Regulation Of Defense Research Articles

Transcriptomic and Phenotypic Responses of CucumberTrichome Density to Silver Nitrate and Sodium Thiosulfate Application

Cucumber (Cucumis sativus L.) is one of the most widely cultivated crops worldwide and is valued for its nutritional, economic, and ecological benefits. The regulation of defense mechanisms against herbivores, along with osmotic loss and environmental regulation, is greatly affected by trichomes in cucumbers. In this study, we attempted to characterize trichomes and examined fruit physiological and transcriptome profiles by RNA sequencing in cucumber breeding lines 6101-4 and 5634-1 at three stages of fruit development through foliar application with a combination of silver nitrate (AgNO3) and sodium thiosulfate (Na2S2O3) in comparison to non-treated controls. Notable increases in the number of trichomes and altered forms were observed for both inbred cultivars 6101-4 and 5634-1 against foliar application of chemical substances. RNA-seq analysis was performed to identify differentially expressed genes (DEGs) involved in multiple pathways in cucumber trichome formation. The enrichment of differentially expressed transcripts showed that foliar application upregulated the expression of many stress-responsive and trichome-associated genes including plant hormone signal transduction, sesquiterpenoid and triterpenoid biosynthesis, and the mitogen-activated protein kinase (MAPK) signaling pathway. The dominant regulatory genes, such as allene oxide synthase (AOS) and MYB1R1 transcription factor, exhibited significant modulations in their expression in response to chemical application. The RNA-seq results were further confirmed by RT-PCR-based analysis, which revealed that after chemical application, the dominant regulatory genes, such as allene oxide synthase (AOS), PTB 19, MYB1R1, bHLH62-like, MADS-box transcription factor, and salicylic acid-binding protein 2-like, were differentially expressed, implying that these DEGs involved in multiple pathways are involved the positive regulation of the initiation and development of trichomes in C. sativus. A comparison of trichome biology and associated gene expression regulation in other plant species has shown that silver nitrate (AgNO3) and sodium thiosulfate (Na2S2O3) are also responsible for hormonal and signaling pathway regulation. This study improves our knowledge of the molecular mechanisms involved in C. sativus trichome development. It also emphasizes the possibility of utilizing chemical composition to modulate C. sativus trichome-related characteristics of C. sativus, leading to the improvement of plant defense mechanisms as well as environmental adaptation.

Genetically-encoded targeted protein degradation technology to remove endogenous condensation-prone proteins and improve crop performance

Effective modulation of gene expression in plants is achievable through tools like CRISPR and RNA interference, yet methods for directly modifying endogenous proteins remain lacking. Here, we identify the E3 ubiquitin ligase E3TCD1 and develope a Targeted Condensation-prone-protein Degradation (TCD) strategy. The X–E3TCD1 fusion protein acts as a genetically engineered degrader, selectively targeting endogenous proteins prone to condensation. For example, a transgenic E3TCD1 fusion with Teosinte branched 1 (TB1) degrades the native TB1 protein, resulting in increased tiller numbers in rice. Additionally, conditional degradation of the negative defense regulator Early Flowering 3 via a pathogen-responsive ProTBF1-uORFsTBF1 cassette enhances rice blast resistance without affecting flowering time in the absence of pathogen. Unlike prevailing targeted protein degradation strategies, the TCD system does not rely on small molecules, antibodies, or genetic knock-in fusion tags, demonstrating its promise as a transgene-based approach for optimizing crop performance.

CFTR as a therapeutic target for severe lung infection.

Lung infection is one of the leading causes of morbidity and mortality worldwide. Even with appropriate antibiotic and antiviral treatment, mortality in hospitalized patients often exceeds 10%, highlighting the need for the development of new therapeutic strategies. Of late, cystic fibrosis transmembrane conductance regulator (CFTR) is - in addition to its well-established roles in the lung airway and extrapulmonary organs - increasingly recognized as a key regulator of alveolar homeostasis and defense. In the alveolar epithelium, CFTR mediates alveolar fluid secretion and liquid homeostasis; in the microvascular endothelium, CFTR maintains vascular barrier function. CFTR also contributes to alveolar immunity. Yet, in lung infection, diverse molecular mechanisms reduce CFTR abundance and otherwise impair its function, promoting alveolar inflammation, edema, and cell death. Preservation or restoration of CFTR function by CFTR modulator drugs thus presents a promising avenue to combat lung infection in a pathogen-independent manner.

Cu2+ mediates the oxidation of the transcription factor MscA to regulate the antioxidant defense of mycobacteria.

Copper (Cu), a trace element with redox activity, is both essential and toxic to living organisms. Its redox properties make it a cofactor for a variety of proteins, but it also causes oxidative stress, hence the need to maintain intracellular copper homeostasis. However, the role of copperin the regulation of antioxidant defense in bacteria remains unclear, and the involved transcription factors remain to be explored. In this study, we identified a novel transcription factor, MscA, that responded directly to Cu2+ to regulate the antioxidant defense of mycobacteria. Cu2+ directly bound to MscA to mediate oxidation and inhibit the DNA binding activity of MscA, subsequently downregulating the expression of antioxidant gene cluster to increase the accumulation of reactive oxygen speciesin mycobacteria, ultimately leading to oxidative damage to mycobacteria. Therefore, we firstly reported that the Cu2+ responsive transcription factor regulated the antioxidant defense in bacteria. This finding firstly and directly links the function of Cu2+ to the antioxidant defense of bacteria, and provides a new insight into bacterial antioxidant defense.

Functional Analysis of Durum Wheat GASA1 Protein as a Biotechnological Alternative Against Plant Fungal Pathogens and a Positive Regulator of Biotic Stress Defense.

Plants are frequently challenged by a variety of microorganisms. To protect themselves against harmful invaders, they have evolved highly effective defense mechanisms, including the synthesis of numerous types of antimicrobial peptides (AMPs). Snakins are such compounds, encoded by the GASA (Gibberellic Acid-Stimulated Arabidopsis) gene family, and are involved in the response to biotic and abiotic stress. Here, we examined the function of the newly identified TdGASA1 gene and its encoded protein in Triticum durum subjected to different biotic stress-related simulants, such as mechanical injury, methyl jasmonate (MeJA), indole-3-acetic acid (IAA), salicylic acid (SA), hydrogen peroxide (H2O2), as well as infection with pathogenic fungi Fusarium graminearum and Aspergillus niger. We found that in durum wheat, TdGASA1 transcripts were markedly increased in response to these stress simulants. Isolated and purified TdGASA1 protein exhibited significant antifungal activity in the growth inhibition test conducted on eight species of pathogenic fungi on solid and liquid media. Transgenic Arabidopsis lines overexpressing TdGASA1 obtained in this study showed higher tolerance to detrimental effects of H2O2, MeJA, and ABA treatment. In addition, these lines exhibited resistance to Fusarium graminearum and Aspergillus niger, which was linked to a marked increase in antioxidant activity in the leaves under stress conditions. This resistance was correlated with the upregulation of pathogenesis-related genes (AtPDF1.2a, AtERF1, AtVSP2, AtMYC2, AtPR1, AtACS6, AtETR1, and AtLOX2) in the transgenic lines. Overall, our results indicate that TdGASA1 gene and its encoded protein respond ubiquitously to a range of biotic stimuli and seem to be crucial for the basal resistance of plants against pathogenic fungi. This gene could therefore be a valuable target for genetic engineering to enhance wheat resistance to biotic stress.

Role of Micronutrients in Preventing Chronic Diseases: A Review

Micronutrients, including vitamins and minerals, are essential nutrients required in small quantities but play a critical role in the prevention and management of chronic diseases. These nutrients are integral to various physiological processes, such as immune function, antioxidant defense, enzyme activity, and gene regulation. Deficiencies in key micronutrients, such as Vitamin D, iron, zinc, and folate, have been linked to increased risks of cardiovascular diseases, diabetes, neurodegenerative disorders, and cancer. Recent research highlights the synergistic effects of micronutrients, where combined nutrient intake enhances bioavailability and effectiveness, emphasizing the need for diverse dietary patterns like the Mediterranean diet. Advances in nutrigenomics and personalized nutrition have shown promise in tailoring dietary interventions based on individual genetic profiles, optimizing the preventive impact of micronutrients. The development of functional foods and biofortification of crops presents sustainable solutions to combat micronutrient deficiencies in resource-limited populations. Emerging trends in supplementation, such as high-dose Vitamin C in cancer therapy and magnesium in managing metabolic syndrome, indicate potential therapeutic roles for micronutrients beyond basic nutrition. However, challenges remain, particularly in assessing nutrient bioavailability and addressing confounding factors in epidemiological studies. Ethical considerations in clinical trials and the limitations of current research methodologies call for more comprehensive, long-term studies to better understand the complex interactions between micronutrients and chronic disease prevention. Sustainable agricultural practices and policies focused on enhancing the micronutrient content of foods are important for addressing global nutrition challenges. As research progresses, leveraging new technologies for more accurate nutrient assessment and targeted interventions will be essential in reducing the global burden of chronic diseases. By integrating scientific advancements with public health strategies, there is potential to improve population health outcomes through optimized micronutrient intake. This comprehensive approach highlights the importance of focusing on both individual dietary interventions and broader food system changes to effectively harness the benefits of micronutrients in chronic disease prevention and management.

MiR472 Deficiency Enhances Arabidopsis thaliana Defense Without Reducing Seed Production.

After having co-existed in plant genomes for at least 200 million years, the products of microRNA (miRNA) and nucleotide-binding leucine-rich repeat protein (NLR) genes formed a regulatory relationship in the common ancestor of modern gymnosperms and angiosperms. From then on, DNA polymorphisms occurring at miRNA target sequences within NLR transcripts must have been compensated by mutations in the corresponding mature miRNA sequence. The potential evolutionary advantage of such regulation remains largely unknown and might be related to two nonexclusive scenarios: (i) miRNA-dependent regulation of NLR levels might prevent defense mis-activation with negative effects on plant growth and reproduction or (ii) reduction of active miRNA levels in response to pathogen-derived molecules (pathogen-associated molecular patterns [PAMPs] and silencing suppressors) might rapidly release otherwise silent NLR transcripts for rapid translation and thereby enhance defense. Here, we used Arabidopsis thaliana plants deficient for miR472 function to study the impact of releasing its NLR targets on plant growth and reproduction and on defense against the fungal pathogen Plectosphaerella cucumerina. We show that miR472 regulation has a dual role, participating both in the tight regulation of plant defense and growth. MIM472 lines, with reduced active miR472, are more resistant to pathogens and, correlatively, have reduced relative growth compared with wild-type plants, although the end of their reproductive phase is delayed, exhibiting higher adult biomass and similar seed yield as the wild-type. Our study highlights how negative consequences of defense activation might be compensated by changes in phenology and that miR472 reduction is an integral part of plant defense responses. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Negative Regulators of the Basal Defence WRKY7, WRKY11 and WRKY17 Modulate the Jasmonic Acid Pathway and an Alternative Splicing Regulatory Network in Response to Pseudomonas syringae in Arabidopsis thaliana

ABSTRACTIn Arabidopsis thaliana, the transcription factors WRKY7, WRKY11 and WRKY17 act as negative defence regulators against Pseudomonas syringae pv. tomato (Pst) DC3000. However, their coordinated regulation of gene expression has yet to be fully explored. In this study, we conducted a transcriptomic analysis on the triple mutant wrky7/11/17 in response to Pst DC3000 at 0, 3 and 24 h post‐inoculation (hpi). Our results suggest that at early infection stages (0 and 3 hpi), WRKY7, WRKY11 and WRKY17 significantly repress a group of genes involved in signal perception and transduction, including receptor‐like kinases. Furthermore, at later stages of interaction (24 hpi), these transcription factors induce genes related to the biosynthesis and signalling of the jasmonic acid (JA) pathway. Further infection experiments with Pst DC3000 in plants treated with methyl jasmonate (a JA analogue) and infections with Botrytis cinerea, a pathogen against which JA‐mediated responses are crucial for effective defence, support this proposal. Moreover, we analysed the role of WRKY7, WRKY11 and WRKY17 in alternative splicing regulation. A comparison between differentially expressed (DEG) and spliced (DAS) genes revealed that over 80% of DAS events do not occur in conjunction with overall changes in gene expression. Alternative splicing events were found in genes with functions in splicing and the JA pathway, such as ALY4, PRP40A, JAZ3 and JAZ10. These results suggest that WRKY7, WRKY11 and WRKY17 can also participate in this layer of gene expression regulation to modulate immunity negatively.

Multi-targeted olink proteomics analyses of cerebrospinal fluid from patients with aneurysmal subarachnoid hemorrhage

BackgroundThe complexity of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) may require the simultaneous analysis of variant types of protein biomarkers to describe it more accurately. In this study, we analyzed for the first time the alterations of cerebrospinal fluid (CSF) proteins in patients with aSAH by multi-targeted Olink proteomics, aiming to reveal the pathophysiology of DCI and provide insights into the diagnosis and treatment of aSAH.MethodsSix aSAH patients and six control patients were selected, and CSF samples were analyzed by Olink Proteomics (including 96-neurology panel and 96-inflammation panel) based on Proximity Extension Assay (PEA). Differentially expressed proteins (DEPs) were acquired and bioinformatics analysis was performed.ResultsPCA analysis revealed better intra- and inter-group reproducibility of CSF samples in the control and aSAH groups. 23 neurology-related and 31 inflammation-relevant differential proteins were identified. In the neurology panel, compared to controls, the up-regulated proteins in the CSF of SAH patients predominantly included macrophage scavenger receptor 1 (MSR1), siglec-1, siglec-9, cathepsin C (CTSC), cathepsin S (CTSS), etc. Meanwhile, in the inflammation group, the incremental proteins mainly contained interleukin-6 (IL-6), MCP-1, CXCL10, CXCL-9, TRAIL, etc. Cluster analysis exhibited significant differences in differential proteins between the two groups. GO function enrichment analysis hinted that the differential proteins pertinent to neurology in the CSF of SAH patients were mainly involved in the regulation of defense response, vesicle-mediated transport and regulation of immune response; while the differential proteins related to inflammation were largely connected with the cellular response to chemokine, response to chemokine and chemokine-mediated signaling pathway. Additionally, in the neurology panel, KEGG enrichment analysis indicated that the differential proteins were significantly enriched in the phagosome, apoptosis and microRNAs in cancer pathway. And in the inflammation panel, the differential proteins were mainly enriched in the chemokine signaling pathway, viral protein interaction with cytokine and cytokine receptor and toll-like receptor signaling pathway.ConclusionsThese identified differential proteins reveal unique pathophysiological characteristics secondary to aSAH. Further characterization of these proteins and aberrant pathways in future research could enable their application as potential therapeutic targets and biomarkers for DCI after aSAH.

ZmICE1a regulates the defence-storage trade-off in maize endosperm.

The endosperm of cereal grains feeds the entire world as a major food supply; however, little is known about its defence response during endosperm development. The Inducer of CBF Expression 1 (ICE1) is a well-known regulator of cold tolerance in plants. ICE1 has a monocot-specific homologue that is preferentially expressed in cereal endosperms but with an unclear regulatory function. Here we characterized the function of monocot-specific ZmICE1a, which is expressed in the entire endosperm, with a predominant expression in its peripheral regions, including the aleurone layer, subaleurone layer and basal endosperm transfer layer in maize (Zea mays). Loss of function of ZmICE1a reduced starch content and kernel weight. RNA sequencing and CUT&Tag-seq analyses revealed that ZmICE1a positively regulates genes in starch synthesis while negatively regulating genes in aleurone layer-specific defence and the synthesis of indole-3-acetic acid and jasmonic acid (JA). Exogenous indole-3-acetic acid and JA both induce the expression of numerous defence genes, which show distinct spatial-specific expression in the basal endosperm transfer layer and subaleurone layer, respectively. Moreover, we dissected a JA-ZmJAZ9-ZmICE1a-MPI signalling axis involved in JA-mediated defence regulation. Overall, our study revealed ZmICE1a as a key regulator of endosperm defence response and a coordinator of the defence-storage trade-off in endosperm development.

Alteration in microbes changed the contents of oviposition-deterrent pheromones on the Spodoptera litura egg surface.

Microorganisms symbiotic with insects, whether permanently or temporarily, play a crucial role in the nutrition, development, reproduction, defence, and metamorphosis regulation. In some Lepidoptera, oviposition-deterrent pheromones (ODPs) on egg surface were used by pregnant females to modify the behaviour of conspecifics to avoid excessive competition for limited resources. In this study, we constructed four different Spodoptera litura groups, including, OH, OA, SH, and OA, which either feed on different hosts or grow in different environments. The 16S rDNA libraries of microbes from the egg surface of the four groups were constructed and sequenced. According to alpha and beta diversity indices, the microbes in environments and diets considerably influenced the richness, diversity, and community compositions of the microbiota on egg surfaces. The quantity of the main ODP components and the corresponding oviposition-deterrent activity among four groups were significantly differed among the four groups. The result of this study revealed that altering of microbes in environments or diets considerably changed the contents of ODP and oviposition-deterrent activity. As ODPs impart oviposition-deterrent activity towards closely related species, the findings of this study suggest that we should pay more attention to the role of symbiotic microorganisms in changing the ability of insects, especially sympatric species, to occupy the optimal niche when developing novel pest-control strategies.

Construction and biological activity of metallothionein fused with ELP

Metallothionein (MT) plays a significant role in heavy metal removal, antioxidant defense, and immune regulation. The current predominant approach for obtaining natural MT is extraction from tissue, which often entails complex procedures resulting in limited yields. In recent years, researchers have adopted the strategy of fusing labels such as GST or His for the heterologous expression of MT. However, a challenge in industrial production arises from the subsequent removal of these labels, which often leads to a significant reduction in the yield. The fusion with elastin-like polypeptides (ELPs) offers a promising solution for achieving soluble expression of the target protein, while providing a simple and fast purification process. In this study, ELP was fused with MT, which significantly up-regulated the soluble expression of MT. The fusion protein ELP-MT with the purity above 97% was obtained efficiently and simply by inverse transition cycling (ITC). ELP-MT exhibited a remarkable 2,2'-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) ammonium salt (ABTS) scavenging activity, with the half maximal inhibitory concentration (IC50) of 0.77 μmol/L, which was 53.7 times that of the vitamin E derivative Trolox. In addition, the fusion protein demonstrated strong 1,1-diphenyl-2-trinitrohydrazine (DPPH) scavenging ability. Furthermore, ELP-MT had no toxicity to the proliferation and promoted the adhesion and migration of NIH/3T3 cells. All these results indicated that ELP-MT had good biocompatibility. We constructed the fusion protein ELP-MT combining the unique properties of MT and elastin, laying a technical foundation for the large-scale production of recombinant MT and facilitating the applications in food, health supplement, and cosmetic industries.

Genome-Wide Association Study and Marker Development for Fusarium Oxysporum Root Rot Resistance in Soybean.

Fusarium oxysporum root rot (FORR) is an important disease threatening soybean production. The development of marker-assisted selection (MAS) molecular markers will help accelerate the disease resistance breeding process and achieve the breeding goal of improving soybean disease resistance. This study evaluated the FORR disease resistance of 356 soybean germplasm accessions (SGAs) and screened resistance-related loci using genome-wide association analysis (GWAS) to develop molecular markers for MAS. A total of 1,355,930 high-quality SNPs were analyzed, 150 SNP sites significantly associated with FORR resistance were identified, and these sites were distributed within 41 QTLs. Additionally, 240 candidate genes were screened near these QTL regions, involving multiple functions such as hormone metabolism, signal transduction, stress defense, and growth regulation. Cleaved amplified polymorphic sequence (CAPS) and Kompetitive Allele-Specific PCR (KASP) molecular markers were developed based on candidate genes with significant SNP loci and beneficial haplotypes. The CAPS markers, S15_50486939-CAPS1 and S15_50452626-CAPS2, can effectively distinguish resistant and sensitive genotypes through enzyme digestion. The KASP marker is based on S07_19078765-G/T and exhibits a genotype clustering pattern consistent with disease resistance, demonstrating its application value in breeding. The CAPS and KASP markers developed in this study can provide reliable tools for MAS in FORR disease-resistant varieties. The research results will help reveal the genetic structure of FORR disease resistance and provide support for efficient breeding.

RNA-seq analysis reveals genes associated with Macrophomina phaseolina-induced host senescence in soybean

BackgroundCharcoal rot of soybean is caused by the hemibiotrophic fungus Macrophomina phaseolina, a global crop destroyer and an important pathogen in the midwestern USA. The quantitative nature of host resistance and the complexity of the soybean-M. phaseolina interaction at the molecular level have hampered resistance breeding. A previous study showed that L-ascorbic acid (LAA) pre-treatment before M. phaseolina inoculation reduced charcoal rot lesion length in excised soybean stems. This study aimed to elucidate the genetic underpinnings of M. phaseolina-induced senescence and the mitigating effects of ascorbic acid on this physiological process within the same pathosystem.ResultsRNA was sequenced from M. phaseolina-resistant and -susceptible soybean genotypes following M. phaseolina inoculation, LAA, and hydrogen peroxide (H2O2)—an oxidative stress inducer—application followed by inoculation. More genes were down-regulated in the resistant and susceptible genotypes than up-regulated when the M. phaseolina-inoculated treatments were compared to mock-inoculated control treatments. Gene ontology (GO) term and KEGG pathways analysis detected M. phaseolina-induced up-regulation of receptor-like kinase genes. In contrast, many genes related to antioxidants, defense, and hormonal pathways were down-regulated in both genotypes. LAA pre-treatment induced genes related to photosynthesis and reactive oxygen species responses in both genotypes. H2O2 pre-treatment following inoculation up-regulated many stress-response genes, while hormone signal transduction and photosynthesis-related genes were down-regulated in both genotypes.ConclusionsResults revealed transcriptional variation and genes associated with M. phaseolina-induced senescence in soybean. Ascorbic acid induced many photosynthetic genes, suggesting a complex regulation of defense and immunity in the plant against the hemibiotroph. Soybean plants also exhibited enhanced stress responsiveness when treated with H2O2 followed by inoculation with M. phaseolina. This study will broaden more research avenues related to transcriptional regulation during the M. phaseolina-soybean interaction and the potential role of receptor-like kinases, oxidative stress-responsive genes, ethylene-mediated signaling and enhanced photosynthetic gene expression when mounting host resistance to this important soybean pathogen.

Exploring the Updated Roles of Ferroptosis in Liver Diseases: Mechanisms, Regulators, and Therapeutic Implications.

Ferroptosis, a newly discovered mode of cell death, is a type of iron-dependent regulated cell death characterized by intracellular excessive lipid peroxidation and imbalanced redox. As the liver is susceptible to oxidative damage and the abnormal iron accumulation is a major feature of most liver diseases, studies on ferroptosis in the field of liver diseases are of great interest. Studies show that targeting the key regulators of ferroptosis can effectively alleviate or even reverse the deterioration process of liver diseases. System Xc- and glutathione peroxidase 4 are the main defense regulators of ferroptosis, while acyl-CoA synthetase long chain family member 4 is a key enzyme causing peroxidation in ferroptosis. Generally speaking, ferroptosis should be suppressed in alcoholic liver disease, non-alcoholic fatty liver disease, and drug-induced liver injury, while it should be induced in liver fibrosis and hepatocellular carcinoma. In this review, we summarize the main regulators involved in ferroptosis and then the mechanisms of ferroptosis in different liver diseases. Treatment options of drugs targeting ferroptosis are further concluded. Determining different triggers of ferroptosis can clarify the mechanism of ferroptosis occurs at both physiological and pathological levels.

Gene Expression Profiling of the Peritumoral Immune Cell Infiltrate of Penile Squamous Cell Carcinomas.

Penile carcinomas are rare tumors in Europe and need further investigations due to their inferior prognosis in late tumor stages. The presence of disparate immune cell infiltrates was observed in these tumors, which were subsequently demonstrated to give rise to divergent tumor prognoses. The objective was to further characterize this immune cell infiltrate with the use of immunohistochemistry and RNA expression. A total of twelve well-characterized cases of penile squamous cell carcinomas with known infection status by human papillomavirus (HPV) and p16 status were assessed. The cases were classified according to their morphological characteristics, including those exhibiting a pronounced peritumoral immune cell infiltrate and those with less peritumoral immune cell infiltration. The generation of RNA expression data was conducted using the nCounter® PanCancer Immune Profiling Panel. Computational models were employed to calculate the proportions of immune cells. To corroborate the findings, an immunohistochemical analysis was conducted using antibodies against CD20, CD3, CD4, CD8, MUM1, CD68, and CD117. Our cases were clustered according to the immune cell infiltrate detected via histology in a group with less immune cell infiltrate density and in a group with increased immune cell infiltrate density. Generally, all immune cells showed an increased amount in the group with pronounced immune cell infiltrate density. The clusters were found to relate to cell functions, the complement system, cytotoxicity, pathogen defense, regulation, and T-cell functions. In cases exhibiting a pronounced immune cell infiltrate, the top three genes that exhibited the greatest upregulation were GZMA, MICB, and GNLY. No relationship to HPV infection status was demonstrated. Immunohistochemistry validated the data gained via RNA expression and showed a correlation with EPIC and Cibersort. The clustering of cases based on immune cell infiltrate density revealed significant distinctions between groups with lower and higher immune cell infiltrate density. The group with increased immune cel infiltrate density showed a greater abundance of immune cells, aligning with key functions like cytotoxicity, pathogen defense, and T-cell regulation. Among these cases, the genes GZMA, MICB, and GNLY were significantly upregulated, suggesting their involvement in an increased immune response. The role of HPV infection status in our cases with regard to the peritumoral immune cell infiltrate remains inconclusive.

Inborn errors of immunity reveal the molecular requirements for the generation and maintenance of human IL-9 expressing cells.

CD4+ T cells play essential roles in adaptive immunity. Distinct CD4+ T-cell subsets-TH1, TH2, TH17, TH22, T follicular helper, and regulatory T cells-have been identified, and their contributions to host defense and immune regulation are increasingly well defined. IL-9-producing TH9 cells were first described in 2008 and appear to play both protective and pathogenic roles in human immunity. However, key requirements for generating human TH9 cells remain incompletely defined. We sought to define signaling pathways that regulate IL-9 production by human CD4+ T cells. Human naive and memory CD4+ T cells were cultured under different conditions, and the molecular mechanisms regulating IL-9 induction were determined by assessing the ability of CD4+ T cells from a broad range of patients (n= 92) with pathogenic variants in key immune genes (n= 21) to differentiate into IL-9+ cells. We identified 2 culture conditions that yielded IL-9-expressing cells from naive CD4+ T cells and amplified IL-9 production by invivo-generated memory CD4+ T cells: TGF-β plus IL-4 (ie, TH9 polarizing condition), and the combination of IL-21, IL-23, IL-6, IL-1β, and TGF-β (ie, TH17 polarizing condition). Combining these conditions had a synergistic effect in generating IL-9+CD4+ T cells. IL-9 induction required STAT3-activating cytokines as well as intact signaling via the T-cell receptor and STAT5. Importantly, IL-9 induction was restrained by IFN-γ/STAT1 and IL-10. Our findings revealed critical molecules involved in inducing/restraining IL-9 production by human CD4+ Tcells, thereby identifying pathways that could be targeted to modulate IL-9 in health and disease.

Intracellular metabolome elucidates the time-of-day-dependent response to hydrogen peroxide in salmonid gill epithelial cells

The internal timekeeping system regulates the daily cycle of physiological and behavioural changes in living organisms. This rhythmic phenomenon also influences cellular responses to reactive oxygen species, such as hydrogen peroxide (H2O2). However, the temporal interaction between H2O2 and fish mucosal cells is not well understood. This study examined the temporal variations of immunological and physiological responses to H2O2 in salmonid gill cells using the RTgill-W1 cell line. The results showed that gene expression levels varied during a 24-h cycle but did not exhibit rhythmicity. The presence of a 12-h light-dark cycle (12L:12D) signal increased gene expression levels compared to a 24-h dark cycle (0L:24D). To investigate whether the time of day affects the defences in gills, cells were exposed to H2O2 at two different times (Zeitgebertime 2, ZT2, or ZT14). Although significant expression changes were observed in genes related to stress and NF-κB signalling, only a limited time-dependent pattern of response to H2O2 was observed. The intracellular metabolome of gill cells was primarily composed of organic acid and derivatives, organoheterocyclic compounds, benzoids, organic oxygen and nitrogen compounds. Exposure to H2O2 at ZT2 led to significant changes in the metabolome compared to the control group, while no such changes were observed at ZT14. Within the control groups, the concentrations of 11 metabolites significantly varied between ZT2 and ZT14, with higher levels at ZT14. These metabolites were involved in arginine biosynthesis, amino acid metabolism, and nitrogen metabolism. In contrast, the level of 26 metabolites significantly varied between ZT2 and ZT14 in H2O2-exposed groups, with lower levels at ZT14. Comparing control and H2O2-exposed groups at ZT2, 38 metabolites were affected, primarily organic acid and derivatives and organic oxygen compounds. Functional annotation revealed that these altered metabolites were involved in 15 different pathways, with valine, leucine, and isoleucine biosynthesis being the most affected. This study reveals the presence of a time-dependent response to H2O2 in salmonid gill cells, which is reflected in the intracellular metabolome. The findings provide new insights into the temporal regulation of mucosal defences in fish.

Differential regulation of glucosinolate-myrosinase mediated defense determines host-aphid interaction in Indian mustard Brassica juncea L.

India's oilseed economy falls short of self-sufficiency and is supplemented by huge imports every year. Increasing national productivity of the major oilseeds is confronted with yield losses due to diverse biotic and abiotic stresses. The productivity of Indian mustard (Brassica juncea Linnaeus), belonging to the family Brassicaceae, is significantly reduced due to damage caused by mustard aphids (Lipaphis erysimi Kaltenbach, Hemiptera: Aphididae). Rapid colonization by the nymphs makes it difficult to protect the crop through agrochemicals. Aphids release effector molecules to modulate the host-defence responses. Glucosinolates (GSLs) extensively found in Brassicaceae family, are hydrolysed by myrosinase into toxic compounds that deter herbivore insects. Here, we investigated the differential activation of the glucosinolate-myrosinase pathway in mustard manifesting susceptibility and resistance to different aphid species. Mustard plants were challenged by two different aphid species mustard aphid and cowpea aphid (Aphis craccivora Koch, Hemiptera: Aphididae) leading to complete host-susceptibility in one case and resistance in the other, respectively. Differential regulation of the GSL biosynthetic pathway and myrosinase activity was assessed by gene expression study and ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC- QToF-ESL-MS). Gene expression study identified selective transcriptional attenuation of the key GSL biosynthetic and myrosinase gene in mustard when challenged with mustard aphid. In contrary, the activation of GSL biosynthetic genes in conjunction with myrosinase at the transcriptional level was profound in mustard, when challenged with cowpea aphid. UPLC-MS analysis showed higher turnover in the hydrolysis of glucosinolates by myrosinase which led to concomitant generation of glucose as byproduct in response to cowpea aphid in mustard plants. GSL-myrosinase pathway is specifically attenuated by the successful aphid species in mustard and thus plays a pivotal role in determining the outcome of the B. juncea-aphid interaction. The results open up a new genetic modification strategy for developing resistance against aphids.

Genotype-Dependent Variations in Oxidative Stress Markers and Bioactive Proteins in Hereford Bulls: Associations with DGAT1, LEP, and SCD1 Genes.

The objective of this study is to assess the influence of genetic polymorphisms in DGAT1, LEP, and SCD1 on the oxidative stress biomarkers and bioactive protein levels in Hereford bulls. A total of sixty-eight bulls were analyzed at 22 months of age to assess growth metrics and carcass quality, with a focus on polymorphisms in these genes. The key markers of oxidative stress, including malondialdehyde (MDA), and the activities of antioxidant enzymes such as glutathione reductase (GluRed), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were measured, alongside bioactive compounds like taurine, carnosine, and anserine. The results show that the TT genotype of DGAT1 is linked to significantly higher MDA levels, reflecting increased lipid peroxidation, but is also associated with higher GluRed and GPx activities and elevated levels of taurine, carnosine, and anserine, suggesting an adaptive response to oxidative stress. The LEP gene analysis revealed that the CC genotype had the highest MDA levels but also exhibited increased GPx and SOD activities, with the CT genotype showing the highest SOD activity and the TT genotype the highest total antioxidant status (TAS). The SCD1 AA genotype displayed the highest activities of GluRed, GPx, and SOD, indicating a more effective antioxidant defence, while the VA genotype had the highest MDA levels and the VV genotype showed lower MDA levels, suggesting protective effects against oxidative damage. These findings highlight genotype specific variations in the oxidative stress markers and bioactive compound levels, providing insights into the genetic regulation of oxidative stress and antioxidant defences, which could inform breeding strategies for improving oxidative stress resistance in livestock and managing related conditions.