Mitochondrial calcium uniporter promotes mitophagy by regulating the PINK1/Parkin pathway in caerulein­treated pancreatic ductal epithelial cells in vitro.

The mitochondrial calcium uniporter (MCU) is a major protein for the uptake of mitochondrial calcium to regulate intracellular energy metabolism, including processes such as mitophagy. The present study investigated the effect of the MCU on mitophagy in pancreatic ductal epithelial cells (PDECs) in acute pancreatitis (AP) in vitro. The normal human PDECs (HPDE6-C7) were treated with caerulein (CAE) to induce AP-like changes, with or without ruthenium red to inhibit the MCU. The mitochondrial membrane potentials (MMPs) and mitochondrial Ca2+ levels were analyzed by fluorescence. The expression levels of MCU, LC3, p62, and translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20), putative kinase 1 (PINK1), and Parkin were measured by western blotting and immunofluorescence. Mitophagy was observed by confocal fluorescence microscopy and transmission electron microscopy. The results showed that CAE increased the MCU protein expression, mitochondrial Ca2+ levels, MMP depolarization and the protein expression of mitophagy markers including the LC3II/I ratio, PINK1, and Parkin. CAE decreased the protein expression of p62 and TOMM20, and promoted the formation of mitophagosomes in HPDE6-C7 cells. Notably, changes in these markers were reversed by inhibiting the MCU. In conclusion, an activated MCU may promote mitophagy by regulating the PINK1/Parkin pathway in PDECs in AP.

[Network Meta-analysis of efficacy and safety of Chinese patent medicines in treatment of ankylosing spondylitis].

To evaluate the efficacy and safety of Chinese patent medicines in the treatment of ankylosing spondylitis(AS) by frequency network Meta-analysis. Randomized controlled trials(RCTs)of Chinese patent medicines for AS were retrieved from CNKI, Wanfang, VIP, CBM, PubMed, EMbase and Cochrane Library databases from the time of database establishment to January 2021. The quality of the included RCTs was evaluated according to the Cochrane bias risk standard, and the data was analyzed by RevMan 5.3 and Stata/MP 15.1. A total of 12 kinds of Chinese patent medicines in 55 RCTs were included. According to Meta-analysis, in term of the effectiveness, the top three optimal medication regimens were Biqi Capsules, Yishen Juanbi Pills and Yaobitong Capsules combined with western medicine. The top three interventions to reduce the erythrocyte sedimentation rate(ESR)were Yishen Juanbi Pills, Xianling Gubao Capsules and Fufang Xuanju Capsules combined with western medicine. The top three interventions to reduce the C-reactive protein(CRP)were Biqi Capsules, Xianling Gubao Capsules and Fufang Xuanju Capsules combined with western medicine. In terms of the safety, top three optimal medication regimens were Total Glucosides of Paeony Capsules, Yishen Juanbi Pills, and Wangbi Tablets combined with western medicine. This network Meta-analysis suggests that Chinese patent medicines combined with conventional western medicine can effectively improve the joint pain symptoms of AS patients and reduce the acute inflammatory indicators, with high safety. However, the literature included in this study is generally of low methodological quality, and the conclusion needs to be verified by high-quality research.

Gelsolin impairs barrier function in pancreatic ductal epithelial cells by actin filament depolymerization in hypertriglyceridemia-induced pancreatitis in vitro.

Gelsolin (GSN) is a calcium-regulated actin-binding protein that can sever actin filaments. Notably, actin dynamics affect the structure and function of epithelial barriers. The present study investigated the role of GSN in the barrier function of pancreatic ductal epithelial cells (PDECs) in hypertriglyceridemia-induced pancreatitis (HTGP). The human PDEC cell line HPDE6-C7 underwent GSN knockdown and was treated with caerulein (CAE) + triglycerides (TG). Intracellular calcium levels and the actin filament network were analyzed under a fluorescence microscope. The expression levels of GSN, E-cadherin, nectin-2, ZO-1 and occludin were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. Ultrastructural changes in tight junctions were observed by transmission electron microscopy. Furthermore, the permeability of PDECs was analyzed by fluorescein isothiocyanate-dextran fluorescence. The results revealed that CAE + TG increased intracellular calcium levels, actin filament depolymerization and GSN expression, and increased PDEC permeability by decreasing the expression levels of E-cadherin, nectin-2, ZO-1 and occludin compared with the control. Moreover, changes in these markers, with the exception of intracellular calcium levels, were reversed by silencing GSN. In conclusion, GSN may disrupt barrier function in PDECs by causing actin filament depolymerization in HTGP in vitro.

[Antagonistic effect of modified Dahuang Zhechong Granule against the apoptosis of epididymal spermatogenic cells in varicocele rats based on Nrf2/HO-1 pathways].

OBJECTIVE: To investigate the effects of modified Dahuang Zhechong Granule (DZG) on the epididymal tissue of varicocele (VC) rats and the expressions of the nuclear factor erythroid 2 (NF-E2)-related factor (Nrf2) and heme oxygenase-1 (HO-1) protein. METHODS: Sixty SD rats were randomly divided into six groups of an equal number: sham operation, VC model control, aescuven forte (AF) and low-, medium- and high-dose DZG. The VC model was established by ligation of the left renal vein with the Turner's method, followed by intragastrical administration of normal saline to the rats in the sham operation and VC model control groups, AF Tablets at 54 mg/kg to those in the AF group, and modified DZG at 0.6, 1.2 and 2.4 g/ml to those in the low-, medium- and high-dose DZG groups respectively, all once daily for 8 weeks. Then, all the animals were sacrificed and their left epididymides harvested for examination of semen quality, observation of local ultrastructural changes, measurement of the apoptosis of spermatogenic cells by Annexin V-FITC, and determination of the expressions of Nrf2 and HO-1 in the epididymal tissue by immunohistochemistry. RESULTS: Evident pathological damage was observed in the left epididymal tissue of the VC model controls, with significantly reduced numbers of spermatogenic cells and sperm at all levels, partially destroyed cellular structure, and disappearance of some subcellular structures such as the lysosome, mitochondrion, endoplasmic reticulum, nucleus and cell membrane, which were all improved to some extent in the DZG and AF group. Sperm concentration and motility in the left epididymis were significantly higher in the medium- and high-dose DZG and AF groups than in the VC model controls (P < 0.05), even more significantly in the high-dose DZG than in the AF group (P < 0.05). The apoptosis rate of spermatogenic cells was markedly higher in the VC model control than in the sham operation group (P < 0.05), but lower in the medium- and high-dose DZG and AF groups than in the VC model controls (P < 0.05). Immunohistochemistry showed positive expressions of Nrf2 and HO-1 proteins, brown, scattered and with a low luminance of the cells, in the left epididymis tissue of the VC model control rats, but with a significantly higher cell luminance in the high-dose DZG and AF groups. CONCLUSIONS: Modified Dahuang Zhechong Granule can effectively repair pathological damage to the epididymis of varicocele rats, increase the expressions of Nrf2 and HO-1 proteins, antagonize the apoptosis of spermatogenic cells and provide a favorable condition for sperm maturation.

Proteome analysis reveals a systematic response of cold-acclimated seedlings of an exotic mangrove plant Sonneratia apetala to chilling stress.

Low temperature in winter was the most crucial abiotic stress that limits the mangrove afforestation northward. Previous study demonstrated that Sonneratia apetala initially transplanted to high latitude area exhibited a stronger plasticity of cold tolerance. To clarify the underlying mechanism, the physiological and proteomic responses to chilling stress were investigated in S. apetala leaves. Our results found that cold-acclimated seedlings had lower relative electrolyte leakage and MDA content than non-acclimated seedlings. On the contrary, higher chlorophyll content and photosynthetic capacity were observed in cold-acclimated seedlings. With proteomic analyses, the differentially accumulated proteins (DAPs) involved in ROS scavenging, photosynthesis and energy metabolism, carbohydrate metabolism, cofactor biosynthesis, and protein folding were suggested to play important roles in enhancing the cold tolerance of S. apetala. However, the down-regulation DAPs were suggested as a tradeoff between plant growth and chilling response. By the protein-protein interaction analyses, translation elongation factor G, chlorophyll A-B binding protein and ascorbate peroxidase 1 were suggested as the important regulators in cold-acclimated S. apetala seedlings under chilling stress. Based on the above results, a schematic diagram describing the mechanism of cold tolerance of exotic mangrove species S. apetala that was achieved by cold acclimation was presented in this study. SIGNIFICANCE: The major environmental factor limits the mangrove afforestation northward is the low temperature in winter. Previous study reported that Sonneratia apetala grew in high latitude exhibited a higher cold tolerance than that in low latitude, which was suggested as a result of cold acclimation. To further understand "how cold acclimation enhance the cold tolerance in S. apetala", the response of S. apetala subjected to chilling stress with or without cold acclimation was investigated in this study at the physiological and proteomic aspects. Our physiological results showed that S. apetala seedlings treated with cold acclimation exhibited a higher tolerance under chilling stress than that without cold acclimation. By using the comparative proteomic approaches and bioinformatic analyses, various biological processes were suggested to play an important role in enhancing the cold tolerance of S. apetala under chilling stress, such as ROS scavenging, photosynthesis and energy metabolism, carbohydrate metabolism, cofactor biosynthesis, and protein folding. Among these differentially accumulated proteins, translation elongation factor G (eEF-G), chlorophyll A-B binding protein (CAB) and ascorbate peroxidase 1 (APX1) were identified as the hub proteins function in coordinated regulating ROS scavenging, photosynthesis and protein biosynthesis in chloroplast and subsequently enhanced the cold tolerance of S. apetala under chilling stress. Our results provided a further understanding of cold acclimation in improving the cold tolerance in exotic mangrove species S. apetala.

Long non-coding RNAs in biology and female reproductive disorders.

Accumulating data from large-scale transcriptome studies have identified a class of poorly understood non-protein-coding RNAs, including microRNAs, piwi-interacting RNAs (piRNAs), and long non-coding RNAs (lncRNAs), and a number of studies suggest that lncRNAs modulate the expression of protein-coding genes in a variety of tissues and organs by altering chromatin modification, transcription, mRNA decay, protein subcellular localization, and other key processes. Although much work still remains to identify the roles of lncRNAs in reproduction-related systems, they are likely to exert widespread effects during these processes. In this review, we highlight our emerging understanding of how lncRNAs regulate gene expression, and we discuss the physiological role of this new class of molecular regulators in neurobiology, cardiology, endocrinology, metabolism, muscle biology, and female reproductive disorders.

[Intervention effect of Modified Dahuang Zhechong Granule on epididymal morphological changes in experimental varicocele rats].

OBJECTIVE: To explore the effect of Modified Dahuang Zhechong Granule (MDZG) on the development and maturation of epididymal sperm in experimental varicocele (VC) rats. METHODS: Sixty SD male rats were randomly divided into six groups of equal number, sham operation, VC model, Aescuven forte, and low-, medium- and high-dose MDZG. The model of left VC was made by the Turner method in all the rats except those of the sham operation group, followed by treatment with 0.9% normal saline for the animals in the sham operation and VC model groups, Aescuven forte tablets at 54 mg per kg of the body weight for those in the Aescuven forte group, and MDZG at 0.6, 1.2 and 2.4 g/ml for those in the low-, medium- and high-dose MDZG groups, all administered intragastrically qd for 8 successive weeks. Then, all the rats were sacrificed and their left epididymides harvested for examination of the quality of the epididymal sperm and the local microscopic and ultrastructural changes of the epididymal tissue. RESULTS: The VC model rats showed significant apoptosis of the epididymal sperm cells, interstitial edema, microvascular dilatation, degeneration and degeneration of the epithelial cells, degeneration of some principal cells and basal cell vacuoles, and immature spermatids in the lumen. Sperm motility was significantly increased in the Aescuven forte and low-, medium- and high-dose MDZG groups as compared with the VC models (P <0.01). Both sperm concentration and motility were markedly higher in the high-dose MDZG than in the Aescuven forte group (P <0.05). Remarkable apoptosis of epididymal sperm cells was observed in the microenvironment of sperm development in the VC models, which exhibited no statistically significant difference from that in the rats of the medium- and high-dose MDZG groups. CONCLUSIONS: Experimental varicocele induced local apoptosis of epididymal sperm cells, interstitial edema and microvascular dilatation in the rat epididymis, while Modified Dahuang Zhechong Granule could improve the stability of epididymal sperm maturation and contribute to their development.

Exotic Spartina alterniflora invasion increases CH4 while reduces CO2 emissions from mangrove wetland soils in southeastern China.

Mangroves are critical in global carbon budget while vulnerable to exotic plant invasion. Spartina alterniflora, one of typical salt marsh plant grows forcefully along the coast of China, has invaded the native mangrove habitats in Zhangjiang Estuary. However, the effects of S. alterniflora invasion on soil carbon gases (CH4 and CO2) emission from mangroves are not fully understood. Accordingly, we conducted a field experiment to investigate the soil CH4 and CO2 emission during growing seasons in 2016 and 2017 at four adjacent wetlands, namely bare mudflat (Mud), Kandelia obovata (KO), Avicennia marina (AM) and S. alterniflora (SA). Potential methane production (PMP), potential methane oxidation (PMO), functional microbial abundance and soil biogeochemical properties were measured simultaneously. Our results indicate that S. alterniflora invasion could dramatically increase soil CH4 emissions mainly due to the enhancement in PMP which facilitated by soil EC, MBC, TOC and mcrA gene abundance. Additionally, S. alterniflora invasion decreases soil CO2 emission. Both heterotrophic microbial respiration (16S rRNA) and methane oxidation (pmoA and ANME-pmoA) are responsible for CO2 emission reduction. Furthermore, S. alterniflora invasion greatly increases GWP by stimulating CH4 emissions. Thus, comparing with mangroves, invasive S. alterniflora significantly (p < 0.001) increases CH4 emission while reduces CO2 emission.

Gq activity- and ß-arrestin-1 scaffolding-mediated ADGRG2/CFTR coupling are required for male fertility.

Luminal fluid reabsorption plays a fundamental role in male fertility. We demonstrated that the ubiquitous GPCR signaling proteins Gq and ß-arrestin-1 are essential for fluid reabsorption because they mediate coupling between an orphan receptor ADGRG2 (GPR64) and the ion channel CFTR. A reduction in protein level or deficiency of ADGRG2, Gq or ß-arrestin-1 in a mouse model led to an imbalance in pH homeostasis in the efferent ductules due to decreased constitutive CFTR currents. Efferent ductule dysfunction was rescued by the specific activation of another GPCR, AGTR2. Further mechanistic analysis revealed that ß-arrestin-1 acts as a scaffold for ADGRG2/CFTR complex formation in apical membranes, whereas specific residues of ADGRG2 confer coupling specificity for different G protein subtypes, this specificity is critical for male fertility. Therefore, manipulation of the signaling components of the ADGRG2-Gq/ß-arrestin-1/CFTR complex by small molecules may be an effective therapeutic strategy for male infertility.

The feasibility of establishing classification system for ovarian function.

The occurrence, development, and decline of ovarian function are the foundation in women's whole life stages, which reflect the process beginning from embryo formation to the aging. Correct assessment of ovarian function is significant for evaluating the potential reproductive ability and predicting the age of menopause, as well as providing both individualized and proper treatment and preventive care based on physiological characteristics of women in different phases. Ovarian reserve (OR) is used to predict the potential fertility of women by evaluating the follicles and the quantity and quality of eggs. Currently, there are multiple indexes used to evaluate ovarian reserve, including anti-Millerian hormone (AMH), follicle-stimulating hormone (FSH), estradiol (E2), inhibin B, antral follicle count (AFC), etc. Although some scholars combine multiple indexes to evaluate the ovarian function, these indexes are far less accurate, detailed, and comprehensive. To find an ideal method for evaluation of ovarian reserve is the hotspot in research of reproductive endocrine. The present authors, for the first time, put forward a classification system of ovarian reserve function after summarizing numerous cases. It can both accurately and effectively evaluate the ovarian function quantitatively. It is of great help in making clinical decisions and of great significance in future development.

Analysis of progesterone receptor membrane component 1 mutation in Han Chinese women with premature ovarian failure.

The gene PGRMC1 is highly expressed in the granulose and luteal cells of rodent and primate ovaries. Its role in anti-apoptosis and regulating cell-cycle progression suggests a role in regulating follicle growth. The hypothesis is supported by the study in mice and studies in Sweden. In this study, the coding exons of PGRMC1 were sequenced among 196 Chinese women with premature ovarian failure (POF) and 200 controls, and one novel missense mutation was identified (C.556C>T, p. Pro186Ser) in the POF group and one novel SNP (C.533C>T, p. Trh177Ile) was identified in both groups. The mutation is not considered causative because protein prediction did not indicate a deleterious effect. It is concluded that coding mutations of PGRMC1 do not seem to be a common cause of the disease in Han Chinese women. Future studies in larger cohorts from other ethnic groups are necessary to establish the role of PGRMC1 in POF.

[Replacement of androgen receptor gene causes complete androgen insensitivity in a large family].

OBJECTIVE: To confirm the clinical diagnosis of complete androgen insensitivity syndrome (CAIS) by molecular genetic testing in a large family. METHODS: PCR was performed to amplify the coding region of androgen gene, followed by direct sequencing in the patients with CAIS and relatives in this family. RESULTS: A missense mutation Arg773His was identified in the patients (homozygous) and carriers (heterozygous). CONCLUSIONS: Mutation Arg773His in the AR gene leads to CAIS in this family. Molecular genetic testing of CAIS facilitates not only prenatal genetic diagnosis but also preimplantation genetic diagnosis and offers genetic counseling for future pregnancies to abandon the transmission of the mutated X chromosome to the coming generation.

Seasonal variation in communities of ammonia-oxidizing bacteria based on polymerase chain reaction - denaturing gradient gel electrophoresis in a biofilm reactor for drinking water pretreatment.

The diversity and variation of total and active ammonia-oxidizing bacteria in a full-scale aerated submerged biofilm reactor for drinking water pretreatment were characterized by clone libraries and denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA and its gene during a whole year. Sequences obtained from clone libraries affiliated with the Nitrosomonas oligotropha lineage and the Nitrosomonas communis lineage. An uncultured subgroup of Nitrosomonas communis lineage was also detected. Seasonal variations in both total and active ammonia-oxidizing bacteria communities were observed in the DGGE profiles, but an RNA-based analysis reflected more obvious dynamic changes in ammonia-oxidizer community than a DNA-based approach. Statistical study based on canonical correspondence analysis showed that a community shift of active ammonia oxidizers was significantly influenced by temperature and pH, but no significant correlation was found between environmental variables and total ammonia-oxidizer community shift.

Population dynamics of ammonia-oxidizing bacteria in an aerated submerged biofilm reactor for micropolluted raw water pretreatment.

Population dynamics of ammonia-oxidizing bacteria (AOB) in a full-scale aerated submerged biofilm reactor for micropolluted raw water pretreatment was investigated using molecular techniques for a period of 1 year. The ammonia monooxygenase (amoA) gene fragments were amplified from DNA and RNA extracts of biofilm samples. Denaturing gradient gel electrophoresis (DGGE) profile based on the amoA messenger RNA approach exhibited a more variable pattern of temporal dynamics of AOB communities than the DNA-derived approach during the study. Phylogenetic analysis of excised DGGE bands revealed three AOB groups affiliated with the Nitrosomonas oligotropha lineage, Nitrosomonas communis lineage, and an unknown Nitrosomonas group. The population size of betaproteobacterial AOB, quantified with 16S ribosomal RNA gene real-time polymerase chain reaction assay, ranged from 6.63 x 10(5) to 2.67 x 10(9) cells per gram of dry biofilm and corresponded to 0.23-1.8% of the total bacterial fraction. Quantitative results of amoA gene of the three specific AOB groups revealed changes in competitive dominance between AOB of the N. oligotropha lineage and N. communis lineage. Water temperature is shown to have major influence on AOB population size in the reactor by the statistic analysis, and a positive correlation between AOB cell numbers and ammonia removal efficiency is suggested (r = 0.628, P < 0.05).

Investigation of total bacterial and ammonia-oxidizing bacterial community composition in a full-scale aerated submerged biofilm reactor for drinking water pretreatment in China.

The community composition of total bacteria and ammonia-oxidizing bacteria in a full-scale aerated submerged biofilm reactor for drinking water pretreatment was characterized by analysis of 16S rRNA gene and the functional gene amoA, respectively. Sampling was performed in February and in July. 16S rRNA gene clone libraries revealed 13 bacterial divisions. At both sampling dates, the majority of clone sequences were related to the Alpha- and Betaproteobacteria. A minor proportion belonged to the following groups: Gammaproteobacteria, Deltaproteobacteria, Nitrospira, Firmicutes, Acidobacteria, Verrucomicrobia, Actinobacteria, Planctomycetes, Chloroflexi, Gemmatimonadetes and the Cytophaga-Flavobacterium-Bacteroides group. Some sequences related to bacteria owning high potential metabolic capacities were detected in both samples, such as Rhodobacter-like rRNA gene sequences. Surveys of cloned amoA genes from the two biofilm samples revealed ammonia-oxidizing bacterial sequences affiliated with the Nitrosomonas oligotropha lineage, Nitrosomonas communis lineage. An unknown Nitrosomonas group of amoA gene sequences was also detected.

Synthesis and transformation of [difluoro(phenylseleno)methyl]- trimethylsilane.

[reaction: see text] A novel and efficient strategy was developed to synthesize [difluoro(phenylseleno)methyl]trimethylsilane (PhSeCF(2)TMS, 2), which was further utilized as a nucleophilic difluoromethylating reagent to incorporate the difluoro(phenylseleno)methyl (PhSeCF(2)) group into carbonyl compounds in good yields. The resulting PhSeCF(2)-containing alcohols 3 could be conveniently converted into corresponding difluoromethyl alcohols 4 by a radical deselenylation.

Asymmetric synthesis of both enantiomers of anti-4,4,4-trifluorothreonine and 2-amino-4,4,4-trifluorobutanoic acid.

A short and efficient enantioselective synthesis of both enantiomers of anti-4,4,4-trifluorothreonine and 2-amino-4,4,4-trifluorobutanoic acid was successfully developed. Trifluoromethylation of benzyl-protected bromoalkene 4 provided key intermediate trifluoromethylated trans-disubstituted alkene 2 in good yield. The sequence then involved Sharpless asymmetric dihydroxylation, nucleophilic opening of cyclic sulfate with NaN(3), palladium-catalyzed selective hydrogenation, and oxidation.