Roles of AGD2a in Plant Development and Microbial Interactions of Lotus japonicus

Arabidopsis AGD2 (Aberrant Growth and Death2) and its close homolog ALD1 (AGD2-like defense response protein 1) have divergent roles in plant defense. We previously reported that modulation of salicylic acid (SA) contents by ALD1 affects numbers of nodules produced by Lotus japonicus, but AGD2′s role in leguminous plants remains unclear. A combination of enzymatic analysis and biological characterization of genetic materials was used to study the function of AGD2 (LjAGD2a and LjAGD2b) in L. japonicus. Both LjAGD2a and LjAGD2b could complement dapD and dapE mutants of Escherichia coli and had aminotransferase activity in vitro. ljagd2 plants, with insertional mutations of LjAGD2, had delayed flowering times and reduced seed weights. In contrast, overexpression of LjAGD2a in L. japonicus induced early flowering, with increases in seed and flower sizes, but reductions in pollen fertility and seed setting rates. Additionally, ljagd2a mutation resulted in increased expression of nodulin genes and corresponding increases in infection threads and nodule numbers following inoculation with Rhizobium. Changes in expression of LjAGD2a in L. japonicus also affected endogenous SA contents and hence resistance to pathogens. Our results indicate that LjAGD2a functions as an LL-DAP aminotransferase and plays important roles in plant development. Moreover, LjAGD2a activates defense signaling via the Lys synthesis pathway, thereby participating in legume–microbe interaction.     

Biomimetic Nanosponges Enable the Detoxification of Vibrio vulnificus Hemolysin

Vibrio vulnificus (V. vulnificus) infection-associated multiple antibiotic resistance has raised serious public health concerns. Recently, nanosponges (NSs) have been expected to provide innovative platforms for addressing antibacterial and drug-resistant challenges by targeting various pore-forming toxins (PFTs). In the present study, we constructed NSs to explore the effects and possible mechanism of recombinant V. vulnificus hemolysin (rVvhA)-induced injuries. In vitro, NSs significantly reversed rVvhA-induced apoptosis and necrosis, and improved toxin-induced intracellular reactive oxygen species (ROS) production, adenosine triphosphate (ATP) depletion, and apoptosis signaling pathway disruption. To explore the clinical translation potential of NSs, we established VvhA-induced septicemia and wound infection mouse models, respectively, and further found NSs could notably attenuate rVvhA-induced acute toxicity and septicemia-associated inflammation, as well as local tissue damage. In a conclusion, NSs showed excellent protective effects against rVvhA-induced toxicity, thus providing useful insights into addressing the rising threats of severe V. vulnificus infections.     

Differential MicroRNA Expression in Porcine Endometrium Related to Spontaneous Embryo Loss during Early Pregnancy

Litter size is an important indicator to measure the production capacity of commercial pigs. Spontaneous embryo loss is an essential factor in determining sow litter size. In early pregnancy, spontaneous embryo loss in porcine is as high as 20–30% during embryo implantation. However, the specific molecular mechanism underlying spontaneous embryo loss at the end of embryo implantation remains unknown. Therefore, we comprehensively used small RNA sequencing technology, bioinformatics analysis, and molecular experiments to determine the microRNA (miRNA) expression profile in the healthy and arresting embryo implantation site of porcine endometrium on day of gestation (DG) 28. A total of 464 miRNAs were identified in arresting endometrium (AE) and healthy endometrium (HE), and 139 differentially expressed miRNAs (DEMs) were screened. We combined the mRNA sequencing dataset from the SRA database to predict the target genes of these miRNAs. A quantitative real-time PCR assay identified the expression levels of miRNAs and mRNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on differentially expressed target genes of DEMs, mainly enriched in epithelial development and amino acids metabolism-related pathways. We performed fluorescence in situ hybridization (FISH) and the dual-luciferase report gene assay to confirm miRNA and predicted target gene binding. miR-205 may inhibit its expression by combining 3′-untranslated regions (3′ UTR) of tubulointerstitial nephritis antigen-like 1 (TINAGL1). The resulting inhibition of angiogenesis in the maternal endometrium ultimately leads to the formation of arresting embryos during the implantation period. This study provides a reference for the effect of miRNA on the successful implantation of pig embryos in early gestation.     

Possible Role of GnIH as a Novel Link between Hyperphagia-Induced Obesity-Related Metabolic Derangements and Hypogonadism in Male Mice

Gonadotropin-inhibitory hormone (GnIH) is a reproductive inhibitor and an endogenous orexigenic neuropeptide that may be involved in energy homeostasis and reproduction. However, whether GnIH is a molecular signal link of metabolism and the reproductive system, and thus, regulates reproductive activity as a function of the energy state, is still unknown. In the present study, we investigated the involvement of GnIH in glycolipid metabolism and reproduction in vivo, and in the coupling between these two processes in the testis level. Our results showed that chronic intraperitoneal injection of GnIH into male mice not only increased food intake and altered meal microstructure but also significantly elevated body mass due to the increased mass of liver and epididymal white adipose tissue (eWAT), despite the loss of testicular weight. Furthermore, chronic intraperitoneal administration of GnIH to male mice resulted in obesity-related glycolipid metabolic derangements, showing hyperlipidemia, hyperglycemia, glucose intolerance, and insulin resistance through changes in the expression of glucose and lipid metabolism-related genes in the pancreas and eWAT, respectively. Interestingly, the expression of GnIH and GPR147 was markedly increased in the testis of mice under conditions of energy imbalance, such as fasting, acute hypoglycemia, and hyperglycemia. In addition, chronic GnIH injection markedly inhibited glucose and lipid metabolism of mice testis while significantly decreasing testosterone synthesis and sperm quality, inducing hypogonadism. These observations indicated that orexigenic GnIH triggers hyperphagia-induced obesity-related metabolic derangements and hypogonadism in male mice, suggesting that GnIH is an emerging candidate for coupling metabolism and fertility by involvement in obesity and metabolic disorder-induced reproductive dysfunction of the testes.     

Integrated Analysis of Microarray,Small RNA,and Degradome Datasets Uncovers the Role of MicroRNAs in Temperature-Sensitive Genic Male Sterility in Wheat

Temperature-sensitive genic male sterile (TGMS) line Beijing Sterility 366 (BS366) has been utilized in hybrid breeding for a long time, but the molecular mechanism underlying male sterility remains unclear. Expression arrays, small RNA, and degradome sequencing were used in this study to explore the potential role of miRNA in the cold-induced male sterility of BS366. Microspore observation showed defective cell plates in dyads and tetrads and shrunken microspores at the vacuolated stage. Differential regulation of Golgi vesicle transport, phragmoplast formation, sporopollenin biosynthesis, pollen exine formation, and lipid metabolism were observed between cold and control conditions. Pollen development was significantly represented in the 352 antagonistic miRNA-target pairs in the integrated analysis of miRNA and mRNA profiles. The specific cleavage of ARF17 and TIR1 by miR160 and miR393 were found in the cold-treated BS366 degradome, respectively. Thus, the cold-mediated miRNAs impaired cell plate formation through repression of Golgi vesicle transport and phragmoplast formation. The repressed expression of ARF17 and TIR1 impaired pollen exine formation. The results of this study will contribute to our understanding of the roles of miRNAs in male sterility in wheat.     

Tetrandrine Inhibits Skeletal Muscle Differentiation by Blocking Autophagic Flux

Tetrandrine is well known to act as a calcium channel blocker. It is a potential candidate for a tumor chemotherapy drug without toxicity. Tetrandrine inhibits cancer cell proliferation and induces cell death through apoptosis and autophagy. As cancer patients usually experience complications with sarcopenia or muscle injury, we thus assessed the effects of tetrandrine on skeletal muscle cells. We report in this study that a low dose of tetrandrine (less than 5 μM) does not affect the proliferation of C2C12 myoblasts, but significantly inhibits myogenic differentiation. Consistently, tetrandrine inhibited muscle regeneration after BaCl₂-induced injury. Mechanistic experiments showed that tetrandrine decreased the p-mTOR level and increased the levels of LC3 and SQSTM1/p62 during differentiation. Ad-mRFP-GFP-LC3B transfection experiments revealed that the lysosomal quenching of GFP signals was suppressed by tetrandrine. Furthermore, the levels of DNM1L/Drp1, PPARGA1 and cytochrome C (Cyto C), as well as caspase 3 activation and ROS production, were decreased following tetrandrine administration, indicating that the mitochondrial network signaling was inhibited. Our results indicate that tetrandrine has dual effects on autophagic flux in myoblasts during differentiation, activation in the early stage and blockade in the late stage. The ultimate blocking of autophagic flux by tetrandrine led to the disruption of mitochondria remodeling and inhibition of myogenic differentiation. The inhibitory effects of tetrandrine on skeletal muscle differentiation may limit its application in advanced cancer patients. Thus, great attention should be paid to the clinical use of tetrandrine for cancer therapy.     

Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus

In microbiological research, it is important to understand the time course of each step in a pathogen’s lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity over time without sampling cells or culture supernatants for analyzing the virus replication. Subgenomic replicon experiments with hepatitis C virus (HCV) showed that transient translation and genome replication can be detected separately, with the first peak of translation observed at 3–4 h and replication beginning around 20 h after viral RNA introduction into cells. From the bioluminescence data set measured every 30 min (48 measurements per day), the initial rates of translation and replication were calculated, and their capacity levels were expressed as the sums of the measured signals in each process, which correspond to the areas on the kinetics graphs. The comparison of various HuH-7-derived cell lines showed that the bioluminescence profile differs among cell lines, suggesting that both translation and replication capacities potentially influence differences in HCV susceptibility. The effects of RNA mutations within the 5′ UTR of the replicon on viral translation and replication were further analyzed in the system developed, confirming that mutations to the miR-122 binding sites primarily reduce replication activity rather than translation. The newly developed real-time monitoring system should be applied to the studies of various viruses and contribute to the analysis of transitions and progression of each process of their life cycle.