Cells

22 pages, 2801 KiB

Open AccessReview

Metabolic Implications of Immune Checkpoint Proteins in Cancer

by Elizabeth R. Stirling, Steven M. Bronson, Jessica D. Mackert, Katherine L. Cook, Pierre L. Triozzi and David R. Soto-Pantoja

Cells 2022, 11(1), 179; https://doi.org/10.3390/cells11010179 - 5 Jan 2022

Cited by 21 | Viewed by 5973

Abstract

Expression of immune checkpoint proteins restrict immunosurveillance in the tumor microenvironment; thus, FDA-approved checkpoint inhibitor drugs, specifically PD-1/PD-L1 and CTLA-4 inhibitors, promote a cytotoxic antitumor immune response. Aside from inflammatory signaling, immune checkpoint proteins invoke metabolic reprogramming that affects immune cell function, autonomous [...] Read more.

Expression of immune checkpoint proteins restrict immunosurveillance in the tumor microenvironment; thus, FDA-approved checkpoint inhibitor drugs, specifically PD-1/PD-L1 and CTLA-4 inhibitors, promote a cytotoxic antitumor immune response. Aside from inflammatory signaling, immune checkpoint proteins invoke metabolic reprogramming that affects immune cell function, autonomous cancer cell bioenergetics, and patient response. Therefore, this review will focus on the metabolic alterations in immune and cancer cells regulated by currently approved immune checkpoint target proteins and the effect of costimulatory receptor signaling on immunometabolism. Additionally, we explore how diet and the microbiome impact immune checkpoint blockade therapy response. The metabolic reprogramming caused by targeting these proteins is essential in understanding immune-related adverse events and therapeutic resistance. This can provide valuable information for potential biomarkers or combination therapy strategies targeting metabolic pathways with immune checkpoint blockade to enhance patient response. Full article

(This article belongs to the Special Issue Metabolic Interactions in Tumor Microenvironment (TME))

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Figure 1

Figure 1
Immune checkpoint proteins regulate metabolic signaling on T cells. (A) Interactions with cancer or antigen-presenting cells can modulate T cell metabolism (B) PD-L1 binding to PD-1, regulates fatty acid oxidation on T cells and limits glutamine metabolism by reduction of SNAT1/2 (C) Activation of CTLA-4 inhibits glycolysis within activated effector T cells inhibiting PI3K/AKT signaling, reduction of glucose uptake by inhibition of GLUT-1. Full article ">Figure 2
Stimulatory checkpoint proteins regulate metabolic signaling. (A) CD28 activation promotes PI3K/Akt/mTORC1 pathway signaling, increasing glycolysis and mitochondrial metabolism. (B) ICOS/ICOSL activation increases glucose uptake and metabolism through mTOR activation. (C) GITR activation can stimulate the TCA cycle by metabolism lipid, glucose, and other nutrient stores (D) 4-1BB activation increases GLUT-1 expression to enhance glycolysis and fatty acid metabolism through LKB1/AMPK/ACC pathway activation. Full article ">Figure 3
Regulation of metabolic signaling of immune checkpoint inhibitors on cancer cells. (A) PD-L1 engagement results in activation of glycolysis and activation of the PI3K/Akt/mTOR pathway. (B) Antibodies to PD-L1 are known to inhibit its metabolic signaling. (C) PD-1 on cancer cells limits Akt and ERK1/2 signaling regulating cancer cell proliferation. Full article ">Figure 4
Metabolic signaling and regulation of PD-L1 expression. (A) Glucose (GLU) starvation and activation of AMPK results in the degradation of PD-L1 and a decrease in its expression. (B) Glutamine (GLN) depletion activates EGFR and MAPK signaling, resulting in the upregulation of PD-L1 during hypoxia. Full article ">

18 pages, 3983 KiB

Open AccessProtocol

Isolation and Propagation of Human Corneal Stromal Keratocytes for Tissue Engineering and Cell Therapy

by Nur Zahirah binte M. Yusoff, Andri K. Riau, Gary H. F. Yam, Nuur Shahinda Humaira binte Halim and Jodhbir S. Mehta

Cells 2022, 11(1), 178; https://doi.org/10.3390/cells11010178 - 5 Jan 2022

Cited by 23 | Viewed by 3744

Abstract

The human corneal stroma contains corneal stromal keratocytes (CSKs) that synthesize and deposit collagens and keratan sulfate proteoglycans into the stromal matrix to maintain the corneal structural integrity and transparency. In adult corneas, CSKs are quiescent and arrested in the G0 phase of [...] Read more.

The human corneal stroma contains corneal stromal keratocytes (CSKs) that synthesize and deposit collagens and keratan sulfate proteoglycans into the stromal matrix to maintain the corneal structural integrity and transparency. In adult corneas, CSKs are quiescent and arrested in the G0 phase of the cell cycle. Following injury, some CSKs undergo apoptosis, whereas the surviving cells are activated to become stromal fibroblasts (SFs) and myofibroblasts (MyoFBs), as a natural mechanism of wound healing. The SFs and MyoFBs secrete abnormal extracellular matrix proteins, leading to corneal fibrosis and scar formation (corneal opacification). The issue is compounded by the fact that CSK transformation into SFs or MyoFBs is irreversible in vivo, which leads to chronic opacification. In this scenario, corneal transplantation is the only recourse. The application of cell therapy by replenishing CSKs, propagated in vitro, in the injured corneas has been demonstrated to be efficacious in resolving early-onset corneal opacification. However, expanding CSKs is challenging and has been the limiting factor for the application in corneal tissue engineering and cell therapy. The supplementation of serum in the culture medium promotes cell division but inevitably converts the CSKs into SFs. Similar to the in vivo conditions, the transformation is irreversible, even when the SF culture is switched to a serum-free medium. In the current article, we present a detailed protocol on the isolation and propagation of bona fide human CSKs and the morphological and genotypic differences from SFs. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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Figure 1
Overview of human corneal stromal keratocyte (CSK) cell culture procedure. In the propagation phase, the culture medium is supplemented with 0.5% fetal bovine serum (FBS) to support the proliferation of CSKs, which are otherwise quiescent. In the stabilization phase, the FBS is removed from the culture medium to allow the cells to regain bona fide CSK phenotypes. Full article ">Figure 2
Still photographs of the human corneal tissue dissection. (A–D) The first dissection steps involve the removal of the corneal epithelial and endothelial cells, and trabecular meshwork from the corneas, by scraping with a surgical blade no. 10. (E,F) The corneal stroma is then separated from the scleral tissue by cutting ~2 mm from the sclera. (G–I) Finally, the corneal stroma is cut into smaller pieces by leaving the edges of each piece still attached to the adjacent pieces. Full article ">Figure 3
Proliferative capacity of corneal stromal keratocytes (CSKs), activated CSKs (A-CSKs), and stromal fibroblasts (SFs). The representative images of CSKs (A), A-CSKs (B), and SFs (C) were captured from cells at P5, which were expanded from the same donor. (D) The proliferative capacity (indicated by Ki-67-positive cells/total number of cells × 100%) of the A-CSKs was 5.6 ± 6.1%. On day 14, following medium switching to serum-free conditions, the proliferative capacity of CSKs was 0%. The SFs had a significantly higher proliferation rate of 20.1 ± 7.2% compared to both the CSKs (p = 3.55 × 10−8) and the activated CSKs (p = 3.78 × 10−5). Group comparisons were statistically determined using one-way ANOVA and Tukey comparison tests. Scale bars = 100 μm. Full article ">Figure 4
Morphology of corneal stromal keratocytes (CSKs), activated CSKs (A-CSKs), and stromal fibroblasts (SFs). The representative images were captured from cells at P5, which were expanded from the same donor. (A–F) Brightfield images at low and high magnification revealed the loss of thin, dendritic morphology and long cellular processes, typically seen in the CSKs, in the SFs. The SFs also featured larger cell bodies compared to the CSKs and A-CSKs. (G–I) Phalloidin staining showed the stellate morphology of the CSKs, which was progressively lost in the A-CSK and SF cell culture. Scale bars = 100 μm. Full article ">Figure 5
Protein expression of corneal stromal keratocytes (CSKs), activated CSKs (A-CSKs), and corneal fibroblasts (SFs). (A,D,G,J) Typical CSK markers, such as ALDH1A1, ALDH3A1, keratocan, and lumican were strongly expressed in the CSKs following 14 days of culture media switching to serum-free conditions. (B,E,H,K) In the propagation medium, the A-CSKs exhibited an attenuated expression of the CSK markers. (C,F,I,L) In contrast, in medium supplemented with 5% FBS, the SFs did not express or express only a little of the CSK markers. (M,N,O) All three cell types were not immunoreactive with α-smooth muscle actin (α-SMA), the cell marker of corneal stromal myofibroblasts (see inset in pane O). Scale bars = 50 μm. Full article ">Figure 6
Gene expression of corneal stromal keratocytes (CSKs), activated CSKs (A-CSKs), and corneal fibroblasts (SFs). The gene expression was detected using real time-polymerase chain reaction. Similar to the protein expression, CSK-associated genes, such as ALDH1A1 (A), ALDH3A1 (B), KERA (C), and LUM (D) were strongly expressed in the CSKs following 14 days of culture media switching to serum-free conditions and were significantly upregulated when compared to the A-CSKs and SFs. (E) The corneal stromal myofibroblast (MyoFB)-associated gene, ACTA2, was significantly downregulated in the CSKs, A-CSKs, and SFs compared to the MyoFBs. For the analysis of differentially expressed genes, CSKs was used as the reference group for comparison, whereas GAPDH was used as the housekeeping gene. Group comparisons were statistically determined using one-way ANOVA and Tukey comparison tests. Full article ">

18 pages, 6420 KiB

Open AccessArticle

Bioengineered Cystinotic Kidney Tubules Recapitulate a Nephropathic Phenotype

by Elena Sendino Garví, Rosalinde Masereeuw and Manoe J. Janssen

Cells 2022, 11(1), 177; https://doi.org/10.3390/cells11010177 - 5 Jan 2022

Cited by 5 | Viewed by 3009

Abstract

Nephropathic cystinosis is a rare and severe disease caused by disruptions in the CTNS gene. Cystinosis is characterized by lysosomal cystine accumulation, vesicle trafficking impairment, oxidative stress, and apoptosis. Additionally, cystinotic patients exhibit weakening and leakage of the proximal tubular segment of the [...] Read more.

Nephropathic cystinosis is a rare and severe disease caused by disruptions in the CTNS gene. Cystinosis is characterized by lysosomal cystine accumulation, vesicle trafficking impairment, oxidative stress, and apoptosis. Additionally, cystinotic patients exhibit weakening and leakage of the proximal tubular segment of the nephrons, leading to renal Fanconi syndrome and kidney failure early in life. Current in vitro cystinotic models cannot recapitulate all clinical features of the disease which limits their translational value. Therefore, the development of novel, complex in vitro models that better mimic the disease and exhibit characteristics not compatible with 2-dimensional cell culture is of crucial importance for novel therapies development. In this study, we developed a 3-dimensional bioengineered model of nephropathic cystinosis by culturing conditionally immortalized proximal tubule epithelial cells (ciPTECs) on hollow fiber membranes (HFM). Cystinotic kidney tubules showed lysosomal cystine accumulation, increased autophagy and vesicle trafficking deterioration, the impairment of several metabolic pathways, and the disruption of the epithelial monolayer tightness as compared to control kidney tubules. In particular, the loss of monolayer organization and leakage could be mimicked with the use of the cystinotic kidney tubules, which has not been possible before, using the standard 2-dimensional cell culture. Overall, bioengineered cystinotic kidney tubules recapitulate better the nephropathic phenotype at a molecular, structural, and functional proximal tubule level compared to 2-dimensional cell cultures. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of Nephropathic Cystinosis)

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16 pages, 28830 KiB

Open AccessArticle

Targeting PGM3 as a Novel Therapeutic Strategy in KRAS/LKB1 Co-Mutant Lung Cancer

by Hyunmin Lee, Feng Cai, Neil Kelekar, Nipun K. Velupally and Jiyeon Kim

Cells 2022, 11(1), 176; https://doi.org/10.3390/cells11010176 - 5 Jan 2022

Cited by 11 | Viewed by 4879

Abstract

In non-small-cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and tumor suppressor STK11 (also known as LKB1) confer an aggressive malignant phenotype, an unfavourability towards immunotherapy, and overall poor prognoses in patients. In a previous study, we showed that murine KRAS/LKB1 [...] Read more.

In non-small-cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and tumor suppressor STK11 (also known as LKB1) confer an aggressive malignant phenotype, an unfavourability towards immunotherapy, and overall poor prognoses in patients. In a previous study, we showed that murine KRAS/LKB1 co-mutant tumors and human co-mutant cancer cells have an enhanced dependence on glutamine-fructose-6-phosphate transaminase 2 (GFPT2), a rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP), which could be targeted to reduce survival of KRAS/LKB1 co-mutants. Here, we found that KRAS/LKB1 co-mutant cells also exhibit an increased dependence on N-acetylglucosamine-phosphate mutase 3 (PGM3), an enzyme downstream of GFPT2. Genetic or pharmacologic suppression of PGM3 reduced KRAS/LKB1 co-mutant tumor growth in both in vitro and in vivo settings. Our results define an additional metabolic vulnerability in KRAS/LKB1 co-mutant tumors to the HBP and provide a rationale for targeting PGM3 in this aggressive subtype of NSCLC. Full article

(This article belongs to the Special Issue New Aspects of Targeting Cancer Metabolism in Therapeutic Approach)

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27 pages, 14903 KiB

Open AccessArticle

Accelerated Growth, Differentiation, and Ploidy with Reduced Proliferation of Right Ventricular Cardiomyocytes in Children with Congenital Heart Defect Tetralogy of Fallot

by Tatyana V. Sukhacheva, Roman A. Serov, Natalia V. Nizyaeva, Artem A. Burov, Stanislav V. Pavlovich, Yulia L. Podurovskaya, Maria V. Samsonova, Andrey L. Chernyaev, Aleksandr I. Shchegolev, Alexei I. Kim, Leo A. Bockeria and Gennady T. Sukhikh

Cells 2022, 11(1), 175; https://doi.org/10.3390/cells11010175 - 5 Jan 2022

Cited by 7 | Viewed by 3105

Abstract

The myocardium of children with tetralogy of Fallot (TF) undergoes hemodynamic overload and hypoxemia immediately after birth. Comparative analysis of changes in the ploidy and morphology of the right ventricular cardiomyocytes in children with TF in the first years of life demonstrated their [...] Read more.

The myocardium of children with tetralogy of Fallot (TF) undergoes hemodynamic overload and hypoxemia immediately after birth. Comparative analysis of changes in the ploidy and morphology of the right ventricular cardiomyocytes in children with TF in the first years of life demonstrated their significant increase compared with the control group. In children with TF, there was a predominantly diffuse distribution of Connexin43-containing gap junctions over the cardiomyocytes sarcolemma, which redistributed into the intercalated discs as cardiomyocytes differentiation increased. The number of Ki67-positive cardiomyocytes varied greatly and amounted to 7.0–1025.5/10⁶ cardiomyocytes and also were decreased with increased myocytes differentiation. Ultrastructural signs of immaturity and proliferative activity of cardiomyocytes in children with TF were demonstrated. The proportion of interstitial tissue did not differ significantly from the control group. The myocardium of children with TF under six months of age was most sensitive to hypoxemia, it was manifested by a delay in the intercalated discs and myofibril assembly and the appearance of ultrastructural signs of dystrophic changes in the cardiomyocytes. Thus, the acceleration of ontogenetic growth and differentiation of the cardiomyocytes, but not the reactivation of their proliferation, was an adaptation of the immature myocardium of children with TF to hemodynamic overload and hypoxemia. Full article

(This article belongs to the Section Cell Proliferation and Division)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Distribution of CMCs of different ploidy classes in patients with TF and the control group. Comparison of ploidy (2c:4c:8c:16c:32c) of patients with TF and the control group (A). The ploidy of the CMCs in children with TF was significantly higher than the CMCs in the control groups (median, Mann-Whitney test, p < 0.05) (B). Comparison of CMCs ploidy classes in patients with TF and controls (C). Change in the ratio of different ploidy classes in patients with TF in different age groups: up to six months, 7–12 months, more than 13 months (D). Comparison of the proportion of binucleated CMCs in the group of children with TF and the control group (median, Mann-Whitney test, p < 0.05) (E). Inverse correlation of CMCs ploidy in children with TF with Nakata index (r = −0.44; p = 0.034) (F). In the myocardium of children under six months of age with TF: CMCs ploidy correlated inversely with age (r = −0.73; p = 0.007) (G). The myocardium of patients with a high percentage of diploid CMCs differentiated faster—the number of CMCs filled with myofibrils increased in it (r = 0.68; p = 0.015) (H) and the number of CMCs not filled with myofibrils decreased (r = −0.68; p = 0.014) (I). Full article ">Figure 2
RV myocardium of patients with TF and in the control group. In the myocardium of patients with TF: significant variability of the CMCs diameter was revealed: on average, from 7.5 ± 2.4 μm (patient with TF, 10 months) (A) and 10.0 ± 1.5 μm (patient with TF, 5 months) (B) up to 10.8 ± 1.5 μm (patient with TF, 8 months) (C). In the control group: the diameter was 4.7–7.7 microns (D,E). Hematoxylin and eosin (×400) bar 20 µm. The diameter of the CMCs of children with TF significantly exceeded <a href="#cells-11-00175-t001" class="html-table">Table 1</a>. (F). In children with TF over 6 months of age: The proportion of CMCs filled with myofibrils increased with age (r = 0.49; p = 0.003) (G), a high-pressure gradient on the pulmonary artery provoked accelerated differentiation with a decrease in the number of CMCs with sarcoplasm not filled with myofibrils (r = −0.39; p = 0.04) (H). Full article ">Figure 3
Connexin-43-containing (Cx43) gap junctions (GJs) in RV CMCs of patients with TF and the control group. In the myocardium of children with TF: Cx43-containing GJs were located both in the intercalated discs (A) and on the lateral sides, along the perimeter of the sarcolemma of the CMCs (B); a large number of binucleated CMCs. Immunohistochemical staining (×200), bar 10 µm. At the ultrastructural level, lateral GJs were found at the border of two CMCs in the form of concentric structures, following the bends of the CMCs sarcolemma (arrows) (C). In the control group: point Cx43-containing GJs were diffusely located along the perimeter of the sarcolemma (D). In the differentiated CMCs filled with myofibrils, the Cx43-containing GJs were localized predominantly in the intercalated discs (E). Immunohistochemical staining (×200), bar 10 µm. The relative length of lateral Cx43-containing GJs was significantly higher in the myocardium of patients with TF than in controls (Mann-Whitney test, p < 0.05) (F). The relative length of Cx43-containing GJs on the lateral surfaces of the CMCs decreased with age (r = −0.45; p = 0.0003) (G). The lateral arrangement of Cx43-containing GJs was characteristic of immature CMCs with incomplete differentiation, not filled with myofibrils (H). Lateral Cx43-containing GJs were less frequently observed in TF patients with low blood oxygen saturation, which suggested their accelerated differentiation (r = 0.34; p = 0.016) (I). In children with TF under six months of age, the lateral arrangement of Cx43-containing GJs was inversely correlated with blood oxygen saturation (r = −0.76; p = 0.028) (J) and positively correlated with a history of dyspnea-cyanotic attacks (r = 0.58; p = 0.002) (K). Full article ">Figure 4
Ultrastructure of immature RV CMCs of children with TF. CMCs of an immature phenotype with electron-transparent sarcoplasm, not filled with myofibrils, with many small mitochondria and vacuoles in the sarcoplasm. Myofibrils were located in a thin layer along the periphery of the sarcoplasm, bar 5 µm (A). In the CMCs of a more differentiated phenotype, myofibrils filled most of the sarcoplasm, in the perinuclear zone there were mitochondria, glycogen granules, single lipofuscin granules, bar 5 µm (B). Assembly of myofibrils in the sarcoplasm of the CMC, bar 1 µm. (C). T-system channels at the level of Z-bands of organizing myofibrils, bar 0,5 µm (D). Paired centrioles, cisterns, and vesicles of the Golgi apparatus in the perinuclear zone of the CMC, bar 0.5 µm (E). Myelin figures, vacuole in the perinuclear zone of the CMC, bar 1 µm. (F). In children with TF under six months of age: the assembly of myofibrils was more common in the CMCs of patients with a low gradient on the pulmonary artery (r = −0.52; p = 0.01) (G); signs of dystrophic changes in ultrastructure (myelin figures, phagosomes) were found in the CMCs of children with low hemoglobin levels (r = −0.54; p = 0.025) (H), in the myocardium with a large number of multinucleated CMCs (r = 0.77; p = 0.006) (I), in differentiated CMCs with a small relative length of lateral Cx43-containing GJs (r = −0.47; p = 0.32) (J). Full article ">Figure 5
Interstitial tissue of the RV myocardium of children with TF and the control group. An insignificant proportion of interstitial tissue in the perivascular zone of the myocardium in children with TF (A,B) and the control group (C,D). Masson trichrome (×200), bar 10 µm. The proportion of interstitial tissue in the myocardium of children with TF and the control group did not differ significantly (E) (median, Mann-Whitney test, p > 0.05). CD34-positive capillaries and small vessels in the myocardial interstitium of a patient with TF. Immunohistochemical staining (×400), bar 20 µm (F). In children with TF over 6 months of age: the proportion of interstitial tissue correlated with age (r = 0.38; p = 0.03) (G) and increased with an increase in the number of differentiated CMCs, the sarcoplasm of which was filled with myofibrils (myofibril-free zones were 10%)(r = 0.64; p = 0.0001) (H). The density of CD34-positive small vessels and capillaries directly correlated with LVEF (r = 0.64; p = 0.003) (I). Full article ">Figure 6
Proliferative-active Ki67+/Sarc α-actin+ CMC in the RV myocardium of patients with TF. In multinucleated CMC, all nuclei were Ki67-positive (arrows) (A). Alexa 488—anti-rabbit antibody to Ki67 (B); Alexa 546—anti-mouse antibody to Sarcomeric α-Actin (C); DAPI nuclei staining (D). Immunoconfocal microscopy, bar 10 мкм. Patient with TF, five months. Ki67-positive CMCs were detected in the myocardium with a large proportion of interstitial tissue (r = 0.33; p = 0.27) (E); in the myocardium with a smaller diameter of the CMCs (r = −0.31; p = 0.02) (F); in the myocardium with a greater relative length of lateral Cx43-containing GJs in the CMCs (r = 0.29; p = 0.02) (G). Full article ">

6 pages, 37108 KiB

Open AccessCommentary

Lymphoma versus Carcinoma and Other Collaborations

by Karen Pulford

Cells 2022, 11(1), 174; https://doi.org/10.3390/cells11010174 - 5 Jan 2022

Viewed by 1887

Abstract

David Mason started his research career at a time when lymphoma diagnosis was based primarily on cellular morphology, clinical symptoms and special cytochemical stains using formalin fixed tissue sections. There were occasions, however, where the morphology was unhelpful, such as in the case [...] Read more.

David Mason started his research career at a time when lymphoma diagnosis was based primarily on cellular morphology, clinical symptoms and special cytochemical stains using formalin fixed tissue sections. There were occasions, however, where the morphology was unhelpful, such as in the case of anaplastic or poorly differentiated tumours, where a distinction between lymphoma and a non-haematopoietic tumour was often problematical. Accurate diagnosis became even more important with the developments in the clinical staging of lymphoma and the availability of more effective treatments. One way forward to improve diagnosis was to use immunohistochemistry to study the antigens expressed by the tumor cells. Full article

(This article belongs to the Special Issue Microscope Based Technologies from Clinical Application to Cell Biology: A Themed Issue in Memory of Prof. Kevin C. Gatter and Prof. David Y. Mason)

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22 pages, 4308 KiB

Open AccessArticle

Dermatan Sulfate Affects Breast Cancer Cell Function via the Induction of Necroptosis

by Grzegorz Wisowski, Adam Pudełko, Krystyna Olczyk, Monika Paul-Samojedny and Ewa M. Koźma

Cells 2022, 11(1), 173; https://doi.org/10.3390/cells11010173 - 5 Jan 2022

Cited by 6 | Viewed by 2480

Abstract

Dermatan sulfate (DS) is widespread in the extracellular matrix (ECM) of animal tissues. This glycosaminoglycan is characterized by a variable structure, which is reflected in the heterogeneity of its sulfation pattern. The sulfate groups are responsible for the binding properties of DS, which [...] Read more.

Dermatan sulfate (DS) is widespread in the extracellular matrix (ECM) of animal tissues. This glycosaminoglycan is characterized by a variable structure, which is reflected in the heterogeneity of its sulfation pattern. The sulfate groups are responsible for the binding properties of DS, which determine an interaction profile of this glycan. However, the detailed role of DS in biological processes such as the neoplasm is still poorly understood. The aim of the study was to assess the effects of the structural variants of DS on breast cancer cells. We found that DS isoforms from normal and fibrotic fascia as well as from intestinal mucosa were able to quickly induce oxidative stress in the cytoplasm and affect the mitochondrial function in luminal breast cancer cells. Moreover, the variants caused the necroptosis of the cells most likely via the first of these mechanisms. This death was responsible for a reduction in the viability and number of breast cancer cells. However, the dynamics and intensity of all of the DS variants-triggered effects were strongly dependent on the cell type and the structure of these molecules. The most pronounced activity was demonstrated by those variants that shared structural features with the DS from the tumor niche. Full article

(This article belongs to the Special Issue Tumor Microenvironment and Cancer Cells—Key Interactions in Cancer Progression)

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19 pages, 7538 KiB

Open AccessReview

Role and Involvement of TENM4 and miR-708 in Breast Cancer Development and Therapy

by Giulia Peppino, Federica Riccardo, Maddalena Arigoni, Elisabetta Bolli, Giuseppina Barutello, Federica Cavallo and Elena Quaglino

Cells 2022, 11(1), 172; https://doi.org/10.3390/cells11010172 - 5 Jan 2022

Cited by 5 | Viewed by 3652

Abstract

Teneurin 4 (TENM4) is a transmembrane protein that is codified by the ODZ4 gene and is involved in nervous system development, neurite outgrowth, and neuronal differentiation. In line with its involvement in the nervous system, TENM4 has also been implicated in several mental [...] Read more.

Teneurin 4 (TENM4) is a transmembrane protein that is codified by the ODZ4 gene and is involved in nervous system development, neurite outgrowth, and neuronal differentiation. In line with its involvement in the nervous system, TENM4 has also been implicated in several mental disorders such as bipolar disorder, schizophrenia, and autism. TENM4 mutations and rearrangements have recently been identified in a number of tumors. This, combined with impaired expression in tumors, suggests that it may potentially be involved in tumorigenesis. Most of the TENM4 mutations that are observed in tumors occur in breast cancer, in which TENM4 plays a role in cells’ migration and stemness. However, the functional role that TENM4 plays in breast cancer still needs to be better evaluated, and further studies are required to better understand the involvement of TENM4 in breast cancer progression. Herein, we review the currently available data for TENM4′s role in breast cancer and propose its use as both a novel target with which to ameliorate patient prognosis and as a potential biomarker. Moreover, we also report data on the tumorigenic role of miR-708 deregulation and the possible use of this miRNA as a novel therapeutic molecule, as miR-708 is spliced out from TENM4 mRNA. Full article

(This article belongs to the Special Issue Emerging Targets and Therapeutic Strategies in Cancer)

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Figure 1
Schematic representation of the ODZ4 gene and of the TENM4 protein structure: (A) The ODZ4 gene counts 34 exons (NCBI Gene ID: 26011, updated on 23 November 2021). Two microRNA miR-5579 and miR-708 are situated in the first intron of the ODZ4 gene and spliced out. (B) TENM4 protein structure comprehend different domains: an intracellular domain with a nuclear localization sequence and an SH3 binding domain, a transmembrane domain, and an extracellular domain composed by eight EGF repeats, an NHL, and YD domains. “Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>, accessed on 26 November 2021”. Full article ">Figure 2
Analysis of ODZ4 genomic alterations in breast cancer patients: (A) ODZ4 alteration frequency observed by analyzing two breast cancer data sets: TCGA (Dataset A) and Metabric (Dataset B). The analyses were performed using the cBioPortal tool (<a href="http://www.cbioportal.org" target="_blank">www.cbioportal.org</a>, accessed on 30 November 2021), an open-access, open-source resource for interactive exploration of multidimensional cancer genomics data sets. (B) Overall survival (upper panel) and relapse free status (lower panel) of breast cancer patients that displayed unaltered ODZ4 (blue line) and genomic ODZ4 alterations (red line). Statistical analysis was performed using the Log Rank Test. Full article ">Figure 3
TENM4 mRNA expression relevance in breast cancer patients. Correlation between Relapse Free Survival (RFS) and Overall Survival (OS) in Triple Negative Breast Cancer (TNBC) (A,E), HER2-positive (B,F), Progesterone Receptor (PR)-positive (C,G), Estrogen Receptor (ER)-positive (D,H), and Grade 3 (I,J) with high (red) or low (black) TENM4 mRNA expression in the tumor. Full article ">Figure 4
Mir-708 relevance in TNBC patients. Correlation between Overall Survival (OS) in TNBC and high (red) or low (black) miR-708 expression in the tumor. Full article ">Figure 5
TENM4 expression in HER2-positive breast cancer cells and tumors: (A) Immunoblot of TENM4 and loading control protein (Actin) that compares HER2-positive TUBO epithelial cells (ep), first-passage tumorspheres (P1) and second-passage tumorspheres (P2). (B) Immunoblot of TENM4 and loading control protein (Vinculin) that compares HER2-positive mammary tumors from 9- (NeuT9wA and B), 14- (NeuT14wA and B), and 24- (NeuT24wA and B) week-old BALB-neuT mice. A total of 80 µg of TUBO cells and BALB-neuT tumor lysates were separated via electrophoresis in a 7.5% Mini-Protean TGX precast gel (Bio-Rad) and transferred onto an Immobilon-P PVDF membrane. Following blocking with 5% non-fat dry milk, the membrane was incubated with sheep anti-TENM4 (1 µg/mL, Cat#AF6320, R&D Systems, Minneapolis, MN, USA), with mouse anti-β-Actin (1:200, Clone AC-15, Santa Cruz Biotechnology), and with mouse anti-Vinculin (1:8000, produced in-house). Full article ">

14 pages, 1515 KiB

Open AccessReview

Lithium and Erectile Dysfunction: An Overview

by Mohammad Sheibani, Mehdi Ghasemi and Ahmad Reza Dehpour

Cells 2022, 11(1), 171; https://doi.org/10.3390/cells11010171 - 5 Jan 2022

Cited by 13 | Viewed by 9171

Abstract

Lithium has been a mainstay of therapy for patients with bipolar disorders for several decades. However, it may exert a variety of adverse effects that can affect patients’ compliance. Sexual and erectile dysfunction has been reported in several studies by patients who take [...] Read more.

Lithium has been a mainstay of therapy for patients with bipolar disorders for several decades. However, it may exert a variety of adverse effects that can affect patients’ compliance. Sexual and erectile dysfunction has been reported in several studies by patients who take lithium as monotherapy or combined with other psychotherapeutic agents. The exact mechanisms underlying such side effects of lithium are not completely understood. It seems that both central and peripheral mechanisms are involved in the lithium-related sexual dysfunction. Here, we had an overview of the epidemiology of lithium-related sexual and erectile dysfunction in previous clinical studies as well as possible pathologic pathways that could be involved in this adverse effect of lithium based on the previous preclinical studies. Understanding such mechanisms could potentially open a new avenue for therapies that can overcome lithium-related sexual dysfunction and improve patients’ adherence to the medication intake. Full article

(This article belongs to the Collection Feature Papers in 'Cells of the Nervous System' Section)

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11 pages, 1520 KiB

Open AccessReview

Muscle and Bone Impairment in Infantile Nephropathic Cystinosis: New Concepts

by Dieter Haffner, Maren Leifheit-Nestler, Candide Alioli and Justine Bacchetta

Cells 2022, 11(1), 170; https://doi.org/10.3390/cells11010170 - 5 Jan 2022

Cited by 7 | Viewed by 3475

Abstract

Cystinosis Metabolic Bone Disease (CMBD) has emerged during the last decade as a well-recognized, long-term complication in patients suffering from infantile nephropathic cystinosis (INC), resulting in significant morbidity and impaired quality of life in teenagers and adults with INC. Its underlying pathophysiology is [...] Read more.

Cystinosis Metabolic Bone Disease (CMBD) has emerged during the last decade as a well-recognized, long-term complication in patients suffering from infantile nephropathic cystinosis (INC), resulting in significant morbidity and impaired quality of life in teenagers and adults with INC. Its underlying pathophysiology is complex and multifactorial, associating complementary, albeit distinct entities, in addition to ordinary mineral and bone disorders observed in other types of chronic kidney disease. Amongst these long-term consequences are renal Fanconi syndrome, hypophosphatemic rickets, malnutrition, hormonal abnormalities, muscular impairment, and intrinsic cellular bone defects in bone cells, due to CTNS mutations. Recent research data in the field have demonstrated abnormal mineral regulation, intrinsic bone defects, cysteamine toxicity, muscle wasting and, likely interleukin-1-driven inflammation in the setting of CMBD. Here we summarize these new pathophysiological deregulations and discuss the crucial interplay between bone and muscle in INC. In future, vitamin D and/or biotherapies targeting the IL1β pathway may improve muscle wasting and subsequently CMBD, but this remains to be proven. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of Nephropathic Cystinosis)

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Figure 1
Clinical presentation and causes of CMBD. Figure from Hohenfellner et al., with permission [<a href="#B18-cells-11-00170" class="html-bibr">18</a>]. Full article ">Figure 2
Serum levels of phosphate (A), calcium (B), intact parathyroid hormone (iPTH, (C)), and 1,25(OH)2D3 (D) in children with infantile nephropathic cystinosis (INC) and CKD controls as estimated glomerular filtration rate (eGFR) and after kidney transplantation (KTX). Gray box plots indicate INC patients; white box plots indicate CKD controls. Horizontal continuous and broken lines in (A) indicate the mean and upper and lower normal range; horizontal broken lines in (B,C) indicate the upper and lower normal range; horizontal broken lines in (D) indicate the PTH target range recommended by KDOQI; a, b, and c indicate p < 0.05, p < 0.01 and p < 0.001 versus healthy children, respectively. SDS, standard deviation score. Figure from Ewert et al., with permission [<a href="#B13-cells-11-00170" class="html-bibr">13</a>]. Full article ">Figure 3
Circulating levels of intact (A) and total (B) fibroblast growth factor 23, soluble Klotho (C), bone alkaline phosphatase (D), tartrate-resistant acid phosphatase 5b (E), osteoprotegerin (F) and sclerostin (G) in children with infantile nephropathic cystinosis (INC), and CKD controls at various stages of CKD and after kidney transplantation (KTX): Gray box plots indicate INC patients while white box plots indicate CKD controls. Horizontal continuous and broken lines indicate the mean, upper, and lower normal range; a, b, and c indicate p < 0.05, p < 0.01, and p < 0.001 versus healthy children, respectively. eGFR, estimated glomerular filtration rate (eGFR); SDS, standard deviation score; iFGF23, intact fibroblast growth factor 23; BAP, bone alkaline phosphatase; TRAP5b, tartrate-resistant acid phosphatase 5b; OPG, osteoprotegerin; sKlotho, soluble Klotho. Figure from Ewert et al. with permission [<a href="#B13-cells-11-00170" class="html-bibr">13</a>]. Full article ">

21 pages, 1463 KiB

Open AccessFeature PaperReview

LRRK2 at Striatal Synapses: Cell-Type Specificity and Mechanistic Insights

by Patrick D. Skelton, Valerie Tokars and Loukia Parisiadou

Cells 2022, 11(1), 169; https://doi.org/10.3390/cells11010169 - 5 Jan 2022

Cited by 11 | Viewed by 4009

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease with a similar clinical presentation and progression to idiopathic Parkinson’s disease, and common variation is linked to disease risk. Recapitulation of the genotype in rodent models causes abnormal dopamine release and increases the [...] Read more.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease with a similar clinical presentation and progression to idiopathic Parkinson’s disease, and common variation is linked to disease risk. Recapitulation of the genotype in rodent models causes abnormal dopamine release and increases the susceptibility of dopaminergic neurons to insults, making LRRK2 a valuable model for understanding the pathobiology of Parkinson’s disease. It is also a promising druggable target with targeted therapies currently in development. LRRK2 mRNA and protein expression in the brain is highly variable across regions and cellular identities. A growing body of work has demonstrated that pathogenic LRRK2 mutations disrupt striatal synapses before the onset of overt neurodegeneration. Several substrates and interactors of LRRK2 have been identified to potentially mediate these pre-neurodegenerative changes in a cell-type-specific manner. This review discusses the effects of pathogenic LRRK2 mutations in striatal neurons, including cell-type-specific and pathway-specific alterations. It also highlights several LRRK2 effectors that could mediate the alterations to striatal function, including Rabs and protein kinase A. The lessons learned from improving our understanding of the pathogenic effects of LRRK2 mutations in striatal neurons will be applicable to both dissecting the cell-type specificity of LRRK2 function in the transcriptionally diverse subtypes of dopaminergic neurons and also increasing our understanding of basal ganglia development and biology. Finally, it will inform the development of therapeutics for Parkinson’s disease. Full article

(This article belongs to the Collection Feature Papers in 'Cells of the Nervous System' Section)

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Figure 1
Single-cell LRRK2 mRNA levels. Single-cell LRRK2 mRNA expression data from dropviz.org for selected types of striatal neurons and dopaminergic midbrain projection neurons. (A) Top: LRRK2 mRNA expression in global clusters defining striatal neuron types. LRRK2 mRNA is more abundant in SPNs than in striatal interneurons. Bottom: Subclusters selected on the basis of their putative identity with well-characterized subpopulations of striatal neurons (i.e., excluding eSPNs and clusters which had been identified as non-striatal neurons). Cell cluster identities are as described by Saunders et al., except Neurogliaform interneurons, which was assigned based on the expression of NPY but not parvalbumin, somatostatin, or Nos1, as defined by Tepper et al. [<a href="#B62-cells-11-00169" class="html-bibr">62</a>]. Some subclusters of fast-spiking interneurons and SPNs do not correspond to a well-characterized cell type and are referred to by the subcluster name. Consistent with immunohistochemical evidence by Mandemakers et al. [<a href="#B57-cells-11-00169" class="html-bibr">57</a>], LRRK2 mRNA is elevated in lateral and striosomal dSPNs. These data can be accessed in the context of the full dataset at [<a href="http://dropviz.org/?stateid=8afe26552f34f746" target="_blank">http://dropviz.org/?stateid=8afe26552f34f746</a>] (accessed on 20 October 2021). (B) Top: LRRK2 mRNA expression in global clusters defining neuron types in the substantia nigra. Bottom: LRRK2 mRNA levels in the subclusters constituting the “Th” (Tyrosine hydroxylase-expressing) global cluster, i.e., dopaminergic neurons of the SNc and VTA. Cell types are as defined by Saunders et al. LRRK2 mRNA expression is low, but detectable, across dopaminergic subtypes. These data can be accessed in the context of the full dataset at [<a href="http://dropviz.org/?stateid=8c997a178c82063c" target="_blank">http://dropviz.org/?stateid=8c997a178c82063c</a>] (accessed on 20 October 2021). Abbreviations: PV: parvalbumin; TH: tyrosine hydroxylase; Pnoc: prepronociceptin; SST: somatostatin; CIN: cholinergic interneuron; eSPN: eccentric SPN [<a href="#B56-cells-11-00169" class="html-bibr">56</a>]; LTS: low-threshold spiking; FS: fast-spiking; IEG: immediate early gene; Rora: RAR-related orphan receptor A; Gad2: glutamic acid decarboxylase 2. Full article ">Figure 2
Pathway-specific effect of LRRK2 on synaptic AMPARs. The R1441C mutation induces an enhanced phosphorylation of GluR1 by PKA, leading to the increased insertion and retention of AMPA receptors into the synaptic membrane of dSPNs, but not iSPNs [<a href="#B78-cells-11-00169" class="html-bibr">78</a>]. This figure was created using biorender.com. Full article ">Figure 3
Candidate AKAP-like helix in the LRRK2 ROC domain. The sequence of the ROC domain is presented with alpha helices identified from sequence analysis highlighted in green. A candidate amphipathic helix was identified within the ROC domain of LRRK2 and is highlighted by the black lines leading to the inset image of the helical wheel. This helix contains the clinically relevant residue R1441 (circled in red). In the legend the “+” indicates resides belonging to a hydrophobic face when a 3–11 helix is generated. The “*” indicates residues belonging to the hydrophobic face when an alpha helix is modeled. The “#” identifies residues that belong to the hydrophobic face when either a 3–11 or alpha helix is generated. Full article ">Figure 4
Effectors of LRRK2 postsynaptic function. Depiction of mechanisms by which LRRK2 affects synaptic function and downstream LRRK2 effectors that affect synapse function. (1) An interaction between the PKA PKARIIβ subunit and LRRK2 ROC domain acts as an AKAP, anchoring LRRK2 in dendritic shafts. The R1441C mutation impairs this interaction, resulting in increased translocation of PKA into dendritic spines. (2) Mutation- and cell-type-specific alteration of AMPAR trafficking in dendritic spines. The R1441C mutation increases GluR1 phosphorylation and AMPAR insertion into the membrane specifically in direct-pathway SPNs, increasing synapse strength. (3) The LRRK2 substrate Rab8 is essential for trafficking newly translated AMPARs to the synapse. (4) LRRK2 kinase activity modulates the translation (by affecting the efficiency with which ribosomes translate mRNAs with complex 5′ UTRs), and the function of voltage-gated calcium channels (VGCCs), resulting in increased calcium influx. (5) LRRK2 phosphorylates the ribosomal S15 subunit, promoting the increased translation of mRNAs. This figure was created using biorender.com. Full article ">

15 pages, 2491 KiB

Open AccessArticle

In Vitro Anticancer Screening and Preliminary Mechanistic Study of A-Ring Substituted Anthraquinone Derivatives

by Ibrahim Morgan, Ludger A. Wessjohann and Goran N. Kaluđerović

Cells 2022, 11(1), 168; https://doi.org/10.3390/cells11010168 - 5 Jan 2022

Cited by 15 | Viewed by 3407

Abstract

Anthraquinone derivatives exhibit various biological activities, e.g., antifungal, antibacterial and in vitro antiviral activities. They are naturally produced in many fungal and plant families such as Rhamnaceae or Fabaceae. Furthermore, they were found to have anticancer activity, exemplified by mitoxantrone and pixantrone, and [...] Read more.

Anthraquinone derivatives exhibit various biological activities, e.g., antifungal, antibacterial and in vitro antiviral activities. They are naturally produced in many fungal and plant families such as Rhamnaceae or Fabaceae. Furthermore, they were found to have anticancer activity, exemplified by mitoxantrone and pixantrone, and many are well known redox-active compounds. In this study, various nature inspired synthetic anthraquinone derivatives were tested against colon, prostate, liver and cervical cancer cell lines. Most of the compounds exhibit anticancer effects against all cell lines, therefore the compounds were further studied to determine their IC₅₀-values. Of these compounds, 1,4-bis(benzyloxy)-2,3-bis(hydroxymethyl)anthracene-9,10-dione (4) exhibited the highest cytotoxicity against PC3 cells and was chosen for a deeper look into its mechanism of action. Based on flow cytometry, the compound was proven to induce apoptosis through the activation of caspases and to demolish the ROS/RNS and NO equilibrium in the PC3 cell line. It trapped cells in the G2/M phase. Western blotting was performed for several proteins related to the effects observed. Compound 4 enhanced the production of PARP and caspase-3. Moreover, it activated the conversion of LC3A/B-I to LC3A/B-II showing that also autophagy plays a role in its mechanism of action, and it caused the phosphorylation of p70 s6 kinase. Full article

(This article belongs to the Special Issue Crosstalk of Autophagy and Apoptosis)

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21 pages, 4891 KiB

Open AccessArticle

Carnosic Acid Attenuates the Free Fatty Acid-Induced Insulin Resistance in Muscle Cells and Adipocytes

by Danja J. Den Hartogh, Filip Vlavcheski, Adria Giacca, Rebecca E. K. MacPherson and Evangelia Tsiani

Cells 2022, 11(1), 167; https://doi.org/10.3390/cells11010167 - 5 Jan 2022

Cited by 18 | Viewed by 3702

Abstract

Elevated blood free fatty acids (FFAs), as seen in obesity, impair insulin action leading to insulin resistance and Type 2 diabetes mellitus. Several serine/threonine kinases including JNK, mTOR, and p70 S6K cause serine phosphorylation of the insulin receptor substrate (IRS) and have been [...] Read more.

Elevated blood free fatty acids (FFAs), as seen in obesity, impair insulin action leading to insulin resistance and Type 2 diabetes mellitus. Several serine/threonine kinases including JNK, mTOR, and p70 S6K cause serine phosphorylation of the insulin receptor substrate (IRS) and have been implicated in insulin resistance. Activation of AMP-activated protein kinase (AMPK) increases glucose uptake, and in recent years, AMPK has been viewed as an important target to counteract insulin resistance. We reported previously that carnosic acid (CA) found in rosemary extract (RE) and RE increased glucose uptake and activated AMPK in muscle cells. In the present study, we examined the effects of CA on palmitate-induced insulin-resistant L6 myotubes and 3T3L1 adipocytes. Exposure of cells to palmitate reduced the insulin-stimulated glucose uptake, GLUT4 transporter levels on the plasma membrane, and Akt activation. Importantly, CA attenuated the deleterious effect of palmitate and restored the insulin-stimulated glucose uptake, the activation of Akt, and GLUT4 levels. Additionally, CA markedly attenuated the palmitate-induced phosphorylation/activation of JNK, mTOR, and p70S6K and activated AMPK. Our data indicate that CA has the potential to counteract the palmitate-induced muscle and fat cell insulin resistance. Full article

(This article belongs to the Special Issue Free Fatty Acids and Pathogenesis of Diabetes Mellitus)

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12 pages, 1695 KiB

Open AccessArticle

Engineered Liposomes Protect Immortalized Immune Cells from Cytolysins Secreted by Group A and Group G Streptococci

by Hervé Besançon, Yu Larpin, Viktoria S. Babiychuk, René Köffel and Eduard B. Babiychuk

Cells 2022, 11(1), 166; https://doi.org/10.3390/cells11010166 - 5 Jan 2022

Cited by 1 | Viewed by 2467

Abstract

The increasing antibiotic resistance of bacterial pathogens fosters the development of alternative, non-antibiotic treatments. Antivirulence therapy, which is neither bacteriostatic nor bactericidal, acts by depriving bacterial pathogens of their virulence factors. To establish a successful infection, many bacterial pathogens secrete exotoxins/cytolysins that perforate [...] Read more.

The increasing antibiotic resistance of bacterial pathogens fosters the development of alternative, non-antibiotic treatments. Antivirulence therapy, which is neither bacteriostatic nor bactericidal, acts by depriving bacterial pathogens of their virulence factors. To establish a successful infection, many bacterial pathogens secrete exotoxins/cytolysins that perforate the host cell plasma membrane. Recently developed liposomal nanotraps, mimicking the outer layer of the targeted cell membranes, serve as decoys for exotoxins, thus diverting them from attacking host cells. In this study, we develop a liposomal nanotrap formulation that is capable of protecting immortalized immune cells from the whole palette of cytolysins secreted by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis—important human pathogens that can cause life-threatening bacteremia. We show that the mixture of cholesterol-containing liposomes with liposomes composed exclusively of phospholipids is protective against the combined action of all streptococcal exotoxins. Our findings pave the way for further development of liposomal antivirulence therapy in order to provide more efficient treatment of bacterial infections, including those caused by antibiotic resistant pathogens. Full article

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Different types of immune cells display different sensitivities to filtered bacterial supernatants. The GAS ATCC 19165 supernatant is more toxic to THP-1 and to Jurkat cells than to Raji cells (a). The GAS 50362 supernatant is more toxic to Jurkat cells compared to THP-1 cells; Raji cells are the most resistant (b). The GGS ATCC 12394 supernatant shows comparable toxicity towards Raji and Jurkat cells; THP-1 cells are the most resistant (c). The GGS 5804 supernatant is more toxic to Raji cells compared to Jurkat cells; THP-1 cells are the most resistant (d). Full article ">Figure 2
Neutralization of the cytotoxins secreted by GAS and GGS by cholesterol-containing liposomes. Ch:Sm-liposomes (Ch = 66 mol/%) completely neutralized cytolytic activities of GAS supernatants (LD>90) towards all immune cell lines used in the current study (a,b). Ch:Sm-liposomes provided full protection to THP-1 cells against the GGS ATCC 12394 supernatant (LD>90), but only partial protection for Jurkat and Raji cells. Protection levels differ significantly between all cell types, p-values < 0.0005 (c). Ch:Sm-liposomes provided no protection against the GGS 5804 supernatant (LD>90) for any cell type (d). Ch:Sm-liposomes provided partial protection against the GGS 5804 supernatant for THP-1 cells (~35% of ~LD90, p-value < 0.005), Jurkat cells (~40% of ~LD70, p-value < 0.008), and Raji cells (~15% of ~LD85, p-value < 0.005) (e). Error bars = mean ± SD; N ≥ 3. Full article ">Figure 3
Inhibition of cytolysins secreted by GAS and GGS by liposomes composed of 18:1/18:1 PC. 18:1/18:1 PC-liposomes displayed no protection towards any immune cell line used in the current study treated with either of the GAS supernatants (a,b). No protection for THP-1 or Jurkat cell lines and partial protection for the Raji cell line were observed against the GGS ATCC 12394 supernatant (~20% of ~LD95, p-value < 0.03) (c). No protection for Jurkat cells and partial protection for THP-1 cells (~30% of ~LD90, p-value < 0.005) and Raji cells (~45% of ~LD85, p-value < 0.006) were observed against the GGS 5804 supernatant (d). Errors bars = mean ± SD; N ≥ 3. Full article ">Figure 4
Inhibition of the cytolysins secreted by GGS by a liposomal mixture composed of Ch:Sm and 18:1/18:1 PC-liposomes. The combination of Ch:Sm-liposomes with 18:1/18:1 PC-liposomes completely protects immune cells against GGS ATCC 12394 cytolysins (a). Against GGS 5804 cytolysins, full protection is reached by THP-1 and Jurkat cells, but it is only partial for Raji cells (b). Full article ">

13 pages, 2999 KiB

Open AccessArticle

Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase

by Peter Kolesar, Karel Stejskal, David Potesil, Johanne M. Murray and Jan J. Palecek

Cells 2022, 11(1), 165; https://doi.org/10.3390/cells11010165 - 4 Jan 2022

Cited by 10 | Viewed by 3455 | Correction

Abstract

Structural Maintenance of Chromosomes (SMC) complexes are important for many aspects of the chromosomal organization. Unlike cohesin and condensin, the SMC5/6 complex contains a variant RING domain carried by its Nse1 subunit. RING domains are characteristic for ubiquitin ligases, and human NSE1 has [...] Read more.

Structural Maintenance of Chromosomes (SMC) complexes are important for many aspects of the chromosomal organization. Unlike cohesin and condensin, the SMC5/6 complex contains a variant RING domain carried by its Nse1 subunit. RING domains are characteristic for ubiquitin ligases, and human NSE1 has been shown to possess ubiquitin-ligase activity in vitro. However, other studies were unable to show such activity. Here, we confirm Nse1 ubiquitin-ligase activity using purified Schizosaccharomyces pombe proteins. We demonstrate that the Nse1 ligase activity is stimulated by Nse3 and Nse4. We show that Nse1 specifically utilizes Ubc13/Mms2 E2 enzyme and interacts directly with ubiquitin. We identify the Nse1 mutation (R188E) that specifically disrupts its E3 activity and demonstrate that the Nse1-dependent ubiquitination is particularly important under replication stress. Moreover, we determine Nse4 (lysine K181) as the first known SMC5/6-associated Nse1 substrate. Interestingly, abolition of Nse4 modification at K181 leads to suppression of DNA-damage sensitivity of other SMC5/6 mutants. Altogether, this study brings new evidence for Nse1 ubiquitin ligase activity, significantly advancing our understanding of this enigmatic SMC5/6 function. Full article

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Figure 1
S. pombe Nse1 promotes in vitro ubiquitination together with Ubc13/Mms2. (A) Nse1 stimulates ubiquitin-chain formation when combined with Ubc13/Mms2. S. pombe E1 (Uba1), indicated E2s, biotinylated ubiquitin, ATP, and MgCl2 were incubated in the presence or absence of Nse1/3/4 trimer for 1 h at 37 °C. The mixture was separated on 12% SDS–PAGE followed by Western blotting and visualization of biotinylated ubiquitin using Streptavidin-HRP. Numbers on the left indicate molecular weights of protein standards (in kDa). (B) Nse1/3/4 trimer promotes ubiquitination more efficiently than Nse1/3 dimer or Nse1 alone, and deletion of its vRING domain ablates this activity. In vitro ubiquitination assay was performed using E1, Ubc13/Mms2, biotinylated ubiquitin, indicated E3, and analyzed as in (A). Full article ">Figure 2
Nse1 mutation R188E disrupts its ubiquitin ligase activity. (A) Nse1–Nse4 interaction analyzed by yeast two-hybrid assay is decreased by Nse1 vRING removal, but not by its R188E mutation. Plasmids carrying indicated genes or corresponding empty vectors were co-transformed into the S. cerevisiae PJ69–4a strain, grown to OD600 ~ 1, fivefold serially diluted and spotted on solid media lacking leucine (L), tryptophan (W), histidine (H), or adenine (A) in absence or presence of 1 mM 3-aminotriazole (1AT), as depicted. Cells were grown for 3 days at 30 °C and scanned. (B) Alignment of the conserved vRING domain sequences of Nse1 from different species: S. pombe (S.p.), S. cerevisiae (S.c.), Dictyostelium discoideum (D.d.), Caenorhabditis elegans (C.e.), Drosophila melanogaster (D.m.), Xenopus laevis (X.l.), Monodelphis domestica (M.d.), Mus musculus (M.m.), Homo sapiens sapiens (H.s.). Amino acid shading represents the following conserved amino acids: violet—Zn2+-coordinating cysteins and histidines; dark green—hydrophobic and aromatic; red—basic. Red arrows on top indicate amino acids mutated in (D) and their corresponding numbers in S. pombe. (C) The Nse1 vRING domain (dark violet)—Ubc13 (grey) interaction model based on the crystal structure of the human TRIM21-Ubc13 [<a href="#B26-cells-11-00165" class="html-bibr">26</a>]. The Nse1-R188 residue is red labeled. (D) Nse1 R188E mutation impairs its ability to promote ubiquitination in vitro. E1, Ubc13/Mms2, biotinylated ubiquitin, and indicated Nse1 variants were incubated with ATP and MgCl2 for 1 h at 37 °C and analyzed as in <a href="#cells-11-00165-f001" class="html-fig">Figure 1</a>A. (E) Nse1(97–178) interacts with ubiquitin in the yeast two-hybrid assay. S. cerevisiae PJ69–4a transformants were analyzed as in (A). Full article ">Figure 3
Nse1-R188E ubiquitin ligase mutant shows increased sensitivity to HU, MMS, and synthetic phenotypes with smc5/6 mutants. (A) Nse1-R188E strain is sensitive to HU and MMS. The indicated S. pombe strains were grown to OD600 ~ 1, tenfold serially diluted, spotted onto rich media with the designated amounts of HU, MMS, or UV dose, and incubated at 28 °C for 3 days. (B) Nse1-R188E mutant shows synthetic lethality with smc6-74 and severe growth defects with smc6-X and nse6Δ. Double mutants were analyzed by tetrad dissection of S. pombe diploid strains resulting from crosses between the strain carrying the nse1-R188E mutation and the indicated smc5/6 mutants. Red rectangles mark double mutants. Full article ">Figure 4
Nse4-K181R mutation partially suppresses the sensitivities of smc6-X, smc6-74, and nse3-R254E to DNA-damaging agents and HU. (A) Sensitivity of depicted smc5/6 mutants to UV, HU, and MMS is partially suppressed by nse4-K181R. (B) Nse4-K181R does not suppress the sensitivity of the nse1-R188E strain. (C) Suppression of smc6-X sensitivity by nse4-K181R is dependent on Ubc13. The indicated S. pombe strains were grown to OD600 ~ 1, tenfold serially diluted, spotted onto rich media with specified amounts of DNA-damaging agents, and incubated at 28 °C for 3 days. Full article ">Figure 5
Model for Nse1–Ubc13~Ub complex. The Ubc13 E2 enzyme (grey) is charged with ubiquitin (blue) and binds the Nse1-vRING domain (dark violet). Ubiquitin is localized in the vicinity of the Nse1-WHB domain (light violet), and their interaction may enhance the ubiquitination process. Full article ">

29 pages, 3204 KiB

Open AccessEditor’s ChoiceReview

Overview of Polyamines as Nutrients for Human Healthy Long Life and Effect of Increased Polyamine Intake on DNA Methylation

by Kuniyasu Soda

Cells 2022, 11(1), 164; https://doi.org/10.3390/cells11010164 - 4 Jan 2022

Cited by 39 | Viewed by 10419

Abstract

Polyamines, spermidine and spermine, are synthesized in every living cell and are therefore contained in foods, especially in those that are thought to contribute to health and longevity. They have many physiological activities similar to those of antioxidant and anti-inflammatory substances such as [...] Read more.

Polyamines, spermidine and spermine, are synthesized in every living cell and are therefore contained in foods, especially in those that are thought to contribute to health and longevity. They have many physiological activities similar to those of antioxidant and anti-inflammatory substances such as polyphenols. These include antioxidant and anti-inflammatory properties, cell and gene protection, and autophagy activation. We have first reported that increased polyamine intake (spermidine much more so than spermine) over a long period increased blood spermine levels and inhibited aging-associated pathologies and pro-inflammatory status in humans and mice and extended life span of mice. However, it is unlikely that the life-extending effect of polyamines is exerted by the same bioactivity as polyphenols because most studies using polyphenols and antioxidants have failed to demonstrate their life-extending effects. Recent investigations revealed that aging-associated pathologies and lifespan are closely associated with DNA methylation, a regulatory mechanism of gene expression. There is a close relationship between polyamine metabolism and DNA methylation. We have shown that the changes in polyamine metabolism affect the concentrations of substances and enzyme activities involved in DNA methylation. I consider that the increased capability of regulation of DNA methylation by spermine is a key of healthy long life of humans. Full article

(This article belongs to the Special Issue Epigenetic Mechanisms of Longevity and Aging)

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32 pages, 2696 KiB

Open AccessEditor’s ChoiceArticle

Co-Expression Analysis of microRNAs and Proteins in Brain of Alzheimer’s Disease Patients

by Callum N. Watson, Ghazala Begum, Emma Ashman, Daniella Thorn, Kamal M. Yakoub, Moustafa Al Hariri, Ali Nehme, Stefania Mondello, Firas Kobeissy, Antonio Belli and Valentina Di Pietro

Cells 2022, 11(1), 163; https://doi.org/10.3390/cells11010163 - 4 Jan 2022

Cited by 10 | Viewed by 3872

Abstract

Alzheimer’s disease (AD) is the most common form of dementia globally; however, the aetiology of AD remains elusive hindering the development of effective therapeutics. MicroRNAs (miRNAs) are regulators of gene expression and have been of growing interest in recent studies in many pathologies [...] Read more.

Alzheimer’s disease (AD) is the most common form of dementia globally; however, the aetiology of AD remains elusive hindering the development of effective therapeutics. MicroRNAs (miRNAs) are regulators of gene expression and have been of growing interest in recent studies in many pathologies including AD not only for their use as biomarkers but also for their implications in the therapeutic field. In this study, miRNA and protein profiles were obtained from brain tissues of different stage (Braak III-IV and Braak V-VI) of AD patients and compared to matched controls. The aim of the study was to identify in the late stage of AD, the key dysregulated pathways that may contribute to pathogenesis and then to evaluate whether any of these pathways could be detected in the early phase of AD, opening new opportunity for early treatment that could stop or delay the pathology. Six common pathways were found regulated by miRNAs and proteins in the late stage of AD, with one of them (Rap1 signalling) activated since the early phase. MiRNAs and proteins were also compared to explore an inverse trend of expression which could lead to the identification of new therapeutic targets. These results suggest that specific miRNA changes could represent molecular fingerprint of neurodegenerative processes and potential therapeutic targets for early intervention. Full article

(This article belongs to the Special Issue MicroRNA-Mediated Silencing Pathways in the Nervous System and Neurological Diseases)

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Figure 1
Heatmap showing the expression of 54 differentially regulated microRNAs in Alzheimer’s disease. Fifty-four microRNAs were found using Mann–Whitney statistical analysis across the 2 different comparisons, Control vs. Braak stage III-IV and Control vs. Braak stage V-VI. A p-value of less than 0.05 was considered significant. Hierarchal clustering is present on the left with colours relevant to their group. This was made using heatmap.2 in the R programming language with complete linkage, and Euclidean distance used to compute. Full article ">Figure 2
Heatmap showing the expression of 174 differentially regulated proteins in Alzheimer’s disease across the 2 compariosns Control vs. Braak stage IiI–IV and Control vs. Braak stage V-VI. A p-value of less than 0.05 was considered significant. Hierarchal clustering is present on the left with colours relevant to their group. This was made using heatmap.2 in the R programming language with complete linkage, and Euclidean distance used to compute. Full article ">Figure 3
Braak stage III-IV pathways analyses. Full article ">Figure 4
Braak stage V-VI pathways analyses. Full article ">Figure 5
DE-miRNA pathway studio analyses in Braak III-IV and Braak V-VI. The pathological processes triggered by Braak III-IV DE-miRNA (panel A) and Braak V-VI DE-miRNA (panel C). The related pathways of Braak III-IV DE-miRNA (panel B) and Braak V-VI DE-miRNA (panel D). Full article ">Figure 6
DE-protein pathway studio analyses in Braak III-IV and Braak V-VI. AD pathways generated by DE-proteins in Braak III-IV (panel A) and Braak V-VI DE-miRNA (panel B). Full article ">

13 pages, 1668 KiB

Open AccessArticle

N-Glycosylation Facilitates 4-1BB Membrane Localization by Avoiding Its Multimerization

by Ruoxuan Sun, Alyssa Min Jung Kim, Allison A. Murray and Seung-Oe Lim

Cells 2022, 11(1), 162; https://doi.org/10.3390/cells11010162 - 4 Jan 2022

Cited by 8 | Viewed by 3259

Abstract

Leveraging the T cell immunity against tumors represents a revolutionary type of cancer therapy. 4-1BB is a well-characterized costimulatory immune receptor existing on activated T cells and mediating their proliferation and cytotoxicity under infectious diseases and cancers. Despite the accumulating interest in implementing [...] Read more.

Leveraging the T cell immunity against tumors represents a revolutionary type of cancer therapy. 4-1BB is a well-characterized costimulatory immune receptor existing on activated T cells and mediating their proliferation and cytotoxicity under infectious diseases and cancers. Despite the accumulating interest in implementing 4-1BB as a therapeutic target for immune-related disorders, less is known about the pattern of its intracellular behaviors and regulations. It has been previously demonstrated that 4-1BB is heavily modified by N-glycosylation; however, the biological importance of this modification lacks detailed elucidation. Through biochemical, biophysical, and cell-biological approaches, we systematically evaluated the impact of N-glycosylation on the ligand interaction, stability, and localization of 4-1BB. We hereby highlighted that N-glycan functions by preventing the oligomerization of 4-1BB, thus permitting its membrane transportation and fast turn-over. Without N-glycosylation, 4-1BB could be aberrantly accumulated intracellularly and fail to be sufficiently inserted in the membrane. The N-glycosylation-guided intracellular processing of 4-1BB serves as the potential mechanism explicitly modulating the “on” and “off” of 4-1BB through the control of protein abundance. Our study will further solidify the understanding of the biological properties of 4-1BB and facilitate the clinical practice against this promising therapeutic target. Full article

(This article belongs to the Special Issue Mechanism of Anti-tumor Immunity of Cells and Immunotherapy)

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Figure 1

Figure 1
4-1BB is a heavily N-glycosylated protein with two modified sites. (A) Alignment of mammalian 4-1BB sequence around the N-glycosylation sites. The N-X-S/T motif is outlined by the red square. (B) Immunoblotting analysis of 4-1BB undergoing deglycosylation treatment by PNGase F. (C) MALDI-MS profiling of permethylated N-glycans released from PNGase F-treated recombinant 4-1BB protein. The masses of indicated glycan species represents the [M + Na+] values. SP, signal peptide; ECD, extracellular domain; TM, transmembrane domain; ICD, intracellular domain; CDR, cysteine-rich domain; EV, empty vector; 4-1BB (G), N-glycosylated 4-1BB; 4-1BB (NG), non-N-glycosylated 4-1BB. Full article ">Figure 2
The impact of N-glycosylation on 4-1BB/4-1BBL interaction. (A) The relative level of bound 4-1BBL to PNGase F-treated and untreated 4-1BB protein (n = 3). (B) Octet analysis calculating the binding affinity of glycosylated (black) and deglycosylated 4-1BB (red) to 4-1BBL along with the KD values. N.S., not significant (Two-tailed student’s t-test). Full article ">Figure 3
The regulation of cell surface level of 4-1BB by N-glycosylation. (A) Jurkat cells were transduced with lentivirus encoding 4-1BB WT and glycosylation-deficient mutant including N138Q, N149Q and 2NQ, respectively. The membrane levels of 4-1BB in each group were compared by flow cytometry. The percentages of positive events were included in the plots. (B) The quantitative results of membrane 4-1BB level in each group in (A) (n = 3). (C) The immunoblotting analysis of total 4-1BB from cells in (A). N.S., not significant; ***, p < 0.001 (Two-tailed student’s t-test); MFI, mean fluorescence intensity. Full article ">Figure 4
N-glycosylation plays an essential role in determining 4-1BB stability. (A) HEK293T cells were transfected with Flag-tagged 4-1BB WT and 2NQ followed by CHX-chase assay to compare their stability. (B) The quantification of the remaining 4-1BB at each time point (n = 3). The percentage values of band intensity were normalized to time 0 h. 50% degradation is indicated by the dashed line. (C) Immunoblotting analysis of the MG-132 effect on 4-1BB WT and 2NQ. HEK293T/4-1BB WT and HEK293T/4-1BB 2NQ cells were treated by 10 μM MG-132 for 0, 4, and 8 h followed by immunoblotting assay. (D) Comparison of polyubiquitination level between WT and 2NQ 4-1BB. Flag-tagged 4-1BB and HA-tagged Ub were co-transfected to HEK293T cells followed by in vivo ubiquitination assay 48 h after transfection. IP, immunoprecipitates. **, p < 0.01; ***, p < 0.001 (Two-tailed student’s t-test). Full article ">Figure 5
Oligomerization is associated with augmented stability of unglycosylated 4-1BB. (A) Non-reducing SDS-PAGE uncovered the formation of dimerized, trimerized, and oligomerized 4-1BB 2NQ. The bands that exist in the presence of DTT were considered monomers. The white circle, triangle, square, and star represent the monomerized, dimerized, trimerized, and oligomerized form of 4-1BB 2NQ. (B) 4-1BB 2NQ failed to form multimers when lacking the key cysteine residue C121. (C) HEK293T cells were transfected with Flag-tagged 4-1BB WT, C121A, 2NQ, and 2NQ/C121A followed by CHX-chase assay to compare their stability. (D) The quantification of the remaining 4-1BB at each time point (n = 3). The percentage values of band intensity were normalized to time 0 h. 50% degradation is indicated by the dashed line. Two-tailed student’s t-test was performed to compare the values on 8 h point. N.S., not significant; **, p < 0.01. Full article ">Figure 6
C121-mediated multimerization governs the stability and localization of unglycosylated 4-1BB. (A) Jurkat cells were transduced with lentivirus encoding 4-1BB WT, C121A, 2NQ, and 2NQ/C121A, respectively. The membrane levels of 4-1BB in each group were compared by flow cytometry. The percentages of positive events were included in the plots. (B) The quantitative results of membrane 4-1BB level in each group in (A) (n = 3). (C) The immunoblotting analysis of total 4-1BB from each group of cells in (A). N.S., not significant; ***, p < 0.001 (Two-tailed student’s t-test); MFI, mean fluorescence intensity. Full article ">

22 pages, 7188 KiB

Open AccessArticle

Cell Type-Selective Loss of Peroxisomal β-Oxidation Impairs Bipolar Cell but Not Photoreceptor Survival in the Retina

by Daniëlle Swinkels, Yannick Das, Sai Kocherlakota, Stefan Vinckier, Eric Wever, Antoine H.C. van Kampen, Frédéric M. Vaz and Myriam Baes

Cells 2022, 11(1), 161; https://doi.org/10.3390/cells11010161 - 4 Jan 2022

Cited by 13 | Viewed by 4153

Abstract

Retinal degeneration is a common feature in peroxisomal disorders leading to blindness. Peroxisomes are present in the different cell types of the retina; however, their precise contribution to retinal integrity is still unclear. We previously showed that mice lacking the central peroxisomal β-oxidation [...] Read more.

Retinal degeneration is a common feature in peroxisomal disorders leading to blindness. Peroxisomes are present in the different cell types of the retina; however, their precise contribution to retinal integrity is still unclear. We previously showed that mice lacking the central peroxisomal β-oxidation enzyme, multifunctional protein 2 (MFP2), develop an early onset retinal decay including photoreceptor cell death. To decipher the function of peroxisomal β-oxidation in photoreceptors, we generated cell type selective Mfp2 knockout mice, using the Crx promotor targeting photoreceptors and bipolar cells. Surprisingly, Crx-Mfp2^−/− mice maintained photoreceptor length and number until the age of 1 year. A negative electroretinogram was indicative of preserved photoreceptor phototransduction, but impaired downstream bipolar cell signaling from the age of 6 months. The photoreceptor ribbon synapse was affected, containing free-floating ribbons and vesicles with altered size and density. The bipolar cell interneurons sprouted into the ONL and died. Whereas docosahexaenoic acid levels were normal in the neural retina, levels of lipids containing very long chain polyunsaturated fatty acids were highly increased. Crx-Pex5^−/− mice, in which all peroxisomal functions are inactivated in photoreceptors and bipolar cells, developed the same phenotype as Crx-Mfp2^−/− mice. In conclusion, the early photoreceptor death in global Mfp2^−/− mice is not driven cell autonomously. However, peroxisomal β-oxidation is essential for the integrity of photoreceptor ribbon synapses and of bipolar cells. Full article

(This article belongs to the Special Issue Peroxisome Biogenesis and Protein Targeting Mechanisms)

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41 pages, 1737 KiB

Open AccessReview

Multifactorial Mechanism of Sarcopenia and Sarcopenic Obesity. Role of Physical Exercise, Microbiota and Myokines

by Jan Bilski, Piotr Pierzchalski, Marian Szczepanik, Joanna Bonior and Jerzy A. Zoladz

Cells 2022, 11(1), 160; https://doi.org/10.3390/cells11010160 - 4 Jan 2022

Cited by 89 | Viewed by 18834

Abstract

Obesity and ageing place a tremendous strain on the global healthcare system. Age-related sarcopenia is characterized by decreased muscular strength, decreased muscle quantity, quality, and decreased functional performance. Sarcopenic obesity (SO) is a condition that combines sarcopenia and obesity and has a substantial [...] Read more.

Obesity and ageing place a tremendous strain on the global healthcare system. Age-related sarcopenia is characterized by decreased muscular strength, decreased muscle quantity, quality, and decreased functional performance. Sarcopenic obesity (SO) is a condition that combines sarcopenia and obesity and has a substantial influence on the older adults’ health. Because of the complicated pathophysiology, there are disagreements and challenges in identifying and diagnosing SO. Recently, it has become clear that dysbiosis may play a role in the onset and progression of sarcopenia and SO. Skeletal muscle secretes myokines during contraction, which play an important role in controlling muscle growth, function, and metabolic balance. Myokine dysfunction can cause and aggravate obesity, sarcopenia, and SO. The only ways to prevent and slow the progression of sarcopenia, particularly sarcopenic obesity, are physical activity and correct nutritional support. While exercise cannot completely prevent sarcopenia and age-related loss in muscular function, it can certainly delay development and slow down the rate of sarcopenia. The purpose of this review was to discuss potential pathways to muscle deterioration in obese individuals. We also want to present the current understanding of the role of various factors, including microbiota and myokines, in the process of sarcopenia and SO. Full article

(This article belongs to the Collection Molecular Mechanisms of Exercise and Healthspan)

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Figure 1

Figure 1
Potential pathogenic mechanisms of age-related sarcopenia and sarcopenic obesity. Full article ">Figure 2
Diagram illustrating myostatin, and IGF-1 pathway interactions. Myostatin’s effects require both Smad2 and Smad3, which block muscle differentiation. Smad2 and 3 activations are both required for myostatin’s inhibitory effects on Akt. IGF-1 released in response to exercise can counteract myostatin’s effects. Full article ">Figure 3
A general outline of the functioning of the Hippo pathway. Diagram presents central axis of pathway consisting of ‘core kinases’ MST1/2, LATS1/2, their up-stream modulators: Merlin/NF2, Kibra/WWC1/2 and effector part of the pathway—YAP (Yes-associated protein encoded by YAP1). What is worth mentioning is active ‘core kinases’ phosphorylate YAP resulting in its cytoplasmic sequestration and down-regulation of the pathway activity. A detailed description of the pathway functioning is in the text of the article. MST1/2 (mammalian sterile 20-like kinases), LATS (large tumor suppressor kinases), Vgll1-4 (vestigial-like, Vito, Tondu), Tead1-4 (TEA/ATTS domain/TEF/scalloped), Merlin/NF2 (neurofibromatosis type 2), WW45/Sav1 (adaptor proteins Salvador homologue1), MOBKL1A (Mps-one binder kinase activator 1), TAOK1, thousand-and-one amino acids kinase 1, Amot (angiomotin). FOXO3a (Forkhead box O3). Full article ">Figure 4
Involvement of Hippo signaling in muscle cells development. Active— non phosphorylated YAP nuclear translocation results in direct activation of co-activation of TEAD dependent genes regulating muscle cells such as α-actin, Mef2c and Myogin. For details, please refer to the text. Full article ">Figure 5
Diagram presenting dynamics of changes in expression of YAP, MyoD and myogenin during activation of satellite cells division, conversion to myoblast and differentiation to myofiber. Based on the information summarized in the article, Hippo pathway might be considered an additional important mediator of balance between development and differentiation of muscle cells. Full article ">Figure 6
Myokines linked to age-related changes, their release during exercise, and putative mechanisms of action. More information on the listed myokines is described in specific paragraphs. Full article ">

33 pages, 50489 KiB

Open AccessReview

Aptamer-Enabled Nanomaterials for Therapeutics, Drug Targeting and Imaging

by Mengping Liu, Lin Wang, Young Lo, Simon Chi-Chin Shiu, Andrew B. Kinghorn and Julian A. Tanner

Cells 2022, 11(1), 159; https://doi.org/10.3390/cells11010159 - 4 Jan 2022

Cited by 46 | Viewed by 7035

Abstract

A wide variety of nanomaterials have emerged in recent years with advantageous properties for a plethora of therapeutic and diagnostic applications. Such applications include drug delivery, imaging, anti-cancer therapy and radiotherapy. There is a critical need for further components which can facilitate therapeutic [...] Read more.

A wide variety of nanomaterials have emerged in recent years with advantageous properties for a plethora of therapeutic and diagnostic applications. Such applications include drug delivery, imaging, anti-cancer therapy and radiotherapy. There is a critical need for further components which can facilitate therapeutic targeting, augment their physicochemical properties, or broaden their theranostic applications. Aptamers are single-stranded nucleic acids which have been selected or evolved to bind specifically to molecules, surfaces, or cells. Aptamers can also act as direct biologic therapeutics, or in imaging and diagnostics. There is a rich field of discovery at the interdisciplinary interface between nanomaterials and aptamer science that has significant potential across biomedicine. Herein, we review recent progress in aptamer-enabled materials and discuss pending challenges for their future biomedical application. Full article

(This article belongs to the Special Issue Frontiers in Aptamers)

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26 pages, 24241 KiB

Open AccessArticle

Altered White Matter and microRNA Expression in a Murine Model Related to Williams Syndrome Suggests That miR-34b/c Affects Brain Development via Ptpru and Dcx Modulation

by Meitar Grad, Ariel Nir, Gilad Levy, Sari Schokoroy Trangle, Guy Shapira, Noam Shomron, Yaniv Assaf and Boaz Barak

Cells 2022, 11(1), 158; https://doi.org/10.3390/cells11010158 - 4 Jan 2022

Cited by 10 | Viewed by 4291

Abstract

Williams syndrome (WS) is a multisystem neurodevelopmental disorder caused by a de novo hemizygous deletion of ~26 genes from chromosome 7q11.23, among them the general transcription factor II-I (GTF2I). By studying a novel murine model for the hypersociability phenotype associated with [...] Read more.

Williams syndrome (WS) is a multisystem neurodevelopmental disorder caused by a de novo hemizygous deletion of ~26 genes from chromosome 7q11.23, among them the general transcription factor II-I (GTF2I). By studying a novel murine model for the hypersociability phenotype associated with WS, we previously revealed surprising aberrations in myelination and cell differentiation properties in the cortices of mutant mice compared to controls. These mutant mice had selective deletion of Gtf2i in the excitatory neurons of the forebrain. Here, we applied diffusion magnetic resonance imaging and fiber tracking, which showed a reduction in the number of streamlines in limbic outputs such as the fimbria/fornix fibers and the stria terminalis, as well as the corpus callosum of these mutant mice compared to controls. Furthermore, we utilized next-generation sequencing (NGS) analysis of cortical small RNAs’ expression (RNA-Seq) levels to identify altered expression of microRNAs (miRNAs), including two from the miR-34 cluster, known to be involved in prominent processes in the developing nervous system. Luciferase reporter assay confirmed the direct binding of miR-34c-5p to the 3’UTR of PTPRU—a gene involved in neural development that was elevated in the cortices of mutant mice relative to controls. Moreover, we found an age-dependent variation in the expression levels of doublecortin (Dcx)—a verified miR-34 target. Thus, we demonstrate the substantial effect a single gene deletion can exert on miRNA regulation and brain structure, and advance our understanding and, hopefully, treatment of WS. Full article

(This article belongs to the Special Issue Pathophysiological Mechanism of Neurodevelopmental Disorders)

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26 pages, 6589 KiB

Open AccessArticle

Recognition and Chaperoning by Pex19, Followed by Trafficking and Membrane Insertion of the Peroxisome Proliferation Protein, Pex11

by Katarzyna M. Zientara-Rytter, Shanmuga S. Mahalingam, Jean-Claude Farré, Krypton Carolino and Suresh Subramani

Cells 2022, 11(1), 157; https://doi.org/10.3390/cells11010157 - 4 Jan 2022

Cited by 5 | Viewed by 3299

Abstract

Pex11, an abundant peroxisomal membrane protein (PMP), is required for division of peroxisomes and is robustly imported to peroxisomal membranes. We present a comprehensive analysis of how the Pichia pastoris Pex11 is recognized and chaperoned by Pex19, targeted to peroxisome membranes and inserted [...] Read more.

Pex11, an abundant peroxisomal membrane protein (PMP), is required for division of peroxisomes and is robustly imported to peroxisomal membranes. We present a comprehensive analysis of how the Pichia pastoris Pex11 is recognized and chaperoned by Pex19, targeted to peroxisome membranes and inserted therein. We demonstrate that Pex11 contains one Pex19-binding site (Pex19-BS) that is required for Pex11 insertion into peroxisomal membranes by Pex19, but is non-essential for peroxisomal trafficking. We provide extensive mutational analyses regarding the recognition of Pex19-BS in Pex11 by Pex19. Pex11 also has a second, Pex19-independent membrane peroxisome-targeting signal (mPTS) that is preserved among Pex11-family proteins and anchors the human HsPex11γ to the outer leaflet of the peroxisomal membrane. Thus, unlike most PMPs, Pex11 can use two mechanisms of transport to peroxisomes, where only one of them depends on its direct interaction with Pex19, but the other does not. However, Pex19 is necessary for membrane insertion of Pex11. We show that Pex11 can self-interact, using both homo- and/or heterotypic interactions involving its N-terminal helical domains. We demonstrate that Pex19 acts as a chaperone by interacting with the Pex19-BS in Pex11, thereby protecting Pex11 from spontaneous oligomerization that would otherwise cause its aggregation and subsequent degradation. Full article

(This article belongs to the Special Issue Peroxisome Biogenesis and Protein Targeting Mechanisms)

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18 pages, 2603 KiB

Open AccessArticle

Pathologic Proteolytic Processing of N-Cadherin as a Marker of Human Fibrotic Disease

by Paul Durham Ferrell, Kristianne Michelle Oristian, Everett Cockrell and Salvatore Vincent Pizzo

Cells 2022, 11(1), 156; https://doi.org/10.3390/cells11010156 - 4 Jan 2022

Cited by 7 | Viewed by 3732

Abstract

Prior research has implicated the involvement of cell adhesion molecule N-cadherin in tissue fibrosis and remodeling. We hypothesize that anomalies in N-cadherin protein processing are involved in pathological fibrosis. Diseased tissues associated with fibrosis of the heart, lung, and liver were probed for [...] Read more.

Prior research has implicated the involvement of cell adhesion molecule N-cadherin in tissue fibrosis and remodeling. We hypothesize that anomalies in N-cadherin protein processing are involved in pathological fibrosis. Diseased tissues associated with fibrosis of the heart, lung, and liver were probed for the precursor form of N-cadherin, pro-N-cadherin (PNC), by immunohistochemistry and compared to healthy tissues. Myofibroblast cell lines were analyzed for cell surface pro-N-cadherin by flow cytometry and immunofluorescent microscopy. Soluble PNC products were immunoprecipitated from patient plasmas and an enzyme-linked immunoassay was developed for quantification. All fibrotic tissues examined show aberrant PNC localization. Cell surface PNC is expressed in myofibroblast cell lines isolated from cardiomyopathy and idiopathic pulmonary fibrosis but not on myofibroblasts isolated from healthy tissues. PNC is elevated in the plasma of patients with cardiomyopathy (p ≤ 0.0001), idiopathic pulmonary fibrosis (p ≤ 0.05), and nonalcoholic fatty liver disease with cirrhosis (p ≤ 0.05). Finally, we have humanized a murine antibody and demonstrate that it significantly inhibits migration of PNC expressing myofibroblasts. Collectively, the aberrant localization of PNC is observed in all fibrotic tissues examined in our study and our data suggest a role for cell surface PNC in the pathogenesis of fibrosis. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of Fibrosis: From Wound Healing Studies to Clinical Management of Fibrotic Diseases)

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Graphical abstract
Full article ">Figure 1
PNC is aberrantly expressed in fibrotic heart, lung, and liver tissues. (A) Representative images of stained tissues from explanted failed human hearts (n = 20, ischemic and non-ischemic etiology), lungs (n = 10, IPF etiology) and livers (n = 40, NAFLD-cirrhosis etiology) show positive expression and aberrant localization of PNC (Brown stain). Corresponding representative images of stained normal human heart (n = 24), lung (n = 18), and liver (n = 32) show lack of aberrantly expressed PNC at the tissue level. Perinuclear staining, consistent with normal N-cadherin processing can be seen in the healthy cardiac tissue. Scale bar = 100 µm. (B) Graphical representation of human samples analyzed in this study. Each column represents a single human sample, annotated by color to indicate the organ system of relevance to this study (top row), whether the patient had fibrosis (second row), the reported sex of the patient (third row), the age of the patient (fourth row) and the method by which the sample was analyzed in this study (fourth row). In cases where demographic data is unknown, unavailable, or unreported, a white bar with black hatch is shown. n = 257 total samples analyzed. Full article ">Figure 2
PNC is localized to the cell surface of myofibroblasts. Myofibroblasts from heart and lung tissues were stained and analyzed by flow cytometry using m-α-PNC mAb 10A10, excluding debris and dead cells via gating and 7AAD exclusion using Flowjo software. Myofibroblasts from fibrosis origins stain positive for cell surface PNC: (A) CF-DCM, cardiac myofibroblasts from dilated cardiomyopathy; Chi-Squared = 177.5; SE Dymax % Positive = 48.0. LL97A, IPF; Chi-Squared = 93.4; SE Dymax % Positive = 54.8, and LL29, IPF; Chi-Squared = 113.5; SE Dymax % Positive = 51.0. PNC was not detected on the surface of primary normal human cardiac myofibroblasts from healthy donor, NHCF; Chi-Squared = 3.47; SE Dymax % Positive = 8.54, primary normal human lung myofibroblasts from healthy donor, NHLF; Chi-Squared = 0; SE Dymax % Positive = 0, or immortalized CCD-16Lu lung myofibroblasts from healthy donor; Chi-Squared = 0; SE Dymax % Positive = 0. Mouse IgG1 isotype control (blue shaded) was compared to m-α-PNC mAb (unshaded). Results are representative of 3 independent experiments, n = 3. For all flow cytometry experiments, Chi-squared ≥ 4 is statistically significant. (B) Cell surface proteins were isolated from myofibroblasts and immunoblotted for N-cadherin, PNC, and Na,k-ATPase α-1 (ATP1A1) cell surface compartment loading control. PNC and N-Cadherin lysates were normalized to the ATP1A1 cell surface loading control for each sample and are reported as a relative value below each band. Full article ">Figure 3
PNC is localized to cellular protrusions. Fixed, unpermeabilized myofibroblasts from fibrotic and healthy tissues were immunostained for m-α-PNC mAb 10A10 (red) and DAPI (blue). PNC is localized to cellular protrusions on pathological myofibroblasts DCM-CF, LL29, and LL97A (arrows). PNC is not expressed on the surface of NHCF, NHLF, or CCD-16Lu isolated from healthy donor. Full article ">Figure 4
Feasibility and development of a PNC ELISA. (A) Soluble PNC product was immunoprecipitated from LL29 conditioned media and patient plasma from pooled healthy donors, IPF patients, NAFLD-Cirrhosis patients and cardiomyopathy patients using m-α-PNC mAb 10A10 compared to mouse IgG1 isotype control (mIgG1) and immunoblotted. (B) Recombinant prodomain analyte was serially diluted in duplicate starting at 100 ng/mL in standard diluent and measured by ELISA to determine the range of the standard. (C) Linearity of endogenous analyte was measured. Plasma from patients with heart failure was serially diluted 1:3, 1:7, 1:15, 1:31 in standard diluent and sPNC was measured by ELISA in duplicate. All linear regression r2 values were calculated to be within acceptable range (r2 ≥ 0.99). (D,E) Healthy donor plasma was diluted in standard diluent to indicated dilutions (1:1, 1:3, 1:7, 1:15) then spiked with recombinant prodomain analyte at 10 ng/mL (D) and 5 ng/mL (E) and analyzed by ELISA. Recovery of analyte was quantified and compared to back calculation of the standard. All dilutions were within the consensus range of 20 percent ± the standard back calculations at concentrations 10 ng/mL and 5 ng/mL. (F) Plasma was assayed for sPNC from healthy controls (n = 26), patients with IPF (n = 9), NAFLD with cirrhosis (n = 12), and cardiomyopathy (n = 9). Ordinary one-way ANOVA analysis with Dunnett’s multiple comparisons test was performed to determine significance (* p ≤ 0.05, **** p ≤ 0.0001). Full article ">Figure 5
FN1 is a potential PNC binding partner. (A) Medium binding ELISA plates were coated with human fibronectin, collagen type I, or collagen type III, then blocked and incubated with his-tagged recombinant N-cadherin prodomain. After washing unbound prodomain, prodomain binding to the immobilized substrate was measured using a biotinylated his-tag specific monoclonal antibody and streptavidin-HRP for detection. Assay was performed in quadruplicate and is representative of at least 3 independent experiments. (B) Representative image of a cardiac myofibroblast isolated from failed cardiac explant tissue showing colocalization of PNC and FN1 immunostained for PNC (red), FN1 (green) and DAPI (blue). Yellow indicates PNC/FN1 colocalization (Merge). (C) Medium binding ELISA plates were coated with fibronectin, blocked, then incubated with either recombinant prodomain of N-cadherin or prodomain in combination with mouse IgG1 isotype control, human IgG4 isotype control, m-α-PNC mAb 10A10 or h-α-PNC mAb HC5LC4. Bound recombinant prodomain was detected using anti-his-tag monoclonal antibody in technical duplicates and representative of at least 3 independent experiments (n = 3). Ordinary one-way ANOVA analysis with Tukey’s multiple comparisons test was performed to determine significance (* p ≤ 0.05, ** p ≤ 0.01). Full article ">Figure 6
Cell surface PNC has a role in myofibroblast migration. Transwell permeable supports with a 6.5 mm polycarbonate membrane and 8 µm pores were coated with human fibronectin and used to separate the upper and lower chambers of a 24-well cell culture plate to measure migration of cells across the membrane (n = 4). Pathological myofibroblast migration is significantly reduced by h-α-PNC mAb (HC5LC4) and recombinant prodomain of N-cadherin (rPro) after 5 h; (A) DCM-CF, dilated cardiomyopathy myofibroblasts (C) LL29, IPF myofibroblasts. No significant effect on myofibroblasts isolated from healthy tissues was observed; (D) immortalized CCD-16Lu lung myofibroblasts from healthy donor (B) NHCF, normal human cardiac myofibroblasts. Two tailed T-test assuming Gaussian distribution analysis was performed to determine significance. (* p ≤ 0.05, ** p ≤ 0.01). Full article ">Figure 7
AUROC analysis of plasma PNC for specificity and sensitivity. Normal donor samples (n= 26) were compared to samples from cardiomyopathy (Red, n = 9), NAFLD-Cirrhosis (Green, n = 12) and IPF (Yellow, n = 9) patients. To determine tissue-agnostic AUROC, all fibrotic samples were combined (Black, n= 30) and compared to normal donor samples. AUROC = area under the receiver operating characteristics curve. Full article ">

25 pages, 5004 KiB

Open AccessArticle

Extracellular Vesicles Derived from Bone Marrow in an Early Stage of Ionizing Radiation Damage Are Able to Induce Bystander Responses in the Bone Marrow

by Dávid Kis, Ilona Barbara Csordás, Eszter Persa, Bálint Jezsó, Rita Hargitai, Tünde Szatmári, Nikolett Sándor, Enikő Kis, Katalin Balázs, Géza Sáfrány and Katalin Lumniczky

Cells 2022, 11(1), 155; https://doi.org/10.3390/cells11010155 - 4 Jan 2022

Cited by 7 | Viewed by 3067

Abstract

Ionizing radiation (IR)-induced bystander effects contribute to biological responses to radiation, and extracellular vesicles (EVs) play important roles in mediating these effects. In this study we investigated the role of bone marrow (BM)-derived EVs in the bystander transfer of radiation damage. Mice were [...] Read more.

Ionizing radiation (IR)-induced bystander effects contribute to biological responses to radiation, and extracellular vesicles (EVs) play important roles in mediating these effects. In this study we investigated the role of bone marrow (BM)-derived EVs in the bystander transfer of radiation damage. Mice were irradiated with 0.1Gy, 0.25Gy and 2Gy, EVs were extracted from the BM supernatant 24 h or 3 months after irradiation and injected into bystander mice. Acute effects on directly irradiated or EV-treated mice were investigated after 4 and 24 h, while late effects were investigated 3 months after treatment. The acute effects of EVs on the hematopoietic stem and progenitor cell pools were similar to direct irradiation effects and persisted for up to 3 months, with the hematopoietic stem cells showing the strongest bystander responses. EVs isolated 3 months after irradiation elicited no bystander responses. The level of seven microRNAs (miR-33a-3p, miR-140-3p, miR-152-3p, miR-199a-5p, miR-200c-5p, miR-375-3p and miR-669o-5p) was altered in the EVs isolated 24 hour but not 3 months after irradiation. They regulated pathways highly relevant for the cellular response to IR, indicating their role in EV-mediated bystander responses. In conclusion, we showed that only EVs from an early stage of radiation damage could transmit IR-induced bystander effects. Full article

(This article belongs to the Special Issue Low Dose Radiation Effects on the Homeostasis and Activation of Immune Cells)

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20 pages, 3421 KiB

Open AccessArticle

Comprehensive Molecular Landscape of Cetuximab Resistance in Head and Neck Cancer Cell Lines

by Izabela N. F. Gomes, Renato J. da Silva-Oliveira, Luciane Sussuchi da Silva, Olga Martinho, Adriane F. Evangelista, André van Helvoort Lengert, Letícia Ferro Leal, Viviane Aline Oliveira Silva, Stéphanie Piancenti dos Santos, Flávia Caroline Nascimento, André Lopes Carvalho and Rui Manuel Reis

Cells 2022, 11(1), 154; https://doi.org/10.3390/cells11010154 - 4 Jan 2022

Cited by 14 | Viewed by 5278

Abstract

Cetuximab is the sole anti-EGFR monoclonal antibody that is FDA approved to treat head and neck squamous cell carcinoma (HNSCC). However, no predictive biomarkers of cetuximab response are known for HNSCC. Herein, we address the molecular mechanisms underlying cetuximab resistance in an in [...] Read more.

Cetuximab is the sole anti-EGFR monoclonal antibody that is FDA approved to treat head and neck squamous cell carcinoma (HNSCC). However, no predictive biomarkers of cetuximab response are known for HNSCC. Herein, we address the molecular mechanisms underlying cetuximab resistance in an in vitro model. We established a cetuximab resistant model (FaDu), using increased cetuximab concentrations for more than eight months. The resistance and parental cells were evaluated for cell viability and functional assays. Protein expression was analyzed by Western blot and human cell surface panel by lyoplate. The mutational profile and copy number alterations (CNA) were analyzed using whole-exome sequencing (WES) and the NanoString platform. FaDu resistant clones exhibited at least two-fold higher IC₅₀ compared to the parental cell line. WES showed relevant mutations in several cancer-related genes, and the comparative mRNA expression analysis showed 36 differentially expressed genes associated with EGFR tyrosine kinase inhibitors resistance, RAS, MAPK, and mTOR signaling. Importantly, we observed that overexpression of KRAS, RhoA, and CD44 was associated with cetuximab resistance. Protein analysis revealed EGFR phosphorylation inhibition and mTOR increase in resistant cells. Moreover, the resistant cell line demonstrated an aggressive phenotype with a significant increase in adhesion, the number of colonies, and migration rates. Overall, we identified several molecular alterations in the cetuximab resistant cell line that may constitute novel biomarkers of cetuximab response such as mTOR and RhoA overexpression. These findings indicate new strategies to overcome anti-EGFR resistance in HNSCC. Full article

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18 pages, 3844 KiB

Open AccessReview

The Impact of Exercise on Telomere Length, DNA Methylation and Metabolic Footprints

by Sandra Haupt, Tobias Niedrist, Harald Sourij, Stephan Schwarzinger and Othmar Moser

Cells 2022, 11(1), 153; https://doi.org/10.3390/cells11010153 - 4 Jan 2022

Cited by 9 | Viewed by 8282

Abstract

Aging as a major risk factor influences the probability of developing cancer, cardiovascular disease and diabetes, amongst others. The underlying mechanisms of disease are still not fully understood, but research suggests that delaying the aging process could ameliorate these pathologies. A key biological [...] Read more.

Aging as a major risk factor influences the probability of developing cancer, cardiovascular disease and diabetes, amongst others. The underlying mechanisms of disease are still not fully understood, but research suggests that delaying the aging process could ameliorate these pathologies. A key biological process in aging is cellular senescence which is associated with several stressors such as telomere shortening or enhanced DNA methylation. Telomere length as well as DNA methylation levels can be used as biological age predictors which are able to detect excessive acceleration or deceleration of aging. Analytical methods examining aging are often not suitable, expensive, time-consuming or require a high level of technical expertise. Therefore, research focusses on combining analytical methods which have the potential to simultaneously analyse epigenetic, genomic as well as metabolic changes. Full article

(This article belongs to the Special Issue Metabolic Inflammation and Cellular Immunity)

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Biological age correlates with the chronological age of a person and depends, among other things, on genetic predisposition, phenotypic changes and epigenetic changes. Nutritional, environmental, psychosocial and other lifestyle (exercise, weight) factors have the potential to both delay and accelerate the aging process and thus modulate risk of diseases. Specific biomarkers could serve as biological age predictors for risk assessment of age-specific diseases and thus influence the aging process positively as well as negatively. This could allow the detection of differences in the risk of age-related diseases for individuals of the same chronological age and identify pathways that have the potential to target these negative epigenetic changes. Full article ">Figure 2
Telomeres act as protective caps at the end of chromosomes. They consist of a sequence of 6 nucleotides (G-strand: 5′-TTAGGG-3′; C-strand: 3′-AATCCC-5′) repeated several thousand times. Telomeres loses length with each cell division. To prevent excessive shortening, they are protected by shelterin. Shelterin is a protein complex consisting of six subunits: telomere repeat binding factor 1 (TRF1), telomere repeat binding factor 2 (TRF2), protection of telomere 1 (POT1), repressor/activator protein 1 (RAP1), TRF1- and TRF2-interacting nuclear protein 2 (TIN2) and tripeptidyl peptidase 1 (TPP1). The 3’ end of the G-rich strand extends over the end of the C-rich strand (5’ end) of the telomere. Given sufficient length of the telomere, this forms the t-loop, which overlaps with the double-stranded 5’ end, building the D-loop protecting the telomeres (c). During replication, the G- and C-strands are open. Telomerase can bind to the G-strand to add telomeric repeats preventing the cell from damage (b). Loss of function and degradation of the shelterin complex leading to DNA damage occur more frequently during aging and cause the cell to stop dividing (a). Full article ">Figure 3
Normal cells can be induced to become senescent, which can induce paracrine senescence. Together with a decline in immune function, this could induce accumulation of senescent cells. In the elderly this accumulation contributes to increased risk in developing age-related diseases. Full article ">

17 pages, 5275 KiB

Open AccessArticle

The Negative Regulative Roles of BdPGRPs in the Imd Signaling Pathway of Bactrocera dorsalis

by Ping Zhang, Zhichao Yao, Shuai Bai and Hongyu Zhang

Cells 2022, 11(1), 152; https://doi.org/10.3390/cells11010152 - 4 Jan 2022

Cited by 5 | Viewed by 2296

Abstract

Peptidoglycan recognition proteins (PGRPs) are key regulators in insects’ immune response, functioning as sensors to detect invading pathogens and as scavengers of peptidoglycan (PGN) to reduce immune overreaction. However, the exact function of PGRPs in Bactrocera dorsalis is still unclear. In this study, [...] Read more.

Peptidoglycan recognition proteins (PGRPs) are key regulators in insects’ immune response, functioning as sensors to detect invading pathogens and as scavengers of peptidoglycan (PGN) to reduce immune overreaction. However, the exact function of PGRPs in Bactrocera dorsalis is still unclear. In this study, we identified and functionally characterized the genes BdPGRP-LB, BdPGRP-SB₁ and BdPGRP-SC₂ in B. dorsalis. The results showed that BdPGRP-LB, BdPGRP-SB₁ and BdPGRP-SC₂ all have an amidase-2 domain, which has been shown to have N-Acetylmuramoyl-l-Alanine amidase activity. The transcriptional levels of BdPGRP-LB and BdPGRP-SC₂ were both high in adult stages and midgut tissues; BdPGRP-SB₁ was found most abundantly expressed in the 2nd instar larvae stage and adult fat body. The expression of BdPGRP-LB and BdPGRP-SB₁ and AMPs were significantly up-regulated after injury infected with Escherichia coli at different time points; however, the expression of BdPGRP-SC₂ was reduced at 9 h, 24 h and 48 h following inoculation with E. coli. By injection of dsRNA, BdPGRP-LB, BdPGRP-SB₁ and BdPGRP-SC₂ were knocked down by RNA-interference. Silencing of BdPGRP-LB, BdPGRP-SB₁ and BdPGRP-SC₂ separately in flies resulted in over-activation of the Imd signaling pathway after bacterial challenge. The survival rate of the ds-PGRPs group was significantly reduced compared with the ds-egfp group after bacterial infection. Taken together, our results demonstrated that three catalytic PGRPs family genes, BdPGRP-LB, BdPGRP-SB₁ and BdPGRP-SC₂, are important negative regulators of the Imd pathway in B. dorsalis. Full article

(This article belongs to the Collection Feature Papers in ‘Cellular Immunology’)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Amino acid sequence alignment of BdPGRPs with that of homologous genes in other insect species. (A) Multiple alignments of PGRP-LB. BdPGRP-LB was aligned with Bactrocera latifrons PGRP-LB (XP_018789449.1), Bactrocera oleae PGRP-LB (XP_014091181.2), Zeugodacus cucurbitae PGRP-LB (XP_011197144.1), Ceratitis capitate PGRP-LB (XP_004518089.1), Rhagoletis zephyria PGRP-LB (XP_017470705.1), Rhagoletis pomonella PGRP-LB (XP_036322481.1), Aedes aegypti PGRP-LB (XP_021709443.1), Drosophila melanogaster PGRP-LB (NP_731575.1), Bombyx mori PGRP-LB (XP_012548100.1), Musca domestica PGRP-LB (XP_005180889.1), and Glossina fuscipes PGRP-LB (ACI22620.1). (B) Multiple alignments of PGRP-SB. BdPGRP-SB1 was aligned with B. latifrons PGRP-SB (XP_018789286.1), B. oleae PGRP-SB (XP_014099773.1), Z. cucurbitae PGRP-SB (XP_011181375.1), C. capitate PGRP-SB (XP_004537949.1), R. zephyria PGRP-SB (XP_017486043.1), R. pomonella PGRP-SB (XP_036336342.1), D. melanogaster PGRP-SB (CAD89135.1), M. domestica PGRP-SB (NP_001295929.1), and B. mori PGRP-SB (XP_004929843.1). (C) Multiple alignments of PGRP-SC2. BdPGRP-SC2 was aligned with B. latifrons PGRP-SC2 (XP_018798904.1), B. oleae PGRP-SC2 (XP_014085196.2), C. capitate PGRP-SC2 (XP_004520319.1), Z. cucurbitae PGRP-SC2 (XP_011180165.1), R. pomonella PGRP-SC2 (XP_036334551.1), M. domestica PGRP-SC2 (XP_005184140.3), D. melanogaster PGRP-SC2 (CAD89184.1), A. aegypti PGRP-SC2 (XP_011492940.1), and B. mori PGRP-SC2 (XP_004929814.1). The identical amino acids are shown against a black background; 75% conserved amino acids are shown against a pink background; 50% conserved amino acids are shown against a blue background. The signal peptides are indicated by dashed lines. The amidase domains are indicated by solid lines. Black arrows indicate the amino acid residues required for the recognition of DAP-type peptidoglycan. Grey arrows indicate the amino acid residues required for Zn2+ binding. White arrows indicate the amino acid residues required for amidase activity. Full article ">Figure 2
Expression profiles of BdPGRPs in B.dorsalis. (A) Relative expression of BdPGRP-LB at different development stages. (B) Relative expression of BdPGRP-SB1 at different development stages. (C) Relative expression of BdPGRP-SC2 at different development stages. (D) Relative expression of BdPGRP-LB from different tissue samples. (E) Relative expression of BdPGRP-SB1 from different tissue samples. (F) Relative expression of BdPGRP-SC2 from different tissue samples. B. dorsalis was collected at various developmental stages: 1 L, 1st instar larvae; 2 L, 2nd instar larvae; 3 L, 3rd instar larvae; EP, early pupal stage; LP, late pupal stage; EA, newly emergence adults; LA, late adult stage. Different adult tissues were collected: HD, head; MG, midgut; HG, hindgut; MT, Malpighian tube; FB, fatbody; OV, ovary; TE, testis. Multiple comparisons were carried out with one-way analysis of variance (ANOVA) and Turkey’s test in SPSS 16.0. Different lower-case letters indicate a significant difference at the level of p < 0.05 and a confidence interval of 95%. The relative gene expression data were analyzed using a 2−ΔΔCT method and the data were normalized to reference gene Rpl32. Full article ">Figure 3
Responses of Dpt and BdPGRPs to opportunistic pathogen E. coli challenges. Relative expression of Dpt (A), BdPGRP-LB (B), BdPGRP-SB1 (C), and BdPGRP-SC2 (D) after infection with E. coli at different time points, respectively. The data are expressed as mean ± SEM and the mean refers to the average of four biological replicates for each sample. Statistical analysis was based on Student’s t-test. * p < 0.05; ** p < 0.01; *** p < 0.001. The relative gene expression data were analyzed using a 2−ΔΔCT method and the data were normalized to reference gene Rpl32. Full article ">Figure 4
Off-target detection after dsRNA injection. (A) Influence of silencing BdPGRP-LB on expression of BdPGRP-SB1 and BdPGRP-SC2. (B) Influence of silencing BdPGRP- SB1 on expression of BdPGRP-LB and BdPGRP-SC2. (C) Influence of silencing BdPGRP- SC2 on expression of BdPGRP-LB and BdPGRP-SB1. All error bars represent the SEM of the mean of three independent biological replicates. Statistical analysis was based on Student’s t-test. * p < 0.05; ** p < 0.01; NS, no significant difference; p > 0.05. The relative gene expression data were analyzed using a 2−ΔΔCT method and the data were normalized to reference gene Rpl32. Full article ">Figure 5
RNA interference efficiency of BdPGRPs. Relative expression of PGRP-LB (A), PGRP-SB1 (B), and PGRP-SC2 (C) after dsRNA injection at different time points with whole body samples. All error bars represent the SEM of the mean of three independent biological replicates. Statistical analysis was based on Student’s t-test. * p < 0.05; ** p < 0.01. The relative gene expression data were analyzed using a 2−ΔΔCT method and the data were normalized to reference gene Rpl32. Full article ">Figure 6
Antimicrobial peptide gene expression in BdPGRPs RNAi flies after bacterial challenges. (A,B) Injury infection with E. coli induced a higher Diptericin (Dpt) expression in BdPGRPs RNAi flies than in the ds-egfp dsRNA injection flies. The data are expressed as the mean ± SEM, and the mean refers to the average of at least three replicates for each sample. Statistical analysis was based on Student’s t-test. * p < 0.05; ** p < 0.01. The relative gene expression data were analyzed using a 2−ΔΔCT method and the data were normalized to reference gene Rpl32. Full article ">Figure 7
Survival rate of B. dorsalis after BdPGRPs RNAi followed by E. coli infection. (A) Three BdPGRPs were knocked down separately. (B) Three BdPGRPs were knocked down at the same time. Statistical analysis was based on Log-rank (Mantel–Cox) test (* p < 0.05). Full article ">

14 pages, 2707 KiB

Open AccessArticle

Characterization of Oxygen Levels in an Uninfected and Infected Human Blood-Cerebrospinal-Fluid-Barrier Model

by Alexander Martens, Nicole de Buhr, Hiroshi Ishikawa, Horst Schroten and Maren von Köckritz-Blickwede

Cells 2022, 11(1), 151; https://doi.org/10.3390/cells11010151 - 4 Jan 2022

Cited by 2 | Viewed by 2437

Abstract

The host–pathogen interaction during meningitis can be investigated with blood-cerebrospinal-fluid-barrier (BCSFB) cell culture models. They are commonly handled under atmospheric oxygen conditions (19–21% O₂), although the physiological oxygen conditions are significantly lower in cerebrospinal fluid (CSF) (7–8% O₂). We [...] Read more.

The host–pathogen interaction during meningitis can be investigated with blood-cerebrospinal-fluid-barrier (BCSFB) cell culture models. They are commonly handled under atmospheric oxygen conditions (19–21% O₂), although the physiological oxygen conditions are significantly lower in cerebrospinal fluid (CSF) (7–8% O₂). We aimed to characterize oxygen levels in a Streptococcus (S.) suis-infected BCSFB model with transmigrating neutrophils. A BCSFB model with human choroid plexus epithelial cells growing on transwell-filters was used. The upper “blood”-compartment was infected and blood-derived neutrophils were added. S. suis and neutrophils transmigrated through the BCSFB into the “CSF”-compartment. Here, oxygen and pH values were determined with the non-invasive SensorDish^® reader. Slight orbital shaking improved the luminescence-based measurement technique for detecting free oxygen. In the non-infected BCSFB model, an oxygen value of 7% O₂ was determined. However, with S. suis and transmigrating neutrophils, the oxygen value significantly decreased to 2% O₂. The pH level decreased slightly in all groups. In conclusion, we characterized oxygen levels in the BCSFB model and demonstrated the oxygen consumption by cells and bacteria. Oxygen values in the non-infected BCSFB model are comparable to in vivo values determined in pigs in the CSF. Infection and transmigrating neutrophils decrease the oxygen value to lower values. Full article

(This article belongs to the Collection Advances in Cell Culture and Tissue Engineering)

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Figure 1
Experimental setup of the BCSFB model under shaking conditions. (A) The experimental setup of the BCSFB model is displayed. Human choroid plexus epithelial cells (HIBCPP) form the BCSFB barrier and divided the model into an upper “blood” compartment and a lower “CSF” compartment. Sensor spots for oxygen and pH measurement are inside the CSF compartment. (B) The number of transmigrated neutrophils was quantified inside the CSF compartment at the end of the six-hour experiment. The shaking setup did not influence the transmigration. One example of an immunofluorescence microscopy picture of the choroid plexus cells grown on a filter shows a transmigrating neutrophil (arrow). Scale bar 10 µm; blue = DNA, gray = phalloidin, green = LL-37, red = myeloperoxidase; single channels and overview pictures are presented in <a href="#cells-11-00151-f0A3" class="html-fig">Figure A3</a>. (C) The host–pathogen interaction of neutrophils and S. suis were not influenced regarding the neutrophil killing capacity of S. suis. All data are presented as mean ± SD of three independent experiments. Statistical differences were analyzed with one-tailed paired Student’s t-test (* p < 0.05, ** p < 0.01). Full article ">Figure 2
Choroid plexus epithelial cells (HIBCPP), neutrophils, and S. suis consume oxygen over time. The oxygen level was determined in the CSF compartment in a shaking setup. (A) The oxygen level did not change in media, whereas with HIBCPP cells, the oxygen decreased over time. (B) Non-activated neutrophils did not consume a high level of oxygen, whereas S. suis significantly consumed oxygen. (C) The host–pathogen interaction of HIBCPP cells, transmigrating neutrophils, and S. suis significantly consumed oxygen. All data are presented as mean ± SD of three independent experiments. Statistical differences were analyzed in A with a one-tailed paired Student’s t-test in each group at 0 h. Statistical differences were analyzed in B and C with a one-way ANOVA at each time-point (p < 0.0001), followed by Tukey multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Full article ">Figure 3
The pH in the CSF compartment is slightly decreased by S. suis and interacting neutrophils after a six-hour incubation period. The pH level was determined in the CSF compartment in a shaking setup. (A) The pH decrease in media and with HIBCPP cells is comparable. (B) No significant influence on pH was detected in the absence of HIBCPP cells. (C) The host–pathogen interaction of HIBCPP cells, transmigrating neutrophils, and S. suis significantly decreased the pH slightly after six hours. All data are presented as mean ± SD of three independent experiments. Statistical differences were analyzed in A with a one-tailed paired Student’s t-test in each group at 0 h. Statistical differences were analyzed in B and C with a one-way ANOVA at each time-point (C: p2h = 0.002; p6h = 0.02), followed by Tukey multiple comparisons in (C) (* p <0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure A1
A shaking setup of the BCSFB model is obligatory to reduce bacterial-related false reduced oxygen levels inside the CSF compartment. (A) The dextran flux rate was determined as an indicator of barrier integrity. No cells on the filter (TEER = 0 Ω x cm2) led to a dextran flux rate of 100%. With increasing TEER values, the dextran flux rate decreased. Dextran flow rates lower than 5% indicate increasing barrier integrity of the HIBCPP layer. At the start of all experiments, only filters with TEER values of the experiment in the gray marked range were included. (B) The oxygen partial pressures were compared in a non-shaking and shaking setup during S. suis infection. After a 2-h incubation period with S. suis, significantly lower oxygen partial pressure was measured in the non-shaking setup (** p ≤ 0.01). (C) The determined bacterial number remained constant between the two setups. (D) Two areas were defined inside the well to quantify S. suis around the sensor spot (S) and in the outer circle. The area of “S” and a defined extra area = “E” were named “A1”. The rest of the well was named “A2”. (E) Significant different numbers of S. suis were reisolated from the two defined areas with the two shaking setups. The total amount of bacteria was comparable (* p < 0.05). (F) The TEER was not influenced by the shaking setup during an infection experiment with transmigrating neutrophils. Full article ">Figure A2
S. suis significantly decreased the barrier integrity after seven hours. The TEER values of S. suis-infected HIBCPP filters were measured before infection (0 h) and after 4, 7, 20, and 24 h (h). The filters were infected and incubated for 24 h with and without antibiotic treatment. (A) A significant decrease in TEER was detectable after 7 h S. suis infection in the absence of antibiotics. After 20 h, the low TEER reflected a loss of barrier integrity. (B) Antibiotic treatment after 4 h did not lead to a TEER value after 24 h in a range higher than 380 Ω x cm2. The mean value ± SD of independent experiments is presented. Each dot represents one filter. Statistical differences were analyzed with one-way ANOVA (p < 0.0001), followed by Dunnett’s multiple comparison test in A and Tukey’s multiple comparison test in B, (** p < 0.01, *** p < 0.001, **** p < 0.0001). Full article ">Figure A3
LL-37 and myeloperoxidase signals identify a transmigrating neutrophil through the BCSFB. Immunofluorescence microscopy was conducted to detect transmigrating neutrophils. Filters were stained and imaged from the “CSF” side. White squares highlight the area of magnification. Settings were adjusted to the respective isotype control; blue = DNA, green = LL-37, red = myeloperoxidase (MPO) and gray = phalloidin). Scale bar overview = 50 µm; scale bar zoom = 10 µm. Full article ">

22 pages, 1842 KiB

Open AccessArticle

The Molecular Subtype of Adult Acute Lymphoblastic Leukemia Samples Determines the Engraftment Site and Proliferation Kinetics in Patient-Derived Xenograft Models

by Anna Richter, Catrin Roolf, Anett Sekora, Gudrun Knuebel, Saskia Krohn, Sandra Lange, Vivien Krebs, Bjoern Schneider, Johannes Lakner, Christoph Wittke, Christoph Kiefel, Irmela Jeremias, Hugo Murua Escobar, Brigitte Vollmar and Christian Junghanss

Cells 2022, 11(1), 150; https://doi.org/10.3390/cells11010150 - 3 Jan 2022

Cited by 4 | Viewed by 2709

Abstract

In acute lymphoblastic leukemia (ALL), conventional cell lines do not recapitulate the clonal diversity and microenvironment. Orthotopic patient-derived xenograft models (PDX) overcome these limitations and mimic the clinical situation, but molecular stability and engraftment patterns have not yet been thoroughly assessed. We herein [...] Read more.

In acute lymphoblastic leukemia (ALL), conventional cell lines do not recapitulate the clonal diversity and microenvironment. Orthotopic patient-derived xenograft models (PDX) overcome these limitations and mimic the clinical situation, but molecular stability and engraftment patterns have not yet been thoroughly assessed. We herein describe and characterize the PDX generation in NSG mice. In vivo tumor cell proliferation, engraftment and location were monitored by flow cytometry and bioluminescence imaging. Leukemic cells were retransplanted for up to four passages, and comparative analyses of engraftment pattern, cellular morphology and genomic hotspot mutations were conducted. Ninety-four percent of all samples were successfully engrafted, and the xenograft velocity was dependent on the molecular subtype, outcome of the patient and transplantation passage. While BCR::ABL1 blasts were located in the spleen, KMT2A-positive cases had higher frequencies in the bone marrow. Molecular changes appeared in most model systems, with low allele frequency variants lost during primary engraftment. After the initial xenografting, however, the PDX models demonstrated high molecular stability. This protocol for reliable ALL engraftment demonstrates variability in the location and molecular signatures during serial transplantation. Thorough characterization of experimentally used PDX systems is indispensable for the correct analysis and valid data interpretation of preclinical PDX studies. Full article

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Engraftment-influencing parameters. Every dot represents a separate animal. (A) Comparison of the initial weight and weight at experiment termination of all 101 mice used for tumor cell expansion. Weight gain is indicated by green dots, while weight loss up to 10% and 20% is displayed in yellow and red, respectively. (B–H) Influence of clinical–pathological parameters on tumor cell engraftment and proliferation in the first PDX passage. (B) Only patients with confirmed date of death or a recent checkup no older than twelve months were included in the analysis to investigate the influence of patient survival. Samples of unknown status or when the last contact was longer than one year ago were excluded. n = 19 mice, mean ± standard deviation, Mann–Whitney test. (C) Influence of patient sex on engraftment velocity. n = 30 mice, mean ± standard deviation, Mann–Whitney test. (D) Correlation of patient age at sample collection with engraftment kinetics. n = 30 mice (Spearman’s correlation coefficient r). (E) Influence of sample origin (bone marrow or peripheral blood) on engraftment velocity. n = 29 mice, mean ± standard deviation, Mann–Whitney test. (F) Influence of the molecular subtype of the primary tumor on engraftment speed. Patients with BCR::ABL1 or KMT2A translocations and additional aberrations were only considered for the BCR::ABL1 and KMT2A cohorts, respectively. n = 30 mice, mean ± standard deviation, no statistical evaluation due to limited sample numbers in subgroups. (G) Correlation of the number of cells injected and the engraftment velocity. n = 30 mice (Spearman’s correlation coefficient r). (H) Influence of mouse sex on engraftment speed. n = 30 mice, mean ± standard deviation, Mann–Whitney test. Full article ">Figure 2
Influence of the molecular subtype on the engraftment sites. All 101 animals used for cell expansion irrespective of the engraftment passage were considered for these analyses. (A,B) Bone marrow and spleen infiltration were determined by a flow cytometric analysis of human C45+/CD19+, C45+/CD5+ and CD45+/CD7+ blasts isolated from animals at the end of the expansion experiment. Kruskal–Wallis and post-hoc Dunn’s multiple comparisons test. (C) Influence of the molecular subtype on the spleen weight. Kruskal–Wallis test. (D,E) Correlation analysis between the spleen weight and spleen infiltration (D) and spleen and bone marrow blast frequency (E). Each dot represents a single animal, and mice engrafted with the same patient material are displayed in the same color. Similar color shades indicate the same molecular subtype. Spearman’s correlation value r. Full article ">Figure 3
Evaluation of tumor engraftment and growth kinetics. (A) Longitudinal quantification of the bioluminescence (full lines) and circulating blast frequency (dotted lines) of two representative animals. (B) Corresponding bioluminescence images of mice depicted in <a href="#cells-11-00150-f003" class="html-fig">Figure 3</a>A. (C) Influence of serial transplantation on the engraftment velocity. Samples with BCR::ABL1, KMT2A and T-ALL are painted in green, red and grey, respectively. Flow cytometric determination of the tumor cell frequency in peripheral blood was performed throughout the observation period. Each line represents an individual animal, and mice engrafted within the same passage are displayed in the same color. Earlier passages are depicted in darker colors. A line printed in bold indicates that the following passage was derived from the indicated animal. (D) Flow cytometric analysis of bone marrow (BM) and spleen infiltration upon termination of the experiment. Each dot represents an individual mouse. Mean ± standard deviation. Full article ">Figure 4
Presence of single nucleotide variants in the KMT2A-positive primary samples and consecutive PDX passages. Only somatic or likely somatic variants detected using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Thermo Fisher Scientific) are summarized irrespectively of their pathogenicity. Two individual mice were analyzed in passage 2 of patients #0122 and #0134 and depicted by separate lines. Only cells from one of those mice were used for the subsequent xenografting to generate passage 3. P, passage. Full article ">

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Cells, Volume 11, Issue 1 (January-1 2022) – 179 articles

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