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Cowpea (Vigna unguiculata) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in Vigna unguiculata is dramatically hampered by the lack of mutant resources and efficient tools for gene inactivation in vivo. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We applied the CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the representative symbiotic nitrogen fixation (SNF) gene in Vigna unguiculata. Our customized guide RNAs (gRNAs) targeting symbiosis receptor-like kinase (SYMRK) achieved ~67% mutagenic efficiency in hairy-root-transformed plants, and nodule formation was completely blocked in the mutants with both alleles disrupted. Various types of mutations were observed near the PAM region of the respective gRNA. These results demonstrate the applicability of the CRISPR/Cas9 system in Vigna unguiculata, and therefore should significantly stimulate functional genomics analyses of many important agronomical traits in this unique crop legume.

Introduction As a lytic inflammatory cell death, pyroptosis has been recently described but has not been unequivocally elucidated in diabetic nephropathy (DN). VX‐765 is a safe and effective inhibitor of caspase‐1, that was well tolerated in a phase II clinical trial in patients with epilepsy, but its application in DN is still undefined. Materials and Methods Immunoblot, co‐immunoprecipitation, confocal microscope and flow cytometry were used to analyze the effects of glucose on pyroptosis in renal tubular epithelia (HK‐2). In vitro, selective caspase‐1 inhibitors VX‐765 and Z‐YVAD‐FMK were administered. Pyroptosis and fibrogenesis were determined by immunoblot, ELISA, cytotoxicity assay and flow cytometry. In vivo, diabetic mice were administered with 100 mg/kg VX‐765. Renal function, pathological changes, and the expressions of NLRC4, GSDMD, IL‐1β, collagen I, fibronectin and CD45 in renal cortex were evaluated. Results We identified NLRC4 as a sensor for caspase‐1 activation. Moreover, we provided morphological and molecular evidence for pyroptosis in glucose‐stressed tubular cells, including ballooned cell membrane, caspase‐1 immunoreactivity, GSDMD cleavage, and the release of inflammatory cytokine and cellular contents. All these effects were prevented by treatment with VX‐765 or Z‐YVAD‐FMK, confirming that caspase‐1 effectively regulates the occurrence of pyroptosis in HK‐2 cells. In vivo, treatment of diabetic animals with VX‐765 ameliorated renal function, suppressed inflammatory cell infiltration and pyroptosis‐associated protein expression, and mitigated tubulointerstitial fibrosis. Conclusions This work revealed that caspase‐1‐mediated pyroptosis drives renal inflammation and fibrosis in diabetes. Our results are the first demonstration of VX‐765 representing a promising therapeutic opportunity for alleviating the progression of DN. VX‐765 represents a promising therapeutic opportunity for alleviating the progression of diabetic nephropathy via inhibiting caspase‐1‐mediated pyroptosis, renal inflammation and fibrosis.

Diabetic nephropathy (DN) is one of the main causes of end-stage renal disease (ESRD). Existing treatments cannot control the progression of diabetic nephropathy very well. In diabetic nephropathy, Many monocytes and macrophages infiltrate kidney tissue. However, the role of these cells in the pathogenesis of diabetic nephropathy has not been fully elucidated. In this study, we analyzed patient kidney biopsy specimens, diabetic nephropathy model animals. Meanwhile, we cocultured cells and found that in diabetic nephropathy, damaged intrinsic renal cells (glomerular mesangial cells and renal tubular epithelial cells) recruited monocytes/macrophages to the area of tissue damage to defend against and clear cell damage. This process often involved the activation of different types of macrophages. Interestingly, the infiltrating macrophages were mainly M1 (CD68+iNOS+) macrophages. In diabetic nephropathy, crosstalk between the Notch pathway and NF-κB signaling in macrophages contributed to the polarization of macrophages. Hyperpolarized macrophages secreted large amounts of inflammatory cytokines and exacerbated the inflammatory response, extracellular matrix secretion, fibrosis, and necroptosis of intrinsic kidney cells. Additionally, macrophage depletion therapy with clodronate liposomes and inhibition of the Notch pathway in macrophages alleviated the pathological changes in kidney cells. This study provides new information regarding diabetic nephropathy-related renal inflammation, the causes of macrophage polarization, and therapeutic targets for diabetic nephropathy.

Chlamydomonas reinhardtii, a model green alga for expressing foreign proteins, faces challenges in multigene expression and enhancing protein expression level in the chloroplast. To address these challenges, we compared heterologous promoters, terminators and intercistronic expression elements (IEEs). We transformed Chlamydomonas chloroplast with a biolistic approach to introduce vectors containing the NanoLuc expression unit regulated by Chlamydomonas or tobacco promoters and terminators. We observed that tobacco promoters PrbcL and PpsbA could not effectively regulate protein expression, whereas tobacco terminators TrbcL and Trps16 did not affect the expression of Nluc protein. Further exploration of IEEs specific to Chlamydomonas revealed that Cr‐IEE2 had a minor effect on both upstream and downstream protein expression, whereas Cr‐IEE5 significantly influenced downstream protein expression. In contrast, tobacco IEE was found to be unsuitable for driving protein expression in Chlamydomonas. Additionally, VOR element and Rep protein derived from beet curly top geminivirus were able to form a minichromosome in Chlamydomonas chloroplast, and this system could enhance protein expression level compared to the traditional method of site‐specific integration in the plastome. This study highlights the potential of IEEs and minichromosome in facilitating heterologous protein expression in Chlamydomonas chloroplast. This study compared representative tobacco and Chlamydomonas promoters/terminators, intercistronic expression elements (IEEs), formation of a minichromosome and their effects on the heterologous protein expression in Chlamydomonas chloroplast. Results showed that the tobacco promoters and IEE do not function in Chlamydomonas, and VOR‐Rep‐mediated minichromosome could dramatically enhance protein expression levels in the algal chloroplast.

The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus.

Long noncoding RNAs (lncRNAs) cover a large class of transcribed RNA molecules that are more than 200 nucleotides in length. An increasing number of studies have shown that lncRNAs control gene expression through different mechanisms and play important roles in a range of biological processes including growth, cell differentiation, proliferation, apoptosis, and invasion. TUG1 was originally discovered in a genomic screen of taurine-treated mouse retinal cells. Previous evidences pointed out that lncRNA TUG1 could inhibit apoptosis and the release of inflammatory factors, improve mitochondrial function, thereby protecting cells from damage, and showing a protective role of TUG1 in diseases. Given that TUG1 has multiple targets and can interfere with multiple steps in the oncogenic process, it has been proposed as a therapeutic target. In this review, we summarize the research progress of lncRNA TUG1 in kidney diseases in the past 8 years, and discuss its related molecular mechanisms.

The aim of our study was to investigate the role of autophagy, a homeostatic process involved in the lysosomal degradation of damaged cell organelles and proteins, in regulating the survival of mesangial cells treated with advanced glycation end products (AGEs). In the present study, AGEs induced mitochondrial depolarization and led to mitochondrial-dependent apoptosis in mesangial cells, as shown by the loss of the mitochondrial membrane potential; increased Bax processing; increased Caspase-9, Caspase-3 and PARP cleavage; and decreased Bcl-2 expression. Meanwhile, AGEs also triggered autophagy flux in mesangial cells, as confirmed by the presence of autophagic vesicles, the conversion of LC3II/LC3I and the increase/decrease in Beclin-1/p62 expression. Interestingly, this study reported apparent apoptosis and autophagy that were dependent on reactive oxygen species (ROS) production. Scavenging ROS with N -acetyl- l -cysteine could prevent the appearance of the autophagic features and reverse AGE-induced apoptosis. Moreover, AGE-triggered mitophagy, which was confirmed by the colocalization of autophagosomes and mitochondria and Parkin translocation to mitochondria, played a potential role in reducing ROS production in mesangial cells. Additionally, inhibition of autophagy significantly enhanced AGE-induced cell apoptosis. Taken together, our data suggest that ROS were the mediators of AGE-induced mesangial cell apoptosis and that autophagy was likely to be the mechanism that was triggered to repair the ROS-induced damage in the AGE-treated cells and thereby promote cell survival. This study provides new insights into the molecular mechanism of autophagy involved in AGE-induced apoptosis in mesangial cells.

Bacterial microcompartments (BMCs) are intracellular structures for compartmentalizing specific metabolic pathways in bacteria. As a unique type of BMCs, carboxysomes utilize protein shells to sequester ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase for efficient carbon dioxide (CO2) fixation. This study aims to reconstruct an α-carboxysome in Saccharomyces cerevisiae and investigate its metabolic effects. Here, genes of the cso operon from Halothiobacillus neapolitanus, Calvin cycle-related enzyme phosphoribulokinase (PRK) from Spinacia oleracea, and two S. cerevisiae chaperone genes, HSP60 and HSP10, were introduced into S. cerevisiae. The engineered yeast strain demonstrated assembled and enzymatically active Rubisco, significant increase in ethanol production and reduction in the byproduct glycerol. Formation of the α-carboxysome structures was observed after purification by sucrose density gradient centrifugation. The engineered yeast strain harboring functional α-carboxysome has the potential for enhancing bioethanol production.

Background Age-related kidney impairment, characterized by tubular epithelial cell senescence and renal fibrosis, poses a significant global public health threat. Although N6-methyladenosine (m6A) methylation is implicated in various pathological processes, its regulatory mechanism in kidney aging remains unclear. Methods An m6A-mRNA epitranscriptomic microarray was performed to identify genes with abnormal m6A modifications in aged human kidney tissues. Histological, immunohistochemical, and immunofluorescent staining, western blot, and RT-qPCR were employed to examine the biological functions of targeted genes and m6A methyltransferases both in vivo and in vitro. RNA immunoprecipitation, chromatin immunoprecipitation, ribosomal immunoprecipitation, and luciferase reporter assays were used to investigate the specific interactions between m6A methyltransferases, targeted genes, and their downstream signals. Results Significantly lower m6A modification levels were observed in aged human kidney tissues. GLIS1, identified as a “metabolic remodeling factor,” showed significantly reduced protein levels with abnormal m6A modifications. The downregulation of GLIS1 induced cell senescence and renal fibrosis by shifting metabolic remodeling from fatty acid oxidation (FAO) to glycolysis. Additionally, the methylated GLIS1 mRNA was regulated by the abnormal expression of METTL3 and YTHDF1. Silencing METTL3/YTHDF1 weakened the translation of GLIS1 and disrupted the balance between FAO and glycolysis. Conclusions Our findings suggest that the m6A modification of GLIS1, activated by METTL3 and reduced in a YTHDF1-dependent manner, leads to kidney aging by regulating the metabolic shift from FAO to glycolysis. This mechanism provides a promising therapeutic target for kidney aging.

To explore the N6-methyladenosine (m A) modification mechanism of taurine upregulated gene 1 (TUG1) and whether methyltransferase 3 (METTL3) can promote peroxisome proliferators-activated receptor γ coactivator 1 alpha (PGC-1α) transcription and alleviate mitochondrial dysfunction. high glucose (HG)-treated HK-2 cell models and db/db mice models injected with rAAV-METTL3 the tail vein were established. The expression levels were determined by RT-qPCR, western blot, and immunohistochemical staining. RNA m A modification was analyzed by the RNase Mazf. The biochemical indicators of mice were detected by enzyme-linked immunosorbent assay. Cell apoptosis was detected by flow cytometry. Histopathological staining was performed to evaluate kidney injury. mtDNA content, mitochondrial complex activity, and ATP were detected by RT-qPCR and detection kits, respectively, per the manufacturer's instructions. Mitochondrial reactive oxygen species production in HK-2 cells incubated with MitoSOX Red and mitochondrial morphology were observed under a fluorescence microscope and transmission electron microscope, respectively. Molecular interactions were verified through RNA immunoprecipitation, RNA pull-down, and dual-luciferase reporter gene assay. METTL3 and TUG1 expression levels decreased in the kidneys of diabetic mice and HG-treated HK-2 cells. Mechanistically, METTL3-mediated m A modification increased the stability of TUG1 in an insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2)-dependent manner. METTL3-mediated m A modification of TUG1 promotes PGC-1α activation, thereby alleviating mitochondrial dysfunction in HG-treated HK-2 cells and db/db mice. Moreover, METTL3 overexpression alleviated kidney injury in db/db mice. METTL3 targets TUG1/PGC-1α and ameliorates mitochondrial dysfunction in diabetic nephropathy in an IGF2BP2-dependent manner.