Comparison of the Effects of LDPE and PBAT Film Residues on Soil Microbial Ecology.

The plastic film is extensively applied with limited recycling, leading to the long-run residue accumulation in soil, which offers a distinctive habitat for microorganisms, and creates a plastisphere. In this study, traditional low-density polyethylene (LDPE) plastic film and biodegradable polybutylene adipate terephthalate (PBAT) plastic film materials were selected to test their effects on soil microbial ecology. Based on high-throughput sequencing, compared to the soil environment, the alpha-diversity of bacterial communities in plastisphere was lower, and the abundance of Actinobacteria increased. Plastic film residues, as bacterial habitats, exhibited greater heterogeneity and harbor unique bacterial communities. The communities were distinguished between plastisphere and soil environment by means of a random-forest (RF) machine-learning model. Prominent distinctions emerged among bacterial functions between soil environment and plastisphere, especially regarding organics degradation. The neutral model and null model indicated that the constitution of bacterial communities was dominated by random processes except in LDPE plastisphere. The bacterial co-occurrence network of the plastisphere exhibited higher complexity and modularity. This study contributes to our comprehending of characteristics of plastisphere bacterial communities in soil environment and the associated ecological risks of plastic film residues accumulation.

Unraveling DDIT4 in the VDR-mTOR pathway: a novel target for drug discovery in diabetic kidney disease.

Introduction: Diabetic kidney disease (DKD) necessitates innovative therapeutic strategies. This study delves into the role of DNA damage-inducing transcription factor 4 (DDIT4) within the VDR-mTOR pathway, aiming to identify a novel target for DKD drug discovery. Methods: Transcriptome data from the Gene Expression Omnibus Database were analyzed to assess the expression of mTOR and VDR expression in human renal tissues. Clinical samples from DKD patients and minimal change disease (MCD) controls were examined, and a DKD animal model using 20-week-old db/db mice was established. DDIT4 plasmid transfection was employed to modulate the VDR-mTOR pathway, with its components evaluated using immunohistochemistry, real-time quantitative PCR (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Results: Changes in the expression of the VDR-mTOR pathway were observed in both DKD patients and the animal model. Overexpression of DDIT4 increased VDR expression and decreased levels of mTOR, p70s6k, and 4E-BP1. Furthermore, DDIT4 treatment regulated autophagy by upregulating LC3I expression and downregulating LC3II expression. Notably, DDIT4 alleviated oxidative stress by reducing the levels of lipid peroxidation product MDA, while simultaneously increasing the levels of superoxide dismutase (SOD) and glutathione (GSH), underscoring the role of DDIT4 in the pathological process of DKD and its potential as a therapeutic target. Conclusion: Unraveling DDIT4's involvement in the VDR-mTOR pathway provides insights for innovative DKD drug discovery, emphasizing its potential as a therapeutic target for future interventions.

Unraveling the interplay of ferroptosis and immune dysregulation in diabetic kidney disease: a comprehensive molecular analysis.

BACKGROUND: Diabetic kidney disease (DKD) is a primary microvascular complication of diabetes with limited therapeutic effects. Delving into the pathogenic mechanisms of DKD and identifying new therapeutic targets is crucial. Emerging studies reveal the implication of ferroptosis and immune dysregulation in the pathogenesis of DKD, however, the precise relationship between them remains not fully elucidated. Investigating their interplay is pivotal to unraveling the pathogenesis of diabetic kidney disease, offering insights crucial for targeted interventions and improved patient outcomes. METHODS: Integrated analysis, Consensus clustering, Machine learning including Generalized Linear Models (GLM), RandomForest (RF), Support Vector Machine (SVM) and Extreme Gradient Boosting (xGB), Artificial neural network (ANN) methods of DKD glomerular mRNA sequencing were performed to screen DKD-related ferroptosis genes.CIBERSORT, ESTIMATE and ssGSEA algorithm were used to assess the infiltration of immune cells between DKD and control groups and in two distinct ferroptosis phenotypes. The ferroptosis hub genes were verified in patients with DKD and in the db/db spontaneous type 2 diabetes mouse model via immunohistochemical and Western blotting analyses in mouse podocyte MPC5 and mesangial SV40-MES-13 cells under high-glucose (HG) conditions. RESULTS: We obtained 16 differentially expressed ferroptosis related genes and patients with DKD were clustered into two subgroups by consensus clustering. Five ferroptosis genes (DUSP1,ZFP36,PDK4,CD44 and RGS4) were identified to construct a diagnostic model with a good diagnosis performance in external validation. Analysis of immune infiltration revealed immune heterogeneity between DKD patients and controls.Moreover, a notable differentiation in immune landscape, comprised of Immune cells, ESTIMATE Score, Immune Score and Stromal Score was observed between two FRG clusters. GSVA analysis indicated that autophagy, apoptosis and complement activation can participate in the regulation of ferroptosis phenotypes. Experiment results showed that ZFP36 was significantly overexpressed in both tissue and cells while CD44 was on the contrary.Meanwhile,spearman analysis showed both ZFP36 and CD44 has a strong correlation with different immune cells,especially macrophage. CONCLUSION: The regulation of the immune landscape in DKD is significantly influenced by the focal point on ferroptosis. Newly identified ferroptosis markers, CD44 and ZFP36, are poised to play essential roles through their interactions with macrophages, adding substantial value to this regulatory landscape.

Population structure and adaptability analysis of Schizothorax o'connori based on whole-genome resequencing.

BACKGROUND: Schizothorax o'connori is an endemic fish distributed in the upper and lower reaches of the Yarlung Zangbo River in China. It has experienced a fourth round of whole gene replication events and is a good model for exploring the genetic differentiation and environmental adaptability of fish in the Qinghai-Tibet Plateau. The uplift of the Qinghai-Tibet Plateau has led to changes in the river system, thereby affecting gene exchange and population differentiation between fish populations. With the release of fish whole genome data, whole genome resequencing has been widely used in genetic evolutionary analysis and screening of selected genes in fish, which can better elucidate the genetic basis and molecular environmental adaptation mechanisms of fish. Therefore, our purpose of this study was to understand the population structure and adaptive characteristics of S. o'connori using the whole-genome resequencing method. RESULTS: The results showed that 23,602,746 SNPs were identified from seven populations, mostly distributed on chromosomes 2 and 23. There was no significant genetic differentiation between the populations, and the genetic diversity was relatively low. However, the Zangga population could be separated from the Bomi, Linzhi, and Milin populations in the cluster analysis. Based on historical dynamics analysis of the population, the size of the ancestral population of S. o'connori was affected by the late accelerated uplift of the Qinghai Tibet Plateau and the Fourth Glacial Age. The selected sites were mostly enriched in pathways related to DNA repair and energy metabolism. CONCLUSION: Overall, the whole-genome resequencing analysis provides valuable insights into the population structure and adaptive characteristics of S. o'connori. There was no obvious genetic differentiation at the genome level between the S. o'connori populations upstream and downstream of the Yarlung Zangbo River. The current distribution pattern and genetic diversity are influenced by the late accelerated uplift of the Qinghai Tibet Plateau and the Fourth Ice Age. The selected sites of S. o'connori are enriched in the energy metabolism and DNA repair pathways to adapt to the low temperature and strong ultraviolet radiation environment at high altitude.

Improved characterization of aminoglycoside penetration into human lung epithelial lining fluid via population pharmacokinetics.

Aminoglycosides are important treatment options for serious lung infections, but modeling analyses to quantify their human lung epithelial lining fluid (ELF) penetration are lacking. We estimated the extent and rate of penetration for five aminoglycosides via population pharmacokinetics from eight published studies. The area under the curve in ELF vs plasma ranged from 50% to 100% and equilibration half-lives from 0.61 to 5.80 h, indicating extensive system hysteresis. Aminoglycoside ELF peak concentrations were blunted, but overall exposures were moderately high.

Population pharmacokinetics and humanized dosage regimens matching the peak, area, trough, and range of amikacin plasma concentrations in immune-competent murine bloodstream and lung infection models.

Amikacin is an FDA-approved aminoglycoside antibiotic that is commonly used. However, validated dosage regimens that achieve clinically relevant exposure profiles in mice are lacking. We aimed to design and validate humanized dosage regimens for amikacin in immune-competent murine bloodstream and lung infection models of Acinetobacter baumannii. Plasma and lung epithelial lining fluid (ELF) concentrations after single subcutaneous doses of 1.37, 13.7, and 137 mg/kg of body weight were simultaneously modeled via population pharmacokinetics. Then, humanized amikacin dosage regimens in mice were designed and prospectively validated to match the peak, area, trough, and range of plasma concentration profiles in critically ill patients (clinical dose: 25-30 mg/kg of body weight). The pharmacokinetics of amikacin were linear, with a clearance of 9.93 mL/h in both infection models after a single dose. However, the volume of distribution differed between models, resulting in an elimination half-life of 48 min for the bloodstream and 36 min for the lung model. The drug exposure in ELF was 72.7% compared to that in plasma. After multiple q6h dosing, clearance decreased by ~80% from the first (7.35 mL/h) to the last two dosing intervals (~1.50 mL/h) in the bloodstream model. Likewise, clearance decreased by 41% from 7.44 to 4.39 mL/h in the lung model. The humanized dosage regimens were 117 mg/kg of body weight/day in mice [administered in four fractions 6 h apart (q6h): 61.9%, 18.6%, 11.3%, and 8.21% of total dose] for the bloodstream and 96.7 mg/kg of body weight/day (given q6h as 65.1%, 16.9%, 10.5%, and 7.41%) for the lung model. These validated humanized dosage regimens and population pharmacokinetic models support translational studies with clinically relevant amikacin exposure profiles.

Optimal assessment of the glomerular filtration rate in older chinese patients using the equations of the Berlin Initiative Study.

AIM: To evaluate the performances of the various estimated glomerular filtration rate (eGFR) equations of the Chronic Kidney Disease Epidemiology Collaboration, the Berlin Initiative Study (BIS), and the Full Age Spectrum (FAS) in older Chinese. METHODS: This study enrolled Chinese adults aged ≥ 65 years who underwent GFR measurements (via 99Tcm-DTPA renal dynamic imaging) in our hospital from 2011 to 2022. Using the measured glomerular filtration rate (mGFR) as the reference, we derived the bias, precision, accuracy, and consistency of each equation. RESULTS: We enrolled 519 participants, comprising 155 with mGFR ≥ 60 mL/min/1.73 m2 and 364 with mGFR < 60 mL/min/1.73 m2. In the total patients, the BIS equation based on creatinine and cystatin C (BIScr-cys) exhibited the lowest bias [median (95% confidence interval): 1.61 (0.77-2.18)], highest precision [interquartile range 11.82 (10.32-13.70)], highest accuracy (P30: 81.12%), and best consistency (95% limit of agreement: 101.5 mL/min/1.73 m2). In the mGFR ≥ 60 mL/min/1.73 m2 subgroup, the BIScr-cys and FAS equation based on creatinine and cystatin C (FAScr-cys) performed better than the other equations; in the mGFR < 60 mL/min/1.73 m2 subgroup, all equations exhibited relatively large deviations from the mGFR. Of all eight equations, the BIScr-cys performed the best. CONCLUSIONS: Although no equation was fully accurate in the mGFR < 60 mL/min/1.73 m2 subgroup, the BIScr-cys (of the eight equations) assessed the eGFRs of the entire population best. A new equation is urgently required for older Chinese and even East Asians, especially those with moderate-to-severe renal insufficiency.

Roughness induced current reversal in fractional hydrodynamic memory.

The existence of a corrugated surface is of great importance and ubiquity in biological systems, exhibiting diverse dynamic behaviors. However, it has remained unclear whether such rough surface leads to the current reversal in fractional hydrodynamic memory. We investigate the transport of a particle within a rough potential under external forces in a subdiffusive media with fractional hydrodynamic memory. The results demonstrate that roughness induces current reversal and a transition from no transport to transport. These phenomena are analyzed through the subdiffusion, Peclet number, useful work, input power, and thermodynamic efficiency. The analysis reveals that transport results from energy conversion, wherein time-dependent periodic force is partially converted into mechanical energy to drive transport against load, and partially dissipated through environmental absorption. In addition, the findings indicate that the size and shape of ratchet tune the occurrence and disappearance of the current reversal, and control the number of times of the current reversal occurring. Furthermore, we find that temperature, friction, and load tune transport, resonant-like activity, and enhanced stability of the system, as evidenced by thermodynamic efficiency. These findings may have implications for understanding dynamics in biological systems and may be relevant for applications involving molecular devices for particle separation at the mesoscopic scale.

Sensitivity of Pharmacokinetics to Differences in the Particle Size Distribution for Formulations of Locally Acting Mometasone Furoate Suspension-Based Nasal Sprays.

To assess bioequivalence of locally acting suspension-based nasal sprays, the U.S. FDA currently recommends a weight-of-evidence approach. In addition to in vitro and human pharmacokinetic (PK) studies, this includes a comparative clinical endpoint study to ensure equivalent bioavailability of the active pharmaceutical ingredient (API) at the site of action. The present study aimed to assess, within an in vitro/in vivo correlation paradigm, whether PK studies and dissolution kinetics are sensitive to differences in drug particle size for a locally acting suspension-based nasal spray product. Two investigational suspension-based nasal formulations of mometasone furoate (MF-I and MF-II; delivered dose: 180 µg) differed in API particle size and were compared in a single-center, double-blind, single-dose, randomized, two-way crossover PK study in 44 healthy subjects with oral charcoal block. Morphology-directed Raman spectroscopy yielded volume median diameters of 3.17 µm for MF-I and 5.50 µm for MF-II, and dissolution studies showed that MF-II had a slower dissolution profile than MF-I. The formulation with larger API particles (MF-II) showed a 45% smaller Cmax and 45% smaller AUC0-inf compared to those of MF-I. Systemic bioavailability of MF-I (2.20%) and MF-II (1.18%) correlated well with the dissolution kinetics, with the faster dissolving formulation yielding the higher bioavailability. This agreement between pharmacokinetics and dissolution kinetics cross-validated both methods and supported their use in assessing potential differences in slowly dissolving suspension-based nasal spray products.

Comparative Population Dynamics of Schizothorax wangchiachii (Cyprinidae: Schizothoracinae) in the Middle Reaches of the Yalong River and the Upper Reaches of the Jinsha River, China.

To explore the differences in the growth characteristics and population dynamics of Schizothorax wangchiachii populations in the Jinsha River (JSR) and the Yalong River (YLR), samples were collected in the upper reaches of the JSR (n = 230) from 2019 to 2020 and the middle reaches of the YLR (n = 187) from 2017 to 2018. In the JSR and YLR populations, the age range was 11 and 12 years old, respectively, and the best growth equation was the Von Bertalanffy equation. The comparative analysis of the two populations showed that the growth coefficient, initial sexual maturity age and age at first capture of the YLR population were greater than those of the JSR population. Comparing the mortality rates of the two groups, we found that the YLR population had the higher female mortality rate (0.658 years-1) and the lower male mortality rate (0.453 years-1). Our assessment of the three natural mortality rates showed that the Fcur of both male and female populations was greater than F25%, indicating that both populations were in an overexploited state. Therefore, we suggest considering the two groups as separate protection units and implementing management measures such as ecological regulation, restoration of tributary habitat and strengthening of fishing ban monitoring to protect their resources.

Neutrophil extracellular trap is an important connection between hemodialysis and acute myocardial infarction.

The incidence of acute myocardial infarction (AMI) in hemodialysis (HD) patients is high and the prognosis is extremely poor. However, the potential connection between HD and AMI, and its regulatory mechanisms remain unclear. In this study, the gene expression profiles of HD (GSE15072) and AMI (GSE66360) were downloaded from the Gene Expression Omnibus database, common differentially expressed genes (DEGs) were obtained using the limma R package, the biological functions were analyzed according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, machine learning was conducted to identify hub genes. Receiver operating characteristic curves and gene set enrichment analyses were used to explore the characters and biological function of hub genes, networks were used for candidate identification of transcription factor (TF), microRNA (miRNA), and drug. After a total of 255 common DEGs were selected, GO and KEGG analyses indicated that neutrophil extracellular trap (NET) may be a potential connection between HD and AMI, LILRB2, S100A12, CYBB, ITGAM, and PPIF were finally identified as hub genes. The area under curve of LILRB2, S100A12, and PPIF was higher than 0.8 in both datasets. Networks show the relationship between hub genes, TF, and miRNA, also the relationship between potential drugs and protein. In conclusion, NETs may be the potential connection between AMI and HD. The potential hub gene, signaling pathways, and drugs provided by this study may contribute to future AMI prevention and intervention in HD patients.

A back propagation neural network approach to estimate the glomerular filtration rate in an older population.

BACKGROUND: The use of creatinine-based glomerular filtration rate (GFR)-estimating equations to evaluate kidney function in elderly individuals does not appear to offer any performance advantages. We therefore aimed to develop an accurate GFR-estimating tool for this age group. METHODS: Adults aged ≥ 65 years who underwent GFR measurement by technetium-99 m-diethylene triamine pentaacetic acid (99mTc-DTPA) renal dynamic imaging were included. Data were randomly split into a training set containing 80% of the participants and a test set containing the remaining 20% of the subjects. The Back propagation neural network (BPNN) approach was used to derive a novel GFR estimation tool; then we compared the performance of the BPNN tool with six creatinine-based equations (Chronic Kidney Disease-Epidemiology Collaboration [CKD-EPI], European Kidney Function Consortium [EKFC], Berlin Initiative Study-1 [BIS1], Lund-Malmö Revised [LMR], Asian modified CKD-EPI, and Modification of Diet in Renal Disease [MDRD]) in the test cohort. Three equation performance criteria were considered: bias (difference between measured GFR and estimated GFR), precision (interquartile range [IQR] of the median difference), and accuracy P30 (percentage of GFR estimates that are within 30% of measured GFR). RESULTS: The study included 1,222 older adults. The mean age of both the training cohort (n = 978) and the test cohort (n = 244) was 72 ± 6 years, with 544 (55.6%) and 129 (52.9%) males, respectively. The median bias of BPNN was 2.06 ml/min/1.73 m2, which was smaller than that of LMR (4.59 ml/min/1.73 m2; p = 0.03), and higher than that of the Asian modified CKD-EPI (-1.43 ml/min/1.73 m2; p = 0.02). The median bias between BPNN and each of CKD-EPI (2.19 ml/min/1.73 m2; p = 0.31), EKFC (-1.41 ml/min/1.73 m2; p = 0.26), BIS1 (0.64 ml/min/1.73 m2; p = 0.99), and MDRD (1.11 ml/min/1.73 m2; p = 0.45) was not significant. However, the BPNN had the highest precision IQR (14.31 ml/min/1.73 m2) and the greatest accuracy P30 among all equations (78.28%). At measured GFR < 45 ml/min/1.73 m2, the BPNN has highest accuracy P30 (70.69%), and highest precision IQR (12.46 ml/min/1.73 m2). The biases of BPNN and BIS1 equations were similar (0.74 [-1.55-2.78] and 0.24 [-2.58-1.61], respectively), smaller than any other equation. CONCLUSIONS: The novel BPNN tool is more accurate than the currently available creatinine-based GFR estimation equations in an older population and could be recommended for routine clinical use.

Central and peripheral lung deposition of fluticasone propionate dry powder inhaler formulations in humans characterized by population pharmacokinetics.

This study aimed to gain an in-depth understanding of the pulmonary fate of three experimental fluticasone propionate (FP) dry powder inhaler formulations which differed in mass median aerodynamic diameters (MMAD; A-4.5 µm, B-3.8 µm and C-3.7 µm; total single dose: 500 µg). Systemic disposition parameter estimates were obtained from published pharmacokinetic data after intravenous dosing to improve robustness. A biphasic pulmonary absorption model, with mucociliary clearance from the slower absorption compartment, and three systemic disposition compartments was most suitable. Rapid absorption, presumably from peripheral lung, had half-lives of 6.9 to 14.6 min. The peripherally deposited dose (12.6 µg) was significantly smaller for formulation A-4.5 µm than for the other formulations (38.7 and 39.3 µg for B-3.8 µm and C-3.7 µm). The slow absorption half-lives ranged from 6.86 to 9.13 h and were presumably associated with more central lung regions, where mucociliary clearance removed approximately half of the centrally deposited dose. Simulation-estimation studies showed that a biphasic absorption model could be reliably identified and that parameter estimates were unbiased and reasonably precise. Bioequivalence assessment of population pharmacokinetics derived central and peripheral lung doses suggested that formulation A-4.5 µm lacked bioequivalence compared to the other formulations both for central and peripheral doses. In contrast, the other fomulations were bioequivalent. Overall, population pharmacokinetics holds promise to provide important insights into the pulmonary fate of inhalation drugs, which are not available from non-compartmental analysis. This supports the assessment of the pulmonary bioequivalence of fluticasone propionate inhaled formulations through pharmacokinetic approaches, and may be helpful for discussions on evaluating alternatives to clinical endpoint studies.

Individual Components of Polymyxin B Modeled via Population Pharmacokinetics to Design Humanized Dosage Regimens for a Bloodstream and Lung Infection Model in Immune-Competent Mice.

Polymyxin B is a "last-line-of-defense" antibiotic approved in the 1960s. However, the population pharmacokinetics (PK) of its four main components has not been reported in infected mice. We aimed to determine the PK of polymyxin B1, B1-Ile, B2, and B3 in a murine bloodstream and lung infection model of Acinetobacter baumannii and develop humanized dosage regimens. A linear 1-compartment model, plus an epithelial lining fluid (ELF) compartment for the lung model, best described the PK. Clearance and volume of distribution were similar among the four components. The bioavailability fractions were 72.6% for polymyxin B1, 12.0% for B1-Ile, 11.5% for B2, and 3.81% for B3 for the lung model and were similar for the bloodstream model. While the volume of distribution was comparable between both models (17.3 mL for the lung and ~27 mL for the bloodstream model), clearance was considerably smaller for the lung (2.85 mL/h) compared to that of the bloodstream model (5.59 mL/h). The total drug exposure (AUC) in ELF was high due to the saturable binding of polymyxin B presumably to bacterial lipopolysaccharides. However, the modeled unbound AUC in ELF was ~16.7% compared to the total drug AUC in plasma. The long elimination half-life (~4 h) of polymyxin B enabled humanized dosage regimens with every 12 h dosing in mice. Daily doses that optimally matched the range of drug concentrations observed in patients were 21 mg/kg for the bloodstream and 13 mg/kg for the lung model. These dosage regimens and population PK models support translational studies for polymyxin B at clinically relevant drug exposures.

An Analysis of Environment-, Individual-, and Tumor-Related Factors Impacting Hypovitaminosis D in Patients with Malignant Tumors.

Vitamin D (VD) plays a regulatory role in tumor occurrence and development, although the factors influencing serum 25-hydroxyvitamin D3 (25(OH)D3) levels in patients with cancer have not been studied. Therefore, we aimed to evaluate circulating levels of 25(OH)D3 and factors influencing the VD status in patients with malignant tumors. Adult patients with malignant tumors who had undergone assessments of serum 25(OH)D3 at Beijing Cancer Hospital from January 2019 to July 2022 were included. A multiple logistic regression model was applied to explore the associations of patient characteristics, environment, and disease characteristics with 25(OH)D3 levels. Among the 1,076 included patients, the median 25(OH)D3 serum concentration was 16.25 ng/mL. VD deficiency and the combined VD insufficiency and sufficiency were observed in 811 (75.37%) and 265 (24.63%) patients, respectively. Latitude, season, sex, body mass index, and type of cancer were associated with VD concentration/status in patients with malignant tumors. 25(OH)D3 concentrations were significantly higher in patients with thyroid cancer and significantly lower in patients receiving tumor-related treatment than in untreated patients. Surprisingly, we observed 25(OH)D3 serum concentration was lower in patients receiving nutritional supplementation than in those receiving no nutritional supplements. Patients with malignant tumors are at high risk of VD deficiency.

Expression Patterns and Gonadotropin Regulation of the TGF-ß II Receptor (Bmpr2) during Ovarian Development in the Ricefield Eel Monopterus albus.

Bmpr2 plays a central role in the regulation of reproductive development in mammals, but its role during ovarian development in fish is still unclear. To ascertain the function of bmpr2 in ovarian development in the ricefield eel, we isolated and characterized the bmpr2 cDNA sequence; the localization of Bmpr2 protein was determined by immunohistochemical staining; and the expression patterns of bmpr2 in ovarian tissue incubated with FSH and hCG in vitro were analyzed. The full-length bmpr2 cDNA was 3311 bp, with 1061 amino acids encoded. Compared to other tissues, bmpr2 was abundantly expressed in the ovary and highly expressed in the early yolk accumulation (EV) stages of the ovary. In addition, a positive signal for Bmpr2 was detected in the cytoplasm of oocytes in primary growth (PG) and EV stages. In vitro, the expression level of gdf9, the ligand of bmpr2, in the 10 ng/mL FSH treatment group was significantly higher after incubation for 4 h than after incubation for different durations. However, bmpr2 expression in the 10 ng/mL FSH treatment group at 2 h, 4 h and 10 h was significantly lower. Importantly, the expression level of bmpr2 and gdf9 in the 100 IU/mL hCG group had similar changes that were significantly decreased at 4 h and 10 h. In summary, Bmpr2 might play a pivotal role in ovarian growth in the ricefield eel, and these results provide a better understanding of the function of bmpr2 in ovarian development and the basic data for further exploration of the regulatory mechanism of gdf9 in oocyte development.

Cyt-C Mediated Mitochondrial Pathway Plays an Important Role in Oocyte Apoptosis in Ricefield Eel (Monopterus albus).

Apoptosis plays a key role in the effective removal of excessive and defective germ cells, which is essential for sequential hermaphroditism and sex change in vertebrates. The ricefield eel, Monopterus albus is a protogynous hermaphroditic fish that undergoes a sequential sex change from female to male. Previous studies have demonstrated that apoptosis is involved in sex change in M. albus. However, the apoptotic signaling pathway is unclear. In the current study, we explored the underlying mechanism of apoptosis during gonadal development and focused on the role of the mitochondrial apoptosis signaling pathway in sex change in M. albus. Flow cytometry was performed to detect apoptosis in gonads at five sexual stages and ovary tissues exposed to hydrogen peroxide (H2O2) in vitro. Then the expression patterns of key genes and proteins in the mitochondrial pathway, death receptor pathway and endoplasmic reticulum (ER) pathway were examined. The results showed that the apoptosis rate was significantly increased in the early intersexual stage and then decreased with the natural sex change from female to male. Quantitative real-time PCR revealed that bax, tnfr1, and calpain were mainly expressed in the five stages. ELISA demonstrated that the relative content of cytochrome-c (cyt-c) in the mitochondrial pathway was significantly higher than that of caspase8 and caspase12, with a peak in the early intersexual stage, while the levels of caspase8 and caspase12 peaked in the late intersexual stage. Interestingly, the Pearson's coefficient between cyt-c and the apoptosis rate was 0.705, which suggests that these factors are closely related during the gonadal development of M. albus. Furthermore, the cyt-c signal was found to be increased in the intersexual stage by immunohistochemistry. After incubation with H2O2, the mRNA expression of mitochondrial pathway molecules such as bax, apaf-1, and caspase3 increased in ovary tissues. In conclusion, the present results suggest that the mitochondrial apoptotic pathway may play a more important role than the other apoptotic pathways in sex change in M. albus.

Identification, characterization and functional analysis of gonadal long noncoding RNAs in a protogynous hermaphroditic teleost fish, the ricefield eel (Monopterus albus).

BACKGROUND: An increasing number of long noncoding RNAs (lncRNAs) have been found to play important roles in sex differentiation and gonad development by regulating gene expression at the epigenetic, transcriptional and posttranscriptional levels. The ricefield eel, Monopterus albus, is a protogynous hermaphroditic fish that undergoes a sequential sex change from female to male. However, the roles of lncRNA in the sex change is unclear. RESULTS: Herein, we performed RNA sequencing to analyse lncRNA expression patterns in five different stages of M. albus development to investigate the roles of lncRNAs in the sex change process. A total of 12,746 lncRNAs (1503 known lncRNAs and 11,243 new lncRNAs) and 2901 differentially expressed lncRNAs (DE-lncRNAs) were identified in the gonads. The target genes of the DE-lncRNAs included foxo1, foxm1, smad3, foxr1, camk4, ar and tgfb3, which were mainly enriched in signalling pathways related to gonadal development, such as the insulin signalling pathway, MAPK signalling pathway, and calcium signalling pathway. We selected 5 highly expressed DE-lncRNAs (LOC109952131, LOC109953466, LOC109954337, LOC109954360 and LOC109958454) for full length amplification and expression pattern verification. They were all expressed at higher levels in ovaries and intersex gonads than in testes, and exhibited specific time-dependent expression in ovarian tissue incubated with follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). The results of quantitative real-time PCR (qRT-PCR) analysis and a dual-luciferase assay showed that znf207, as the gene targeted by LOC109958454, was expressed in multiple tissues and gonadal developmental stages of M. albus, and its expression was also inhibited by the hormones FSH and hCG. CONCLUSIONS: These results provide new insights into the role of lncRNAs in gonad development, especially regarding natural sex changes in fish, which will be useful for enhancing our understanding of sequential hermaphroditism and sex changes in the ricefield eel (M. albus) and other teleosts.

Complement Deposition Predicts Worsening Kidney Function and Underlines the Clinical Significance of the 2010 Renal Pathology Society Classification of Diabetic Nephropathy.

Objectives: Converging evidence points towards a role of the complement system in the pathogenesis of diabetic nephropathy (DN). The classification system of diabetic kidney lesions devised by the Renal Pathology Society (RPS) in 2010 are based on the pathogenic process of DN. Therefore, we investigated the correlation between glomerular C3 deposits and RPS DN classification and the combined deleterious effects thereof on kidney function. Methods: The study analyzed data from 217 diabetic patients who underwent renal biopsy between 2010 and 2021 and were found to have DN as the only glomerular disease. C3 deposition was considered positive if the glomerular C3 immunofluorescence intensity was at the trace or ≥1+ level. We divided DN into five glomerular lesion classes and separately evaluated the degree of interstitial and vascular involvement. The primary outcome was the composite of a ≥50% decline from the initial estimated glomerular filtration rate, end-stage renal disease, and death. Results: None of the patients were classified into class I, and few were classified into classes IIa (7.8%) and IV (9.2%). Most patients were classified as IIb (30.9%) and III (52.1%). C3 deposition was detected in 53.9% of patients. Multivariate logistic regression analysis showed that DN class was significantly correlated with C3 deposits [odds ratio, 1.59; 95% confidence interval (CI), 1.08-2.36; p = 0.02). During a median follow-up of 22 months, 123 (56.7%) patients reached the composite outcome. The endpoints occurred more frequently in patients with C3 deposition (69.2 vs. 42%) compared with those without C3 deposition. Patients with C3 deposition in either class IIb [hazards ratio (HR), 3.9 (95% CI, 1.14-13.17) vs. 2.46 (95% CI, 0.68-8.89)] or III [HR, 4.98 (95% CI, 1.53-16.23) vs. 2.63 (95% CI, 0.77-9.0)] had a higher risk of adverse kidney outcomes than those without C3 deposition. The prognostic accuracy of the combination of DN class and C3 deposits at 1 and 3 years was higher than that for DN class only. Conclusions: Complement deposition together with DN class predicts more rapid deterioration of kidney function in DN, which underlines the clinical significance of the DN phenotype according to the RPS classification.

Activation of Complement Pathways in Kidney Tissue May Mediate Tubulointerstitial Injury in Diabetic Nephropathy.

Introduction: Key genes involved in tubulointerstitial injury may influence the development and progression of diabetic nephropathy (DN). We investigated whether complement-related genes are linked to the mechanism underlying tubulointerstitial injury in DN. Methods: We analyzed the microarray data of 17 tubulointerstitial tissue samples from DN patients and 21 normal controls from the Gene Expression Omnibus. A gene co-expression network was constructed, and genes were divided into modules by weighted gene co-expression network analysis (WGCNA). We also investigated the association of C3 and C1q deposits in kidney tissues with a composite outcome of end-stage renal disease or a 50% reduction in the estimated glomerular filtration rate (eGFR) in DN patients. Finally, we performed immunohistochemical analyses of C3, C1q, C5b-9, mannose-binding lectin (MBL), and factor B in kidney tissues. Results: Nine co-expression modules were constructed using 12,075 genes from the 38 human tubulointerstitial tissue samples. Black module with more genes was positively correlated with tubulointerstitial injury in DN. C3, one of the top 10 genes in tubulointerstitial injury, was verified in an independent dataset; C3 was significantly overexpressed in tubulointerstitial tissue from patients with DN compared to the normal controls. The mRNA level of C3 in renal tubulointerstitium was negatively correlated with eGFR in DN patients (r = -0.75; p = 0.001). Analysis of the follow-up data of 54 DN patients demonstrated that codeposits of C3 and C1q in kidney tissues were independently associated with the renal outcome in DN (hazard ratio, 2.3, 95% confidence interval, 1.01-5.2, p < 0.05). Immunohistochemical analysis showed that patients with higher C1q, C3, C5b-9, MBL, or factor B expression in renal tubulointerstitium were more likely to progress to kidney failure. Conclusion: Local complement activation of the classical, lectin and alternative pathways appears linked to tubulointerstitial injury and disease progression in DN.