Search Results (192)

The barbel chub (Squaliobarbus curriculus) exhibits remarkable resistance to grass carp reovirus (GCRV), a devastating pathogen in aquaculture. To reveal the molecular basis of this resistance, we investigated complement factor D (DF)—a rate-limiting serine protease governing alternative complement pathway activation. Molecular cloning revealed that the barbel chub DF (ScDF) gene encodes a 1251-bp cDNA sequence translating into a 250-amino acid protein. Crucially, bioinformatic characterization identified a unique N-glycosylation site at Asn¹³⁹ in ScDF, representing a structural divergence absent in grass carp (Ctenopharyngodon idella) DF (CiDF). While retaining a conserved Tryp_SPc domain harboring the catalytic triad (His⁶¹, Asp¹⁰⁹, and Ser²⁰⁴) and substrate-binding residues (Asp¹⁹⁸, Ser²¹⁹, and Gly²²¹), sequence and phylogenetic analyses confirmed ScDF’s evolutionary conservation, displaying 94.4% amino acid identity with CiDF and clustering within the Cyprinidae. Expression profiling revealed constitutive ScDF dominance in the liver, and secondary prominence was observed in the heart. Upon GCRV challenge in S. curriculus kidney (SCK) cells, ScDF transcription surged to a 438-fold increase versus uninfected controls at 6 h post-infection (hpi; p < 0.001)—significantly preceding the 168-hpi response peak documented for CiDF in grass carp. Functional validation showed that ScDF overexpression suppressed key viral capsid genes (VP2, VP5, and VP7) and upregulated the interferon regulator IRF9. Moreover, recombinant ScDF protein incubation induced interferon pathway genes and complement C3 expression. Collectively, ScDF’s rapid early induction (peaking at 6 hpi) and multi-pathway coordination may contribute to barbel chub’s GCRV resistance. These findings may provide molecular insights into the barbel chub’s high GCRV resistance compared to grass carp and novel perspectives for anti-GCRV breeding strategies in fish. Full article

Background: In the adaptive immune response of the dromedary (Camelus dromedarius, Camdro), the T cell receptor (TR) repertoire of the gamma–delta (γδ) T cells is unusually diversified both by somatic hypermutation in rearranged TR gamma (TRG) and delta (TRD) genes and by the diversity in sequence and length of the third complementarity-determining region (CDR3) of the TRD chain. Methods: The purpose was to investigate, in the absence of 3D structures, the role of Camdro γδ T cells, focusing on the binding interactions at the interface between the V-gamma and V-delta domains, and in complex with the CD1D, a major histocompatibily class I (MH1)-like glycoprotein presenting lipid antigen in association with B2M. A combination of hypermutated TRG dromedary cDNA clones was paired with TRD clones bearing very long, long, or short CDR3s, all isolated from the spleen of a single animal. Results: The 3D models of the Camdro TRG_TRD/CD1D_B2M complexes were inferred using the Homo sapiens 3D structure and the ImMunoGeneTics (IMGT) numbering for V, C, and G domains, and investigated for binding interactions at the interface of the paired V-gamma_V-delta and at the interface with CD1D. Our results suggest that transcripts with long CDR3s may derive from a population of CD1D-restricted γδ T cells. Both the CD1D G-alpha1-like and G-alpha-2 like domain helices were contacted by both the V-gamma and V-delta CDR-IMGT loops. Conclusions: Our findings further emphasize the similarity between the γδ T cells population we analyzed in Camelus dromedarius and the CD1D-restricted γδ NKT cells in Homo sapiens. Full article

Autolysis in sea cucumber has long been a threat to raw material storage and product processing. The involvement of endogenous cysteine protease in sea cucumber autolysis has been proved extendedly. However, as an essential part of the mechanism of autolysis, the role of its endogenous inhibitor has seldom been reported. To investigate the role of cysteine protease inhibitors in the early stage of hypoxic exposure-induced autolysis, a novel cystatin gene (SjCyt) belonging to the subfamily of cystatin C was cloned from Apostichopus japonicus by homology cloning and rapid amplification of cDNA ends. The affinity of SjCyt to cysteine protease (cathepsin L and cathepsin B) was investigated by molecular dynamics simulations. Pertinent metrics, including the root mean square deviation, radius of gyration, Gibbs free energy, binding free energy, and bond-forming frequency, showed that the conformation of SjCyt–SjCL was more stable and confirmed a stronger interaction of SjCyt with cathepsin L than with cathepsin B. Thus, cathepsin L (SjCL) was selected to further study its co-expression with SjCyt over a period of 9 h at an early stage of hypoxic exposure. Quantitative RT-qPCR revealed a ubiquitous transcriptional profile of SjCyt and SjCL in all the tested tissues, with the highest abundance in the dorsal epidermis, tube feet, and coelomocytes. Temporal transcription of them showed an overall up-regulated co-expression in the dorsal epidermis and tube feet. However, up-regulated SjCyt and down-regulated SjCL were observed at the protein level. Further immunofluorescence double labeling also found increased staining of SjCyt and SjCyt–SjCL complexes and decreased SjCL. Additionally, recombinant SjCyt was prepared and demonstrated an evident autolysis-inhibiting effect. The results of this study indicated that the anti-autolytic regulation of SjCyt functions at the very early stage of hypoxic exposure, exerting effects at both the transcriptional and translational levels. The above finding offers new insights into the mechanisms of sea cucumber autolysis. Full article

Glyoxalase I (GLYI) is a key enzyme that detoxifies methylglyoxal, a toxic byproduct of glycolysis, and is essential for plant pollination. However, the genome-wide identification and functional analysis of GLYI in Brassica rapa L. (B. rapa) remain limited. This study identified 17 BrGLYI genes (BrGLYI1–BrGLYI17) from the B. rapa genome. The self-compatible line 039-1 and the self-incompatible line GAU-28-5 were used as experimental materials, and Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) was performed to examine the effect of BrGLYI genes on self-compatibility in winter B. rapa. Preliminary results showed that BrGLYI13 exhibited significant tissue specificity, with higher expression in the flowers of 039-1 compared to GAU-28-5. The open reading frame of BrGLYI13 (852 bp) was cloned from both 039-1 and GAU-28-5 cDNA, with no base mutations observed between the two lines. RT-qPCR revealed higher BrGLYI13 expression in the stigma of 039-1 compared to GAU-28-5. Based on the functional conservation and sequence homology, BrGLYI13 is speculated to play a similar role to that of AtGLYI3 in methylglyoxal detoxification and stress response. Furthermore, the knockout of AtGLYI3 resulted in reduced silique lengths and seed numbers. These findings suggest that BrGLYI13 is involved in the self-compatibility response in B. rapa and promotes the silique length and seed number in the Arabidopsis mutant, providing a basis for further research on the mechanisms of self-compatibility in B. rapa. Full article

The thioredoxin (Trx) system is one of the most significant systems in living organisms as it regulates cellular redox reactions and plays a pivotal protective role within the cell by promoting redox homeostasis. Trx and thioredoxin reductase (TrxR) are the core oxidoreductases of the Trx system. In this study, the novel full-length cDNAs of LvTrx2 and LvTrxR were cloned from Litopenaeus vannamei. The ORFs of LvTrx2 and LvTrxR were 453 bp and 1785 bp, encoding polypeptides consisting of 150 and 596 amino acids. Sequence alignment analysis revealed that the amino acid sequence of LvTrx2 shared a high degree of identity (93%) with that of Penaeus chinensis, while in LvTrxR, it exhibited a similarity level of 95% with previously submitted Penaeus chinensis and Penaeus monodon sequences. Regarding tissue-specific expression patterns, LvTrx2 showed its highest expression levels in hepatopancreas and gill. For LvTrxR, the highest expression was observed in gill followed by hepatopancreas and intestine. During exposure to ammonia-N, there was a significant upregulation in the relative mRNA levels of LvTrx2 and LvTrxR in hepatopancreas and gill, with the peak values occurring at 24 h or 48 h of exposure. After LPS injection, the LvTrx2 and LvTrxR transcripts in hepatopancreas and gill had different upregulated levels. These findings suggest that LvTrx2 and LvTrxR play pivotal roles in enhancing stress resistance and bolstering antibacterial defense mechanisms in L. vannamei. To explore the roles, LvTrx2 expression was knocked down in vivo to verify the defense mechanism against 4-NP stress. LvTrx2 silencing in 4-NP-challenged shrimp could significantly induce the gene expression of antioxidant-related genes (except for LvTrxR) and aggravate the oxidative damage of lipids. This study suggests that the Trx system is involved in regulating the antioxidant processes, and LvTrx2 and LvTrxR play a vital role in defense responses against environmental stress. Full article

5-Hydroxytryptamine (5-HT) plays a vital role in the reproductive process of vertebrates and is also present in many invertebrates. The cDNA of the Sepiella japonica 5-HT₆ receptor (Sj5-HT₆r) was first cloned by RACE (Rapid Amplification of cDNA Ends). The length was 1450 bp, and the predicted open reading frame (ORF) was 1116 bp, which encoded 371 amino acids. Sequence characteristics analysis showed that Sj5-HT₆r shares a high degree of identity with 5-HT₆r from other cephalopods and forms a sister branch to bivalves. Subcellular localization showed that Sj5-HT₆r protein was localized on the HEK293T cell membrane surface. Quantitative Real-time PCR (qPCR) analysis demonstrated that Sj5-HT₆r was highly expressed in reproductive organs of both sexes. In particular, transcripts with significant expression were observed at stage III of female gonadal development in tissues of the ovary and nidamental gland, and at stage IV in tissues of the accessory nidamental gland. In situ hybridization (ISH) experiment results indicated that Sj5-HT₆r mRNA was primarily distributed in all regions of the optic lobes except the plexiform zone. These results may provide a basis for the future exploration of the reproductive regulation of 5-HT and 5-HT₆ receptors in S. japonica. Full article

Retroviruses, like other RNA viruses, mutate at very high rates and exist as genetically heterogeneous populations. The error-prone activity of viral reverse transcriptase (RT) is largely responsible for the observed variability, most notably in HIV-1. In addition, RTs are widely used in biotechnology to detect RNAs and to clone expressed genes, among many other applications. The fidelity of retroviral RTs has been traditionally analyzed using enzymatic (gel-based) or reporter-based assays. However, these methods are laborious and have important limitations. The development of next-generation sequencing (NGS) technologies opened the possibility of obtaining reverse transcription error rates from a large number of sequences, although appropriate protocols had to be developed. In this review, we summarize the developments in this field that allowed the determination of RNA-dependent DNA synthesis error rates for different RTs (viral and non-viral), including methods such as PRIMER IDs, REP-SEQ, ARC-SEQ, CIR-SEQ, SMRT-SEQ and ROLL-SEQ. Their advantages and limitations are discussed. Complementary DNA (cDNA) synthesis error rates obtained in different studies, using RTs and RNAs of diverse origins, are presented and compared. Future improvements in methodological pipelines will be needed for the precise identification of mutations in the RNA template, including modified bases. Full article

Vitellogenins (Vtgs) are key yolk precursor proteins in fish, serving as critical indicators of gonadal maturation in females and reliable biomarkers for detecting xeno-oestrogenic pollution, particularly through their expression in juveniles or males. The vtg gene family comprises multiple subtypes that are species-specific, necessitating precise characterisation and quantification for effective use as biomarkers in studies on estrogenic endocrine-disrupting chemicals (EEDCs). In this study, we successfully cloned and characterised the full-length cDNAs of three vtg subtypes (vtgAa, vtgAb, and vtgC) from Scatophagus argus. Differential expression analysis revealed that vtgAb exhibited the highest responsiveness to 17α-ethynylestradiol (EE2) exposure, with a 3-fold increase in vivo at 10.0 μg/g EE2 and a 30-fold increase in vitro at 10⁻⁷ mol/L EE2. The expression patterns were dose- and time-dependent, with peak expression observed 72 h post-exposure. While in vivo assays indicated moderate upregulation, in vitro experiments demonstrated significantly higher expression, attributed to direct hepatocyte interaction with EE2. These findings confirm vtgAb as the most responsive subtype to oestrogen exposure in S. argus and highlight the species’ tolerance to EE2, as compared to more sensitive species like Danio rerio. This study shows the evolutionary conservation of vtg transcripts across teleost species and reinforces the importance of subtype-specific characterisation to advance their application as biomarkers for EEDCs, with significant implications for environmental monitoring and pollution regulation. Full article

The determinate inflorescence trait of Brassica napus L. is associated with various desirable agricultural characteristics. BnTFL1s (BnaA10.TFL1 and BnaC09.TFL1), which encode the transcription factor TERMINAL FLOWER 1 (TFL1), have previously been identified as candidate genes controlling this trait through map-based cloning. However, the mechanism underlying the effects of the BnTFL1 proteins remains unclear. Further, proteins generally interact with each other to fulfill their biological functions. The objective of this study was to construct a cDNA library of the shoot apical meristem (SAM) of B. napus and screen for proteins that interact with BnTFL1s, to better understand its mechanism of action. The recombination efficiency of the yeast two-hybrid (Y2H) library that we constructed was 100%, with insertion fragment lengths ranging from 750 to 2000 bp and a capacity of approximately 1.44 × 10⁷ CFUs (colony-forming units), sufficient for screening protein interactions. Additionally, the bait vector pGBKT7-BnTFL1s was transformed into yeast cells alongside positive and negative controls, demonstrating no toxicity to the yeast cells and no self-activation. This bait was used to screen the SAM cDNA library of B. napus, ultimately identifying two BnTFL1s-interacting proteins: 14-3-3-like protein GF14 omega GRF2. These interactions were verified through one-to-one interaction experiments. This study provides a foundation for further research on the biological functions of the BnTFL1s genes and their regulatory role in inflorescence formation in B. napus, while providing a reference for studying similar mechanisms in other plants. Full article

Rice lesion mimic mutants are important materials for studying the mechanisms of cell death. In-depth research on these mutants can provide insights into the molecular mechanisms underlying rice growth and development, offering a theoretical basis for crop improvement. In this study, rice variety Wuyunjing 21 (WYJ21) was mutagenized with ethyl methanesulfonate to obtain a lesion mimic mutant, lmm28. Unlike wild-type (WT) plants, the lmm28 mutant exhibits brown lesions on the leaves starting from the early tillering stage. The size of the lesions increases as the plant grows. Additionally, the lmm28 mutant shows significantly reduced plant height, tiller number, number of effective panicles, seed setting rate, and 1000-grain weight compared to the WT. Leaf staining of the mutant revealed an accumulation of reactive oxygen species and cell death in the lesion leaves. Transmission electron microscopy images showed that, in the lmm28 mutant, the nuclear boundaries in leaf cells became indistinct and damage to the chloroplast membrane structures was observed, with thylakoid disorganization occurring in some chloroplasts. Genetic analysis and map-based cloning localized the candidate gene of the mutant to a 167.79 kb region on chromosome 5. After analyzing the annotated genes within this region, the candidate gene was preliminarily identified as OsBON3. Sequencing analysis revealed that, in lmm28, a base change from GT to GC occurred at the 5′ splice junction of the 15th intron of OsBON3. Further analysis, using cDNA amplification of exons 14–16 followed by sequencing, showed that the mutation at the splice recognition site caused the incorrect splicing of OsBON3 pre-mRNA, leading to an increased number of transcripts in lmm28. The transcript containing an inserted intron is present at much higher levels than the normal transcript, which may lead to a reduction in the protein levels containing the functional vWA domain. Therefore, the vWA domain of OsBON3 is likely crucial for maintaining ROS homeostasis in rice and plays a key role in regulating its growth and development. Full article

The previously available coding region for the spiny dogfish (Squalus acanthias) AQP3-2 gene was amplified from cDNAs using PCR. Agarose gel electrophoresis gave a band of the AQP3-2 coding region, as well as multiple smaller splice variant bands. The main AQP3-2 band and the largest and most fluorescently intense pair of these splice variant bands were cloned and sequenced. Amplifications were performed on a range of tissue cDNAs, but AQP3-2 was only expressed in the kidney and brain. Quantitative PCR amplifications using pre-existing kidney cDNA from an environmental salinity acclimation experiment showed that the abundance of mRNA from both the main AQP3-2 transcript and the largest splice variant (Splice Variant 1) was lower in 120% seawater (SW) acclimated fish, although only the values for Splice Variant 1 were statistically significant. A custom-made affinity-purified rabbit polyclonal AQP3-2 antibody was produced, and this gave four bands of around the correct sizes (which were 27 and 32 kDa) for the complete AQP3-2 and Splice Variant 1 proteins. Two of the bands may have been N-glycosylated forms of these proteins. Other bands were also present on the Western blot. No bands were present when the antibody was pre-blocked by the peptide antigen. In tissue sections of the dogfish kidney, immunohistochemical localization experiments showed that AQP3-2 was expressed in the early distal tubule (EDT) and late distal tubule (LDT) nephron segments. The results suggest that AQP3-2 may be involved in cell volume regulation in the EDT and water and urea absorption in the LDT nephron segment. Full article

Mycobacterium avium subsp. paratuberculosis (MAP) is known to cause paratuberculosis. One notable protein, MAP3773c, plays a critical role in iron metabolism as a transcription factor. This study aims to investigate the binding affinity of MAP3773c to the chromatin of the Ferroportin1 (FPN1) gene in murine macrophage J774 A.1. We conducted a sequence alignment to identify potential interaction sites for MAP3773c. Following this, we used in silico analysis to predict binding interactions, complemented by electrophoretic mobility shift assay (EMSA) to confirm in vitro binding of MAP3773c. The map3773c gene was cloned into the pcDNA3.1 vector, with subsequent expression analysis carried out via Western blotting and real-time PCR. Chromatin immunoprecipitation (CHiP) assays were performed on transfected macrophages to confirm binding in the native chromatin context. Our in silico and in vitro analysis indicated that MAP3773c interacts with two binding motifs within the FPN1 coding region. The ChiP results provided additional validation, demonstrating the binding of MAP3773c to the FPN1 chromatin through successful amplification of the associated chromatin fragment via PCR. Our study demonstrated that MAP3773c binds to FPN1 and provides insight into the role of MAP3773c and its effect on host iron transport. Full article

Background: Schwann cells (SCs) and their plasticity contribute to the peripheral nervous system’s capacity for nerve regeneration after injury. The Egr2/Krox20 promoter antisense RNA (Egr2-AS) recruits chromatin remodeling complexes to inhibit Egr2 transcription following peripheral nerve injury. Methods: RNA-seq and ATAC-seq were performed on control cells, Lenti-GFP-transduced cells, and cells overexpressing Egr2-AS (Lenti-AS). Egr2 AS-RNA was cloned into the pLVX-DsRed-Express2-N1 lentiviral expression vector (Clontech, Mountain View, CA, USA), and the levels of AS-RNA expression were determined. Ezh2 and Wdr5 were immunoprecipitated from rat SCs and RT-qPCR was performed against AS-Egr2 RNA. ChIP followed by DNA purification columns was used to perform qPCR for relevant promoters. Hi-C, HiC-DC+, R, Bioconductor, and TOBIAS were used for significant and differential loop analysis, identifications of COREs and CORE-promotor loops, comparisons of TF activity at promoter sites, and identification of site-specific TF footprints. OnTAD was used to detect TADs, and Juicer was used to identify A/B compartments. Results: Here we show that a Neuregulin-ErbB2/3 signaling axis mediates binding of the Egr2-AS to YY1^Ser184 and regulates its expression. Egr2-AS modulates the chromatin accessibility of Schwann cells and interacts with two distinct histone modification complexes. It binds to EZH2 and WDR5 and enables targeting of H3K27me3 and H3K4me3 to promoters of Egr2 and C-JUN, respectively. Expression of the Egr2-AS results in reorganization of the global chromatin landscape and quantitative changes in the loop formation and contact frequency at domain boundaries exhibiting enrichment for AP-1 genes. In addition, the Egr2-AS induces changes in the hierarchical TADs and increases transcription factor binding scores on an inter-TAD loop between a super-enhancer regulatory hub and the promoter of mTOR. Conclusions: Our results show that Neuregulin-ErbB2/3-YY1 regulates the expression of Egr2-AS, which mediates remodeling of the chromatin landscape in Schwann cells. Full article

The 18-exon human TKFC gene codes for dual-activity triokinase and FMN cyclase (TKFC) in an ORF, spanning from exon 2 to exon 18. In addition to TKFC-coding transcripts (classified as tkfc type by their intron-17 splice), databases contain evidence for alternative TKFC transcripts, but none of them has been expressed, studied, and reported in the literature. A novel full-ORF transcript was cloned from brain cDNA and sequenced (accession no. DQ344550). It results from an alternative 3′ splice-site in intron 17. The cloned cDNA contains an ORF also spanning from exon 2 to exon 18 of the TKFC gene but with a 56-nt insertion between exons 17 and 18 (classified as tkfc_ins56 type). This insertion introduces an in-frame stop, and the resulting ORF codes for a shorter TKFC variant, which, after expression, is enzymatically inactive. TKFC intron-17 splicing was found to be differentially expressed in human tissues. In a multiple-tissue northern blot using oligonucleotide probes, the liver showed a strong expression of the tkfc-like splice of intron 17, and the heart preferentially expressed the tkfc_ins56-like splice. Through a comparison to global expression data from massive-expression studies of human tissues, it was inferred that the intestine preferentially expresses TKFC transcripts that contain neither of those splices. An analysis of transcript levels quantified by RNA-Seq in the GTEX database revealed an exception to this picture due to the occurrence of a non-coding short transcript with a tkfc-like splice. Altogether, the results support the occurrence of potentially relevant transcript variants of the TKFC gene, differentially expressed in human tissues. (This work is dedicated in memoriam to Professor Antonio Sillero, 1938–2024, for his lifelong mentoring and his pioneering work on triokinase). Full article

The aim of the present study was to investigate possible differences in the sensitivity of HNSCC cells to known EMT regulators. Three HNSCC cell lines (UM-SCC-1, -3, -22B) and the HaCaT control keratinocyte cell line were exposed to transforming growth factor beta 1 (TGF-β1), a known EMT master regulator, and the cellular response was evaluated by real-time cell analysis (RTCA), Western blot, quantitative PCR, flow cytometry, immunocytochemistry, and the wound closure (scratch) assay. Targeted sequencing on 50 cancer-related genes was performed using the Cancer Hotspot Panel v2. Mutant, and wild type SMAD4 cDNA was used to generate recombinant SMAD4 constructs for expression in mammalian cell lines. The most extensive response to TGF-β1, such as cell growth and migration, β-actin expression, or E-cadherin (CDH1) downregulation, was seen in cells with a more epithelial phenotype. Lower response correlated with higher basal p-TGFβ RII (Tyr424) levels, pointing to a possible autocrine pre-activation of these cell lines. Targeted sequencing revealed a homozygous SMAD4 mutation in the UM-SCC-22B cell line. Furthermore, PCR cloning of SMAD4 cDNA from the same cell line revealed an additional SMAD4 transcript with a 14 bp insertion mutation, which gives rise to a truncated SMAD4 protein. Overexpression of this mutant SMAD4 protein in the highly epithelial control cell line HaCaT resulted in upregulation of TGF-β1 and vimentin. Consistent with previous reports, the invasive and metastatic potential of HNSCC tumor cells appears associated with the level of autocrine secretion of EMT regulators such as TGF-β1, and it could be influenced by exogenous EMT cytokines such as those derived from immune cells of the tumor microenvironment. Furthermore, mutant SMAD4 appears to be a significant contributor to the mesenchymal transformation of HNSCC cells. Full article