BackgroundKinesin superfamily proteins are microtubule-based molecular motors essential for the intracellular transport of various cargos, including organelles, proteins, and RNAs. However, their exact roles during mammalian oocyte meiosis have not been fully clarified.ResultsHerein, we investigated the critical events during porcine oocyte meiotic maturation with the treatment of Eg5-specific inhibitor monastrol. We found that Eg5 inhibition resulted in oocyte meiotic failure by displaying the poor expansion of cumulus cells and reduced rate of polar body extrusion. In the meantime, the spindle assembly and chromosome alignment were compromised, accompanied by the decreased level of acetylated α-tubulin, indicative of less stable microtubules. Impaired actin dynamics and mitochondria integrity were also observed in Eg5-inhibited oocytes. Additionally, inhibition of Eg5 caused the abnormal distribution of cortical granules and ovastacin, a cortical granule component, potentially leading to the fertilization failure.ConclusionsOur findings reveal that Eg5 possesses an important function in porcine oocyte meiotic progression by regulating the organelle dynamics and arrangement.

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Increasing evidence suggests that epigenetic dysfunction may influence the stability of normal pregnancy. The ten-eleven translocation (TET) family and 5-hydroxymethylcytosine (5-hmC) were found to be linked with epigenetic reprogramming. The present study aimed to examine the expression of the TET family and 5-hmC in the villi of human embryos and compared their expression between normal pregnancy and early pregnancy loss (EPL). Embryonic villi were collected from normal pregnant women (control) experiencing medical abortion and from EPL patients at gestation ages of 6, 7 and 8 weeks. The mRNAs of TET family were analysed using quantitative polymerase chain reaction (qPCR), and TET proteins using Western blotting and immunohistochemical analysis. The MethylFlash™ Kit was used to quantify the absolute amount of 5-methylcytosine (5-mC) and 5-hmC. Our results showed that the expression of the TETs and 5-hmC in the normal villus decreased with increasing gestational age. Immunohistochemistry revealed that the TET proteins were expressed in the cytoplasm of trophoblasts and their expression was the highest in the 6-week tissue samples, which was consistent with the qPCR and Western blot results. The expression of TET1, TET2, and TET3 was lower in the villi in EPL group than in normal pregnancy group (P<0.05 for all). It was concluded that the TET family and 5-hmC are critical in epigenetic reprogramming of human embryo. The findings also suggest that a deficiency of TETs in the villus might be associated with human EPL.

Antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) have been detected in human-impacted habitats, especially in densely populated cities. The Qinghai–Tibet Plateau is located far from the heavily populated regions of China, and Tibetan residents have distinct dietary habits and gut microbes. Antibiotic-resistance monitoring in the Tibetan population is rare. Here, we collected stool samples from Tibetan outpatients with diarrhea. From 59 samples, 48 antibiotic-resistant Enterobacteriaceae isolates were obtained, including 19 extended-spectrum beta lactamase (ESBL)-producing isolates from 16 patients and 29 polymyxin-resistant isolates from 22 patients. Either ESBL or mcr genes were found in 17 Escherichia coli isolates, approximately 58.8% of which were multidrug-resistant, and ten incompatible plasmid types were found. The gene blaCTX-M was a common genotype in the ESBL-producing E. coli isolates. Four E. coli isolates contained mcr-1. The same mcr-1-carrying plasmid was found in distinct E. coli isolates obtained from the same sample, thus confirming horizontal transmission of mcr-1 between bacteria. Genomic clustering of E. coli isolates obtained from Lhasa, with strains from other regions providing evidence of clone spreading. Our results reveal a strong presence of ARB and ARGs in Tibetan outpatients with diarrhea, implying that ARB and ARGs should be monitored in the Tibetan population.

Mycobacterium tuberculosis (MTB), one of the major bacterial pathogens for lethal infectious diseases, is capable of surviving within the phagosomes of host alveolar macrophages; therefore, host genetic variations may alter the susceptibility to MTB. In this study, to identify host genes exploited by MTB during infection, genes were non-selectively inactivated using lentivirus-based antisense RNA methods in RAW264.7 macrophages, and the cells that survived virulent MTB infection were then screened. Following DNA sequencing of the surviving cell clones, 26 host genes affecting susceptibility to MTB were identified and their pathways were analyzed by bioinformatics analysis. In total, 9 of these genes were confirmed as positive regulators of collagen α-5(IV) chain (Col4a5) expression, a gene encoding a type IV collagen subunit present on the cell surface. The knockdown of Col4a5 consistently suppressed intracellular mycobacterial viability, promoting the survival of RAW264.7 macrophages following mycobacterial infection. Furthermore, Col4a5 deficiency lowered the pH levels of intracellular vesicles, including endosomes, lysosomes and phagosomes in the RAW264.7 cells. Finally, the knockdown of Col4a5 post-translationally increased microsomal vacuolar-type H+-ATPase activity in macrophages, leading to the acidification of intracellular vesicles. Our findings reveal a novel role for Col4a5 in the regulation of macrophage responses to mycobacterial infection and identify Col4a5 as a potential target for the host-directed anti-mycobacterial therapy.

Background: Tibetan antelope (Rhinopithecus), blue sheep (Pseudois nayauris), and plateau pika (Ochotona curzoniae) are wild animals living on the Qinghai-Tibet Plateau. There have been no reports of naturally-occurring transmissible spongioform encephalopathies (TSEs) involving these animals. Furthermore, the PRNP genes have not been described in the literature. Methods: The PRNP genes from 21 Tibetan antelopes, 4 blue sheep, and 3 plateau pikas were obtained and sequenced. The recombinant proteins were then prepared. Using scrapie strains (263K, 139A, ME7, and S15) as the seeds, the reactivity of the PrP proteins from sheep (rSheepPrP25-234) and pika (rPikaPrP23-230) were tested using real-time quaking-induced conversion (RT-QuIC). Protein misfolding cyclic amplification (PMCA) tests of the brain homogenates from domestic sheep and rabbits were performed with the seeds of strains 263K and ME7. Results: The PRNP genes of bovids were 771 bp long and encoded 256 amino acids (aa), showing 100% homology with the wild-type sheep prion protein (PrP) aa sequence. The PRNP gene of pika was 759 bp long and encoded 252 amino acids, showing 92.1% homology with the aa sequence of domestic rabbits. The sheep and pika proteins revealed positive reactions in 10-5 diluted seeds. Only rPikaPrP23-230 produced positive curves in 10-7 diluted seeds. The PMCA tests failed to produce proteinase K (PK)-resistant PrP (PrPres). Conclusion: This is the first description of PRNP genes and PrP aa sequences of Tibetan antelope, blue sheep, and plateau pike from the Qinghai-Tibet Plateau. In the presence of rodent prions, the PrPs of sheep and pika efficiently induce fibrillation in RT-QuIC, but do not generate PrPres in PMCA. Our results indicate that pika, as one of the important links in the Qinghai-Tibet Plateau biological chain, may play an important role in the prion circulation. Pika PrP deserves further analysis for its potential application value in assays for human prion disease.

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