Comparison of Hydrostatic Filtration Dialysis with Ultracentrifugation Methods for the Identification and Proteomic Profiling of Urinary Extracellular Vesicles.

BACKGROUND: Urinary extracellular vesicles (UEVs) carry rich markers of their parent cells, so they can serve as possible biomarkers of kidney diseases. METHODS: In this study, we isolated urinary extracellular vesicles from five individuals using a simple, clinically applicable method called hydrostatic filtration dialysis (HFD) and compared it to the gold-standard ultracentrifuga-tion (UC) with transmission electron microscopy (TEM). We also employed a proteomic approach using pooled human urine samples from the same five individuals to profile the protein composition of UEVs to evaluate the effectiveness of these two methods. RESULTS: Notably, using TEM, we found that all isolations contained 0 - 400 nm vesicles with the traditionally reported morphology, although the TEM results showed that the UEVs isolated from HFD compared to those from UC are larger and more extensive. We obtained a total of 2,564 UEV proteins in the two methods. We showed a large overlap (2,185 > 85%) between the proteins identified by both isolation methods. The result also showed that the obtained proteins in extracellular vesicles, which are isolated with these methods, are consistent with the results in currently available databases. However, in the associated gene ontologies, the enriched proteins found by the two methods showed some differences. CONCLUSIONS: The HFD method is clinically feasible and allows large-scale protein profiling of UEV biomarkers. The results of this study also provide valuable UEV protein data from the methodological comparison, which might be valuable to other researchers.

Analysis of codon usage patterns in Morus notabilis based on genome and transcriptome data.

Codons play important roles in regulating gene expression levels and mRNA half-lives. However, codon usage and related studies in multicellular organisms still lag far behind those in unicellular organisms. In this study, we describe for the first time genome-wide patterns of codon bias in Morus notabilis (mulberry tree), and analyze genome-wide codon usage in 12 other species within the order Rosales. The codon usage of M. notabilis was affected by nucleotide composition, mutation pressure, nature selection, and gene expression level. Translational selection optimal codons were identified and highly expressed genes of M. notabilis tended to use the optimal codons. Genes with higher expression levels have shorter coding region and lower amino acid complexity. Housekeeping genes showed stronger translational selection, which, notably, was not caused by the large differences between the expression level of housekeeping genes and other genes.

Proteomics-based analysis of potential therapeutic targets in patients with peritoneal dialysis-associated peritonitis.

BACKGROUND: Peritoneal dialysis-associated peritonitis (PDAP) is the most common complication in peritoneal dialysis patients. We propose screening for characteristic expressed proteins in the dialysate of PDAP patients to provide clues for the diagnosis of PDAP and its therapeutic targets. METHODS: Dialysate samples were collected from patients with a first diagnosis of PDAP (n = 15) and from patients who had not experienced peritonitis (Control, n = 15). Data-independent acquisition (DIA) proteomic analysis was used to screen for differentially expressed proteins (DEPs). Co-expression networks were constructed via weighted gene co-expression network analysis (WGCNA) for detection of gene modules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used for functional annotation of DEPs and gene modules. Hub proteins were validated using the parallel reaction monitoring (PRM) method. RESULTS: A total of 142 DEPs in the dialysate of PDAP patients were identified. 70 proteins were upregulated and 72 proteins were downregulated. GO and KEGG analysis showed that DEPs were mainly enriched in cell metabolism, glycolysis/glycogenesis and hypoxia-inducible factor-1 signaling pathway. Subsequently, a co-expression network was constructed and four gene modules were detected. Myeloperoxidase (MPO) and myeloperoxidase (HP) were the key proteins of the blue and turquoise modules, respectively. Additionally, PRM analysis showed that the expression of MPO and HP was significantly upregulated in the PDAP group compared to the non-peritonitis group, which was consistent with our proteomics data. CONCLUSION: MPO and HP were differentially expressed in the dialysate of PDAP patients and may be potential diagnostic and therapeutic targets for PDAP.

Comparison of Efficacy and Safety of Acupuncture and Moxibustion in Acute Phase and Non-acute Phase of Bell's Palsy: a meta-analysis.

OBJECTIVE: To evaluate the efficacy and safety of acupuncture and moxibustion for bell's palsy in the acute phase compared with the non-acute phase. METHODS: Computer retrieval of PubMed, Embase, The Cochrane Library, Web of Science, China National Knowledge Internet (CNKI), Wanfang data, were conducted. According to the inclusion and exclusion criteria, the quality of literature was evaluated, and useful data was extracted. All statistical analyses were performed by RevMan5.3 software. RESULTS: 17 eligible RCTs with a total of 2644 patients were included in this meta-analysis. The meta-analysis results demonstrated the cure rate of acupuncture and moxibustion for Bell's palsy in the acute phase were lower than that in the non-acute phase (P<0.05). The time to cure of acupuncture and moxibustion for Bell's palsy in the acute phase was shorter than that in the non-acute phase (P<0.05), and the incidence of sequelae during the treatment period of acupuncture and moxibustion for Bell's palsy in the acute phase were lower than that in non-acute phase (P<0.05). CONCLUSIONS: Acupuncture and moxibustion were safe and effective stimulation for Bell's palsy in the acute phase compared with the non-acute phase, improving the cure rate of Bell's palsy, shorten the time to cure, and reduce the occurrence of sequelae. However, more multicenter RCTs with a large sample number and high quality should verify the conclusion mentioned above.

Association of Lipid Parameters with the Risk of Chronic Kidney Disease: A Longitudinal Study Based on Populations in Southern China.

OBJECTIVE: To investigate which plasma lipid parameters are useful for detecting chronic kidney disease (CKD) in a Chinese population without known CKD or renal impairment. METHODS: This was a prospective study. In southern Chinese cities from 2012 to 2013, a total of 1037 subjects aged ≥ 18 years old received a survey. Logistic regression and multiple linear regression analyses were performed. The lipid parameters studied included total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), non-high-density lipoprotein cholesterol (nHDL-C), TG/HDL-C ratio, TC/HDL-C ratio and nHDL-C/HDL-C ratio. RESULTS: After adjusting for confounding factors, the fourth percentile of logTG/HDL-C was observed to be an independent risk factor for CKD (OR = 2.453, P < 0.001), and the highest quantile of the logTG/HDL-C ratio was associated with a higher prevalence of CKD (P < 0.05). This risk was reduced when the model was adjusted with Insulin resistance (IR) (OR = 2.034, P < 0.05). In the group of women, glucose metabolism disorders, high uric acid, and obesity, this risk was increased. Multiple regression models showed that log TG and nonHDL-C/HDL-C were negatively correlated with eGFR (P < 0.05), while log TG and TC were positively correlated with logACR (P < 0.05). The area under the curve (ROC) of lgTG/HDL was 0.623 (p < 0.001). CONCLUSION: The serum logTG/HDL-C ratio is the only suitable predictor of CKD, and IR may be the mechanism. This risk needs to be controlled in a specific population. Log TG and nonHDL-C/HDL-C were negatively correlated with eGFR, while log TG and TC were positively correlated with logACR.

Visceral Adiposity Index and Chronic Kidney Disease in a Non-Diabetic Population: A Cross-Sectional Study.

PURPOSE: To investigate the correlation between visceral obesity and pathogenesis of chronic kidney disease (CKD) among non-diabetic individuals, and to evaluate the potential of visceral adiposity index (VAI) as a predictor of CKD. PATIENTS AND METHODS: From December 2017 to March 2018, 1877 non-diabetic participants (male n=699, female n=1208) in southern China were recruited for a cross-sectional survey. Males and females were divided into four groups according to gender-specific quartiles of VAI scores. A logistic regression model was established to analyze the correlation between visceral adiposity index and CKD. RESULTS: Visceral adiposity index was positively correlated with CKD and was negatively associated with estimated glomerular filtration rate (eGFR). Using group one as the control, odds ratios (ORs) were calculated to determine the risk of developing CKD as VAI increased (male: group four 2.73 [P<0.005]; female: Group three 1.76 [P<0.05], Group four 2.88 [P<0.005]). When related factors such as history of hypertension, smoking, alcohol use, and physical inactivity were normalized in the logistic model before calculation, ORs became 2.73 (male: P<0.05), and 2.18 (female: P<0.05), respectively. The results differed after normalizing further for systolic blood pressure (SBP), diastolic blood pressure (DBP), hypersensitive c-reactive protein (hsCRP), interleukin-6 (IL-6), homocysteine (Hcy), superoxide dismutase (SOD), and retinol-binding protein (RBP). There were no significant differences in ORs among the female groups. CONCLUSION: Visceral adiposity index was significantly associated with CKD in non-diabetic individuals. It may be a good predictor of the pathogenesis of CKD and was dependent on hsCRP, IL-6, Hcy, SOD, RBP, and blood pressure levels in females and males with VAI scores of 1.41 and higher. Visceral adiposity index may be used to predict CKD in males with VAI less than 0.983.

Lymphocyte To High-Density Lipoprotein Ratio As A New Indicator Of Inflammation And Metabolic Syndrome.

PURPOSE: Metabolic syndrome (MetS), which is a global public health problem, is a state of chronic low-grade inflammation. This study looked at the changes in hematological parameters and the predictive value of the lymphocyte to high-density lipoprotein cholesterol (HDL-C) ratio (LHR) as a new index in subjects with and without MetS in coastal cities in southern China. PATIENTS AND METHODS: In this cross-sectional study, there were 852 participants (n = 598 with MetS and n = 254 without MetS). MetS was defined in accordance with the National Cholesterol Education Program, Adult Treatment Panel III (NCEP-ATP III) criteria. RESULTS: MetS was positively correlated with white blood cell count, total lymphocyte count, neutrophil count, red blood cell count, hematocrit, hemoglobin, and high-sensitivity C-reactive protein levels (p<0.05). In addition, there was a positive correlation between LHR and the number of metabolic risk factors for MetS. In a logistic regression analysis, LHR (odds ratio: 4.117; 95% CI: 2.766-6.309; p<0.001) was an independent predictor of MetS. When a receiver operating characteristic (ROC) curve analysis was used to assess the value of LHR for predicting MetS, the area under the curve yielded a cut-off value of 1.657, with a sensitivity of 65% and a specificity of 64% (p<0.0001). CONCLUSION: In summary, MetS can involve changes in blood parameters, and LHR may be a useful marker of inflammation to assess the presence and severity of MetS.

[Value of podocalyxin levels in urinary extracellular vesicles for diagnosis of diabetic nephropathy].

OBJECTIVE: To explore the value of detecting podocalyxin (PCX) level in urinary extracellular vesicles for the diagnosis of diabetic nephropathy. METHODS: This study was conducted among 57 diabetic patients admitted during the period from March to September, 2017, including 34 with uncomplicated diabetics and 23 with diabetic nephropathy; 21 patients with other types of nephropathy and 11 healthy individuals were also included to serve as the controls. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to verify the separation of urinary extracellular vesicles. The molecular markers of extracellular vesicles (TSG101 and podocalyxin [PCX]) were detected using Western blotting. PCX levels in extracellular vesicles were also detected using ELISA. RESULTS: TEM reveal the presence of numerous extracellular vesicles in the urine with intact morphology and different sizes, and most of them were below 300 nm in diameter as shown by NTA. TSG101 expression was detected in the samples from all the 4 groups. Positive expression of PCX was detected in the samples from patients with diabetic nephropathy but not in the other groups. In patients with diabetic nephropathy, the mean PCX levels (3.27±2.30 ng/µmol)was significantly higher than those in the healthy control group (1.22±0.36 ng/µmol), uncomplicated diabetes group (2.22±1.29 ng/µmol) and nephropathy group (1.24±0.45 ng/µmol). CONCLUSIONS: PCX level in urinary extracellular vesicles is significantly increased in patients with diabetic nephropathy, suggesting the value of PCX as a potential marker for clinical diagnosis of diabetic nephropathy.

Endocannabinoid 2-Arachidonoylglycerol Suppresses LPS-Induced Inhibition of A-Type Potassium Channel Currents in Caudate Nucleus Neurons Through CB1 Receptor.

Inflammation plays a pivotal role in the pathogenesis of many diseases in the central nervous system. Caudate nucleus (CN), the largest nucleus in the brain, is also implicated in many neurological disorders. 2-Arachidonoylglycerol (2-AG), the most abundant endogenous cannabinoid, has been shown to exhibit neuroprotective effects through its anti-inflammatory action from some proinflammatory stimuli. However, the neuroprotective mechanism of 2-AG is complex and has not been fully understood. A-type K(+) channels critically regulate neuronal excitability and have been demonstrated to be associated with some nervous system diseases. The aim of this study was to explore whether A-type K(+) channels were involved in neurotoxicity of lipopolysaccharides (LPS) and the neuroprotective mechanism of 2-AG in CN neurons. Whole cell patch clamp recording was used to investigate the influence of LPS on the function of A-type K(+) channels and its modulation by 2-AG in primary cultured rat CN neurons. Our findings showed that in cultured CN neurons, LPS significantly decreased the A-type potassium currents (I A) in a voltage-insensitive way. The further data demonstrated that an elevation of 2-AG levels by directly applying exogenous 2-AG or inhibiting monoacylglycerol lipase (MAGL) to prevent 2-AG hydrolysis was capable of suppressing the LPS-induced inhibition of IA and the action of 2-AG is mediated through CB1 receptor-dependant way. The study provides a better understanding of inflammation-related neurological disorders and suggests the therapeutic potential for 2-AG for the treatment of these diseases.

Effect of Homocysteine on Voltage-Gated Sodium Channel Currents in Primary Cultured Rat Caudate Nucleus Neurons and Its Modulation by 2-Arachidonylglycerol.

Homocysteine (Hcy) is an important risk factor for Alzheimer's disease (AD) and other neurodegenerative diseases. Caudate nucleus (CN), the largest nucleus in the brain, is also implicated in many neurological disorders. 2-Arachidonoylglycerol (2-AG), the most abundant endogenous cannabinoid, has been shown to exhibit neuroprotective effects from many stimuli in the central nervous system (CNS). Furthermore, it has been reported that voltage-gated sodium channels (VGSCs) are the common targets of many neuronal damages and drugs. However, it is still not clear whether VGSCs are involved in the neurotoxicity of Hcy and the neuroprotective effect of 2-AG in CN neurons. In the present study, whole-cell patch clamp recording was used to invest the action of Hcy on sodium currents in primary cultured rat CN neurons and its modulation by 2-AG. The results showed that in cultured CN neurons, pathological concentration of Hcy (100 µM) significantly increased the voltage-gated sodium currents (I(Na)) and produced a hyperpolarizing shift in the activation-voltage curve of I(Na). The further data demonstrated 2-AG is capable of suppressing elevation of Hcy-induced increase in I(Na) and hyperpolarizing shift of activation curves most partly through CB1 receptor-dependent way. Our study provides a better understanding of Hcy-associated neurological disorders and suggests the therapeutic potential for 2-AG for the treatment of these diseases.

Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling.

AIMS: URB602 is a selective inhibitor of monoacylglycerol lipase (MAGL), a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl glycerol (2-AG). It has been described that URB602 significantly enhances depolarization-induced increases in 2-AG. A high level of homocysteine (Hcy) is a modifiable risk factor for developing Alzheimer's disease (AD). The aim of this study was to investigate the protective effects of URB602 on Hcy-induced impairments underlying its cellular and molecular mechanism in primary cultured caudate nucleus (CN) neurons. MAIN METHODS: The expressions of cyclooxygenase-2 (COX-2), ERK1/2, NF-κB and IκB-α as well as cleaved caspase-3 and p-Bcl-2 in Hcy-, URB602 or SR1 (a selective inhibitor of CB1 receptor)-treated primary cultured neurons in CN were measured by immunoblotting technique and neurotoxicity assays were performed by using Hoechst staining. KEY FINDINGS: The MAGL inhibitor URB602 exerted a neuroprotective effect on Hcy-induced impairment through suppression of cyclooxygenase-2 (COX-2) elevation and ERK1/2 and NF-κB phosphorylation as well as suppressions of IκB-α degradation in a CB1 receptor-dependent way. Moreover, anti-neuronal impairments of URB602 were mediated by modulating down-regulation of cleaved caspase-3 expression and up-regulation of p-Bcl-2 expression in a CB1 receptor-dependent manner in primary cultured CN neurons. SIGNIFICANCE: These data suggest that the MAGL inhibitor is a promising therapeutic target for some neurodegenerative disorders, such as AD, via the COX-2 signaling pathway.

BmECM25, from the silkworm Bombyx mori, is an extracellular matrix protein.

BmECM25 (previously reported as BmVMP25) was previously predicted as a gene encoding the vitelline membrane protein in silkworm, Bombyx mori. In this study, we investigated the detail temporal and spatial patterns of BmECM25 protein. Western blot results showed that BmECM25 was expressed in the follicular epithelium cells from stages -6 to +1, and was then secreted into the oocytes. However, the abundance of BmECM25 decreased during the subsequent oogenesis and finally disappeared in the mature follicles. Immunofluorescence detection showed that BmECM25 locates inside the VM layer and forms a discontinuous layer. These features of BmECM25 suggest that it is an oocyte membrane matrix protein, not a vitelline membrane protein.

Retrotransposon "Qian" mediated segmental duplication in silkworm, Bombyx mori.

Transposable elements constitute a large fraction of the eukaryotic genomes. They have the potential to alter genome structure and play a major role in genome evolution. Here, we report a segmental duplication mediated by a novel long terminal repeat (LTR) retrotransposon as the cause of an egg-shell recessive lethal mutant (l-em mutant) in silkworm (Bombyx mori). The segmental duplication resulted in the duplication of six genes and the disruption of two genes. Disruption of BmEP80 (B. mori egg protein 80), a gene encoding a major egg-shell structure protein, is likely responsible for the lethal water-loss phenotype in the l-em/l-em mutant. Our data revealed that BmEP80 is present in the inner egg-shell layer and plays important roles in resistance to water efflux form eggs. A novel LTR retrotransposon (named as "Qian") was identified and the model for the Qian-mediated chromosomal segmental duplication was proposed. Detail biochemical and genomic analyses on the l-em mutant offer an opportunity to demonstrate that an LTR retrotransposon could trigger duplication of a chromosomal segment (∼96.3 kb) and confer novel phenotype.