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DDX3X regulates the translation of a subset of human transcripts containing complex 5' untranslated regions (5' UTRs). In this study, we developed the helicase activity reporter for translation (HART), which uses DDX3X-sensitive 5' UTRs to measure DDX3X-mediated translational activity in cells. To directly measure RNA structure in DDX3X-dependent mRNAs, we used SHAPE-MaP to determine the secondary structures present in DDX3X-sensitive 5' UTRs and then used HART to investigate how sequence alterations influence DDX3X sensitivity. Additionally, we identified residues 38-44 as potential mediators of DDX3X's interaction with the translational machinery. HART revealed that both DDX3X's association with the translational machinery and its helicase activity are required for its function in promoting the translation of DDX3X-sensitive 5' UTRs. These findings suggest DDX3X plays a crucial role in regulating translation through its interaction with the translational machinery during ribosome scanning and establish the HART reporter as a robust, lentivirally encoded, colorimetric measurement of DDX3X-dependent translation in cells.

Cryoelectron microscopy (cryo-EM) clarified the quaternary structure of the DNA complex of coactivator-bound estrogen receptor alpha (ERα), revealing the adjacency of the N-terminal domain (NTD) and C-terminal ligand-binding domain (LBD). ERα-NTD and LBD constitute activation function 1 (AF-1) and activation function 2 (AF-2), respectively. These domains are essential for transcription activation. Their spatial proximity was judged to be essential for ERα to recruit the SRC coactivator proteins. In the present study, we first evaluated untethered free ERα-NTD(AF-1) [residues 1–180] and its-truncated desNTD(AF-1)-ERα [residues 181–595] in a luciferase reporter gene assay. ERα-NTD(AF-1) was completely inactive, whereas desNTD(AF-1)-ERα exhibited 66% activity of wild-type ERα. Surprisingly, ERα-NTD(AF-1) was found to inhibit desNTD(AF-1)-ERα markedly. Therefore, assuming that ERα-NTD(AF-1) must also inhibit wild-type full-length ERα, we co-expressed ERα-NTD(AF-1) and full-length ERα. As expected, ERα-NTD(AF-1) inhibited ERα in a dose-dependent manner, but non-competitively for 17β-estradiol. When their intracellular transport was examined immunocytochemically, ERα-NTD(AF-1) showed a distinct translocation from the cytoplasm to the nucleus, despite being expressed solely in the cytoplasm without full-length ERα. This nuclear translocation was attributable to a direct interaction between ERα-NTD(AF-1) and full-length ERα consisting of the nuclear localization signal. The present results demonstrated that, in full-length ERα, the N-terminally tethered NTD(AF-1) domain collaborates with the C-terminal LBD(AF-2) for coactivator recruitment. [ABSTRACT FROM AUTHOR]

The CRISPR system finds extensive application in molecular biology, but its continuous activity can yield adverse effects. Leveraging programmable CRISPR/Cas9 function via nano-device mediation effectively mitigates these drawbacks. The integration of RNA-sensing platforms into CRISPR thus empowers it as a potent tool for processing internal cell data and modulating gene activity. Here, an intelligent guide RNA—a cis-repressed gRNA synthetic circuit enabling efficient recognition of specific trigger RNAs—is developed. This platform carries two toehold switches and includes an inhibited CrRNA sequence. In this system, the presence of cognate trigger RNA promotes precise binding to the first toehold site, initiating a cascade that releases CrRNA to target a reporter gene (luciferase) in this study. Decoupling the CrRNA segment from the trigger RNA enhances the potential of this genetic logic circuit to respond to specific cellular circumstances, offering promise as a synthetic biology platform. Developed 'IngRNA,' an intelligent CRISPR/Cas9 system with RNA-sensing toehold switches, enabling precise gene modulation by activating CrRNA in response to specific trigger RNAs. Demonstrated effective luciferase targeting in various systems. [ABSTRACT FROM AUTHOR]

We genetically manipulated HaCaT cells, a spontaneously immortalised normal keratinocyte cell line, to stably express two different coloured luciferase reporter genes, driven by interleukin 8 (IL‐8) and ubiquitin‐C (UBC) promoters, respectively. Subsequently, we generated a three‐dimensional (3D) skin‐like in vitro composite (SLIC) utilising these cells, with the objective of monitoring bioluminescence emitted from the SLIC. This SLIC was generated on non‐woven silica fibre membranes in differentiation medium. Immunohistochemical analyses of skin differentiation markers in the SLIC revealed the expression of keratins 2 and 10, filaggrin, and involucrin, indicating mature skin characteristics. This engineered SLIC was employed for real‐time bioluminescence monitoring, allowing the assessment of time‐ and dose‐dependent responses to UV stress, as well as to hydrophilic and hydrophobic chemical loads. Notably, evaluation of responses to hydrophobic substances has been challenging with conventional 2D cell culture methods, suggesting the need for a new approach, which this technology could address. Our observations suggest that engineered SLIC with constitutively expressing reporters driven by selected promoters which are tailored to specific objectives, significantly facilitates assays exploring the physiological functions of skin cells based on genetic response mechanisms. It also highlights new avenues for evaluating the physiological impacts of various compounds designed for topical application to human skin. [ABSTRACT FROM AUTHOR]

Annotation of newly sequenced genomes frequently includes genes, but rarely covers important non-coding genomic features such as the cis-regulatory modules--e.g., enhancers and silencers--that regulate gene expression. Here, we begin to remedy this situation by developing a workflow for rapid initial annotation of insect regulatory sequences, and provide a searchable database resource with enhancer predictions for 33 genomes. Using our previously developed SCRMshaw computational enhancer prediction method, we predict over 2.8 million regulatory sequences along with the tissues where they are expected to be active, in a set of insect species ranging over 360 million years of evolution. Extensive analysis and validation of the data provides several lines of evidence suggesting that we achieve a high true-positive rate for enhancer prediction. One, we show that our predictions target specific loci, rather than random genomic locations. Two, we predict enhancers in orthologous loci across a diverged set of species to a significantly higher degree than random expectation would allow. Three, we demonstrate that our predictions are highly enriched for regions of accessible chromatin. Four, we achieve a validation rate in excess of 70% using in vivo reporter gene assays. As we continue to annotate both new tissues and new species, our regulatory annotation resource will provide a rich source of data for the research community and will have utility for both small-scale (single gene, single species) and large-scale (many genes, many species) studies of gene regulation. In particular, the ability to search for functionally related regulatory elements in orthologous loci should greatly facilitate studies of enhancer evolution even among distantly related species. [ABSTRACT FROM AUTHOR]

Focused ultrasound ablation surgery (FUAS) is a minimally invasive treatment option that has been utilized in various tumors. However, its clinical advancement has been hindered by issues such as low safety and efficiency, single image guidance mode, and postoperative tumor residue. To address these limitations, this study aimed to develop a novel multi-functional gas-producing engineering bacteria biological targeting cooperative system. Pulse-focused ultrasound (PFUS) could adjust the ratio of thermal effect to non-thermal effect by adjusting the duty cycle, and improve the safety and effectiveness of treatment.The genetic modification of Escherichia coli (E.coli) involved the insertion of an acoustic reporter gene to encode gas vesicles (GVs), resulting in gas-producing E.coli (GVs-E.coli) capable of targeting tumor anoxia. GVs-E.coli colonized and proliferated within the tumor while the GVs facilitated ultrasound imaging and cooperative PFUS. Additionally, multifunctional cationic polyethyleneimine (PEI)-poly (lactic-co-glycolic acid) (PLGA) nanoparticles (PEI-PLGA/EPI/PFH@Fe3O4) containing superparamagnetic iron oxide (SPIO, Fe3O4), perfluorohexane (PFH), and epirubicin (EPI) were developed. These nanoparticles offered synergistic PFUS, supplementary chemotherapy, and multimodal imaging capabilities.GVs-E.coli effectively directed the PEI-PLGA/EPI/PFH@Fe3O4 to accumulate within the tumor target area by means of electrostatic adsorption, resulting in a synergistic therapeutic impact on tumor eradication.In conclusion, GVs-E.coli-mediated multi-functional nanoparticles can synergize with PFUS and chemotherapy to effectively treat tumors, overcoming the limitations of current FUAS therapy and improving safety and efficacy. This approach presents a promising new strategy for tumor therapy. [ABSTRACT FROM AUTHOR]

Background: Comprehensive treatment of gastric cancer (GC) is progressing, but the rapid proliferation and metastasis of GC remains a cause of high recurrence and mortality rates. In this study we investigated GC-associated circRNA tending to yield more insight into the mechanisms of gastric cancer development. Methods: We detected the expression levels of circTSN in GC tissues and cell lines using qRT-PCR. The circular structure of circTSN was confirmed by Sanger sequencing, agarose gel electrophoresis and RNase R. A series of cell functional experiments were employed to investigate the implication of circTSN aberrant expression on the proliferation and metastasis of GC cells. The predicted binding domain between circTSN and miR-1825 was analyzed by luciferase reporter gene analysis. Meanwhile, subcutaneous tumor xenografts in nude mice were used to validate the role of circTSN in vivo. Results: It was found that RNA levels of circTSN were significantly elevated in GC tissues and cell lines, which was also confirmed to contain a closed-loop structure. CCK8, clone formation, EdU, transwell and in vivo experiments indicated that the highly expressed circTSN was involved in the proliferation and metastasis process of GC. In addition, circTSN modulates the expression of SLC38A2 by sequence-specific binding to miR-1825. Conclusion: This study identified that circTSN, which is highly expressed in GC, was able to contribute to the proliferation and metastasis of GC cell through miR-1825/SLC38A2 axis and this might provide a new candidate target for the precision treatment of GC. [ABSTRACT FROM AUTHOR]

This article discusses a study on creating broad-spectrum resistance to bacterial blight in rice using targeted insertion. The researchers used CRISPR/Cas9 gene editing to insert effector binding elements (EBEs) into rice genes, resulting in resistance against diverse strains of Xanthomonas oryzae pv. oryzae (Xoo), the bacteria that causes bacterial blight. The study found that incorporating multiple EBEs into rice genes increased resistance to a greater number of Xoo strains. The researchers also used genome sequencing to identify novel EBEs and demonstrated the effectiveness of sequencing-guided EBE deployment in enhancing resistance. The study suggests that this programmable immunity system could help counter resistance breakdown in rice varieties. [Extracted from the article]

Inflammatory diseases of the intestinal tract in piglets severely impair the economic performance of pig farms. Pig milk exosomes can encapsulate miRNAs which can then enter the piglet intestine to play an immunomodulatory role. Previously, we comparatively analyzed and identified exosomal miRNAs in the colostrum and mature milk of Bamei and Landrace pigs, and we screened for ssc-miR-22-3p, which is associated with inflammation and immune response; however, the role played by ssc-miR-22-3p in the immune response in IPEC-J2 cells is not yet clear. In this study, we first constructed a pig intestinal inflammatory response model using Lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (Poly (I:C)), and we investigated the role of ssc-miR-22-3p targeting MAPK14 in the regulation of LPS and Poly (I:C)-induced inflammatory injury in IPEC-J2 cells by RT-qPCR, cell counting kit-8 (CCK-8), EdU staining, lactate dehydrogenase (LDH) activity assay, and dual luciferase reporter gene assay. We successfully established LPS and Poly (I:C)-induced cell damage models in IPEC-J2 cells. The immune response of IPEC-J2 cells was stimulated by induction of IPEC-J2 cells at 10 μg/mL LPS and 20 μg/mL Poly (I:C) for 24 h. Overexpression of ssc-miR-22-3p decreased cytokine expression and promoted cell viability and proliferation. The functional enrichment analysis revealed that ssc-miR-22-3p targets genes enriched in the pathways of negative regulation of inflammatory response and bacterial invasion of epithelial cells. The validity of the binding site of ssc-miR-22-3p to MAPK14 was tested by a dual luciferase reporter gene. Pig milk exosome ssc-miR-22-3p promotes cell viability and proliferation by targeting MAPK14, and it alleviates LPS and Poly (I:C)-induced inflammatory responses in IPEC-J2 cells. [ABSTRACT FROM AUTHOR]

Studying influenza A viruses (IAVs) requires secondary experimental procedures to detect the presence of the virus in infected cells or animals. The ability to generate recombinant (r)IAV using reverse genetics techniques has allowed investigators to generate viruses expressing foreign genes, including fluorescent and luciferase proteins. These rIAVs expressing reporter genes have allowed for easily tracking viral infections in cultured cells and animal models of infection without the need for secondary approaches, representing an excellent option to study different aspects in the biology of IAV where expression of reporter genes can be used as a readout of viral replication and spread. Likewise, these reporter-expressing rIAVs provide an excellent opportunity for the rapid identification and characterization of prophylactic and/or therapeutic approaches. To date, rIAV expressing reporter genes from different viral segments have been described in the literature. Among those, rIAV expressing reporter genes from the viral NS segment have been shown to represent an excellent option to track IAV infection in vitro and in vivo, eliminating the need for secondary approaches to identify the presence of the virus. Here, we summarize the status on rIAV expressing traceable reporter genes from the viral NS segment and their applications for in vitro and in vivo influenza research. [ABSTRACT FROM AUTHOR]

Genome-wide association studies have identified a locus on chromosome 10q22, where many co-inherited single nucleotide polymorphisms (SNPs) are associated with atrial fibrillation (AF). This study seeks to identify the impact of this locus on gene expression at the transcript isoform level in human left atria and to gain insight into potential causal variants. Bulk RNA sequencing was analyzed to identify myozenin 1 (MYOZ1) and synaptopodin 2-like (SYNPO2L) transcript isoforms and the association of common SNPs in this region with transcript isoform expression levels. Chromatin marks were used to suggest candidate regulatory SNPs in this region. Protein amino acid changes were examined for predicted functional consequences. Transfection of MYOZ1 and two SYNPO2L isoforms were performed to localize their encoded proteins in cardiomyocytes derived from stem cells. We identified one MYOZ1 transcript isoform and four SYNPO2L transcript isoforms, two of which encode proteins, while the other two encode long noncoding RNAs (lncRNAs). The risk allele of the strongest AF susceptibility SNP on chromosome 10q22 is associated with decreased MYOZ1 expression and increased expression of the two SNYPO2L lncRNA isoforms. There are many SNPs co-inherited with the top AF-associated SNP due to linkage disequilibrium (LD), including rs11000728, which we propose as the MYOZ1 regulatory SNP, confirmed by reporter gene transfection. In addition, this LD block includes three missense SNPs in the SYNPO2L gene, with the minor protective haplotype predicted to be detrimental to protein function. MYOZ1 and both protein isoforms of SYNPO2L were localized to the sarcomere. This is a complex locus with the potential for several SNPs in a haplotype to alter AF susceptibility by opposing effects on MYOZ1 and SYNPO2L lncRNA expression, along with effects on SYNPO2L protein function. [ABSTRACT FROM AUTHOR]

Interventional chemotherapy is a common operation in the clinical treatment of liver cancer. The aim of this study was to investigate the expression and molecular mechanism of serum miR‐4746‐5p in liver cancer patients before and after interventional chemotherapy. The levels of miR‐4746‐5p and CDKN1C in serum samples from liver cancer patients were detected using real‐time fluorescence quantitative polymerase chain reaction. Receiver operating characteristic curves revealed the diagnostic value of miR‐4746‐5p in tumors. Differences in clinical indicators between liver cancer patients and healthy controls were assessed using Pearson correlation analysis. Luciferase reporter gene assays confirmed the targeted interaction between miR‐4746‐5p and CDKN1C. In vitro cellular assays were validated by Cell Counting Kit‐8, Transwell assay, and chemoresistance assay. Serum miR‐4746‐5p levels were increased in liver cancer patients but were downregulated after chemotherapy intervention. CDKN1C expression showed the opposite trend. Low levels of miR‐4746‐5p mediated cell growth and metastasis by targeting and negatively regulating CDKN1C expression, while silencing CDKN1C restored cell activity. Inhibition of miR‐4746‐5p reduced chemoresistance, while downregulation of CDKN1C affected cell sensitivity. miR‐4746‐5p may be a potential therapeutic factor for liver cancer diagnosis and interventional chemotherapy. [ABSTRACT FROM AUTHOR]

Background: Osteosarcoma is the most common primary bone malignancy, with a strong tendency towards local invasion and metastasis. The SRY-Box Transcription Factor 1 (SOX1) gene, a member of the HMG-box family of DNA-binding transcription factors, plays a crucial role in embryogenesis and tumorigenesis. However, its role in osteosarcoma, particularly in relation to metastatic potential, is not well understood. Methods: The GSE14359 dataset containing five samples of conventional osteosarcoma and four samples of lung metastatic osteosarcoma was obtained from the Gene Expression Omnibus (GEO) database and analyzed for differential gene expression using the R language. Gene expression was detected using qPCR and Western blotting. Transcriptional activity was assessed by Luciferase reporter gene assays, and cell metastatic ability was assessed by migration and invasion assays. Results: The study demonstrated that SOX1 binds to a specific response element within the Transmembrane 4 Superfamily Member 12 (TSPAN12) promoter, upregulating TSPAN12 and its associated inflammatory pathways. Silencing TSPAN12 markedly reduces SOX1-mediated osteosarcoma cell invasion and inflammatory response, while TSPAN12 overexpression reverses these effects in SOX1-suppressed cells. Conclusion: In this study, our findings elucidate SOX1's role in enhancing osteosarcoma metastasis via TSPAN12 upregulation, offering new insights into the molecular mechanisms of osteosarcoma progression. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Does RXFP2 disruption impair male fertility? SUMMARY ANSWER We identified biallelic variants in RXFP2 in patients with male infertility due to spermatogenic arrest at the spermatid stage, supporting a role of RXFP2 in human spermatogenesis, specifically in germ cell maturation. WHAT IS KNOWN ALREADY Since RXFP2, the receptor for INSL3, plays a crucial role in testicular descent during prenatal development, biallelic variants lead to bilateral cryptorchidism, as described in four families to date. While animal models have also suggested a function in spermatogenesis, the postnatal functions of RXFP2 and its ligand INSL3, produced in large amounts by the testes from puberty throughout adulthood, are largely unknown. STUDY DESIGN, SIZE, DURATION A family with two male members affected by impaired fertility due to spermatogenic maturation arrest and a history of bilateral cryptorchidism underwent clinical, endocrinological, histological, genomic, in vitro cellular, and in silico investigations. PARTICIPANTS/MATERIALS, SETTING, METHODS The endocrinological and histological findings were correlated with publicly available single-cell RNA sequencing (scRNA-seq) data. The genomic defects have been characterized using long-read sequencing and validated with in silico modeling and an in vitro cyclic AMP reporter gene assay. MAIN RESULTS AND THE ROLE OF CHANCE An intragenic deletion of exon 1–5 of RXFP2 (NM_130806.5) was detected in trans with a hemizygous missense variant c.229G>A, p.(Glu77Lys). The p.(Glu77Lys) variant caused no clear change in cell surface expression or ability to bind INSL3, but displayed absence of a cAMP signal in response to INSL3, indicating a loss-of-function. Testicular biopsy in the proband showed a maturation arrest at the spermatid stage, corresponding to the highest level of RXFP2 expression in scRNA-seq data, thereby providing a potential explanation for the impaired fertility. LIMITATIONS, REASONS FOR CAUTION Although this is so far the only study of human cases that supports the role of RXFP2 in spermatogenic maturation, this is corroborated by several animal studies that have already demonstrated a postnatal function of INSL3 and RXFP2 in spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS This study corroborates RXFP2 as gene implicated in autosomal recessive congenital bilateral cryptorchidism due to biallelic variants, rather than autosomal-dominant cryptorchidism due to monoallelic RXFP2 variants. Our findings also support that RXFP2 is essential in human spermatogenesis, specifically in germ cell maturation, and that biallelic disruption can cause male infertility through spermatogenic arrest at the spermatid stage. STUDY FUNDING/COMPETING INTEREST(S) Funding was provided by the Bellux Society for Pediatric Endocrinology and Diabetology (BELSPEED) and supported by a Research Foundation Flanders (FWO) senior clinical investigator grant (E.D.B. 1802220N) and a Ghent University Hospital Special Research Fund grant (M.C. FIKO-IV institutional fund). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Gas vesicles (GVs) are gas-filled microbial organelles formed by unique 3-nm thick, amphipathic, force-bearing protein shells, which can withstand multiple atmospheric pressures and maintain a physically stable air bubble with megapascal surface tension. However, the molecular process of GV assembly remains elusive. To begin understanding this process, we have devised a high-throughput in vivo assay to determine the interactions of all 11 proteins in the pNL29 GV operon. Complete or partial deletions of the operon establish interdependent relationships among GV proteins during assembly. We also examine the tolerance of the GV assembly process to protein mutations and the cellular burdens caused by GV proteins. Clusters of GV protein interactions are revealed, proposing plausible protein complexes that are important for GV assembly. We anticipate our findings will set the stage for designing GVs that efficiently assemble in heterologous hosts during biomedical applications. Synopsis: Growing interest in biomedical applications for gas vesicles (GVs) demands an understanding of their structure and assembly, but little is known to date on the roles of individual gas vesicle proteins (Gvps) as assembly factors. This study utilizes modular, parallelized protein-protein interaction screening to elucidate key interactions and interdependencies of Gvps during GV assembly. Out of 66 possible Gvp-to-Gvp interactions, 28 formed a dense protein-protein interaction network. Selective inclusion of other Gvps led to gain or loss of protein-protein interactions, revealing interdependence during GV assembly. Specific subnetworks of Gvps are revealed that may be linked to different stages of GV assembly. A number of Gvps show tolerance for fusion proteins during GV assembly, unveiling a degree of plasticity with implications for GV engineering. Iburg at al develop a high-throughput in vivo screen to construct a comprehensive map of the interactions among all 11 proteins of the pNL29 gas vesicle operon. [ABSTRACT FROM AUTHOR]

Summary: Reproduction in angiosperms relies on the precise growth of pollen tubes, facilitating the delivery of sperm cells to the ovule for double fertilization. LATERAL ORGAN BOUNDARIES DOMAIN10 (LBD10), a plant‐specific transcription factor, plays a pivotal role in Arabidopsis pollen development.Here, we uncovered LBD10's function in sustaining pollen tube growth and integrity.The lbd10 mutant exhibited elevated levels of reactive oxygen species (ROS) and hydrogen peroxide (H2O2) in both pollen grains and tubes, leading to compromised pollen tube growth. The inhibition of ROS synthesis and scavenging of excess ROS with an antioxidant treatment each alleviated these defects in lbd10. The lbd10 mutant displayed reduced flavonol accumulation in both pollen grains and tubes. All the altered phenotypes of lbd10 were complemented by expressing LBD10 under its native promoter. Exogenous application of flavonoids recused the defects in pollen tube growth and integrity in lbd10, along with reducing the excess levels of ROS and H2O2. LBD10 directly binds the promoters of key flavonol biosynthesis genes in chromatin and promotes reporter gene expression in Arabidopsis mesophyll protoplasts.Our findings indicate that LBD10 modulates ROS homeostasis by transcriptionally activating genes crucial for flavonol biosynthesis, thereby maintaining pollen tube growth and integrity. [ABSTRACT FROM AUTHOR]

Chronic prostatitis-induced excessive inflammation and oxidative stress (OS) damage substantially affect men's quality of life. However, its treatment remains a major clinical challenge. Therefore, the identification of drugs that can decrease chronic prostatitis and oxidative stress targets is urgent and essential. CXCR4 is a classic chemokine receptor that is crucially associated with the occurrence and development of inflammation. This investigation aimed to elucidate how CXCR4 affects prostatitis regression and progression. The effect of CXCR4 on chronic prostatitis was evaluated by HE staining, immunohistochemistry, immunofluorescence, PCR, and TUNEL analyses. Furthermore, CXCR4 influence on metabolism was also evaluated by monitoring body weight, body temperature, food intake, and LC/MS. Additionally, chromatin immunoprecipitation, Western blot, and double luciferase reporter gene assays were carried out to elucidate the mechanism by which CXCR4 modulates Fads2 transcription by PPARγ. Lastly, ROS, DHE, mito-tracker, and ATP were utilized to validate the α-linolenic acid's protective effect against OS in prostate epithelial cells. It was revealed that the inhibition of CXCR4 can effectively alleviate prostatitis in mice. Furthermore, downregulating CXCR4 expression can markedly reduce the inflammatory cell infiltration in mouse prostates, decrease the elevated levels of DNA damage markers,MDA and 4-HNE, and mitigate apoptosis of prostatic epithelial cells. Moreover, treatment of CXCR4 knockdown mice with a PPARγ inhibitor revealed different degrees of changes in the above phenotypes. Mechanistically, the PPARγ protein translocates to the nucleus and serves as a transcription factor to regulate Fads2 expression, thereby altering PUFA metabolism. Additionally, in vitro experiments indicated that α-linolenic acid can effectively alleviate OS damage and RWPE-1 cell apoptosis by protecting mitochondrial function and enhancing the antioxidant capacity of prostatic epithelial cells. In conclusion, reducing the levels of CXCR4 can alleviate inflammation and OS damage in chronic prostatitis. [Display omitted] • CXCR4 affects OS and PUFA metabolism in CP/CPPS. • PPARγ transcriptionally regulates Fads2. • ALA alleviates OS in RWPE-1 cells. [ABSTRACT FROM AUTHOR]

Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species. [ABSTRACT FROM AUTHOR]

Promoters are powerful tools for breeding new varieties using transgenic technology. However, the low and unstable expression of target genes is still a limiting factor in Larix kaempferi (Lamb.) Carr (Japanese larch) genetic transformation. In this study, we analyzed L. kaempferi transcriptome data, screened out highly expressed genes, cloned their promoters, and constructed plant expression vectors containing the β-glucuronidase (GUS) reporter gene driven by these promoters. Recombinant vectors were introduced into the L. kaempferi embryogenic callus by means of the Agrobacterium-mediated transient or stable genetic transformation method, and the promoter activity was then determined by measuring GUS expression and its enzyme activity in the transformed materials. Four highly expressed genes were identified: L. kaempferi Zhang Chen Yi-1 (LaZCY-1), Zhang Chen Yi-2 (LaZCY-2), Translationally Controlled Tumor Protein (LaTCTP), and ubiquitin (LaUBQ). The 2000 bp fragments upstream of ATG in these sequences were cloned as promoters and named pLaZCY-1, pLaZCY-2, pLaTCTP, and pLaUBQ. Semi-quantitative and quantitative RT-PCR analyses of transient genetic transformation materials showed that all four promoters could drive GUS expression, indicating that they have promoter activities. Semi-quantitative and quantitative RT-PCR analyses and the histochemical staining of stable genetic transformation materials showed that the pLaUBQ promoter had higher activity than the other three L. kaempferi promoters and the CaMV35S promoter. Thus, the pLaUBQ promoter was suggested to be used in larch genetic transformation. [ABSTRACT FROM AUTHOR]

Objective: Evaluation of Nepeta cataria as a host with specific endogenous metabolite background for transient expression and metabolic engineering of secondary biosynthetic sequences. Results: The reporter gene gfp::licBM3 as well as three biosynthetic genes leading to the formation of the cannabinoid precursor olivetolic acid were adopted to the modular cloning standard GoldenBraid, transiently expressed in two chemotypes of N. cataria and compared to Nicotiana benthamiana. To estimate the expression efficiency in both hosts, quantification of the reporter activity was carried out with a sensitive and specific lichenase assay. While N. benthamiana exhibited lichenase activity of 676 ± 94 μmol g−1 s−1, N. cataria cultivar '1000', and the cultivar 'Citriodora' showed an activity of 37 ± 8 μmol g−1 s−1 and 18 ± 4 μmol g−1 s−1, respectively. Further, combinatorial expression of genes involved in cannabinoid biosynthetic pathway acyl-activating enzyme 1 (aae1), olivetol synthase (ols) and olivetolic acid cyclase (oac) in N. cataria cv. resulted presumably in the in vivo production of olivetolic acid glycosides. Conclusion: Nepeta cataria is amenable to Agrobacterium-mediated transient expression and could serve as a novel chassis for the engineering of secondary metabolic pathways and transient evaluation of heterologous genes. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults with the lowest survival rates five years post-diagnosis. Oncolytic viruses (OVs) selectively target and damage cancer cells, and for this reason they are being investigated as new therapeutic tools also against GBM. Methods: An oncolytic herpes simplex virus type 1 (oHSV-1) with deletions in the γ34.5 neurovirulence gene and the US12 gene, expressing enhanced green fluorescent protein (EGFP-oHSV-1) as reporter gene was generated and tested for its capacity to infect and kill the murine GL261 glioblastoma (GBM) cell line. Syngeneic mice were orthotopically injected with GL261cells. Seven days post-implantation, EGFP-oHSV-1 was administered intratumorally. Twenty-one days after parental tumor challenge in the opposite brain hemisphere, mice were sacrified and their brains were analysed by immunohistochemistry to assess tumor presence and cell infiltrate. Results: oHSV-1 replicates and induces cell death of GL261 cells in vitro. A single intracranial injection of EGFP-oHSV-1 in established GL261 tumors significantly prolongs survival in all treated mice compared to placebo treatment. Notably, 45% of treated mice became long-term survivors, and rejected GL261 cells upon rechallenge in the contralateral brain hemisphere, indicating an anamnestic antitumoral immune response. Post-mortem analysis revealed a profound modification of the tumor microenvironment with increased infiltration of CD4 + and CD8 + T lymphocytes, intertumoral vascular collapse and activation and redistribution of macrophage, microglia, and astroglia in the tumor area, with the formation of intense fibrotic tissue suggestive of complete rejection in long-term survivor mice. Conclusions: EGFP-oHSV1 demonstrates potent antitumoral activity in an immunocompetent GBM model as a monotherapy, resulting from direct cell killing combined with the stimulation of a protective adaptive immune response. These results open the way to possible application of our strategy in clinical setting. [ABSTRACT FROM AUTHOR]

Feline infectious peritonitis (FIP) is a lethal, immune-mediated disease in cats caused by feline infectious peritonitis virus (FIPV), a biotype of feline coronavirus (FCoV). In contrast to feline enteric coronavirus (FECV), which exclusively infects enterocytes and causes diarrhea, FIPV specifically targets macrophages, resulting in the development of FIP. The transmission and infection mechanisms of this complex, invariably fatal disease remain unclear, with no effective vaccines or approved drugs for its prevention or control. In this study, a full-length infectious cDNA clone of the wild-type FIPV WSU79-1149 strain was constructed to generate recombinant FIPV (rFIPV-WT), which exhibited similar growth kinetics and produced infectious virus titres comparable to those of the parental wild-type virus. In addition, the superfold green fluorescent protein (msfGFP) and Renilla luciferase (Rluc) reporter genes were incorporated into the rFIPV-WT cDNA construct to generate reporter rFIPV-msfGFP and rFIPV-Rluc viruses. While the growth characteristics of the rFIPV-msfGFP virus were similar to those of its parental rFIPV-WT, the rFIPV-Rluc virus replicated more slowly, resulting in the formation of smaller plaques than did the rFIPV-WT and rFIPV-msfGFP viruses. In addition, by replacing the S, E, M, and ORF3abc genes with msfGFP and Rluc genes, the replicon systems repFIPV-msfGFP and repFIPV-Rluc were generated on the basis of the cDNA construct of rFIPV-WT. Last, the use of reporter recombinant viruses and replicons in antiviral screening assays demonstrated their high sensitivity for quantifying the antiviral effectiveness of the tested compounds. This integrated system promises to significantly streamline the investigation of virus replication within host cells, enabling efficient screening for anti-FIPV compounds and evaluating emerging drug-resistant mutations within the FIPV genome. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR‐associated nuclease 9 (Cas9)‐mediated gene editing technology has revolutionized the study of fundamental biological questions in various insects. Diverse approaches have been developed to deliver the single‐guide RNA (sgRNA) and Cas9 to the nucleus of insect embryos or oocytes to achieve gene editing, including the predominant embryonic injection methods and alternative protocols through parental ovary delivery. However, a systematic comparative study of these approaches is limited, especially within a given insect. Here, we focused on revealing the detailed differences in CRISPR/Cas9‐mediated gene editing between the embryo and ovary delivery methods in the beetle Tribolium castaneum, using the cardinal and tyrosine hydroxylase (TH) as reporter genes. We demonstrated that both genes could be efficiently edited by delivering Cas9/sgRNA ribonucleoproteins to the embryos by microinjection, leading to the mutant phenotypes and indels in the target gene sites. Next, the Cas9/sgRNA complex, coupled with a nanocarrier called Branched Amphiphilic Peptide Capsules (BAPC), were delivered to the ovaries of parental females to examine the efficacy of BAPC‐mediated gene editing. Although we observed that a small number of beetles’ progeny targeting the cardinal exhibited the expected white‐eye phenotype, unexpectedly, no target DNA indels were found following subsequent sequencing analysis. In addition, we adopted a novel approach termed “direct parental” CRISPR (DIPA‐CRISPR). However, we still failed to find gene‐editing events in the cardinal or TH gene‐targeted insects. Our results indicate that the conventional embryonic injection of CRISPR is an effective method to initiate genome editing in T. castaneum. However, it is inefficient by the parental ovary delivery approach. [ABSTRACT FROM AUTHOR]

Caenorhabditis elegans males undergo sex-specific tail tip morphogenesis (TTM) under the control of the DM-domain transcription factor DMD-3. To find genes regulated by DMD-3, we performed RNA-seq of laser-dissected tail tips. We identified 564 genes differentially expressed (DE) in wild-type males versus dmd-3(-) males and hermaphrodites. The transcription profile of dmd-3(-) tail tips is similar to that in hermaphrodites. For validation, we analyzed transcriptional reporters for 49 genes and found male-specific or malebiased expression for 26 genes. Only 11 DE genes overlapped with genes found in a previous RNAi screen for defective TTM. GO enrichment analysis of DE genes finds upregulation of genes within the unfolded protein response pathway and downregulation of genes involved in cuticle maintenance. Of the DE genes, 40 are transcription factors, indicating that the gene network downstream of DMD-3 is complex and potentially modular. We propose modules of genes that act together in TTM and are co-regulated by DMD-3, among them the chondroitin synthesis pathway and the hypertonic stress response. [ABSTRACT FROM AUTHOR]

Presbycusis, also referred to as age-related hearing loss (ARHL), is a multifaceted condition caused by the natural aging process affecting the auditory system. Genome-wide association studies (GWAS) in human populations can identify potential genes linked to ARHL. Despite this, our knowledge of the biochemical and molecular mechanisms behind the condition remains incomplete. This study aims to evaluate a potential protective tool for ARHL treatment by comparing human blood-based target gene-miRNA associations regulated in ARHL. To identify promising target genes for ARHL, we utilized an mRNA assay. To determine the role of miRNA in ARHL, we investigated the expression profile of miRNA in whole blood in ARHL patients with real-time polymerase chain reaction (RT-qPCR). A reporter gene assay was performed to confirm the regulation of candidate genes by microRNA. Through RT-qPCR validation analysis, we finally confirmed the relationship between ARHL and the role of the interferon-gamma (IFNG) gene. This gene can be regarded as an age-related gene. Through gene ontology (GO) analysis, it has been found that these genes are enriched in pathways related to apoptosis. Among them, IFNG induces an inflammatory response, apoptotic cell death, and cellular senescence. We found that miR-409-3p downregulates the expression of the IFNG in vitro. In addition, the downregulation of the IFNG by miRNA 409-3p promoted cell apoptosis and suppressed proliferation. In conclusion, our study produced gene signatures and associated microRNA regulation that could be a protective key for ARHL patients. IFNG genes and miR-409-3p should be investigated for their usefulness as a new biomarker for treatment modality. [ABSTRACT FROM AUTHOR]

Background Genome-wide association studies have identified several nonfunctional tag single-nucleotide polymorphisms (SNPs) associated with severe malaria. We hypothesized that causal SNPs could play a significant role in severe malaria by altering promoter or enhancer activity. Here, we sought to identify such regulatory SNPs. Methods SNPs in linkage disequilibrium with tagSNPs associated with severe malaria were identified and were further annotated using FUMA. Then, SNPs were prioritized using the integrative weighted scoring method to identify regulatory ones. Gene reporter assays were performed to assess the regulatory effect of a region containing candidates. The association between SNPs and severe malaria was assessed using logistic regression models in a Senegalese cohort. Results Among 418 SNPs, the best candidates were rs116525449 and rs79644959, which were in full disequilibrium between them, and located within the ARL14 promoter. Our gene reporter assay results revealed that the region containing the SNPs exhibited cell-specific promoter or enhancer activity, while the SNPs influenced promoter activity. We detected an association between severe malaria and those 2 SNPs using the overdominance model and we replicated the association of severe malaria with the tagSNP rs116423146. Conclusions We suggest that these SNPs regulate ARL14 expression in immune cells and the presentation of antigens to T lymphocytes, thus influencing severe malaria development. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is a high‐mortality cancer. Docetaxel (DCT) combined with second‐generation anti‐androgens is considered the golden standard therapy for PCa, whose application is limited for DCT resistance (DR). Therefore, exploring the mechanism of DR is of great importance. In this study, PCa cell lines of PC3 and DU145 were employed, and DR cells were constructed by treatment with graded DCT. CircSLC4A7, miR‐1205, and microtubule‐associated protein tau (MAPT) transfections were established. Cell counting kit‐8 assay was performed to evaluate the cell activity and IC50 of DCT. After being treated with DCT, DR was assessed by colony formation assay, flow cytometry analysis, and terminal transferase‐mediated UTP nick end‐labeling assay. Real‐time quantitative PCR and western blotting analysis evaluated the expression levels of genes. The dual‐luciferase reporter gene assay verified the miR‐1205 binding sites with circSLC4A7 and MAPT. An animal experiment was performed to assess the tumor growth influenced by circSLC4A7. After conducting DR cells and isolated exosomes, we found that not only co‐culture with DR cells but also treatment with DR cells' exosomes would promote the DR of normal cells. Moreover, circSLC4A7 was highly expressed in DR cells and their exosomes. CircSLC4A7 overexpression enhanced DR, represented as raised IC50 of DCT, increased colony formation, and decreased cell apoptosis after DCT treatment, while circSLC4A7 knockdown had the opposite effect. MiR‐1205 was confirmed as a circSLC4A7‐sponged miRNA and miR‐1205 inhibitor reversed the effect of sh‐circSLC4A7. MAPT was further identified as a target of miR‐1205 and had a similar effect with circSLC4A7. The effect of circSLC4A7 on DR was also confirmed by xenograft experiments. Collectively, circSLC4A7 in resistant‐cells‐derived exosomes promotes DCT resistance of PCa via miR‐1205/MAPT axis, which may provide a new treatment strategy for DR of PCa. [ABSTRACT FROM AUTHOR]

Genetic transformation and regeneration procedures in Saintpaulia are dependent on different parameters, which limit the genetic manipulation efficiency. We focused on introducing an efficient model for gene transformation and regeneration after that, via RSM. Two commercial cultivars were transformed by the GUS reporter gene via Agrobacterium-mediated method. Results showed that variables infection time, BA and IAA concentrations, and cultivar type significantly (p ≤ .05) affected gene transformation. According to the highest desirability (0.94) of the obtained model, the highest transformation rate was achieved (68.7%) by 15 s dip in bacterial suspension followed by regeneration steps in the medium supplemented with 100 µg/l BA + 1000 µg/l IAA. Subsequently, the given model validity was evaluated by a target gene (etr1-1). The highest rate of etr1-1 transformation (76.25%) was achieved by 30 s infection time in the proposed medium containing 100 µg/l BA + 1000 µg/l IAA, according to the model. Genomic and reverse transcription PCRs confirmed that GUS, nptII and etr1-1 genes were successfully integrated and expressed in both cultivars at each transformation. Our results indicate that optimising the time of infection and media using the RSM design could greatly improve the accuracy and feasibility of a model for achieving a high rate of genetic transformation. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the inhibitory effect of antimicrobial peptide merecidin on triple-negative breast cancer (TNBC) and the mechanism of inhibiting epithelial-mesenchymal transformation (EMT) by regulating miR-30d-5p/vimentin. Methods: TNBC cell lines (MDA-MB-231, MDA-MB-468) were treated with merecidin to assess proliferation, migration, invasion ability, and EMT. Confocal laser localization was used to examine the role of merecidin and TNBC cells. The relationship between merecidin and miR-30d-5p was determined through RT-qPCR and dual-luciferase reporter gene, and the relationship between merecidin and vimentin was verified through pulling down the experiment. The effects of miR-30d-5p on the migration and invasion ability of TNBC cells were confirmed through scratch and transwell experiments. Vimentin levels, tumor volume, shape, size, and weight were observed in the MDA-MB-231 subcutaneous tumor model in nude mice. Results: merecidin inhibited the proliferation, migration, invasion, and EMT of TNBC cells. merecidin was primarily located in the cytoplasm of TNBC cells, and the expression of miR-30d-5p was low in TNBC cells. merecidin significantly up-regulated the expression of miR-30d-5p. miR-30d-5p negatively regulated vimentin. merecidin could bind to vimentin in vitro. miR-30d-5p inhibited the migration and invasion ability of TNBC cells, while vimentin promoted their migration and invasion ability. Down-regulation of miR-30d-5p or overexpression of vimentin partially counteracted the inhibitory effects of merecidin on TNBC cell migration, invasion ability, and EMT. In nude mouse tumor models, merecidin significantly suppressed tumor growth. Conclusion: Merecidin effectively blocks the EMT process and inhibits the migration and invasion of TNBC cells by regulating miR-30d-5p/vimentin. [ABSTRACT FROM AUTHOR]

Background: Rosmarinic acid (RA), a natural phenolic acid, exhibits promising anti-cancer properties. The abnormal expression of microRNA (miRNA) regulates the gene expression and plays a role as an oncogenic or tumor suppressor in TNBC. However, the biological role of RA in miR-30a-5p on BCL2L11 during MDA-MB-231 induced breast cancer stem-like cells (BCSCs) progression and its regulatory mechanism have not been elucidated. Objective: To investigate whether RA inhibited the silencing effect of miR-30a- 5p on the BCL2L11 gene and promoted apoptosis in BCSCs. Materials and Methods: We assessed the migration, colony formation, proliferation, cell cycle, and apoptosis of BCSCs after RA treatment using the wound-healing assay, colony formation assay, CCK-8 assay, and flowcytometry, respectively. The expression of mRNA and protein levels of BCL-2, Bax, BCL2L11, and P53 genes in BCSCs after RA treatment was obtained by real-time polymerase chain reaction and Western blot. DifferentialmiRNA expression in BCSCswas analyzed by high-throughput sequencing. Targetscan was utilized to predict the targets of miR-30a-5p. The dual luciferase reporter system was used for validation of the miR-30a-5p target. Results:Wound-healing assay, colony formation assay,CCK-8 assay, and cell cycle assay results showed that RA inhibited migration, colony formation and viability of BCSCs, and cell cycle arrest in the G0-G1 phase. At the highest dose of RA, we noticed cell atrophy, while the arrest rate at 100 μg/mL RA surpassed that at 200 μg/mL RA. Apoptotic cells appeared early (Membrane Associated Protein V FITC+, PI-) or late (Membrane Associated Protein V FITC+, PI+) upon administration of 200 µg/mL RA, Using high-throughput sequencing to compare the differences in miRNA expression, we detected downregulation ofmiR-30a-5p expression, and the results of dual luciferase reporter gene analysis indicated that BCL2L11 was a direct target of miR-30a-5p. Conclusion: RA inhibited the silencing effect of miR-30a-5p on the BCL2L11 gene and enhanced apoptosis in BCSCs. [ABSTRACT FROM AUTHOR]

Background: Our previous genome‑wide association studies (GWAS) have suggested rs912304 in 14q12 as a suggestive risk variant for type 1 diabetes (T1D). However, the association between this risk region and T1D subgroups and related clinical risk features, the underlying causal functional variant(s), putative candidate gene(s), and related mechanisms are yet to be elucidated. Methods: We assessed the association between variant rs912304 and T1D, as well as islet autoimmunity and islet function, stratified by the diagnosed age of 12. We used epigenome bioinformatics analyses, dual luciferase reporter assays, and expression quantitative trait loci (eQTL) analyses to prioritize the most likely functional variant and potential causal gene. We also performed functional experiments to evaluate the role of the causal gene on islet function and its related mechanisms. Results: We identified rs912304 as a risk variant for T1D subgroups with diagnosed age ≥ 12 but not < 12. This variant is associated with residual islet function but not islet-specific autoantibody positivity in T1D individuals. Bioinformatics analysis indicated that rs912304 is a functional variant exhibiting spatial overlaps with enhancer active histone marks (H3K27ac and H3K4me1) and open chromatin status (ATAC-seq) in the human pancreas and islet tissues. Luciferase reporter gene assays and eQTL analyses demonstrated that the biallelic sites of rs912304 had differential allele-specific enhancer activity in beta cell lines and regulated STXBP6 expression, which was defined as the most putative causal gene based on Open Targets Genetics, GTEx v8 and Tiger database. Moreover, Stxbp6 was upregulated by T1D-related proinflammatory cytokines but not high glucose/fat. Notably, Stxbp6 over-expressed INS-1E cells exhibited decreasing insulin secretion and increasing cell apoptosis through Glut1 and Gadd45β, respectively. Conclusions: This study expanded the genomic landscape regarding late-onset T1D risk and supported islet function mechanistically connected to T1D pathogenesis. [ABSTRACT FROM AUTHOR]

Sepsis-induced cardiomyopathy (SIC) is described as a reversible myocardial depression that occurs in patients with septic shock. Increasing evidence shows that microRNA-194-5p (miR-194-5p) participates in the regulation of oxidative stress, mitochondrial dysfunction, and apoptosis and its expression is associated with the occurrence and progression of cardiovascular disease; however, the effects of miR-194-5p in SIC are still unclear. This study explores whether miR-194-5p could modulate SIC by affecting oxidative stress, mitochondrial function, and apoptosis. Experimental septic mice were induced by intraperitoneal injection of lipopolysaccharide (LPS) in C57BL/6J mice. The biological role of miR-194-5p in SIC in vivo was investigated using cardiac echocardiography, ELISA, western blot, qRT-PCR, transmission electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, bioinformatics analysis, and dual-luciferase reporter gene assay. Our major finding is that miR-194-5p antagomir mitigates sepsis-induced cardiac dysfunction, inflammation, oxidative stress, apoptosis and mitochondrial dysfunction in the hearts of septic mice, while miR-194-5p agomir triggers the opposite effects. Furthermore, dual-specificity phosphatase 9 (DUSP9) is a direct target of miR-194-5p and the cardioprotective effects of miR-194-5p antagomir on cardiac dysfunction, inflammation, apoptosis, mitochondrial dysfunction and oxidative stress are abolished through inhibiting DUSP9. Therefore, miR-194-5p inhibition could mitigate SIC via DUSP9 in vivo and the novel miR-194-5p/DUSP9 axis might be the potential treatment targets for SIC patients. [ABSTRACT FROM AUTHOR]

In female eutherian mammal development, X-chromosome inactivation (XCI) of one of the two X chromosomes is initiated early. Understanding the relationship between the initiation of XCI and cell fate is critical for understanding early female development and requires a system that can monitor XCI in single living cells. Traditional embryonic stem cells (ESCs) used for XCI studies often lose X chromosomes spontaneously during culture and differentiation, making accurate monitoring difficult. Additionally, most XCI assessment methods necessitate cell disruption, hindering cell fate tracking. We developed the Momiji (version 2) ESC line to address these difficulties, enabling real-time monitoring of X-chromosome activity via fluorescence. We inserted green and red fluorescent reporter genes and neomycin and puromycin resistance genes into the two X chromosomes of PGK12.1 ESCs, creating a female ESC line that retains two X chromosomes more faithfully during differentiation. Momiji (version 2) ESCs exhibit a more stable XX karyotype than other ESC lines, including the parental PGK12.1 line. This new tool offers valuable insights into the relationship between XCI and cell fate, improving our understanding of early female development. [ABSTRACT FROM AUTHOR]

Objective：To investigate the effect and mechanism of miR-217 targeted regulation of extracellular signal-regulated kinase 2 (ERK2) expression on activity and immune escape of non-small cell lung cancer cells (NSCLC) . Methods：qRT-PCR was used to detect expression levels of miR-217 and ERK2 mRNA in NSCLC tissues, adjacent tissues, and HLF-1, A549 and HCC827 cell lines. Analyzed prognosis and survival status of NSCLC patients with different miR-217 expression level. Bioinformatics and dual luciferase gene reporting experiments were used to analyze the targeting relationship between miR-217 and ERK2. Cultivated NSCLC A549 cells and divided them into NC group, miR-217 inhibitor group, miR-217 mimic group, miR-217 mimic+ERK2 NC group and miR-217 mimic+ERK2 group. Except for the NC group without any treatment, all other groups were transfected with corresponding plasmids to analyze the proliferation activity and immune escape status of A549 cells in each group, and clarified the mechanism of action. Results：Compared with adjacent tissues, expression of miR-217 in NSCLC tissue was decreased, while expression of ERK2 mRNA was increased (P<0.05) . Compared with human normal lung fibroblast HLF-1 cell lines, expression of miR-217 in NSCLC cell lines A549 and HCC827 were decreased, while expression of ERK2 mRNA was increased (P<0.05) . Analysis of the relationship between miR-217 and prognosis of NSCLC patients based on Kaplan-Meier Plotter database showed that low expression of miR-217 was associated with poor prognosis of patients (HR=0.90, P=0.033) . Dual fluorescein reporter genes showed matching sequences between the 3'UTR regions of miR-217 and ERK2. miR-217 mimic fragment could inhibit ERK2-WT signal, but had no effect on ERK2-MUT. Compared with NC group, cell proliferation activity, PD-L1 and PD-L2 mRNA expression levels of miR-217 inhibitor group were inreased, while CD8+T cell activity was decreased, and cell proliferation activity, PD-L1 and PD-L2 mRNA expression levels of miR- 217 mimic group were decreased, while CD8+T cell activity was increased (P<0.05) . Compared with miR-217 mimic group, cell proliferation activity, CD8+T cell activity, PD-L1 and PD-L2 mRNA expression levels of miR-217 mimic+ERK2 NC group had no significant changes (P>0.05), cell proliferation activity, PD-L1 and PD-L2 mRNA expression levels of miR-217 mimic+ERK2 group were increased, while CD8+T cell activity was decreased (P<0.05) . Conclusion：Overexpression of miR-217 can reduce the activity of NSCLC cell A549, inhibit the expression of PD-L1, activate CD8+T cells in tumor microenvironment, and then inhibit immune escape, which may play a role by targeting ERK2. [ABSTRACT FROM AUTHOR]

Objective: To investigate the potential mechanism of miR-1298-5p in non-small cell lung cancer (NSCLC) to regulate the MSH2 gene and its effect on the biological behavior of tumor cells and the tumor immune microenvironment. Methods: Bioinformatics was used to identify the key genes and miRNA involved in NSCLC. Cell proliferation was detected by CCK-8 assay, and cell invasion and migration were detected by Transwell assay. Levels of inflammatory factors were detected by ELISA assay. Western blot was used to measure the expression of MSH2 in cells, and fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the expressions of miR-1298-5p and MSH2 gene in NSCLC cells. Dual luciferase reporter gene assay was performed to verify the targeting relationship between miR-1298-5p and MSH2. Spearman correlation analysis was performed to correlate miR-1298-5p with immune cells and immune factors in the tumor immune microenvironment. Results: The level of miR-1298-5p was down-regulated in NSCLC cells compared with normal lung tissue cells. miR-1298-5p overexpression inhibited the proliferation, migration and invasion of NSCLC cells. MSH2 was confirmed to be a target gene of miR-1298-5p using luciferase reporter gene assay. Furthermore, downregulation of miR-1298-5p in NSCLC cells could be reversed by silencing MSH2. miR-1298-5p expression levels were negatively correlated with the levels of Treg, IL-10, and TGF-β, and positively correlated with the levels of CD3+T, CD4+T, CD8+T, NK cells, IL-2, and IFN-γ. Conclusion: miR-1298-5p negatively regulates MSH2 to inhibit the proliferation, invasion and migration of NSCLC cells and improve the tumor immune microenvironment. [ABSTRACT FROM AUTHOR]

MoMo30 is an antiviral protein isolated from aqueous extracts of Momordica balsamina L. (Senegalese bitter melon). Previously, we demonstrated MoMo30's antiviral activity against HIV-1. Here, we explore whether MoMo30 has antiviral activity against the COVID-19 virus, SARS-CoV-2. MLV particles pseudotyped with the SARS-CoV-2 Spike glycoprotein and a Luciferase reporter gene (SARS2-PsV) were developed from a three-way co-transfection of HEK293-T17 cells. MoMo30's inhibition of SARS2-PsV infection was measured using a luciferase assay and its cytotoxicity using an XTT assay. Additionally, MoMo30's interactions with the variants and domains of Spike were determined by ELISA. We show that MoMo30 inhibits SARS2-PsV infection. We also report evidence of the direct interaction of MoMo30 and SARS-CoV-2 Spike from WH-1, Alpha, Delta, and Omicron variants. Furthermore, MoMo30 interacts with both the S1 and S2 domains of Spike but not the receptor binding domain (RBD), suggesting that MoMo30 inhibits SARS-CoV-2 infection by inhibiting fusion of the virus and the host cell via interactions with Spike. [ABSTRACT FROM AUTHOR]

RNA virus polymerases carry out multiple functions necessary for successful genome replication and transcription. A key tool for molecular studies of viral RNA-dependent RNA polymerases (RdRps) is a 'minigenome' or 'minireplicon' assay, in which viral RdRps are reconstituted in cells in the absence of full virus infection. Typically, plasmids expressing the viral polymerase protein(s) and other co-factors are co-transfected, along with a plasmid expressing an RNA encoding a fluorescent or luminescent reporter gene flanked by viral untranslated regions containing cis-acting elements required for viral RdRp recognition. This reconstitutes the viral transcription/replication machinery and allows the viral RdRp activity to be measured as a correlate of the reporter protein signal. Here, we report on the development of a 'first-generation' plasmid-based minigenome assay for species A rotavirus using a firefly luciferase reporter gene. [ABSTRACT FROM AUTHOR]

The highly abundant and stable antiviral small RNA derived from honeysuckle, known as miR2911, has been shown to play a key role in inhibiting influenza virus infection and SARS-CoV-2 infection. However, whether miR2911 inhibits the replication of porcine reproductive and respiratory syndrome virus (PRRSV) remains unknown. Hence, this study investigated the mechanisms underlying the action of miR2911 during PRRSV infection. Six targets of miR2911 within the PRRSV orf1 (Nsp2: 2459 to 2477, 1871 to 1892, 954 to 977, and 1271 to 1292; Nsp1: 274 to 296 and 822 to 841) were successfully identified by using the miRanda v1.0b software. The miR2911 target sequence was analyzed by target sequence comparison, and only individual base mutations existed in different prevalent strains, and the miR2911 target region was highly conserved among different strains. Subsequently, through the dual luciferase reporter gene assay and miR2911 overexpression assay, it was demonstrated that miR2911 significantly inhibits the replication of PRRSV by targeting regions of PRRSV Nsp1 and Nsp2. These findings offer new insights for the development of novel anti-PRRSV drugs. [ABSTRACT FROM AUTHOR]

Genetic constructs must be delivered selectively to target tissues and intracellular compartments at the necessary concentrations in order to achieve the maximum therapeutic effect in gene therapy. Development of targeted carriers for non-viral delivery of nucleic acids into cells, including those in muscle, which is one of the most challenging tissues to transfect in vivo, remains a topical issue. We have studied ternary complexes of plasmid DNA and an arginine–histidine-rich peptide-based carrier coated with a glutamate–histidine-rich polymer bearing skeletal muscle targeting peptide (SMTP) for the gene delivery to muscle tissue. The relaxation of the ternary complexes after polyanion treatment was assessed using the ethidium bromide displacement assay. The developed polyplexes were used to transfect C2C12 myoblasts in full-media conditions, followed by analysis of their toxic properties using the Alamar Blue assay and expression analysis of lacZ and GFP reporter genes. After delivering plasmids containing the GFP and lacZ genes into the femoral muscles of mdx mice, which are model of Duchenne muscular dystrophy, GFP fluorescence and β-galactosidase activity were detected. We observed that the modification of ternary polyplexes with 10 mol% of SMTP ligand resulted in a 2.3-fold increase in lacZ gene expression when compared to unmodified control polyplexes in vivo. Thus, we have demonstrated that the developed DNA/carrier complexes and SMTP-modified coating are nontoxic, are stable against polyanion-induced relaxation, and can provide targeted gene delivery to muscle cells and tissues. The results of this study are useful for a range of therapeutic applications, from immunization to amelioration of inherited neuromuscular diseases. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 belongs to the family Coronaviridae and carries a single-stranded positive-sense RNA genome. During coronavirus (CoV) replication, defective or defective interfering RNAs that lack a large portion of the genome often emerge. These defective RNAs typically carry the necessary RNA elements that are required for replication and packaging. We identified the minimum requirement of the 5′ proximal region necessary for viral RNA replication by using artificially generated SARS-CoV-2 minigenomes. The minigenomes consist of the 5′-proximal region, an open reading frame (ORF) that encodes a fusion protein consisting of the N-terminal of viral NSP1 and a reporter gene, and the 3′ untranslated region of the SARS-CoV-2 genome. We used a modified SARS-CoV-2 variant to support replication of the minigenomes. A minigenome carrying the 5′ proximal 634 nucleotides replicated, whereas those carrying shorter than 634 nucleotides did not, demonstrating that the entire 265 nt-long 5′ untranslated region and N-terminal portion of the NSP1 coding region are required for the minigenome replication. Minigenome RNAs carrying a specific amino acid substitution or frame shift insertions in the partial NSP1 coding sequence abrogated minigenome replication. Introduction of synonymous mutations in the minigenome RNAs also affected the replication efficiency of the minigenomes. These data suggest that the expression of the N-terminal portion of NSP1 and the primary sequence of the 5′ proximal 634 nucleotides are important for minigenome replication. IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, is highly transmissible and continues to have a significant impact on public health and the global economy. While several vaccines mitigate the severe consequences of SARS-CoV-2 infection, mutant viruses with reduced reactivity to current vaccines continue to emerge and circulate. This study aimed to identify the minimal 5′ proximal region of SARS-CoV-2 genomic RNA required for SARS-CoV-2 defective RNA replication and investigate the importance of an ORF encoded in these defective RNAs. Identifying cis-acting replication signals of SARS-CoV-2 genomic RNA is critical for the development of antivirals that target these signals. Additionally, replication-competent defective RNAs can serve as therapeutic reagents to interfere with SARS-CoV-2 replication. Our findings provide valuable insights into the mechanisms of SARS-CoV-2 RNA replication and the development of reagents that suppress SARS-CoV-2 replication. [ABSTRACT FROM AUTHOR]

Most rare diseases are caused by mutations and can have devastating consequences. Precise gene editing by CRISPR/Cas is an exciting possibility for helping these patients, if no irreversible developmental defects have occurred. To optimize gene editing therapy, reporter mice for gene editing have been generated which, by expression of reporter genes, indicate the efficiency of precise and imprecise gene editing. These mice are important tools for testing and comparing novel gene editing methodologies. This review provides a comprehensive overview of reporter mice for gene editing which all have been used for monitoring CRISPR/Cas-mediated gene editing involving DNA double-strand breaks (DSBs). Furthermore, we discuss how reporter mice can be used for quickly checking genetic alterations by base editing (BE) or prime editing (PE). [ABSTRACT FROM AUTHOR]

The prolactin (PRL) hormone is a major regulator of mammary gland development and lactation. However, it remains unclear whether and how PRL contributes to mammary epithelial cell proliferation and secretion. The Boer and Macheng black crossbred goats are superior in reproduction, meat, and milk, and are popular in Hubei province. To elucidate the mechanisms of PRL on mammary growth and lactation, to improve the local goat economic trade, we have performed studies on these crossbred goats during pregnancy and early lactation, and in goat mammary epithelial cells (GMECs). Here, we first found that the amino acid transporters of SNAT1 and SNAT2 expression in vivo and in vitro were closely associated with PRL levels, the proliferation and secretion of GMECs; knockdown and over-expression of SNAT1/2 demonstrated that PRL modulated the proliferation and lactation of GMECs through regulating SNAT1/2 expression. Transcriptome sequencing and qPCR assays demonstrated the effect of PRL on the transcriptional regulation of SNAT1 and SNAT2 in GMECs. Dual-luciferase reporter gene assays further verified that the binding of the potential PRL response element in the SNAT1/2 promoter regions activated SNAT1/2 transcription after PRL stimulation. Additionally, silencing of either PRLR or STAT5 nearly abolished PRL-stimulated SNAT1/2 promoter activity, suggesting PRLR–STAT5 signaling is involved in the regulation of PRL on the transcriptional activation of SNAT1/2. These results illustrated that PRL modulates the proliferation and secretion of GMECs via PRLR–STAT5-mediated regulation of the SNAT1/2 pathway. This study provides new insights into how PRL affects ruminant mammary development and lactation through regulation of amino acid transporters. [ABSTRACT FROM AUTHOR]

Intestinal inflammation and compromised barrier function are critical factors in the pathogenesis of gastrointestinal disorders. This study aimed to investigate the role of miR-192-5p in modulating intestinal epithelial barrier (IEB) integrity and its association with autophagy. A DSS-induced colitis model was used to assess the effects of miR-192-5p on intestinal inflammation. In vitro experiments involved cell culture and transient transfection techniques. Various assays, including dual-luciferase reporter gene assays, quantitative real-time PCR, Western blotting, and measurements of transepithelial electrical resistance, were performed to evaluate changes in miR-192-5p expression, Rictor levels, and autophagy flux. Immunofluorescence staining, H&E staining, TEER measurements, and FITC-dextran analysis were also used. Our findings revealed a reduced expression of miR-192-5p in inflamed intestinal tissues, correlating with impaired IEB function. Overexpression of miR-192-5p alleviated TNF-induced IEB dysfunction by targeting Rictor, resulting in enhanced autophagy flux in enterocytes (ECs). Moreover, the therapeutic potential of miR-192-5p was substantiated in colitis mice, wherein increased miR-192-5p expression ameliorated intestinal inflammatory injury by enhancing autophagy flux in ECs through the modulation of Rictor. Our study highlights the therapeutic potential of miR-192-5p in enteritis by demonstrating its role in regulating autophagy and preserving IEB function. Targeting the miR-192-5p/Rictor axis is a promising approach for mitigating gut inflammatory injury and improving barrier integrity in patients with enteritis. NEW & NOTEWORTHY: We uncover the pivotal role of miR-192-5p in fortifying intestinal barriers amidst inflammation. Reduced miR-192-5p levels correlated with compromised gut integrity during inflammation. Notably, boosting miR-192-5p reversed gut damage by enhancing autophagy via suppressing Rictor, offering a potential therapeutic strategy for fortifying the intestinal barrier and alleviating inflammation in patients with enteritis. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T-cell products are commonly generated using lentiviral vector (LV) transduction. Optimal final formulation buffer (FFB) supporting LV stability during cryostorage is crucial for cost-effective manufacturing. To identify the ideal LV FFB composition for ex vivo CAR-T production, primary human T cells were transduced with vesicular stomatitis virus G-protein (VSV-G) -pseudotyped LVs (encoding a reporter gene or an anti-CD19-CAR). The formulations included phosphate-buffered saline (PBS), HEPES, or X-VIVOTM 15, and stabilizing excipients. The functional and viral particle titers and vector copy number were measured after LV cryopreservation and up to 24 h post-thaw incubation. CAR-Ts were produced with LVs in selected FFBs, and the resulting cells were characterized. Post-cryopreservation, HEPES-based FFBs provided higher LV functional titers than PBS and X-VIVOTM 15, and 10% trehalose-20 mM MgCl 2 improved LV transduction efficiency in PBS and 50 mM HEPES. Thawed vectors remained stable at +4°C, while a ≤ 25% median decrease in the functional titer occurred during 24 h at room temperature. Tested excipients did not enhance LV post-thaw stability. CAR-Ts produced using LVs cryopreserved in 10% trehalose- or sucrose-20 mM MgCl 2 in 50 mM HEPES showed comparable transduction rates, cell yield, viability, phenotype, and in vitro functionality. A buffer consisting of 10% trehalose-20 mM MgCl 2 in 50 mM HEPES provided a feasible FFB to cryopreserve a VSV-G -pseudotyped LV for CAR-T-cell production. The LVs remained relatively stable for at least 24 h post-thaw, even at ambient temperatures. This study provides insights into process development, showing LV formulation data generated using the relevant target cell type for CAR-T-cell manufacturing. [ABSTRACT FROM AUTHOR]

SUMMARY: RNA‐guided endonucleases originating from the bacterial CRISPR/Cas system are a versatile tool for targeted gene editing. To determine the functional relevance of a gene of interest, deletion of the entire open reading frame (ORF) by two independent double‐strand breaks (DSBs) is particularly attractive. This strategy greatly benefits from high editing efficiency, which is strongly influenced by the Cas endonuclease version used. We developed two reporter switch‐on assays, for quantitative comparison and optimization of Cas constructs. The assays are based on four components: (i) A reporter gene, the mRNA of which carries a hairpin (HP) loop targeted by (ii) the endoribonuclease Csy4. Cleavage of the mRNA at the HP loop by Csy4 abolishes the translation of the reporter. Csy4 was used as the target for full deletion. (iii) A Cas system targeting sites flanking the Csy4 ORF with a 20‐bp spacer either side to preferentially detect full‐deletion events. Loss of functional Csy4 would lead to reporter gene expression, allowing indirect quantification of Cas‐mediated deletion events. (iv) A reference gene for normalization. We tested these assays on Nicotiana benthamiana leaves and Lotus japonicus calli induced on hypocotyl sections, using Firefly luciferase and mCitrine as reporter genes and Renilla luciferase and hygromycin phosphotransferase II as reference genes, respectively. We observed a >90% correlation between reporter expression and full Csy4 deletion events, demonstrating the validity of these assays. The principle of using the Csy4–HP module as Cas target should be applicable to other editing goals including single DSBs in all organisms. Significance Statement: Cas endonuclease systems have revolutionized genome editing but rapid quantitative assays for the optimization of this process that circumvent the need for DNA extraction from individual plants are rare or lacking. Here, we describe two quantitative reporter switch‐on assays for the direct side‐by‐side comparison of the editing efficiency of different Cas systems, like Cas9 and Cas12a, the principles of which can be applied for optimizing gene editing in any plant species. [ABSTRACT FROM AUTHOR]

Gliomas are the most prevalent primary malignant brain tumors worldwide. Growing evidences indicate that circular RNAs (circRNAs) play an important role in the regulation of biological behavior of tumors. We aimed to investigate the role and mechanism of circVCAN in glioma. RNase R treatment was utilized to assess the cyclic properties of circVCAN. CircVCAN, miR‐488‐3p, and myocyte enhancer factor 2C (MEF2C) levels in glioma tissues and cells were detected by reverse transcription real‐time polymerase chain reaction (RT‐qPCR), and the localization of them in glioma cells was determined with fluorescence in situ hybridization. Furthermore, a variety of biologically functional assessments were used to validate the role of circVCAN in glioma. The regulatory mechanisms of circVCAN, miR‐488‐3p, and MEF2C were further confirmed by double luciferase reporter gene assay, RNA immunoprecipitation and RNA pull‐down assay, and the binding of MEF2C to JAGGED1 was revealed by chromatin immunoprecipitation. Additionally, a xenograft tumor model was constructed to demonstrate the effect of circVCAN on tumor growth in vivo. Our results indicated that circVCAN was more stable than its linear RNA and was significantly upregulated in gliomas. CircVCAN overexpression stimulated glioma cells to proliferate and metastasize, but circVCAN silencing exerted the opposite effect. Meanwhile, silencing circVCAN inhibited tumor growth in vivo. Moreover, we found that circVCAN interacted with miR‐488‐3p to regulate MEF2C expression, and miR‐488‐3p inhibition or MEF2C overexpression reversed the inhibitory effect on malignant bio‐behaviors mediated by circVCAN knockdown in glioma cells. MEF2C promoted the transcription of JAGGED1, and circVCAN knockdown reduced the binding between MEF2C and JAGGED1. Collectively, circVCAN is a carcinogenic circRNA in glioma, and the circVCAN/miR‐488‐3p/MEF2C‐JAGGED1 axis could serve as a potential target for the management of glioma. [ABSTRACT FROM AUTHOR]

Extra spindle-polar body like 1 (ESPL1) is associated with the development of a variety of cancers, including bladder cancer, and is closely related to chemoresistance. In this study, we aimed to reveal the role of ESPL1 in bladder cancer progression and cisplatin (DDP) resistance. First, ESPL1 was found to be highly expressed in tumor tissues and cells of bladder cancer, and more highly expressed in cisplatin resistant tumor tissues or cells. The binding of PAX2 in ESPL1 promoter region was predicted by Jaspar database and verified by Ch-IP analysis and the luciferase reporter gene assay. Next, cisplatin-resistant T24 cells (T24/DDP) were established and transfected with ESPL1 siRNA (si-ESPL1) or overexpression vector (pcDNA-ESPL1) or co-transfected with PAX2 siRNA (si-PAX2) or overexpression vector (pcDNA-PAX2), and then treated with DDP or AG490, an inhibitor of JAK2. The results showed that silencing ESPL1 significantly reduced T24/DDP cell viability, colony formation and invasion, enhanced sensitivity to DDP, and induced cell apoptosis. Silencing PAX2 decreased ESPL1 expression, enhanced sensitivity to DDP, and induced apoptosis of T24/DDP cells, and inhibited activation of JAK2/STAT3 pathway. Overexpressing ESPL1 reversed the effect of PAX2 silencing on T24/DDP cells, while AG490 counteracted the reversal effect of overexpressing ESPL1. Finally, a xenograft tumor model was established and found that silencing ESPL1 or DDP treatment inhibited tumor growth, while silencing ESPL1 combined with DDP treatment had the best effect. In summary, this study suggested that PAX2-mediated ESPL1 transcriptional activation enhanced cisplatin resistance in bladder cancer by activating JAK2/STAT3 pathway. [ABSTRACT FROM AUTHOR]

Renal fibrosis (RF) is a typical pathological presentation of end‐stage chronic kidney disease (CKD) and autosomal dominant polycystic kidney disease (ADPKD). However, the precise regulatory mechanisms governing this re‐expression process remain unclear. Differentially expressed microRNAs (miRNAs) associated with RF were screened by microarray analysis using the Gene Expression Omnibus (GEO) database. The miRNAs upstream of the genes in question were predicted using the miRWalk database. The miRNAs involved in the two GEO data sets were intersected to identify key miRNAs; their regulatory pathways were investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Subsequently, the effects and the underlying mechanisms of target miRNA on RF were examined in a unilateral ureteral obstruction (UUO)‐induced mice renal fibrotic model and a transforming growth factor‐β1 (TGF‐β1)‐induced tubular epithelium (HK‐2) fibrotic cell model. In total, 109 and 32 differentially expressed miRNAs were identified in the GSE133530 and GSE80247 data sets, respectively. GREM1 was identified as a hub gene, where its 2196 upstream miRNAs were predicted; miR‐574‐5p was found to be downregulated and closely related to fibrosis after data set intersection and enrichment analyses, thus was selected for further investigation. A differential expression heatmap (GSE162794) showed that miR‐542‐5p was downregulated. The expression of GREM1 mRNA was upregulated, whereas that of miR‐542‐5p was downregulated in UUO mice and fibrotic HK‐2 cells as compared with the relevant controls. The binding site of miR‐542‐5p was predicted at the 3'UTR region of GREM1 and was confirmed by subsequent dual luciferase reporter gene assay. Western blot analysis showed that Gremlin‐1 and Fibronectin were significantly upregulated after induction of TGF‐β1; when miR‐542‐5p was overexpressed or GREM1 mRNA was interfered, the upregulations of Gremlin‐1 and Fibronectin were significantly reduced. Our research demonstrates that miR‐542‐5p plays a critical role in the progression of RF, and thus may be a promising therapeutic target for CKD and ADPKD. [ABSTRACT FROM AUTHOR]

MicroRNA (miR)-200b-3p has been associated with many tumors, but its involvement in pituitary adenoma is unclear. This study investigated the molecular mechanism underlying miR-200b-3p regulation in pituitary adenomas to provide a theoretical basis for treatment. Bioinformatics was used to analyze pituitary adenoma-related genes and screen new targets related to RECK and miRNA. As well, the relationship between miR-200b-3p and RECK protein was verified using a double-luciferase reporter gene assay. The expression of miR-200b-3p in clinical samples was analyzed by in situ hybridization. Transfection of the miR-200b-3p inhibitor and small interfering-RECK (si-RECK) was verified by qPCR. GH3 cell viability and proliferation were detected using CCK8 and EdU assays. Apoptosis was detected by flow cytometry and western blotting. Wound healing and Transwell assays were used to detect cell migration and invasion. The effects of miR-200b-3p and RECK on GH3 cells were verified using salvage experiments. miR-200b-3p was highly expressed in pituitary tumor tissue. Inhibitors of miR-200b-3p inhibited cell proliferation promoted cell apoptosis, inhibited invasion and migration, and inhibited the expression of matrix metalloproteinases. Interestingly, miR-200b-3p negatively regulated RECK. The expression of RECK in pituitary adenoma tissues was lower than that in neighboring tissues. Si-RECK rescued the function of miR-200b-3p inhibitors in the above cellular behaviors, and miR-200b-3p accelerated the development of pituitary adenoma by negatively regulating RECK expression. In summary, this study investigated the molecular mechanism by which miR-200b-3p regulates the progression of pituitary adenoma through the negative regulation of RECK. The findings provide a new target for the treatment of pituitary adenoma. [ABSTRACT FROM AUTHOR]

The von Hippel-Lindau tumor suppressor protein (VHL), an E3 ubiquitin ligase, functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors. Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway, although the specific molecular mechanisms remain unclear. Herein, the roles of mandarin fish (Siniperca chuatsi) VHL (scVHL) in the NF-κB signaling pathway and mandarin fish ranavirus (MRV) replication were explored. The transcription of scVHL was induced by immune stimulation and MRV infection, indicating a potential role in innate immunity. Dual-luciferase reporter gene assays and reverse transcription quantitative PCR (RT-qPCR) results demonstrated that scVHL evoked and positively regulated the NF-κB signaling pathway. Treatment with NF-κB signaling pathway inhibitors indicated that the role of scVHL may be mediated through scIKKα, scIKKβ, scIκBα, or scp65. Co-immunoprecipitation (Co-IP) analysis identified scIκBα as a novel target protein of scVHL. Moreover, scVHL targeted scIκBα to catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway. Following MRV infection, NF-κB signaling remained activated, which, in turn, promoted MRV replication. These findings suggest that scVHL not only positively regulates NF-κB but also significantly enhances MRV replication. This study reveals a novel function of scVHL in NF-κB signaling and viral infection in fish. [ABSTRACT FROM AUTHOR]