Regulation Of Transcriptional Activity Research Articles

Discovery and validation of Hsa-microRNA-3665 promoter methylation as a potential biomarker for the prognosis of esophageal squaous cell carcinoma.

Methylation of microRNA (miRNA) promoters associated with diseases is a common epigenetic mechanism in the development of various human cancers. However, its relationship with prognosis in esophageal squamous cell carcinoma (ESCC) remains unclear. This study aims to explore the association between the methylation level of has-miR-3665 promoter and prognosis in ESCC. Human miRNA data were downloaded from miRbase, and we identified CpG islands of these human miRNAs by genomics browser analysis. MiRNA methylation levels were detected by methylation-specific high-resolution melting. Gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to explore the molecular mechanism of hsa-miR-3665. Cox regression analysis was used to investigate prognostic factors. The overall survival rate was predicted by a nomogram. We found that 88 human miRNAs had promoter methylatio, of which 15 miRNAs were found to be epigenetically regulated in ESCC cells compared with their normal counterparts, including hsa-miR-3665. Meanwhile, hsa-miR-3665 expression was significantly lower in ESCC tumour tissue than in adjacent tissue (P = 0.03). GO and KEGG analyses demonstrated that the target genes are involved in protein transport, transcription regulator activity, MAPK and RAS signaling pathway. High hsa-miR-3665 promoter methylation levels were associated with a poor prognosis (HR = 3.89, 95% CI 1.11 ~ 13.55). Moreover, a nomogram incorporating the hsa-miR-3665 methylation level and clinical factors presented a good performance for predicting survival in the training and validation tests, with C-indices of 0.748 and 0.751, respectively. High hsa-miR-3665 promoter methylation levels may be a potential biomarker for the progression of ESCC.

Clinical spectrum and molecular basis in 19 Chinese patients with 46, XY disorder of sexual development caused by NR5A1 mutations

BackgroundNuclear receptor subfamily 5 group A member 1 (NR5A1) plays pivotal roles in steroidogenesis and gonadal development. 46, XY disorder of sexual development (DSD) caused by NR5A1 mutations is a rare genetic condition. This study aimed to provide a comprehensive analysis of the clinical characteristics and molecular defects observed in 19 Chinese patients with NR5A1 variants, including assessing the deleterious effects of novel variants in vitro and evaluating their functional impact on the gonad and adrenal glands in vivo.Materials and methodsSubjects with NR5A1 variants were identified from 223 Chinese 46, XY DSD patients via next-generation sequencing. In-silico analysis and functional assays were performed to evaluate the transcriptional activity, expression levels and nuclear localization of novel NR5A1 variants. The histological structure of the gonads was evaluated via immunohistochemistry (IHC).ResultsPatients with NR5A1 gene variants presented with serious conditions, including micropenis, cryptorchidism, azoospermia, and radiological abnormalities of the spleen. Five novel NR5A1 variants were identified, including heterozygous p.Y5*, p.Q42E and p.L359_L363del, as well as copy number variation (CNV) of chr9:127213317–127570245_del and an exon 6 duplication. A total of 63.2% (12/19) of patients harbored additional variants other than NR5A1. Defective transcriptional regulatory activities and abnormal protein expression levels were observed in NR5A1 variants. The reduced levels of DHEA-S and 11-oxygenated steroids indicate a mild impairment in adrenal function among certain patients. The IHC analysis of the testis revealed intact expression levels of SOX9 in Sertoli cells, while significant differences were observed in the expression pattern of CYP17A1 in Leydig cells among patients. The preserved maturation of adult Leydig cells in the patients may trigger spontaneous puberty.ConclusionsPatients with NR5A1 mutations exhibit complex phenotypes. The observed clinical heterogeneity may be attributed to oligogenic mutations, dysregulated Leydig cell function, as well as the impaired ability to modulate the transcription of target genes.

DRN facilitates WUS transcriptional regulatory activity by chromatin remodeling to regulate shoot stem cell homeostasis in Arabidopsis.

Shoot stem cells, harbored in the shoot apical meristem (SAM), play key roles during post-embryonic development of Arabidopsis and function as the origin of plant aerial tissues. Multiple transcription factors are involved in the sophisticated transcriptional regulation of stem cell homeostasis, with the WUSCHEL (WUS)/CLAVATA3 (CLV3) negative feedback loop playing a central role. WUS acts as a master regulator in maintaining stem cells through its transcriptional regulatory activity including repressive and activating abilities. Although the interaction between WUS and TOPLESS confers the repressive activity of WUS in transcriptional control, the mechanism by which WUS activates gene expression remains elusive. Here, we showed that DORNRÖSCHEN competitively interacts with WUS and disturbs the WUS homodimer, which recruits BRAHMA to activate CLV3 expression via nucleosome depletion for maintaining the stem cell pool.

Single-cell transcriptomics reveals IRF7 regulation of the tumor microenvironment in isocitrate dehydrogenase wild-type glioma.

Mutations in isocitrate dehydrogenase (IDH) are important markers of glioma prognosis. However, few studies have examined the gene expression regulatory network (GRN) in IDH-mutant and wild-type gliomas. In this study, single-cell RNA sequencing and spatial transcriptome sequencing were used to analyze the GRN of cell subsets in patients with IDH-mutant and wild-type gliomas. Through gene transcriptional regulation analysis, we identified the M4 module, whose transcription factor activity is highly expressed in IDH wild-type gliomas compared to IDH-mutants. Enrichment analysis revealed that these genes were predominantly expressed in microglia and macrophages, with significant enrichment in interferon-related signaling pathways. Interferon regulatory factor 7 (IRF7), a transcription factor within this pathway, showed the highest percentage of enrichment and was primarily localized in the core region of wild-type IDH tumors. A machine-learning prognostic model identified novel subgroups within the wild-type IDH population. Additionally, IRF7 was shown to promote the proliferation and migration of T98G and U251 cells in vitro, and its knockdown affected glioma cell proliferation in vivo. This study systematically established the regulatory mechanism of IDH transcriptional activity in gliomas at the single-cell level and drew a corresponding cell map. The study presents a transcriptional regulatory activity map for IDH wild-type gliomas, involving single-cell RNA sequencing and spatial transcriptomics to identify gene regulatory networks, machine learning models for IDH subtyping, and experimental validation, highlighting the role of IRF7 in glioma progression.

E242-E261 region of MYC regulates liquid-liquid phase separation and tumor growth by providing negative charges

MYC is one of the most extensively studied oncogenic proteins and is closely associated with the occurrence and progression of many tumors. Previous studies have shown that MYC regulates cell fate through its liquid-liquid phase separation (LLPS) mechanism, which is dependent on two disordered domains within its N-terminal transcriptional activation regions. In this study, we revealed that the negatively charged conserved region (E242-E261) of the MYC protein controls its condensation formation and irreversible aggregation through multivalent electrostatic interactions (MEIs). Furthermore, deletion or mutation of the E242-E261 amino acids in the MYC protein enhances the transcriptional function of MYC by altering its aggregation capacity and subsequently promoting cancer cell proliferation. The discovery of the negatively charged region and its regulatory action on the phase separation of MYC provides a new understanding on the aggregation and function of MYC.

Discovery of a Small-Molecule Inhibitor Targeting the Biofilm Regulator BrpT in Vibrio vulnificus.

Vibrio vulnificus, an opportunistic human pathogen, employs biofilm formation as a key survival and virulence mechanism. BrpT, a transcriptional regulator, is essential for V. vulnificus biofilm development by regulating the expression of biofilm-related genes. In this study, we aimed to identify a small molecule inhibitor of BrpT to combat V. vulnificus biofilm formation. High-throughput screening of 7,251 compounds using an Escherichia coli reporter strain carrying the arabinose-inducible brpT gene and a BrpT-activated promoter fused to the luxCDABE operon identified a hit compound, BTI (BrpT Inhibitor). BTI potently inhibited BrpT activity in V. vulnificus (EC50 of 6.48 μM) without affecting bacterial growth or host cell viability. Treatment with BTI significantly reduced the expression of the BrpT regulon and impaired biofilm formation and colony rugosity in V. vulnificus, thus increasing its susceptibility to antibiotics. In vitro biochemical analyses revealed that BTI directly binds to BrpT and inhibits its transcriptional regulatory activity. The identification of BTI as a specific inhibitor of BrpT that effectively diminishes V. vulnificus biofilm formation provides a promising foundation for the development of novel anti-biofilm strategies, with the potential to address the growing challenge of antibiotic resistance and improve the treatment of biofilm-associated infections.

Comparative transcriptome analysis of Aspergillus niger revealed its biocontrol mechanisms in response to the guava wilt pathogen Fusarium oxysporum f. sp. psidii

Wilt is one of the major destructive diseases in guava cultivation worldwide. Biocontrol agent Aspergillus niger has been recognized for its efficacy against various plant pathogens including guava wilt disease. This biocontrol fungus has been widely studied for its mycoparasitic and antibiosis mechanisms. Still, a notable gap exists in the literature regarding the molecular mechanisms and biosynthetic pathways underlying biocontrol activity when challenged with guava wilt pathogen. In this investigation, we conducted comprehensive global RNA sequencing, which generated a total of 35.16 GB data, comprising 18–22 million reads having 52–54 % GC content. Over 55,464 transcripts displayed differential expression profiles during the interaction of biocontrol and the pathogen, out of that 5285 transcripts were expressed at FC2 and Pval 0.05. They were mainly associated with catalytic activity (48.45 %), protein or DNA binding activity (44.9 %), transporter activity (8.2 %), and transcription regulator activity (5.5 %). In the biological process category, 36.5 % and 34.7 % of transcripts were linked to "metabolic" and "cellular processes," respectively. The upregulated DEGs were predominantly linked to hydrolytic activity and secondary metabolite production, encompassing AB hydrolases, cyanate hydratase, Isochorismatase hydrolase, galacturan 1,4-alpha-galacturonidase B, Esterase/Lipase, MFS monosaccharide transporter, ATP-binding cassette transporter, polyamine transporter 3, endoglucanase A, and non-ribosomal siderophore peptide synthase (SidC). Our investigation highlighted that the biocontrol mechanism primarily involves the active participation of hydrolytic enzymes, transporters, transcription factors, and secondary metabolites. Additionally, the data were validated using RT-qPCR, affirming the consistency of RNA-seq findings. This study represents the inaugural effort to elucidate the biocontrol mechanism of A. niger against F. oxysporum f. sp. psidii using the RNA-seq approach. The findings shed light on the antagonistic mechanisms of A. niger at the molecular level, enhancing our comprehension of A. niger as a biocontrol agent and deepening our exploration of biocontrol and pathogen interactions.

IGFBP7 promotes the proliferation and differentiation of primary myoblasts and intramuscular preadipocytes in chicken

Though it is well known that insulin-like growth factor (IGF) binding protein 7 (IGFBP7) plays an important role in myogenesis and adipogenesis in mammals, its impact on the proliferation, differentiation, and lipid deposition in chicken primary myoblasts (CPM) and intramuscular preadipocytes remains unexplored. In the present study, we firstly examined the correlation between SNPs within the genomic sequence of the IGFBP7 gene and carcass and blood chemical traits in a F2 resource population by genetic association analysis, and found that a significant correlation between the SNP (4_49499525) located in the intron region of IGFBP7 and serum high-density lipoproteins (HDL). We then examined the expression patterns of IGFBP7 across different stages of proliferation and differentiation in CPMs and intramuscular preadipocytes via qPCR, and explored the biological functions of IGFBP7 through gain- and loss-of-function experiments and a range of techniques including qPCR, CCK-8, EdU, flow cytometry, Western blot, immunofluorescence, and Oil Red O staining to detect the proliferation, differentiation, and lipid deposition in CPMs and intramuscular preadipocytes. We ascertained that the expression levels of the IGFBP7 gene increased as cell differentiation progresses in CPMs and intramuscular preadipocytes, and that IGFBP7 promotes the proliferation and differentiation of these cells, as well as facilitates intracellular lipid deposition. Furthermore, we investigated the regulatory mechanism of IGFBP7 expression by using co-transfection strategy and dual-luciferase reporter assay, and discovered that the myogenic transcription factors (MRF), myoblast determination factor (MyoD) and myogenin (MyoG), along with the adipocyte-specific transcription factor (TF) CCAAT/enhancer-binding protein α (C/EBPα), can bind to the core transcription activation region of the IGFBP7 promoter located 500 bp upstream from the transcription start site, thereby promoting IGFBP7 transcription and expression. Taken together, our study underscores the role of IGFBP7 as a positive regulator for myogenesis and adipogenesis, while also elucidating the functional and transcriptional regulatory mechanisms of IGFBP7 in chicken skeletal muscle development and intramuscular adipogenesis.

TaNAC1 boosts powdery mildew resistance by phosphorylation-dependent regulation of TaSec1a and TaCAMTA4 via PP2Ac/CDPK20.

The integrity of wheat (Triticum aestivum) production is increasingly jeopardized by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), particularly amid the vicissitudes of climate change. Here, we delineated the role of a wheat transcription factor, TaNAC1, which precipitates cellular apoptosis and fortifies resistance against Bgt. Utilizing BiFC, co-immunoprecipitation, protein quantification, luciferase report assays, we determined that cytoplasmic TaNAC1-7A undergoes phosphorylation at the S184/S258 sites by TaCDPK20, facilitating its nuclear translocation. This migration appears to prime further phosphorylation by TaMPK1, thereby enhancing transcriptional regulatory activity. Notably, the apoptotic activity of phosphorylated TaNAC1-7A is negatively modulated by the nuclear protein phosphatase PP2Ac. Furthermore, activation of TaNAC1 phosphorylation initiates transcription of downstream genes TaSec1a and TaCAMTA4, through binding to the C[T/G]T[N7]A[A/C]G nucleic acid motif. Suppression of TaNAC1, TaCDPK20, and TaMPK1 in wheat compromises its resistance to Bgt strain E09, whereas overexpression of TaNAC1 and silencing of PP2Ac markedly elevate resistance levels. Our results reveal the pivotal role of TaNAC1 in basal resistance which is mediated by its effects on homotypic fusion, vacuolar protein sorting, and the expression of defense-related genes. The findings highlight the potential through targeting TaNAC1 and its regulators as a strategy for improving wheat's resistance to fungal pathogens.

Dicranopteris dichotoma rhizosphere-derived Bacillus sp. MQB12 acts as an enhancer of plant growth via increasing phosphorus utilization, hormone synthesis, and rhizosphere microbial abundance

In recent years, microbial inoculants have showed a great potential to replace chemical fertilizers as a new generation of soil amendment agents, however, the understanding of their effects on nutrient cycling within plants and rhizosphere microbial diversity are still limited. In this study, the rhizosphere growth-promoting bacteria MQB12 was used to inoculate Vigna radiata to evaluate the effects of external inoculants on plant transcriptomics and rhizosphere soil microbial diversity. Enrichment analysis using GO and KEGG revealed significant enrichment in DNA-binding transcription factor activity, transcriptional regulatory factor activity, and phenylpropanoid biosynthesis among the differentially expressed genes. MQB12 inoculation positively responded to phosphorus starvation response, increased the expression of phosphorus starvation response genes (PHT/PAP), enhanced the synthesis of ethylene and salicylic acid to cope with external stress, and improved the expression of plant disease resistance genes to strengthen the disease resistance of plants to pathogens. At the same time, microbial diversity analysis further revealed the positive effect of MQB12 inoculum. MQB12 inoculum enriched beneficial flora, improved flora abundance, changed the structure and diversity of V. radiata rhizosphere microbial community, enhanced the interconnections between the flora, and positively promoted growth. MQB12 was found to adjust the microflora of the rhizosphere, which subsequently changed the environment for plant colonization. This change led to the enrichment of beneficial bacteria and removal of pathogenic bacteria, which positively affected the internal pathways of plants. Additionally, changes in gene expression levels of plants resulted in the formation of different phenotypes and various metabolites, further influencing the formation of rhizosphere microbial communities through close contact between roots and soil. This study provides new insights into the effects of microbial agents on plant growth and root environment construction and is conducive to the further development and application of microbial agents.Graphical

Chrom-seq identifies RNAs at chromatin marks.

Chromatin marks are associated with transcriptional regulatory activities. However, very few lncRNAs have been characterized with the role in regulating epigenetic marks, largely due to the technical difficulty in identifying chromatin-associating RNA. Current methods are largely limited by the availability of ChIP-grade antibody and the crosslinking, which generates high noise. Here, we developed a method termed Chrom-seq to efficiently capture RNAs associated with various chromatin marks in living cells. Chrom-seq jointly applies highly specific chromatin mark reader with APEX2, which catalyzes the oxidation of biotin-aniline to label the adjacent RNAs for isolation by streptavidin-coated beads. Using the readers of mCBX7/dPC, mCBX1, and mTAF3, we detected RNA species significantly associated with H3K27me3, H3K9me3, and H3K4me3, respectively. We demonstrated that Chrom-seq outperformed other equivalent methods in terms of sensitivity, efficiency, and cost of practice. It provides an antibody-free approach to systematically map RNAs at chromatin marks with potential regulatory roles in epigenetic events.

Elucidating the molecular mechanisms in phytohormones-induced alleviation of postharvest physiological deterioration in cassava tuberous roots

Cassava is a staple food crop in tropical regions, serving as a significant source of carbohydrates. However, rapid postharvest physiological deterioration (PPD) significantly limits the shelf life and utilization of cassava. Despite its importance, the overlapping molecular mechanisms underlying phytohormones-induced PPD alleviation in cassava tuberous roots remain unknown. In this study, the phytohormones ethephon (ET), gibberellic acid (GA), and methyl jasmonate (MeJA) were applied to cassava tuberous roots after harvest. These treatments led to significantly reduced H2O2 accumulation and alleviation of PPD. Transcriptomic analysis revealed shared functions, including transcriptional regulation and oxidoreductase activity, among differentially expressed genes (DEGs) affected by ET, GA, or MeJA. A total of 305 genes encoding antioxidant enzymes were systematically identified, with 151 responsive to phytohormone treatments across ten families. Notably, the expression of 10 Glutathione S-transferase (GST) genes was induced by all three phytohormone treatments, while their expression was suppressed in the absence of phytohormones during PPD. By using co-expression network analysis, DNA-binding motif analysis, and experimental validation using yeast one-hybrid and dual-luciferase assays, a regulatory network involving MeMYB102 and MeZAT11 transcription factors (TFs) that directly promote the expression of MeGST42, MeGST43, MeGST44, and MeGST46 was established. Overall, this study enhances the understanding of the overlapping molecular mechanisms underlying phytohormones-induced alleviation of PPD in cassava, thereby providing genetic resources for developing PPD-resistant cassava.

Navβ2 Intracellular Fragments Contribute to Aβ1-42-Induced Cognitive Impairment and Synaptic Deficit Through Transcriptional Suppression of BDNF.

A pathological hallmark of Alzheimer's disease (AD) is the region-specific accumulation of the amyloid-beta protein (Aβ), which triggers aberrant neuronal excitability, synaptic impairment, and progressive cognitive decline. Previous works have demonstrated that Aβ pathology induced aberrant elevation in the levels and excessive enzymatic hydrolysis of voltage-gated sodium channel type 2 beta subunit (Navβ2) in the brain of AD models, accompanied by alteration in excitability of hippocampal neurons, synaptic deficits, and subsequently, cognitive dysfunction. However, the mechanism is unclear. In this research, by employing cell models treated with toxic Aβ1-42 and AD mice, the possible effects and potential mechanisms induced by Navβ2. The results reveal that Aβ1-42 induces remarkable increases in Navβ2 intracellular domain (Navβ2-ICD) and decreases in both BDNF exons and protein levels, as well as phosphorylated tropomyosin-related kinase B (pTrkB) expression in cells and mice, coupled with cognitive impairments, synaptic deficits, and aberrant neuronal excitability. Administration with exogenous Navβ2-ICD further enhances these effects induced by Aβ1-42, while interfering the generation of Navβ2-ICD and/or complementing BDNF neutralize the Navβ2-ICD-conducted effects. Luciferase reporter assay verifies that Navβ2-ICD regulates BDNF transcription and expression by targeting its promoter. Collectively, our findings partially elucidate that abnormal enzymatic hydrolysis of Navβ2 induced by Aβ1-42-associated AD pathology leads to intracellular Navβ2-ICD overload, which may responsible to abnormal neuronal excitability, synaptic deficit, and cognition dysfunction, through its transcriptional suppression on BDNF. Therefore, this work supplies novel evidences that Navβ2 plays crucial roles in the occurrence and progression of cognitive impairment of AD by transcriptional regulatory activity of its cleaved ICD.

ROK family regulator NagC promotes prodigiosin biosynthesis independent of N-acetylglucosamine in Serratia sp. ATCC 39006.

The ROK family transcription factor NagC is an important global regulator in the γ-proteobacteria. A large number of genes involved in the transport and metabolism of sugars, as well as those associated with biofilm formation and pathogenicity, are regulated by NagC. In all of these regulations, the transcriptional regulatory activity of NagC responds to the supply of GlcNAc in the environment. Here, we found for the first time that NagC can regulate antibiotic biosynthesis, whose transcriptional regulatory activity is independent of GlcNAc. This suggests that NagC may respond to more signals and regulate more physiological processes in Gram-negative bacteria.

Single-cell and spatial multiomic inference of gene regulatory networks using SCRIPro.

The burgeoning generation of single-cell or spatial multiomic data allows for the characterization of gene regulation networks (GRNs) at an unprecedented resolution. However, the accurate reconstruction of GRNs from sparse and noisy single-cell or spatial multiomic data remains challenging. Here, we present SCRIPro, a comprehensive computational framework that robustly infers GRNs for both single-cell and spatial multi-omics data. SCRIPro first improves sample coverage through a density clustering approach based on multiomic and spatial similarities. Additionally, SCRIPro scans transcriptional regulator (TR) importance by performing chromatin reconstruction and in silico deletion analyses using a comprehensive reference covering 1,292 human and 994 mouse TRs. Finally, SCRIPro combines TR-target importance scores derived from multiomic data with TR-target expression levels to ensure precise GRN reconstruction. We benchmarked SCRIPro on various datasets, including single-cell multiomic data from human B-cell lymphoma, mouse hair follicle development, Stereo-seq of mouse embryos, and Spatial-ATAC-RNA from mouse brain. SCRIPro outperforms existing motif-based methods and accurately reconstructs cell type-specific, stage-specific, and region-specific GRNs. Overall, SCRIPro emerges as a streamlined and fast method capable of reconstructing TR activities and GRNs for both single-cell and spatial multi-omic data. SCRIPro is available at https://github.com/wanglabtongji/SCRIPro. Supplementary data are available at Bioinformatics online.

Transcriptome analysis of East Asian common octopus, Octopus sinensis, paralarvae.

The genes involved in cephalopod development and their association with hatching and survival during early life stages have been extensively studied. However, few studies have investigated the paralarvae transcriptome of the East Asian common octopus (Octopus sinen sis). This study aimed to identify the genes related to embryonic development and hatching in O. sinensis using RNA sequencing (RNA-seq) and verify the genes most relevant to different embryonic stages. RNA samples from hatched and 25days post-hatching (dph) O. sinensis paralarvae were used to construct cDNA libraries. Clean reads from individual samples were aligned to the reference O. sinensis database to identify the differentially expressed genes (DEGs) between the 0- and 25-dph paralarvae libraries. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to supplement the RNA-seq data for embryogenic developmental stages. A total of 12,597 transcripts were annotated and 5,468 DEGs were identified between the 0- and 25-dph O. sinensis paralarvae, including 2,715 upregulated and 2,753 downregulated transcripts in the 25-dph paralarvae. Several key DEGs were related to transmembrane transport, lipid biosynthesis, monooxygenase activity, lipid transport, neuropeptide signaling, transcription regulation, and protein-cysteine S-palmitoyltransferase activity during the post-hatching development of O. sinensis paralarvae. RT-qPCR analysis further revealed that SLC5A3A, ABCC12, and NPC1 transcripts in 20 and/or 30days post-fertilization (dpf) embryos were significantly higher (p < 0.05) than those in 10-dpf embryos. Transcriptome profiles provide molecular targets to understand the embryonic development, hatching, and survival of O. sinensis paralarvae, and enhance octopus production.

Biocompatibility characterisation of CMOS-based Lab-on-Chip electrochemical sensors for in vitro cancer cell culture applications

Lab-on-Chip electrochemical sensors, such as Ion-Sensitive Field-Effect Transistors (ISFETs), are being developed for use in point-of-care diagnostics, such as pH detection of tumour microenvironments, due to their integration with standard Complementary Metal Oxide Semiconductor (CMOS) technology. With this approach, the passivation of the CMOS process is used as a sensing layer to minimise post-processing, and Silicon Nitride (Si3N4) is the most common material at the microchip surface. ISFETs have the potential to be used for cell-based assays however, there is a poor understanding of the biocompatibility of microchip surfaces. Here, we quantitatively evaluated cell adhesion, morphogenesis, proliferation and mechano-responsiveness of both normal and cancer cells cultured on a Si3N4, sensor surface. We demonstrate that both normal and cancer cell adhesion decreased on Si3N4. Activation of the mechano-responsive transcription regulators, YAP/TAZ, are significantly decreased in cancer cells on Si3N4 in comparison to standard cell culture plastic, whilst proliferation marker, Ki67, expression markedly increased. Non-tumorigenic cells on chip showed less sensitivity to culture on Si3N4 than cancer cells. Treatment with extracellular matrix components increased cell adhesion in normal and cancer cell cultures, surpassing the adhesiveness of plastic alone. Moreover, poly-l-ornithine and laminin treatment restored YAP/TAZ levels in both non-tumorigenic and cancer cells to levels comparable to those observed on plastic. Thus, engineering the electrochemical sensor surface with treatments will provide a more physiologically relevant environment for future cell-based assay development on chip.

The draft genome of Nitzschia closterium f. minutissima and transcriptome analysis reveals novel insights into diatom biosilicification

BackgroundNitzschia closterium f. minutissima is a commonly available diatom that plays important roles in marine aquaculture. It was originally classified as Nitzschia (Bacillariaceae, Bacillariophyta) but is currently regarded as a heterotypic synonym of Phaeodactylum tricornutum. The aim of this study was to obtain the draft genome of the marine microalga N. closterium f. minutissima to understand its phylogenetic placement and evolutionary specialization. Given that the ornate hierarchical silicified cell walls (frustules) of diatoms have immense applications in nanotechnology for biomedical fields, biosensors and optoelectric devices, transcriptomic data were generated by using reference genome-based read mapping to identify significantly differentially expressed genes and elucidate the molecular processes involved in diatom biosilicification.ResultsIn this study, we generated 13.81 Gb of pass reads from the PromethION sequencer. The draft genome of N. closterium f. minutissima has a total length of 29.28 Mb, and contains 28 contigs with an N50 value of 1.23 Mb. The GC content was 48.55%, and approximately 18.36% of the genome assembly contained repeat sequences. Gene annotation revealed 9,132 protein-coding genes. The results of comparative genomic analysis showed that N. closterium f. minutissima was clustered as a sister lineage of Phaeodactylum tricornutum and the divergence time between them was estimated to be approximately 17.2 million years ago (Mya). CAFF analysis demonstrated that 220 gene families that significantly changed were unique to N. closterium f. minutissima and that 154 were specific to P. tricornutum, moreover, only 26 gene families overlapped between these two species. A total of 818 DEGs in response to silicon were identified in N. closterium f. minutissima through RNA sequencing, these genes are involved in various molecular processes such as transcription regulator activity. Several genes encoding proteins, including silicon transporters, heat shock factors, methyltransferases, ankyrin repeat domains, cGMP-mediated signaling pathways-related proteins, cytoskeleton-associated proteins, polyamines, glycoproteins and saturated fatty acids may contribute to the formation of frustules in N. closterium f. minutissima.ConclusionsHere, we described a draft genome of N. closterium f. minutissima and compared it with those of eight other diatoms, which provided new insight into its evolutionary features. Transcriptome analysis to identify DEGs in response to silicon will help to elucidate the underlying molecular mechanism of diatom biosilicification in N. closterium f. minutissima.

Integrative Single-Cell Analysis of Cardiomyopathy Identifies Differences in Cell Stemness and Transcriptional Regulatory Networks among Fibroblast Subpopulations.

Cardiomyopathy encompasses a broad spectrum of diseases affecting myocardial tissue, characterized clinically by abnormalities in cardiac structure, heart failure, and/or arrhythmias. Clinically heterogeneous, major types include dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RM), ischemic cardiomyopathy (ICM), among which DCM is more prevalent, while ICM exhibits higher incidence and mortality rates. Myocardial injury during cardiomyopathy progression may lead to myocardial fibrosis. Failure to intervene early and inhibit the process of myocardial fibrosis may culminate in heart failure. Cardiac fibroblasts constitute crucial cellular components determining the extent and quality of myocardial fibrosis, with various subpopulations exerting diverse roles in cardiomyopathy progression. Despite this, understanding of the cellular plasticity and transcriptional regulatory networks of cardiac fibroblasts in cardiomyopathy remains limited. Therefore, in this study, we conducted comprehensive single-cell analysis of cardiac fibroblasts in cardiomyopathy to explore differences in cellular plasticity and transcriptional regulatory networks among fibroblast subpopulations, with the aim of providing as many useful references as possible for the diagnosis, prognosis, and treatment of cardiomyopathy. Cells with mitochondrial gene expression comprising >20% of total expressed genes were excluded. Differential expression genes (DEGs) and stemness genes within cardiac fibroblast subpopulations were subjected to Gene Ontology (GO) analysis of biological processes (BP) and AUCell analysis. Monocle software was employed to analyze the pseudo-temporal trajectory of cardiac fibroblasts in cardiomyopathy. Additionally, the Python package SCENIC was utilized to assess enrichment of transcription factors and activity of regulators within cardiac fibroblast subpopulations in cardiomyopathy. Following batch effect correction, 179,927 cells were clustered into 32 clusters, designated as T_NK cells, endothelial cells, myeloid cells, fibroblasts, pericytes, SMCs, CMs, proliferating cells, EndoCs, and EPCs. Among them, 8148 fibroblasts were further subdivided into 4 subpopulations, namely C0 THBS4+ Fibroblasts, C1 LINC01133+ Fibroblasts, C2 FGF7+ Fibroblasts, and C3 AGT + Fibroblasts. Results from GO_BP and AUCell analyses suggest that C3 AGT + Fibroblasts may be associated with immune response activation, protein transport, and myocardial contractile function, correlating with disease progression in cardiomyopathy. Transcription factor enrichment analysis indicates that FOS is the most significant TF in C3 AGT + Fibroblasts, also associated with the M1 module, possibly implicated in protein hydrolysis, intracellular DNA replication, and cell proliferation. Moreover, correlation analysis of transcriptional regulatory activity between fibroblast subpopulations reveals a more pronounced heterogeneity within C3 AGT + Fibroblasts in cardiomyopathy. C3 AGT + Fibroblasts exhibit increased sensitivity towards adverse outcomes in cardiomyopathy, such as myocardial fibrosis and impaired cardiac contractile function, compared to other cardiac fibroblast subpopulations. The differential cellular plasticity and transcriptional regulatory activity between C3 AGT + Fibroblasts and other subgroups offer new perspectives for targeting fibroblast subpopulation activity to treat cardiomyopathy. Additionally, stemness genes EPAS1 and MYC, along with the regulator FOS, may play roles in modulating the biological processes of cardiac fibroblasts in cardiomyopathy.

Decoding the effect of photoperiodic cues in transducing kisspeptin-melatonin circuit during the pubertal onset in common carp.

This study unravels the intricate interplay between photoperiod, melatonin, and kisspeptin to orchestrate the pubertal onset of Common carp. Female fingerlings exposed to long days (LD) exhibited a hormonal crescendo, with upregulated hypothalamic-pituitary-ovarian (HPO) axis genes (kiss1, kiss1r, kiss2, gnrh2, gnrh3) and their downstream targets (lhr, fshr, ar1, esr1). However, the expression of the melatonin receptor (mtnr1a) diminished in LD, suggesting a potential inhibitory role. This hormonal symphony was further amplified by increased activity of key transcriptional regulators (gata1, gata2, cdx1, sp1, n-myc, hoxc8, plc, tac3, tacr3) and decreased expression of delayed puberty genes (mkrn1, dlk1). In contrast, short days (SD) muted this hormonal chorus, with decreased gnrh gene and regulator expression, elevated mtnr1a, and suppressed gonadal development. In in-vitro, estradiol mimicked the LD effect, boosting gnrh and regulator genes while dampening mtnr1a and melatonin-responsive genes. Conversely, melatonin acted as a conductor, downregulating gnrh and regulator genes and amplifying mtnr1a. Our findings illuminate the crucial roles of melatonin and kisspeptin as opposing forces in regulating pubertal timing. LD-induced melatonin suppression allows the kisspeptin symphony to flourish, triggering GnRH release and, ultimately, gonadal maturation. This delicate dance between photoperiod, melatonin, and kisspeptin orchestrates common carp's transition from juvenile to reproductive life.