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The immune system’s ability to detect and eliminate transformed cells is a critical factor in suppressing cancer development. However, immune surveillance in tumors is often disrupted by various immune escape mechanisms, many of which remain poorly understood. The Natural Killer Group 2D (NKG2D) receptor is an activating receptor expressed on natural killer (NK) cells and cytotoxic T lymphocytes. It can recognize and bind with varying affinities to a wide range of structurally diverse ligands, including MHC class I chain-related proteins A and B (MICA and MICB) and members of the ULBP family (ULBP1-6). The expression of these ligands plays a crucial role in immune antitumor responses and cancer immunoevasion mechanisms. Some evidence suggests that functional polymorphisms in the NKG2D receptor and the genes encoding its ligands significantly influence HLA-independent cancer immunosurveillance. Consequently, the NKG2D-NKG2D ligands (NKG2DLs) axis represents a promising target for developing novel therapeutic strategies. This review aims to provide a general overview of the role of NKG2D and its ligands in various malignancies and explore their potential in advancing personalized cancer treatment protocols. Full article

(This article belongs to the Section Cancer Immunology and Immunotherapy)

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Boxplot comparisons of the expression of the KLRK1 gene in human blood cells. Data are from the HemaExplorer project [<a href="#B14-immuno-05-00009" class="html-bibr">14</a>] and are available through the BloodSpot server (<a href="https://www.bloodspot.eu/" target="_blank">https://www.bloodspot.eu/</a> (accessed on 31 January 2025)) [<a href="#B15-immuno-05-00009" class="html-bibr">15</a>]. Abbreviations: “HSC-BM”—Bone marrow hematopoietic stem cells; “early HPC_BM”—early bone marrow hematopoietic progenitors; “CMP”—common myeloid progenitors; “GMP”—granulocyte monocyte progenitors; “MEP”—megakaryocyte-erythroid progenitors; “PM_BM”—bone marrow promyelocytes; “MY_BM”—bone marrow myelocytes; “PMN_BM”—bone marrow polymorphonuclear cells; “PMN_PB”—peripheral blood polymorphonuclear cells. Full article ">Figure 2
Expression of the two major NKG2D ligands based on transcriptomic data in selected malignancies. (A) MICA gene. (B) MICB gene. Data were analyzed and plotted using the TNMplot (<a href="https://tnmplot.com/" target="_blank">https://tnmplot.com/</a>) server (accessed on 15 January 2025) [<a href="#B51-immuno-05-00009" class="html-bibr">51</a>]. Left-sided boxplots represent expression in normal tissues whereas the right-sided—in tumor tissue. The names of tumors subtypes in red and with an asterisk (*) represent comparisons using the Mann–Whitney test with p < 0.05 and expression > 10 in tumor or normal tissue. TNMplot uses transcriptomic data from the following databases: GEO, GTex, TCGA, and TARGET [<a href="#B51-immuno-05-00009" class="html-bibr">51</a>]. Abbreviations: “AML”—acute myeloid leukemia, “Lung_AC”—lung adenocarcinoma; “Lung_SC”—lung squamous cell carcinoma; “Renal_CC”—renal clear cell carcinoma; “Uterus_CS”—squamous cell cervical cancer; “Uterus_EC”—endometrial cancer. Full article ">Figure 3
Summary of the main known mechanisms for immune escape by cancer cells involving the NKG2D-NKG2DLs axis. Full article ">

27 pages, 7555 KiB

Open AccessArticle

Cylindracin, a Fruiting Body-Specific Protein of Cyclocybe cylindracea, Represses the Egg-Laying and Development of Caenorhabditis elegans and Drosophila melanogaster

by Yamato Kuratani, Akira Matsumoto, Ayako Shigenaga, Koji Miyahara, Keisuke Ekino, Noriaki Saigusa, Hiroto Ohta, Makoto Iwata and Shoji Ando

Toxins 2025, 17(3), 118; https://doi.org/10.3390/toxins17030118 (registering DOI) - 1 Mar 2025

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Abstract

Mushrooms are a valuable source of bioactive compounds to develop efficient, secure medicines and environmentally friendly agrochemicals. Cylindracin is a small cysteine-rich protein that is specifically expressed in the immature fruiting body of the edible mushroom Cyclocybe cylindracea. Recombinant protein (rCYL), comprising the C-terminal cysteine-rich domain of cylindracin, inhibits the hyphal growth and conidiogenesis of filamentous fungi. Here, we show that rCYL represses the egg-laying and development of Caenorhabditis elegans and Drosophila melanogaster. The feeding of rCYL at 16 µM reduced the body volume of C. elegans larvae to approximately 60% when compared to the control. At the same concentration, rCYL repressed the frequencies of pupation and emergence of D. melanogaster to 74% and 40%, respectively, when compared to the control. In virgin adult flies, feeding of rCYL at 47 µM substantially repressed the frequency of egg-laying, and the pupation and emergence of the next generation, especially for females. These inhibitory effects of rCYL gradually disappeared after ceasing the ingestion of rCYL. The use of fluorescence-labeled rCYL revealed that the protein accumulates specifically at the pharynx cuticles of C. elegans. In D. melanogaster, fluorescence-labeled rCYL was detected primarily in the midguts and to a lesser degree in the hindguts, ovaries, testes, and malpighian tubules. rCYL was stable against trypsin, chymotrypsin, and pepsin, whereas it did not inhibit proteolytic and glycolytic enzymes in vitro. rCYL oligomerized and formed amyloid-like aggregates through the binding to heparin and heparan sulfate in vitro. These results suggest that rCYL has potential as a new biocontrol agent against pests. Full article

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Inhibitory effect of rCYL on the development of nematodes. (a) Synchronized eggs were placed on an NGM plate seeded with Escherichia coli, containing recombinant cylindracin (rCYL) or bovine serum albumin (BSA) as a control, and incubated in triplicate per condition. After 24, 48, 72, and 96 h, the body volumes (nl) of nematodes on each medium were measured as previously described [<a href="#B30-toxins-17-00118" class="html-bibr">30</a>] (n = 6). The mean values ± standard deviations (SD) were plotted. Statistically significant differences from the control were determined using Student’s t-test and are indicated as * p < 0.05, and ** p < 0.01. (b) Representative images of nematodes reared on medium containing BSA or rCYL after 96 h incubation. Full article ">Figure 2
Inhibitory effect of rCYL on the fecundity of Drosophila. (a) Virgin male and female adult flies of G1 generation were separately cultivated on BSA–medium (BSA at 0.3 mg/mL) or rCYL–medium (rCYL at 0.3 mg/mL) for 53 h. The adult flies reared on rCYL–medium are shown in red. (b) Five different combinations (I)–(V) of the virgin G1 male and female adult flies were prepared on fresh BSA–medium or rCYL–medium, and then allowed to mate freely and lay eggs on each medium for 62 h. (c) After transferring the G1 adult flies in the combinations (I)–(V) to (f), G2 (second-generation) eggs on each medium were counted. The mean value of the G2 eggs is shown on the top of each column. (d) After 7 days, G2 pupae on each medium were counted. G2 pupae that formed belatedly were also counted, and the total numbers of the G2 pupae on each medium are shown. (e) After 3 days, G2 adult flies emerged from the pupae were counted. (f) After transferring from (c), the G1 adult flies in the five combinations were allowed to mate freely and lay eggs on fresh BSA–medium. Their combinations were numbered afresh as (1)–(5). (g) After 3 days, the G1 adult flies in the five combinations were removed and the eggs (referred to as G2-2 eggs) laid on the BSA–medium were continuously incubated. (h) After 9 days, G2-2 adult flies emerged on each medium were counted. (i) Ten randomly selected G2-2 male and female adult flies in the combinations (1)–(5) in (h) were transferred onto fresh BSA–medium, and allowed to mate freely and lay eggs. (j) After 3 days, the G2-2 adult flies were removed. G3 (third-generation) eggs laid on the BSA–medium were continuously incubated. (k) After 12 days, G3 adult flies emerged on each medium were counted. All experiments were performed in triplicate per condition, and the mean values ± SD are shown. Statistically significant differences from the control were determined using Student’s t-test and are indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001. Full article ">Figure 3
Localization of fluorescently labeled rCYL at the pharynx in nematodes. Adult nematodes were incubated on medium containing 0.2 mg/mL FITC-rCYL or 0.2 mg/mL FITC-BSA. (a) The nematode fed on FITC-BSA showed a faint fluorescent signal along the intestine (left, phase-contrast; right, fluorescence). Scale bar, 50 µm. (b) The nematode fed on FITC-rCYL exhibited intense fluorescence signals along the pharynx, in addition to a faint signal along the intestine. Scale bar, 50 µm. (c) Schematic presentation of the pharynx of C. elegans. The cuticle that lines the pharynx is indicated in red. (d) Phase-contrast image of the anterior part of the nematode that was fed on FITC-rCYL and then stained with Calcofluor white (CFW). Scale bar, 20 µm. (e) Fluorescence image detecting CFW in the same part of the nematode as in (d). CFW is a chitin probe. Scale bar, 20 µm. (f) Fluorescence image detecting FITC-rCYL in the same part of the nematode as in (d). Scale bar, 20 µm. (g) Images (e) and (f) were merged. FITC-rCYL localized at the structure that was stained with CFW. Scale bar, 20 µm. Full article ">Figure 4
Localization of fluorescently labeled rCYL in Drosophila. Stereo-microscopic (a) and fluorescence (b) images of a female adult fly that was fed on medium containing FITC-BSA and dissected are shown, as a control. In (c–h), stereo-microscopic images (c,e) and fluorescence images (d,f–h) of female adult flies that were fed on medium containing FITC-rCYL and dissected, are shown. In (d,f), an intense fluorescent signal at the midgut (MG) and weak signals at the foregut (FG), hindgut (HG), crop (CR), ovary (OV), and malpighian tubules (MT) were observed. In (g,h), the ovary (OV) in (f) was expanded. Unfertilized eggs (UE) were also detected by a fluorescent signal. In (i,j), a stereo-microscopic image (i) and fluorescence image (j) of a male adult fly that was fed on medium containing FITC-BSA and dissected are shown as a control. In (k–o), stereo-microscopic images (k,n) and fluorescence images (l,m,o) of male adult flies that were fed on medium containing FITC-rCYL are shown. In (l,m,o), an intense fluorescent signal at the midgut (MG) and weak fluorescent signals at the foregut (FG), hindgut (HG), crop (CR), testis (TE), and malpighian tubules (MT) were observed. The image of the midgut in (l) is expanded in (m), revealing fluorescent particles (arrowheads) in the midgut. In (p,q), a stereo-microscopic image (p) and fluorescence image (q) of eggs laid by adult flies fed on medium containing FITC-BSA are shown as a control. In (r–u), stereo-microscopic images (r,t) and fluorescence images (s,u) of eggs laid by adult flies fed on medium containing FITC-rCYL are shown. Weak, unevenly distributed fluorescence signals (s) or, in some cases, multiple fluorescence bands around the center of the eggs (u) were observed. In (v,w), a stereo-microscopic image (v) and fluorescence image (w) of larvae that hatched from eggs laid by flies fed on medium containing FITC-BSA are shown as a control. In (x–z), a stereo-microscopic image (x) and fluorescence images (y,z) of larvae that hatched from the eggs of adult flies fed on medium containing FITC-rCYL and then grew on rCYL–medium are shown. In (y,z), an intense fluorescent signal at the midgut (MG) and weak fluorescent signals at the foregut (FG), hindgut (HG), and malpighian tubules (MT) were observed. Full article ">Figure 5
Fluorescence microscopic observation of the midgut contents. The adult flies fed on FITC-BSA (a) or FITC-rCYL (b,c) were dissected, fixed with paraformaldehyde, and then observed. In contrast to (a), fluorescent contents (arrows) were observed in the midgut (MG in b,c). The larvae fed on FITC-rCYL were also dissected, fixed, and then observed (d). The region with a broken line in (d) was expanded and brightened in (e) to clearly reveal the midgut (MG) tissue and the fluorescent contents. In (e), due to the partial breakdown of the midgut tissue, the contents were observed to be composed of many fluorescent particles (arrows). (f) The midgut of the adult fly that was fed on FITC-rCYL and then fasted in PBS showed no fluorescent contents. Asterisk: air bubble. Scale bars in (a–c,e,f): 200 µm. Scale bar in (d): 400 µm. Full article ">Figure 6
rCYL is stable against proteolytic enzymes but does not inhibit proteolytic and glycolytic enzymes. (a) rCYL was incubated with bovine trypsin (Tsin), α-chymotrypsin (Csin), and porcine pepsin (Psin), and the reaction mixtures were resolved by SDS-PAGE. As a control (C), rCYL was incubated in a buffer without enzymes. rCYL was stable against proteolytic reactions by the enzymes (n = 2). By contrast, rCYL, which was first reduced and denatured by treatment with dithiothreitol at 100 °C (DTT-treated rCYL), was completely hydrolyzed by the proteases (n = 2). (b) rCYL was preincubated with bovine trypsin and then mixed with a trypsin-specific substrate Bz-L-Arg-pNA, and the proteolytic reaction was monitored calorimetrically for liberated pNA. rCYL did not inhibit proteolysis of the substrate by trypsin (n = 3). (c,d) α-Amylase from Bacillus amyloliquefaciens or glucoamylase from Aspergillus niger were preincubated with rCYL or the same amount of buffer (Control), and then mixed with starch. The liberated oligosaccharides and glucose were detected using the Bernfeld method [<a href="#B44-toxins-17-00118" class="html-bibr">44</a>]. rCYL did not inhibit these glycolytic enzymes (n = 3). Full article ">Figure 7
Protein cross-linking of rCYL. (a) rCYL was cross-linked via incubation with bis-N-succinimidyl-(nonaethylene glycol) ester [BS(PEG)9], and the reaction mixture was resolved by SDS-PAGE. The protein bands were detected using Coomassie brilliant blue staining. By increasing the concentration of BS(PEG)9 from 5 µM to 500 µM, a band representing the rCYL dimer was observed at ~13 kDa (indicated with an asterisk). When the cross-linker was employed at 5 mM, the protein bands disappeared from the gel. (b) As the concentration of rCYL increased in the presence of BS(PEG)9, two protein bands were observed at ~13 kDa (indicated with an asterisk) and ~24 kDa (indicated with the double asterisks), which represent the rCYL dimer and the rCYL tetramer, respectively. Full article ">Figure 8
rCYL binding assay to heparin and heparan sulfate. (a) FITC-rCYL at 0.25 mg/mL was incubated with 100 µL of porcine–heparin agarose resin in 20 mM sodium phosphate buffer for 1 h. After washing the resin, the resin was incubated in 20 mM sodium phosphate (indicated as “Buffer only”), 20 mM sodium phosphate containing 0.5 M NaCl (“0.5 M NaCl”), 20 mM sodium phosphate containing 1 M NaCl (“1 M NaCl”), 20 mM sodium phosphate containing 1 M NaCl plus 4 M urea (“1 M NaCl + 4 M urea”), or 20 mM sodium phosphate containing 1 M NaCl plus 8 M urea (“1 M NaCl + 8 M urea”) for 10 min. After washing the resin, the fluorescence intensity of the resin was measured. (b–e) Representative fluorescence images of the heparin resins that were recovered after incubation in 20 mM sodium phosphate (indicated as “Buffer only”) (b), in the same buffer containing 0.5 M NaCl (“0.5 M NaCl”) (c), in the same buffer containing 1 M NaCl (“1 M NaCl”) (d), or in the same buffer containing 1 M NaCl plus 8 M urea (“1 M NaCl + 8 M urea”) (e). Scale bars, 200 µm. (f–i) rCYL at 0.4 mg/mL was incubated with 4 mg/mL heparin (f,g) or heparan sulfate (HS) (h,i) in 20 mM phosphate buffer containing 150 mM NaCl for 30 min, and was then incubated with 5 µM thioflavin T. Representative images of amyloid aggregates that emit green fluorescence are shown. Scale bars, 20 µm. Full article ">

15 pages, 4150 KiB

Open AccessArticle

Ubiquitin Ligase Gene OsPUB57 Negatively Regulates Rice Blast Resistance

by Jian Zhang, Qiang Du, Yugui Wu, Mengyu Shen, Furong Gao, Zhilong Wang, Xiuwen Xiao, Wenbang Tang and Qiuhong Chen

Plants 2025, 14(5), 758; https://doi.org/10.3390/plants14050758 (registering DOI) - 1 Mar 2025

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Abstract

The ubiquitination and degradation of proteins are widely involved in plant biotic and abiotic stress responses. E3 ubiquitin ligases play an important role in the ubiquitination of specific proteins. In this study, we identified the function of a U-box E3 ubiquitin ligase gene OsPUB57 in rice. Expression analyses revealed that OsPUB57 was mainly expressed in the aboveground part of rice. Drought, salt, cold, JA (jasmonic acid), PAMPs (pathogen-associated molecular patterns) or Magnaportheoryzae treatment could significantly suppress the expression of OsPUB57 in rice. Compared with wild-type plants, OsPUB57-overexpressing plants showed a decrease in resistance to M. oryzae, while the mutant plants exhibited an enhancement of M. oryzae resistance. The expression level detection indicated that OsPUB57 negatively regulates rice blast resistance, probably by down-regulating the expression of the defense-related genes OsPR1a and OsAOS2. This study provides a candidate gene for the genetic improvement of rice blast resistance. Full article

(This article belongs to the Section Plant Protection and Biotic Interactions)

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Sequence analyses of OsPUB57 and its homologous sequences. (A) Sequence alignment of OsPUB57 and its homologous sequences from T. turgidum (VAH34870.1), T. dicoccoides (XP_037480658.1), T. aestivum (XP_044458441.1), T. urartu (EMS46603.1), H. vulgare (XP_044967372.1), B. distachyon (XP_010230025.2), S. bicolor (KAG0550666.1), and M. lutarioriparius (CAD6211570.1). The U-box domain (black line) and kinase domain (red line) are underlined; (B) Phylogenetic tree of OsPUB57 and its homologs. Full article ">Figure 2
Expression profile analysis of OsPUB57 in different rice tissues and under different stress treatments. (A) Expression levels of OsPUB57 in different tissues at the seedling and heading stages; (B–D) The expression profile of OsPUB57 under different treatments. Error bars are standard deviations based on three replicates. Analysis of significant differences in expression levels between the control and treatment samples was conducted at each time point (* represents p < 0.05). Full article ">Figure 3
Expression profile of OsPUB57 in rice under different phytohormone treatments. (A) MeJA treatments and (B) SA treatments. Error bars are standard deviations based on three replicates. Analyses of significant differences in expression levels between the control and treatment samples were conducted at each time point (* represents p < 0.05). Full article ">Figure 4
The expression level of OsPUB57 in response to M. oryzae inoculation (A) and PAMP treatments (B). Error bars are standard deviations based on three replicates. Analyses of significant differences in expression levels between the control and treatment samples were conducted at each time point (* represents p < 0.05). Full article ">Figure 5
Rice blast resistance identification of OsPUB57-overexpressing plants (OX-11, OX-12 and OX-17) and the WT plants (Nipponbare). (A) The expression level of OsPUB57; (B) Rice blast resistance phenotypes. Bar = 1 cm; (C) Relative lesion area; (D) Relative fungal growth. Error bars indicate standard deviations from three replicates. Asterisks represent significant differences compared with the WT (* represents p < 0.05). Full article ">Figure 6
Rice blast resistance evaluation of the mutant plants (M-1, M-22 and M-35) of OsPUB57 and the WT plants (Nipponbare). (A) Rice blast resistance phenotypes. Bar = 1 cm; (B) Relative lesion area; (C) Relative fungal growth. Error bars indicate standard deviations from three replicates. Asterisks represent significant differences compared with the WT (* represents p < 0.05). Full article ">Figure 7
The expression levels of two defense-related genes (OsAOS2 and OsPR1a) in the mutant plants of OsPUB57 before and after inoculation with M. oryzae. (A) The expression level of OsAOS2. (B) The expression level of OsPR1a. Error bars indicate standard deviations from three replicates. Asterisks represent significant differences compared with the WT at each time point (* represent p < 0.05). Full article ">

14 pages, 3861 KiB

Open AccessArticle

The Reliable Detection of Homocysteine Using a Biosensor Based on Recombinant Cystathionine β-Synthase and Nanoporous Gold

by Zihan Huang, Yan Gao, Lei Zhang, Ting Cai, Ruijun Liu and Xia Wang

Microorganisms 2025, 13(3), 559; https://doi.org/10.3390/microorganisms13030559 (registering DOI) - 1 Mar 2025

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Abstract

Given the essential roles of homocysteine (Hcy) and the interference of cysteine in effectively monitoring human health, this study proposed a synergistic effect strategy that combines the unique structural and functional properties of nanoporous gold (NPG) with the selective recognition capability of a recombinant cystathionine β-synthase (CBS) for the sensitive and specific detection of Hcy. The CBS protein with specific catalytic activity for Hcy was successfully produced in recombinant Escherichia coli BL21 (pET-30a-cbs) using the cbs gene from Pseudomonas aeruginosa PAO1. The electrochemical mechanism demonstrated that the electrooxidation of H₂S, a catalytic product of the CBS, was an irreversibly surface-controlled process on the CBS/NPG/GCE electrode surface. The electrochemical detection of Hcy exhibited excellent linearity, with a high sensitivity reaching 10.43 µA mM⁻¹ cm⁻² and a low detection limit of 1.31 µM. Furthermore, the CBS/NPG/GCE biosensor was successfully used to detect Hcy in urine samples with strong anti-interference capability and high selectivity (relative standard deviation less than 2.81%), while effectively reducing the interference from cysteine. These results confirmed that the proposed CBS/NPG/GCE electrochemical sensor achieved specific, sensitive, and reliable rapid detection of homocysteine, making it highly promising for practical applications in clinical treatment and health assessment. Full article

(This article belongs to the Collection Feature Papers in Environmental Microbiology)

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The schematic construction of recombinant expression vector (pET-30a-cbs) and electrophoresis images of PCR amplification and double digestion. (A) The PCR amplification and electrophoresis analysis for the target gene cbs. (B) The schematic construction of recombinant cloning plasmid (pEASY-blunt-cbs). (C) The schematic construction of recombinant expression vector (pET-30a-cbs). Lanes a and c show the DNA marker, lane b shows the PCR product of the cbs gene, and lanes d and e display the double digestion of the pET-30a and pET-30a-cbs plasmids, respectively. Full article ">Figure 2
(A) The protein electrophoretogram of the crude enzyme solutions from induced E. coli BL21 (pET-30a) (lane b) and E. coli BL21 (pET-30a-cbs) (lane c), purified CBS enzyme solution (lane d), and protein marker (lane a). (B) Lead acetate test results for the crude enzyme solutions from E. coli BL21 (pET-30a) (i) and E. coli BL21 (pET-30a-cbs) (ii) and purified CBS enzyme solution (iii), reacting with 5 mM of Hcy + 5 mM of Cys. Full article ">Figure 3
(A) The CV curves of GCE (a) and CBS/NPG/GCE (c) in PBS (50 mM, pH 8.0), as well as GCE (b) and CBS/NPG/GCE (d) in PBS (50 mM, pH 8.0) containing 200 µM of Hcy and 200 µM of Cys. (B) The CV curves of the CBS/NPG/GCE at different scan rates ranging from 10 to 200 mV s−1 in PBS (50 mM, pH 8.0) containing 150 μM of sodium sulfide. The inset profile shows the peak current density versus scan rate. Full article ">Figure 4
(A) Effect of the working potential on the current density response of the NPG/GCE in PBS (50 mM, pH 8.0) containing 100 µM sodium sulfide. (B) Amperometric i-t responses of the CBS/NPG/GCE upon successive additions of Hcy to PBS (50 mM, pH 8.0) containing 200 µM of Cys at the working potential of −0.1 V. (C) The linear relationship between the current density (μA cm−2) and the Hcy concentration (μM). (D) Effect of common compounds in urine on Hcy detection. Full article ">Scheme 1
Schematic diagram of the construction for CBS/NPG/GCE electrode (A) and the principle of Hcy detection (B). Full article ">

11 pages, 406 KiB

Open AccessReview

Molecular Biomarkers in Neurological Diseases: Advances in Diagnosis and Prognosis

by Athena Myrou, Konstantinos Barmpagiannos, Aliki Ioakimidou and Christos Savopoulos

Int. J. Mol. Sci. 2025, 26(5), 2231; https://doi.org/10.3390/ijms26052231 (registering DOI) - 1 Mar 2025

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Abstract

Neurological diseases contribute significantly to disability and mortality, necessitating improved diagnostic and prognostic tools. Advances in molecular biomarkers at genomic, transcriptomic, epigenomic, and proteomic levels have facilitated early disease detection. Notably, neurofilament light chain (NfL) serves as a key biomarker of neurodegeneration, while liquid biopsy techniques enable non-invasive monitoring through exosomal tau, α-synuclein, and inflammatory markers. Artificial intelligence (AI) and multi-omics integration further enhance biomarker discovery, promoting precision medicine. A comprehensive literature review was conducted using PubMed, Scopus, and Web of Science to identify studies (2010–2024) on molecular biomarkers in neurodegenerative and neuroinflammatory disorders. Key findings on genomic mutations, transcriptomic signatures, epigenetic modifications, and protein-based biomarkers were analyzed. The findings highlight the potential of liquid biopsy and multi-omics approaches in improving diagnostic accuracy and therapeutic stratification. Genomic, transcriptomic, and proteomic markers demonstrate utility in early detection and disease monitoring. AI-driven analysis enhances biomarker discovery and clinical application. Despite advancements, challenges remain in biomarker validation, standardization, and clinical implementation. Large-scale longitudinal studies are essential to ensure reliability. AI-powered multi-omics analysis may accelerate biomarker application, ultimately improving patient outcomes in neurological diseases. Full article

(This article belongs to the Section Molecular Neurobiology)

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Contribution of different biomarkers in liquid biopsy. Full article ">

14 pages, 2970 KiB

Open AccessArticle

Disorders of Iron Metabolism: A “Sharp Edge” of Deoxynivalenol-Induced Hepatotoxicity

by Haoyue Guan, Yujing Cui, Zixuan Hua, Youtian Deng, Huidan Deng and Junliang Deng

Metabolites 2025, 15(3), 165; https://doi.org/10.3390/metabo15030165 (registering DOI) - 1 Mar 2025

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Abstract

Background/Objectives: Deoxynivalenol (DON), known as vomitoxin, is one of the most common mycotoxins produced by Fusarium graminearum, with high detection rates in feed worldwide. Ferroptosis is a novel mode of cell death characterized by lipid peroxidation and the accumulation of reactive oxygen species. Although it has been demonstrated that DON can induce ferroptosis in the liver, the specific mechanisms and pathways are still unknown. The aim of this experiment was to investigate that DON can induce iron metabolism disorders in the livers of mice, thereby triggering ferroptosis and causing toxic damage to the liver. Methods: Male C57 mice were treated with DON at a 5 mg/kg BW concentration as an in vivo model. After sampling, organ coefficient monitoring, liver function test, histopathological analysis, liver Fe²⁺ content test, and oxidative stress-related indexes were performed. The mRNA and protein expression of Nrf2 and its downstream genes were also detected using a series of methods including quantitative real-time PCR, immunofluorescence double-labeling, and Western blotting analysis. Results: DON can cause damage to the liver of a mouse. Specifically, we found that mouse livers in the DON group exhibited pathological damage in cell necrosis, inflammatory infiltration, cytoplasmic vacuolization, elevated relative liver weight, and significant changes in liver function indexes. Meanwhile, the substantial reduction in the levels of glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) in the DON group indicated that DON also caused oxidative stress in the liver. Notably, DON exposure increased the levels of Fe²⁺ and Malondialdehyde (MDA) in the liver, which provides strong evidence for the occurrence of iron metabolism and ferroptosis disorders. Most importantly, mRNA and protein expression of Nrf2, an important pathway for iron metabolism and ferroptosis, along with its downstream genes, heme oxygenase (HO-1), quinone oxidoreductase (NQO1), glutathione peroxidase (GPX4), and solute carrier gene (SLC7a11), were significantly inhibited in the DON group. Conclusions: Based on our results, the Nrf2 pathway is closely associated with DON-induced iron metabolism disorders and ferroptosis in mouse livers, suggesting that maintaining hepatic iron homeostasis and activating the Nrf2 pathway may be a potential target for mitigating DON hepatotoxicity in the future. Full article

(This article belongs to the Special Issue Animal Nutritional Metabolism and Toxicosis Disease)

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Assessment of liver injury in mice. (A) Visceral coefficients of the control and DON groups (n = 12, values represent ± SEMs). (B) Detection of Fe2+ content in mouse liver tissue (n = 12, values represent ± SEMs). (C) Assessment of mouse liver function-related indexes, including ALB, ALP, ALT, AST, GLB, and T-bil (n = 12, values represent ± SEMs). (D) Histopathological analysis of H&E-stained mouse livers (magnification ×20, bar = 50 μm). Black arrows are inflammatory cell infiltration, yellow arrows are cell necrosis, and green arrows are cytoplasmic vacuolization. No obvious lesions were seen in the control group. * (p < 0.05) vs. Control, *** (p < 0.001) vs. Control. Full article ">Figure 2
Changes in oxidative stress-related indices in mouse liver. Activities of SOD, GSH, T-AOC, MDA, and CAT in mouse liver cells (n = 12, values represent ± SEMs), * (p < 0.05) vs. Control, ** (p < 0.01) vs. Control. Full article ">Figure 3
mRNA expression levels of the ferroptosis signature pathway and Nrf2 and its downstream pathways in mouse livers (n = 12, values represent ± SEMs), ** (p < 0.01) vs. Control, *** (p < 0.001) vs. Control. Full article ">Figure 4
Immunofluorescence double-staining results of the signature pathway of ferroptosis as well as Nrf2 and its downstream pathway in mouse livers (magnification ×10, bar = 100 μm). (A) Diagram of double-staining results of GPX4 (red) + COX-2 (green) in mouse livers. (B) Double-staining results of mouse liver NQO1 (red) + HO-1 (green). (C) Diagram of double-staining results of mouse liver Nrf2 (red) + SLC7a11 (green). (D) Immunofluorescence staining fluorescence intensity number values (n = 3, values represent ± SEMs), * (p < 0.05) vs. Control, ** (p < 0.01) vs. Control. Full article ">Figure 5
Results of immunoblotting analysis of ferroptosis signature pathways as well as Nrf2 and its downstream pathways in mouse livers. (A) Nrf2, NQO1, HO-1, GPX4, SLC7a11, COX-2, and β-actin protein blot bands. (B) Nrf2, NQO1, HO-1, GPX4, SLC7a11, and COX-2 protein expression levels (n = 3, values represent ± SEMs), * (p < 0.05) vs. Control, ** (p < 0.01) vs. Control. Full article ">

19 pages, 5049 KiB

Open AccessArticle

Graphene-Based Far-Infrared Therapy Promotes Adipose Tissue Thermogenesis and UCP1 Activation to Combat Obesity in Mice

by Jinshui Zhang, Shuo Li, Xin Cheng, Xiaocui Tan, Yingxian Shi, Guixin Su, Yulong Huang, Yang Zhang, Rui Xue, Jingcao Li, Qiongyin Fan, Huajin Dong, Yun Deng and Youzhi Zhang

Int. J. Mol. Sci. 2025, 26(5), 2225; https://doi.org/10.3390/ijms26052225 (registering DOI) - 28 Feb 2025

Viewed by 213

Abstract

Hyperthermia (HT) has broad potential for disease treatment and health maintenance. Previous studies have shown that far-infrared rays (FIRs) at 8–10 μm can potentially reduce inflammation, oxidative stress, and gut microbiota imbalance. However, the effects of FIR HT on energy metabolism require further investigation. To investigate the effects of graphene-FIR HT therapy on diet-induced obesity and their regulatory mechanisms in energy metabolism disorders. After 8 weeks of hyperthermia, mice fed standard chow or a high-fat diet (HFD) underwent body composition analysis. Energy expenditure was measured using metabolic cages. The protein changes in adipose tissue were detected by molecular technology. Graphene-FIR therapy effectively mitigated body fat accumulation, improved dyslipidemia, and impaired liver function while enhancing insulin sensitivity. Furthermore, graphene-FIR therapy increased V_O2, V_CO2, and EE levels in HFD mice to exhibit enhanced metabolic activity. The therapy activated the AMPK/PGC-1α/SIRT1 pathway in adipose tissue, increasing the expression of uncoupling protein 1 (UCP1) and glucose transporter protein four (GLUT4), activating the thermogenic program in adipose tissue, and improving energy metabolism disorder in HFD mice. In short, graphene-FIR therapy represents a comprehensive approach to improving the metabolic health of HFD mice. Full article

(This article belongs to the Section Molecular Endocrinology and Metabolism)

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Figure 1

Figure 1
Specific FIR generated by graphene-based devices. (A) SEM image of graphene film; (B) Raman spectrum of the graphene FIR; (C) SEM image of carbon fiber film; (D) Raman spectrum of the carbon fiber FIR; (E) FIR emission spectra of graphene and carbon fiber (graphene: 30 W, carbon fiber: 30 W); (F) the absorption peak of the human body surface was approximately 8.0 µm; (G) the FIR emission peak of the graphene was approximately 8.03 µm, and the carbon fiber was approximately 7.685 µm. Full article ">Figure 2
Graphene-FIR therapy induces thermogenesis and increases blood flow in vivo. (A) Schematic representation of thermal imaging of human supraclavicular area and temperature changes before or after Gra-FIR therapy. Data are displayed as mean ± SEM; n = 10, * p < 0.05, ** p < 0.01 VS baseline. The selected part of the black box is the temperature detection area. (B,C) Schematic diagram of thermal imaging and statistical analysis of temperature changes in mice after Gra-FIR or Car-FIR. (D–F) A schematic diagram of rectal temperature measurement in mice and statistical analysis of rectal temperature changes before and after the Gra-FIR or Car-FIR therapy. (G–I) Representative images and statistical analysis of blood perfusion in BAT and iWAT of mice after Gra-FIR or Car-FIR therapy. The site selected by the black circle box was the blood flow detection area. Data are displayed as mean ± SEM; n = 7, ** p < 0.01 VS NC group; ## p < 0.01 VS Gra-FIR group. Full article ">Figure 3
Graphene-FIR therapy can improve HFD-induced weight gain. (A) Schematic of the experimental design. (B) Schematic diagram of the FIR device. (C) Line chart of mice body weight in each group (n = 7–10). (D) The total body fat and mass content of each group (n = 6). (E) Representative images of liver, BAT, and iWAT in each group. (F) Organ weight in each group (n = 7–10). (G) HE staining of liver, BAT, and iWAT (200X; n = 3). Scale-bar: 50 μm. Data are displayed as mean ± SEM. * p < 0.05, ** p < 0.01 VS NC group; # p < 0.05, ## p < 0.01 VS HFD group. Full article ">Figure 4
Graphene-FIR therapy can improve dyslipidemia and insulin resistance in HFD mice. (A–F) The serum levels of TG, TC, LDL-C, HDL-C, ALT, and AST. (G,H) GTT and area under the curve. (I,J) ITT and area under the curve. (K) Fasting blood glucose levels. (L–N) Serum levels of leptin, insulin, and adiponectin. (O) HOMA-IR index. Data are displayed as mean ± SEM; n = 6–10, * p < 0.05, ** p < 0.01; # p < 0.05, ## p < 0.01 VS HFD group. Full article ">Figure 5
Graphene-FIR therapy has no impact on energy intake but increases EE in HFD mice, (A,B) Daily food intake of each group. (C) Energy intake of mice. (D,E) O2 consumption (VO2). (F,G) CO2 release (VCO2). (H,I) Energy expenditure. (J,K) Respiratory exchange rate (RER). (L) Core temperature. (M–P) Predicted metabolic rate (MR) either in light or dark. (Q,R) Activity distance within 24 h. (S,T) Number of activities within 24 h. Data are displayed as mean ± SEM; n = 6–10, * p < 0.05, ** p < 0.01, ns represents no significant difference. Full article ">Figure 6
Graphene-FIR therapy could activate adipose tissue thermogenesis and activate the AMPK/SIRT1/PGC-1α pathway in HFD mice. (A–E) Representative protein bands and relative protein expressions of AMPK, PGC-1α, SIRT1, UCP1, and GLUT4. β-actin was used as a loading control. (F–J) The mRNA expression of thermogenic genes. Data are displayed as mean ± SEM; n = 6, * p < 0.05, ** p < 0.01. Full article ">

21 pages, 4529 KiB

Open AccessArticle

Enhancing Physicochemical and Piezoelectric Properties of Eggshell Membrane Proteins by Ultrasonic-Assisted Enzymes for Food and Sensor Applications

by Xinhua Liang, Honglian Cong, Gaoming Jiang and Haijun He

Int. J. Mol. Sci. 2025, 26(5), 2190; https://doi.org/10.3390/ijms26052190 - 28 Feb 2025

Viewed by 83

Abstract

This research sought to explore the impact of ultrasonic pretreatment on the physicochemical characteristics of proteins derived from eggshell membranes through enzymatic extraction. Response surface methodology (RSM) and Box-Behnken design were employed to identify the ideal conditions for the extraction process. The optimal parameters determined were enzyme usage at 4.2%, pH level at 2.4, a solid-to-solvent ratio of 1:20 g/mL, and an extraction time of 21.5 h. The eggshell membrane was pretreated by ultrasound before pepsin hydrolysis under optimized conditions. The findings indicated that the hydrolyzed products subjected to ultrasonic pretreatment exhibited enhanced solubility, surface hydrophobicity, water and oil retention, foaming characteristics, and emulsifying ability compared to the untreated hydrolyzed products. Furthermore, the piezoelectric properties of the protein with ultrasonic pretreatment were also significantly improved. Additionally, the protein-based piezoelectric device displayed excellent sensing performance and was successfully applied for human motion detection and precise identification of different pressure positions. These findings indicate that ultrasound has great potential to improve the physicochemical quality of eggshell membrane proteins, providing a theoretical basis and research approach for food protein modification and the preparation of green electronic devices. Full article

(This article belongs to the Section Physical Chemistry and Chemical Physics)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
The three-dimensional (3D) response surface graphs and two-dimensional (2D) contour plots exhibiting the interactive effects of enzyme usage (a); pH (b); solid-to-solvent ratio (c); and time (d) on EY. Interaction between (a,a′) enzyme usage and pH; (b,b′) enzyme usage and solid-to-solvent ratio; (c,c′) enzyme usage and time; (d,d′) pH and solid-to-solvent ratio; (e,e′) pH and time; (f,f′) solid-to-solvent ratio and time. Full article ">Figure 2
The functional properties of different samples. (a) solubility and H0; (b) WHC and OHC; (c) FC and FS; (d) EAI and ESI. Mean value ± standard deviations of three independent experiments were shown (n = 3). Different letters labeled mean significant difference (p < 0.05). Full article ">Figure 3
(a) FTIR spectra of ESMP, PSC, and UPSC; (b) FTIR of the range of 650 to 2200 cm−1 for the samples; (c–e) FTIR amide I regions of ESMP, PSC, UPSC, and deconvolution of amide I bands into individual peaks; (f) The secondary structure contents of ESMP, PSC, and UPSC. Full article ">Figure 4
SEM image of different samples. (a,b) ESM; (c) ESM with enzyme treatment; (d) ESM with ultrasonic-assisted enzyme treatment; (e) PSC, and (f) UPSC. Full article ">Figure 5
Mechanical properties of different samples. (a) Stress-strain curves; (b) The maximum tensile stress; (c) Breaking strain; (d) Young’s modulus of different samples. Mean ± SD of three independent experiments are shown (n = 3). Full article ">Figure 6
Piezoelectric performance of all samples. (a) The voltages and (b) currents of different samples; (c) Piezoelectric voltages and (d) currents at 1.5 Hz under different applied forces (5–40 N); (e) Piezoelectric voltages and (f) currents under 10 N at different frequencies (0.3–1.5 Hz); (g,h) Stability test of the device under 3000 working cycles. Full article ">Figure 7
Applications of the device in detecting various human motions. (a) Diagram of the test on the human body; (b–e) The signals corresponding to finger bending, wrist bending, elbow bending, and knee bending; (f) Schematic diagram of a 4 × 4 pixel sensing array; (g) The voltages corresponding to spatial pressure distributions. Full article ">

12 pages, 473 KiB

Open AccessArticle

Novel Methods for the Analysis of Serum NET Remnants: Evaluation in Patients with Severe COVID-19

by Francesco Pisani, Caterina Porciani, Cristina Croia, Valentina Pucino, Agostino Virdis, Ilaria Puxeddu, Paola Migliorini and Federico Pratesi

Int. J. Mol. Sci. 2025, 26(5), 2221; https://doi.org/10.3390/ijms26052221 - 28 Feb 2025

Viewed by 114

Abstract

Neutrophil extracellular traps (NETs) are web-like structures composed of chromatin and proteins from neutrophil granules. Several studies highlight the heterogeneity of NETs, underscoring the challenges associated with their detection. In patients with COVID-19, high levels of NET fragments, called NET remnants, are detected in the circulation but also in alveoli and bronchioles. NET remnants are usually measured as complexes of DNA and myeloperoxidase (DNA−MPO). Taking advantage of proteomic data on NET composition, we developed new solid-phase assays to detect NET remnants, measuring complexes of DNA with alpha enolase (DNA−eno) or calprotectin (DNA−cal). The two assays were compared with the DNA−MPO test for the detection of in vitro-generated NET and serum NET remnants; all of them showed similar sensitivity in the detection of in vitro-generated NET. In an analysis of 40 patients with severe COVID-19 and 25 healthy subjects, the results of the three assays were highly correlated, and all detected significantly higher levels of NET remnants in patient sera. Moreover, the level of NET remnants correlated with impaired gas exchange and increased with the progressive decline of pulmonary function. The proposed assays thus represent a novel tool with which to evaluate NETosis; using antibodies to different NET constituents may allow their fingerprinting in different disorders. Full article

(This article belongs to the Section Molecular Immunology)

12 pages, 1067 KiB

Open AccessReview

The Dual Role of cGAS-STING Signaling in COVID-19: Implications for Therapy

by Daniele Castro di Flora, João Paulo Zanardini Lara, Aline Dionizio and Marília Afonso Rabelo Buzalaf

Cells 2025, 14(5), 362; https://doi.org/10.3390/cells14050362 (registering DOI) - 28 Feb 2025

Viewed by 128

Abstract

The progression of COVID-19 involves a sophisticated and intricate interplay between the SARS-CoV-2 virus and the host’s immune response. The immune system employs both innate and adaptive mechanisms to combat infection. Innate immunity initiates the release of interferons (IFNs) and pro-inflammatory cytokines, while the adaptive immune response involves CD4+ Th lymphocytes, B lymphocytes, and CD8+ Tc cells. Pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPS) and damage-associated molecular patterns (DAMPs), activating the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) signaling pathway, a crucial component of the innate immune response to SARS-CoV-2. This pathway fulfills a dual function during infection. In the early phase of infection, the virus can suppress cGAS-STING signaling to avoid immune detection. However, in the late stages, the activation of this pathway may trigger excessive inflammation and tissue damage, exacerbating disease severity. Modulating the cGAS-STING pathway, whether through agonists like dimeric amidobenzimidazole (diABZI) or inhibitors targeting viral proteins, such as 3CLpro, for example, offers a promising approach for personalized therapy to control the immune response and mitigate severe inflammation, ultimately improving clinical outcomes in patients with severe COVID-19. Full article

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Figure 1
Innate immune response activation by RNA and DNA sensors. MAVS and STING are activated by viral RNA or cytosolic DNA, activating kinases IKK and TBK1. These, in turn, phosphorylate the adaptor proteins (MAVS or STING), which recruit IRF3, allowing its phosphorylation by TBK1. Phosphorylated IRF3 suffers dimerization and induces IFN in the nucleus. Non-continuous arrows indicate recruitment and activation. Modified from Liu et al. [<a href="#B12-cells-14-00362" class="html-bibr">12</a>]. Reproduced with permission. Full article ">Figure 2
The dual role of cGAS-STING signaling in COVID-19 and potential therapeutic targeting. In the early phase of infection, the virus can suppress cGAS-STING signaling to evade immune detection. Thus, cGAS-STING agonists are beneficial at this stage in order to reduce viral replication and control infection. However, in the late stages, activation of this pathway can lead to excessive inflammation and tissue damage, exacerbating disease severity. In this case, therapy might antagonize cGAS-STING. Modified from Elahi et al. Downward arrow indicates reduction [<a href="#B55-cells-14-00362" class="html-bibr">55</a>]. Full article ">

24 pages, 1273 KiB

Open AccessReview

Recent Advances in Electrochemical Biosensors for Neurodegenerative Disease Biomarkers

by Mingyu Bae, Nayoung Kim, Euni Cho, Taek Lee and Jin-Ho Lee

Biosensors 2025, 15(3), 151; https://doi.org/10.3390/bios15030151 - 28 Feb 2025

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Abstract

Neurodegenerative diseases, such as Parkinson’s disease (PD) and Alzheimer’s disease (AD), represent a growing global health challenge with overlapping biomarkers. Key biomarkers, including α-synucleins, amyloid-β, and Tau proteins, are critical for accurate detection but are often assessed using conventional methods like enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), which are invasive, costly, and time-intensive. Electrochemical biosensors have emerged as promising tools for biomarker detection due to their high sensitivity, rapid response, and potential for miniaturization. The integration of nanomaterials has further enhanced their performance, improving sensitivity, specificity, and practical application. To this end, this review provides a comprehensive overview of recent advances in electrochemical biosensors for detecting neurodegenerative disease biomarkers, highlighting their strengths, limitations, and future opportunities. By addressing the challenges of early diagnosis, this work aims to stimulate interdisciplinary innovation and improve clinical outcomes for neurodegenerative disease patients. Full article

(This article belongs to the Special Issue Novel Graphene-Based Biosensors for Biomedical Applications)

18 pages, 6647 KiB

Open AccessArticle

Genome-Wide Identification and Functional Characterization of the Glycosyltransferase 43 (GT43) Gene Family in Sorghum bicolor for Biofuel Development: A Comprehensive Study

by Rehana Rehana, Muhammad Anwar, Sarmad Frogh Arshad, Muhammad Usman and Imran Ahmad Khan

Processes 2025, 13(3), 709; https://doi.org/10.3390/pr13030709 (registering DOI) - 28 Feb 2025

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Abstract

Sorghum (Sorghum bicolor) is an essential bioenergy crop. Cellulosic and non-cellulosic polysaccharides, which can be transformed into biofuels, comprise most of its biomass. Many glycosyltransferases (GT) families, including GT43, are involved in the biosynthesis of xylan in plants’ primary and secondary cells. In this study, the GT43 gene family was identified, and its secondary structure and a three-dimensional (3D) model were constructed. Additionally, subcellular localization, detection of motifs, and analyses of its phylogenetic tree, physiochemical properties, protein–protein interaction network, gene structure, functional domain, gene duplication, Cis-acting elements, sequence logos, multiple sequence alignment, and gene expression profiles were performed based on RNA-sequence analyses. As a result, eleven members of the GT43 gene family were identified, and the phylogenetic tree of the GT43 gene family showed that all GT43 genes had evolutionary relationships with sorghum. Analyses of gene structure, motifs, sequence logos, and multiple sequence alignment showed that all members of the GT43 protein family were highly conserved. Subcellular localization showed all members of the GT43 protein family were localized in different compartments of sorghum. The secondary structure of the GT43 genes comprised different percentages of α-helices, random coils, β-turns, and extended strands. The tertiary structure model showed that all GT43 proteins had similar 3D structures. The results of the current study indicated that members of the GT43 gene family (Sobic.010G238800, Sobic.003G254700, and Sobic.001G409100) were highly expressed in internodes of the sorghum plant, based on RNA-Sequencing. The framework used in this study will be valuable for advancing research aligned with modern technology requirements and for enhancing understanding of the relationships among GT43 genes in Sorghum bicolor. Full article

(This article belongs to the Special Issue Biomass, Coal and Other Solid Fuels: Thermal Conversion, Emission and Pollutant Control)

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18 pages, 874 KiB

Open AccessArticle

Identification of Subtle Differences in the Physiological Quality of Commercial Soybean Seed Lots Using Shotgun Proteomics During Germination

by Fellipe Ramos Sampaio, Irma Yuliana Mora-Ocampo, Fredy Davi Albuquerque Silva, Kevein Ruas Oliveira, Carlos Priminho Pirovani and Rafael Marani Barbosa

Agronomy 2025, 15(3), 609; https://doi.org/10.3390/agronomy15030609 - 28 Feb 2025

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Abstract

Soybean seeds with similar germination rates may exhibit subtle differences in physiological quality, influencing field performance and storage longevity. This study used a shotgun proteomics approach to characterize the proteomic profile of two commercial soybean seed lots (higher- and lower-quality) during germination, aiming to identify biomarkers associated with vigor and deterioration. Proteins were analyzed across three germination phases: imbibition (Phase I, 0.5 h), metabolic activation (Phase II, 20 h), and radicle protrusion (Phase III, 51 h). A total of 777 proteins were identified, and of these differentially abundant proteins (DAPs), the following totals were detected: 12 in Phase I, 17 in Phase II, and 28 in Phase III. In Phase I, ribosomal proteins were more abundant in high-quality seeds, indicating efficient translation and preparation for germination. Conversely, in Phase III, low-quality seeds showed increased levels of storage proteins and stress-response proteins, including alcohol dehydrogenase (ADH), heat shock proteins, and annexins, reflecting delayed germination and more deterioration. These findings highlight the dynamic nature of protein expression during germination and demonstrate the potential of proteomics to detect subtle differences in physiological quality. The identified biomarkers provide insights for seed quality assessment and offer practical applications for improving classification and management of commercial soybean seed lots. Full article

(This article belongs to the Section Plant-Crop Biology and Biochemistry)

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Figure 1
Tetrazolium test in soybean cultivar M8808 seed lots, quantifying viability, vigor, and moisture, mechanical and stink bug damage in seeds. (*) t test (p < 0.01). Full article ">Figure 2
The imbibition curve of two soybean cultivar M8808 seed lots (B and E). Full article ">Figure 3
Proteins identified by mass spectrometry. he number of proteins identified in soybean seeds during the germination stages, which went through the periods of false discovery rate (FDR) < 1%, Score > 5, and Scored Peak Intensity (SPI) > 60%. Full article ">

20 pages, 5109 KiB

Open AccessArticle

Unveiling the Potential Role of Dhurrin in Sorghum During Infection by the Head Smut Pathogen Sporisorium reilianum f. sp. reilianum

by Coumba Fall, Seunghyun Lim, Ezekiel Ahn, Sunchung Park, Louis K. Prom and Clint W. Magill

Plants 2025, 14(5), 740; https://doi.org/10.3390/plants14050740 - 28 Feb 2025

Viewed by 185

Abstract

The cyanogenic glucoside dhurrin is found in sorghum and has been reported for its role in defense against biotic and abiotic stresses, both involving hydrogen cyanide (HCN) release. The fungus Sporisorium reilianum f. sp. reilianum (SRS) causes sorghum head smut and the infection occurs at the seedling stage, later resulting in panicle loss. Here, the focus was to determine the role of dhurrin in sorghum’s reaction against SRS infection. We investigated the genomic basis of HCN potential (HCNp) variation and its relationship with seedlings’ response to SRS inoculation, along with other sorghum traits, and the expression of dhurrin biosynthetic genes in SRS-inoculated young sorghum. Genome-wide association studies (GWAS) using HCNp scores showed significant single nucleotide polymorphisms (SNPs) on chromosomes harboring the dhurrin biosynthetic and catabolic genes but not in proximity. Significant hits were also detected in or near genes encoding proteins involved in plant defense/resistance against biotic stresses. Correlation analyses showed a strong positive relationship between average HCNp scores and latent period in SRS-inoculated sorghum seedlings. RT-qPCR revealed that the dhurrin biosynthetic genes were upregulated in the leaves of the head smut resistant line BTx635 up to two days after SRS inoculation. Our results suggest the involvement of dhurrin in sorghum’s protection against SRS. Full article

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17 pages, 6111 KiB

Open AccessArticle

Botanical Origin, Phytochemical Profile, and Antioxidant Activity of Bee Pollen from the Mila Region, Algeria

by Nassiba Boulfous, Hakima Belattar, Roberto Ambra, Gianni Pastore and Asma Ghorab

Antioxidants 2025, 14(3), 291; https://doi.org/10.3390/antiox14030291 - 28 Feb 2025

Viewed by 108

Abstract

Bee pollen is a complex mixture of floral pollen, and nectar fused substances from bee saliva. It is well known for its high content of proteins, carbohydrates, lipids, vitamins, and phenolic compounds, among various other physiologically active components. Its composition varies significantly depending on its botanical sources and environmental conditions. This study investigates the relationship between the botanical origins, chemical compositions, and antioxidant activities of 15 bee pollen samples collected from different areas in the Mila region of northeastern Algeria. The botanical origins were identified using a palynological method, categorizing 11 samples as monofloral and the rest as polyfloral. The total phenolic and flavonoid contents were measured, and their antioxidant capacities were evaluated through DPPH radical scavenging assay, reducing power assay (FRAP), and total antioxidant capacity (TAC). HPLC analysis was conducted to measure 17 phenolic compounds. The data indicated that the total phenolic content (TPC) and flavonoid content (TFC) ranged from 7.72 ± 0.29 to 23.49 ± 1.48 mg GAE/g and from 1.48 ± 0.00 to 5.57 ± 0.27 mg QE/g of pollen, respectively. The variations in the concentration of bioactive compounds among samples led to significant differences in their antioxidant activities: DPPH (IC₅₀: 1.12 ± 0.15 to 0.21 ± 0.00 mg/mL), FRAP (EC₅₀: 0.06 ± 0.00 to 0.29 ± 0.00 mg/mL), and TAC (262.17 ± 3.41 to 677.14 ± 12.81 EAA mg/100 g of bee pollen), with the most active samples being monofloral types from Cistus type and Brassica type. A strong correlation was observed between TPC, TFC, and antioxidant activity. Among the 17 tested compounds, only coumaric acid, rutin, myricetin, naringenin, resveratrol, and kaempferol were detected. In conclusion, both monofloral and polyfloral bee pollen samples represent a rich source of polyphenols with significant antioxidant potential. Full article

(This article belongs to the Special Issue Antioxidant and Antimicrobial Activity of Honey Bee Products—2nd Edition)

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Figure 1
Geographic distribution origin by municipality (in blue) of the bee pollen samples collected the Mila region (northeastern Algeria). Created with ArcGIS 10.8. Full article ">Figure 2
Microscopic appearance of the different pollen types found in Algerian pollen samples. (a) Brassica type. (b) Cistus type. (c) Hedysarum coronarium. (d) Erica sp. (e) Eucalyptus sp. (f) Apiaceae. (g) Pimpinella anisum. (h) Liliaceae. (i) Aster type. (j) Helianthus annuus. (k) Carduus type. (l) Taraxacum type. (m) Tamarix sp. (n) Rubus sp. (o) Quercus sp. (p) Olea europaea. Full article ">Figure 3
Chromatogram of the standards (A). As an example, the chromatogram of sample 9, zoomed with RT > 40 min (B). Full article ">Figure 4
Pearson Correlation shown by the parameters studied in bee pollen samples from different botanical origins. Full article ">

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