[Chemical Speciation, Influencing Factors, and Regression Model of Heavy Metals in Farmland of Typical Carbonate Area with High Geological Background, Southwest China].

The speciation of heavy metals in soil is an important factor determining their bioavailability and toxicity, and it is crucial for the scientific assessment of ecological risks posed by heavy metals in soils of typical carbonate areas with high geological background in southwest China. In order to investigate the distribution of speciation of heavy metals in soils of carbonate rock with high geological background, we selected a typical carbonate rock distribution area in Guizhou Province and used the second national soil survey plots as sampling units. A total of 309 topsoil samples were collected from farmland. The improved Tessier seven-step sequential extraction method was used to analyze the seven chemical forms of heavy metals:water-soluble (F1); exchangeable (F2); carbonate-bound (F3); weakly organic-bound (F4); iron-manganese oxide-bound (F5); strongly organic-bound (F6); and residual (F7) forms of arsenic (As), cadmium (Cd), copper (Cu), mercury (Hg), nickel (Ni), lead (Pb), and zinc (Zn). The study found that the residual forms of heavy metals As, Cu, Hg, Ni, Pb, and Zn in the soil accounted for more than 50%, the effective components (F1-F3) accounted for less than 5%, and the potential biological effective components (F4-F6) were less than 45%, indicating low reactivity and low ecological risk. The effective and potentially bioavailable components of Cd accounted for 55.49% and 29.37%, respectively, which were much higher than those of other heavy metals. The ecological risk based on the speciation of heavy metals in the soil was much lower than that based on the total content of heavy metals. The stepwise regression equations could effectively establish the relationship between the bioavailable and potentially bioavailable fractions of Cd, Cu, and Pb and their influencing factors. Total heavy metal contents and pH value were important factors influencing the speciation of heavy metals in soils of carbonate rock with high geological background areas. The enrichment of heavy metal elements in the residual fraction was influenced by long-term zinc smelting activities and the weathering of carbonate rocks into soil. Soil organic matter (OM) and oxide content had a relatively small influence on the speciation of heavy metals in the soil.

Gut microbiota and transcriptome dynamics in every-other-day fasting are associated with neuroprotection in rats with spinal cord injury.

Introduction: Every-other-day fasting (EODF) is a classical intermittent fasting (IF) mode with neuroprotective effects that promotes motor function recovery after spinal cord injury (SCI) in rats. However, its dynamic effects on the gut microbiota and spinal cord transcriptome remain unknown. Methods: In this study, 16S rRNA sequencing and RNA-seq analysis were used to investigate the effects of ad libitum (AL) and EODF dietary modes on the structural characteristics of rat gut microbiota in rats and the spinal cord transcriptome at various time points after SCI induction. Results: Our results showed that both dietary modes affected the bacterial community composition in SCI rats, with EODF treatment inducing and suppressing dynamic changes in the abundances of potentially anti-inflammatory and pro-inflammatory bacteria. Furthermore, the differentially expressed genes (DEGs) enriched after EODF intervention in SCI rats were associated with various biological events, including immune inflammatory response, cell differentiation, protein modification, neural growth, and apoptosis. In particular, significant spatiotemporal differences were apparent in the DEGs associated with neuroprotection between the EODF and AL interventions. These DGEs were mainly focused on days 1, 3, and 7 after SCI. The relative abundance of certain genera was significantly correlated with DEGs associated with neuroprotective effects in the EODF-SCI group. Discussion: Our results showed that EODF treatment may exert neuroprotective effects by modulating the transcriptome expression profile following SCI in rats. Furthermore, gut microbiota may be partially involved in mediating these effects.

Dietary Restriction May Attenuate the Expression of Cell Death-Related Proteins in Rats with Acute Spinal Cord Injury.

OBJECTIVE: There is currently no effective treatment for spinal cord injuries (SCIs). Previous studies have shown that every-other-day fasting (EODF), a dietary restriction method, can reduce SCI size and promote motor function recovery, making it a potential novel treatment. However, the mechanism that underlies the positive impact of EODF on SCI remains unclear. Caspase-dependent apoptosis and necroptosis, which involve receptor-interacting protein kinase (RIPK), drive the loss of nerve cells and restrict motor function recovery after SCI. Dietary restriction has a significant inhibitory effect on Caspase and RIPK expression. This study aimed to investigate whether the EODF diet achieves a neuroprotective effect by inhibiting Caspase-dependent apoptosis and RIPK-dependent necroptosis after SCI. METHODS: The model rats underwent EODF for 4 weeks before SCI or started EODF diet immediately after SCI. Immunoblotting and immunohistochemical analyses were used to assess the impact of the intervention on protein expression. Apoptosis in the spinal cord was detected by TdT-mediated dUTP nick-end labeling. RESULTS: Immunoblotting analysis results revealed that the levels of both RIPK1 and RIPK3 proteins in the injury zone were reduced at 6, 12, and 24 hours and at 3 and 7 days after SCI, respectively. Immunohistochemistry results showed that EODF reduced the expression of Caspase-3 and Bax proteins, while prophylactic EODF decreased the rate of apoptosis detected by TdT-mediated dUTP nick-end labeling within 3 days after SCI. CONCLUSIONS: These findings indicate that the mechanism by which EODF exerts neuroprotective effects may be related to the simultaneous inhibition of apoptosis and necroptosis in SCI.

TCF3 Regulates the Proliferation and Apoptosis of Human Spermatogonial Stem Cells by Targeting PODXL.

Spermatogonial stem cells (SSCs) are the initial cells for the spermatogenesis. Although much progress has been made on uncovering a number of modulators for the SSC fate decisions in rodents, the genes mediating human SSCs remain largely unclear. Here we report, for the first time, that TCF3, a member of the basic helix-loop-helix family of transcriptional modulator proteins, can stimulate proliferation and suppress the apoptosis of human SSCs through targeting podocalyxin-like protein (PODXL). TCF3 was expressed primarily in GFRA1-positive spermatogonia, and EGF (epidermal growth factor) elevated TCF3 expression level. Notably, TCF3 enhanced the growth and DNA synthesis of human SSCs, whereas it repressed the apoptosis of human SSCs. RNA sequencing and chromatin immunoprecipitation (ChIP) assays revealed that TCF3 protein regulated the transcription of several genes, including WNT2B, TGFB3, CCN4, MEGF6, and PODXL, while PODXL silencing compromised the stem cell activity of SSCs. Moreover, the level of TCF3 protein was remarkably lower in patients with spermatogenesis failure when compared to individuals with obstructive azoospermia with normal spermatogenesis. Collectively, these results implicate that TCF3 modulates human SSC proliferation and apoptosis through PODXL. This study is of great significance since it would provide a novel molecular mechanism underlying the fate determinations of human SSCs and it could offer new targets for gene therapy of male infertility.

Trace the profile and function of circular RNAs in Sertoli cell only syndrome.

Studies increasingly show the involvement of circular RNAs (circRNAs) in several diseases. This study aims to explore the circRNA expression pattern in the testicular tissues of patients with Sertoli only cell syndrome (SCOS) and their potential functions. High throughput circRNA microarray analysis indicated that 399 circRNAs were upregulated and 1195 were down-regulated (fold change >2, P < 0.05) in SCOS relative to obstructive azoospermia (OA). The hsa_circRNA_101222, hsa_circRNA_001387, hsa_circRNA_001153, hsa_circRNA_101373 and hsa_circRNA_103864 were validated by qRT-PCR. Furthermore, the hosting genes of the differentially expressed circRNAs (DEcircRNAs) were enriched in biological processes related to cell cycle and intercellular communication. Also, the overlapping genes between the hosting genes of SCOS-related DEcircRNAs and those highly expressed in Sertoli cells of non-obstructive azoospermia (NOA) were enriched in immune cell development and cell communication. Taken together, aberrantly expressed circRNAs likely mediate SCOS development by regulating the function of Sertoli cells and the spermatogenic microenvironment.

Characterization, isolation, and culture of spermatogonial stem cells in Macaca fascicularis.

Spermatogonial stem cells (SSCs) have great applications in both reproductive and regenerative medicine. Primates including monkeys are very similar to humans with regard to physiology and pathology. Nevertheless, little is known about the isolation, the characteristics, and the culture of primate SSCs. This study was designed to identify, isolate, and culture monkey SSCs. Immunocytochemistry was used to identify markers for monkey SSCs. Glial cell line-derived neurotrophic factor family receptor alpha-1 (GFRA1)-enriched spermatogonia were isolated from monkeys, namely Macaca fascicularis (M. fascicularis), by two-step enzymatic digestion and magnetic-activated cell sorting, and they were cultured on precoated plates in the conditioned medium. Reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and RNA sequencing were used to compare phenotype and transcriptomes in GFRA1-enriched spermatogonia between 0 day and 14 days of culture, and xenotransplantation was performed to evaluate the function of GFRA1-enriched spermatogonia. SSCs shared some phenotypes with rodent and human SSCs. GFRA1-enriched spermatogonia with high purity and viability were isolated from M. fascicularis testes. The freshly isolated cells expressed numerous markers for rodent SSCs, and they were cultured for 14 days. The expression of numerous SSC markers was maintained during the cultivation of GFRA1-enriched spermatogonia. RNA sequencing reflected a 97.3% similarity in global gene profiles between 0 day and 14 days of culture. The xenotransplantation assay indicated that the GFRA1-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-KitW/W (W) mutant mice. Collectively, GFRA1-enriched spermatogonia are monkey SSCs phenotypically both in vitro and in vivo. This study suggests that monkey might provide an alternative to human SSCs for basic research and application in human diseases.

Hsa-miR-1908-3p Mediates the Self-Renewal and Apoptosis of Human Spermatogonial Stem Cells via Targeting KLF2.

Spermatogenesis depends on precise epigenetic and genetic regulation of spermatogonial stem cells (SSCs). However, it remains largely unknown about the roles and mechanisms of small noncoding RNA in regulating the self-renewal and apoptosis of human SSCs. Notably, we have found that Homo sapiens-microRNA (hsa-miR)-1908-3p is expressed at a higher level in human spermatogonia than pachytene spermatocytes. MiR-1908-3p stimulated cell proliferation and DNA synthesis of the human SSC line. Allophycocyanin (APC) Annexin V and propidium iodide staining, determined by flow cytometric analysis and TUNEL assays, showed that miR-1908-3p inhibited early and late apoptosis of the human SSC line. Furthermore, Kruppel-like factor 2 (KLF2) was predicted and verified as the target of miR-1908-3p, and, significantly, KLF2 silencing resulted in the increase of proliferation and DNA synthesis, as well as reduction of apoptosis of the human SSC line. Moreover, KLF2 silencing ameliorated the decrease in the proliferation and DNA synthesis and the enhancement in the apoptosis of the human SSC line caused by miR-1908-3p inhibition. Collectively, these results implicate that miR-1908-3p stimulates the self-renewal and suppresses the apoptosis of human SSCs by targeting KLF2. This study thus provides a novel epigenetic regulatory mechanism underlying the fate determinations of human SSCs, and it offers new endogenous targets for treating male infertility.

Testicular biopsies microarray analysis reveals circRNAs are involved in the pathogenesis of non-obstructive azoospermia.

Circular RNAs (circRNAs) have been reported to be involved in many diseases. But there is no report on circRNAs in non-obstructive azoospermia (NOA). The purpose of this paper is to explore the circular RNA expression profile and potential functions of circRNAs in NOA patients. We first preformed circRNA expression profiling analysis using a circRNA microarray in testicular samples from NOA and obstructive azoospermia (OA) patients. CircRNAs were validated by qRT-PCR. Bioinformatics analysis were used to construct the ceRNA network. GO and KEGG enrichment analysis were performed by using DAVID. Microarray analysis identified 82 differentially expressed circRNAs in NOA specimens. The differential expression of hsa_circRNA_402130, hsa_circRNA_072697, hsa_circRNA_030050, hsa_circRNA_100812 and hsa_circRNA_406168 was confirmed by qRT-PCR. Enrichment analysis revealed the association of hsa_circRNA_402130 and hsa_circRNA_072697 with multiple signaling pathways. The data indicated that circRNAs were significantly dysregulated in NOA specimens and might involve in the pathogenesis of NOA.

A network-based approach to identify DNA methylation and its involved molecular pathways in testicular germ cell tumors.

Background: Testicular germ cell tumors (TGCT) is the most common testicular malignancy threaten young male reproductive health. This study aimed to identify aberrantly methylated-differentially expressed genes and pathways in TGCT by comprehensive bioinformatics analysis. Methods: Data of gene expression microarrays (GSE3218, GSE18155) and gene methylation microarrays (GSE72444) were collected from GEO database. Integrated analysis acquired aberrantly methylated-genes. Functional and pathway enrichment analysis were performed using DAVID database. Protein-protein interaction (PPI) network was constructed by STRING and App Mcode was used for module analysis. GEPIA platform and DiseaseMeth database were used for confirming the expression and methylation levels of hub genes. Finally, Human Protein Atlas database was performed to evaluate the prognostic significance. Results: Totally 604 hypomethylation-high expression and 147 hypermethylation-low genes were identified. The high expressed genes were enriched in biological processes of cell proliferation and migration. The top 8 hub genes of PPI network were GAPDH, VEGFA, PTPRC, RIPK4, MMP9, CSF1R, KRAS and FN1. After validation in GEPIA platform, all hub genes were elevated in TGCT tissues. Only MMP9, CSF1R and PTPRC showed hypomethylation-high expression status, which predicted the poor outcome of TGCT patients. Conclusion: Our study indicated possible aberrantly methylated-differentially expressed genes and pathways in TGCT by bioinformatics analysis, which may provide novel insights for unraveling pathogenesis of TGCT.

PLZFposc-KITpos-delineated A1-A4-differentiating spermatogonia by subset and stage detection upon Bouin fixation.

While hallmarks of rodent spermatogonia stem cell biomarkers' heterogeneity have recently been identified, their stage and subset distributions remain unclear. Furthermore, it is currently difficult to accurately identify subset-specific SSC marker distributions due to the poor nuclear morphological characteristics associated with fixation in 4% paraformaldehyde. In the present study, testicular cross-sections and whole-mount samples were Bouin fixed to optimize nuclear resolution and visualized by immunohistochemistry (IHC) and immunofluorescence (IF). The results identified an expression pattern of PLZFhighc-KITpos in A1 spermatogonia, while A2-A4-differentiating spermatogonia were PLZFlowc-KITpos. Additionally, this procedure was used to examine asymmetrically expressing GFRA1 and PLZF clones, asymmetric Apr and false clones were distinguished based on the presence or absence of TEX14, a molecular maker of intercellular bridges, despite having identical nuclear morphology and intercellular distances that were <25 µm. In conclusion, this optimized Bouin fixation procedure facilitates the accurate identification of spermatogonium subsets based on their molecular profiles and is capable of distinguishing asymmetric and false clones. Therefore, the findings presented herein will facilitate further morphological and functional analysis studies and provide further insight into spermatogonium subtypes.

[Study on the method for the determination of trace boron, molybdenum, silver, tin and lead in geochemical samples by direct current arc full spectrum direct reading atomic emission spectroscopy (DC-Arc-AES)].

The method for the determmation of trace boron, molybdenum, silver, tin and lead in geochemical samples by direct current are full spectrum direct reading atomic emission spectroscopy (DC-Arc-AES) was established. Direct current are full spectrum direct reading atomic emission spectrometer with a large area of solid-state detectors has functions of full spectrum direct reading and real-time background correction. The new electrodes and new buffer recipe were proposed in this paper, and have applied for national patent. Suitable analytical line pairs, back ground correcting points of elements and the internal standard method were selected, and Ge was used as internal standard. Multistage currents were selected in the research on current program, and each current set different holding time to ensure that each element has a good signal to noise ratio. Continuous rising current mode selected can effectively eliminate the splash of the sample. Argon as shielding gas can eliminate CN band generating and reduce spectral background, also plays a role in stabilizing the are, and argon flow 3.5 L x min(-1) was selected. Evaporation curve of each element was made, and it was concluded that the evaporation behavior of each element is consistent, and combined with the effects of different spectrographic times on the intensity and background, the spectrographic time of 35s was selected. In this paper, national standards substances were selected as a standard series, and the standard series includes different nature and different content of standard substances which meet the determination of trace boron, molybdenum, silver, tin and lead in geochemical samples. In the optimum experimental conditions, the detection limits for B, Mo, Ag, Sn and Pb are 1.1, 0.09, 0.01, 0.41, and 0.56 microg x g(-1) respectively, and the precisions (RSD, n=12) for B, Mo, Ag, Sn and Pb are 4.57%-7.63%, 5.14%-7.75%, 5.48%-12.30%, 3.97%-10.46%, and 4.26%-9.21% respectively. The analytical accuracy was validated by national standards and the results are in agreement with certified values. The method is simple, rapid, is an advanced analytical method for the determination of trace amounts of geochemical samples' boron, molybdenum, silver, tin and lead, and has a certain practicality.

Osteogenic differentiated periodontal ligament stem cells maintain their immunomodulatory capacity.

Periodontal ligament stem cells (PDLSCs) have great potential for regenerating periodontal ligament tissue, which is involved in attaching teeth to the underlying alveolar bone. Recently, PDLSCs were characterized as having both low immunogenicity and profound immunomodulation abilities. Further, transplanted PDLSCs differentiate into osteoblasts in vivo. In the present study, we investigated the immunological characteristics of osteogenic differentiated PDLSCs. We found that PDLSCs expressed mesenchymal stem cells markers, including STRO-1 and CD146, but were negative for CD14, CD34 and CD45. RT-PCR indicated that NCAM1, MSX1 and S100A4 were expressed in PDLSCs. The cells underwent osteogenic and adipogenic differentiation when cultured in defined medium. Osteogenic differentiated PDLSCs failed to stimulate allogeneic T cell proliferation and suppressed phytohaemagglutinin-triggered T cell proliferation. Indomethacin, an inhibitor of prostaglandin E2 (PGE2) production, restored the T cell proliferation inhibited by osteogenic differentiated PDLSCs. These data confirm that osteogenic differentiated PDLSCs have low immunogenicity and demonstrate that they suppress T cell proliferation in vitro through secretion of PGE2.

[Determination of high content of tin in geochemical samples by solid emission spectrometry].

A method for the determination of high content of tin in geochemical samples by solid emission spectrometry was presented. The dedicated high content tin spectrum standard series was developed. K2S2O7, NaF, Al2O3 and carbon powder were used as buffers and Ge was used as internal standard, and the ratio of sample/matrix/buffer is 1 : 1 : 2. A weak sensitive line (Sn 242. 170 0 nm) was used as the analytical line. The technologies of vertical electrodes, AC arc overlap spectrograph, interception of the exposure, quantitative computer translation spectrum and background correction were used. The determination range is 100-22 350 microg x g(-1), the detection limit is 16.64 microg x g(-1), and the precision is (RSD, n = 12) 4.11%-6.46%. The accuracy of the method has been verified by determination of high content of tin in national geochemical standard samples and the results are in agreement with certified value. The method can be used for measurement directly without dilution of high content of tin in geochemical samples, and it greatly improved the detection upper limit for the determination of tin with solid emission spectroscopy and has certain practical value.

Expression of cystic fibrosis transmembrane conductance regulator in rat ovary.

The protein expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl(-) channel, in ovarian stimulated premature female rat ovary during a cycle of follicle development and corpus luteum formation was investigated. Animals were injected with 10 U pregnant Mare's serum gonadotropin (PMSG) and subsequently 10 U hCG 48 h later. Time-dependent immunohistochemistry and Western blotting experiments were performed before and 24, 48, 72 h after hCG treatment. The immunohistochemistry revealed that administration of PMSG stimulated the CFTR expression in thecal cell layer and granulosa cell layer of mature follicles 48 h post injection, coincident with the PMSG-induced peak in follicular estradiol. However, the expression of CFTR in the granulose lutein cell layer and thecal lutein cell layer was time-dependently reduced following hCG injection, in accordance with the gradually increased progestogen level during luteum corpus formation. Western blotting analysis demonstrated that rat ovarian tissue expressed the special CFTR band at 170 kD. It is concluded that cAMP-dependent Cl(-) channels are involved in regulation of follicle development and luteum formation.

Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Rat Ovary / 华中科技大学学报（医学）（英德文版）

Summary: The protein expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in ovarian stimulated premature female rat ovary during a cycle of follicle development and corpus luteum formation was investigated. Animals were injected with 10 U pregnant Mare's serum gonadotropin (PMSG) and subsequently 10U hCG 48h later. Time-dependent immunohistochemistry and Western blotting experiments were performed before and 24, 48, 72h after hCG treatment. The immnnohistochemistry revealed that administration of PMSG stimulated the CFTR expression in theeal cell layer and granulosa cell layer of mature follicles 48 h post injection, coincident with the PMSG-induced peak in follicular estradiol. However, the expression of CFTR in the granuiose lutein cell layer and theeal lutein cell layer was time-dependently reduced following hCG injection, in accordance with the gradually increased progestogen level during luteum corpus formation. Western blotting analysis demonstrated that rat ovarian tissue expressed the special CFTR band at 170kD. It is concluded that cAMP-dependent Cl- channels are involved in regulation of follicle development and luteum formation.