Copies Of Sequence Research Articles

A review of the genetic background in complicated WT1-related disorders.

The Wilms tumor 1 (WT1) gene was first identified in 1990 as a strong candidate for conferring a predisposition to Wilms tumor. The WT1 protein has four zinc finger structures (DNA binding domain) at the C-terminus, which bind to transcriptional regulatory sequences on DNA, and acts as a transcription factor. WT1 is expressed during kidney development and regulates differentiation, and is also expressed in glomerular epithelial cells after birth to maintain the structure of podocytes. WT1-related disorders are a group of conditions associated with an aberrant or absent copy of the WT1 gene. This group of conditions encompasses a wide phenotypic spectrum that includes Denys-Drash syndrome (DDS), Frasier syndrome (FS), Wilms-aniridia-genitourinary-mental retardation syndrome, and isolated manifestations of nephropathy or Wilms tumor. The genotype-phenotype correlation is becoming clearer: patients with missense variants in DNA binding sites including C2H2 sites manifest DDS and develop early-onset and rapidly developing end-stage kidney disease. A deeper understanding of the genotype-phenotype correlation has also been obtained in DDS, but no such correlation has been observed in FS. The incidence of Wilms tumor is higher in patients with DDS and exon-truncating variants than in those with non-truncating variants. Here, we briefly describe the genetic background of this highly complicated WT1-related disorders.

Investigation on the molecular mechanism of SPA interference with osteogenic differentiation of bone marrow mesenchymal stem cells

Binding of Staphylococcus aureus protein A (SPA) to osteoblasts induces apoptosis and inhibits bone formation. Bone marrow-derived mesenchymal stem cells (BMSCs) have the ability to differentiate into bone, fat and cartilage. Therefore, it was important to analyze the molecular mechanism of SPA on osteogenic differentiation. We introduced transcript sequence data to screen out differentially expressed genes (DEGs) related to SPA-interfered BMSC. Protein–protein interaction (PPI) network of DEGs was established to screen biomarkers associated with SPA-interfered BMSC. Receiver operating characteristic (ROC) curve was plotted to evaluate the ability of biomarkers to discriminate between two groups of samples. Finally, we performed GSEA and regulatory analysis based on biomarkers. We identified 321 DEGs. Subsequently, 6 biomarkers (Cenpf, Kntc1, Nek2, Asf1b, Troap and Kif14) were identified by hubba algorithm in PPI. ROC analysis showed that six biomarkers could clearly discriminate between normal differentiated and SPA-interfered BMSC. Moreover, we found that these biomarkers were mainly enriched in the pyrimidine metabolism pathway. We also constructed '71 circRNAs-14 miRNAs-5 mRNAs' and '10 lncRNAs-5 miRNAs-2 mRNAs' networks. Kntc1 and Asf1b genes were associated with rno-miR-3571. Nek2 and Asf1b genes were associated with rno-miR-497-5p. Finally, we found significantly lower expression of six biomarkers in the SPA-interfered group compared to the normal group by RT-qPCR. Overall, we obtained 6 biomarkers (Cenpf, Kntc1, Nek2, Asf1b, Troap, and Kif14) related to SPA-interfered BMSC, which provided a theoretical basis to explore the key factors of SPA affecting osteogenic differentiation.

O-150 Identification and validation of RPL7 and RCCD1 as potential biomarkers associated with immune infiltration in patients with thin endometrium

Abstract Study question For patients with thin endometrium (TE), what potential genes could be utilized for exploring the underlying mechanisms and clinical therapy? Summary answer Ribosomal protein L7 (RPL7) and RCC1 domain containing 1 (RCCD1) were suggested as specific biomarkers for TE. What is known already TE is an important contributor to embryo implantation failure and female infertility. However, the pathogenesis and mechanism of TE are not clear, and consequently, there is a paucity of data on effective targeted treatments for patients with TE. Study design, size, duration Endometrial cells were collected from 18 women with TE and 18 normal controls for transcriptional sequencing. Experimental validation was based on other 10 endometrium samples, consisting of 6 TE samples and 4 controls. Participants/materials, setting, methods After performing transcriptional sequencing, differentially expressed genes (DEGs) were extracted between the two groups via bioinformatic analysis. Based on DEGs, we applied functional enrichment and LASSO analysis to identify potential biomarkers, whose expressions were then validated by quantitative real-time PCR and western blot. Immune infiltration as well as the correlation with immune cells was also explored in the study. Main results and the role of chance RPL7 and RCC1 were identified and validated to be potential biomarkers, which exhibited an overexpression in TE at both mRNA and protein levels. An abnormal immune environment was found in TE, and correlation analysis showed that the two candidate genes correlated with atypical regulation of immune cells. Limitations, reasons for caution It might not be adequate to evaluate TE by only considering the immunologic aspects. Moreover, prospective studies are required to determine whether RPL7 and RCC1 may be used for early clinical diagnosis and prediction of pregnancy outcomes. Wider implications of the findings Further understanding and better knowledge of the functions of RPL7 and RCCD1 can promote the development of targeted therapeutics to improve clinical outcomes of patients with TE. Trial registration number not applicable

Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein.

R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, precise RNA-mediated insertion of transgenes (PRINT), relies on the codelivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here, we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand the current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.

Optimizing molecular assays for glyphosate and ALS-inhibitor resistance diagnostics in four weed species

Herbicide-resistant weeds pose a threat to food production in modern agriculture, causing US$32 billion in crop production losses worldwide. In Michigan, highly troublesome and widespread weeds include waterhemp, Palmer amaranth, common ragweed, and horseweed, with accessions that are resistant to glyphosate (Group 9) and ALS-inhibitors (Group 2), major herbicide sites of action utilized in soybean and corn cropping systems. Molecular assays for rapid resistance diagnostics to confirm the in-field status of herbicide resistance can assist with more effective, timely, and proactive management. In this research, we developed and tested PCR-based assays to identify target site resistance mechanisms to both herbicide groups through Sanger sequencing and EPSPS copy number variation. Nine different SNPs were identified in five ALS positions known to confer herbicide resistance among all species surveyed. Pro197Ser was the most frequent in horseweed and common ragweed accessions, whereas Trp574Leu was the predominant mutation in Palmer amaranth and waterhemp. Four horseweed accessions contained the Pro106Ser mutation in the EPSPS gene, which confers resistance to glyphosate. Additionally, waterhemp and Palmer amaranth had 2–7 and 20–160 copies of EPSPS, respectively. The assays were validated by comparing genotyping of several field-collected accessions of unknown resistance status with known resistant and susceptible accessions. The efficacy of genotyping assays was >98% and required only two days, confirming that molecular assays are a robust tool for rapid resistance diagnostics. These assays can help growers evaluate herbicide resistance status in weed populations within the same growing season, allowing them to adopt effective management practices.

Detecting haplotype-specific transcript variation in long reads with FLAIR2

BackgroundRNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, the use of short-read RNA-seq has limited the community’s ability to interrogate both forms of RNA variation simultaneously.ResultsWe employ long-read sequencing technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We develop a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generate nanopore data with high sequence accuracy from H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We apply our workflow to identify key inosine isoform associations to help clarify the prominence of ADAR in tumorigenesis.ConclusionsUltimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.

Electroacupuncture activates the peroxisome proliferators-activated receptor pathway to improve thephenotype of cerebral palsy.

This study explores the efficacy of electroacupuncture (EA) in treating cerebral palsy (CP) in Sprague-Dawley (SD) pups, specifically CP animal models, and its molecular mechanisms. Gait analysis and Y-maze were used to detect the improvement of motor ability and cognitive function of CP rats after EA treatment. Transcription sequencing was used to determine the key pathway for EA to improve the symptoms of CP. PPAR agonists were used to verify the causal relationship between the pathway and the improvement of CP phenotype. The motor ability and cognitive function of CP pups were improved after EA treatment. The results of transcriptome sequencing suggest that the improvement of CP phenotype may be caused by the activation of PPAR pathway. PPAR pathway is widely activated in the epithelium of CP pups treated with EA, which is verified by qPCR. Rosiglitazone (Ros), a PPAR agonist, can improve CP phenotype while activating PPAR pathway, which proves the causal relationship between PPAR pathway activation and CP phenotype improvement. Our study demonstrated behavioral improvements and enhanced cognitive functions in CP models after EA treatment by activating PPAR pathway, suggesting new perspectives for CP rehabilitation, and providing theoretical support for acupuncture treatment of CP.

Transcriptomic analysis reveals nanoplastics-induced apoptosis, autophagy and immune response in Litopenaeus vannamei

Increasing attention is being paid to the toxic physiological effects of nanoplastics (NPs) on aquatic organisms. However, few studies have systematically evaluated the regulatory mechanisms of NPs on immune response in crustaceans. In this study, a 28-day chronic exposure experiment was conducted in which shrimps were exposed to various 80-nm polystyrene NPs concentrations (0, 0.1, 1, 5 and 10 mg/L). Transcriptomic analysis was used to investigate the regulatory mechanisms of NPs in immune response of Litopenaeus vannamei. With increasing NPs concentration, the total hemocyte count (THC) content decreased, while phagocytosis rate (PR) and respiratory burst (RB) showed trends of first rising and then falling. High concentration (10 mg/L) of NPs caused the destruction of hepatopancreas tissue structure, the shedding of microvilli, the increase number of hepatocyte apoptosis and autophagy structure. With increasing NPs concentration, the lysozyme (Lys), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities first increased and then decrease, while contents of lipid peroxidation and malondialdehyde increased; the expression levels of Toll, MyD88, GPx, SOD, proPO, Lys, and ALF generally increased at first and then decreased. Transcriptional sequencing analysis showed that the pathway of differentially expressed genes in KEGG enrichment mainly included lysosome (ko04142), apoptosis (ko04210) pathways, indicating that the NPs mainly affected the immune regulatory mechanism. Further analysis by Gene Set Enrichment Analysis (GSEA) showed that the up-regulation pathways of NPs activation mainly included immune response-related pathways such as mitochondrial autophagy, DNA repair, autophagosomes signaling pathway. Our results indicated that NPs exposure induced oxidative stress, apoptosis and autophagy in shrimps. This study provides a basis for further understanding of the mechanisms of antioxidant immune regulation by NPs in shrimp and may serve as a reference for healthy ecological culture of shrimp.

Comparison of the diagnostic significance of cerebrospinal fluid metagenomic next-generation sequencing copy number variation analysis and cytology in leptomeningeal malignancy

BackgroundDiagnosis and monitoring of leptomeningeal malignancy remain challenging, and are usually based on neurological, radiological, cerebrospinal fluid (CSF) and pathological findings. This study aimed to investigate the diagnostic performance of CSF metagenomic next-generation sequencing (mNGS) and chromosome copy number variations (CNVs) analysis in the detection of leptomeningeal malignancy.MethodsOf the 51 patients included in the study, 34 patients were diagnosed with leptomeningeal malignancies, and 17 patients were diagnosed with central nervous system (CNS) inflammatory diseases. The Sayk’s spontaneous cell sedimentation technique was employed for CSF cytology. And a well-designed approach utilizing the CSF mNGS-CNVs technique was explored for early diagnosis of leptomeningeal malignancy.ResultsIn the tumor group, 28 patients were positive for CSF cytology, and 24 patients were positive for CSF mNGS-CNVs. Sensitivity and specificity of CSF cytology were 82.35% (95% CI: 66.83-92.61%) and 94.12% (95% CI: 69.24-99.69%). In comparison, sensitivity and specificity of CSF mNGS-CNV were 70.59% (95% CI: 52.33-84.29%) and 100% (95% CI: 77.08-100%). There was no significant difference in diagnostic consistency between CSF cytology and mNGS-CNVs (p = 0.18, kappa = 0.650).ConclusionsCSF mNGS-CNVs tend to have higher specificity compared with traditional cytology and can be used as a complementary diagnostic method for patients with leptomeningeal malignancies.

Structural complexity of the white flower locus in Chrysanthemum

ABSTRACT The white flower trait in Chrysanthemum is attributed to the carotenoid cleavage dioxygenase 4a gene (CCD4a), a key domestication gene that contributed to generation of various flower colours in modern chrysanthemum cultivars. This trait is believed to be controlled by a single dominant locus. We studied the structure of this CCD4a locus utilising the whole genome sequence information of Chrysanthemum makinoi, a diploid white flower species, and found six homologous sequences of CCD4a, two of which being functional, and arranged in tandem in a 586-kb region on Chromosome 7. A qPCR analysis disclosed that white flower Chrysanthemum species possess at least two and as many as eight copies of CCD4a homologous sequences per monoploid genome, indicating high polymorphism of the CCD4a region. A detailed analysis of the structural diversity of the CCD4a region in Chrysanthemum could provide significant insights into the domestication trajectory of the cultivated chrysanthemum.

CapTrap-seq: a platform-agnostic and quantitative approach for high-fidelity full-length RNA sequencing

Long-read RNA sequencing is essential to produce accurate and exhaustive annotation of eukaryotic genomes. Despite advancements in throughput and accuracy, achieving reliable end-to-end identification of RNA transcripts remains a challenge for long-read sequencing methods. To address this limitation, we develop CapTrap-seq, a cDNA library preparation method, which combines the Cap-trapping strategy with oligo(dT) priming to detect 5’ capped, full-length transcripts. In our study, we evaluate the performance of CapTrap-seq alongside other widely used RNA-seq library preparation protocols in human and mouse tissues, employing both ONT and PacBio sequencing technologies. To explore the quantitative capabilities of CapTrap-seq and its accuracy in reconstructing full-length RNA molecules, we implement a capping strategy for synthetic RNA spike-in sequences that mimics the natural 5’cap formation. Our benchmarks, incorporating the Long-read RNA-seq Genome Annotation Assessment Project (LRGASP) data, demonstrate that CapTrap-seq is a competitive, platform-agnostic RNA library preparation method for generating full-length transcript sequences.

Transcriptome-Based Analysis of the Mechanism of Action of Metabolic Disorders Induced by Waterborne Copper Stress in Coilia nasus.

To reveal the effects of waterborne copper stress on gene expression changes, molecular pathways, and physiological functions in Coilia nasus, juvenile fish were equally divided into two experimental groups, and the copper levels were 1.61 ± 0.03 mg/L (copper-exposed group) and 0 mg/L (control group), respectively. After 4 h, gill tissue samples were collected for transcript sequencing analysis, and two libraries were constructed from the copper treatment group (Cu) and the control group (C) and sequenced using Illumina sequencing technology. The results showed that approximately 40.2-46.0 M clean reads were obtained from each library, and the percentage of uniquely mapped transcripts ranged from 80.57 to 84.93%. A total of 3915 differentially expressed genes (DEGs) were identified under waterborne copper stress, among which 1300 genes were up-regulated, and 2615 genes were down-regulated. Twelve DEGs were randomly selected for quantitative RT-PCR (qRT-PCR) analysis, and the results confirmed that the transcriptome analysis was reliable. Furthermore, the DEGs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and the results showed that most of the DEGs were involved in metabolic pathways, including steroid biosynthesis, glutathione metabolism, and peroxisome proliferator-activated receptor (PPAR) signaling pathways. Furthermore, due to the waterborne copper levels, gsk-3β was significantly up-regulated, while other metabolism-related genes (tor, pi3k, lpl, aqp7, fabp3) were significantly down-regulated. In addition, the copper-exposed group significantly reduced the expression of some immunity genes (ifn-γ, stat1, cxcl10, and tgf-β), and enhanced the expression of il-1β and tnf-α. In summary, these results indicated that copper causes metabolic disorders and insufficient energy supply in the body, and induces oxidative stress, which results in reduced immune functions.

Low-abundant but highly transcriptionally active uncharacterised Nitrosomonas drive ammonia-oxidation in the Brouage mudflat, France

Exploring differences in nitrification within adjacent sedimentary structures of ridges and runnels on the Brouage mudflat, France, we quantified Potential Nitrification Rates (PNR) alongside amoA genes and transcripts. PNR was lower in ridges (≈1.7 fold-lower) than runnels, despite higher (≈1.8 fold-higher) ammonia-oxidizing bacteria (AOB) abundance. However, AOB were more transcriptionally active in runnels (≈1.9 fold-higher). Sequencing of amoA genes and transcripts revealed starkly contrasting profiles with transcripts from ridges and runnels dominated (≈91 % in ridges and ≈98 % in runnels) by low abundant (≈4.6 % of the DNA community in runnels and ≈0.8 % in ridges) but highly active phylotypes. The higher PNR in runnels was explained by higher abundance of this group, an uncharacterised Nitrosomonas sp. cluster. This cluster is phylogenetically similar to other active ammonia-oxidizers with worldwide distribution in coastal environments indicating its potential, but previously overlooked, contribution to ammonia oxidation globally. In contrast DNA profiles were dominated by highly abundant but low-activity clusters phylogenetically distinct from known Nitrosomonas (Nm) and Nitrosospira (Ns). This cluster is also globally distributed in coastal sediments, primarily detected as DNA, and often classified as Nitrosospira or Nitrosomonas. We therefore propose to classify this cluster as Ns/Nm. Our work indicates that low abundant but highly active AOB could be responsible for the nitrification globally, while the abundant AOB Ns/Nm may not be transcriptionally active, and as such account for the lack of correlation between rate processes and gene abundances often reported in the literature. It also raises the question as to what this seemingly inactive group is doing?

Integrating Iso-seq and RNA-seq data for the reannotation of the greater amberjack genome

The greater amberjack is a very important fishery species with high commercial value, and it is distributed worldwide. Transcriptome-based studies on S. dumerili have been limited by an inadequate reference genome and a lack of well-annotated full-length transcripts. In this study, a total of 12 tissues from juvenile and adult fish both sexes were collected for next-generation RNA sequencing (RNA-seq) and full-length isoform sequencing (Iso-seq). For Iso-seq, a total of 163,218, 149,716, and 189,169 high-quality unique transcript sequences were obtained, with an N50 of 5,441, 5,255, and 5,939, from juvenile, adult male and adult female S. dumerili, respectively. We integrated the Iso-seq and RNA-seq data to construct a comprehensive gene annotation and systematically profiled the dynamics of gene expression across the 12 tissues. Our gene models had greater detail and accuracy than those from NCBI and Ensembl, with more precise polyA locations. These resources serve as a foundation for functional genomic studies and provide valuable insights into the molecular mechanisms underlying the development, reproduction and commercial traits of amberjack.

Misclassification of a frequent variant from PMS2CL pseudogene as a PMS2 loss of function variant in Brazilian patients.

PMS2, a Lynch Syndrome gene, presents challenges in genetic testing due to the existence of multiple pseudogenes. This study aims to describe a series of cases harboring a variant in the PMS2CL pseudogene that has been incorrectly assigned to PMS2 with different nomenclatures. We reviewed data from 647 Brazilian patients who underwent multigene genetic testing at a single center to identify those harboring the PMS2 V1:c.2186_2187delTC or V2:c.2182_2184delACTinsG variants, allegedly located at PMS2 exon 13. Gene-specific PCR and transcript sequencing was performed. Among the 647 individuals, 1.8% (12) carried the investigated variants, with variant allele frequencies ranging from 15 to 34%. By visually inspecting the alignments, we confirmed that both V1 and V2 represented the same variant and through gene-specific PCR and PMS2 transcript analysis, we demonstrated that V1/V2 is actually located in the PMS2CL pseudogene. Genomic databases (ExAC and gnomAD) report an incidence of 2.5 - 5.3% of this variant in the African population. Currently, V1 is classified as "uncertain significance" and V2 as "conflicting" in ClinVar, with several laboratories classifying them as "pathogenic". We identified a frequent African PMS2CL variant in the Brazilian population that is misclassified as a PMS2 variant. It is likely that V1/V2 have been erroneously assigned to PMS2 in several manuscripts and by clinical laboratories, underscoring a disparity-induced matter. Considering the limitations of short-read NGS differentiating between certain regions of PMS2 and PMS2CL, using complementary methodologies is imperative to provide an accurate diagnosis.

The effect of exogenous melatonin on waterlogging stress in Clematis.

Clematis is the queen of the vines, being an ornamental plant with high economic value. Waterlogging stress reduces the ornamental value of the plant and limits its application. Melatonin plays an important role in plant resistance to abiotic stresses. In this study, the physiological responses and gene expression levels of two wild species, namely, Clematis tientaiensis and Clematis lanuginosa, and two horticultural varieties, namely, 'Sen-No-Kaze' and 'Viva Polonia,' under waterlogging stress were analyzed to determine the effect of melatonin on waterlogging tolerance. The results showed that the waterlogging tolerances of C. lanuginosa and 'Sen-No-Kaze' were relatively poor, but were significantly improved by concentrations of 100 μmol·L-1 and 50 μmol·L-1 melatonin. C. tientaiensis and 'Viva Polonia' had relatively strong tolerance to waterlogging, and this was significantly improved by 200 μmol·L-1 melatonin. Under waterlogging stress, the relative conductivity and H2O2 content of Clematis increased significantly; the photosynthetic parameters and chlorophyll contents were significantly decreased; photosynthesis was inhibited; the contents of soluble protein and soluble sugars were decreased. Effective improvement of waterlogging tolerance after exogenous melatonin spraying, the relative conductivity was decreased by 4.05%-27.44%; the H2O2 content was decreased by 3.84%-23.28%; the chlorophyll content was increased by 35.59%-103.36%; the photosynthetic efficiency was increased by 25.42%-45.86%; the antioxidant enzyme activities of APX, POD, SOD, and CAT were increased by 28.03%-158.61%; the contents of proline, soluble protein, and soluble sugars were enhanced, and cell homeostasis was improved. Transcription sequencing was performed on wild Clematis with differences in waterlogging tolerance, and nine transcription factors were selected that were highly correlated with melatonin and that had the potential to improve waterlogging tolerance, among which LBD4, and MYB4 were significantly positively correlated with the antioxidant enzyme system, and bHLH36, DOF36, and WRKY4 were significantly negatively correlated. Photosynthetic capacity was positively correlated with DOF36 and WRKY4 while being significantly negatively correlated with MYB4, MOF1, DOF47, REV1 and ABR1. Melatonin could enhance the flooding tolerance of Clematis by improving photosynthetic efficiency and antioxidant enzyme activity. This study provides an important basis and reference for the application of melatonin in waterlogging-resistant breeding of Clematis.

Ckip-1 3’UTR alleviates prolonged sleep deprivation induced cardiac dysfunction by activating CaMKK2/AMPK/cTNI pathway

Sleep deprivation (SD) has emerged as a critical concern impacting human health, leading to significant damage to the cardiovascular system. However, the underlying mechanisms are still unclear, and the development of targeted drugs is lagging. Here, we used mice to explore the effects of prolonged SD on cardiac structure and function. Echocardiography analysis revealed that cardiac function was significantly decreased in mice after five weeks of SD. Real-time quantitative PCR (RT-q-PCR) and Masson staining analysis showed that cardiac remodeling marker gene Anp (atrial natriuretic peptide) and fibrosis were increased, Elisa assay of serum showed that the levels of creatine kinase (CK), creatine kinase-MB (CK-MB), ANP, brain natriuretic peptide (BNP) and cardiac troponin T (cTn-T) were increased after SD, suggesting that cardiac remodeling and injury occurred. Transcript sequencing analysis indicated that genes involved in the regulation of calcium signaling pathway, dilated cardiomyopathy, and cardiac muscle contraction were changed after SD. Accordingly, Western blotting analysis demonstrated that the cardiac-contraction associated CaMKK2/AMPK/cTNI pathway was inhibited. Since our preliminary research has confirmed the vital role of Casein Kinase-2 -Interacting Protein-1 (CKIP-1, also known as PLEKHO1) in cardiac remodeling regulation. Here, we found the levels of the 3’ untranslated region of Ckip-1 (Ckip-1 3’UTR) decreased, while the coding sequence of Ckip-1 (Ckip-1 CDS) remained unchanged after SD. Significantly, adenovirus-mediated overexpression of Ckip-1 3’UTR alleviated SD-induced cardiac dysfunction and remodeling by activating CaMKK2/AMPK/cTNI pathway, which proposed the therapeutic potential of Ckip-1 3’UTR in treating SD-induced heart disease.

Transcriptomic profiling and gene network analysis revealed regulatory mechanisms of bract development in Bougainvillea glabra

BackgroundBracts are important for ornamental plants, and their developmental regulation process is complex; however, relatively little research has been conducted on bracts. In this study, physiological, biochemical and morphological changes in Bougainvillea glabra leaves, leaf buds and bracts during seven developmental periods were systematically investigated. Moreover, transcriptomic data of B. glabra bracts were obtained using PacBio and Illumina sequencing technologies, and key genes regulating their development were screened.ResultsScanning electron microscopy revealed that the bracts develop via a process involving regression of hairs and a color change from green to white. Transcriptome sequencing revealed 79,130,973 bp of transcript sequences and 45,788 transcripts. Differential gene expression analysis revealed 50 expression patterns across seven developmental periods, with significant variability in transcription factors such as BgAP1, BgFULL, BgCMB1, BgSPL16, BgSPL8, BgDEFA, BgEIL1, and BgBH305. KEGG and GO analyses of growth and development showed the involvement of chlorophyll metabolism and hormone-related metabolic pathways. The chlorophyll metabolism genes included BgPORA, BgSGR, BgPPH, BgPAO and BgRCCR. The growth hormone and abscisic acid signaling pathways involved 44 and 23 homologous genes, and coexpression network analyses revealed that the screened genes BgAPRR5 and BgEXLA1 are involved in the regulation of bract development.ConclusionsThese findings improve the understanding of the molecular mechanism of plant bract development and provide important guidance for the molecular regulation and genetic improvement of the growth and development of ornamental plants, mainly ornamental bracts.

Whole transcriptome analysis to identify non-coding RNA regulators and hub genes in sperm of non-obstructive azoospermia by microarray, single-cell RNA sequencing, weighted gene co-expression network analysis, and mRNA-miRNA-lncRNA interaction analysis

BackgroundThe issue of male fertility is becoming increasingly common due to genetic differences inherited over generations. Gene expression and evaluation of non-coding RNA (ncRNA), crucial for sperm development, are significant factors. This gene expression can affect sperm motility and, consequently, fertility. Understanding the intricate protein interactions that play essential roles in sperm differentiation and development is vital. This knowledge could lead to more effective treatments and interventions for male infertility.Materials and methodsOur research aim to identify new and key genes and ncRNA involved in non-obstructive azoospermia (NOA), improving genetic diagnosis and offering more accurate estimates for successful sperm extraction based on an individual’s genotype.ResultsWe analyzed the transcript of three NOA patients who tested negative for genetic sperm issues, employing comprehensive genome-wide analysis of approximately 50,000 transcript sequences using microarray technology. This compared gene expression profiles between NOA sperm and normal sperm. We found significant gene expression differences: 150 genes were up-regulated, and 78 genes were down-regulated, along with 24 ncRNAs up-regulated and 13 ncRNAs down-regulated compared to normal conditions. By cross-referencing our results with a single-cell genomics database, we identified overexpressed biological process terms in differentially expressed genes, such as “protein localization to endosomes” and “xenobiotic transport.” Overrepresented molecular function terms in up-regulated genes included “voltage-gated calcium channel activity,” “growth hormone-releasing hormone receptor activity,” and “sialic acid transmembrane transporter activity.” Analysis revealed nine hub genes associated with NOA sperm: RPL34, CYB5B, GOL6A6, LSM1, ARL4A, DHX57, STARD9, HSP90B1, and VPS36.ConclusionsThese genes and their interacting proteins may play a role in the pathophysiology of germ cell abnormalities and infertility.

Transcriptional inhibition after irradiation occurs preferentially at highly expressed genes in a manner dependent on cell cycle progression.

In response to DNA double strand damage, ongoing transcription is inhibited to facilitate accurate DNA repair while transcriptional recovery occurs after DNA repair is complete. However, the mechanisms at play and identity of the transcripts being regulated in this manner are unclear. In contrast to the situation following UV damage, we found that transcriptional recovery after ionizing radiation (IR) occurs in a manner independent of the HIRA histone chaperone. Sequencing of the nascent transcripts identified a programmed transcriptional response, where certain transcripts and pathways are rapidly downregulated after IR, while other transcripts and pathways are upregulated. Specifically, most of the loss of nascent transcripts occurring after IR is due to inhibition of transcriptional initiation of the highly transcribed histone genes and the rDNA. To identify factors responsible for transcriptional inhibition after IR in an unbiased manner, we performed a whole genome gRNA library CRISPR / Cas9 screen. Many of the top hits in our screen were factors required for protein neddylation. However, at short times after inhibition of neddylation, transcriptional inhibition still occurred after IR, even though neddylation was effectively inhibited. Persistent inhibition of neddylation blocked transcriptional inhibition after IR, and it also leads to cell cycle arrest. Indeed, we uncovered that many inhibitors and conditions that lead to cell cycle arrest in G1 or G2 phase also prevent transcriptional inhibition after IR. As such, it appears that transcriptional inhibition after IR occurs preferentially at highly expressed genes in cycling cells.