SPIDR is required for homologous recombination during mammalian meiosis.

Meiotic recombinases RAD51 and DMC1 mediate strand exchange in the repair of DNA double-strand breaks (DSBs) by homologous recombination. This is a landmark event of meiosis that ensures genetic diversity in sexually reproducing organisms. However, the regulatory mechanism of DMC1/RAD51-ssDNA nucleoprotein filaments during homologous recombination in mammals has remained largely elusive. Here, we show that SPIDR (scaffold protein involved in DNA repair) regulates the assembly or stability of RAD51/DMC1 on ssDNA. Knockout of Spidr in male mice causes complete meiotic arrest, accompanied by defects in synapsis and crossover formation, which leads to male infertility. In females, loss of Spidr leads to subfertility; some Spidr-/- oocytes are able to complete meiosis. Notably, fertility is rescued partially by ablation of the DNA damage checkpoint kinase CHK2 in Spidr-/- females but not in males. Thus, our study identifies SPIDR as an essential meiotic recombination factor in homologous recombination in mammals.

A millimeter water-in-oil droplet as an alternative back exchange prevention strategy for hydrogen/deuterium exchange mass spectrometry of peptides/proteins.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a versatile bioanalytical technique for protein analysis. Since the reliability of HDX-MS analysis considerably depends on the retention of deuterium labels in the post-labeling workflow, deuterium/hydrogen (D/H) back exchange prevention strategies, including decreasing the pH, temperature, and exposure time to protic sources of the deuterated samples, are widely adopted in the conventional HDX-MS protocol. Herein, an alternative and effective back exchange prevention strategy based on the encapsulation of a millimeter droplet of a labeled peptide solution in a water-immiscible organic solvent (cyclohexane) is proposed. Cyclohexane was used to prevent the undesirable uptake of water by the droplet from the atmospheric vapor through the air-water interface. Using the pepsin digest of deuterated myoglobin, our results show that back exchange kinetics of deuterated peptides is retarded in a millimeter droplet as compared to that in the bulk solution. Performing pepsin digestion directly in a water-in-oil droplet at room temperature (18-21 °C) was found to preserve more deuterium labels than that in the bulk digestion with an ice-water bath. Based on the present findings, it is proposed that keeping deuterated peptides in the form of water-in-oil droplets during the post-labelling workflow will facilitate the preservation of deuterium labels on the peptide backbone and thereby enhance the reliability of the H/D exchange data.

MiRNA-224-5p regulates the defective permeability barrier in sensitive skin by targeting claudin-5.

BACKGROUND: Sensitive skin is hypersensitive to various external stimuli and a defective epidermal permeability barrier is an important clinical feature of sensitive skin. Claudin-5 (CLDN5) expression levels decrease in sensitive skin. This study aimed to explore the impact of CLDN5 deficiency on the permeability barrier in sensitive skin and the regulatory role of miRNAs in CLDN5 expression. MATERIALS AND METHODS: A total of 26 patients were retrospectively enrolled, and the CLDN5 expression and permeability barrier dysfunction in vitro were assessed. Then miRNA-224-5p expression was also assessed in sensitive skin. RESULTS: Immunofluorescence and electron microscopy revealed reduced CLDN5 expression, increased miR-224-5p expression, and disrupted intercellular junctions in sensitive skin. CLDN5 knockdown was associated with lower transepithelial electrical resistance (TEER) and Lucifer yellow penetration in keratinocytes and organotypic skin models. The RNA-seq and qRT-PCR results indicated elevated miR-224-5p expression in sensitive skin; MiR-224-5p directly interacted with the 3`UTR of CLDN5, resulting in CLDN5 deficiency in the luciferase reporter assay. Finally, miR-224-5p reduced TEER in keratinocyte cultures. CONCLUSION: These results suggest that the miR-224-5p-induced reduction in CLDN5 expression leads to impaired permeability barrier function, and that miR-224-5p could be a potential therapeutic target for sensitive skin.

Lactobacillus paracasei ZFM54 alters the metabolomic profiles of yogurt and the co-fermented yogurt improves the gut microecology of human adults.

Gut microbiota imbalance could lead to various diseases, making it important to optimize the structure of flora in adults. Lactobacillus paracasei ZFM54 is a bacteriocin and folic acid producing Lactobacillus strain. Herein ZFM54 was used as the potentialy probiotic bacterium to ferment milk together with a yogurt starter. We optimized the fermentation conditions and the obtained yogurts were then subjected to volatile and non-volatile metabolome analysis, showing that ZFM54 cannot only improve the acidity, water holding capacity and live lactic acid bacteria counts, but also improve many volatile acid contents and increase some beneficial non-volatile metabolites such as N-ethyl glycine and L-Lysine, endowing the yogurt with more flavor and better function. The regulatory effects of the co-fermented yogurt on intestinal microecology of volunteers were investigated by 16S rRNA sequencing and short-chain fatty acids (SCFAs) analysis after a continuous consuming the yogurt of 2-week, showing better effect to increase the relative abundance of beneficial bacteria such as Ruminococcus and Alistipes, decrease harmful bacteria (Escherichia-Shigella and Enterobacter), and enhance the production of SCFAs (acetate, propionate and butyric acid) than the control yogurt. In conclusion, L. paracasei ZFM54 can significantly improve the health benefits of yogurt, laying the foundation for its commercial application in improving gut microbiota.

[Identification and visual analysis of ginsenosides in multi-steamed roots of Panax quinquefolium based on UPLC-Q-TOF-MS/MS and MALDI-MSI].

This study aims to investigate the component variations and spatial distribution of ginsenosides in Panax quinquefolium roots during repeated steaming and drying. Ultra performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS) was employed to identify the ginsenosides in the root extract. Matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI) was employed to visualize the spatial distribution and spatiotemporal changes of prototype ginsenosides and metabolites in P. quinquefolium roots. The UPLC results showed that 90 ginsenosides were identified during the steaming process of the roots, and polar ginsenosides were converted into low polar or non-polar ginsenosides. The content of prototype ginsenosides decreased, while that of rare ginsenosides increased, which included 20(S/R)-ginsenoside Rg_3, 20(S/R)-ginsenoside Rh_2, and ginsenosides Rk_1, Rg_5, Rs_5, and Rs_4. MALDI-MSI results showed that ginsenosides were mainly distributed in the epidermis and phloem. As the steaming times increased, ginsenosides were transported to the xylem and medulla. This study provides fundamental information for revealing the changes of biological activity and pharmacological effect of P. quinquefolium roots that are caused by repeated steaming and drying and gives a reference for expanding the application scope of this herbal medicine.

Peptide Oxidation Induced by Liquid-Solid Contact Electrification as Revealed in Liquid Microjunction-Surface Sampling Probe Mass Spectrometry.

Reproducible peptide oxidation was observed using a homebuilt liquid microjunction-surface sampling probe (LMJ-SSP) platform for analyzing peptide standards. Although electrochemical oxidation and corona discharges have previously been associated with analyte oxidation in electrospray ionization (ESI) and ESI-related ambient ionization mass spectrometry (MS) methods, they were unlikely the causes for the peptide oxidation observed in the LMJ-SSP studies. A systematic investigation demonstrated that analyte oxidation was induced during the droplet drying on a solid surface through liquid-solid electrification processes. To minimize unwanted analyte oxidation, the water content in the sample solution should be decreased and the use of hydroxyl-functionalized substrates, such as glass slides, should be avoided. In addition, if water is an essential solvent component, adding an antioxidant, such as ascorbic acid, to the sample solution before droplet evaporation on the solid surface could lower the percentage of analyte oxidation. The present findings apply to all the MS methods that involve drying microliters of sample solution onto a suitable substrate in their sample preparation protocols.

Spatially Resolved Metabolomics Combined with the 3D Tumor-Immune Cell Coculture Spheroid Highlights Metabolic Alterations during Antitumor Immune Response.

The metabolic cross-talk between tumor and immune cells plays key roles in immune cell function and immune checkpoint blockade therapy. However, the characterization of tumor immunometabolism and its spatiotemporal alterations during immune response in a complex tumor microenvironment is challenging. Here, a 3D tumor-immune cell coculture spheroid model was developed to mimic tumor-immune interactions, combined with mass spectrometry imaging-based spatially resolved metabolomics to visualize tumor immunometabolic alterations during immune response. The inhibition of T cells was simulated by coculturing breast tumor spheroids with Jurkat T cells, and the reactivation of T cells can be monitored through diminishing cancer PD-L1 expressions by berberine. This system enables simultaneously screening and imaging discriminatory metabolites that are altered during T cell-mediated antitumor immune response and characterizing the distributions of berberine and its metabolites in tumor spheroids. We discovered that the transport and catabolism of glutamine were significantly reprogrammed during the antitumor immune response at both metabolite and enzyme levels, corresponding to its indispensable roles in energy metabolism and building new biomass. The combination of spatially resolved metabolomics with the 3D tumor-immune cell coculture spheroid visually reveals metabolic interactions between tumor and immune cells and possibly helps decipher the role of immunometabolic alterations in tumor immunotherapy.

The emerging role of extracellular vesicles in the testis.

Extracellular vesicles (EVs) are nano-sized membrane-bounded particles, released by all cells and capable of transporting bioactive cargoes, proteins, lipids, and nucleic acids, to regulate a variety of biological functions. Seminal plasma is enriched in EVs, and extensive evidence has revealed the role of EVs (e.g. prostasomes and epididymosomes) in the male genital tract. Recently, EVs released from testicular cells have been isolated and identified, and some new insights have been generated on their role in maintaining normal spermatogenesis and steroidogenesis in the testis. In the seminiferous tubules, Sertoli cell-derived EVs can promote the differentiation of spermatogonial stem cells (SSCs), and EVs secreted from undifferentiated A spermatogonia can inhibit the proliferation of SSCs. In the testicular interstitium, EVs have been identified in endothelial cells, macrophages, telocytes, and Leydig cells, although their roles are still elusive. Testicular EVs can also pass through the blood-testis barrier and mediate inter-compartment communication between the seminiferous tubules and the interstitium. Immature Sertoli cell-derived EVs can promote survival and suppress the steroidogenesis of Leydig cells. Exosomes isolated from macrophages can protect spermatogonia from radiation-induced injury. In addition to their role in intercellular communication, testicular EVs may also participate in the removal of aberrant proteins and the delivery of antigens for immune tolerance. EVs released from testicular cells can be detected in seminal plasma, which makes them potential biomarkers reflecting testicular function and disease status. The testicular EVs in seminal plasma may also affect the female reproductive tract to facilitate conception and may even affect early embryogenesis through modulating sperm RNA. EVs represent a new type of intercellular messenger in the testis. A detailed understanding of the role of testicular EV may contribute to the discovery of new mechanisms causing male infertility and enable the development of new diagnostic and therapeutic strategies for the treatment of infertile men.

Identification of raffinose family oligosaccharides in processed Rehmannia glutinosa Libosch using matrix-assisted laser desorption/ionization mass spectrometry image combined with machine learning.

RATIONALE: Currently, research on oligosaccharides primarily focuses on the physiological activity and function, with a few studies elaborating on the spatial distribution characterization and variation in the processing of Rehmannia glutinosa Libosch. Thus, imaging the spatial distributions and dynamic changes in oligosaccharides during the steaming process is significant for characterizing the metabolic networks of R. glutinosa. It will be beneficial to characterize the impact of steaming on the active ingredients and distribution patterns in different parts of the plant. METHODS: A highly sensitive matrix-assisted laser desorption/ionization mass spectrometry image (MALDI-MSI) method was used to visualize the spatial distribution of oligosaccharides in processed R. glutinosa. Furthermore, machine learning was used to distinguish the processed R. glutinosa samples obtained under different steaming conditions. RESULTS: Imaging results showed that the oligosaccharides in the fresh R. glutinosa were mainly distributed in the cortex and xylem. As steaming progressed, the tetra- and pentasaccharides were hydrolyzed and diffused gradually into the tissue section. MALDI-MS profiling combined with machine learning was used to identify the processed R. glutinosa samples accurately at different steaming intervals. Eight algorithms were used to build classification machine learning models, which were evaluated for accuracy, precision, recall, and F1 score. The linear discriminant analysis and random forest models performed the best, with prediction accuracies of 0.98 and 0.97, respectively, and thus can be considered for identifying the steaming durations of R. glutinosa. CONCLUSIONS: MALDI-MSI combined with machine learning can be used to visualize the distribution of oligosaccharides and identify the processed samples after steaming for different durations. This can enhance our understanding of the metabolic changes that occur during the steaming process of R. glutinosa; meanwhile, it is expected to provide a theoretical reference for the standardization and modernization of processing in the field of medicinal plants.

Use of group IIB metal ions as charge carriers for collision-induced dissociation of glycopeptide and glycan.

RATIONALE: Dissociation of biomolecules by tandem mass spectrometry (MS/MS) generates a variety of fragment ions which provide useful information for the structural characterization of biomolecules. Different fragmentation strategies result in different mass spectra for the same molecule and thus provide distinct features. Charge carriers play important roles in determining the dissociation pathways of the target precursor ions. The use of various transition metals ions as charge carriers of glycopeptide and glycan might provide additional structural information and needs to be investigated. METHODS: A 9.4 T SolariX FTICR mass spectrometer was used for collision-induced dissociation (CID) of glycopeptide and glycan. Group IIB metal ions, including Zn2+ , Cd2+ and Hg2+ , were used as charge carriers. Glycopeptide NLTK-M5 G2 and glycan G1F were used as the model systems. RESULTS: For Zn2+ - and Cd2+ -adducted species, cross-ring cleavages, glycosidic cleavages and cleavages along the peptide backbone could be obtained. There is a high degree of similarity in their CID spectra with that of Mg2+ ion-adducted glycopeptide species. For Hg2+ -adducted species, only glycosidic cleavages were observed in high abundance. The formation of doubly-charged ions (M2+ ) and a series of [f-H]+ fragments indicated unique dissociation pathways for Hg2+ -adducted glycopeptide. CONCLUSIONS: Zn2+ and Cd2+ -adducted glycopeptide species produced similar dissociation CID spectra, whereas Hg2+ -adducted species produced significantly different CID spectra. Similar CID dissociation features were also observed for Group IIB metal ions adducted glycan species. These results demonstrated that different metal ions might be used to tune the dissociation behaviors of glycopeptides and glycans.

Prenatal exposure to fine particulate matter and newborn anogenital distance: a prospective cohort study.

BACKGROUND: Considerable attention has been paid to reproductive toxicity of fine particulate matter (PM2.5). However, the relationship between prenatal PM2.5 exposure and anogenital distance (AGD) has not been well studied. We aim to investigate the potential effects of prenatal exposure to PM2.5 on newborn AGD. METHODS: Prenatal PM2.5 exposure of 2332 participates in Shanghai (2013-2016) was estimated using high-performance machine learning models. Anoscrotal distance (AGDas) in male infants and anofourchette distance (AGDaf) in female infants were measured by well-trained examiners within 3 days after birth. We applied multiple linear regression models and multiple informant models to estimate the association between prenatal PM2.5 exposure and AGD. RESULTS: Multiple linear regression models showed that a 10 µg/m3 increase in PM2.5 exposure during full pregnancy, the second and third trimesters was inversely associated with AGDas (adjusted beta = - 1.76, 95% CI: - 2.21, - 1.31; - 0.73, 95% CI: - 1.06, - 0.40; and - 0.52; 95% CI: - 0.87, - 0.18, respectively) in males. A 10 µg/m3 increase in PM2.5 exposure during the full pregnancy, the first, second, and third trimesters was inversely associated with AGDaf (adjusted beta = - 4.55; 95% CI: - 5.18, - 3.92; - 0.78; 95% CI: - 1.10, - 0.46; - 1.11; 95% CI: - 1.46, - 0.77; - 1.45; 95% CI: - 1.78, - 1.12, respectively) in females after adjusting for potential confounders. Multiple informant models showed consistent but slightly attenuated associations. CONCLUSION: Our study observed a significant association between gestational PM2.5 exposure during pregnancy and shortened AGD in newborns, and provided new evidence on potential reproductive toxicity of prenatal PM2.5 exposure.

Mixed infection of three nontuberculous mycobacteria species identified by metagenomic next-generation sequencing in a patient with peritoneal dialysis-associated peritonitis: a rare case report and literature review.

BACKGROUND: Peritonitis caused by nontuberculous mycobacteria (NTM) is an infrequent but important complication in patients undergoing peritoneal dialysis (PD). There has been no report of mixed infections with multiple NTM. Peritoneal dialysis-associated peritonitis (PDAP) caused by Mycobacterium abscessus is more common than that caused by M. smegmatis and M. goodii. CASE PRESENTATION: This case concerns a patient with PDAP caused by gram-positive bacilli, which could not be identified at the species level in successive detections of initial peritoneal effluent. Later, M. smegmatis was detected with no sensitivity results in bacterial culture. However, metagenomic next-generation sequencing (mNGS) and first whole-genome sequences indicated that there were three species coexisting in the culture, including M. smegmatis (24,708 reads), M. abscessus (9224 reads), and M. goodii (8305 reads). This is the first case of PDAP with specific evidence that conventional detection methods isolated a poorly pathogenic NTM, whereas mNGS and first whole-genome sequences identified multiple NTM. Pathogenic bacteria might not be detected using conventional methods due to their lower abundance. This case report is the first description of mixed infections with more than two species of NTM during PDAP. CONCLUSIONS: PDAP caused by multiple NTM is rare, and the diagnosis is difficult. When NTM are isolated by conventional tests in patients who are suspected of infection, clinicians should be vigilant, and further tests should be performed to determine the presence of rare or even previously unknown bacteria, for which the quantity is relatively low, but the pathogenicity is high. The rare pathogen may be a primary agent in causing such complications.

Identification of a novel pyroptosis-related gene signature in human spermatogenic dysfunction.

PURPOSE: To reveal the underlying roles that pyroptosis-related genes (PRGs) played in human spermatogenic dysfunction. METHODS: One discovery set and three validation sets were employed to inspect the previously reported 33 PRGs in the human testis with different status of spermatogenesis. PRGs that differentially expressed in all sets were considered as key differentially expressed pyroptosis-related genes (PR-DEGs). The relationships between key PR-DEGs and samples' clinicopathological, therapeutic, and immune patterns were respectively studied. Single-cell RNA sequencing (scRNS-seq) analyses were conducted to show the expression changes and related mechanisms of key PR-DEGs at a single-cell resolution. RESULTS: CASP4 and GPX4 were identified as two key PR-DEGs. These two genes were significantly dysregulated in spermatogenic dysfunctional samples, but with opposite tendency. CASP4 was negatively correlated with Johnsen scores but positively correlated with follicle-stimulating hormone (FSH) levels (all p < 0.05), while GPX4 exhibited significant positive correlations with Johnsen scores and negative relevance with FSH. For treatments, both molecules showed a prospective value of being predictors for sperm retrieval surgeries. Moreover, CASP4 and GPX4 were potential immunoregulators in the testicular immune microenvironment and showed significant correlations to testicular macrophages and mast cell infiltration. In scRNA-seq analyses, GPX4 was highly expressed in germ cells, which therefore suffered a sharp reduction with the loss of germ cells in spermatogenic dysfunction. On the other hand, CASP4 were basically somatic cell-derived, and the proportion of CASP4-positive Leydig cells significantly increased in disease testes (p = 0.0001). CONCLUSION: In all, we revealed two key PRGs of human testes that might be functional in spermatogenic dysfunction.

Association of embryo aneuploidy and sperm DNA damage in unexplained recurrent implantation failure patients under NGS-based PGT-A cycles.

PURPOSE: Recurrent implantation failure (RIF) is one of the most common conditions affecting In Vitro Fertilization (IVF)/Intracytoplasmic sperm injection (ICSI) outcomes. Aneuploidy embryos, one of the main types of embryos-related factors, was reported to be a major contributor to RIF. The present study aimed to examine the association between sperm DNA fragmentation index (DFI) and outcomes of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) in unexplained RIF patients. METHODS: This study analyzed 119 couples with unexplained RIF who underwent 119 PGT-A cycles between January, 2017 and March, 2022. The 119 males were divided into 3 groups according to their sperm DFI levels: Group1 (low, DFI ≤ 15%, n = 50), Group2 (medium, 15% < DFI < 30%, n = 41) and Group3 (high, DFI ≥ 30%, n = 28). Sperm DFI was measured by sperm chromatin structure analysis (SCSA) technique. Trophectoderm biopsy on day 5 or 6 were performed with NGS technique. The following outcomes of PGT-A were analyzed and compared: fertilization, good-quality embryos, aneuploidy rate, miscarriage, live birth and newborn defects. RESULTS: The component of aneuploidy embryos was significantly higher in high DFI group (42.71%) than that of medium group (28.39%) and low group (27.80%). The miscarriage rate of high DFI group (27.27%) and medium group (14.29%) is significantly higher than that of low group (0.00%). No significant differences were found regarding fertility, good-quality embryo rate, pregnancy rate, live birth rate or newborn defects among three groups. CONCLUSION: The sperm DNA damage is associated with blastocyst aneuploidy and miscarriage rate in unexplained RIF cases. Embryo selection by PGT-A and efforts to decrease sperm DFI before IVF/ICSI treatments should be considered for those male patients with high DFI.

The regulation function of intestinal microbiota by folate-producing Lactiplantibacillus plantarum LZ227.

BACKGROUND: Folic acid is a class of B vitamins that have the function of improving intestinal microbiota. RESULT: Lactiplantibacillus plantarum LZ227, which is a highly folate-producing strain, was used as the research object, and the folic acid produced by LZ227 was further identified by liquid chromatography-mass spectrometry, and the structural diversity, community composition, abundance difference, and short-chain fatty acids content in fermentation broth were studied by the manure slurry fermentation model. The results showed that the folic acid produced by LZ227 was 5-methyltetrahydrofolate. CONCLUSION: LZ227 can increase the intestinal microbial diversity in the folate-free state, regulate the intestinal flora, increase the abundance of Firmicutes in the intestinal flora, and inhibit the abundance of Bacteroidetes. LZ227 can inhibit the growth of Alistipes, Parabacteroides, and Bacteroides in the intestine. LZ227 significantly reduced the acetic acid content and significantly increased the butyric acid content in the folate-free case. © 2023 Society of Chemical Industry.

Whole-exome sequencing of a large Chinese azoospermia and severe oligospermia cohort identifies novel infertility causative variants and genes.

Rare coding variants have been proven to be one of the significant factors contributing to spermatogenic failure in patients with non-obstructive azoospermia (NOA) and severe oligospermia (SO). To delineate the molecular characteristics of idiopathic NOA and SO, we performed whole-exome sequencing of 314 unrelated patients of Chinese Han origin and verified our findings by comparing to 400 fertile controls. We detected six pathogenic/likely pathogenic variants and four variants of unknown significance, in genes known to cause NOA/SO, and 9 of which had not been earlier reported. Additionally, we identified 20 novel NOA candidate genes affecting 25 patients. Among them, five (BRDT, CHD5, MCM9, MLH3 and ZFX) were considered as strong candidates based on the evidence obtained from murine functional studies and human single-cell (sc)RNA-sequencing data. These genetic findings provide insight into the aetiology of human NOA/SO and pave the way for further functional analysis and molecular diagnosis of male infertility.

Paper-Based Flow Sensor for the Detection of Hyaluronidase via an Enzyme Hydrolysis-Induced Viscosity Change in a Polymer Solution.

Hyaluronidase (HAase) is implicated in inflammation, cancer development, and allergic reaction. The detection of HAase is significantly important in clinical diagnosis and medical treatment. Herein, we propose a new principle for the development of equipment-free and label-free paper-based flow sensors based on the enzymatic hydrolysis-induced viscosity change in a stimuli-responsive polymer solution, which increases the water flow distance on the pH indicator paper. The detection of HAase is demonstrated as an example. This facile and versatile method can overcome the potential drawbacks of traditional hydrogel-based sensors, including complex preparation steps, slow response time, or low sensitivity. Moreover, it can also avoid the use of specialized instruments, labeled molecules, or functionalized nanoparticles in the sensors developed using the polymer solutions. Using this strategy, the detection of HAase is achieved with a limit of detection as low as 0.2 U/mL. Also, it works well in human urine. Additionally, the detection of tannic acid, which is an inhibitor of HAase, is also fulfilled. Overall, a simple, efficient, high-throughput, and low-cost detection method is developed for the rapid and quantitative detection of HAase and its inhibitor without the use of labeled molecules, synthetic particles, and specialized instruments. As only minimal reagents of HAase, HA, and paper are used, it is very promising in the development of commercial kits for point-of-care testing.

Exosomes released from Sertoli cells contribute to the survival of Leydig cells through CCL20 in rats.

Reciprocal communication between Sertoli and Leydig cells occurs in the testes; however, the detailed mechanisms involved are not completely understood. Exosomes can communicate within neighboring or distant cells to regulate cell function. Our aim was to determine whether exosomes released from Sertoli cells can regulate the survival of Leydig cells. We found that exosomes released from rat primary Sertoli cells could be internalized by Leydig cells in vitro, and promote the survival of Leydig cells, as assessed by optical density at 450 nm, compared to untreated control (mean ± SD: 0.95 ± 0.04 vs 0.79 ± 0.03, P < 0.05). When the exosomes were injected into the interstitial area of rat testis, they could also be internalized by Leydig cells in vivo. To investigate if exosomes released from Sertoli cells can reach Leydig cells in vivo, exosomes were injected into the efferent duct, from where they entered the interstitial space from seminiferous tubules, which indicated that they may cross the blood-testis barrier (BTB). Further in vitro studies found that exosomes released from Sertoli cells significantly increased CC-chemokine ligand 20 (Ccl20) mRNA (mean ± SD: 2.79 ± 0.08 vs 0.98 ± 0.04, P < 0.01) and protein (mean ± SD: 1.08 ± 0.06 vs 0.53 ± 0.05 ng/ml, P < 0.01) levels in Leydig cells, compared to the untreated Leydig cells. CCL20 promoted the phosphorylation of AKT (protein kinase B) in Leydig cells, compared to untreated control (mean ± SD: 0.074 ± 0.002 vs 0.051 ± 0.002, P < 0.01). In conclusion, our results demonstrated that exosomes released by Sertoli cells may cross the BTB and promote the survival of Leydig cells. The findings may add new evidence for Sertoli-Leydig cell communication.

Inconsistency analysis between metagenomic next-generation sequencing results of cerebrospinal fluid and clinical diagnosis with suspected central nervous system infection.

BACKGROUND: Recently, with the rapid progress of metagenomic next-generation sequencing (mNGS), inconsistency between mNGS results and clinical diagnoses has become more common. There is currently no reasonable explanation for this, and the interpretation of mNGS reports still needs to be standardised. METHODS: A retrospective analysis was conducted on 47 inpatients with suspected central nervous system (CNS) infections, and clinical data were recorded. The final diagnosis was determined by an expert group based on the patient's clinical manifestation, laboratory examination, and response to treatment. mNGS results were compared with the final diagnosis, and any inconsistencies that occurred were investigated. Finally, the credibility of mNGS results was evaluated using the integral approach, which consists of three parts: typical clinical features, positive results with the traditional method, and cerebrospinal fluid cells ≥ 100 (× 106/L) or protein ≥ 500 mg/L, with one point for each item. RESULTS: Forty-one patients with suspected CNS infection were assigned to infected (ID, 31/41, 75.61%) and non-infected groups (NID, 10/41, 24.39%) after assessment by a panel of experts according to the composite diagnostic criteria. For mNGS-positive results, 20 of the 24 pathogens were regarded as contaminants when the final score was ≤ 1. The remaining 11 pathogens detected by mNGS were all true positives, which was consistent with the clinical diagnosis when the score was ≥ 2. For mNGS negative results, when the score was ≥ 2, the likelihood of infection may be greater than when the score is ≤ 1. CONCLUSION: The integral method is effective for evaluating mNGS results. Regardless of whether the mNGS result was positive or negative, the possibility of infection was greater when the score was ≥ 2. A negative mNGS result does not necessarily indicate that the patient was not clinically infected, and, therefore, clinical features are more important.

Fate and ecological risks of current-use pesticides in seawater and sediment of the Yellow Sea and East China Sea.

With the frequent use of chemical pesticides, the current-use pesticides (CUPs) emerge and concentrate in the sea. The partition between the sediment and seawater is essential for understanding the environmental fate of CUPs. However, there is little research on this topic. In the present study, seventeen CUPs were screened in seawater and sediment samples collected from the Yellow Sea and East China Sea. Total concentration of 17 CUPs in surface seawater samples ranged from 9.5 to 267.3 ng/L, with 6 CUPs presenting 100% detection frequency. Carbendazim, tricyclazole, tebuconazole, atrazine and imidacloprid accounted for >80% of all CUPs, which was due to their large application in the local agriculture and fishing activities. Higher concentration sites were located near the shore and Yangtze river estuary, indicating intense human activities and riverine input that elevated the level of CUPs in marginal sea. The pesticides in seawater were mainly found in the surface followed by the bottom layer, which indicated that atmospheric deposition and re-suspension played key roles for their vertical distribution characteristics. The high fugacity fraction ratios (ff > 0.5) indicated the non-equilibrium state of pesticides that might have been transferred from sediment to seawater at most sites. These 17 detectable pesticides in seawater were at low levels, presenting ignorable or low toxic effects to aquatic organisms.