The metabolomics changes in epididymal lumen fluid of CABS1 deficient male mice potentially contribute to sperm deformity.

Introduction: Epididymal lumen fluids provides a stable microenvironment for sperm maturation. Ca2+ binding protein CABS1 is known to maintain structural integrity of mouse sperm flagella during epididymal transit of sperm. Besides, CABS1 was reported to contain anti-inflammatory peptide sequences and be present in both human saliva and plasma. However, little is known about the role of CABS1 in regulation of the microenvironment of epididymal lumen fluids. Methods: To further confirm the role of CABS1 in epididymis, we identified the expression of CABS1 in epididymal lumen fluids. Moreover, high performance liquid chromatography, coupled with tandem mass spectrometry technique was used to analyze the metabolic profiles and in vivo microperfusion of the cauda epididymis and inductively coupled plasma mass spectrometry (ICP-MS) assays was used to detect the concentration of metal ion of mouse cauda epididymal lumen fluids in CABS1 deficient and normal mice. Results: The results showed that CABS1 is present in epididymal lumen fluids, and the concentration of calcium in epididymal lumen fluids is not changed in Cabs1-/- male mice. Among 34 differential metabolites identified in cauda epididymis, 21 were significantly upregulated while 13 were significantly downregulated in KO cauda epididymis. Pathway analysis identified pyrimidine metabolism, inositol phosphate metabolism, arachidonic acid metabolism, purine metabolism and histidine metabolism as relevant pathways in cauda epididymis. Discussion: The perturbations of mitochondrial dysfunction and inflammation may be the crucial reason for the poor performance of Cabs1-/- sperm.

Advancement and Potential Applications of Epididymal Organoids.

The epididymis, a key reproductive organ, is crucial for sperm concentration, maturation, and storage. Despite a comprehensive understanding of many of its functions, several aspects of the complex processes within the epididymis remain obscure. Dysfunction in this organ is intricately connected to the formation of the microenvironment, disruptions in sperm maturation, and the progression of male infertility. Thus, elucidating the functional mechanisms of the epididymal epithelium is imperative. Given the variety of cell types present within the epididymal epithelium, utilizing a three-dimensional (3D) in vitro model provides a holistic and practical framework for exploring the multifaceted roles of the epididymis. Organoid cell culture, involving the co-cultivation of pluripotent or adult stem cells with growth factors on artificial matrix scaffolds, effectively recreates the in vivo cell growth microenvironment, thereby offering a promising avenue for studying the epididymis. The field of epididymal organoids is relatively new, with few studies focusing on their formation and even fewer detailing the generation of organoids that exhibit epididymis-specific structures and functions. Ongoing challenges in both clinical applications and mechanistic studies underscore the importance of this research. This review summarizes the established methodologies for inducing the in vitro cultivation of epididymal cells, outlines the various approaches for the development of epididymal organoids, and explores their potential applications in the field of male reproductive biology.

Reducing air pollution does not necessarily reduce related adults' mortality burden: Variations in 177 countries with different economic levels.

Prolonged exposure to PM2.5 is associated with increased mortality. However, reducing air pollution concentrations does not necessarily reduce the related burden of deaths. Here, we aim to estimate the variations in PM2.5-related mortality due to contributions from key factors - PM2.5 concentration, population exposure, and healthcare levels - for 177 countries from 2000 to 2018 at the 1-km grid scale according to the Global Mortality Exposure Model (GEMM) model. We find that global reductions in PM2.5-related deaths mainly come from high and upper-middle income countries, where lowered air pollutant concentration and better healthcare can offset mortality burdens caused by increasing exposed populations. Changes in population exposure to PM2.5 contribute the most (54 %) to change in global related deaths over the examined period, followed by changes in healthcare (-42 %) and pollution concentrations (4 %). The impacts vary across countries and regions within them due to other drivers, which are significantly influenced by development status. Policies aiming at reducing PM2.5 associated health risks need to account for country-specific balances of these key socioeconomic drivers.

Smart seq2 revealed distinct molecular responses during in vitro porcine oocyte maturation before or after the addition of mogroside V.

Oocyte maturation involves both nuclear and cytoplasmic maturation. Mogroside V (MV) has been shown to enhance nuclear maturation, mitochondrial content, and developmental potential of porcine oocyte during in vitro maturation (IVM). However, the impact of MV on cytoplasmic maturation and its underlying mechanisms are not understood. This study aimed to assess the effect of MV on cytoplasmic maturation. Germinal vesicle (GV) oocytes treated with MV exhibited a noticeable increase in cortical granules (CGs) formation. Additionally, MV enhanced the expression of NNAT and improved glucose uptake in mature oocytes. Further insights were gained through Smart-seq2 analysis of RNA isolated from 100 oocytes. A total of 11,274 and 11,185 transcripts were identified in oocytes treated with and without MV, respectively. Among quantified genes, 438 differentially expressed genes (DEGs) were identified for further analysis. Gene Ontology (GO) enrichment analysis indicated that these DEGs were primarily involved in DNA repair regulation, cellular response to DNA damage, intracellular components, and organelles. Furthermore, the DEGs were significantly enriched in three KEGG pathways: fatty acid synthesis, pyruvate metabolism, and WNT signalling. To validate the results, lipid droplets (LD) and triglyceride (TG) were examined. MV led to an increase in the accumulation of LD and TG production in mature oocytes. These findings suggest that MV enhances cytoplasmic maturation by promoting lipid droplet synthesis. Overall, this study provides valuable insights into the mechanisms through which MV improves oocyte quality during IVM. The results have significant implications for research in livestock reproduction and offer guidance for future studies in this field.

Detection and differentiation of seven porcine respiratory pathogens using a multiplex ligation-dependent probe amplification assay.

Respiratory diseases due to viral or bacterial agents, either alone or in combination, cause substantial economic burdens to the swine industry worldwide. Rapid and reliable detection of causal pathogens is crucial for effective epidemiological surveillance and disease management. This research aimed to employ the multiplex ligation-dependent probe amplification (MLPA) assay for simultaneous detection of seven distinct pathogens causing respiratory problems in swine, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV2), Pasteurella multocida, Actinobacillus pleuropneumoniae, and Glässerella parasuis. The results indicated no probe cross-reactivity among the seven target agents with other swine pathogens. The detection limits ranged from 5 to 34 copies per assay for the target organisms. The MLPA assay was evaluated with 88 samples and compared to real-time or multiplex PCR for the target pathogens. The MLPA assay demonstrated high relative test sensitivities (100 %) and reasonable to good relative specificities at 62.5 %, 95.1 %, 86.8 %, and 97.6 % for PRRSV, P. multocida, G. parasuis, and PCV2, respectively, relative to comparator PCR assays. In 71 samples where MLPA and comparator PCR assays matched exactly, infections were detected in 64 samples (90.1 %), with PRRSV being the most commonly found virus and 50.7 % of the samples showing co-infection with two to five of the pathogens. This approach serves as a valuable tool for conducting differential diagnoses and epidemiological investigations of pathogen prevalence within swine populations.

Rapamycin protects Sertoli cells against BPA-induced autophagy disorders.

Bisphenol A (BPA) is a well-known environmental contaminant that can negatively impact reproductive function. Disruption of autophagy is implicated in BPA-induced cell injury, the specific molecular mechanisms through which BPA affects autophagy in Sertoli cells are still unknown. In the present study, TM4 cells were exposed to various doses of BPA (10, 100, and 200 µM), and the results indicated that BPA exposure led to the accumulation of autophagosomes, this change was accompanied by increased expression of p-mTOR and decreased expression of Atg12, a protein involved in regulating autophagy initiation. Additionally, BPA exposure upregulated the expression levels of p62, a protein involved in autophagic degradation. The inhibition of autophagy initiation and autophagic degradation contributes to the accumulation of autophagosomes. Further studies showed that BPA exposure didn't affect the expression of the lysosome protein LAMP1; however, decreased cytoplasmic retention of acridine orange in TM4 cells may explain the disruption of autophagy. The role of rapamycin and chloroquine (CQ), an autophagy inhibitor that impairs lysosomal degradation also confirmed the effect of BPA on autophagy regulation. Specifically, rapamycin can protect Sertoli cells against BPA-induced cell injury by promoting autophagy. These findings contribute to our understanding of the mechanisms underlying reproductive toxicity caused by BPA.

A novel core-shell up-conversion nanoparticles immunochromatographic assay for the detection of deoxynivalenol in cereals.

Deoxynivalenol (DON) toxin is a type B group of trichothecene mycotoxins mainly originating from specific Fusarium fungi, seriously harming human and livestock health. Herein, a novel core-shell up-conversion nanoparticles immunochromatographic assay (CS-UCNPs-ICA) was developed for deoxynivalenol based on the competitive reaction principle. By exploiting the fluorescence intensity of the T and C lines of CS-UCNPs-ICA, the concentrations of DON were obtained sensitively and precisely under optimized conditions in 5 min with a detection limit of 0.1 ng/mL. The CS-UCNPs-ICA strips only specifically detect DON and its derivatives (3-Ac-DON and 15-Ac-DON), with no cross-reaction with other mycotoxins. The low CV values illustrated a modest intra- and inter-assay variation, confirming the superior precision of this method. In the spiked experiment, the mean recoveries of corn and wheat ranged from 94.74% to 100.90% and 96.21%-104.81%, respectively. Furthermore, the approach generated results that were in good agreement with data from HPLC and ELISA analyses of naturally contaminated feed and cereals, confirming that the significant advantages of proposed strips were their high practicality, rapidness, and simplicity. Therefore, the CS-UCNPs-ICA strips platform serves as a promising candidate for developing new approaches for rapid testing or high throughput screening from DON in food products.

Magnetic dendritic mesoporous silica nanoparticles based integrated platform for rapid and efficient analysis of saccharides.

BACKGROUND: As an essential compound in living organism, saccharides have attracted enormous attentions from scientists in various fields. Understanding the distribution of saccharides in various samples is of great scientific importance. However, the low signal response and lack of specific recognition technology of saccharides and the complex matrix of samples make the analysis of saccharides a very challenge task. Thus, the development of a simple and straightforward strategy for the analysis of saccharides would represent a great contribution to the field. RESULTS: In this study, by employing the sulfonyl functionalized magnetic dendritic mesoporous silica nanoparticles as the substrate, we develop an integrated platform for analysis of saccharides. The construction of the platform mainly relied on multi-functional boronic acid, which serves as separation and derivation ligands at the same time. In the general procedure, the boronic acid is first immobilized onto the surface of substrate, then the selective enrichment of saccharides can be realized via boronate affinity separation. Finally, by the rational choice of the solution, we are able to elute the labelled complex (boronic acid-saccharide) from the substrate, which can be direct subjected to HPLC-UV analysis. The reliable precision (<15 %), accuracy (80-100 %), reproducibility (<10 %), improved sensitivity (20x) and limited time-consuming (down to minutes) of the proposed platform are experimentally demonstrated. SIGNIFICANCE AND NOVELTY: The successful quantification of different saccharides (alditols, glucose) in real samples is achieved. The proposed strategy is not only straightforward and fast, but also avoid the requirement of special equipment. With these attractive features, we believe that this strategy will greatly prompt the analysis of saccharides in various samples (eg. food, pharmaceutics and biosamples).

In situ digestion-assisted multi-template imprinted nanoparticles for efficient analysis of protein phosphorylation.

A new general approach called in situ digestion-assisted multi-template imprinting is proposed for preparation of phospho-specific molecularly imprinted nanoparticles. Through the novel templating strategy and controllable imprinting process, imprinted nanoparticles specific to the intact phosphoprotein and its phosphopeptides were synthesized. The prepared imprinted nanoparticles exhibited excellent specificity (cross reactivity < 10%), high affinity (10-6 M), high efficiency (47.5%), and good generality (both intact phosphoprotein and phosphopeptides). We also realized the fine tuning of the recognition at peptide level of the imprinted nanoparticles by adjusting the imprinting time. Based on the selective enrichment of the imprinted nanoparticles, the MS identification of both the intact phosphoprotein (Tau) and phosphopeptides (angiotensin II and peptides of Tau) in real complex samples could be achieved. Therefore, we believe that the in situ digestion-assisted multi-template imprinting strategy holds promising future in both phosphorylation analysis and proteomics applications.

Novel mutations of TEX11 are associated with non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) affects 10% of infertile men worldwide, and genetic studies revealed that there are plenty of monogenic mutations that responsible for a part of idiopathic NOA cases. Testis-expressed gene 11 (TEX11) is an X-linked meiosis-specific gene, many pathogenic variants in TEX11 have been detected in NOA patients, and the deficiency of this gene can cause abnormal meiotic recombination and chromosomal synapsis. However, many NOA-affected cases caused by TEX11 mutation remain largely unknown. This study reported three novel TEX11 mutations (exon 5, c.313C>T: p.R105*), (exon 7, c.427A>C: p.K143Q) and (exon 29, c.2575G>A: p.G859R). Mutations were screened using whole-exome sequencing (WES) and further verified by amplifying and sequencing the specific exon. Histological analysis of testicular biopsy specimens revealed a thicker basement membrane of the seminiferous tubules and poorly developed spermatocytes, and no post-meiotic round spermatids or mature spermatozoa were observed in the seminiferous tubules of patients with TEX11 mutation. Conclusion: This study presents three novel variants of TEX11 as potential infertility alleles that have not been previously reported. It expanded the variant spectrum of patients with NOA, which also emphasizes the necessity of this gene screening for the clinical auxiliary diagnosis of patients with azoospermia.

Hydrogen sulfide protects Sertoli cells against toxicant Acrolein-induced cell injury.

Acrolein (ACR), a highly toxic α,ß-unsaturated aldehyde, is considered to be a common mediator behind the reproductive injury induced by various factors. However, the understanding of its reproductive toxicity and prevention in reproductive system is limited. Given that Sertoli cells provide the first-line defense against various toxicants and that dysfunction of Sertoli cell causes impaired spermatogenesis, we, therefore, examined ACR cytotoxicity in Sertoli cells and tested whether hydrogen sulfide (H2S), a gaseous mediator with potent antioxidative actions, could have a protective effect. Exposure of Sertoli cells to ACR led to cell injury, as indicated by reactive oxygen species (ROS) generation, protein oxidation, P38 activation and ultimately cell death that was prevented by antioxidant N-acetylcysteine (NAC). Further studies revealed that ACR cytotoxicity on Sertoli cells was significantly exacerbated by the inhibition of H2S-synthesizing enzyme cystathionine γ-lyase (CSE), while significantly suppressed by H2S donor Sodium hydrosulfide (NaHS). It was also attenuated by Tanshinone IIA (Tan IIA), an active ingredient of Danshen that stimulated H2S production in Sertoli cells. Apart from Sertoli cells, H2S also protected the cultured germ cells from ACR-initiated cell death. Collectively, our study characterized H2S as endogenous defensive mechanism against ACR in Sertoli cells and germ cells. This property of H2S could be used to prevent and treat ACR-related reproductive injury.

Management of gestational weight gain in obese or overweight women based on resting energy expenditure: A pilot cohort study.

Resting energy expenditure (REE) comprises 60% of total energy expenditure and variations may be associated with gestational weight gain (GWG). This study aims to explore the usability and feasibility of REE guided intervention for GWG in obese and overweight women. We conducted a prospective cohort study in LuHe Hospital of Capital Medical University in Beijing, China between May 1, 2017 and May 31, 2018. Obese/overweight women who had routine prenatal care visit at 10 to 13 weeks of gestation, were recruited after written informed consent was obtained. The intervention group (those women who were recruited between January 1 and May 31, 2018) used REE calculated daily total energy to manage GWG, while the control group (those women who were recruited between May 1 and December 31, 2017) used prepregnancy body mass index calculated daily total energy to manage GWG. GWG and daily total energy between the 2 groups were recorded from 10 to 13 weeks of gestation to delivery. A total of 68 eligible women (35 in intervention group and 33 in control group) were included in the final analysis. Daily total energy in the intervention group increased less than the control group, especially from 2nd trimester to 3rd trimester (1929.54 kcal/d vs. 2138.33 kcal/d). The variation of daily total energy from 1st trimester to 3rd trimester in the intervention group was lower than the control group (226.17 kcal/d vs 439.44 kcal/d). Overall GWG of the intervention group (13.45 kg) was significantly lower than the control group (18.20 kg). The percentage of excess-GWG in the intervention group (31.42%) was also significantly lower than the control (57.57%). Findings from our pilot study suggest that diet recommendation basting on REE may improve management of GWG in obese/overweight women.

The impacts of urban structure on PM2.5 pollution depend on city size and location.

Many cities across the world face the challenge of severe fine particulate matter (PM2.5) pollution. Among the many factors that affect PM2.5 pollution, there is an increasing interest in the impacts of urban structure. However, quantifying these impacts in China has been difficult due to differences of study area and scale in existing research, as well as limited sample sizes. Here, we conducted a continental study focusing on 301 prefectural cities in mainland China to investigate the effects of urban structure, including urban size and urban compactness, on PM2.5 concentrations. Based on PM2.5 raster and land cover data, we used quantile regression and a general multilinear model to estimate the effects and relative contributions of urban size and urban compactness on urban PM2.5 pollution, with explicit consideration for pollution level, urban size and geographical location. We found: (1) nationwide, the larger and more compact that cities were, the heavier the PM2.5 pollution tended to be. Additionally, this relationship became stronger with increasing levels of pollution. (2) In general, urban size played a more important role than urban form, and there were no significant interactive effects between the two metrics on urban PM2.5 concentrations at the national scale. (3) The impacts of urban size and form varied by city size and geographical location. The impacts of urban size were only significant for small or medium-large cities but not for large cities. Among large cities, only urban form had a significantly positive effect on urban PM2.5 concentrations. The further north and west that cities were, the more dependent PM2.5 pollution was on urban form, whereas the further south and east that cities were, the greater the impact of urban size. These results provide insights into how urban design and planning can be used to alleviate air pollution.

Cabs1 Maintains Structural Integrity of Mouse Sperm Flagella during Epididymal Transit of Sperm.

The calcium-binding protein spermatid-associated 1 (Cabs1) is a novel spermatid-specific protein. However, its function remains largely unknown. In this study, we found that a long noncoding RNA (lncRNA) transcripted from the Cabs1 gene antisense, AntiCabs1, was also exclusively expressed in spermatids. Cabs1 and AntiCabs1 knockout mice were generated separately (using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 methods) to investigate their functions in spermatogenesis. The genetic loss of Cabs1 did not affect testicular and epididymal development; however, male mice exhibited significantly impaired sperm tail structure and subfertility. Ultrastructural analysis revealed defects in sperm flagellar differentiation leading to an abnormal annulus and disorganization of the midpiece-principal piece junction, which may explain the high proportion of sperm with a bent tail. Interestingly, the proportion of sperm with a bent tail increased during transit in the epididymis. Furthermore, Western blot and immunofluorescence analyses showed that a genetic loss of Cabs1 decreased Septin 4 and Krt1 and increased cyclin Y-like 1 (Ccnyl1) levels compared with the wild type, suggesting that Cabs1 deficiency disturbed the expression of cytoskeleton-related proteins. By contrast, AntiCabs1-/- mice were indistinguishable from the wild type regarding testicular and epididymal development, sperm morphology, concentration and motility, and male fertility. This study demonstrates that Cabs1 is an important component of the sperm annulus essential for proper sperm tail assembly and motility.

Generation of Transgenic Cloned Buffalo Embryos Harboring the EGFP Gene in the Y Chromosome Using CRISPR/Cas9-Mediated Targeted Integration.

Sex control technology is of great significance in the production of domestic animals, especially for rapidly breeding water buffalo (bubalus bubalis), which served as a research model in the present study. We have confirmed that a fluorescence protein integrated into the Y chromosome is fit for sexing pre-implantation embryos in the mouse. Firstly, we optimized the efficiency of targeted integration of exogenous gene encoding enhanced green fluorescent protein (eGFP) and mCherry in Neuro-2a cells, mouse embryonic stem cells, mouse embryonic cells (NIH3T3), buffalo fetal fibroblast (BFF) cells. The results showed that a homology arm length of 800 bp on both sides of the target is more efficient that 300 bp or 300 bp/800 bp. Homology-directed repair (HDR)-mediated knock-in in BFF cells was also significantly improved when cells were supplemented with pifithrin-µ, which is a small molecule that inhibits the binding of p53 to mitochondria. Three pulses at 250 V resulted in the most efficient electroporation in BFF cells and 1.5 µg/mL puromycin was found to be the optimal concentration for screening. Moreover, Y-Chr-eGFP transgenic BFF cells and cloned buffalo embryos were successfully generated using CRISPR/Cas9-mediated gene editing combined with the somatic cell nuclear transfer (SCNT) technique. At passage numbers 6-8, the growth rate and cell proliferation rate were significantly lower in Y-Chr-eGFP transgenic than in non-transgenic BFF cells; the expression levels of the methylation-related genes DNMT1 and DNMT3a were similar; however, the expression levels of the acetylation-related genes HDAC1, HDAC2, and HDAC3 were significantly higher (p < 0.05) in Y-Chr-eGFP transgenic BFF cells compared with non-transgenic cells. Y-Chr-eGFP transgenic BFFs were used as donors for SCNT, the results showed that eGFP reporter is suitable for the visualization of the sex of embryos. The blastocyst rates of cloned buffalo embryos were similar; however, the cleavage rates of transgenic cloned embryos were significantly lower compared with control. In summary, we optimized the protocol for generating transgenic BFF cells and successfully generated Y-Chr-eGFP transgenic embryos using these cells as donors.

AEG-1 aggravates inflammation via promoting NALP3 inflammasome formation in murine endometriosis lesions.

Endometriosis (EMs) is one of the most common gynaecological diseases in women of childbearing age. Astrocyte elevated gene-1 (AEG-1) is associated with the invasion, migration, apoptosis and prognosis of various cancers. However, the roles of AEG-1 in EMs and its corresponding molecular mechanism are still unknown. In this study, animal models of EMs were established and mice were divided into two groups (n = 10): Sham group and EMs group. The EMs cells were isolated from EMs model. The AEG-1 gene was knocked down by shRNA, while the SOCS1 gene was knocked down by siRNA. Histological changes, AEG-1 expression in tissues and inflammatory factors level were detected by H&E staining, immunohistochemistry and ELISA, respectively. RT-qPCR and western blotting were used to determine the expression level of related proteins. The present study found AEG-1 was up-regulated in the EMs model. Enhanced AEG-1 promoted inflammatory cell infiltration, and elevated the levels of IL-1ß, IL-6, and TNF-α in EM group (p < 0.05). Besides, AEG-1 overexpression promoted the expression of NALP3, ASC and Cleaved-caspase-1, while decreased SOCS1 level (p < 0.05). Decrease of SOCS1 further promoted the formation of NALP3 inflammasome. The inhibitory effect of AEG-1 on SOCS1 was weakened after the addition of MG-132 (p < 0.01). Furthermore, silencing AEG-1 alone increased SOCS1 level, decreased the levels of inflammatory cytokines, thereby inhibited the formation of NALP3 inflammasome. All these results demonstrated that AEG-1 aggravated inflammation via promoting NALP3 inflammasome formation in murine endometriosis lesions.

Identification of the Sex of Pre-implantation Mouse Embryos Using a Marked Y Chromosome and CRISPR/Cas9.

Although numerous attempts have been made to alter the sex ratio of the progeny of mammals, the limitations of current technologies have prevented their widespread use in farm animals. The presence or absence of a Y chromosome determines whether a mammalian embryo develops as a male or female, and non-invasive genetic reporters such as fluorescence protein markers have been intensively applied in a variety of fields of research. To develop a non-invasive and instantaneous method for advance determination of the sex of embryos, we developed a Y chromosome-linked eGFP mouse line that stably expresses green fluorescent protein under the control of the CAG promoter. The development of the CRISPR/Cas9 system has made it easy to deliver an exogenous gene to a specific locus of a genome, and linking a tracer to the Y chromosome has simplified the process of predicting the sex of embryos collected by mating a Y-Chr-eGFP transgenic male with a wild-type female. XY embryos appeared green, under a fluorescence microscope, and XX embryos did not. Y chromosome-linked genes were amplified by nested PCR to further confirm the accuracy of this method, and the simultaneous transplantation of green and non-green embryos into foster mothers indicated that 100% accuracy was achieved by this method. Thus, the Y-Chr-eGFP mouse line provides an expeditious and accurate approach for sexing pre-implantation embryos and can be efficiently used for the pre-selection of sex.

Spatiotemporal variation in PM2.5 concentrations and their relationship with socioeconomic factors in China's major cities.

The air quality issues caused by extreme haze episodes in China have become increasingly serious in recent years. In particular, fine particulate matter (PM2.5) has become the major component of haze with many adverse impacts and has therefore become of great concern to scientists, government, and the general public in China. This study investigates the spatiotemporal variation in PM2.5 in 269 Chinese cities from 2015 to 2016 and its associations with socioeconomic factors to identify the possible strategies for PM2.5 pollution mitigation. Specifically, we first quantified the spatial pattern of PM2.5 concentrations in both 2015 and 2016, and then changes between the two years. Next, we examined the relationship between socioeconomic factors and PM2.5 concentrations and changes. The results showed that most cities in eastern China experienced decreases in PM2.5 concentration, although most of these cities already had high PM2.5 pollution level. Cities with low PM2.5 concentrations experienced increases in PM2.5 concentrations and were mostly located in southern and southwestern China. The PM2.5 concentration was the highest in winter, followed by in spring, autumn and summer; for changes in PM2.5 concentrations, the highest magnitude of decrease occurred in summer, followed by the decreases in winter, autumn and spring. Cities with high PM2.5 concentrations tended to be clustered, but the clustered characteristics were not clearly related to the changes in PM2.5 concentrations. The relationship between PM2.5 concentration and urban size was an inverse U-shaped curve, suggesting the existence of the Environmental Kuznets Curve for air quality in China. Population density and secondary industry share are the keys factors relating to air pollution control. In comparison to other cities, most moderately developed cities had a greater magnitude of decrease in PM2.5 concentrations and the key factor for pollution improvement was industrial structure; however, smaller cities tended to have a greater increase in PM2.5 concentrations and population density was the most important influencing factor. As a result, for air pollution control in China, specific regulations should be carried out according to different regions and different developmental stages based on the locations of cities.

Analyzing bovine OCT4 and NANOG enhancer activity in pluripotent stem cells using fluorescent protein reporters.

Green fluorescent protein (GFP) reporters controlled by the regulatory region of OCT4 and NANOG-two master regulators for pluripotency are widely used in studies of pluripotent stem cell establishment and embryo development. Alongside the challenge in establishing bovine pluripotent stem cells, the application of bovine-specific gene reporters has rarely been explored. Using lentivirus-based GFP reporter, we investigated the upstream regulatory regions of bovine OCT4 and NANOG. These reporters show activity in both naïve- and primed-state pluripotency when infected into mouse and human embryonic stem cells (ESCs), respectively. Consistent with what is found in humans and mice, the bovine OCT4-distal enhancer (bOCT4-DE) but not the proximal enhancer (bOCT4-PE) region is preferentially activated in naïve-state pluripotency. Furthermore, the bOCT4-DE region is silenced upon conversion of naive-state ESCs into primed-state epiblast stem cells (EpiSCs). Co-infection of mouse fibroblasts with the reprograming factors for induced pluripotent stem cell (iPSC) induction leads to the generation of GFP positive colonies, demonstrating that these GFP reporters can serve as live indicators for induced pluripotent cell establishment. We further proved that the bovine OCT4 distal enhancer is active in bovine blastocysts. We established the lentiviral-based fluorescent reporters controlled by bovine OCT4 and NANOG enhancer sequences. These reporter constructs show activity in naïve- and primed-pluripotent states. These reporters may serve as versatile tools for bovine ESC/iPSC generation and identification, as well as for developmental studies of bovine embryos.

Role of BCL2L10 in regulating buffalo (Bubalus bubalis) oocyte maturation.

It has been reported that BCL2L10 is abundantly and specifically expressed in adult human and mouse oocytes and played a very important role in oocytes maturation and early embryonic development. This study is to investigate the expression pattern of BCL2L10 in buffalo ovaries and its effect on the in vitro maturation of buffalo oocytes, so as to dissect mechanism of oocytes maturation and provide theoretical guidance for improvement of the in vitro maturation of buffalo oocytes. The results showed that BCL2L10 gene was enriched in ovary and the expression of BCL2L10 was oocyte specific and up-regulated during oocyte maturation. BCL2L10 protein and mRNA were detectable in buffalo early embryos, upregulated at 2-cell to 8-cell stages and down-regulated in the later stages. Knockdown of BCL2L10 by RNA interference resulted in a significant decrease in the maturation rate (33.5%) and cleavage rate (37.52%) of buffalo oocytes coupled with up-regulation of apoptosis-related gene Caspase-9. We concluded that BCL2L10 is a candidate associated with buffalo oocyte maturation.