Upstream Regulator Research Articles

The MYB Transcription Factor GmMYB78 Negatively Regulates Phytophthora sojae Resistance in Soybean.

Phytophthora root rot is a devastating disease of soybean caused by Phytophthora sojae. However, the resistance mechanism is not yet clear. Our previous studies have shown that GmAP2 enhances sensitivity to P. sojae in soybean, and GmMYB78 is downregulated in the transcriptome analysis of GmAP2-overexpressing transgenic hairy roots. Here, GmMYB78 was significantly induced by P. sojae in susceptible soybean, and the overexpressing of GmMYB78 enhanced sensitivity to the pathogen, while silencing GmMYB78 enhances resistance to P. sojae, indicating that GmMYB78 is a negative regulator of P. sojae. Moreover, the jasmonic acid (JA) content and JA synthesis gene GmAOS1 was highly upregulated in GmMYB78-silencing roots and highly downregulated in overexpressing ones, suggesting that GmMYB78 could respond to P. sojae through the JA signaling pathway. Furthermore, the expression of several pathogenesis-related genes was significantly lower in GmMYB78-overexpressing roots and higher in GmMYB78-silencing ones. Additionally, we screened and identified the upstream regulator GmbHLH122 and downstream target gene GmbZIP25 of GmMYB78. GmbHLH122 was highly induced by P. sojae and could inhibit GmMYB78 expression in resistant soybean, and GmMYB78 was highly expressed to activate downstream target gene GmbZIP25 transcription in susceptible soybean. In conclusion, our data reveal that GmMYB78 triggers soybean sensitivity to P. sojae by inhibiting the JA signaling pathway and the expression of pathogenesis-related genes or through the effects of the GmbHLH122-GmMYB78-GmbZIP25 cascade pathway.

THOC7-AS1/OCT1/FSTL1 axis promotes EMT and serves as a therapeutic target in cutaneous squamous cell carcinoma

BackgroundTHOC7-AS1 and FSTL1 expression are frequently upregulated in cutaneous squamous cell carcinoma (cSCC). However, their molecular biological mechanisms remain elusive and their potential as therapeutic targets needs urgent exploration.MethodsHuman tissue samples were used to evaluate clinical parameters. In vitro and in vivo experiments assessed biological functions. Quantitative PCR, western blot, immunohistochemistry, immunocytochemistry, immunoprecipitation, RNA fluorescence in situ hybridization, RNA pull-down, RNA immunoprecipitation, silver staining, chromatin immunoprecipitation, dual luciferase reporter assays etc. were utilized to explore the molecular biological mechanisms.ResultsWe found FSTL1 is an oncogene in cSCC, with high expression in tumor tissues and cells. Its elevated expression closely associates with tumor size and local tissue infiltration. In vitro and in vivo, high FSTL1 expression promotes cSCC proliferation, migration and invasion, facilitating malignant behaviors. Mechanistically, FSTL1 interacts with ZEB1 to promote epithelial-to-mesenchymal transition (EMT) in cSCC cells. Exploring upstream regulation, we found THOC7-AS1 can interact with OCT1, which binds the FSTL1 promoter region and promotes FSTL1 expression, facilitating cSCC progression. Finally, treating tumors with THOC7-AS1 antisense oligonucleotides inhibited cSCC proliferative and migratory abilities, delaying tumor progression.ConclusionsThe THOC7-AS1/OCT1/FSTL1 axis regulates EMT and promotes tumor progression in cSCC. This study provides clues and ideas for cSCC targeted therapy.

Wnt, glucocorticoid and cellular prion protein cooperate to drive a mesenchymal phenotype with poor prognosis in colon cancer

BackgroundThe mesenchymal subtype of colorectal cancer (CRC), associated with poor prognosis, is characterized by abundant expression of the cellular prion protein PrPC, which represents a candidate therapeutic target. How PrPC is induced in CRC remains elusive. This study aims to elucidate the signaling pathways governing PrPC expression and to shed light on the gene regulatory networks linked to PrPC.MethodsWe performed in silico analyses on diverse datasets of in vitro, ex vivo and in vivo models of mouse CRC and patient cohorts. We mined ChIPseq studies and performed promoter analysis. CRC cell lines were manipulated through genetic and pharmacological approaches. We created mice combining conditional inactivation of Apc in intestinal epithelial cells and overexpression of the human prion protein gene PRNP. Bio-informatic analyses were carried out in two randomized control trials totalizing over 3000 CRC patients.ResultsIn silico analyses combined with cell-based assays identified the Wnt-β-catenin and glucocorticoid pathways as upstream regulators of PRNP expression, with subtle differences between mouse and human. We uncover multiple feedback loops between PrPC and these two pathways, which translate into an aggravation of CRC pathogenesis in mouse. In stage III CRC patients, the signature defined by PRNP-CTNNB1-NR3C1, encoding PrPC, β-catenin and the glucocorticoid receptor respectively, is overrepresented in the poor-prognosis, mesenchymal subtype and associates with reduced time to recurrence.ConclusionsAn unleashed PrPC-dependent vicious circle is pathognomonic of poor prognosis, mesenchymal CRC. Patients from this aggressive subtype of CRC may benefit from therapies targeting the PRNP-CTNNB1-NR3C1 axis.

STAM2 negatively regulates the MyD88-mediated NF-κB signaling pathway in miiuy croaker, Miichthys miiuy

Signal transducing adapter molecule 2 (STAM2), a member of the Signal Transducing Adapter Molecule (STAM) family, is a protein with significant implications in diverse signaling pathways and endocytic membrane trafficking. However, the role of the STAM2, especially in fish, remains largely unknown. In this study, we discovered that STAM2 negatively regulates the NF-κB signaling pathway, and its inhibitory effect is enhanced upon LPS induction. Our study confirmed that STAM2 can enhance the degradation of myeloid differentiation primary-response protein 88 (MyD88), an upstream regulator of NF-κB pathway. Furthermore, the UIM domain of STAM2 is important for the inhibition of MyD88. Mechanistically, STAM2 inhibits the NF-κB signaling pathway by targeting the MyD88 autophagy pathway. In addition, we showed that STAM2 promotes the proliferation of Vibrio harveyi. In summary, our study reveals that STAM2 inhibits NF-κB signaling activation and mediates innate immunity in teleost via the autophagy pathway.

The AGAMOUS-LIKE 16-GENERAL REGULATORY FACTOR 1 module regulates axillary bud outgrowth via catabolism of abscisic acid in cucumber.

Lateral branches are important components of shoot architecture and directly affect crop yield and production cost. Although sporadic studies have implicated abscisic acid (ABA) biosynthesis in axillary bud outgrowth, the function of ABA catabolism and its upstream regulators in shoot branching remain elusive. Here, we showed that the MADS-box transcription factor AGAMOUS-LIKE 16 (CsAGL16) is a positive regulator of axillary bud outgrowth in cucumber (Cucumis sativus). Functional disruption of CsAGL16 led to reduced bud outgrowth, whereas overexpression of CsAGL16 resulted in enhanced branching. CsAGL16 directly binds to the promoter of the ABA 8'-hydroxylase gene CsCYP707A4 and promotes its expression. Loss of CsCYP707A4 function inhibited axillary bud outgrowth and increased ABA levels. Elevated expression of CsCYP707A4 or treatment with an ABA biosynthesis inhibitor largely rescued the Csagl16 mutant phenotype. Moreover, cucumber General Regulatory Factor 1 (CsGRF1) interacts with CsAGL16 and antagonizes CsAGL16-mediated CsCYP707A4 activation. Disruption of CsGRF1 resulted in elongated branches and decreased ABA levels in the axillary buds. The Csagl16 Csgrf1 double mutant exhibited a branching phenotype resembling that of the Csagl16 single mutant. Therefore, our data suggest that the CsAGL16-CsGRF1 module regulates axillary bud outgrowth via CsCYP707A4-mediated ABA catabolism in cucumber. Our findings provide a strategy to manipulate ABA levels in axillary buds during crop breeding to produce desirable branching phenotypes.

Menin Deficiency Induces Autism-Like Behaviors by Regulating Foxg1 Transcription and Participates in Foxg1-Related Encephalopathy.

FOXG1 syndrome is a developmental encephalopathy caused by FOXG1 (Forkhead box G1) mutations, resulting in high phenotypic variability. However, the upstream transcriptional regulation of Foxg1 expression remains unclear. This report demonstrates that both deficiency and overexpression of Men1 (protein: menin, a pathogenic gene of MEN1 syndrome known as multiple endocrine neoplasia type 1) lead to autism-like behaviors, such as social defects, increased repetitive behaviors, and cognitive impairments. Multifaceted transcriptome analyses revealed that Foxg1 signaling is predominantly altered in Men1 deficiency mice, through its regulation of the Alpha Thalassemia/Mental Retardation Syndrome X-Linked (Atrx) factor. Atrx recruits menin to bind to the transcriptional start region of Foxg1 and mediates the regulation of Foxg1 expression by H3K4me3 (Trimethylation of histone H3 lysine 4)modification. The deficits observed in menin deficient mice are rescued by the over-expression of Foxg1, leading to normalized spine growth and restoration of hippocampal synaptic plasticity. These findings suggest that menin may have a putative role in the maintenance of Foxg1 expression, highlighting menin signaling as a potential therapeutic target for Foxg1-related encephalopathy.

Abstract LB250: Dynamics of transcript alternations and differential splicing in the evolution of acquired MAPKi resistance in melanoma

Abstract The primary therapy for melanoma with a BRAF mutation involves Mitogen-Activated Protein Kinase inhibitors (MAPKi), targeting the dysregulated MAPK signaling pathway. Despite the initial response, many patients often develop acquired resistance. Resistance to MAPKi could be linked to various altered signaling pathways, including reactivation of the MAPK pathway, activation of alternative survival pathways like PI3K/PTEN/AKT, engagement of receptor tyrosine kinases (RTKs) such as PDGFRβ and EGFR, and developmental pathways. While numerous omics studies have aimed to understand the mechanism of acquired resistance, how these pathways are altered remains elusive. To gain further insights into the acquired MAPKi resistance in melanoma, we explored the role of RNA splicing events in this context by analyzing publicly available datasets of pre- and post-MAPKi treated melanoma from patients and in vitro studies using cell lines. We investigated the differential transcript usage (DTU) to detect the specific splice variants that are altered during the development of resistance. We find that the genes involving DTU are enriched in various pathways related to MAPKi resistance, such as the MAPKi signaling pathway, PI3K/AKT pathway, signaling by RTKs, etc. Furthermore, the DTU-centered analysis provides better insights into MAPKi resistance mechanisms compared to standard differential gene expression analysis. Next, we analyzed the developmental splicing signatures during MAPKi resistance development, revealing developmental reprogramming as a key factor in MAPKi resistance. Further, we identified the top 10 transcription factors as potential upstream regulators of the perturbed transcripts, among which five have been previously reported to be involved in various stages of embryonic development. This finding suggests a link between the reactivation of developmental pathways and MAPKi resistance development. Further, by analyzing MAPKi-treated melanoma cell-line models at different time points, such as no-treatment, on-treatment, and at the resistant stage, we observed significant alterations in transcript isoforms during the development of resistance. These changes involve lipid metabolic reprogramming and PI3K/Akt/mTOR signaling, providing further insights into the mechanisms of MAPKi resistance. Our study also uncovers a global shift in transcript profiles towards shorter 3’ and 5’ UTRs in response to MAPKi treatment, suggesting the existence of a master regulatory mechanism influencing global splicing changes during therapy resistance. In summary, our study deciphered the post-transcriptional mechanism coordinating the signaling changes, underscoring the complexity of MAPKi resistance in melanoma, as revealed by the joint analysis of both transcript alterations and splicing events. Citation Format: Sumit Mukherjee, Arashdeep Singh, Hyunjeong Joo, Sumeet Patiyal, Hyungsoo Kim, Lipika R. Pal, Kun Wang, Chi-Ping Day, Ze’ev A. Ronai, Eytan Ruppin, Sridhar Hannenhalli. Dynamics of transcript alternations and differential splicing in the evolution of acquired MAPKi resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB250.

A viral protein competitively bound to rice CIPK23 inhibits potassium absorption and facilitates virus systemic infection in rice.

Potassium (K+) plays a crucial role as a macronutrient in the growth and development of plants. Studies have definitely determined the vital roles of K+ in response to pathogen invasion. Our previous investigations revealed that rice plants infected with rice grassy stunt virus (RGSV) displayed a reduction in K+ content, but the mechanism by which RGSV infection subverts K+ uptake remains unknown. In this study, we found that overexpression of RGSV P1, a specific viral protein encoded by viral RNA1, results in enhanced sensitivity to low K+ stress and exhibits a significantly lower rate of K+ influx compared to wild-type rice plants. Further investigation revealed that RGSV P1 interacts with OsCIPK23, an upstream regulator of Shaker K+ channel OsAKT1. Moreover, we found that the P1 protein recruits the OsCIPK23 to the Cajal bodies (CBs). In vivo assays demonstrated that the P1 protein competitively binds to OsCIPK23 with both OsCBL1 and OsAKT1. In the nucleus, the P1 protein enhances the binding of OsCIPK23 to OsCoilin, a homologue of the signature protein of CBs in Arabidopsis, and facilitates their trafficking through these CB structures. Genetic analysis indicates that mutant in oscipk23 suppresses RGSV systemic infection. Conversely, osakt1 mutants exhibited increased sensitivity to RGSV infection. These findings suggest that RGSV P1 hinders the absorption of K+ in rice plants by recruiting the OsCIPK23 to the CB structures. This process potentially promotes virus systemic infection but comes at the expense of inhibiting OsAKT1 activity.

ASH2L Mediates Epidermal Differentiation and Hair Follicle Morphogenesis through H3K4me3 Modification

The processes of epidermal development in mammals are regulated by complex molecular mechanisms, such as histone modifications. Histone H3 lysine K4 methylation mediated by COMPASS (complex of proteins associated with Set1) methyltransferase is associated with gene activation, but its effect on epidermal lineage development remains unclear. Therefore, we constructed a mouse model of specific ASH2L (COMPASS methyltransferase core subunit) deletion in epidermal progenitor cells and investigated its effect on the development of mouse epidermal lineage. Furthermore, downstream target genes regulated by H3K4me3 were screened using RNA sequencing combined with Cleavage Under Targets and Tagmentation sequencing. Deletion of ASH2L in epidermal progenitor cells caused thinning of the suprabasal layer of the epidermis and delayed hair follicle morphogenesis in newborn mice. These phenotypes may be related to the reduced proliferative capacity of epidermal and hair follicle progenitor cells. ASH2L depletion may also lead to depletion of the epidermal stem cell pools in late mouse development. Finally, genes related to hair follicle development (Shh, Edar, and Fzd6), Notch signaling pathway (Notch2, Notch3, Hes5, and Nrarp), and ΔNp63 were identified as downstream target genes regulated by H3K4me3. Collectively, ASH2L-dependent H3K4me3 modification served as an upstream epigenetic regulator in epidermal differentiation and hair follicle morphogenesis in mice.

Comparison of transcriptomic profiles between HFPO-DA and prototypical PPARα, PPARγ, and cytotoxic agents in mouse, rat, and pooled human hepatocytes.

Like many per- or polyfluorinated alkyl substances (PFAS), toxicity studies with HFPO-DA (ammonium, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate), a short-chain PFAS used in the manufacture of some types of fluorinated polymers, indicate that the liver is the primary target of toxicity in rodents following oral exposure. Although the current weight of evidence supports the PPARα mode of action (MOA) for liver effects in HFPO-DA-exposed mice, alternate MOAs have also been hypothesized including PPARγ or cytotoxicity. To further evaluate the MOA for HFPO-DA in rodent liver, transcriptomic analyses were conducted on samples from primary mouse, rat, and pooled human hepatocytes treated for 12, 24, or 72 h with various concentrations of HFPO-DA, or agonists of PPARα (GW7647), PPARγ (rosiglitazone), or cytotoxic agents (ie, acetaminophen or d-galactosamine). Concordance analyses of enriched pathways across chemicals within each species demonstrated the greatest concordance between HFPO-DA and PPARα agonist GW7647-treated hepatocytes compared with the other chemicals evaluated. These findings were supported by benchmark concentration modeling and predicted upstream regulator results. In addition, transcriptomic analyses across species demonstrated a greater transcriptomic response in rodent hepatocytes treated with HFPO-DA or agonists of PPARα or PPARγ, indicating rodent hepatocytes are more sensitive to HFPO-DA or PPARα/γ agonist treatment. These results are consistent with previously published transcriptomic analyses and further support that liver effects in HFPO-DA-exposed rodents are mediated through rodent-specific PPARα signaling mechanisms as part of the MOA for PPARα activator-induced rodent hepatocarcinogenesis. Thus, effects observed in mouse liver are not appropriate endpoints for toxicity value development for HFPO-DA in human health risk assessment.

Shared genetic aetiology of Alzheimer’s disease and age-related macular degeneration by APOC1 and APOE genes

BackgroundAlzheimer’s disease (AD) and age-related macular degeneration (AMD) share similar pathological features, suggesting common genetic aetiologies between the two. Investigating gene associations between AD and AMD may provide useful insights...

MiR-199a-5p targets DUSP14 to regulate cell proliferation, invasion and stemness in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) shows the highest morbidity among malignant tumors worldwide. Despite improvements of diagnosis and treatment, patient prognosis remains unfavorable. Therefore, there is a need to discover a novel treatment strategy for NSCLC. DUSP14 is related to various cancers as the regulatory factor for cellular processes. However, its specific roles in NSCLC and the upstream modulator remain largely unclear. MethodsDUSP14 expression patterns within the lung cancer patient cohort from TCGA database were analyzed using UALCAN online tool. Different databases including miRDB, starbase, and Targetscan were employed to screen the upstream regulator of DUSP14. DUSP14 and miR-199a-5p expression was determined by qRT-PCR and Western blot techniques. To confirm binding interaction of DUSP14 with miR-199a-5p, we conducted a dual-luciferase reporter assay. Cell viability, migration, and stemness properties were assessed using CCK-8, EdU (5-ethynyl-2′-deoxyuridine) incorporation, transwell invasion, and sphere formation assays. The effect of DUSP14 silencing on tumorigenesis was assessed with the NSCLC cell xenograft mouse model. ResultsOur study discovered that DUSP14 exhibited high expression within NSCLC tumor samples, which is related to the dismal prognostic outcome in NSCLC patients. Silencing DUSP14 impaired NSCLC cell proliferation, migration, and tumor sphere formation. Besides, we identified miR-199a-5p as the upstream regulatory factor for DUSP14, and its expression was negatively related to DUSP14 level within NSCLC tissues. Introducing miR-199a-5p recapitulated the function of DUSP14 silencing in NSCLC cell aggressiveness and stemness. Moreover, knocking down DUSP14 efficiently inhibited tumor formation in NSCLC cells of the xenograft model. ConclusionsOur study suggests that DUSP14 is negatively regulated by miR-199a-5p within NSCLC, whose overexpression is required for sustaining NSCLC cell proliferation, invasion and stemness.

Cellular senescence induced by down-regulation of PTBP1 correlates with exon skipping of mitochondrial-related gene NDUFV3

Abstract As the most prevalent type of alternative splicing in animal cells, exon skipping plays an important role in expanding the diversity of transcriptome and proteome, thereby participating in the regulation of diverse physiological and pathological processes such as development, aging, and cancer. Cellular senescence serving as an anti-cancer mechanism could also contribute to individual aging. Although the dynamic changes of exon skipping during cellular senescence were revealed, its biological consequence and upstream regulator remain poorly understood. Here, by using human foreskin fibroblasts (HFF) replicative senescence as a model, we discovered that splicing factor PTBP1 was an important contributor for global exon skipping events during senescence. Down-regulated expression of PTBP1 induced senescence-associated phenotypes and related mitochondrial functional changes. Mechanistically, PTBP1 binds to the third exon of mitochondrial complex I subunit coding gene NDUFV3 and protects the exon from skipping. We further confirmed that exon skipping of NDUFV3 correlates with and partially contributes to cellular senescence and related mitochondrial functional changes upon PTBP1 knockdown. Together, we revealed for the first time that mitochondrial-related gene NDUFV3 is a new downstream target for PTBP1-regulated exon skipping to mediate cellular senescence and mitochondrial functional changes.

High expression of NXPH4 correlates with poor prognosis, metabolic reprogramming, and immune infiltration in colon adenocarcinoma.

Colon adenocarcinoma (COAD) is a prevalent gastrointestinal malignant disease with high mortality rate, and identification of novel prognostic biomarkers and therapeutic targets is urgently needed. Although neurexophilin 4 (NXPH4) has been investigated in several tumors, its role in COAD remains unclear. The aim of this study was to explore the prognostic value and potential functions of NXPH4 in COAD. The expression of NXPH4 in COAD were analyzed using The Cancer Genome Atlas (TCGA) and datasets from the Gene Expression Omnibus (GEO) database. The prognostic value of NXPH4 was determined using Kaplan-Meier analysis and Cox regression analysis. To investigate the possible mechanism underlying the role of NXPH4 in COAD, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) were employed. The correlation between NXPH4 expression and immune cell infiltration levels was examined thorough single-sample gene set enrichment analysis (ssGSEA). Furthermore, the competing endogenous RNA (ceRNA) regulatory network that may be involved in NXPH4 in COAD was predicted and constructed through a variety of databases. NXPH4 expression was significantly higher in COAD tissue compared with normal colon tissues. Meanwhile, high expression of NXPH4 was associated with poor prognosis in COAD patients. GO-KEGG and GSEA analyses indicated that NXPH4 was associated with glycolysis and hypoxia pathway, and may promote COAD progression and metastasis by modulating metabolic reprogramming. ssGSEA analysis demonstrated that NXPH4 expression also associated with immune infiltration. Furthermore, we identified various microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) as upstream regulators of NXPH4 in COAD. The present study revealed that high expression of NXPH4 is associated with tumor progression, metabolic reprogramming, and immune infiltration. These findings suggest that NXPH4 could serve as a reliable prognostic biomarker and a promising therapeutic target in COAD.

5 Janus Kinase 1/2 inhibition Effect on Cytokine Levels in Tears of Patients with Ocular Graft Versus Host Disease

OBJECTIVES/GOALS: The goal of this study is to identify if ocular graft versus host disease (oGVHD) patients treated with a systemic JAK inhibitor have a change in their tear cytokine profile (a possible bio-marker) and oGVHD score. oGHVD is a severe inflammatory dry eye disease and major cause of morbidity after a hematopoietic stem cell transplant. METHODS/STUDY POPULATION: Janus Kinase (JAK) is an upstream regulator of cytokine production. A JAK 1/2 inhibitor, Ruxolitinib, was recently FDA approved for the treatment of chronic GVHD. We propose that JAK inhibition results incytokine changes in tears and improvement of oGVHD. To study this, we will quantify tear cytokines in patients with oGVHD, with and without systemic JAK inhibition treatment. Patients with ‘definite’ oGVHD based on the international chronic oGVHD diagnostic criteria (ICOGVHD) whom we have collected tears will be grouped based on JAK inhibition treatment. Tear cytokines are analyzed using Iso spark Meteor bulk quantitative proteomic analysis. RESULTS/ANTICIPATED RESULTS: Seven patients were identified from our patient cohort who met inclusion criteria (oGVHD; tears collected while on Ruxolitinib), five patients were identified whom we have collected tears with oGVHD who have not taken ruxolitinib. The following 10 cytokines will be analyzed in the tears by the Iso spark Meteor bulk quantitative proteomic analysis: GM-CSF, IFN-g, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17A, TNF-a. The change in cytokine levels will be compared with the ICOGVHD score, corneal fluorescein staining, schirmers test (measurement of tear production), conjunctival injection score, ocular surface disease index score (validated symptomatic score of dry eye disease). DISCUSSION/SIGNIFICANCE: OGVHD is a major cause of morbidity for patients who undergo a hematopoietic stem cell transplant and is the result of a highly complex immune process including dysregulation of pro-inflammatory cytokines. It is critical to understand the effect of cytokine changes on the eyes to potentially identify a biomarker and possible treatment targets.

Network meta-analysis of the molecular mechanisms and signaling pathways underlying alcohol-induced thymic atrophy.

Thymic atrophy is characterized by loss of thymocytes, destruction of thymic architecture, and a subsequent decrease in naïve T cells with compromised immunity. Thymic atrophy occurs during aging. Environmental factors including alcohol misuse also induce thymic atrophy. Despite the link between alcohol misuse and thymic atrophy, the underlying mechanism is understudied. We aimed to identify molecules and signaling pathways that underly alcohol-induced thymic atrophy during aging. F344 rats were given 3-day binge-ethanol (4.8 g/kg/day; 52% w/v; i.g.) and the thymus was collected and weighed. Molecular mechanisms underlying ethanol-induced thymic atrophy were investigated by network meta-analysis using the QIAGEN Ingenuity Pathway Analysis (IPA). The molecules associated with ethanol were identified from the QIAGEN Knowledge Base (QKB) and those associated with thymic atrophy were identified from QKB and Mouse Genome Informatics (MGI). Aging-mediated Differential Expression Genes (DEGs) from mouse thymocytes were obtained from the Gene Expression Omnibus (GEO) database (GSE132136). The relationship between the molecules and associated signaling pathways were studied using IPA. Binge-ethanol decreased thymic weight in F344 rats. Our meta-analysis using IPA identified molecules commonly shared by ethanol and thymic atrophy through which simulation with ethanol increased thymic atrophy. We then obtained aging-mediated DEGs from the atrophied thymocytes. We found that ethanol contributed to thymic atrophy through modulation of the aging-mediated DEGs. Our network meta-analysis suggests that ethanol may augment thymic atrophy through increased expression of cytokines (e.g., IL-6, IL-17A and IL-33) along with their regulators (e.g., STAT1 and STAT3). Exposure to alcohol may augment thymic atrophy by altering the activity of key inflammatory mediators, such as STAT family members and inflammatory cytokines. These findings provide insights into the signaling pathways and upstream regulators that underly alcohol-induced thymic atrophyduring aging, suggesting that alcohol consumption could prepone thymic atrophy.

Differential proteomics profile of microcapillary networks in response to sound pattern-driven local cell density enhancement

Spatial cell organization and biofabrication of microcapillary networks in vitro has a great potential in tissue engineering and regenerative medicine. This study explores the impact of local cell density enhancement achieved through an innovative sound-based patterning on microcapillary networks formation and their proteomic profile. Human umbilical vein endothelial cells (HUVEC) and human pericytes from placenta (hPC-PL) were mixed in a fibrin suspension. The mild effect of sound-induced hydrodynamic forces condensed cells into architected geometries showing good fidelity to the numerical simulation of the physical process. Local cell density increased significantly within the patterned areas and the capillary-like structures formed following the cell density gradient. Over five days, these patterns were well-maintained, resulting in concentric circles and honeycomb-like structures.Proteomic analysis of the pre-condensed cells cultured for 5 days, revealed over 900 differentially expressed proteins when cells were preassembled through mild-hydrodynamic forces. Gene ontology (GO) enrichment analysis identified cellular components, molecular functions, and biological processes that were up- and down-regulated, providing insights regarding molecular processes influenced by the local density enhancement. Furthermore, we employed Ingenuity Pathway Analysis (IPA) to identify altered pathways and predict upstream regulators. Notably, VEGF-A emerged as one of the most prominent upstream regulators.Accordingly, this study initiates the unraveling of the changes in microcapillary networks at both molecular and proteins level induced by cell condensation obtained through sound patterning. The findings provide valuable insights for further investigation into sound patterning as a biofabrication technique for creating more complex microcapillary networks and advancing in vitro models.

Increased m6A modification of BDNF mRNA via FTO promotes neuronal apoptosis following aluminum-induced oxidative stress

N6-methyladenosine (m6A) RNA modification is a new epigenetic molecular mechanism involved in various biological or pathological processes. Exposure to aluminum (Al) has been considered to promote neuronal apoptosis resulting in cognitive dysfunction, yet whether m6A modification participates in the underlying mechanism remains largely unknown. Here, rats exposed to aluminum-maltolate [Al(mal)3] for 90 days showed impaired learning and memory function and elevated apoptosis, which were related to the increased m6A level and decreased fat mass and obesity-associated protein (FTO, an m6A demethylase) in the hippocampus. Accordingly, similar results presented in PC12 cells following Al(mal)3 treatment and FTO overexpression relieved the increased apoptosis and m6A level in vitro. Next, we identified brain-derived neurotrophic factor (BDNF) as the functional downstream target of FTO in a m6A-dependent manner. Furthermore, it was found that as the onset of aluminum neurotoxicity, oxidative stress may be the upstream regulator of FTO in aluminum-induced apoptosis. Taken together, these results suggest that increased m6A modification of BDNF mRNA via FTO promotes neuronal apoptosis following aluminum-induced oxidative stress.

Identification of miR-29a as a novel biomarker for lumpy skin disease virus exposure in cattle

ABSTRACT Micro RNAs (miRNAs) have been implicated in the regulation of maturation, proliferation, differentiation, and activation of immune cells. In this study, we demonstrated that miR-29a antagonizes IFN-γ production at early times post-LSDV infection in cattle. miR-29a was predicted to target upstream IFN-γ regulators, and its inhibition resulted in enhanced IFN-γ production in sensitized peripheral blood mononuclear cells (PBMCs). Further, stimulation of PBMCs with LSDV antigen exhibited lower levels of miR-29a, concomitant with a potent cell-mediated immune response (CMI), characterized by an increase in LSDV-specific CD8+ T cell counts and enhanced levels of IFN-γ, which eventually facilitated virus clearance. In addition, a few immunocompromised cattle (developed secondary LSDV infection at ~ 6 months) that failed to mount a potent cell-mediated immune response, were shown to maintain higher miR-29a levels. Furthermore, as compared to the sensitized crossbred cattle, PBMCs from sensitized Rathi (a native Indian breed) animals exhibited lower levels of miR-29a along with an increase in CD8+ T cell counts and enhanced levels of IFN-γ. Finally, we analysed that a ≥ 60% decrease in miR-29a expression levels in the PBMCs of sensitized cattle correlated with a potent CMI response. In conclusion, miR-29a expression is involved in antagonizing the IFN-γ response in LSDV-infected cattle and may serve as a novel biomarker for the acute phase of LSDV infection, as well as predicting the functionality of T cells in sensitized cattle. In addition, Rathi cattle mount a more potent CMI response against LSDV than crossbred cattle.

Calmodulin-like protein MdCML15 interacts with MdBT2 to modulate iron homeostasis in apple.

BTB and TAZ domain proteins (BTs) function as specialized adaptors facilitating substrate recognition of the CUL3-RING ubiquitin ligase (CRL3) complex that targets proteins for ubiquitination in reaction to diverse pressures. Nonetheless, knowledge of the molecular mechanisms by which the apple scaffold protein MdBT2 responds to external and internal signals is limited. Here we demonstrate that a putative Ca 2+ sensor, calmodulin-like 15 (MdCML15), acts as an upstream regulator of MdBT2 to negatively modulate its functions in plasma membrane H+-ATPase regulation and iron deficiency tolerance. MdCML15 was identified to be substantially linked to MdBT2, and to result in the ubiquitination and degradation of the MdBT2 target protein MdbHLH104. Consequently, MdCML15 repressed the MdbHLH104 target, MdAHA8's expression, reducing levels of a specific membrane H+-ATPase. Finally, the phenotype of transgenic apple plantlets and calli demonstrated that MdCML15 modulates membrane H+-ATPase-produced rhizosphere pH lowering alongside iron homeostasis through an MdCML15-MdBT2-MdbHLH104-MdAHA8 pathway. Our results provide new insights into the relationship between Ca2+ signaling and iron homeostasis.