Graphene oxide-assisted optimized narrow-thermal-cycling amplification for accurate detection of Salmonella spp.

Salmonella is a rod-shaped, Gram-negative zoonotic pathogen that poses a serious global socioeconomic and public health threat. Rapid and accurate detection of Salmonella spp. is critical for effective control of its infection. In this study, an accurate, sensitive and specific graphene oxide-assisted accelerated strand exchange amplification (GO-ASEA) method for rapid detection of Salmonella spp. was developed and validated. The detection limit of the GO-ASEA method was 8.6 × 101 fg µL-1 of Salmonella genomic DNA or 1 × 101 CFU g-1 of Salmonella in spiked chicken faeces free of pre-enrichment. And the GO-ASEA method could specifically detect Salmonella spp. without cross-reactivity with other enteric pathogens. In addition, the novel method achieved Salmonella detection within 30 min and was validated using 209 clinical samples, showing its good clinical applicability. Therefore, the GO-ASEA method is a new optional tool for the rapid detection of pathogenic microorganisms, which is ideal for food safety monitoring and high-throughput detection.

PPP2R2A affects embryonic implantation by regulating the proliferation and apoptosis of Hu sheep endometrial stromal cells.

Embryonic implantation is a complex reproductive physiological process in mammals. Although several endometrial proteins affecting embryonic implantation have been reported in the past, there are still potential endometrial proteins that have been neglected, and their specific regulatory mechanisms are unclear. This study demonstrated that protein phosphatase 2A regulatory subunit B55α (PPP2R2A) served as a novel regulator in medication of sheep embryonic implantation in vitro. Our results showed that sheep PPP2R2A encoded 447 amino acids and shared 91.74%-92.36% amino acid sequences with its orthologs compared with other species. Meanwhile, PPP2R2A was widely expressed in sheep uterine tissues, and it could regulate the expression levels of key regulators of embryonic implantation in endometrial stromal cells (ESCs). Knockdown of PPP2R2A significantly inhibited cell proliferation by blocking cell cycle transfer G0/G1 into S phase accompanied by downregulation of CDK2, CDK4, CCND1, CCNE1 and upregulation of P21. In contrast to PPP2R2A overexpression, PPP2R2A interference greatly promoted cell apoptosis and the expression of BAX, CASP3, CASP9 and BAX/BCL-2. Taken together, these results suggest that PPP2R2A, as a novel regulatory factor, affects embryonic implantation via regulating the proliferation and apoptosis of Hu sheep ESCs in vitro.

Effects of SPATA6 on proliferation, apoptosis and steroidogenesis of Hu sheep Leydig cells in vitro.

This study aimed to investigate the expression pattern of spermatogenesis associated protein 6 (SPATA6) in Hu sheep testis and to ascertain the effects of SPATA6 on sheep Leydig cells (LCs) function linked to spermatogenesis. In the present study, we detected a 1970 bp cDNA fragment of SPATA6 included a 1467 bp coding sequence which encoded 487 amino acids. Meanwhile, sheep SPATA6 shared 51.70%-97.41% amino acid sequences with its orthologs compared with other species. In addition, SPATA6 was highly expressed in testis and localized in cytoplasm and nucleus of LCs as well as spermatogenic cells at different stages. Compared to the negative control (NC), SPATA6 interference promoted apoptosis of LCs with the increase of BAX/BCL-2 mRNA and protein levels, while the results of SPATA6 overexpression were on the contrary. Meanwhile, cell cycle was blocked at G2/M phase and CDK1 and CCNB1 were down-regulated after SPATA6 interference. SPATA6 overexpression induced cell cycle transfer G0/G1 into S and G2/M phase with upregulation of CDK1, CDK4, CCND1 and CCND2. Moreover, the secretion of testosterone hormone and the expression of StAR in LCs with SPATA6 overexpression were significantly promoted. Overall, our data suggest that SPATA6 is an important functional molecule of spermatogenesis, via regulating the proliferation, apoptosis and testosterone biosynthesis of Hu sheep LCs. These findings will enhance the understanding of the roles of SPATA6 in sheep spermatogenesis.

ZEA exerts toxic effects on reproduction and development by mediating Dio3os in mouse endometrial stromal cells.

Zearalenone (ZEA) and imprinted long noncoding RNAs (lncRNAs) are both closely related to reproduction and development. However, whether they have connections in regulating reproduction and development is not clear yet. The aim of this research is to investigate their relationship. lncRNA microarray was performed to analyze differentially expressed genes, and real-time quantitative polymerase chain reaction (PCR) was used to verify the accuracy of microarray analysis. Meanwhile, the technologies of rapid amplification of cDNA ends, RNA fluorescence in situ hybridization and bioinformatics were adopted to characterize the selected lncRNA. Analysis of lncRNA microarray showed lncRNAs and messenger RNAs related to reproduction and development were significantly differently expressed, and Dio3os was probably the target lncRNA. Then, the experiment of real-time quantitative PCR verified the accuracy of microarray data. Characterization of Dio3os showed Dio3os, an antisense lncRNA with 2312 bp and 15 open reading frames, was enriched in the cytoplasm. Our findings suggest ZEA probably exerts toxic effects on reproduction and development by mediating Dio3os.

Evaluation of deoxynivalenol-induced toxic effects on mouse endometrial stromal cells: Cell apoptosis and cell cycle.

Deoxynivalenol (DON) is a type B trichothecene mycotoxin which has toxic effects on humans and animals. Although DON has been studied in various cell types for its cytotoxicity, there is litter information about the effects of DON on mouse endometrial stromal cells (ESCs). Thus, in this study, we investigated the toxic effects of DON on mouse ESCs and its possible mechanisms. DON inhibited the cell viability in a dose- and time-dependent manner. TUNEL assay results showed that DON caused apoptosis and TUNEL-positive cells increased with increasing DON concentrations in mouse ESCs. Western blot showed that DON significantly increased the expression levels of apoptosis-related protein including Caspase-9, Caspase-3, poly (ADP-ribose) polymerase (PARP) and the ratio of Bax/Bcl-2. After DON treatment, the expression levels of cell cycle-related protein including p38/p-p38, Cdc25C/p-Cdc25C, Cdc2/p-Cdc2 and cyclinB1 were significantly decreased and immunoprecipitation analysis showed that cyclinB1-Cdc2 complex was significantly decreased. However, the combination of SB203580 (p38 specific inhibitor) and DON treatment significantly reversed the depression of Cdc25C/p-Cdc25C, Cdc2/p-Cdc2, cyclinB1 and cyclinB1-Cdc2 complex. Collectively, these data suggest that DON causes apoptosis via mitochondria apoptosis pathway and induces G2 arrest via p38 MAPK signaling pathway in mouse ESCs.

Exploration of ZEA cytotoxicity to mouse endometrial stromal cells and RNA-seq analysis.

Because zearalenone (ZEA) causes harmful influence to animals and widely exists in the world, the researches on ZEA have never stopped. However, the mechanisms of ZEA on proliferation, cycle, and apoptosis in endometrial stromal cells (ESCs) remain poorly defined. Therefore, the purpose of our study was to explore the effects of ZEA to ESCs and demonstrate them by transcriptomic analysis. The results showed that after ZEA treatment, ESCs appeared numerous adverse reactions, and the phenomena of cell viability decrease, DNA replication block and apoptosis were detected by flow cytometry, Annexin V-FITC/PI double-staining method, TUNEL assay, and so on. Then, RNA-seq approach was adopted to prove the validity of above experiments, as expected, the results from different expression genes, gene ontology terms, and KEGG pathway were all consistent with those. Overall, the results suggested that ZEA could cause a series of reactions by cytotoxicity to mouse ESCs, meanwhile there must be some substances and mechanisms protect cells against cytotoxicity damage.

Advances in nanopore sequencing technology.

Much tremendous break through have been obtained in recent years for nanopore sequencing to achieve the goal of $1000 genome. As a method of single molecule sequencing, nanopore sequencing can discriminate the individual molecules of the target DNA strand rapidly due to the current blockages by translocating the nucleotides through a nano-scale pore. Both the protein-pores and solid-state nanopore channels which called single nanopore sequencing have been studied widely for the application of nanopore sequencing technology. This review will give a detail representation to protein nanopore and solid-state nanopore sequencing. For protein nanopore sequencing technology, we will introduce different nanopore types, device assembly and some challenges still exist at present. We will focus on more research fields for solid-state nanopore sequencing in terms of materials, device assembly, fabricated methods, translocation process and some specific challenges. The review also covers some of the technical advances in the union nanopore sequencing, which include nanopore sequencing combine with exonuclease, hybridization, synthesis and design polymer.