Mouse KL2 is a unique MTSE involved in chromosome-based spindle organization and regulated by multiple kinases during female meiosis.

Microtubule-severing enzymes (MTSEs) play important roles in mitosis and meiosis of the primitive organisms. However, no studies have assessed their roles in mammalian meiosis of females, whose abnormality accounts for over 80% of the cases of gamete-originated human reproductive disease. In the current study, we reported that katanin-like 2 (KL2) was the only MTSE concentrating at chromosomes. Furthermore, the knockdown of KL2 significantly reduced chromosome-based increase in the microtubule (MT) polymer, increased aberrant kinetochore-MT (K-MT) attachment, delayed meiosis, and severely affected normal fertility. Importantly, we demonstrated that the inhibition of aurora B, a key kinase for correcting aberrant K-MT attachment, eliminated KL2 from chromosomes completely. KL2 also interacted with phosphorylated eukaryotic elongation factor-2 kinase; they competed for chromosome binding. We also observed that the phosphorylated KL2 was localized at spindle poles, and that KL2 phosphorylation was regulated by extracellular signal-regulated kinase 1/2. In summary, our study reveals a novel function of MTSEs in mammalian female meiosis and demonstrates that multiple kinases coordinate to regulate the levels of KL2 at chromosomes.

Oocytes could rearrange immunoglobulin production to survive over adverse environmental stimuli.

Immunoglobulins are key humoral immune molecules produced and secreted by B lymphocytes at various stages of differentiation. No research has reported whether immunoglobulins are present in the non-proliferative female germ cells-oocytes-and whether they are functionally important for oocyte quality, self-protection, and survival. Herein, we found that IgG was present in the oocytes of immunodeficient mice; the IgG-VDJ regions were highly variable between different oocytes, and H3K27Ac bound and regulated the IgG promoter region. Next, IgG mRNA and protein levels increased in response to LPS, and this increment was mediated by CR2 on the oocyte membrane. Finally, we revealed three aspects of the functional relevance of oocyte IgG: first, oocytes could upregulate IgG to counteract the increased ROS level induced by CSF1; second, oocytes could upregulate IgG in response to injected virus ssRNA to maintain mitochondrial integrity; third, upon bacterial infection, oocytes could secrete IgG, subsequently encompassing the bacteria, thus increasing survival compared to somatic cells. This study reveals for the first time that the female germ cells, oocytes, can independently adjust intrinsic IgG production to survive in adverse environments.

Fidgetin knockdown and knockout influences female reproduction distinctly in mice.

Microtubule-severing proteins (MTSPs), are a family of proteins which use adenosine triphosphate to sever microtubules. MTSPs have been shown to play an important role in multiple microtubule-involved cellular processes. One member of this family, fidgetin ( FIGN), is also involved in male fertility; however, no studies have explored its roles in female fertility. In this study, we found mouse fidgetin is rich within oocyte zona pellucida (ZP) and is the only MTSP member to do so. Fidgetin also appears to interact with all three ZP proteins. These findings prompted us to propose that fidgetin might prevent polyspermy. Results from in vitro maturation oocytes analysis showed that fidgetin knockdown did cause polyspermy. We then deleted all three fidgetin isoforms with CRISPR/Cas9 technologies; however, female mice remained healthy and with normal fertility. Of all mouse MTSPs, only the mRNA level of fidgetin-like 1 ( FIGNL1) significantly increased. Therefore, we assert that fidgetin-like 1 compensates fidgetin's roles in fidgetin knockout female mice.

Gm364 coordinates MIB2/DLL3/Notch2 to regulate female fertility through AKT activation.

Many integral membrane proteins might act as indispensable coordinators in specific functional microdomains to maintain the normal operation of known receptors, such as Notch. Gm364 is a multi-pass transmembrane protein that has been screened as a potential female fertility factor. However, there have been no reports to date about its function in female fertility. Here, we found that global knockout of Gm364 decreased the numbers of primordial follicles and growing follicles, impaired oocyte quality as indicated by increased ROS and γ-H2AX, decreased mitochondrial membrane potential, decreased oocyte maturation, and increased aneuploidy. Mechanistically, Gm364 directly binds and anchors MIB2, a ubiquitin ligase, on the membrane. Subsequently, membrane MIB2 ubiquitinates and activates DLL3. Next, the activated DLL3 binds and activates Notch2, which is subsequently cleaved within the cytoplasm to produce NICD2, the intracellular active domain of Notch2. Finally, NICD2 can directly activate AKT within the cytoplasm to regulate oocyte meiosis and quality.

ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress.

Objective. Chronic stress (CS)-induced abnormal metabolism and other subsequent aspects of abnormality are threatening human health. Little is known regarding whether and how protein post-translational-modifications (PTMs) correlate with abnormal metabolism under CS. The aim of this study was to address this issue and also identify novel key protein PTM. Methods. First, we screened which pan-PTM had significant change between control and CS female mice and whether clinical CS females had similar pan-PTM change. Second, we performed quantitative PTM-omics and metabolomics to verify the correlation between abnormal protein PTMs and atypical metabolism. Third, we performed quantitative phospho-omics to identify the key PTM-regulating enzyme and investigate the interaction between PTM protein and PTM-regulating enzyme. Fourth, we attempted to rectify the abnormal metabolism by correcting the activity of the PTM-regulating enzyme. Finally, we examined whether the selected key protein was also correlated with stress scores and atypical metabolism in clinical women. Results. We initially found that multiple tissues of CS female mice have downregulated pan-crotonylation, and verified that the plasma of clinical CS females also had downregulated pan-crotonylation. Then we determined that ATP5O-K51 crotonylation decreased the most and also caused gross ATP5O decrement, whereas the plasma of CS mice had downregulated phospholipids. Next, downregulating ATP5O crotonylation partially recapitulated the downregulated phospholipid metabolism in CS mice. Next, we verified that HDAC2-S424 phosphorylation determined its decrotonylation activity on ATP5O-K51. Furthermore, correcting HDAC2 hyper-phosphorylation recovered the gross ATP5O level and partially rescued the downregulated phospholipid metabolism in CS mice. Finally, the ATP5O level was also significantly lower and correlated with high stress scores and downregulated phospholipid metabolism in clinical female plasma. Conclusion. This study discovered a novel PTM mechanism involving two distinct types of PTM in CS and provided a novel reference for the clinical precautions and treatments of CS.

Rat BAT xenotransplantation recovers the fertility and metabolic health of PCOS mice.

Polycystic ovarian syndrome (PCOS) is a major severe ovary disorder affecting 5-10% of reproductive women around the world. PCOS can be considered a metabolic disease because it is often accompanied by obesity and diabetes. Brown adipose tissue (BAT) contains abundant mitochondria and adipokines and has been proven to be effective for treating various metabolic diseases. Recently, allotransplanted BAT successfully recovered the ovarian function of PCOS rat. However, BAT allotransplantation could not be applied to human PCOS; the most potent BAT is from infants, so voluntary donors are almost inaccessible. We recently reported that single BAT xenotransplantation significantly prolonged the fertility of aging mice and did not cause obvious immunorejection. However, PCOS individuals have distinct physiologies from aging mice; thus, it remains essential to study whether xenotransplanted rat BAT can be used for treating PCOS mice. In this study, rat-to-mouse BAT xenotransplantation, fortunately, did not cause severe rejection reaction, and significantly recovered ovarian functions, indicated by the recovery of fertility, oocyte quality, and the levels of multiple essential genes and kinases. Besides, the blood biochemical index, glucose resistance, and insulin resistance were improved. Moreover, transcriptome analysis showed that the recovered PCOS F0 mother following BAT xenotransplantation could also benefit the F1 generation. Finally, BAT xenotransplantation corrected characteristic gene expression abnormalities found in the ovaries of human PCOS patients. These findings suggest that BAT xenotransplantation could be a novel therapeutic strategy for treating PCOS patients.

GTPases Arf5 and Arl2 function partially distinctly during oocyte meiosis.

Mammalian female meiosis must be tightly regulated to produce high-quality mature oocytes for subsequent regular fertilization and healthy live birth of the next generation. GTPases control many important signal pathways involved in diverse cellular activities. ADP-ribosylation factor family members (Arfs) in mice possess GTPase activities, and some members have been found to function in meiosis. However, whether other Arfs play a role in meiosis is unknown. In this study, we found that Arl2 and Arf5 are the richest among Arfs in mouse oocytes, and they are more abundant in oocytes than in granular cells. Furthermore, Arl2 and Arf5 depletion both impeded meiotic progression, but by affecting spindles and microfilaments, respectively. Moreover, Arl2 and Arf5 depletion both significantly increased regular reactive oxygen species levels and decreased mitochondrial membrane potential and autophagy, indicating that oocyte quality was damaged by Arl2 and Arf5 depletion. These results suggest that Arl2 and Arf5 are two novel essential GTPases required for oocyte meiosis and quality control.

Downregulation of the ubiquitin ligase KBTBD8 prevented epithelial ovarian cancer progression.

OBJECTIVES: Kelch repeat and BTB domain-containing protein 8, KBTBD8, has been identified as a female fertility factor. However, there have been no reports on the role of KBTBD8 in the progression of epithelial ovarian cancer, EOC. Our study aimed to address this issue. METHODS: We first examine KBTBD8 expression in EOC tissues and cells. Next, we performed RNA sequencing to reveal the overall mechanism. Then we investigated the roles of KBTBD8 in the proliferation, migration, and health status of cultured EOC cells. Finally, we employed tumor xenograft models to evaluate the role of KBTBD8 in vivo. RESULTS: First, KBTBD8 level was significantly higher in EOC tissues and cells. Next, comparative RNA sequencing identified more tumorigenesis-related genes that KBTBD8 might regulate. Then we found that KBTBD8 knockdown significantly decreased EOC cell proliferation, migration, and the activities of multiple tumorigenesis-related kinases. Finally, KBTBD8 knockdown significantly diminished ovarian tumor formation in vivo. CONCLUSION: Proper KBTBD8 level is essential for the healthy growth of ovarian somatic cells, such as ovarian epithelial cells. Excessive KBTBD8 might be a significant impetus for EOC progression. KBTBD8 reduction greatly inhibits EOC proliferation and migration.

Fam70A binds Wnt5a to regulate meiosis and quality of mouse oocytes.

OBJECTIVES: Little is known about the roles of integral membrane proteins beyond channels, carriers or receptors in meiotic oocytes. The transmembrane protein Fam70A was previously identified as a likely "female fertility factor" in Fox3a-knockout mouse ovaries where almost all follicles underwent synchronous activation and the mice became infertile very early. However, whether Fam70A functions in oocyte meiosis remains unknown. Therefore, the present study aimed to address this question. MATERIALS AND METHODS: Co-immunoprecipitation, immunogold labelling-electron microscopy, co-localization and yeast two-hybrid assays were used to verify the interaction. Antibody or small interfering RNA transfection was used to deplete the proteins. Immunofluorescence, immunohistochemistry and live tracker staining were used to examine the localization or characterize phenotypes. Western blot was used to examine the protein level. RESULTS: Fam70A was enriched in oocyte membranes important for normal meiosis. Fam70A depletion remarkably disrupted spindle assembly, chromosome congression and first polar body extrusion, which subsequently increased aneuploidy and abnormal fertilization. Moreover, Fam70A directly bound Wnt5a, the most abundant Wnt member within oocytes. Depletion of either Fam70A or Wnt5a remarkably increased adenomatous polyposis coli (APC), which stabilizes active ß-catenin and microtubules. Consequently, depletion of either Fam70A or Wnt5a remarkably increased p-ß-catenin (inactive form) and acetylated tubulin, while APC knockdown remarkably decreased these two. Furthermore, Fam70A depletion remarkably reduced Akt phosphorylation. CONCLUSIONS: Fam70A regulates meiosis and quality of mouse oocytes through both canonical and non-canonical Wnt5a signalling pathways.

Exploring utility of genomic epidemiology to trace origins of highly pathogenic influenza A/H7N9 in Guangdong.

The first highly pathogenic (HP) influenza A/H7N9 was reported in Guangdong in January 2017. To investigate the emergence and spread of HP A/H7N9 in Guangdong province, we sequenced 297 viruses (58 HP A/H7N9, 19 low pathogenic (LP) A/H7N9, and 220 A/H9N2) during 2016-2017. Our analysis showed that during the fifth wave, three A/H7N9 lineages were co-circulating in Guangdong: the local LP Pearl River Delta (PRD) lineage (13%), the newly imported LP Yangtze River Delta (YRD) lineage (23%), and the HP YRD lineage (64%). Previously circulating YRD-lineage LP during the third wave evolved to the YRD-lineage HP A/H7N9 in Guangdong. All YRD-lineage LP detected during the fifth wave most likely originated from newly imported viruses into Guangdong. Genotype comparison of HP A/H7N9 suggests limited outward spread of HP A/H7N9 to other provinces. The distribution of HP A/H7N9 cleavage site variants on live poultry markets differed from that found in humans, suggesting a V1-type cleavage site may facilitate human infections.

[Bacteriologic profile and drug resistance in children with respiratory infection from 2016 to 2018].

OBJECTIVE: To study the bacteriologic profile and drug resistance of respiratory infection in children, and to provide a basis for etiological diagnosis and rational use of antimicrobial agents. METHODS: A retrospective analysis was performed for 15 047 children who attended the hospital due to respiratory infection from January 2016 to December 2018. Their sputum samples were collected, and the Phoenix-100 automatic microbial identification system was used for the identification and drug sensitivity analysis of the isolated pathogenic bacteria. RESULTS: Of all 17 174 sputum samples detected, there were 2 395 positive samples, with a positive rate of 13.95%; a total of 2 584 strains of pathogenic bacteria were isolated, among which there were 1 577 (61.03%) Gram-negative strains, 967 (37.42%) Gram-positive strains, and 40 (1.55%) fungal strains. The most common pathogen was Haemophilus influenzae (33.90%), followed by Streptococcus pneumoniae (33.55%), Moraxella catarrhalis (19.20%), and Staphylococcus aureus (3.64%). Among the 2 331 children with positive infection, 251 had mixed infection, most commonly with Haemophilus influenzae and Streptococcus pneumoniae. The detection rate of pathogenic bacteria was higher in winter and spring and lower in summer and autumn. There was a significant difference in the detection rate of pathogenic bacteria between different age groups (P<0.05), with the highest detection rate in infants aged 1 month to <1 year. Streptococcus pneumoniae and Staphylococcus aureus had a sensitivity rate of 100% to vancomycin, linezolid, and teicoplanin, and Haemophilus influenzae had a lower sensitivity rate to ampicillin, compound sulfamethoxazole and cefuroxime and a higher sensitivity rate to other drugs. CONCLUSIONS: Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis are the main pathogenic bacteria of respiratory infection in children, and mixed infection is the most common type of infection. The detection rate of pathogenic bacteria varies across seasons and ages. Different pathogenic bacteria have different features of drug resistance, and antibiotics should be selected based on drug sensitivity results.

Antigenic Variation of Avian Influenza A(H5N6) Viruses, Guangdong Province, China, 2014-2018.

Market surveillance showed continuing circulation of avian influenza A(H5N6) virus in live poultry markets in Guangdong Province in 2017, despite compulsory vaccination for avian influenza A(H5Nx) and A(H7N9). We analyzed H5N6 viruses from 2014-2018 from Guangdong Province, revealing antigenic drift and decreased antibody response against the vaccine strain in vaccinated chickens.

Single xenotransplant of rat brown adipose tissue prolonged the ovarian lifespan of aging mice by improving follicle survival.

Prolonging the ovarian lifespan is attractive and challenging. An optimal clinical strategy must be safe, long-acting, simple, and economical. Allotransplantation of brown adipose tissue (BAT), which is most abundant and robust in infants, has been utilized to treat various mouse models of human disease. Could we use BAT to prolong the ovarian lifespan of aging mice? Could we try BAT xenotransplantation to alleviate the clinical need for allogeneic BAT due to the lack of voluntary infant donors? In the current study, we found that a single rat-to-mouse (RTM) BAT xenotransplantation did not cause systemic immune rejection but did significantly increase the fertility of mice and was effective for more than 5 months (equivalent to 10 years in humans). Next, we did a series of analysis including follicle counting; AMH level; estrous cycle; mTOR activity; GDF9, BMP15, LHR, Sirt1, and Cyp19a level; ROS and annexin V level; IL6 and adiponectin level; biochemical blood indices; body temperature; transcriptome; and DNA methylation studies. From these, we proposed that rat BAT xenotransplantation rescued multiple indices indicative of follicle and oocyte quality; rat BAT also improved the metabolism and general health of the aging mice; and transcriptional and epigenetic (DNA methylation) improvement in F0 mice could benefit F1 mice; and multiple KEGG pathways and GO classified biological processes the differentially expressed genes (DEGs) or differentially methylated regions (DMRs) involved were identical between F0 and F1. This study could be a helpful reference for clinical BAT xenotransplantation from close human relatives to the woman.

The ubiquitin ligase KBTBD8 regulates PKM1 levels via Erk1/2 and Aurora A to ensure oocyte quality.

Tight control of energy metabolism is essential for normal cell function and organism survival. PKM (pyruvate kinase, muscle) isoforms 1 and 2 originate from alternative splicing of PKM pre-mRNA. They are key enzymes in oxidative phosphorylation and aerobic glycolysis, respectively, and are essential for ATP generation. The PKM1:PKM2 expression ratio changes with development and differentiation, and may also vary under metabolic stress and other conditions. Until now, there have been no reports about the function and regulation of PKM isozymes in oocytes. Here, we demonstrate that PKM1 or PKM2 depletion significantly disrupts ATP levels and mitochondrial integrity, and exacerbates free-radical generation and apoptosis in mouse oocytes. We also show that KBTBD8, a female fertility factor in the KBTBD ubiquitin ligase family, selectively regulates PKM1 levels through a signaling cascade that includes Erk1/2 and Aurora A kinases as intermediates. Finally, using RNA sequencing and protein network analysis, we identify several regulatory proteins that may be govern generation of mature PKM1 mRNA. These results suggest KBTBD8 affects PKM1 levels in oocytes via a KBTBD8→Erk1/2→Aurora A axis, and may also affect other essential processes involved in maintaining oocyte quality.

A Singular Value Decomposition Method for Tunable Diode Laser Absorption Spectroscopy System to Remove Systematic Noises.

Detection of gas concentration with tunable diode laser absorption spectroscopy (TDLAS) techniques is affected by baseline drift and high-frequency noise. Therefore, how to remove the systematic noises has been a hot spot. This paper analyzes the significance of singular value decomposition (SVD) in TDLAS detection system with two different methods of constructing a matrix, and it discusses the differences of processing results for different noises. The second harmonic signal is arranged in a matrix and decomposed. We select the appropriate threshold and putthose singular values smaller than the threshold into zero, then reconstruct the matrix. Experiments show that SVD method does not require additional system components or pass into the zero gas to subtract background. This method is able to remove noises of TDLAS system quickly and effectively. We found that the method of constructing a hankel matrix is suitable for removing high-frequency noise. However, the method of constructing a continuous-cutoff-signal matrix is suitable for removing baseline drift. For example, we set up a TDLAS system to measure the concentration of NH3 while the noise removal rate of the second harmonic curve is up to 80% with this method.

[Characterization of qnr gene for plasmid-mediated quionlone resistance].

OBJECTIVE: To explore the distribution of qnr gene and broad spectrum p-lactamase (ESBLs) gene in gram-negative bacteria which were isolated from our hospital patients. METHODS: qnr gene in nonrepetitive 129 isolates of Escherichia coli, 10 isolates of Enterobacter cloacac and 29 isolates of K. pneunoniae were detected by polymerase chain reaction (PCR). For qnr gene positive strains, int I, SHV-1, TEM-1, CTX-M, OXA-I , OXA-II , OXA-III, DHA and EBC genes were examined. Plasmid conjugatable test was applied to examine whether qnr gene was located in conjugate plasmid and ERIC-PCR was carried out for DNA homologous analysis. ESBLs detection (according to phenotypic confirmatory test based on National Committee for Clinical Laboratory Standards criteria) and susceptibility test to 16 antibiotics were also performed. RESULTS: qnr gene was found in 6 clinical isolates including 5 strain of E. coli and one strain of E. cloacac, but qnr gene was undetectable in K. pneunoniae isolates. The 6 clinical isolates were suspectible to imipenem but resistance to some other drugs while only 2 isolates of E. coli were susceptible to quinolone. Among the 6 qnr gene-positive strains, all of them belonged to I type integron-positive isolates, 4 isolates of them were TEM-1 producing strains,with only one isolate was OXA-III gene producing strain, and 2 isolates of them were EBC producing strains. Most of them were with 2 ESBLs gene if not more. qnr gene was on transferable plasmids which could be disseminated by clone. CONCLUSION: In Wuhan city, the prevalence of qnr was confirmed. qnr gene were found with some ESBLs gene in the same strains, and qnr gene in suspect strains. The transmission of qnr gene producing strains could be mediated by transferable plasmids or clone, forcing us to make intensive investigation and take effective control measures.

[A case-control study on the risk factors of nosocomial infection caused by imipenem-resistant Pseudomonas aeruginosa].

OBJECTIVE: To explore the risk factors for nosocomial infection caused by imipenem-resistant Pseudomonas aeruginosa (IRPA). METHODS: A case-control study was carried out for the comparison of 2 groups of 'case' patients with 'controlled' patients. The first group of 'case' patients had nosocomial isolation of IRPA, and the second group had imipenem-susceptible Pseudomonas aeruginosa (ISPA). 'Control' patients were selected from the same medical or surgical services from which 'case' patients were receiving care when isolation of IRPA or ISPA occurred. Risk factors analyzed included the use of antimicrobials, comorbid conditions, and demographic variables. IRPA was recovered from 67 patients, and ISPA from 150 patients while the control case were 200 and 159 respectively. All patients were from Renmin Hospital of Wuhan University during Jan 2002 to Dec 2003. Data were analyzed with unconditional logistic regression and principal component analysis. RESULTS: Data from multivariate unconditional logistic regression analysis showed that the independent risk factors for IRPA nosocomial infection were: time at risk (OR = 1.03, 95% CI: 1.01-1.04), imipenem (OR = 4.65, 95% CI: 1.35-11.52), PIP/TAZ (OR = 3.37, 95% CI 1.85-9.43) and quinolones (OR = 1.85, 95% CI: 1.25-5.34) while the third cephalosporins (OR = 2.54, 95% CI: 1.26-5.23) and aminoglycoside antibiotics (OR = 1.86, 95% CI 1.42-3.26) time at risk (OR = 1.05, 95% CI: 1.03-1.05) were associated with isolated ISPA. CONCLUSION: Nosocomial infection of IRPA could be caused by the use of imipenem and other antibiotics, suggesting that to limit the use of imipenem was not sufficient to contain the increasing incidence of IRPA.

Dissemination and spread of CTX-M extended-spectrum beta-lactamases among clinical isolates of Klebsiella pneumoniae in central China.

Of 50 extended-spectrum beta-lactamase-producing (ESBL) isolates of Klebsiella pneumoniae collected from six hospitals in Hubei Province, 20 were found to produce CTX-M ESBLs (40%). Sequence analysis of the six isolates carrying bla(CTX-M) genes revealed that they all harboured bla(CTX-M-3). In the majority of isolates, bla(CTX-M) genes were within large conjugative plasmids. A high degree of diversity of the RAPD types revealed that all the isolates carrying CTX-M were genetically unrelated and horizontal transfer of plasmids was probably the main mechanism of bla(CTX-M) spread. This is the first report of the occurrence of CTX-M-3 ESBLs in central China; previously this enzyme was identified only in Europe. A more comprehensive survey of ESBL types from China is urgently needed.