control group, hematoxylin and eosin, ×400; (<b>C</b>) group I, hematoxylin and eosin, ×100; (<b>D</b>,<b>E</b>) group I, hematoxylin and eosin, ×400; (<b>F</b>) group II, hematoxylin and eosin, ×100; (<b>G</b>,<b>H</b>) group II, hematoxylin and eosin, ×400.</p> Full article ">Figure 1 Cont.
<p>Liver of rats: (<b>A</b>) control group, hematoxylin and eosin, ×100; (<b>B</b>) control group, hematoxylin and eosin, ×400; (<b>C</b>) group I, hematoxylin and eosin, ×100; (<b>D</b>,<b>E</b>) group I, hematoxylin and eosin, ×400; (<b>F</b>) group II, hematoxylin and eosin, ×100; (<b>G</b>,<b>H</b>) group II, hematoxylin and eosin, ×400.</p> Full article ">Figure 2
<p>Results of ICH studies: (<b>A</b>) control group, <span class="html-italic">Ki-67</span>; (<b>B</b>) group I, <span class="html-italic">Ki-67</span>; (<b>C</b>) group II, <span class="html-italic">Ki-67</span>. It can be seen that single cells with reaction results are present in the field of view only in animals of the first experimental group. (<b>D</b>) Control group, <span class="html-italic">Per2</span>; (<b>E</b>) group I, <span class="html-italic">Per2</span>; (<b>F</b>) group II, <span class="html-italic">Per2</span>, ×400.</p> Full article ">Figure 2 Cont.
<p>Results of ICH studies: (<b>A</b>) control group, <span class="html-italic">Ki-67</span>; (<b>B</b>) group I, <span class="html-italic">Ki-67</span>; (<b>C</b>) group II, <span class="html-italic">Ki-67</span>. It can be seen that single cells with reaction results are present in the field of view only in animals of the first experimental group. (<b>D</b>) Control group, <span class="html-italic">Per2</span>; (<b>E</b>) group I, <span class="html-italic">Per2</span>; (<b>F</b>) group II, <span class="html-italic">Per2</span>, ×400.</p> Full article ">
11 pages, 442 KiB  
Article
Coronavirus Disease 2019-Associated Thrombotic Microangiopathy: A Single-Center Experience
by Marija Malgaj Vrečko, Andreja Aleš-Rigler, Špela Borštnar and Željka Večerić-Haler
Int. J. Mol. Sci. 2024, 25(22), 12475; https://doi.org/10.3390/ijms252212475 - 20 Nov 2024
Viewed by 606
Abstract
Coronavirus disease 2019 (COVID-19) can lead to various multisystem disorders, including thrombotic microangiopathy (TMA). We present here eight patients with COVID-19-associated TMA who were treated at our center. Our aim was to summarize the demographic and clinical characteristics of the patients and discuss [...] Read more.
Coronavirus disease 2019 (COVID-19) can lead to various multisystem disorders, including thrombotic microangiopathy (TMA). We present here eight patients with COVID-19-associated TMA who were treated at our center. Our aim was to summarize the demographic and clinical characteristics of the patients and discuss the possible role of COVID-19. One patient presented with thrombotic thrombocytopenic purpura (TTP) and seven with atypical hemolytic–uremic syndrome (aHUS.) Most patients had no obvious symptoms of COVID-19, and TMA occurred after viremia. Two patients had concomitant non-COVID-19-related triggers for TMA: exposure to tacrolimus and everolimus; first presentation of antiphospholipid syndrome. The patient with TTP was treated with therapeutic plasma exchange (TPE), steroids and caplacizumab, resulting in complete hematologic recovery. Six patients with aHUS were treated with TPE with or without steroids, four of whom received a C5 complement inhibitor and one an intravenous immunoglobulin. One patient with aHUS was treated with a C5 complement inhibitor and a steroid. We observed one partial and one complete recovery of renal function, while five patients experienced renal failure. There were no deaths. We believe that COVID-19 may act as a trigger for TMA in patients who have either pre-existing endothelial injury or an underlying predisposition to complement activation, and may also trigger autoimmune diseases. As a consequence of the different underlying pathophysiologies, the treatment of COVID-19-associated TMA requires a specific approach based on the subtype of the syndrome and possible concomitant triggers. Full article
(This article belongs to the Special Issue Recent Advances in Pathophysiology and Immunology Related to SARS-CoV-2 Infection)
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<p>Vascular and glomerular thrombotic microangiopathy in patient with COVID-19-associated TMA, active: in small arteries, endothelium is swollen, lumen is closed, and arterial wall is thickened. Glomerular segmental acute thrombotic microangiopathy with mesangiolysis. Diffuse interstitial edema and bleeding into the interstitium, signs of diffuse tubular damage (hematoxylin and eosin, trichrome staining; 400× magnification). Images provided by Maja Frelih, Institute of Pathology, Medical Faculty, Ljubljana, Slovenia.</p> Full article ">
20 pages, 4799 KiB  
Article
Male Wistar Rats Chronically Fed with a High-Fat Diet Develop Inflammatory and Ionic Transport Angiotensin-(3–4)-Sensitive Myocardial Lesions but Preserve Echocardiographic Parameters
by Thuany Crisóstomo, Rafael Luzes, Matheus Leonardo Lima Gonçalves, Marco Antônio Estrela Pardal, Humberto Muzi-Filho, Glória Costa-Sarmento, Debora B. Mello and Adalberto Vieyra
Int. J. Mol. Sci. 2024, 25(22), 12474; https://doi.org/10.3390/ijms252212474 - 20 Nov 2024
Viewed by 626
Abstract
The central aim of this study was to investigate whether male Wistar rats chronically fed a high-fat diet (HFD) over 106 days present high levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), and Na+ and Ca2+ transport alterations in the [...] Read more.
The central aim of this study was to investigate whether male Wistar rats chronically fed a high-fat diet (HFD) over 106 days present high levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), and Na+ and Ca2+ transport alterations in the left ventricle, together with dyslipidemia and decreased glucose tolerance, and to investigate the influence of Ang-(3–4). The rats became moderately overweight with an expansion of visceral adiposity. Na+-transporting ATPases, sarco-endoplasmic reticulum Ca2+-ATPase (SERCA2a), and the abundance of Angiotensin II receptors were studied together with lipid and glycemic profiles from plasma and left-ventricle echocardiographic parameters fractional shortening (FS) and ejection fraction (EF). IL-6 and TNF-α increased (62% and 53%, respectively), but returned to normal levels with Angiotensin-(3–4) administration after 106 days. Significant lipidogram alterations accompanied a decrease in glucose tolerance. Angiotensin II receptors abundance did not change. (Na+ + K+)ATPase and ouabain-resistant Na+-ATPase were downregulated and upregulated, respectively, but returned to normal values upon Angiotensin-(3–4) administration. SERCA2a lost its ability to respond to excess ATP. Echocardiography showed no changes in FS or EF. We conclude that being overweight causes an increase in Ang-(3–4)-sensitive IL-6 and TNF-α levels, and ion transport alterations in the left ventricle that could evolve into future heart dysfunction. Full article
(This article belongs to the Special Issue Molecular Research in Obesity and Obesity Related Disorders: 2nd Edition)
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<p>Administration of the high-fat diet (HFD) over 104 days starting at 58 days of age leads to a small but significant overweight of male rats. Left panel: body mass at the beginning of different dietary exposures (zero time). Right panel: body mass 104 days later. Diets and days of exposure to the different diets are indicated on the abscissae; <span class="html-italic">n</span> = 30 (CTR rats) and <span class="html-italic">n</span> = 26 (HFD rats). Bars show mean ± SD. Differences between CTR and HFD were assessed on the same day using unpaired Student’s <span class="html-italic">t</span>-test; <span class="html-italic">p</span> value (Day 104) is indicated within the panel.</p> Full article ">Figure 2
<p>Evolution of systolic blood pressure in CTR (<span class="html-italic">n</span> = 30) and HFD (<span class="html-italic">n</span> = 26) rats. The data points show mean ± SD. Differences were assessed using one-way-ANOVA followed by Bonferroni’s test for selected pairs (CTR and HFD rats of the same age). * <span class="html-italic">p</span> &lt; 0.05; **** <span class="html-italic">p</span> &lt; 0.0001. The Ang-(3–4) data at day 106 (green circle in the CTR + Ang-(3–4) group; orange circle in HFD + Ang-(3–4) rats) are reproduced from Crisóstomo et al. [<a href="#B6-ijms-25-12474" class="html-bibr">6</a>] under the terms of Creative Common License CC BY 4.0.</p> Full article ">Figure 3
<p>Evolution of body mass among rats housed in metabolic cages: Effect of Ang-(3–4) administration [4 doses (2 doses per day) on days 104 and 105]. Diets, days of exposure to the different diets at the end of the study, and administration or not of Ang-(3–4) are indicated on the abscissae. CTR (<span class="html-italic">n</span> = 30) and HFD (<span class="html-italic">n</span> = 26) rats were divided into two subgroups. One of them was used to follow body mass evolution without Ang-(3–4) treatment between days 104 and 106 (<span class="html-italic">n</span> = 17 and 12 for CTR and HFD, respectively) (<b>A</b>,<b>B</b>). The other subgroup was used to follow body mass evolution after Ang-(3–4) administration (<span class="html-italic">n</span> = 13 and 14 for CTR and HFD, respectively) (<b>C</b>,<b>D</b>). Bars show mean ± SD. Differences between days 104 and 106 in each subgroup were assessed using paired Student’s <span class="html-italic">t</span>-test; <span class="html-italic">p</span> values are indicated within the panels. Even though the untreated CTR body mass values at 104 days in (<b>A</b>,<b>C</b>) are numerically different after the random formation of the subgroups, and the same applies for untreated HFD rats at 104 days in (<b>B</b>,<b>D</b>), there were no statistical differences within the pairs of CTR and HFD groups (<span class="html-italic">p</span> = 0.0569 and 0.2285, respectively; unpaired Student’s <span class="html-italic">t</span>-test).</p> Full article ">Figure 4
<p>Water intake (<b>A</b>), urinary volume (<b>B</b>), and water balance (<b>C</b>) in 24 h measured on day 106. Measurements were carried out after administering or not 2 doses of Ang-(3–4) on days 104 and 105 (total of 4 doses). Diets and administration or not of Ang-(3–4) are indicated on the abscissae; <span class="html-italic">n</span> = 10–16. Bars show mean ± SD. Differences were assessed using two-way ANOVA followed by Bonferroni’s test; <span class="html-italic">p</span> values are indicated within the panels.</p> Full article ">Figure 5
<p>Augmented visceral fat in HFD rats. Measurements were carried out on day 106 after administering or not 4 doses of Ang-(3–4) (2 each day) on days 104 and 105. Diets and administration or not of Ang-(3–4) are indicated on the abscissae. (<b>A</b>) Epididymal fat. (<b>B</b>) Perirenal fat. Bars show mean ± SD (<span class="html-italic">n</span> = 11–17). Differences were assessed using two-way ANOVA followed by Bonferroni’s test; <span class="html-italic">p</span> values are indicated within the panels.</p> Full article ">Figure 6
<p>Increased proinflammatory cytokines in microsomes of left-ventricle cardiomyocytes from HFD rats: recovery of control levels after Ang-(3–4) administration. (<b>A</b>) Representative Western blots of IL-6 (25 kDa). (<b>B</b>) Representative Western blots of TNF-α (23 kDa). Upper lanes, cytokines; bottom lanes, loading control, GAPDH (37 kDa). (<b>C</b>) Quantification of IL-6 levels. (<b>D</b>) Quantification of TNF-α levels. After administering 4 doses of Ang-(3–4) on days 104 and 105 (2 each day), measurements were carried out on day 106. Diets and administration or not of Ang-(3–4) are indicated above the blots (<b>A</b>,<b>B</b>) and on the abscissae (<b>C</b>,<b>D</b>). Bars show mean ± SD (<span class="html-italic">n</span> = 6 different preparations of cardiac microsomes that were the same for measuring both cytokines). Differences were assessed using two-way ANOVA followed by Bonferroni’s test; <span class="html-italic">p</span> values are indicated within the panels.</p> Full article ">Figure 7
<p>An early atypical lipidogram in overweight rats was encountered after 70 days of exposure to the HFD diet (128 days of life). (<b>A</b>) total cholesterol (TC), (<b>B</b>) high-density lipoproteins (HDL), (<b>C</b>) low-density lipoproteins (LDL), and (<b>D</b>) Triglycerides (TG) were measured in the same plasma samples (<span class="html-italic">n</span> = 10 for CTR and HFD groups). LDL cholesterol was estimated using the empirical formula LDL = TC − HDL − TG/5 [<a href="#B8-ijms-25-12474" class="html-bibr">8</a>]. Diets are indicated on the abscissae. Bars show mean ± SD. Differences were assessed using unpaired Student’s <span class="html-italic">t</span>-test; <span class="html-italic">p</span> values are indicated within the panels.</p> Full article ">Figure 8
<p>Moderate hyperglycemia and impaired glucose tolerance in HFD rats. (<b>A</b>) After 95 days on the different diets (153 days of life), the rats (<span class="html-italic">n</span> = 10 for CTR and HFD) were fasted for 12 h for a first determination of blood glucose (groups indicated on the abscissa). Bars correspond to mean ± SD. The difference was analyzed using unpaired Student’s <span class="html-italic">t</span>-test (<span class="html-italic">p</span> value shown within the panel). (<b>B</b>) After blood collection to determine fasting blood glucose, the rats received glucose by gavage (arrow), and plasma concentrations were measured at the times indicated on the abscissa. The points correspond to mean ± SD. Statistical comparison between means at each time point was performed using the unpaired Student’s <span class="html-italic">t</span>-test, following [<a href="#B9-ijms-25-12474" class="html-bibr">9</a>]. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001. The blue area shows the area under the curve in CTR rats and the pink area shows the extra area under the curve in HFD rats. (<b>C</b>) Quantifying the area under the glucose tolerance curves corresponding to CTR and HFD rats. Bars represent mean ± SD. The difference was investigated using the unpaired Student’s <span class="html-italic">t</span>-test (<span class="html-italic">p</span> value indicated within the panel).</p> Full article ">Figure 9
<p>The abundance of Ang II receptors in microsomes of left ventricle cardiomyocytes in HFD rats remains unmodified at day 106. AT<sub>1</sub>R (<b>A</b>,<b>C</b>) protein levels and AT<sub>2</sub>R (<b>B</b>,<b>D</b>) were measured after 106 days of exposure to the different diets. Ang-(3–4) (4 doses; 2 doses each day) was administered during days 104 and 105. Combinations of diets and treatment or not with Ang-(3–4) are indicated above the blots in (<b>A</b>,<b>B</b>), where 41 and 37 kDa correspond to the molecular masses of Ang II receptors and the loading control GAPDH, respectively. The combinations are also indicated on the abscissae of the receptor’s quantification panels (<b>C</b>,<b>D</b>), which show mean ± SD; <span class="html-italic">n</span> = 7–8. Differences were assessed using two-way ANOVA.</p> Full article ">Figure 10
<p>Opposite profiles of Na<sup>+</sup>-transporting ATPase activities in microsomes of left-ventricle cardiomyocytes from HFD rats. Combinations of diets and treatment or not with Ang-(3–4) are indicated on the abscissae and above the lanes of the representative Western blot. (<b>A</b>) Ouabain-sensitive (Na<sup>+</sup> + K<sup>+</sup>)ATPase activity. (<b>B</b>) Ouabain-resistant, furosemide-sensitive Na<sup>+</sup>-ATPase activity. Bars show mean ± SD; <span class="html-italic">n</span> = 8 in all cases, using different microsome preparations. (<b>C</b>) Representative Western blot of ouabain-sensitive (Na<sup>+</sup> + K<sup>+</sup>)ATPase; 110 kDa and 37 kDa correspond to the molecular masses of the α-catalytic subunit of (Na<sup>+</sup> + K<sup>+</sup>)ATPase and GAPDH, respectively. (<b>D</b>) Quantification of the immunodetections of ouabain-sensitive (Na<sup>+</sup> + K<sup>+</sup>)ATPase corrected by the loading control. Bars show mean ± SD; <span class="html-italic">n</span> = 4 microsome preparations. In (<b>A</b>,<b>B</b>,<b>D</b>), the differences were assessed using two-way ANOVA followed by Bonferroni’s test; <span class="html-italic">p</span> values are indicated within the panels.</p> Full article ">Figure 11
<p>Abnormal kinetics of sarco-endoplasmic reticulum Ca<sup>2+</sup>-ATPase (SERCA2a) in microsomes of left-ventricle cardiomyocytes from HFD rats: partial recovery of the control ATP concentration dependence in Ang-(3–4)-treated HFD animals. (<b>A</b>) Simulations of ATP concentration dependence of SERCA2a between 0.1 and 5 mM using experimental mean values of enzyme velocity (<span class="html-italic">n</span> = 5 different microsome preparations). The curves were generated using equations 1 or 2 (see text). Blue, CTR; pink, HFD; green, CTR + Ang-(3–4); orange, HFD + (Ang-(3–4). (<b>B</b>) V<sub>max</sub> values (nmol P<sub>i</sub> × mg<sup>−1</sup> × min<sup>−1</sup>). (<b>C</b>) K<sub>m ATP</sub> (mM). (<b>D</b>) K<sub>i ATP</sub> (mM). In (<b>B</b>,<b>C</b>,<b>D</b>), bars show mean ± SD (<span class="html-italic">n</span> = 4–5). Combinations of diets and treatment or not with Ang-(3–4) are indicated on the abscissae. Differences were assessed using two-way ANOVA followed by Bonferroni’s test (<b>B</b>,<b>C</b>) and one-way ANOVA followed Bonferroni’s test for selected pairs (<b>D</b>); <span class="html-italic">p</span> values are indicated within the panels.</p> Full article ">Figure 12
<p>Echocardiography at day 106 reveals unchanged structural and functional parameters of the left ventricle behind the metabolic, cellular, and molecular changes found in overweight rats. Measurements of the left-ventricular internal diameter at the end of diastole (LVIDd) and the end of systole (LVIDs) allow calculation of the fractional shortening (FS). Measurements of the left-ventricular end-diastolic volume (EDV) and end-systolic volume (ESV) allow calculation of ejection fraction (EF). Representative echocardiographic recordings of LVIDd (<b>A</b>) and LVIDs (<b>B</b>). Representative echocardiographic recordings of EDV (<b>C</b>) and ESV (<b>D</b>). (<b>E</b>) Ejection fraction calculated as EF (%) = [(EDV − ESV)/EDV] × 100. (<b>F</b>) Fractional shortening calculated as FS (%) = [(LVIDd − LVDIs)/LVIDd] × 100. Bars represent mean ± SD, which were compared using two-way ANOVA followed by Bonferroni’s test (<span class="html-italic">n</span> = 12–17 rats). Combinations of diets and treatment or not with Ang-(3–4) are indicated above the images and on the abscissae of the graphic representations.</p> Full article ">
31 pages, 6624 KiB  
Article
Multi-Spectroscopic and Molecular Modeling Studies of Interactions Between Anionic Porphyrin and Human Serum Albumin
by Tadeusz Strózik, Marian Wolszczak, Maria Hilczer, Magdalena Pawlak, Tomasz Wasiak, Piotr Wardęga, Maksim Ionov and Maria Bryszewska
Int. J. Mol. Sci. 2024, 25(22), 12473; https://doi.org/10.3390/ijms252212473 - 20 Nov 2024
Viewed by 602
Abstract
The subject of this study is the interaction between 5,10,15,20-tetrakis (4-sulfonatophenyl)–porphyrin (TSPP), a potential photosensitizer for photodynamic therapy (PDT) and radiotherapy, and human serum albumin (HSA), a crucial protein in the body. The main objective was to investigate the binding mechanisms, structural changes, [...] Read more.
The subject of this study is the interaction between 5,10,15,20-tetrakis (4-sulfonatophenyl)–porphyrin (TSPP), a potential photosensitizer for photodynamic therapy (PDT) and radiotherapy, and human serum albumin (HSA), a crucial protein in the body. The main objective was to investigate the binding mechanisms, structural changes, and potential implications of these interactions for drug delivery and therapeutic applications. Spectroscopic techniques and computational methods were employed to investigate the mechanism and effects of TSPP binding by HSA. The results suggest the possibility of simultaneous binding of three TSPP ions at binding sites of different affinity within albumin. The estimated values of the binding constant Kb for these sites were in the range of 0.6 to 6.6 μM−1. Laser flash photolysis indicated the stabilization of TSPP in the HSA structure, which resulted in prolonged lifetimes of the excited states (singlet and triplet) of porphyrin. Circular dichroism analysis was used to assess the changes in the secondary and tertiary structures of HSA upon TSPP binding. An analysis of the molecular docking results allowed us to identify the preferred TSPP binding sites within HSA and provided information on the specific interactions of amino acids involved in the stabilization of TSPP–HSA complexes. The estimated free energy of the binding of porphyrin at the three most favorable docking sites found in the HSA structure that was considered native were in the range of −80 to −41 kcal/mol. Finally, thermal unfolding studies showed that TSPP increased the stability of the secondary structure of albumin. All these findings contribute to the understanding of the interactions between TSPP and HSA, offering valuable insights for the development of novel cancer therapy approaches. Full article
(This article belongs to the Section Molecular Biophysics)
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<p>Absorption spectra of TSPP in pH 7 buffer upon addition of different amounts of HSA. Concentration of TSPP is constant and equal to 5.0 µM, HSA concentrations as indicated.</p> Full article ">Figure 2
<p>(<b>a</b>) Absorbance at λ = 423 nm versus total concentration of porphyrin measured for pH ~7 buffer solutions containing: (1) only TSPP, <span class="html-italic">A</span><sub>L</sub>; (2) 2 μM HSA and TSPP, <span class="html-italic">A<sub>obs</sub></span>; (3) 75 μM HSA and TSPP, <span class="html-italic">A</span><sub>LP</sub>. (<b>b</b>) Contribution of the porphyrin in the binding state to the absorbance observed at λ = 423 nm, <span class="html-italic">xA</span><sub>LP</sub>, as a function of <span class="html-italic">C</span><sub>TSPP</sub> The molar fraction <span class="html-italic">x</span> of the binding TSPP molecules is given by Equation (6). The theoretical curve given by Equation (9) is fitted to the experimental data by taking <span class="html-italic">K<sub>d</sub></span>, <span class="html-italic">n</span> and <span class="html-italic">ε<sub>b</sub></span> as adjustable parameters.</p> Full article ">Figure 3
<p>Dependence of [LP]/(<span class="html-italic">C</span><sub>HSA</sub>[L]) on the occupancy of the macromolecule [LP]/<span class="html-italic">C</span><sub>HSA</sub>. [LP] and <span class="html-italic">C</span><sub>HSA</sub> are concentrations of bound porphyrin and HSA, respectively. Experimental data are calculated using the absorption spectra shown in <a href="#ijms-25-12473-f001" class="html-fig">Figure 1</a>. Assuming that there are two different types of independent TSPP binding sites in the albumin, linear fits for experimental points in the range of low (1) and high (2) values of [LP]/<span class="html-italic">C</span><sub>HSA</sub> allow for a rough estimation of the binding parameters: <span class="html-italic">n</span><sub>1</sub>, <span class="html-italic">K<sub>d</sub></span><sub>1</sub>, <span class="html-italic">n</span><sub>2</sub>, and <span class="html-italic">K<sub>d</sub></span><sub>2</sub>.</p> Full article ">Figure 4
<p>Triplet–triplet (T–T) transient absorption spectra for N<sub>2</sub>-saturated buffer solutions containing 3 µM TSPP in the absence (red circles) and in the presence (black circles) of 20 µM HSA recorded 1.7 µs after a laser light pulse of wavelength 351 nm. The spectrum for the TSPP + HSA solution was multiplied by ~1.5 in order to normalize it to the maximum of the T–T band for the TSPP solution. The solid bold lines correspond to the modified absorption spectra of the considered solutions (original spectrum multiplied by −1 and normalized to the minimum of the corresponding transient absorption spectrum).</p> Full article ">Figure 5
<p>Kinetic curves of triplet–triplet absorbance decay recorded at λ = 460 nm for buffer solutions containing TSPP and HSA in the following molar ratios: 1:0 (10:0 µM; curve 1), 1:1 (30:30 µM; curve 2), 2:1 (40:20 µM; curve 3), 2.5:1 (50:20 µM; curve 4), 3:1 (30:10 µM; curve 5), and 4:1 (40:10 µM; curve 6). Kinetic curves were recorded after a pulse of laser light with a wavelength of λ = 351 nm. The solutions were vacuum deaerated. The kinetic curves were registered for 460 nm. Exponential functions of the best fit are superimposed on the experimental curves. Exponential best-fit functions are superimposed on the experimental curves (parameters of the fits are provided in the <a href="#app1-ijms-25-12473" class="html-app">Supplementary Materials Table S1</a>).</p> Full article ">Figure 6
<p>Far UV CD spectra of buffer solution of HSA (0.25 μM) and TSPP; the porphyrin to protein molar ratio, <span class="html-italic">C</span><sub>TSPP</sub>/<span class="html-italic">C</span><sub>HSA</sub>, changes from 0:1 to 150:1, room temperature. Inset: ellipticity measured at <span class="html-italic">λ</span> = 222 nm as a function of the porphyrin concentration in the buffer solution of HSA (0.25 μM).</p> Full article ">Figure 7
<p>Dependence of ellipticity, registered at <span class="html-italic">λ</span> = 222 nm for buffer solutions of HSA (0.5 µM) and TSPP, on the porphyrin concentration, <span class="html-italic">C</span><sub>TSPP</sub>/<span class="html-italic">C</span><sub>HSA</sub> changes from 0:1 to 25:1, room temperature. Values of ellipticity are obtained as averages of three independent measurements, the error bars are shown in the figure. Data points are fitted by straight lines plotted in the figure, and values of the correlation coefficients <span class="html-italic">R</span> are given for each linear fit 1 to 3. The original CD spectra for the data presented in <a href="#ijms-25-12473-f007" class="html-fig">Figure 7</a> have been added to the <a href="#app1-ijms-25-12473" class="html-app">Supplementary Material as Figure S2</a>.</p> Full article ">Figure 8
<p>Near UV CD spectra of the buffer solution of HSA (30 µM) and TSPP; the porphyrin to protein molar ratio, <span class="html-italic">C</span><sub>TSPP</sub>/<span class="html-italic">C</span><sub>HSA</sub>, changes from 0:1 to 5:1, room temperature. Inset: ellipticity measured at <span class="html-italic">λ</span> = 267 nm as a function of the porphyrin concentration in the buffer solution of HSA.</p> Full article ">Figure 9
<p>Overall view of the HSA native structure with three TSPP docking sites found, each marked with a circle: green for site 1, red for site 2, and black for site 3.</p> Full article ">Figure 10
<p>(<b>Upper Part</b>): Interactions between TSPP and amino acid residues within three binding sites of HSA (PDB ID: 1E78) illustrated in 2D using the Schrödinger Ligand Interaction Diagram. The three binding sites correspond to those depicted in <a href="#ijms-25-12473-f009" class="html-fig">Figure 9</a>. Residues are depicted as spheres labeled with their respective residue names and numbers, and colored based on their properties (refer to the legend). Interactions between residues and the ligand are visualized as lines, with each interaction type assigned a specific color (refer to the legend). The ligand’s binding pocket is delineated by a colored line surrounding it, indicating the nearest residue’s property. Hydrophobic residues are shown in green, positively charged residues in blue, negatively charged residues in red, and polar residues in cyan. The exposure of the ligand’s atoms to solvent is marked, indicated by a break in the line outlining the pocket. (<b>Lower Part</b>): The 3D structure of TSPP interacting with amino acid residues within the binding sites. Different types of interactions are highlighted with distinct colors for the marked bonds: yellow for hydrogen bonds, purple for ionic bonds, and brown for π-cation interactions.</p> Full article ">Figure 11
<p>The populations of contacts (interactions) between amino acid residues of albumin (PDB ID: 1E78) and TSPP obtained for the three binding sites during the Desmond MD simulation. The three binding sites correspond to those depicted in <a href="#ijms-25-12473-f009" class="html-fig">Figure 9</a>. Contacts are classified into four types (hydrogen bonds, hydrophobic, ionic, and water bridges) and each interaction type is assigned a color according to the legend.</p> Full article ">Figure 12
<p>Changes in far UV CD spectrum of the HSA buffer solution (<span class="html-italic">C</span><sub>HSA</sub> = 0.5 μM) caused by temperature increase from 25 to 80 °C. Inset: ellipticity recorded for wavelength <span class="html-italic">λ</span> = 222 nm as a function of temperature. The experimental points are fitted by two straight lines. For each linear fit the value of the correlation coefficient <span class="html-italic">R</span> is given.</p> Full article ">Figure 13
<p>Changes in far UV CD spectrum of the HSA (0.5 µM) and TSPP (1.5 µM) buffer solution caused by temperature increase from 25 to 80 °C. Inset: ellipticity recorded for wavelength <span class="html-italic">λ</span> = 222 nm as a function of temperature. The experimental points are fitted by two straight lines. For each linear fit the value of the correlation coefficient <span class="html-italic">R</span> is given.</p> Full article ">Figure 14
<p>Temperature dependence of fluorescence ratio F350/F330 obtained for buffer solutions of porphyrin and protein in a molar ratio <span class="html-italic">C</span><sub>TSPP</sub>/<span class="html-italic">C</span><sub>HSA</sub> varying from 0:1 to 5:1 (black circles). The inset in the first picture shows the density of measurement points provided by the Prometheus NT.48 device. The melting (unfolding) temperatures (Tm) for each solution are determined based on the graph of the first derivative of F350 with respect to the temperature. The experimental data are fitted with a polynomial function, and first derivative (blue line) displays peaks at the points of maximal slope, which correspond to Tm.</p> Full article ">
6 pages, 656 KiB  
Editorial
Vistas in Signaling Pathways Implicated in HSV-1 Reactivation
by Kostas A. Papavassiliou, Amalia A. Sofianidi, Fotios G. Spiliopoulos, Vassiliki A. Gogou and Athanasios G. Papavassiliou
Int. J. Mol. Sci. 2024, 25(22), 12472; https://doi.org/10.3390/ijms252212472 - 20 Nov 2024
Viewed by 525
Abstract
Ancient Greek physicians, including Hippocrates, documented skin conditions resembling herpes as early as 500 before common era (BCE), but it was not until the 1920s that Lowenstein successfully isolated the herpes virus from human lesions, significantly advancing our understanding of the infection [...] [...] Read more.
Ancient Greek physicians, including Hippocrates, documented skin conditions resembling herpes as early as 500 before common era (BCE), but it was not until the 1920s that Lowenstein successfully isolated the herpes virus from human lesions, significantly advancing our understanding of the infection [...] Full article
(This article belongs to the Special Issue Recent Advances in Herpesviruses)
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<p>The current scenery of signaling pathways that contribute to HSV-1 reactivation. NGF deprivation potentiates the neuronal JNK stress pathway, where DLK and JIP3 stimulate JNK, leading to the phosphorylation of histone 3 at serine residue 10 that induces lytic gene expression during phase I of HSV-1 reactivation. During phase II, histone demethylases (LSD1 and JMJD3/UTX) remove repressive chromatin marks from HSV-1 lytic promoters. Neuronal hyperexcitability is a DLK-mediated trigger of HSV-1 reactivation that can be induced by upstream IL-1β release. Axotomy/explant causes a rise in intracellular calcium levels that potentiates adenylyl cyclase and DLK/JIP3, resulting in JNK activation and perhaps the same histone methyl/phospho switch that triggers viral gene expression associated with HSV-1 reactivation under NGF deprivation. Increased intracellular calcium augments histone acetylation on HSV-1 lytic promoters due to the nuclear export of histone deacetylases (HDAC5 and HDAC3). Additionally, following axotomy/explant, the transcriptional cofactor HCF-1 translocates to the nucleus and binds to immediate early viral gene promoters recruiting histone demethylases (LSD1 and JMJD2) and methyltransferases (Set1) to upregulate lytic gene expression. While physiological NGF signaling and endogenous DNA repair mechanisms maintain HSV-1 latency via Akt activation (phosphorylation), endogenous DNA repair inhibition and accumulation of exogenous DNA damage allow the Akt Ser473-specific phosphatase PHLPP1 to dephosphorylate and deactivate Akt, evoking the reactivation of the HSV-1 genome. Wnt/β-catenin signaling promotes HSV-1 reactivation probably via a β-catenin-dependent transcriptional reprogramming event that orchestrates lytic gene expression and virus shedding. Red inhibitory arrows represent points of therapeutic targeting. Akt, protein kinase B; cAMP, cyclic adenosine monophosphate; DLK, dual leucine zipper kinase; H3K9me3, histone 3 lysine 9 trimethylation; H3pS10, phosphorylation of serine 10 of histone H3; HCF-1, host cell factor-1; HDAC5, histone deacetylase 5; HDAC3, histone deacetylase 3; IL-1β, interleukin-1 beta; IL1R, interleukin 1 receptor; ic, intracellular; JIP3, JNK-interacting scaffold protein 3; JMJD2, Jumonji C domain-containing histone lysine demethylase 2; JMJD3, Jumonji C domain-containing histone lysine demethylase 3; JNK, c-Jun N-terminal kinase; LSD1, lysine-specific histone demethylase 1; NHEJ, non-homologous end joining; NGF, nerve growth factor; PHLPP1, PH domain and leucine-rich repeat protein phosphatase 1; PI3K, phosphoinositide 3-kinase; PKA: protein kinase A; Set1, histone-lysine N-methyltransferase and H3 lysine-4 specific protein; S473, serine residue 473; Top2β, topoisomerase 2β; T308, threonine residue 308; UTX, ubiquitously transcribed tetratricopeptide repeat X chromosome. This figure was created using the tools provided by BioRender.com (accessed on 1 November 2024).</p> Full article ">Figure 2
<p>Summary of potential therapeutic targets associated with signaling pathways that promote HSV-1 reactivation. Examples of drugs that inhibit HSV-1 reactivation are included for most therapeutic targets.</p> Full article ">
19 pages, 2288 KiB  
Article
Astrocyte Dysfunction Reflected in Ischemia-Induced Astrocyte-Derived Extracellular Vesicles: A Pilot Study on Acute Ischemic Stroke Patients
by Timea Forró, Doina Ramona Manu, Lucian Barbu-Tudoran and Rodica Bălașa
Int. J. Mol. Sci. 2024, 25(22), 12471; https://doi.org/10.3390/ijms252212471 - 20 Nov 2024
Viewed by 760
Abstract
Extracellular vesicles (EVs) secreted by astrocytes (ADEVs) mediate numerous biological processes, providing insights into damage, repair, and protection following ischemic stroke (IS). This pilot study aimed to broaden the current knowledge on the astrocyte response to ischemia by dynamically assessing the aquaporin-4 (AQP4) [...] Read more.
Extracellular vesicles (EVs) secreted by astrocytes (ADEVs) mediate numerous biological processes, providing insights into damage, repair, and protection following ischemic stroke (IS). This pilot study aimed to broaden the current knowledge on the astrocyte response to ischemia by dynamically assessing the aquaporin-4 (AQP4) and glial cell line-derived neurotrophic factor (GDNF) as cargo proteins of these vesicles in eighteen acute IS patients and nine controls. EV proteins were detected by Western blotting and followed 24 h (D1), 7 days (D7), and one month (M1) after symptoms onset. The post-ischemic ADEV AQP4 and GDNF levels were higher at D1 compared to the control group (p = 0.006 and p = 0.023). Significant differences were observed in ADEV AQP4 during the three evaluated time points (n = 12, p = 0.013) and between D1 and D7 (z = 2.858, p = 0.012), but not in EV GDNF. There was a positive relationship between the severity of stroke at D1 according to the National Institutes of Health Stroke Scale, and ADEV AQP4 at D1 (r = 0.50, p = 0.031), as well as ADEV GDNF at D1 and D7 (r = 0.49, p = 0.035 and r = 0.53, p = 0.021, respectively). The release of EVs with distinct protein profiles can be an attractive platform for the development of biomarkers in IS. Full article
(This article belongs to the Special Issue Molecular, Cellular, and Blood Biomarkers in Acute Ischemic Stroke)
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<p>Representative Western blots for aquaporin-4 (AQP4) showing multiple bands in extracellular vesicle (EV) aliquots of an AIS patient with two key moments that missed one-month follow-up (<b>a</b>) and two patients with three key moments (<b>b</b>,<b>c</b>): one major band at ≈37 kilodaltons (kDa), additional bands at 50 and 75 kDa, suggesting AQP4 dimers or glycosylation, and between 100–150 kDa as possible tetramers. Approximate molecular weight (MW) markers in kDa are labeled adjacent on the left. Abbreviations: TEV—total extracellular vesicles; ADEV—astrocyte-derived extracellular vesicles; D1—24 h; D7—7 days; M1—one month after stroke onset.</p> Full article ">Figure 2
<p>Representative Western blots for glial cell line-derived neurotrophic factor (GDNF) of three AIS patients with three key moments (<b>a</b>,<b>b</b>). Data for patient 2 is presented on separate blots: D1 and D7 TEV (<b>a</b>), D7 ADEV and M1 (<b>b</b>). The blots display multiple bands in EV aliquots: at ≈25 kDa as a monomer, at ≈50 kDa as a dimer, and, additionally, near 75 and 150 kDa MW as a combination of a monomer and dimer<b>.</b> Approximate MW markers in kDa are labeled adjacent on the left.</p> Full article ">Figure 3
<p>AQP4 (<b>a</b>,<b>b</b>) and GDNF (<b>c</b>,<b>d</b>) band intensities in TEVs (<b>a</b>,<b>c</b>) and ADEVs (<b>b</b>,<b>d</b>) of AIS patients and control participants (C). Data are represented as individual value boxplots with median and interquartile range (IQR, Mann–Whitney U test; * <span class="html-italic">p</span>  &lt;  0.05; ** <span class="html-italic">p</span>  &lt;  0.01; ns—not significant).</p> Full article ">Figure 4
<p>AQP4 (<b>a</b>) and GDNF (<b>b</b>) band intensities in ADEVs during the patients’ follow-up: D1, D7, and M1. Data are represented as individual value boxplots with median and IQR (Friedman’s ANOVA, followed by Dunn’s post hoc only for AQP4 (<b>a</b>), as for GDNF, there were no statistically significant differences (<b>b</b>); * <span class="html-italic">p</span>  &lt;  0.05; ns—not significant).</p> Full article ">Figure 5
<p>Bead flow separation data for the anti-tetraspanin antibodies coupled with Exo-FITC staining showing beads with no captured EVs (<b>a</b>) and beads with captured EVs = tetraspanin-positive (CD9, CD63, and CD81) EVs population (<b>b</b>).</p> Full article ">Figure 6
<p>Bead flow separation data for the anti-GLAST antibody coupled with Exo-FITC staining showing beads with no captured EVs (<b>a</b>) and beads with captured EVs = GLAST-positive EVs population (<b>b</b>).</p> Full article ">Figure 7
<p>Transmission (TE, (<b>a</b>,<b>b</b>)) and scanning electron micrographs (SE, (<b>c</b>–<b>e</b>)) of the isolated EVs shown at two different magnifications of 50,000× (<b>a</b>,<b>c</b>) and 150,000× (<b>b</b>,<b>d</b>,<b>e</b>). Multiple vesicles with typical EV morphology can be seen in each image.</p> Full article ">
10 pages, 4414 KiB  
Article
Knockout of OsGAPDHC7 Gene Encoding Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase Affects Energy Metabolism in Rice Seeds
by Jin-Young Kim, Ye-Ji Lee, Hyo-Ju Lee, Ji-Yun Go, Hye-Mi Lee, Jin-Shil Park, Yong-Gu Cho, Yu-Jin Jung and Kwon-Kyoo Kang
Int. J. Mol. Sci. 2024, 25(22), 12470; https://doi.org/10.3390/ijms252212470 - 20 Nov 2024
Viewed by 583
Abstract
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major glycolytic enzyme that plays an important role in several cellular processes, including plant hormone signaling, plant development, and transcriptional regulation. In this study, we divided it into four groups through structural analysis of eight GAPDH genes identified [...] Read more.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major glycolytic enzyme that plays an important role in several cellular processes, including plant hormone signaling, plant development, and transcriptional regulation. In this study, we divided it into four groups through structural analysis of eight GAPDH genes identified in the rice genome. Among them, the expression level of five genes of cytosolic GAPDH was shown to be different for each organ. The mutation induction of the GAPDHC7 gene by the CRISPR/Cas9 system revealed that the 7 bp and 2 bp deletion, early end codon, was used in protein production. In addition, the selected mutants showed lower plant heights compared to the wild-type plants. To investigate the effect on carbohydrate metabolism, the expression of the genes of starch-branched enzyme I (SbeI), sucrose synthase (SS), and 3-phosphoglycer phosphokinase (PGK) increased the expression of the SBeI gene threefold in the knockout lines compared to the wild-type (WT) plant, while the expression of the SS and PGK genes decreased significantly. And the starch and soluble sugar content of the knockout lines increased by more than 60% compared to the WT plant. Also, the free amino acid content was significantly increased in the Gln and Asn contents of the knockout lines compared to the WT plants, while the contents of Gly and Ser were decreased. Our results suggest that OsGAPDHC7 has a great influence on energy metabolism, such as pre-harvested sprouting and amino acid content. Full article
(This article belongs to the Special Issue Genetic Analysis Based on CRISPR/Cas9 Technology: 2nd Edition)
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<p>The phylogenetic tree and sequence alignment of GAPDH proteins. (<b>A</b>) Neighbor-joining phylogenetic tree of the GAPDH family. The phylogenetic analysis was represented using the MEGA 5.1 software. The four groups (I, II, II, IV) are as follows: I, cytosolic <span class="html-italic">GAPDH</span>; II, non-phosphorylated <span class="html-italic">GAPDH</span>; II, chloroplastic <span class="html-italic">GAPDHB</span> subunit; IV, chloroplastic <span class="html-italic">GAPDHA</span> subunit. (<b>B</b>) The predicted Gp_dh_N domain is blue, and the Gp_dh_C domain is green. (<b>C</b>) Multiple sequence alignment of the domain in rice and <span class="html-italic">Arabidopsis</span>.</p> Full article ">Figure 2
<p>The expression patterns of <span class="html-italic">OsGAPDHC</span> genes. qPCR analysis expression of <span class="html-italic">OsGAPDHC</span> genes in four tissues of rice including roots, leaf, stem, and flower. Error bars represent standard deviations calculated in three replicates.</p> Full article ">Figure 3
<p>The <span class="html-italic">OsGAPDHC7</span> gene editing and sanger sequencing analysis. (<b>A</b>) Schematic diagram of the <span class="html-italic">OsGAPDHC7</span> gene with sgRNA designed in the NAD+ binding domain region and C-terminal domain region. (<b>B</b>) Sanger sequencing analysis results in the gene-edited plants. The underline indicates the PAM region. Deletion bases are indicated by “−” and their sizes, and insertion bases are indicated by “+” and their bases. Insertion bases are indicated in blue letters. Red arrows and letters indicate the positions and sequences of sgRNA. (<b>C</b>) Amino acid translation of edited regions. (<b>D</b>) The phenotype of <span class="html-italic">gapdhc7-2</span> and <span class="html-italic">gapdhc7-13</span> mutant lines and WT plants.</p> Full article ">Figure 4
<p>Change in GAPDH activity in the seedling of the <span class="html-italic">gapdhc7-2</span> and <span class="html-italic">gapdhc7-13</span> mutant lines and WT plants. Error bars represent standard deviations calculated in three replicates. Statistical significance was analyzed with Student’s <span class="html-italic">t</span>-test (** <span class="html-italic">p</span> &lt; 0.05, * <span class="html-italic">p</span> &lt; 0.1).</p> Full article ">Figure 5
<p>Changes in gene expression in the <span class="html-italic">gapdhc7-2</span> and <span class="html-italic">gapdhc7-13</span> mutant lines and WT plants. (<b>A</b>) <span class="html-italic">OsGAPDHC7</span> gene expression level assay. (<b>B</b>) Starch branching enzyme I (<span class="html-italic">OsSb</span>e<span class="html-italic">I</span>) expression level assay. (<b>C</b>) Sucrose synthase (<span class="html-italic">OsSS</span>) expression level assay. (<b>D</b>) 3-Phosphoglyceric phosphokinase (<span class="html-italic">PGK</span>) expression level assay. The immature samples were stored at −80 °C for 60 days and used in the experiments. Error bars represent standard deviations calculated in three replicates. Statistical significance was analyzed with Student’s <span class="html-italic">t</span>-test (*** <span class="html-italic">p</span> &lt; 0.01, ** <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 6
<p>Changes in carbohydrate metabolism in <span class="html-italic">gapdhc7-2</span> and <span class="html-italic">gapdhc7-13</span> mutant lines and WT plants. (<b>A</b>) Starch contents analysis. (<b>B</b>) Fructose contents analysis. (<b>C</b>) Glucose contents analysis. (<b>D</b>) Galactose contents analysis. Seeds were stored at room temperature and used in experiments. Error bars represent standard deviations calculated in three replicates. Statistical significance was analyzed with Student’s <span class="html-italic">t</span>-test (*** <span class="html-italic">p</span> &lt; 0.01, ** <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">
17 pages, 1837 KiB  
Article
A Study of the Community Relationships Between Methanotrophs and Their Satellites Using Constraint-Based Modeling Approach
by Maryam A. Esembaeva, Mikhail A. Kulyashov, Fedor A. Kolpakov and Ilya R. Akberdin
Int. J. Mol. Sci. 2024, 25(22), 12469; https://doi.org/10.3390/ijms252212469 - 20 Nov 2024
Viewed by 613
Abstract
Biotechnology continues to drive innovation in the production of pharmaceuticals, biofuels, and other valuable compounds, leveraging the power of microbial systems for enhanced yield and sustainability. Genome-scale metabolic (GSM) modeling has become an essential approach in this field, which enables a guide for [...] Read more.
Biotechnology continues to drive innovation in the production of pharmaceuticals, biofuels, and other valuable compounds, leveraging the power of microbial systems for enhanced yield and sustainability. Genome-scale metabolic (GSM) modeling has become an essential approach in this field, which enables a guide for targeting genetic modifications and the optimization of metabolic pathways for various industrial applications. While single-species GSM models have traditionally been employed to optimize strains like Escherichia coli and Lactococcus lactis, the integration of these models into community-based approaches is gaining momentum. Herein, we present a pipeline for community metabolic modeling with a user-friendly GUI, applying it to analyze interactions between Methylococcus capsulatus, a biotechnologically important methanotroph, and Escherichia coli W3110 under oxygen- and nitrogen-limited conditions. We constructed models with unmodified and homoserine-producing E. coli strains using the pipeline implemented in the original BioUML platform. The E. coli strain primarily utilized acetate from M. capsulatus under oxygen limitation. However, homoserine produced by E. coli significantly reduced acetate secretion and the community growth rate. This homoserine was taken up by M. capsulatus, converted to threonine, and further exchanged as amino acids. In nitrogen-limited modeling conditions, nitrate and ammonium exchanges supported the nitrogen needs, while carbon metabolism shifted to fumarate and malate, enhancing E. coli TCA cycle activity in both cases, with and without modifications. The presence of homoserine altered cross-feeding dynamics, boosting amino acid exchanges and increasing pyruvate availability for M. capsulatus. These findings suggest that homoserine production by E. coli optimizes resource use and has potential for enhancing microbial consortia productivity. Full article
(This article belongs to the Special Issue Biochemical and Molecular Aspects of Bioremediation of Soils with Organic Pollutants by Microorganisms)
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<p>(<b>A</b>) The impact of the oxygen/methane ratio on acetate production predicted by the <span class="html-italic">i</span>McBath model after modifications. (<b>B</b>) The effect of nitrate reduction in the medium on acetate production predicted by the <span class="html-italic">i</span>McBath model following the modifications.</p> Full article ">Figure 2
<p>Schematic representation of the developed pipeline in the BioUML platform [<a href="#B43-ijms-25-12469" class="html-bibr">43</a>] for the reconstruction and analysis of the <span class="html-italic">M. capsulatus</span> and <span class="html-italic">E. coli</span> microbial community models. Initially, the original <span class="html-italic">i</span>McBath and <span class="html-italic">i</span>EC1372_W3110 models were modified using the COBRApy library [<a href="#B50-ijms-25-12469" class="html-bibr">50</a>] and the OptFlux tool [<a href="#B47-ijms-25-12469" class="html-bibr">47</a>] to enable acetate secretion in <span class="html-italic">i</span>McBath and homoserine production in the <span class="html-italic">i</span>EC1372_W3110 model (see <a href="#sec4-ijms-25-12469" class="html-sec">Section 4</a>). These models are combined into a community model using the PyCoMo tool [<a href="#B31-ijms-25-12469" class="html-bibr">31</a>]. Subsequently, modifications to the community model can be performed using the ‘apply_fixed_abundance’ and ‘bounds’ methods with followed up parsimonious flux balance analysis (pFBA) in COBRApy (see <a href="#sec4-ijms-25-12469" class="html-sec">Section 4</a>). Finally, fluxes for cross-feeding metabolites within the community are visualized using ScyNet [<a href="#B52-ijms-25-12469" class="html-bibr">52</a>], where the color shows which metabolites are consumed (turquoise lines) and which are excreted (orange lines) by community members from (and to) the environment.</p> Full article ">Figure 3
<p>Schematic representation of cross-feeding metabolites in the community consisting of <span class="html-italic">M. capsulatus</span> and <span class="html-italic">E. coli</span> under oxygen-limited conditions (<b>A</b>), <span class="html-italic">M. capsulatus</span> and modified <span class="html-italic">E. coli</span> under oxygen-limited conditions (<b>B</b>), <span class="html-italic">M. capsulatus</span> and <span class="html-italic">E. coli</span> under nitrogen-limited conditions (<b>C</b>), and <span class="html-italic">M. capsulatus</span> and modified <span class="html-italic">E. coli</span> under nitrogen-limited conditions (<b>D</b>). <span class="html-italic">M. capsulatus</span> is shown in purple, while <span class="html-italic">E. coli</span> is shown in yellow. Oxygen-limited conditions are highlighted with a green background, whereas nitrogen-limited conditions are represented with a pink background. Metabolites from the medium are indicated in green, and cross-feeding metabolites are shown in pink. Red arrows represent metabolite uptake from the medium, black arrows indicate internal reactions of the models, and blue arrows indicate exchange reactions. The numbers above the blue arrows indicate metabolite flux with units of mmol·gDCW<sup>−1</sup>·h<sup>−1</sup>, with flux values taken from <a href="#app1-ijms-25-12469" class="html-app">Supplementary Table S2</a>.</p> Full article ">
21 pages, 4891 KiB  
Article
BMAL1—Potential Player of Aberrant Stress Response in Q31L Mice Model of Affective Disorders: Pilot Results
by Kristina Smirnova, Tamara Amstislavskaya and Liudmila Smirnova
Int. J. Mol. Sci. 2024, 25(22), 12468; https://doi.org/10.3390/ijms252212468 - 20 Nov 2024
Viewed by 673
Abstract
Dysregulation in the stress-response system as a result of genetical mutation can provoke the manifestation of affective disorders under stress conditions. Mutations in the human DISC1 gene is one of the main risk factors of affective disorders. It was known that DISC1 regulates [...] Read more.
Dysregulation in the stress-response system as a result of genetical mutation can provoke the manifestation of affective disorders under stress conditions. Mutations in the human DISC1 gene is one of the main risk factors of affective disorders. It was known that DISC1 regulates a large number of proteins including BMAL1, which is involved in the regulation of glucocorticoid synthesis in the adrenal glands and the sensitivity of glucocorticoid receptor target genes. Male mice with a point mutation Q31L in the Disc1 gene were exposed to chronic unpredictable stress (CUS), after which the behavioral and physiological stress response assessed. To assess whether there were any changes in BMAL1 in key brain regions involved in the stress response, immunohistochemistry was applied. It was shown that the Q31L mice had an aberrant behavioral response, especially to the 2 weeks of CUS, which was expressed in unchanged motor activity, increased time of social interaction, and alterations in anxiety and fear-related behavior. Q31L males did not show an increase in blood corticosterone levels after CUS and a decrease in body weight. Immunohistochemical analysis in intact Q31L mice revealed a decrease in BMAL1 immunofluorescence in the CA1 hippocampal area and lateral habenula. Thus, the Q31L mutation of the Disc1 gene disrupts behavioral and physiological stress response and the BMAL1 dysregulation may underlie it, so this protein can act as a molecular target for the treatment of affective disorders. Full article
(This article belongs to the Special Issue Advances in Animal Models in Biomedical Research, 2nd Edition)
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<p>Locomotor activity parameters in the OF test. Data presented as the median, Q1 and Q3, minimum and maximum as whiskeys, x—mean; Int—intact animals, CUS-2—animals after two weeks of CUS, CUS-4—animals after four weeks of CUS; *<sup>,#,<span>$</span></sup> <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. *—significant difference between genotypes in subgroups, #—significant difference between CUS subgroup and Int inside one genotype, <span>$</span>—significant difference between CUS-2 and -4 inside one genotype. Sample size: 8–10 mice in each group.</p> Full article ">Figure 2
<p>Anxiety related parameters of the OF test. Data presented as median, Q1 and Q3, minimum and maximum as whiskeys, x—mean; Int—intact animals, CUS-2—animals after two weeks of CUS, CUS-4—animals after four weeks of CUS; * <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, ***<sup>, ###</sup> <span class="html-italic">p</span> &lt; 0.001; *—significant difference between genotypes in subgroups. Sample size: 8–10 mice in each group.</p> Full article ">Figure 3
<p>Anxiety related parameters of the DLC test. Data presented as mean ± SE. Int—intact animals, CUS-2—animals after two weeks of CUS, CUS-4—animals after four weeks of CUS; ** <span class="html-italic">p</span> &lt; 0.01. Sample size: 8–10 mice in each group.</p> Full article ">Figure 4
<p>Freezing in the OF test. Data presented as mean ± SE. Int—intact animals, CUS-2—animals after two weeks of CUS, CUS-4—animals after four weeks of CUS; *<sup>,<span>$</span></sup> <span class="html-italic">p</span> &lt; 0.05, ***<sup>,###</sup> <span class="html-italic">p</span> &lt; 0.001; *—significant difference between genotypes in subgroups, <span>$</span>—significant difference between CUS-2 and -4 inside one genotype. Sample size: 8–10 mice in each group.</p> Full article ">Figure 5
<p>Social preference and social motivation. Data presented as mean ± SE. S2—animals after two weeks of CUS, S4—animals after four weeks of CUS; <sup>@,#,<span>$</span></sup> <span class="html-italic">p</span> &lt; 0.05, **<sup>,@@,##,<span>$</span><span>$</span></sup> <span class="html-italic">p</span> &lt; 0.01, ***<sup>,@@@</sup> <span class="html-italic">p</span> &lt; 0.001; #—significant difference between CUS subgroups and Int inside one genotype, <span>$</span>—significant difference between CUS-2 and -4 inside one genotype, @—significant difference between social object and dummy. Sample size: 8–10 mice in each group.</p> Full article ">Figure 6
<p>Average distance from mice to the intruder. Data presented as mean ± SE. Int—intact animals, CUS-2—animals after two weeks of CUS, CUS-4—animals after four weeks of CUS; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, **<sup>,##,<span>$</span><span>$</span></sup> <span class="html-italic">p</span> &lt; 0.01; #—significant difference between CUS subgroups and Int inside one genotype. Sample size: 8–10 mice in each group.</p> Full article ">Figure 7
<p>Minute immobility changes in the FST. Data presented as median. Int—intact animals, CUS-2—animals after two weeks of CUS, CUS-4—animals after four weeks of CUS; *<sup>,<span>$</span></sup> <span class="html-italic">p</span> &lt; 0.05, **<sup>,##</sup> <span class="html-italic">p</span> &lt; 0.01, <sup>###,<span>$</span><span>$</span><span>$</span></sup> <span class="html-italic">p</span> &lt; 0.001; *—significant difference between genotypes in subgroups, <span>$</span>—first time of high immobility level compared with the first minute in the Q31L group. Sample size: 8–10 mice in each group.</p> Full article ">Figure 8
<p>Common immobile floating during last 4 min of the FST. Data presented as mean ± SE. Int—intact animals, CUS-2—animals after two weeks of CUS, CUS-4—animals after four weeks of CUS; **<sup>,##</sup> <span class="html-italic">p</span> &lt; 0.01. Sample size: 8–10 mice in each group.</p> Full article ">Figure 9
<p>Plasma corticosterone level. Data presented as median, Q1 and Q3, minimum and maximum as whiskeys; X—mean. Int—intact animals, CUS-2—animals after two weeks of CUS, CUS-4—animals after four weeks of CUS; *<sup>,<span>$</span></sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01; *—significant difference between genotypes in subgroups, <span>$</span>—significant difference between CUS-2 and -4 inside one genotype. Sample size: 7–8 mice in each group.</p> Full article ">Figure 10
<p>Body weight changes in mice after CUSs. Data presented as median, Q1 and Q3, minimum and maximum as whiskeys; x—mean. Int—intact animals, CUS-2—animals after two weeks of CUS, CUS-4—animals after four weeks of CUS; **<sup>,##</sup> <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001. Sample size: 8–10 mice in each group.</p> Full article ">Figure 11
<p>BMAL1 immunofluorescence in the hippocampal area. Data presented as mean ± SE. *** <span class="html-italic">p</span> &lt; 0.001. Sample size: 5 mice of each strain.</p> Full article ">Figure 12
<p>Microphotography of the hippocampal area, zoom 20×. DG—dentate gyrus. White boxes—frame where data were obtained.</p> Full article ">Figure 13
<p>(<b>A</b>) BMAL1 immunofluorescence in the habenular region. Data presented as mean ± SE. *** <span class="html-italic">p</span> &lt; 0.001. Sample size: 5 mice of each strain. (<b>B</b>) Microphotography of the habenular area, zoom 20×. MHb—medial habenula, LHb—lateral habenula. White boxes—frame where data were obtained.</p> Full article ">Figure 14
<p>Experimental design. INT—intact animals, CUS-2—animals after two weeks of CUS, CUS-4—animals after four weeks of CUS; in squares—quantity of male mice per each genotype, in rectangles—description of used methods.</p> Full article ">Figure A1
<p>Neuroendocrine components of the stress-response system. Picture based on Smith, S.M. and Vale, W.W. (2006) [<a href="#B20-ijms-25-12468" class="html-bibr">20</a>] Black arrows—neutral or cyclical influences; blue arrows—inhibitory influences; red arrows—activating influences. Abbreviations: AVP—arginine vasopressin; ACTH—adrenocorticotropic hormone; GABA—gamma-aminobutyric acid; GC—glucocorticoids; ArN—arcuate nucleus; DMN—dorsomedial nucleus of the hypothalamus; ILC—infralimbic cortex; CRH—corticotropin releasing hormone; PVN—paraventricular nucleus of the hypothalamus; PLC—perilimbic cortex; POA—preoptic area; PFC—prefrontal cortex; SCN—suprachiasmatic nucleus; NTS—nuclei of the terminal strip; SN—Nucleus of the solitary tract; Psycho. stressors—psychological stressors.</p> Full article ">Figure A2
<p>CUS timetable used in the present study. ** is undistinguished time of stressor ending.</p> Full article ">
23 pages, 2272 KiB  
Review
Effect of Oxidative Stress on Mitochondrial Damage and Repair in Heart Disease and Ischemic Events
by Paweł Kowalczyk, Sebastian Krych, Karol Kramkowski, Agata Jęczmyk and Tomasz Hrapkowicz
Int. J. Mol. Sci. 2024, 25(22), 12467; https://doi.org/10.3390/ijms252212467 - 20 Nov 2024
Viewed by 1088
Abstract
The literature analysis conducted in this review discusses the latest achievements in the identification of cardiovascular damage induced by oxidative stress with secondary platelet mitochondrial dysfunction. Damage to the platelets of mitochondria as a result of their interactions with reactive oxygen species (ROS) [...] Read more.
The literature analysis conducted in this review discusses the latest achievements in the identification of cardiovascular damage induced by oxidative stress with secondary platelet mitochondrial dysfunction. Damage to the platelets of mitochondria as a result of their interactions with reactive oxygen species (ROS) and reactive nitrogen species (RNS) can lead to their numerous ischemic events associated with hypoxia or hyperoxia processes in the cell. Disturbances in redox reactions in the platelet mitochondrial membrane lead to the direct oxidation of cellular macromolecules, including nucleic acids (DNA base oxidation), membrane lipids (lipid peroxidation process) and cellular proteins (formation of reducing groups in repair proteins and amino acid peroxides). Oxidative changes in biomolecules inducing tissue damage leads to inflammation, initiating pathogenic processes associated with faster cell aging or their apoptosis. The consequence of damage to platelet mitochondria and their excessive activation is the induction of cardiovascular and neurodegenerative diseases (Parkinson’s and Alzheimer’s), as well as carbohydrate metabolism disorders (diabetes). The oxidation of mitochondrial DNA can lead to modifications in its bases, inducing the formation of exocyclic adducts of the ethano and propano type. As a consequence, it disrupts DNA repair processes and conduces to premature neoplastic transformation in critical genes such as the p53 suppressor gene, which leads to the development of various types of tumors. The topic of new innovative methods and techniques for the analysis of oxidative stress in platelet mitochondria based on methods such as a nicking assay, oxygen consumption assay, Total Thrombus formation Analysis System (T-Tas), and continuous-flow left ventricular assist devices (CF-LVADs) was also discussed. They were put together into one scientific and research platform. This will enable the facilitation of faster diagnostics and the identification of platelet mitochondrial damage by clinicians and scientists in order to implement adequate therapeutic procedures and minimize the risk of the induction of cardiovascular diseases, including ischemic events correlated with them. A quantitative analysis of the processes of thrombus formation in cardiovascular diseases will provide an opportunity to select specific anticoagulant and thrombolytic drugs under conditions of preserved hemostasis. Full article
(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Cardiovascular Diseases)
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<p>Methods related to platelet analysis (LVADs, T-Tas, nicking assay, and oxygen consumption assay) (own driving).</p> Full article ">Figure 2
<p>Principle of mitochondria dysfunction (own driving) and analyzed by methods of nicking assay and oxygen consumption assay [<a href="#B77-ijms-25-12467" class="html-bibr">77</a>,<a href="#B78-ijms-25-12467" class="html-bibr">78</a>].</p> Full article ">Figure 3
<p>Proposed scheme of carcinogenic factors leading to oxidative stress-induced reactive oxygen species (ROS) and nitrogen (RNS) species; these can trigger lipid peroxidation that yields dialdehydes and alkenals, which cause exocyclic DNA base damage. PUFA, polyunsaturated fatty acid; iNOS, inducible nitric oxide synthase [<a href="#B135-ijms-25-12467" class="html-bibr">135</a>].</p> Full article ">Figure 4
<p>Major pathways for formation of exocyclic propano and etheno DNA adducts resulting from lipid peroxidation products and environmental mutagens/carcinogens [<a href="#B143-ijms-25-12467" class="html-bibr">143</a>] with modifications.</p> Full article ">Figure 5
<p>Oxidative changes inside the mitochondria of blood platelets. <a href="https://biologydictionary.net/mitochondria/" target="_blank">https://biologydictionary.net/mitochondria/</a>, accessed on 1 January 2020 with own modifications.</p> Full article ">
20 pages, 5198 KiB  
Article
Multi-Omics Approaches Uncovered Critical mRNA–miRNA–lncRNA Networks Regulating Multiple Birth Traits in Goat Ovaries
by Weibing Lv, Ren An, Xinmiao Li, Zengdi Zhang, Wanma Geri, Xianrong Xiong, Shi Yin, Wei Fu, Wei Liu, Yaqiu Lin, Jian Li and Yan Xiong
Int. J. Mol. Sci. 2024, 25(22), 12466; https://doi.org/10.3390/ijms252212466 - 20 Nov 2024
Viewed by 715
Abstract
The goat breeding industry on the Tibetan Plateau faces strong selection pressure to enhance fertility. Consequently, there is an urgent need to develop goat lines with higher fertility and adaptability. The ovary, as a key organ determining reproductive performance, is regulated by a [...] Read more.
The goat breeding industry on the Tibetan Plateau faces strong selection pressure to enhance fertility. Consequently, there is an urgent need to develop goat lines with higher fertility and adaptability. The ovary, as a key organ determining reproductive performance, is regulated by a complex transcriptional network involving numerous protein-coding and non-coding genes. However, the molecular mechanisms of the key mRNA–miRNA–lncRNA regulatory network in goat ovaries remain largely unknown. This study focused on the histology and differential mRNA/miRNA/lncRNA between Chuanzhong black goat (CBG, high productivity, multiple births) and Tibetan goat (TG, strong adaptability, single birth) ovaries. Histomorphological analysis showed that the medulla proportion in CBG ovaries was significantly reduced compared to TG. RNA-Seq and small RNA-Seq analysis identified 1218 differentially expressed (DE) mRNAs, 100 DE miRNAs, and 326 DE lncRNAs, which were mainly enriched in ovarian steroidogenesis, oocyte meiosis, biosynthesis of amino acids and protein digestion, and absorption signaling pathways. Additionally, five key mRNA–miRNA–lncRNA interaction networks regulating goat reproductive performance were identified, including TCL1B–novel68_mature–ENSCHIT00000010023, AKAP6–novel475_mature–ENSCHIT00000003176, GLI2–novel68_mature–XR_001919123.1, ITGB5–novel65_star–TCONS_00013850, and VWA2–novel71_mature–XR_001919911.1. Further analyses showed that these networks mainly affected ovarian function and reproductive performance by regulating biological processes such as germ cell development and oocyte development, which also affected the plateau adaptive capacity of the ovary by participating in the individual immune and metabolic capacities. In conclusion, we identified numerous mRNA–miRNA–lncRNA interaction networks involved in regulating ovarian function and reproductive performance in goats. This discovery offers new insights into the molecular breeding of Tibetan Plateau goats and provides a theoretical foundation for developing new goat lines with high reproductive capacity and strong adaptability to the plateau environment. Full article
(This article belongs to the Special Issue Molecular Studies in Endocrinology and Reproductive Biology—Second Edition)
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<p>Morphological and statistical analysis of ovarian tissues. (<b>A</b>,<b>B</b>) H&amp;E staining image of the maximum cross-section of TG (<b>A</b>) and CBG (B) ovaries, scale bar = 1000 μm. (<b>C</b>) The number of follicles in ovarian tissue by statistical analysis. (<b>D</b>) The total area of the maximum cross-section of ovaries. (<b>E</b>,<b>F</b>) The area (<b>E</b>) and proportion (<b>F</b>) of medullary in the maximum cross-section of ovaries. (<b>G</b>) Relative expression levels of reproduction-related marker genes in ovarian tissues. Note: ** represents <span class="html-italic">p</span> &lt; 0.01, * represents <span class="html-italic">p</span> &lt; 0.05, and ns represents <span class="html-italic">p</span> &gt; 0.05.</p> Full article ">Figure 2
<p>DE mRNA screening and functional enrichment analysis between CBG and TG. (<b>A</b>) FPKM density distribution curve. (<b>B</b>) PCA plot. (<b>C</b>) Volcano plot of DE mRNAs. The <span class="html-italic">X</span>-axis was log<sub>2</sub>FoldChange and the <span class="html-italic">Y</span>-axis was −log<sub>10</sub>pValue. (<b>D</b>) Bar chart of DE mRNAs statistics. The <span class="html-italic">x</span>-axis represents the comparison groups, and the y-axis represents the number of differential genes in each group. (<b>E</b>) RT-qPCR validation of DE mRNAs. (<b>F</b>,<b>G</b>) GO enrichment analysis for up-regulated mRNAs (<b>F</b>) and (<b>G</b>) down-regulated mRNAs. The <span class="html-italic">x</span>-axis represented GO term names, and the <span class="html-italic">y</span>-axis represented −log<sub>10</sub>pValue. (<b>H</b>,<b>I</b>) KEGG enrichment analysis for up-regulated mRNAs (<b>H</b>) and (<b>I</b>) down-regulated mRNAs. The <span class="html-italic">X</span>-axis represented the enrichment score.</p> Full article ">Figure 3
<p>DE miRNA screening and functional enrichment analysis. (<b>A</b>) Length distribution of miRNAs. (<b>B</b>) PCA plot. (<b>C</b>) Volcano plot of DE miRNAs. (<b>D</b>) Histogram of DE miRNAs number in these two groups. (<b>E</b>) RT-qPCR validation of DE miRNAs. (<b>F</b>,<b>G</b>) Top 10 bar plots of GO enrichment analysis for target genes of up-regulated miRNAs (<b>F</b>) and down-regulated miRNAs (<b>G</b>). The <span class="html-italic">Y</span>-axis represented GO terms and the <span class="html-italic">X</span>-axis was −log<sub>10</sub>pValue. (<b>H</b>,<b>I</b>) KEGG enrichment analysis for target genes of up-regulated miRNAs (<b>H</b>) and down-regulated miRNAs (<b>I</b>). The <span class="html-italic">X</span>-axis was the enrichment score.</p> Full article ">Figure 4
<p>DE lncRNA screening and functional enrichment analysis in the CBG and BG. (<b>A</b>) Venn diagram of coding potential prediction results for candidate lncRNAs. (<b>B</b>) FPKM density distribution curve. (<b>C</b>) PCA plot. (<b>D</b>) Volcano plot of DE lncRNAs expression. (<b>E</b>) Histogram of DE lncRNAs statistics. (<b>F</b>) RT-qPCR validation of DE lncRNAs. (<b>G</b>,<b>H</b>) GO enrichment analysis for up-regulated lncRNAs (<b>G</b>) and down-regulated lncRNAs (<b>H</b>). The <span class="html-italic">X</span>-axis represented GO term names; The <span class="html-italic">Y</span>-axis represented −log<sub>10</sub>pValue. (<b>I</b>,<b>J</b>) KEGG enrichment analysis for up-regulated lncRNAs (<b>I</b>) and down-regulated lncRNAs (<b>J</b>). The <span class="html-italic">X</span>-axis represented the enrichment score, with larger bubbles indicating more DE lncRNAs, and bubble color ranging between purple, blue, green and red, with smaller <span class="html-italic">p</span> values indicating higher significance. G: GO:0043154： negative regulation of cysteine-type endopeptidase activity. GO:2000480： negative regulation of cytokine-mediated signaling pathway. H: GO:0000981： DNA-binding transcription factor activity, RNA polymerase II-specific. GO:0001077： proximal promoter DNA-binding transcription activator activity, RNA polymerase II-specific. GO:0000978： RNA polymerase II cis-regulatory region sequence-specific DNA binding.</p> Full article ">Figure 5
<p>Co-expression analysis of lncRNAs and mRNAs. (<b>A</b>) GO enrichment analysis of co-expressed DE genes with lncRNAs (top 10). The <span class="html-italic">X</span>-axis represented GO terms, and the <span class="html-italic">Y</span>-axis represented the number of lncRNAs enriched. (<b>B</b>) KEGG enrichment bubble chart of co-expressed DE genes with lncRNAs (top 10). The <span class="html-italic">X</span>-axis represented the enrichment score, with larger bubbles indicating more DE genes in the term, and bubble color ranging from gray to red, reflecting decreasing <span class="html-italic">p</span> -values and increasing significance. CP: Cellular Processes; EIP: Environmental Information Processing; GIP: Genetic Information Processing; HD: Human Diseases; Meta.: Metabolism; OS: Organismal Systems. (<b>C</b>) Analysis of lncRNA trans-acting target genes. The red circles represented lncRNAs, green inverted triangles represented genes, and node size indicated quantity. (<b>D</b>) Analysis of lncRNA cis-acting target genes. The left and right sides of the <span class="html-italic">y</span>-axis represented mRNA and lncRNA, respectively. The <span class="html-italic">x</span>-axis indicated the distance between mRNA and lncRNA, with negative values indicating upstream and positive values indicating downstream. Identical lncRNAs were represented by the same color bar chart. Note: ** represents <span class="html-italic">p</span> &lt; 0.01, * represents <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 6
<p>CeRNA interaction network analysis. (<b>A</b>) Co-expression network analysis of miRNAs and mRNAs. (<b>B</b>) Regulatory network analysis of mRNAs and lncRNAs. (<b>C</b>) Regulatory network analysis of miRNAs and lncRNAs. (<b>D</b>) Regulatory network analysis of mRNAs, miRNAs, and lncRNAs. Red circles represented mRNAs, green triangles represented miRNAs, and blue rounded rectangles represented lncRNAs. The size of the shapes indicated the quantity. (<b>E</b>) GO enrichment analysis of mRNAs in ceRNA. The <span class="html-italic">Y</span>-axis represented GO terms, and the <span class="html-italic">X</span>-axis represented the enrichment score. (<b>F</b>) KEGG enrichment analysis of mRNAs in ceRNA. The <span class="html-italic">X</span>-axis represented the enrichment score, and the <span class="html-italic">Y</span>-axis represented enriched pathways.</p> Full article ">Figure 7
<p>Key mRNA–miRNA–lncRNA regulating goat lambing traits and plateau adaptability.</p> Full article ">
25 pages, 6147 KiB  
Review
The Gut–Heart Axis: Molecular Perspectives and Implications for Myocardial Infarction
by Katherine Rivera, Leticia Gonzalez, Liena Bravo, Laura Manjarres and Marcelo E. Andia
Int. J. Mol. Sci. 2024, 25(22), 12465; https://doi.org/10.3390/ijms252212465 - 20 Nov 2024
Viewed by 1394
Abstract
Myocardial infarction (MI) remains the leading cause of death globally, imposing a significant burden on healthcare systems and patients. The gut–heart axis, a bidirectional network connecting gut health to cardiovascular outcomes, has recently emerged as a critical factor in MI pathophysiology. Disruptions in [...] Read more.
Myocardial infarction (MI) remains the leading cause of death globally, imposing a significant burden on healthcare systems and patients. The gut–heart axis, a bidirectional network connecting gut health to cardiovascular outcomes, has recently emerged as a critical factor in MI pathophysiology. Disruptions in this axis, including gut dysbiosis and compromised intestinal barrier integrity, lead to systemic inflammation driven by gut-derived metabolites like lipopolysaccharides (LPSs) and trimethylamine N-oxide (TMAO), both of which exacerbate MI progression. In contrast, metabolites such as short-chain fatty acids (SCFAs) from a balanced microbiota exhibit protective effects against cardiac damage. This review examines the molecular mediators of the gut–heart axis, considering the role of factors like sex-specific hormones, aging, diet, physical activity, and alcohol consumption on gut health and MI outcomes. Additionally, we highlight therapeutic approaches, including dietary interventions, personalized probiotics, and exercise regimens. Addressing the gut–heart axis holds promise for reducing MI risk and improving recovery, positioning it as a novel target in cardiovascular therapy. Full article
(This article belongs to the Special Issue Molecular Advances and Insights on Diagnosis, Prevention and Therapeutics of Cardiovascular Diseases)
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<p>Host–microorganism interface. (<b>A</b>) Schematic representation of the main components of the intestinal barrier. (<b>B</b>) Junctional complexes linking adjacent epithelial cells in normal and impaired intestinal barrier.</p> Full article ">Figure 2
<p>Complexity of the gut microbiota and its adaptation to different microenvironments in the lower GI tract. Four major bacterial phyla (Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria) are found in different sections of the GI tract. Oxygen levels decrease progressively from the stomach to the colon, reflecting a shift from an aerobic to an anaerobic environment. Population density and mucus thickness both increase from the stomach to the colon, corresponding with higher microbial diversity and density in the large intestine, while pH decreases along the tract, providing favorable conditions for specific bacterial communities in different regions.</p> Full article ">Figure 3
<p>Risk factors in the gut–heart axis in health and disease. In a healthy state (eubiosis), factors like exercise and a fiber- and antioxidant-rich diets support beneficial gut bacteria, boosting SCFA production and limiting harmful compounds like TMA and LPS. Conversely, risk factors such as a Western diet, aging, antibiotics, and pollution lead to gut dysbiosis, where pathogenic bacteria increase inflammatory mediators, impair gut integrity, and raise systemic inflammation and MI risk.</p> Full article ">
28 pages, 1245 KiB  
Review
Remodeling of the Intracardiac Ganglia During the Development of Cardiovascular Autonomic Dysfunction in Type 2 Diabetes: Molecular Mechanisms and Therapeutics
by Anthony J. Evans and Yu-Long Li
Int. J. Mol. Sci. 2024, 25(22), 12464; https://doi.org/10.3390/ijms252212464 - 20 Nov 2024
Viewed by 594
Abstract
Type 2 diabetes mellitus (T2DM) is one of the most significant health issues worldwide, with associated healthcare costs estimated to surpass USD 1054 billion by 2045. The leading cause of death in T2DM patients is the development of cardiovascular disease (CVD). In the [...] Read more.
Type 2 diabetes mellitus (T2DM) is one of the most significant health issues worldwide, with associated healthcare costs estimated to surpass USD 1054 billion by 2045. The leading cause of death in T2DM patients is the development of cardiovascular disease (CVD). In the early stages of T2DM, patients develop cardiovascular autonomic dysfunction due to the withdrawal of cardiac parasympathetic activity. Diminished cardiac parasympathetic tone can lead to cardiac arrhythmia-related sudden cardiac death, which accounts for 50% of CVD-related deaths in T2DM patients. Regulation of cardiovascular parasympathetic activity is integrated by neural circuitry at multiple levels including afferent, central, and efferent components. Efferent control of cardiac parasympathetic autonomic tone is mediated through the activity of preganglionic parasympathetic neurons located in the cardiac extensions of the vagus nerve that signals to postganglionic parasympathetic neurons located in the intracardiac ganglia (ICG) on the heart. Postganglionic parasympathetic neurons exert local control on the heart, independent of higher brain centers, through the release of neurotransmitters, such as acetylcholine. Structural and functional alterations in cardiac parasympathetic postganglionic neurons contribute to the withdrawal of cardiac parasympathetic tone, resulting in arrhythmogenesis and sudden cardiac death. This review provides an overview of the remodeling of parasympathetic postganglionic neurons in the ICG, and potential mechanisms contributing to the withdrawal of cardiac parasympathetic tone, ventricular arrhythmogenesis, and sudden cardiac death in T2DM. Improving cardiac parasympathetic tone could be a therapeutic avenue to reduce malignant ventricular arrhythmia and sudden cardiac death, increasing both the lifespan and improving quality of life of T2DM patients. Full article
(This article belongs to the Special Issue Cellular and Molecular Progression of Cardiovascular Diseases)
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<p>A schematic diagram of the anatomy and physiology of cardiac parasympathetic innervation of the heart. GPA: ganglionic parasympathetic axon, GPN: ganglionic parasympathetic neuron, ACh: acetylcholine, MCR: muscarinic cholinergic receptor, nAChR: nicotinic acetylcholine receptor. Figure was created in <a href="http://BioRender.com" target="_blank">BioRender.com</a>.</p> Full article ">Figure 2
<p>Cellular and Molecular Mechanisms Driving Cardiac Vagal Postganglionic Parasympathetic Neuronal Dysfunction in T2DM. AGE: Advanced glycation end product, ROS: reactive oxygen species, CVP: cardiac vagal postganglionic. Figure was created in BioRender.com.</p> Full article ">
17 pages, 2008 KiB  
Review
Ferroptosis and Its Potential Role in the Physiopathology of Skeletal Muscle Atrophy
by Chen-Chen Sun, Jiang-Ling Xiao, Chen Sun and Chang-Fa Tang
Int. J. Mol. Sci. 2024, 25(22), 12463; https://doi.org/10.3390/ijms252212463 - 20 Nov 2024
Viewed by 808
Abstract
Skeletal muscle atrophy is a major health concern, severely affecting the patient’s mobility and life quality. In the pathological process of skeletal muscle atrophy, with the progressive decline in muscle quality, strength, and function, the incidence of falling, fracture, and death is greatly [...] Read more.
Skeletal muscle atrophy is a major health concern, severely affecting the patient’s mobility and life quality. In the pathological process of skeletal muscle atrophy, with the progressive decline in muscle quality, strength, and function, the incidence of falling, fracture, and death is greatly increased. Unfortunately, there are no effective treatments for this devastating disease. Thus, it is imperative to investigate the exact pathological molecular mechanisms underlying the development of skeletal muscle atrophy and to identify new therapeutic targets. Decreased muscle mass, strength, and muscle fiber cross-sectional area are typical pathological features and manifestations of skeletal muscle atrophy. Ferroptosis, an emerging type of programmed cell death, is characterized by iron-dependent oxidative damage, lipid peroxidation, and reactive oxygen species accumulation. Notably, the understanding of its role in skeletal muscle atrophy is emerging. Ferroptosis has been found to play an important role in the intricate interplay between the pathological mechanisms of skeletal muscle atrophy and its progression caused by multiple factors. This provides new opportunities and challenges in the treatment of skeletal muscle atrophy. Therefore, we systematically elucidated the ferroptosis mechanism and its progress in skeletal muscle atrophy, aiming to provide a comprehensive insight into the intricate relationship between ferroptosis and skeletal muscle atrophy from the perspectives of iron metabolism and lipid peroxidation and to provide new insights for targeting the pathways related to ferroptosis and the treatment of skeletal muscle atrophy. Full article
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
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<p>Regulatory pathways of protein synthesis and degradation. IGF/PI3K/AKT regulates protein homeostasis. IGF/PI3K/AKT/GSK3β and IGF/PI3K/AKT/m-TOR promote protein synthesis. IGF/PI3K/AKT/FOXO suppresses protein degradation. The calcium-dependent calpain system, cysteine–aspartate protease system, mandolin–proteasome pathway, ubiquitin–proteasome system (UPS), and the autophagy–lysosome pathway (ALP) promoting protein degradation.</p> Full article ">Figure 2
<p>Ferroptosis has been implicated in various systemic diseases.</p> Full article ">Figure 3
<p>Signaling pathways that contribute to ferroptosis. (1) Transferrin (TF) binds to the transferrin receptor (TfR) to mediate Fe<sup>3+</sup> entry into cells. The Fe<sup>3+</sup> is reduced to Fe<sup>2+</sup> by the ferriductase STEAP3, and the free Fe<sup>2+</sup> is transported out of the endosome via the divalent metal transporter 1 (DMT1). Increased free Fe<sup>2+</sup> in cells promotes the production of hydroxyl radicals (·OH) and ROS via the Fenton reaction, triggering ferroptosis. (2) Activating the system Xc-GSH-GPX4 pathway inhibits ferroptosis. (3) ACSL4 and LPCAT3 facilitate the conversion of PUFA to PUFA-CoAs, increasing cellular sensitivity to ferroptosis. (4) LOX (such as ALOX15) drives ferroptosis by promoting the peroxidation of PUFA. (5) Erastin inhibits the system Xc-GSH-GPX4, leading to the accumulation of lipid peroxidation and triggering ferroptosis. (6) Erastin inhibits the Keap/Nrf2/Ho-1 pathway, resulting in increased free Fe<sup>2+</sup>. (7) Erastin directly interacts with VDAC2/3 and induces mitochondrial dysfunction, triggering ferroptosis.</p> Full article ">
12 pages, 2319 KiB  
Review
Continuous Use During Disuse: Mechanisms and Effects of Spontaneous Activity of Unloaded Postural Muscle
by Boris S. Shenkman, Vitaliy E. Kalashnikov, Kristina A. Sharlo, Olga V. Turtikova, Roman O. Bokov and Timur M. Mirzoev
Int. J. Mol. Sci. 2024, 25(22), 12462; https://doi.org/10.3390/ijms252212462 - 20 Nov 2024
Viewed by 539
Abstract
In most mammals, postural soleus muscles are involved in the maintenance of the stability of the body in the gravitational field of Earth. It is well established that immediately after a laboratory rat is exposed to conditions of weightlessness (parabolic flight) or simulated [...] Read more.
In most mammals, postural soleus muscles are involved in the maintenance of the stability of the body in the gravitational field of Earth. It is well established that immediately after a laboratory rat is exposed to conditions of weightlessness (parabolic flight) or simulated microgravity (hindlimb suspension/unloading), a sharp decrease in soleus muscle electrical activity occurs. However, starting from the 3rd day of mechanical unloading, soleus muscle electrical activity begins to increase and reaches baseline levels approximately by the 14th day of hindlimb suspension. This phenomenon, observed in the course of rat hindlimb suspension, was named the “spontaneous electrical activity of postural muscle”. The present review discusses spinal mechanisms underlying the development of such spontaneous activity of rat soleus muscle and the effect of this activity on intracellular signaling in rat soleus muscle during mechanical unloading. Full article
(This article belongs to the Special Issue Molecular Insight into Skeletal Muscle Atrophy and Regeneration)
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<p>Alterations in the levels of integrated rat soleus EMG activity before and during 2-week HS. Values are means ± SE. *—significantly different from Control (pre-suspension) level (<span class="html-italic">p</span> &lt; 0.05). Modified from [<a href="#B8-ijms-25-12462" class="html-bibr">8</a>].</p> Full article ">Figure 2
<p>Changes in rat soleus EMG activity and L5 afferent activity (V s<sup>–1</sup>) during 14-day HS. Data are expressed as mean ± SD. *—significant difference from the pre-suspension (CONT) values (<span class="html-italic">p</span> &lt; 0.05). Modified from [<a href="#B9-ijms-25-12462" class="html-bibr">9</a>].</p> Full article ">Figure 3
<p>Regulation of the content of intracellular chloride ions in mature neurons and its effect on the function of GABAA receptor. (<b>a</b>) In intact mature motoneurons, KCC2 activity is higher than NKCC1 activity, resulting in a gradual outflow of chloride ions from the cytoplasm and an increase in concentration of Cl<sup>−</sup> ions on the outer side of the membrane; (<b>b</b>) binding of GABA to its receptors on the membrane surface opens ion channels, resulting in an influx of Cl<sup>−</sup> ions into the motoneuron and subsequent membrane hyperpolarization; (<b>c</b>) in immature motoneurons and after spinal cord injury, higher levels of NKCC1 and lower levels of KCC2 lead to the accumulation of Cl<sup>−</sup> ions at the inner side of the membrane; (<b>d</b>) the opening of ion channels caused by binding of GABA to its receptors causes outflow of Cl<sup>−</sup> ions and membrane depolarization.</p> Full article ">Figure 4
<p>Spinal cord injury may result in reduced expression of KCC2 in motoneuron membranes resulting in a more positive equilibrium potential for chloride ions, a switch in synaptic input from inhibitory to excitatory, motoneuron hyperactivity, and muscle spasticity. Modified from [<a href="#B16-ijms-25-12462" class="html-bibr">16</a>].</p> Full article ">Figure 5
<p>Changes in rat soleus muscle EMG activity and KCC2 content in spinal motoneurons with prochlorperazine and CLP-290 administration during hindlimb suspension (HS). (<b>a</b>) Typical patterns of EMG activity of rat soleus before and during HS with and without prochlorperazine or CLP-290 administration; (<b>b</b>) KCC2 content in rat lumbar spinal cord after administration of prochlorperazine or CLP290; (<b>c</b>) integral EMG activity of rat soleus HS with and without administration of prochlorperazine and CLP-290. C—control, 1HS—7HS—days of hindlimb suspension, 7HS + Phpz—7-day HS with prochlorperazine administration, 7 HS + CLP-290—7-day HS with CLP290 administration, *—significant difference from control (<span class="html-italic">p</span> &lt; 0.05). #—significant difference from HS + Phpz or HS + CLP-290 (<span class="html-italic">p</span> &lt; 0.05). Adapted from [<a href="#B28-ijms-25-12462" class="html-bibr">28</a>,<a href="#B29-ijms-25-12462" class="html-bibr">29</a>].</p> Full article ">Figure 6
<p>Effects of prochlorperazine and CLP-290 administration during HS on PGC1α expression and p70S6K phosphorylation in rat soleus muscle. (<b>a</b>) Changes in PGC1α mRNA expression in rat soleus muscle with prochlorperazine and CLP-290 administration during 7-day hindlimb suspension (HS); (<b>b</b>) Changes in p70S6K (Thr389) phosphorylation in rat soleus muscle with prochlorperazine and CLP-290 administration during 7-day hindlimb suspension. C—control, 7 HS—7 days of hindlimb suspension, 7 HS + Phpz—7-day HS with prochlorperazine administration, 7 HS + CLP-290—7-day HS with CLP290 administration, *—significant difference from control (<span class="html-italic">p</span> &lt; 0.05), #—significant difference from 7 HS (<span class="html-italic">p</span> &lt; 0.05). Adapted from [<a href="#B33-ijms-25-12462" class="html-bibr">33</a>,<a href="#B34-ijms-25-12462" class="html-bibr">34</a>].</p> Full article ">
14 pages, 927 KiB  
Review
Strategies for Modifying Adenoviral Vectors for Gene Therapy
by Anna Muravyeva and Svetlana Smirnikhina
Int. J. Mol. Sci. 2024, 25(22), 12461; https://doi.org/10.3390/ijms252212461 - 20 Nov 2024
Viewed by 893
Abstract
Adenoviral vectors (AdVs) are effective vectors for gene therapy due to their broad tropism, large capacity, and high transduction efficiency, making them widely used as oncolytic vectors and for creating vector-based vaccines. This review also considers the application of adenoviral vectors in oncolytic [...] Read more.
Adenoviral vectors (AdVs) are effective vectors for gene therapy due to their broad tropism, large capacity, and high transduction efficiency, making them widely used as oncolytic vectors and for creating vector-based vaccines. This review also considers the application of adenoviral vectors in oncolytic virotherapy and gene therapy for inherited diseases, analyzing strategies to enhance their efficacy and specificity. However, despite significant progress in this field, the use of adenoviral vectors is limited by their high immunogenicity, low specificity to certain cell types, and limited duration of transgene expression. Various strategies and technologies aimed at improving the characteristics of adenoviral vectors are being developed to overcome these limitations. Significant attention is being paid to the creation of tissue-specific promoters, which allow for the controlled expression of transgenes, as well as capsid modifications that enhance tropism to target cells, which also play a key role in reducing immunogenicity and increasing the efficiency of gene delivery. This review focuses on modern approaches to adenoviral vector modifications made to enhance their effectiveness in gene therapy, analyzing the current achievements, challenges, and prospects for applying these technologies in clinical practice, as well as identifying future research directions necessary for successful clinical implementation. Full article
(This article belongs to the Section Molecular Genetics and Genomics)
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<p>Structure of adenovirus.</p> Full article ">Figure 2
<p>Strategies for modifying adenoviral vectors. (<b>a</b>) Control of transgene expression: use of tissue-specific promoters to confine transgene expression to target cells, enhancing safety and minimizing off-target effects. (<b>b</b>) Changing tropism: capsid pseudotyping, which replaces capsid proteins with those from other adenovirus serotypes, and conjugation with molecular adapters, which binds capsid proteins to specific cell receptors, are used to alter vector specificity and improve the targeting of cell types, thereby enhancing therapeutic efficacy. (<b>c</b>) Reducing immunogenicity: chemical modifications (e.g., PEGylation) or genetic alterations to the capsid to lower immune recognition, allowing for safer repeated administration and prolonged therapeutic effects.</p> Full article ">
19 pages, 5951 KiB  
Article
Biallelic Germline BRCA1 Frameshift Mutations Associated with Isolated Diminished Ovarian Reserve
by Anne Helbling-Leclerc, Marie Falampin, Abdelkader Heddar, Léa Guerrini-Rousseau, Maud Marchand, Iphigenie Cavadias, Nathalie Auger, Brigitte Bressac-de Paillerets, Laurence Brugieres, Bernard S. Lopez, Michel Polak, Filippo Rosselli and Micheline Misrahi
Int. J. Mol. Sci. 2024, 25(22), 12460; https://doi.org/10.3390/ijms252212460 - 20 Nov 2024
Viewed by 1019
Abstract
The use of next-generation sequencing (NGS) has recently enabled the discovery of genetic causes of primary ovarian insufficiency (POI) with high genetic heterogeneity. In contrast, the causes of diminished ovarian reserve (DOR) remain poorly understood. Here, we identified by NGS and whole exome [...] Read more.
The use of next-generation sequencing (NGS) has recently enabled the discovery of genetic causes of primary ovarian insufficiency (POI) with high genetic heterogeneity. In contrast, the causes of diminished ovarian reserve (DOR) remain poorly understood. Here, we identified by NGS and whole exome sequencing (WES) the cause of isolated DOR in a 14-year-old patient. Two frameshift mutations in BRCA1 (NM_007294.4) were found: in exon 8 (c.470_471del; p.Ser157Ter) and in exon 11 (c.791_794del, p.Ser264MetfsTer33). Unexpectedly, the patient presented no signs of Fanconi anemia (FA), i.e., no developmental abnormalities or indications of bone marrow failure. However, high chromosomal fragility was found in the patient’s cells, consistent with an FA diagnosis. RT-PCR and Western-blot analysis support the fact that the c. 791_794del BRCA1 allele is transcribed and translated into a shorter protein (del11q), while no expression of the full-length BRCA1 protein was found. DNA damage response (DDR) studies after genotoxic agents demonstrate normal activation of the early stages of the DDR and FANC/BRCA pathway. This is consistent with the maintenance of residual repair activity for the del11q BRCA1 isoform. Our observation is the first implication of bi-allelic BRCA1 mutations in isolated ovarian dysfunction or infertility in humans, without clinical signs of FA, and highlights the importance of BRCA1 in ovarian development and function. Full article
(This article belongs to the Special Issue Advances in Genetics of Human Reproduction)
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<p>Clinical feature of the family of the proband. Cancer family history including age at the first diagnostic is reported. St. = stomach cancer, Ov. = ovarian cancer, Blad. = bladder cancer, Col. = colorectal cancer, TBT. = temporal benign tumor, Adreno. = adrenocortical adenoma.</p> Full article ">Figure 2
<p>Confirmation of the two <span class="html-italic">BRCA1</span> variants identified in the patient by Sanger Sequencing. The two variants in the patient were confirmed by Sanger sequencing as shown by the two electropherograms centered around the first variant (c.470_471del; p.Ser157Ter) located in exon 8 (left panel) and the second variant (c.791_794del, p.Ser264MetfsTer33) located in exon 11 (right panel). The deleted nucleotides (two and four nucleotides respectively) are highlighted by a red rectangle.</p> Full article ">Figure 3
<p>Chromosomal fragility in response to mitomycin C on metaphase chromosomes. Chromosomal breakages and radial figures are shown, respectively, by red and green arrows.</p> Full article ">Figure 4
<p>Survival curve after genotoxic treatment. (<b>a</b>) Survival curve after mitomycin C (MMC) and (<b>b</b>) Olaparib treatment during the 3 days. (n = 3 for MMC tests; mean ± SEM; GraphPad unpaired <span class="html-italic">t</span> test *** <span class="html-italic">p</span> &lt; 0.001 and n = 1 for Olaparib test).</p> Full article ">Figure 5
<p><span class="html-italic">BRCA1</span> transcripts analysis. (<b>a</b>) Partial genomic organization of <span class="html-italic">BRCA1</span> with both mutations identified in the patient’s DNA. The dashed line at <span class="html-italic">BRCA1</span> c.787 indicate an alternative splice site that yields an in-frame truncated transcript, <span class="html-italic">BRCA1</span> del11q [<a href="#B35-ijms-25-12460" class="html-bibr">35</a>]. (<b>b</b>) Localization of the primers and size of the amplicons generated to distinguish the full-length (FL) and the del11q <span class="html-italic">BRCA1</span> transcripts. (<b>c</b>) <span class="html-italic">BRCA1</span> transcripts: RT-PCR from lymphoblasts cells of a control (WT) and the patient using three pairs of primers amplifying the reference transcript (FL) or the del11q isoform. (<b>d</b>) Relative quantity of <span class="html-italic">BRCA1</span> transcript isoforms from three replicate experiments with two different amplicons for the full-length and del11q transcripts. (mean ± SEM).</p> Full article ">Figure 6
<p>BRCA1 protein analysis. (<b>a</b>) Total protein extract from immortalized lymphoblasts from a control (WT) or the patient were loaded on 3–8% Tris-acetate SDS-PAGE gel and revealed with an anti-BRCA1 antibody recognizing the C terminal extremity. MCM7 is an internal loading control. (<b>b</b>) Relative amounts of BRCA1 protein isoforms from replicate experiments. (n = 7; mean ± SEM).</p> Full article ">Figure 7
<p>DDR and FANC/BRCA pathway activation after genotoxic agents’ overnight treatment. (<b>a</b>) Detection of DSB signaling by γ-H2AX, DNA resection revealed by p-RPA32 expression (p-RPA/RPA total) in patient and control cells after overnight treatment. (<b>b</b>) FANC/BRCA pathway activation revealed by FANCD2 monoubiquitination. (Aph: Aphidicolin at 0.6 µM; HU: Hydroxyurea at 5 mM or MMC: mitomycin C at 200 ng/mL).</p> Full article ">Figure 8
<p>BRCA1 recruitment on chromatin. Soluble (S1) and chromatin fractions (P2) were analyzed by Western-blot (6% and 15% Tris-Glycine SDS-PAGE), after two culture conditions (treated with HU at 5 mM overnight versus untreated). An enrichment of histone H4 in chromatin extracts confirmed the fractionation. Gray bands mask a sample that cannot be publicly presented (<b>A</b>,<b>B</b>) in different acquisition times).</p> Full article ">Figure 9
<p>Cell cycle: (<b>a</b>) cell cycle determined by flux cytometry for each individual by EdU and propidium iodide (PI) staining. (<b>b</b>) Cell distribution by cycle phases (n = 2; mean).</p> Full article ">
18 pages, 10499 KiB  
Article
Oxidative Stress-Associated Alteration of circRNA and Their ceRNA Network in Differentiating Neuroblasts
by Ebrahim Mahmoudi, Behnaz Khavari and Murray J. Cairns
Int. J. Mol. Sci. 2024, 25(22), 12459; https://doi.org/10.3390/ijms252212459 - 20 Nov 2024
Viewed by 581
Abstract
Oxidative stress from environmental exposures is thought to play a role in neurodevelopmental disorders; therefore, understanding the underlying molecular regulatory network is essential for mitigating its impacts. In this study, we analysed the competitive endogenous RNA (ceRNA) network mediated by circRNAs, a novel [...] Read more.
Oxidative stress from environmental exposures is thought to play a role in neurodevelopmental disorders; therefore, understanding the underlying molecular regulatory network is essential for mitigating its impacts. In this study, we analysed the competitive endogenous RNA (ceRNA) network mediated by circRNAs, a novel class of regulatory molecules, in an SH-SY5Y cell model of oxidative stress, both prior to and during neural differentiation, using RNA sequencing and in silico analysis. We identified 146 differentially expressed circRNAs, including 93 upregulated and 53 downregulated circRNAs, many of which were significantly co-expressed with mRNAs that potentially interact with miRNAs. We constructed a circRNA–miRNA–mRNA network and identified 15 circRNAs serving as hubs within the regulatory axes, with target genes enriched in stress- and neuron-related pathways, such as signaling by VEGF, axon guidance, signaling by FGFR, and the RAF/MAP kinase cascade. These findings provide insights into the role of the circRNA-mediated ceRNA network in oxidative stress during neuronal differentiation, which may help explain the regulatory mechanisms underlying neurodevelopmental disorders associated with oxidative stress. Full article
(This article belongs to the Special Issue Targeting Oxidative Stress for Disease)
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<p>Schematic of the oxidative stress cell models in this study. The two oxidative stress models using SH-SY5Y neuroblastoma cells include: pre-differentiation oxidative stress (PD-OS), in which the oxidative condition was introduced before neuronal differentiation; and during-differentiation oxidative stress (DD-OS), in which the oxidative condition was introduced during differentiation. In the next step, the circRNA profile of each model was identified by RNA sequencing using total RNA depleted of ribosomal RNA, followed by bioinformatic analysis.</p> Full article ">Figure 2
<p>Analysis of circRNA differential expression in oxidative stress. Volcano plot generated using fold change values and <span class="html-italic">p</span>-values to illustrate the expression changes of circRNA in oxidative conditions PD-OS (<b>a</b>) and DD-OS (<b>b</b>). Red and green dots indicate significant upregulation and downregulation, respectively, with the vertical lines representing a fold change &gt; 2 and the horizontal line representing a <span class="html-italic">p</span> &lt; 0.05 cutoff. Differentially expressed circRNAs that survived multiple testing correction are labelled on plots. (<b>c</b>) Side-by-side comparison of the expression change from sequencing data and qRT-PCR results for the top 11 differentially expressed circRNAs. All reactions were performed in triplicate. (<b>d</b>) Correlation between the expression change from sequencing data and qRT-PCR results (Spearman’s rank, ρ = 0.74, <span class="html-italic">p</span> = 0.013). (<b>e</b>) Gene Ontology (GO) enrichment analysis for the altered circRNA parental genes in PD-OS. All the significantly enriched GO terms are shown (FDR &lt; 0.05).</p> Full article ">Figure 3
<p>The circRNA-associated ceRNA network in oxidative stress. The potential interaction network of circRNA-miRNA-mRNA. (<b>a</b>) The networks are constructed based on the co-expression between the DE circRNAs and DE mRNAs and the predicted miRNA, with binding sites for each correlated circRNA–mRNA pair. (<b>b</b>) The predicted interaction network in PD-OS includes 454 axes and 244 nodes. (<b>c</b>) The predicted network in DD-OS includes 122 axes and 103 nodes. The circle and the parallelogram represent DE circRNA and DE mRNA, respectively, with red and blue denoting upregulation and downregulation, respectively. The colour intensity associates with the expression fold change. The gray inverted triangles indicate miRNA.</p> Full article ">Figure 3 Cont.
<p>The circRNA-associated ceRNA network in oxidative stress. The potential interaction network of circRNA-miRNA-mRNA. (<b>a</b>) The networks are constructed based on the co-expression between the DE circRNAs and DE mRNAs and the predicted miRNA, with binding sites for each correlated circRNA–mRNA pair. (<b>b</b>) The predicted interaction network in PD-OS includes 454 axes and 244 nodes. (<b>c</b>) The predicted network in DD-OS includes 122 axes and 103 nodes. The circle and the parallelogram represent DE circRNA and DE mRNA, respectively, with red and blue denoting upregulation and downregulation, respectively. The colour intensity associates with the expression fold change. The gray inverted triangles indicate miRNA.</p> Full article ">Figure 4
<p>The enrichment of circRNA-sponged miRNAs in psychiatric disorders. (<b>a</b>) Of the 114 and 60 miRNAs that were predicted to be sponged by DE circRNA in PD-OS and DD-OS, respectively, more than 50% were dysregulated in psychiatric disorders. We observed significant enrichment of circRNA-sponged miRNAs in these diseases for both PD-OS (<b>b</b>) and DD-OS (<b>c</b>) conditions, using Fisher’s exact test.</p> Full article ">Figure 5
<p>Identification of the ceRNA subnetwork. The interaction subnetwork in (<b>a</b>) PD-OS and (<b>b</b>) DD-OS. The subnetwork in PD-OS includes 10 hub circRNAs potentially regulating 57 mRNAs through interacting with 86 miRNAs. In the DD-OS subnetwork with 5 circRNA hubs, 20 mRNAs are regulated through 46 miRNAs. The circle represents circRNA, the parallelogram denotes mRNA, and the inverted triangle indicates miRNA.</p> Full article ">Figure 6
<p>Functional analysis of the ceRNA subnetwork. (<b>a</b>) GO enrichment analysis for the target genes in the ceRNA subnetwork in PD-OS, which showed significantly enriched biological processes (FDR &lt; 0.05). (<b>b</b>) Pathway enrichment analysis of the target genes in PD-OS, which indicated a significant enrichment of the genes in several pathways (FDR &lt; 0.05). Selected GO terms and pathways are shown.</p> Full article ">
19 pages, 6202 KiB  
Article
In Vitro Cell Model Investigation of Alpha-Synuclein Aggregate Morphology Using Spectroscopic Imaging
by Priyanka Swaminathan, Therése Klingstedt, Vasileios Theologidis, Hjalte Gram, Johan Larsson, Lars Hagen, Nina B. Liabakk, Odrun A. Gederaas, Per Hammarström, K. Peter R. Nilsson, Nathalie Van Den Berge and Mikael Lindgren
Int. J. Mol. Sci. 2024, 25(22), 12458; https://doi.org/10.3390/ijms252212458 - 20 Nov 2024
Viewed by 1137
Abstract
Recently, it has been hypothesized that alpha-synuclein protein strain morphology may be associated with clinical subtypes of alpha-synucleinopathies, like Parkinson’s disease and multiple system atrophy. However, direct evidence is lacking due to the caveat of conformation-specific characterization of protein strain morphology. Here we [...] Read more.
Recently, it has been hypothesized that alpha-synuclein protein strain morphology may be associated with clinical subtypes of alpha-synucleinopathies, like Parkinson’s disease and multiple system atrophy. However, direct evidence is lacking due to the caveat of conformation-specific characterization of protein strain morphology. Here we present a new cell model based in vitro method to explore various alpha-synuclein (αsyn) aggregate morphotypes. We performed a spectroscopic investigation of the HEK293 cell model, transfected with human wildtype-αsyn and A53T-αsyn variants, using the amyloid fibril-specific heptameric luminescent oligomeric thiophene h-FTAA. The spectral profile of h-FTAA binding to aggregates displayed a blue-shifted spectrum with a fluorescence decay time longer than in PBS, suggesting a hydrophobic binding site. In vitro spectroscopic binding characterization of h-FTAA with αsyn pre-formed fibrils suggested a binding dissociation constant Kd < 100 nM. The cells expressing the A53T-αsyn and human wildtype-αsyn were exposed to recombinant pre-formed fibrils of human αsyn. The ensuing intracellular aggregates were stained with h-FTAA followed by an evaluation of the spectral features and fluorescence lifetime of intracellular αsyn/h-FTAA, in order to characterize aggregate morphotypes. This study exemplifies the use of cell culture together with conformation-specific ligands to characterize strain morphology by investigating the spectral profiles and fluorescence lifetime of h-FTAA, based upon its binding to a certain αsyn aggregate. This study paves the way for toxicity studies of different αsyn strains in vitro and in vivo. Accurate differentiation of specific alpha-synucleinopathies is still limited to advanced disease stages. However, early subtype-specific diagnosis is of the utmost importance for prognosis and treatment response. The potential association of αsyn aggregates morphotypes detected in biopsies or fluids to disease phenotypes would allow for subtype-specific diagnosis in subclinical disease stage and potentially reveal new subtype-specific treatment targets. Notably, the method may be applied to the entire spectrum of neurodegenerative diseases by using a combination of conformation-specific ligands in a physicochemical environment together with other types of polymorphic amyloid variants and assess the conformation-specific features of various protein pathologies. Full article
(This article belongs to the Section Molecular Biology)
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<p>Emission spectra of h-FTAA binding to PFFs fixed at 1 µM concentration with varying concentrations of h-FTAA from (<b>A</b>) 1125 nM to 4500 nM to (<b>B</b>) 141 nM to 563 nM. The samples were excited at 450 nm and the emission was recorded in the range of 500–700 nm. Each spectrum was baseline corrected using h-FTAA emission in PBS only, respectively. The shaded region in each spectrum represents the standard deviation from triplicates of the varied concentrations of h-FTAA while keeping the concentration of PFFs fixed at 1 µM.</p> Full article ">Figure 2
<p>Binding curve of PFFs (1 μM) vs. h-FTAA concentration (red squares). The blue triangles show the signal obtained from only h-FTAA in PBS. The excitation wavelength was 450 nm, and the spectra were collected as in <a href="#ijms-25-12458-f001" class="html-fig">Figure 1</a>. The dashed curves are simulations where the blue dashed line corresponds to 6% QY of h-FTAA in PBS (<a href="#ijms-25-12458-t001" class="html-table">Table 1</a>). Green dot-dashed: 1-site binding K<sub>d</sub> = 25 nM; QY 30%. Red dashed: two-site model, K<sub>d1</sub> = 100 nM; K<sub>d2</sub> = 300 nM. QY(h-FTAA/PFF-site1) 40%; QY(h-FTAA/PFF-site2) 20%. For details of the two-site model, see [<a href="#B32-ijms-25-12458" class="html-bibr">32</a>].</p> Full article ">Figure 3
<p>Hyperspectral imaging and fluorescence lifetime measurements of PFFs stained with 500 nM h-FTAA. (<b>A</b>) Representative fluorescence image and (<b>B</b>) False-color coded FLIM image of PFFs stained with h-FTAA. The sample was excited at 475 nm and the photons were collected in the 500–700 nm range. The color bar to the right represents the lifetime ranging from 0 ns to 2 ns. (<b>C</b>) Spectral analysis of h-FTAA when it is bound to PFFs, showing emission maxima at approximately 540 nm and 580 nm. The five ROIs (red) used to record the emission spectra are shown in (<b>B</b>). (<b>D</b>) Fluorescence decay time distribution recorded from the FLIM image using the same ROIs (red) that were selected for the spectral analysis in (<b>C</b>). The shaded regions in the plots represent the standard deviation.</p> Full article ">Figure 4
<p>Endogenous αsyn expressed in HEK293 cells after transfection with 4 μg human A53T-αsyn or WT-αsyn. (<b>A</b>) Western blot showing αsyn protein bands at approximately 14 kDa in A53T-αsyn and WT-αsyn HEK293 cells which were probed with mouse anti-αsyn antibody Syn211. Representative immunofluorescence images of (<b>B</b>) A53T-αsyn, (<b>C</b>) WT-αsyn HEK293 cells showing localization of αsyn in cytosol, and (<b>D</b>) Untransfected HEK293 cells showing absence of αsyn, when labeled with mouse anti-αsyn antibody Syn211. Scale bar represents 10 µm.</p> Full article ">Figure 5
<p>Representative fluorescence images of HEK293 cells expressing (<b>A</b>) A53T-αsyn or (<b>B</b>) WT-αsyn, seeded with 500 nM human-αsyn PFFs and stained with 1 μM h-FTAA (green) and 5 µM DRAQ5 (red). (<b>C</b>) Untransfected HEK293 cells were also exposed to PFFs, showing minimal fluorescence from h-FTAA. Scale bar represents 10 μm.</p> Full article ">Figure 6
<p>Representative spectral analysis and lifetime distributions of h-FTAA binding to aggregates in A53T-αsyn-HEK293 and WT-αsyn-HEK293 cells. The samples were excited at 475 nm. (<b>A</b>) Emission spectra and (<b>B</b>) lifetime distributions of h-FTAA binding to aggregates in A53T-αsyn (red) and WT-αsyn-HEK293 (green) cells. Shaded areas correspond to the standard deviation of 5 ROIs.</p> Full article ">Figure 7
<p>Representative differential interference contrast (DIC) and confocal microscopy images showing h-FTAA-stained (green), pS129-probed αsyn aggregates (red) in (<b>A</b>) A53T-αsyn-HEK293 cells and (<b>B</b>) WT-αsyn-HEK293 cells. The samples were excited at 475 nm and 650 nm, respectively. (<b>C</b>) Untransfected HEK293 cells, also seeded with PFFs, show no fluorescence from h-FTAA or the anti-αsyn pS129 antibody, indicating absence of pS129-positive αsyn aggregates. Scale bar represents 25 µm.</p> Full article ">Figure 8
<p>Representative spectral analysis and lifetime distributions of h-FTAA-stained, pS129-labeled aggregates in A53T-αsyn-HEK293 and WT-αsyn-HEK293 cells. The samples were excited at 475 nm and 650 nm for h-FTAA and Alexa Fluor 647, respectively. (<b>A</b>) Emission spectra and (<b>B</b>) life-time distributions for h-FTAA-stained, pS129-labeled aggregates in A53T-αsyn-HEK293 and WT-αsyn-HEK293 cells. Shaded areas correspond to the standard deviation of 5 ROIs.</p> Full article ">
11 pages, 2313 KiB  
Article
Exposure to Radiofrequency Electromagnetic Fields Enhances Melanin Synthesis by Activating the P53 Signaling Pathway in Mel-Ab Melanocytes
by Ju Hwan Kim, Dong-Jun Kang, Jun Young Seok, Mi-Hye Kim, Dong-Seok Kim, Sang-Bong Jeon, Hyung-Do Choi, Jung Ick Moon, Nam Kim and Hak Rim Kim
Int. J. Mol. Sci. 2024, 25(22), 12457; https://doi.org/10.3390/ijms252212457 - 20 Nov 2024
Viewed by 908
Abstract
The skin is the largest body organ that can be physiologically affected by exposure to radiofrequency electromagnetic fields (RF-EMFs). We investigated the effect of RF-EMFs on melanogenesis; Mel-Ab melanocytes were exposed to 1760 MHz radiation with a specific absorption rate of 4.0 W/kg [...] Read more.
The skin is the largest body organ that can be physiologically affected by exposure to radiofrequency electromagnetic fields (RF-EMFs). We investigated the effect of RF-EMFs on melanogenesis; Mel-Ab melanocytes were exposed to 1760 MHz radiation with a specific absorption rate of 4.0 W/kg for 4 h/day over 4 days. Exposure to the RF-EMF led to skin pigmentation, with a significant increase in melanin production in Mel-Ab melanocytes. The phosphorylation level of cAMP response element binding protein (CREB) and the expression of microphthalmia-associated transcription factor (MITF), which regulate the expression of tyrosinase, were significantly increased in Mel-Ab after RF-EMF exposure. Interestingly, the expression of tyrosinase was significantly increased, but tyrosinase activity was unchanged in the RF-EMF-exposed Mel-Ab cells. Additionally, the expression of p53 and melanocortin 1 receptor (MC1R), which regulate MITF expression, was significantly increased. These results suggest that the RF-EMF induces melanogenesis by increasing phospho-CREB and MITF activity. Importantly, when Mel-Ab cells were incubated at 38 °C, the melanin production and the levels of tyrosinase significantly decreased, indicating that the increase in melanin synthesis by RF-EMF exposure is not due to a thermal effect. In conclusion, RF-EMF exposure induces melanogenesis in Mel-Ab cells through the increased expression of tyrosinase via the activation of MITF or the phosphorylation of CREB, which are initiated by the activation of p53 and MC1R. Full article
(This article belongs to the Special Issue Molecular Research Progress of Skin and Skin Diseases)
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<p>Cellular morphology and pigmentation of Mel-Ab melanocytes after RF-EMF exposure. The Mel-Ab cells were cultured for 4 d with or without 1760 MHz radiofrequency electromagnetic field (RF-EMF) exposure (at 4.0 W/kg for 4 h/d) and heat treatment (38 °C). (<b>A</b>) Cellular morphology was observed using a microscope (Olympus, CKX53, Tokyo, Japan). Red arrows indicate darkened Mel-Ab cells. The 200× magnified photos with 100 µm scale bar are shown. (<b>B</b>) The cell pellets of control, RF-EMF-exposed, and 38 °C-incubated Mel-Ab melanocytes (lower panel) and dark intensity of each pellet was quantified using the ImageJ software bundled with 64-bit Java 8 (upper panel). (<b>C</b>) Melanin content was measured in Mel-Ab cells under each condition. (<b>D</b>) Cell viability of Mel-Ab cells under each condition. Data are expressed as mean ± standard error of the mean. * <span class="html-italic">p</span> and # <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> and ## <span class="html-italic">p</span> &lt; 0.01 compared to control (<span class="html-italic">n</span> = 6). RF-EMF, radiofrequency electromagnetic field.</p> Full article ">Figure 2
<p>Expression levels of heat shock proteins in Mel-Ab melanocytes after RF-EMF exposure. Expression of <span class="html-italic">Hsp27</span> (<b>A</b>), <span class="html-italic">Hsp70</span> (<b>B</b>), and <span class="html-italic">Hsp90</span> (<b>C</b>) mRNA transcripts determined by quantitative real-time PCR. The relative mRNA levels of heat shock proteins were calculated by normalizing to the expression of <span class="html-italic">Gapdh</span>, using the 2<sup>−ΔΔCt</sup> method. Data are expressed as mean ± standard error of the mean. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001 compared to control (<span class="html-italic">n</span> = 4). RF-EMF, radiofrequency electromagnetic field.</p> Full article ">Figure 3
<p>Expression levels of tyrosinase and tyrosine hydroxylase were increased by RF-EMF exposure. (<b>a</b>). Expressional quantification of tyrosinase (<b>A</b>) and tyrosine hydroxylase (<b>B</b>) using Western blotting. (<b>b</b>). The graphs show the quantification of protein levels of tyrosinase (<b>A</b>), and tyrosine hydroxylase (<b>B</b>) normalized to that of β-actin. (<b>C</b>). Tyrosinase activity was measured in each condition. The data indicate the mean ± standard error of the mean. Levels of statistical significance were evaluated using one-way ANOVA. *** <span class="html-italic">p</span> &lt; 0.001, * <span class="html-italic">p</span> and # <span class="html-italic">p</span> &lt; 0.05 compared to the control (<span class="html-italic">n</span> = 4). RF-EMF, radiofrequency electromagnetic field; TH, tyrosine hydroxylase.</p> Full article ">Figure 4
<p>Expression levels of p53 and <span class="html-italic">Mc1r</span> were significantly increased by RF-EMF exposure in Mel-Ab melanocytes. (<b>A</b>). Expressional quantification of p53 by Western blotting (<b>a</b>). The graphs show the quantification of protein levels of p53 normalized to those of β-actin (<b>b</b>). (<b>B</b>). The mRNA levels of <span class="html-italic">Mc1r</span> were analyzed by quantitative real-time PCR. The relative mRNA levels of <span class="html-italic">Mc1r</span> were calculated by normalizing to the expression of <span class="html-italic">Gapdh</span> by the 2<sup>-ddCt</sup> method. Data are expressed as the mean ± standard error of the mean. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001 compared to control (<span class="html-italic">n</span> = 3). RF-EMF, radiofrequency electromagnetic field.</p> Full article ">Figure 5
<p>The expression level of p53 was significantly increased by RF-EMF exposure in HaCaT keratinocytes. HaCaT cells were exposed to RF-EMF or treated with 38 °C heat. (<b>A</b>). Expressional quantification of p53 using Western blotting. (<b>B</b>). The graphs show the quantification of protein levels of p53 normalized to those of β-actin. Data are expressed as the mean ± standard error of the mean. * <span class="html-italic">p</span> &lt; 0.05 compared to control (<span class="html-italic">n</span> = 3). RF-EMF, radiofrequency electromagnetic field.</p> Full article ">Figure 6
<p>Expression levels of MITF and phospho-CREB were increased by RF-EMF exposure. Quantification of MITF (<b>A</b>), p-CREB (Ser133), and CREB (<b>B</b>) expression by Western blotting. Data are presented as mean ± standard error of the mean. Statistical significance was evaluated using one-way ANOVA; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 compared to control (<span class="html-italic">n</span> = 6). RF-EMF, radiofrequency electromagnetic field.</p> Full article ">
25 pages, 5814 KiB  
Article
The Generation of Genetically Engineered Human Induced Pluripotent Stem Cells Overexpressing IFN-β for Future Experimental and Clinically Oriented Studies
by Olga Sheveleva, Elena Protasova, Elena Grigor’eva, Nina Butorina, Valeriia Kuziaeva, Daniil Antonov, Victoria Melnikova, Sergey Medvedev and Irina Lyadova
Int. J. Mol. Sci. 2024, 25(22), 12456; https://doi.org/10.3390/ijms252212456 - 20 Nov 2024
Viewed by 708
Abstract
Induced pluripotent stem cells (iPSCs) can be generated from various adult cells, genetically modified and differentiated into diverse cell populations. Type I interferons (IFN-Is) have multiple immunotherapeutic applications; however, their systemic administration can lead to severe adverse outcomes. One way of overcoming the [...] Read more.
Induced pluripotent stem cells (iPSCs) can be generated from various adult cells, genetically modified and differentiated into diverse cell populations. Type I interferons (IFN-Is) have multiple immunotherapeutic applications; however, their systemic administration can lead to severe adverse outcomes. One way of overcoming the limitation is to introduce cells able to enter the site of pathology and to produce IFN-Is locally. As a first step towards the generation of such cells, here, we aimed to generate human iPSCs overexpressing interferon-beta (IFNB, IFNB-iPSCs). IFNB-iPSCs were obtained by CRISPR/Cas9 editing of the previously generated iPSC line K7-4Lf. IFNB-iPSCs overexpressed IFNB RNA and produced a functionally active IFN-β. The cells displayed typical iPSC morphology and expressed pluripotency markers. Following spontaneous differentiation, IFNB-iPSCs formed embryoid bodies and upregulated endoderm, mesoderm, and some ectoderm markers. However, an upregulation of key neuroectoderm markers, PAX6 and LHX2, was compromised. A negative effect of IFN-β on iPSC neuroectoderm differentiation was confirmed in parental iPSCs differentiated in the presence of a recombinant IFN-β. The study describes new IFN-β-producing iPSC lines suitable for the generation of various types of IFN-β-producing cells for future experimental and clinical applications, and it unravels an inhibitory effect of IFN-β on stem cell neuroectoderm differentiation. Full article
(This article belongs to the Special Issue Diversity of Induced Pluripotent Stem Cells)
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<p>The generation of IFNB-overexpressing human iPSC lines. The ORF of the human IFNB gene was inserted into the AAVS1 locus of parental iPSC line K7-4Lf using the CRISPR/Cas9 technology. In brief, K7-iPSCs were transfected with pAAVS1-hPGK-IFNB1 and sgRNA-Cas9 plasmids; successfully transfected clones were selected in a puromycin-containing medium; the selected clones were expanded and screened for the presence of on-target and the absence of off-target inserts. (<b>a</b>) pAAVS1-hPGK-IFNB1 plasmid scheme; (<b>b</b>–<b>d</b>) representative gels showing the results of iPSC screening for on-target and off-target inserts; (<b>b</b>) representative gel showing the results of iPSC screening for the presence of unmodified wild-type AAVS loci (WT AAVS1). C+, positive control, parental K7-iPSCs containing unmodified AAVS1 loci; C−, negative control, a previously generated iPSC line with a proven correct insert of the roGFP2-Orp1 transgene into the AAVS1 locus [<a href="#B47-ijms-25-12456" class="html-bibr">47</a>]; (<b>c</b>) representative gel showing the results of on-target screening in edited iPSCs. C+, iPSC line with a proven correct insert of the roGFP2-Orp1 transgene into the AAVS1 locus [<a href="#B47-ijms-25-12456" class="html-bibr">47</a>]. C−, parental K7-iPSC line; (<b>d</b>) representative gel showing the results of off-target screening in edited iPSCs. C+, pAAVS1-hPGK-IFNB1 plasmid; C−, iPSC line with a proven correct insert into the AAVS1 locus. iPSCs, induced pluripotent stem cells; K7-iPSCs, parental K7-4Lf iPSC line.</p> Full article ">Figure 2
<p>IFNB-iPSC lines display the morphological and phenotypic characteristics of pluripotent cells and a normal karyotype. At least three IFNB-iPSC lines, LA8-iPSCs, LC8-iPSCs, and LE4-iPSCs, were expanded and examined in each of the indicated assays. The results obtained using IFNB-iPSC line LA8 are shown as representative. (<b>a</b>) Light microscopy of LA8-iPSCs and parental K7-iPSCs growing on mouse embryonic fibroblast feeder cells. Note the similar morphology of IFNB-iPSCs and K7-iPSCs. Phase contrast. 10× magnification. (<b>b</b>) The karyogram of LA8-iPSCs. (<b>c</b>) Positive immunohistochemical staining of LA8-iPSCs for alkaline phosphatase. K7-iPSCs were used as a positive control. (<b>d</b>) The expression of pluripotency markers <span class="html-italic">OCT4</span>, <span class="html-italic">SOX2</span>, and <span class="html-italic">NANOG</span> by LA8-iPSCs (RT-qPCR; representative experiment, n = 3; median with 95% CI). (<b>e</b>) The expression of OCT4 (red) and SOX2 (green) pluripotency proteins in LA8-iPSCs. Note similar patterns of protein expression in IFNB-iPSCs and the parental K7-iPSCs. Immunofluorescence staining, confocal microscopy (Zeiss LSM 880 microscope; Carl Zeiss, Jena, Germany). Nuclei were stained with DAPI (blue). In all images, the scale bar is 100 µm. IFNB-iPSCs, iPSCs with a constitutive overexpression of the IFNB gene; LA8-iPSCs, IFNB-iPSC line LA8; LC8-iPSCs, IFNB-iPSC line LC8; LE4-iPSCs, IFNB-iPSC line LE4.</p> Full article ">Figure 3
<p>IFNB-iPSCs express functional IFN-β. IFNB-iPSCs and K7-iPSCs were cultured in parallel and used for RNA isolation; the preparation of cell extracts; and the collection of cell culture supernatants. (<b>a</b>) IFNB-iPSCs display an increased expression of <span class="html-italic">IFNB</span> as compared with the parental K7-iPSC line. RNAs isolated from IFNB-iPSCs and K7-iPSCs were subjected to RT-PCR; <span class="html-italic">IFNB</span> expression values were normalized relative to <span class="html-italic">GAPDH</span>, and fold changes were calculated as relative IFNB mRNA levels in IFNB-iPSCs relative to K7-iPSCs (2<sup>−∆∆Cq</sup>). Dotted line, fold change = 2 (significance threshold). Data are shown as boxes and whiskers with minimal and maximal values (K7-iPSCs, LA8-iPSCs, and LC8-iPSCs, summarized results of at least 3 independent experiments; LE4-iPSCs, one experiment, technical replicates). Note that LA8-iPSCs and LC8-iPSCs bear a homozygous <span class="html-italic">IFNB</span> insertion, whereas LE4-iPSCs are heterozygous. The significance of the differences was determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. (<b>b</b>,<b>c</b>) IFNB-iPSCs express IFN-β at the protein level. (<b>b</b>) IFNB-iPSCs and K7-iPSCs were pelleted, frozen, and analyzed in a Western blot. The graph shows the relative densitometric values of IFN-β normalized to β-actin. (<b>c</b>) The supernatants were collected from IFNB-iPSCs and K7-iPSCs and analyzed using ELISA (the results of two independent experiments; in each of them, K7-iPSCs, LA8-iPSCs, and LC8-iPSCs were cultured in parallel and independently of cells of another experiment). (<b>d</b>) The supernatants of IFNB-iPSCs exhibit IFN-β-like functional activity. The supernatants were obtained from IFNB-iPSC and K7-iPSC cultures and were added to THP-1 macrophage-like cells pre-activated with PMA. Control THP-1 cells were cultured in the absence of iPSC supernatants. THP-1 RNA was isolated from all cultures, and the expressions of ISGs were analyzed in RT-qPCR using <span class="html-italic">RPL27</span> as a housekeeping gene. Data are shown as boxes and whiskers with minimal and maximal values and individual points. The representative results of one out of two experiments are presented. Numbers on the graphs show the FDRs (Benjamini, Krieger, and Yekutieli correction for multiple comparisons). FDRs &lt; 0.05 were considered as significant; for the comparison of IFNB-iPSCs and K7-iPSCs, FDRs &gt; 0.05 are also shown. The differences between LA8-iPSCs and LC8-iPSCs were insignificant. PMA, phorbol 12-myristate-13-acetate.</p> Full article ">Figure 4
<p>The immunofluorescence analysis reveals the expression of endoderm-, mesoderm-, and ectoderm-associated proteins in embryonic bodies spontaneously differentiated from IFNB-iPSCs. IFNB-iPSC lines LA8 and LC8 and parental K7-iPSCs were cultured in low-adhesive conditions to stimulate the formation of embryoid bodies (EBs). On day 15, EBs were transferred to Matrigel-coated coverslips to induce the formation of cell monolayers; 6 days later, the cells were stained with antibodies specific to endoderm, mesoderm, and ectoderm markers and analyzed by confocal microscopy (Zeiss LSM 880 (Carl Zeiss, Jena, Germany)). Scale bar: 50 µm. (<b>a</b>) Immunofluorescence staining of spontaneously differentiated EBs for germ layer markers AFP (endoderm) and ACTA2 (mesoderm). The results obtained using IFNB-iPSC line LA8 and parental K7-iPSCs are shown. Similar results were obtained using IFNB-iPSC line LC8 (<a href="#app1-ijms-25-12456" class="html-app">Supplementary Figure S4</a>). (<b>b</b>) Immunofluorescence staining of spontaneously differentiated EBs for germ layer marker TUBB3 (ectoderm). Two different fields of view for IFNB-EBs (line LA8 as a representative) and K7-EBs are shown. ACTA2, actin alpha 2; EBs, embryoid bodies; TUBB3, tubulin beta-3.</p> Full article ">Figure 5
<p>A disrupted expression of ectoderm-associated genes in embryonic bodies spontaneously differentiated from IFNB-iPSCs. IFNB-iPSC lines LA8 and LC8 and parental K7-iPSCs were cultured in low-adhesive conditions to stimulate the formation of embryoid bodies (EBs). On day 20, RNA was isolated and gene expressions were analyzed using RT-PCR. Whole-cell lysates of EBs were also analyzed by a Western blot. (<b>a</b>–<b>c</b>) Relative expressions of endoderm- (<b>a</b>), mesoderm- (<b>b</b>), and ectoderm- (<b>c</b>) associated markers in iPSCs and 20-day EBs. Data are shown as boxes and whiskers with minimal and maximal values (summarized results of at least 2 independent experiments). The significance of the differences was determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. Figures indicate the FDRs for the main comparisons, irrespectively of their significance (i.e., inter-line comparisons on differentiation day 20 and intra-line comparisons between iPSCs and 20-day EBs). (<b>d</b>) The Western blot analysis of PAX6 protein in IFNB-EBs and K7-EBs (one experiment). The graph shows the relative densitometric values of PAX6 normalized to HSP90.</p> Full article ">Figure 6
<p>Exogenous IFN-β disrupts the expression of ectoderm-associated genes in differentiating parental K7-iPSCs. K7-iPSCs were subjected to spontaneous (<b>a</b>) or directed (<b>b</b>–<b>d</b>) differentiation in the absence or in the presence of recombinant human IFN-β; at the end of the differentiation, the expression of ectoderm-associated markers was analyzed using RT-PCR (housekeeping gene, <span class="html-italic">RPL27</span>) and compared with that observed in original K7-iPSCs. The significance of the differences was determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. (<b>a</b>) Changes in the expression of ectoderm-associated genes in spontaneously differentiated EBs. Gene expression was analyzed on day 20. Data are shown as boxes and whiskers with minimal and maximal values. The summarized results of two independent experiments are shown. (<b>b</b>) Light microscopy of K7-iPSCs differentiating in the absence or in the presence of IFN-β. Made on differentiation days 1 and 7. (<b>c</b>) Changes in the expression of ectoderm-associated genes following the directed differentiation of K7-iPSCs in the STEMdiff™ Trilineage Ectoderm Medium. Gene expression was analyzed on day 7 (as recommended by the manufacturer). Data are shown as boxes and whiskers with minimal and maximal values. The representative results of one out of two experiments are presented. (<b>d</b>) Changes in the expression of the <span class="html-italic">IFNB</span> gene following the directed differentiation of K7-iPSCs in the STEMdiff™ Trilineage Ectoderm Medium (the representative results of one out of two experiments).</p> Full article ">
23 pages, 7668 KiB  
Article
Impact of Reduced Saliva Production on Intestinal Integrity and Microbiome Alterations: A Sialoadenectomy Mouse Model Study
by Kanna Maita, Hisako Fujihara, Mitsuki Matsumura, Moeko Miyakawa, Ryoko Baba, Hiroyuki Morimoto, Ryoko Nakayama, Yumi Ito, Koji Kawaguchi and Yoshiki Hamada
Int. J. Mol. Sci. 2024, 25(22), 12455; https://doi.org/10.3390/ijms252212455 - 20 Nov 2024
Viewed by 660
Abstract
This study investigates the effect of reduced saliva production on intestinal histological structure and microbiome composition using a sialoadenectomy murine model, evaluating differences in saliva secretion, body weight, intestinal histopathological changes, and microbiome alteration using 16S rRNA gene sequencing across three groups (control, [...] Read more.
This study investigates the effect of reduced saliva production on intestinal histological structure and microbiome composition using a sialoadenectomy murine model, evaluating differences in saliva secretion, body weight, intestinal histopathological changes, and microbiome alteration using 16S rRNA gene sequencing across three groups (control, sham, and sialoadenectomy). For statistical analysis, one-way analysis of variance and multiple comparisons using Bonferroni correction were performed. p-values < 0.05 were considered statistically significant. Microbiome analysis was performed using Qiime software. The results show that reduced saliva secretion leads to structural changes in the intestinal tract, including shorter and atrophic villi, deformed Paneth cells, decreased goblet cell density, and immunohistochemical changes in epidermal growth factor and poly(ADP-ribose) polymerase-1, especially at three months after surgery. They also showed significant alterations in the intestinal microbiome, including increased Lactobacillaceae and altered populations of Ruminococcaceae and Peptostreptococcaceae, suggesting potential inflammatory responses and decreased short-chain fatty acid production. However, by 12 months after surgery, these effects appeared to be normalized, indicating potential compensatory mechanisms. Interestingly, sham-operated mice displayed favorable profiles, possibly due to immune activation from minor surgical intervention. This study underscores saliva’s essential role in intestinal condition, emphasizing the “oral–gut axis” and highlighting broader implications for the relationship between oral and systemic health. Full article
(This article belongs to the Special Issue Recent Research on Cell and Molecular Biology)
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<p>Changes in body weight and saliva secretion during the experimental period. (<b>a</b>) Changes in body weight during the experimental period were not significantly different among the three groups. The sialoadenectomy group showed a slower increase in body weight 6 months after surgery, but this did not reach statistical significance. (<b>b</b>) Changes in saliva secretion during the experimental period. The amount of saliva secreted in the sialoadenectomy group was significantly lower than the other two groups during the experimental period. ** <span class="html-italic">p</span> &lt; 0.01; n.s., not significant.</p> Full article ">Figure 2
<p>Morphological changes in intestinal villi. (<b>a</b>) Representative hematoxylin and eosin-stained images of jejunal villi from each group at 3, 6, and 12 months after surgery. (<b>b</b>) Representative hematoxylin and eosin-stained images of ileal villi from each group at 3, 6, and 12 months after surgery. Scale bar: 200 μm.</p> Full article ">Figure 3
<p>Histopathological changes in Paneth cells. Representative hematoxylin and eosin-stained Paneth cells at 3, 6, and 12 months after surgery for each group. The right photo in each column is a magnified view of the red box in the corresponding left photo. The Paneth cells in the sialoadenectomy group showed deformation (arrows) that recovered over time. The control group showed slight deformation 12 months after surgery (arrowheads).</p> Full article ">Figure 4
<p>Analysis of sialoadenectomy effects on the number of goblet cells using d-PAS staining. (<b>a</b>) d-PAS staining of the jejunum (upper) and ileum (lower) at 3, 6, and 12 months after surgery. Scale bar: 200 μm. (<b>b</b>) The d-PAS-positive goblet cell density per unit area in the ileum was significantly decreased in the sialoadenectomy group at 3 months after surgery compared to the sham and control groups, but this difference was not significant at 6 or 12 months after surgery. In contrast, goblet cell density in the jejunum showed no significant differences among the three groups throughout the experimental period. * <span class="html-italic">p</span> &lt; 0.05, n.s., not significant.</p> Full article ">Figure 5
<p>Immunohistochemical analysis of EGF expression in the sialoadenectomy, sham, and control groups. Representative results for each group are shown. (<b>a</b>) EGF expression was detected at the epithelial surface of the jejunum, ileum, and colon in the control (upper row) and sham (middle row) group 3 months after surgery and was relatively weak in the sialoadenectomy group (lower row). Scale bar: 200 μm. (<b>b</b>) Semi-quantitative analysis following conversion to grayscale. The gray values in the sialoadenectomy group were significantly higher (equivalent to decreased EGF expression) compared with the other two groups in the jejunum, ileum, and colon. Values are expressed as mean ± SEM. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 6
<p>Immunohistochemical analysis of anti-PARP-1 antibody at 3 months after surgery. (<b>a</b>) The sialoadenectomy group (lower row) showed significantly more PARP-1-positive cells compared with the control (upper row) and sham (middle row) groups in the jejunum, ileum, and colon. Arrows: PARP-1-positive cells. Scale bar: 200 μm. (<b>b</b>) The gray values were measured using ImageJ (1.54 f) software and compared among the three groups. At 3 months after surgery, the sialoadenectomy group showed significantly higher PARP-1 positivity in the jejunum, ileum, and colon compared with the control and sham groups. Values are expressed as mean ± SEM. ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 7
<p>Microbiome composition across the three groups at 3, 6, and 12 months after surgery. (<b>a</b>) Microbiome composition at the phylum level. No specific phylum level microbiome was consistently detected throughout the experimental period. (<b>b</b>) Microbiome composition at the genus level. Significant differences in relative abundance were observed among the groups at 3, 6, and 12 months after surgery. The genera shown in the figure exhibited statistically significant variations in mean relative abundance, reflecting distinct microbiome characteristics within each group. <span class="html-italic">Lactobacillus</span> was observed in all three groups at 3 and 6 months after surgery, with significant differences across the three groups. However, 12 months after surgery, <span class="html-italic">Lactobacillus</span> was the most abundant microbiota in all three groups, and there was no significant difference across the groups.</p> Full article ">Figure 7 Cont.
<p>Microbiome composition across the three groups at 3, 6, and 12 months after surgery. (<b>a</b>) Microbiome composition at the phylum level. No specific phylum level microbiome was consistently detected throughout the experimental period. (<b>b</b>) Microbiome composition at the genus level. Significant differences in relative abundance were observed among the groups at 3, 6, and 12 months after surgery. The genera shown in the figure exhibited statistically significant variations in mean relative abundance, reflecting distinct microbiome characteristics within each group. <span class="html-italic">Lactobacillus</span> was observed in all three groups at 3 and 6 months after surgery, with significant differences across the three groups. However, 12 months after surgery, <span class="html-italic">Lactobacillus</span> was the most abundant microbiota in all three groups, and there was no significant difference across the groups.</p> Full article ">Figure 8
<p>Within-subject α-diversity. There was no significant difference among the three groups at (<b>a</b>) 3, (<b>b</b>) 6, and (<b>c</b>) 12 months after surgery.</p> Full article ">Figure 9
<p>β-diversity of microbial communities at (<b>a</b>) 3, (<b>b</b>) 6, and (<b>c</b>) 12 months after surgery. Significant differences were observed between control and sialoadenectomy groups at 3 months after surgery and between the sham group and both control and sialoadenectomy groups at 6 months after surgery (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 10
<p>Cladograms plotted from the LEfSe analysis. Taxonomic changes in intestinal microbiota across the three groups at (<b>a</b>) 3, (<b>b</b>) 6, and (<b>c</b>) 12 months after surgery.</p> Full article ">Figure 11
<p>Differentially abundant microbiota across the three groups identified using LEfSe. Panels represent results at (<b>a</b>) 3, (<b>b</b>) 6, and (<b>c</b>) 12 months after surgery. Log-transformed LDA scores are plotted on the x-axis. LDA scores &gt; 4 were considered statistically significant. Bar length indicates the relative influence of each microbiota.</p> Full article ">Figure 12
<p>Possible and plausible mechanisms of the effect of sialoadenectomy on the intestinal microbiome. Sialoadenectomy induces xerostomia (dry mouth), which is associated with oral microbiome dysbiosis, subsequently affecting the intestinal microbiome. Sialoadenectomy also reduces epidermal growth factor (EGF) secretion, which may directly alter the intestinal epithelium, further contributing to intestinal microbiome changes.</p> Full article ">
17 pages, 7325 KiB  
Article
Bioinformatics and Expression Analyses of the TaATLa Gene Subfamily in Wheat (Triticum aestivum L.)
by Yifei Chen, Kexin Zhao, Heng Chen, Luzhen Wang, Shuai Yan, Lei Guo, Jianjun Liu, Haosheng Li, Danping Li, Wenjia Zhang, Xiaoyan Duan, Xiukun Liu, Xinyou Cao and Xin Gao
Int. J. Mol. Sci. 2024, 25(22), 12454; https://doi.org/10.3390/ijms252212454 - 20 Nov 2024
Viewed by 521
Abstract
Amino acids are the main form of nitrogen in plants, and their transport across cell membranes relies on amino acid transporters (AATs). Among the plant AATs, the TaATLa subfamily comprises 18 members, yet the bioinformatics characteristics and functions of TaATLa genes in common [...] Read more.
Amino acids are the main form of nitrogen in plants, and their transport across cell membranes relies on amino acid transporters (AATs). Among the plant AATs, the TaATLa subfamily comprises 18 members, yet the bioinformatics characteristics and functions of TaATLa genes in common wheat remain poorly understood due to their complex genomes. This study performed genomic analyses of TaATLas. These analyses included chromosome distributions, evolutionary relationships, collinearity, gene structures, and expression patterns. An analysis of cis-acting elements and predicted miRNA-TaATLas regulatory networks suggests that TaATLas are regulated by light, hormones, and stress signals. Functional assays revealed that TaATLa6 transports glutamine (Gln), glutamate (Glu), and aspartate (Asp) in yeast. In contrast, TaATLa4 specifically transports Gln and Asp. Furthermore, TaATLas exhibits diverse gene expression patterns, with TaATLa4-7D enhancing yeast heat tolerance in a heterologous expression system, indicating its potential role in adapting to environmental stress by regulating amino acid transport and distribution. This study sheds light on the functional roles of TaATLa genes, with implications for improving nitrogen use in wheat and other crop species. Full article
(This article belongs to the Section Molecular Biology)
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<p>Evolutionary analysis of TaATLas. (<b>a</b>) Phylogenetic tree of ATLa proteins is constructed by the neighbor-joining method using MEGA11 from the following species: Ta, <span class="html-italic">Triticum aestivum</span> L. (18); At, <span class="html-italic">Arabidopsis thaliana</span> L. (5); Os, <span class="html-italic">Oryza sativa</span> L. (6); Td, <span class="html-italic">Triticum dicoccoides</span> L. (11); Tu, <span class="html-italic">Triticum urartu</span> L. (5); Aet, <span class="html-italic">Aegilops tauschii</span> L. (6); and Tt, <span class="html-italic">Triticum turgidum</span> L. (11). Based on the homologous genes of ATLa in wheat, 62 proteins are divided into 7 groups and marked with different colors. (<b>b</b>) Distribution and duplication events of <span class="html-italic">TaATLa</span> genes across the wheat genome. All typical <span class="html-italic">TaATLa</span> genes are mapped to 21 wheat chromosomes in a circle using Circos tool, and segmental duplications are mapped to their respective locations. Gray regions indicate all synteny blocks within the wheat genome, while red lines represent segmental duplications. The chromosome numbers are marked outside of the circle.</p> Full article ">Figure 2
<p>Collinearity analysis of <span class="html-italic">ATLa</span> genes by individually comparing <span class="html-italic">Triticum aestivum</span> with <span class="html-italic">Arabidopsis thaliana</span>, <span class="html-italic">Oryza sativa</span>, <span class="html-italic">Aegilops tauschii</span>, and <span class="html-italic">Triticum dicoccoides</span>. Gray lines in the background represent the collinear blocks of the plant genome and red lines in highlight indicate the syntenic <span class="html-italic">ATLa</span> gene pairs.</p> Full article ">Figure 3
<p>Gene structures and conserved motifs of <span class="html-italic">TaATLa</span> genes, and the prediction of cis-acting elements of <span class="html-italic">TaATLa</span> promoters. (<b>a</b>) The neighbor-joining (NJ) phylogenetic tree was constructed with protein sequences encoded by the longest transcript of <span class="html-italic">TaATLa</span> genes with bootstrap values of 1000 replicates. (<b>b</b>) Distribution of all motifs identified by MEME. Differently coloured frames represent different protein motifs. (<b>c</b>) Gene structures of the 18 <span class="html-italic">TaATLa</span> genes. The green rectangles in gene structures represent the coding sequences (CDSs), and the black lines represent introns. (<b>d</b>) Predicted cis-acting elements of <span class="html-italic">TaATLa</span> promoters by PlantCARE. The different cis-acting elements are represented by differently coloured boxes. Names of cis-acting elements are shown on the right.</p> Full article ">Figure 4
<p>A representation of the regulatory network between the putative miRNAs and their targeted <span class="html-italic">TaATLa</span> genes. Blue boxes represent <span class="html-italic">TaATLa</span> genes and beige boxes represent targeted miRNAs.</p> Full article ">Figure 5
<p>Expression pattern analysis of <span class="html-italic">TaATLa</span> gene subfamily. (<b>a</b>) Heatmap of <span class="html-italic">TaATLas</span> expression in a variety of tissues at different stages. (<b>b</b>) Heatmap of <span class="html-italic">TaATLas</span> expression before and after drought stress, heat stress, and co-drought and heat stress. D_1 and D_6 represent 1 h and 6 h after drought stress treatment of wheat, respectively; H_1 and H_6 represent 1 h and 6 h after heat stress treatment of wheat, respectively; DH_1 and DH_6 represent 1 h and 6 h after co-drought and heat stress treatment of wheat, respectively; CK represents no stress treatment of wheat. The red, white and blue cells represent the highest, medium, and lowest gene expression levels, respectively. The colour scale represents Log<sub>2</sub> expression values.</p> Full article ">Figure 6
<p>Yeast 22Δ10α growth complementation assay with an amino acid as the sole nitrogen source. (<b>a</b>) Images of yeast mutants transformed with <span class="html-italic">TaATLa4-7A</span>, <span class="html-italic">-7B</span>, <span class="html-italic">-7D</span> or empty vector pDR196 growth on YNB solid media were taken after 72 h at 28 °C. The 23344c (wild-type yeast strain) served as positive control. (<b>b</b>) Growth rates of yeast mutants transformed with <span class="html-italic">TaATLa4-7A</span>, <span class="html-italic">-7B</span>, <span class="html-italic">-7D</span> or empty vector pDR196. OD (Ab600) were measured at 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h (n = 3). (<b>c</b>) Images of yeast mutants transformed with <span class="html-italic">TaATLa6-7A</span>, <span class="html-italic">-7B</span>, <span class="html-italic">-7D</span> or empty vector pDR196 growth on YNB solid media were taken after 72 h at 28 °C. (<b>d</b>) Growth rates of yeast mutants transformed with <span class="html-italic">TaATLa6-7A</span>, <span class="html-italic">-7B</span>, <span class="html-italic">-7D</span> or empty vector pDR196.</p> Full article ">Figure 7
<p>Expression of <span class="html-italic">TaATLa4</span> and <span class="html-italic">TaATLa6</span> in yeast to determine their responses under high temperature stress. (<b>a</b>) Images of yeast mutant 22Δ10α transformed with <span class="html-italic">TaATLa4-7A</span>, <span class="html-italic">-7B</span>, <span class="html-italic">-7D</span> or empty vector pDR196 growth on synthetic defined media lacking uracil (SD-Ura) solid medium after 72 h at 28 °C and 39 °C, respectively. (<b>b</b>) Images of yeast mutant 22Δ10α transformed with <span class="html-italic">TaATLa6-7A</span>, <span class="html-italic">-7B</span>, <span class="html-italic">-7D</span> or empty vector pDR196 growth on SD-Ura solid medium after 72 h at 28 °C and 39 °C, respectively.</p> Full article ">
15 pages, 769 KiB  
Review
The Evolving Role of Cannabidiol-Rich Cannabis in People with Autism Spectrum Disorder: A Systematic Review
by Bilal Jawed, Jessica Elisabetta Esposito, Riccardo Pulcini, Syed Khuram Zakir, Matteo Botteghi, Francesco Gaudio, Daniele Savio, Caterina Martinotti, Stefano Martinotti and Elena Toniato
Int. J. Mol. Sci. 2024, 25(22), 12453; https://doi.org/10.3390/ijms252212453 - 20 Nov 2024
Viewed by 1753
Abstract
Autism spectrum disorder (ASD) is a neurological disease and lifelong condition. The treatment gap in ASD has led to growing interest in alternative therapies, particularly in phytocannabinoids, which are naturally present in Cannabis sativa. Studies indicate that treatment with cannabidiol (CBD)-rich cannabis [...] Read more.
Autism spectrum disorder (ASD) is a neurological disease and lifelong condition. The treatment gap in ASD has led to growing interest in alternative therapies, particularly in phytocannabinoids, which are naturally present in Cannabis sativa. Studies indicate that treatment with cannabidiol (CBD)-rich cannabis may possess the potential to improve fundamental ASD symptoms as well as comorbid symptoms. This systematic review aims to assess the safety and efficacy of CBD-rich cannabis in alleviating the symptoms of ASD in both children and adults, addressing the treatment gap and growing interest in CBD as an alternative treatment. A comprehensive literature search was conducted in February 2024 using the PUBMED and Scopus databases while following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The search focused on studies from 2020 onward involving human populations diagnosed with ASD and treated with CBD. Four studies met the inclusion criteria and were analyzed. The review included 353 participants with ASD from studies conducted in Israel, Turkey, and Brazil. The studies varied in design, sample size, dose, and treatment duration. Dosages of CBD were often combined with trace amounts of THC. Improvements were noted in behavioral symptoms, social responsiveness, and communication, but cognitive benefits were less consistent. Adverse effects ranged in severity. Mild effects such as somnolence and decreased appetite were common, while more concerning effects, including increased aggression, led to some cases of treatment discontinuation. CBD-rich cannabis shows promise in improving behavioral symptoms associated with ASD. However, variations in study designs, dosages, and outcome measures highlight the need for standardized assessment tools and further research to understand pharmacological interactions and optimize treatment protocols. Despite the mild adverse effects observed, larger, well-controlled trials are necessary to establish comprehensive safety and efficacy profiles. Full article
(This article belongs to the Section Molecular Neurobiology)
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<p>Study selection according to Preferred Reporting Items for Systematic Reviews and Meta Analysis (PRISMA) for evolving role of cannabidiol rich cannabis in people with autism spectrum disorder.</p> Full article ">
18 pages, 1514 KiB  
Article
Probenecid Inhibits Extracellular Signal-Regulated Kinase and c-Jun N-Terminal Kinase Mitogen-Activated Protein Kinase Pathways in Regulating Respiratory Syncytial Virus Response
by Les P. Jones, Harrison C. Bergeron, David E. Martin, Jackelyn Murray, Fred D. Sancilio and Ralph A. Tripp
Int. J. Mol. Sci. 2024, 25(22), 12452; https://doi.org/10.3390/ijms252212452 - 20 Nov 2024
Viewed by 685
Abstract
We examined the effect of probenecid in regulating the ERK and JNK downstream MAPK pathways affecting respiratory syncytial virus replication. Background: We have previously shown that probenecid inhibits RSV, influenza virus, and SARS-CoV-2 replication in vitro in preclinical animal models and in humans. [...] Read more.
We examined the effect of probenecid in regulating the ERK and JNK downstream MAPK pathways affecting respiratory syncytial virus replication. Background: We have previously shown that probenecid inhibits RSV, influenza virus, and SARS-CoV-2 replication in vitro in preclinical animal models and in humans. In a Phase two randomized, placebo-controlled, single-blind, dose range-finding study using probenecid to treat non-hospitalized patients with symptomatic, mild-to-moderate COVID-19, we previously showed that a 1000 mg twice daily treatment for 5 days reduced the median time to viral clearance from 11 to 7 days, and a 500 mg twice daily treatment for 5 days reduced the time to viral clearance from 11 to 9 days more than the placebo. Methods: In this study, we sought to determine the mechanism of action of the probenecid inhibition of RSV replication in human respiratory epithelial (A549) cells. Results: We show that probenecid inhibits the RSV-induced phosphorylation of JNKs and ERKs and the downstream phosphorylation of c-jun, a component of the AP-1 transcription complex needed for virus replication. The inhibition of JNKs by probenecid reversed the repression of transcription factor HNF-4. Conclusion: The probenecid inhibition of JNK and ERK phosphorylation involves the MAPK pathway that precludes virus replication. Full article
(This article belongs to the Special Issue MAPK Signaling Cascades in Human Health and Diseases 2.0)
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Figure 1
<p>Auto-Western blot analysis of JNK1,2 protein expression and phosphorylation in response to RSV infection of probenecid-treated A549 cells. (<b>A</b>) Fold change post-infection of JNK1,2 following treatment. (<b>B</b>) Auto-Western blot analysis of JNK1,2 protein expression following treatment. A549 cells were treated with 1 μM probenecid in 0.02% DMSO, or 25 μM SP600125 in 0.02% DMSO, or diluent (0.02% DMSO) for 2 h, or given no treatment. Cells were infected with RSV A2 (MOI = 1.0) for 24 hpi before harvesting, or cells were not infected and cultured for 24 h before harvesting. Culture supernatants were removed, and cells washed and subjected to lysis. Total cell lysates were clarified by centrifugation, and total protein concentration was estimated by BCA protein analysis. Cell lysates were transferred to RayBioTech (Atlanta, GA, USA) for auto-Western blot analysis using their validated antibodies for specific antigen detection. All samples were adjusted to 0.2 mg/mL total protein concentration by RayBioTech prior to auto-Western blot analysis. Experiments were performed with independent replicates for each condition tested. Chemiluminescence values from auto-Western blot readout corresponding to specific band densities from each analyte were normalized to β-actin and mean value calculated for replicate samples. Fold change post-infection was determined by dividing mean normalized band density values corresponding to RSV-infected samples by mean normalized band density values corresponding to uninfected control samples.</p> Full article ">Figure 1 Cont.
<p>Auto-Western blot analysis of JNK1,2 protein expression and phosphorylation in response to RSV infection of probenecid-treated A549 cells. (<b>A</b>) Fold change post-infection of JNK1,2 following treatment. (<b>B</b>) Auto-Western blot analysis of JNK1,2 protein expression following treatment. A549 cells were treated with 1 μM probenecid in 0.02% DMSO, or 25 μM SP600125 in 0.02% DMSO, or diluent (0.02% DMSO) for 2 h, or given no treatment. Cells were infected with RSV A2 (MOI = 1.0) for 24 hpi before harvesting, or cells were not infected and cultured for 24 h before harvesting. Culture supernatants were removed, and cells washed and subjected to lysis. Total cell lysates were clarified by centrifugation, and total protein concentration was estimated by BCA protein analysis. Cell lysates were transferred to RayBioTech (Atlanta, GA, USA) for auto-Western blot analysis using their validated antibodies for specific antigen detection. All samples were adjusted to 0.2 mg/mL total protein concentration by RayBioTech prior to auto-Western blot analysis. Experiments were performed with independent replicates for each condition tested. Chemiluminescence values from auto-Western blot readout corresponding to specific band densities from each analyte were normalized to β-actin and mean value calculated for replicate samples. Fold change post-infection was determined by dividing mean normalized band density values corresponding to RSV-infected samples by mean normalized band density values corresponding to uninfected control samples.</p> Full article ">Figure 2
<p>Auto-Western blot analysis of ERK1/2 protein expression and phosphorylation in response to RSV infection of probenecid-treated A549 cells. (<b>A</b>) Fold change post-infection of ERK1,2 following treatment. (<b>B</b>) Auto-Western blot analysis of ERK1,2 protein expression following treatment. A549 cells were treated with 1 μM probenecid in 0.02% DMSO, or 25 μM SP600125 in 0.02% DMSO, or diluent (0.02% DMSO) for 2 h, or given no treatment. Cells were infected with RSV A2 (MOI = 1.0) for 24 hpi before harvesting, or cells were not infected and cultured for 24 h before harvesting. Culture supernatants were removed, and cells washed and subjected to lysis. Total cell lysates were clarified by centrifugation, and total protein concentration was estimated by BCA protein analysis. Cell lysates were transferred to RayBioTech (Atlanta, GA, USA) for auto-Western analysis using their validated antibodies for specific antigen detection. All samples were adjusted to 0.2 mg/mL total protein concentration by RayBioTech prior to auto-Western blot analysis. Experiments were performed with independent replicates for each condition tested. Chemiluminescence values from auto-Western blot readout corresponding to specific band densities from each analyte were normalized to β-actin and mean value calculated for replicate samples. Fold change post-infection was determined by dividing mean normalized band density values corresponding to RSV-infected samples by mean normalized band density values corresponding to uninfected control samples.</p> Full article ">Figure 2 Cont.
<p>Auto-Western blot analysis of ERK1/2 protein expression and phosphorylation in response to RSV infection of probenecid-treated A549 cells. (<b>A</b>) Fold change post-infection of ERK1,2 following treatment. (<b>B</b>) Auto-Western blot analysis of ERK1,2 protein expression following treatment. A549 cells were treated with 1 μM probenecid in 0.02% DMSO, or 25 μM SP600125 in 0.02% DMSO, or diluent (0.02% DMSO) for 2 h, or given no treatment. Cells were infected with RSV A2 (MOI = 1.0) for 24 hpi before harvesting, or cells were not infected and cultured for 24 h before harvesting. Culture supernatants were removed, and cells washed and subjected to lysis. Total cell lysates were clarified by centrifugation, and total protein concentration was estimated by BCA protein analysis. Cell lysates were transferred to RayBioTech (Atlanta, GA, USA) for auto-Western analysis using their validated antibodies for specific antigen detection. All samples were adjusted to 0.2 mg/mL total protein concentration by RayBioTech prior to auto-Western blot analysis. Experiments were performed with independent replicates for each condition tested. Chemiluminescence values from auto-Western blot readout corresponding to specific band densities from each analyte were normalized to β-actin and mean value calculated for replicate samples. Fold change post-infection was determined by dividing mean normalized band density values corresponding to RSV-infected samples by mean normalized band density values corresponding to uninfected control samples.</p> Full article ">Figure 3
<p>Auto-Western blot analysis of c-Jun protein expression and phosphorylation in response to RSV infection of probenecid-treated A549 cells. (<b>A</b>) Fold change post-infection of c-Jun following treatment. (<b>B</b>) Auto-Western blot analysis of c-Jun protein expression following treatment. A549 cells were treated with 1 μM probenecid in 0.02% DMSO, or 25 μM SP600125 in 0.02% DMSO, or diluent (0.02% DMSO) for 2 h, or given no treatment. Cells were infected with RSV A2 (MOI = 1.0) for 24 hpi before harvesting, or cells were not infected and cultured for 24 h before harvesting. Culture supernatants were removed, cells washed, and subjected to lysis. Total cell lysates were clarified by centrifugation, and total protein concentration was estimated by BCA protein analysis. Cell lysates were transferred to RayBioTech (Atlanta, GA, USA) for auto-Western analysis using their validated antibodies for specific antigen detection. All samples were adjusted to 0.2 mg/mL total protein concentration by RayBioTech prior to auto-Western blot analysis. Experiments were performed with independent replicates for each condition tested. Chemiluminescence values from auto-Western blot readout corresponding to specific band densities from each analyte were normalized to β-actin and mean value calculated for replicate samples. Fold change post-infection was determined by dividing mean normalized band density values corresponding to RSV-infected samples by mean normalized band density values corresponding to uninfected control samples.</p> Full article ">Figure 3 Cont.
<p>Auto-Western blot analysis of c-Jun protein expression and phosphorylation in response to RSV infection of probenecid-treated A549 cells. (<b>A</b>) Fold change post-infection of c-Jun following treatment. (<b>B</b>) Auto-Western blot analysis of c-Jun protein expression following treatment. A549 cells were treated with 1 μM probenecid in 0.02% DMSO, or 25 μM SP600125 in 0.02% DMSO, or diluent (0.02% DMSO) for 2 h, or given no treatment. Cells were infected with RSV A2 (MOI = 1.0) for 24 hpi before harvesting, or cells were not infected and cultured for 24 h before harvesting. Culture supernatants were removed, cells washed, and subjected to lysis. Total cell lysates were clarified by centrifugation, and total protein concentration was estimated by BCA protein analysis. Cell lysates were transferred to RayBioTech (Atlanta, GA, USA) for auto-Western analysis using their validated antibodies for specific antigen detection. All samples were adjusted to 0.2 mg/mL total protein concentration by RayBioTech prior to auto-Western blot analysis. Experiments were performed with independent replicates for each condition tested. Chemiluminescence values from auto-Western blot readout corresponding to specific band densities from each analyte were normalized to β-actin and mean value calculated for replicate samples. Fold change post-infection was determined by dividing mean normalized band density values corresponding to RSV-infected samples by mean normalized band density values corresponding to uninfected control samples.</p> Full article ">Figure 4
<p>Auto-Western blot analysis of HNF-4 protein expression and phosphorylation in response to RSV infection of probenecid-treated A549 cells. (<b>A</b>) Fold change post-infection of HNF-4 following treatment. (<b>B</b>) Auto-Western blot analysis of HNF-4 protein expression following treatment. A549 cells were treated with 1 μM probenecid in 0.02% DMSO, or 25 μM SP600125 in 0.02% DMSO, or diluent (0.02% DMSO) for 2 h, or given no treatment. Cells were infected with RSV A2 (MOI = 1.0) for 24 hpi before harvesting, or cells were not infected and cultured for 24 h before harvesting. Culture supernatants were removed, cells washed, and subjected to lysis. Total cell lysates were clarified by centrifugation, and total protein concentration was estimated by BCA protein analysis. Cell lysates were transferred to RayBioTech (Atlanta, GA, USA) for auto-Western blot analysis using their validated antibodies for specific antigen detection. All samples were adjusted to 0.2 mg/mL total protein concentration by RayBioTech prior to auto-Western blot analysis. Experiments were performed with independent replicates for each condition tested. Chemiluminescence values from auto-Western blot readout corresponding to specific band densities from each analyte were normalized to β-actin and mean value calculated for replicate samples. Fold change post-infection was determined by dividing mean normalized band density values corresponding to RSV-infected samples by mean normalized band density values corresponding to uninfected control samples.</p> Full article ">Figure 4 Cont.
<p>Auto-Western blot analysis of HNF-4 protein expression and phosphorylation in response to RSV infection of probenecid-treated A549 cells. (<b>A</b>) Fold change post-infection of HNF-4 following treatment. (<b>B</b>) Auto-Western blot analysis of HNF-4 protein expression following treatment. A549 cells were treated with 1 μM probenecid in 0.02% DMSO, or 25 μM SP600125 in 0.02% DMSO, or diluent (0.02% DMSO) for 2 h, or given no treatment. Cells were infected with RSV A2 (MOI = 1.0) for 24 hpi before harvesting, or cells were not infected and cultured for 24 h before harvesting. Culture supernatants were removed, cells washed, and subjected to lysis. Total cell lysates were clarified by centrifugation, and total protein concentration was estimated by BCA protein analysis. Cell lysates were transferred to RayBioTech (Atlanta, GA, USA) for auto-Western blot analysis using their validated antibodies for specific antigen detection. All samples were adjusted to 0.2 mg/mL total protein concentration by RayBioTech prior to auto-Western blot analysis. Experiments were performed with independent replicates for each condition tested. Chemiluminescence values from auto-Western blot readout corresponding to specific band densities from each analyte were normalized to β-actin and mean value calculated for replicate samples. Fold change post-infection was determined by dividing mean normalized band density values corresponding to RSV-infected samples by mean normalized band density values corresponding to uninfected control samples.</p> Full article ">
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