Serum 25-hydroxyvitamin D concentrations, vitamin D receptor polymorphisms, and risk of infections among individuals with type 2 diabetes: a prospective cohort study.

BACKGROUND: Evidence on the association between serum 25-hydroxyvitamin D [25(OH)D] and infections among patients with type 2 diabetes (T2D), a group susceptible to vitamin D deficiency and infections, is limited. OBJECTIVES: We aimed to examine this association in individuals with T2D, and to evaluate whether genetic variants in vitamin D receptor (VDR) would modify this association. METHODS: This study included 19,851 participants with T2D from UK Biobank. Infections were identified by linkage to hospital inpatient and death registers. Negative binomial regression models were used to estimate incidence rate ratios (IRRs) and 95% confidence intervals (CIs), with adjustment of potential confounders. RESULTS: In patients with T2D, the incidence rate of infections was 29.3/1000 person-years. Compared to those with 25(OH)D of 50.0-74.9 nmol/L, the multivariable-adjusted IRRs and 95% CIs of total infections, pneumonia, gastrointestinal infections and sepsis were 1.44 (1.31, 1.59), 1.49 (1.27, 1.75), 1.47 (1.22, 1.78), and 1.41 (1.14, 1.73), respectively, in patients with 25(OH)D <25.0 nmol/L. Nonlinear inverse associations between 25(OH)D concentrations and the risks of total infections (P-overall <0.001; P-nonlinear = 0.002) and gastrointestinal infections (P-overall <0.001; P-nonlinear = 0.040) were observed, with a threshold effect at ∼50.0 nmol/L. The vitamin D-infection association was not modified by genetic variants in VDR (all P-interaction >0.050). CONCLUSIONS: In patients with T2D, lower serum 25(OH)D concentration (<50 nmol/L) was associated with higher risks of infections, regardless of genetic variants in VDR. Notably, nonlinear inverse associations between 25(OH)D concentrations and the risks of infections were found, with a threshold effect at ∼50.0 nmol/L. These findings highlighted the importance of maintaining adequate vitamin D in reducing the risk of infections in patients with T2D.

Emerging role of TWEAK-Fn14 axis in lupus, a disease related to autoimmunity and fibrosis.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder. Lupus nephritis (LN) is one of the severe clinical implications in SLE, and this was relates to fibrosis in the kidney. As an important marker in the tumor necrosis factor (TNF) superfamily, TNF-like weak inducer of apoptosis (TWEAK) has been given much attention with respect to its role in regulating pro-inflammatory immune response. Fibroblast growth factor-inducible 14 (Fn14), the sole receptor for TWEAK, has been found expressed in different immune and non-immune cells. TWEAK binds to Fn14, and then regulates inflammatory components production via downstream signaling pathways. To date, dysregulated expression of TWEAK, Fn14 has been reported in SLE, LN patients, and in vivo, in vitro studies have discussed the significant role of TWEAK-Fn14 axis in SLE, LN pathogenesis, partly through mediating the fibrosis process. In this review, we will discuss the association of TWEAK-Fn14 axis in lupus. Understanding the relationship will better realize the potential for making TWEAK-Fn14 as a marker for the diseases, and will help to give many clues for targeting them in treatment of lupus in the future.

A review on the efficacy and mechanism of action of Shenkang injection against chronic kidney disease.

Chronic kidney disease (CKD) is one of the most common conditions which significantly increases the risk for serious health outcomes. Epidemiological investigations have shown that CKD has become a serious global health problem. At present, there are no treatments for CKD, thus the need for an effective and safe treatment for this condition. Shenkang Injection (SKI), which is an herbal medication in Chinese Medicine, has been used in the management and treatment of CKD and has achieved favorable therapeutic effects. The purpose of this paper is to review the clinical efficacy, mechanism of action, and safety profile of SKI when used in CKD, and to provide comprehensive potential evidence for its clinical application.

Renal protective effects of astragaloside IV, in diabetes mellitus kidney damage animal models: A systematic review, meta-analysis.

Astragaloside IV (ASIV) is the essential active component of astragalus that has diverse biological activities. Previous research has suggested its potentially beneficial effects on diabetic nephropathies. However, its effects and protective mechanism remain unclear. In this study, we conducted a preclinical systematic review to evaluate the efficacy and potential mechanisms of ASIV in reducing kidney damage in diabetes mellitus (DM) models. Studies were searched from nine databases until January 2020. A random-effects model was used to calculate combined standardised mean difference estimates and 95 % confidence intervals. Risk of bias of studies was assessed using the Systematic Review Center for Laboratory Animal Experimentation risk of bias tool 10-item checklist. RevMan 5.3 software was used for statistical analysis. Twenty-three studies involving 562 animals were included in the meta-analysis. Studies quality scores ranged from 2 to 5. The ASIV group induced a marked decrease in serum creatinine (P < 0.00001), blood urea nitrogen (P < 0.00001), 24-h urine protein (P < 0.00001) and pathological score (P < 0.001) compared with the control group. The determined potential mechanisms of ASIV action were relieving oxidative stress, delaying renal fibrosis, anti-apoptosis and anti-inflammatory action. We conclude that ASIV exerts renal protective effects in animals with DM through multiple signalling pathways.

Inhibition of autophagy enhances adenosine­induced apoptosis in human hepatoblastoma HepG2 cells.

In cancer research, autophagy acts as a double­edged sword: it increases cell viability or induces cell apoptosis depending upon the cell context and functional status. Recent studies have shown that adenosine (Ado) has cytotoxic effects in many tumors. However, the role of autophagy in Ado­induced apoptosis is still poorly understood. In the present study, Ado­induced apoptotic death and autophagy in hepatoblastoma HepG2 cells was investigated and the relationship between autophagy and apoptosis was identified. In the present study, it was demonstrated that Ado inhibited HepG2 cell growth in a time­ and concentration­dependent manner and activated endoplasmic reticulum (ER) stress, as indicated by G0/G1 cell cycle arrest, the increased mRNA and protein levels of GRP78/BiP, PERK, ATF4, CHOP, cleaved caspase­3, cytochrome c and the loss of mitochon-drial membrane potential (ΔΨm). Ado also induced autophagic flux, revealed by the increased expression of the autophagy marker microtubule­associated protein 1 light chain 3­II (LC3­II), Beclin­1, autophagosomes, and the degradation of p62, as revealed by western blot analysis and macrophage­derived chemokine (MDC) staining. Blocking autophagy using LY294002 notably entrenched Ado­induced growth inhibition and cell apoptosis, as demonstrated with the increased expression of cytochrome c and p62, and the decreased expression of LC3­II. Conversely, the autophagy inducer rapamycin alleviated Ado­induced apoptosis and markedly increased the ΔΨm. Moreover, knockdown of AMPK with si­AMPK partially abolished Ado­induced ULK1 activation and mTOR inhibition, and thus reinforced CHOP expression and Ado­induced apoptosis. These results indicated that Ado­induced ER stress resulted in apoptosis and autophagy concurrently. The AMPK/mTOR/ULK1 signaling pathway played a protective role in the apoptotic procession. Inhibition of autophagy may effectively enhance the anticancer effect of Ado in human hepatoblastoma HepG2 cells.

A novel PNPLA6 compound heterozygous mutation identified in a Chinese patient with Boucher­Neuhäuser syndrome.

The combination of cerebellar degeneration, hypogonadotropic hypogonadism and chorioretinal dystrophy defines Boucher­Neuhäuser syndrome (BNS), which has been associated with autosomal­recessive mutations in the patatin­like phospholipase domain containing 6 (PNPLA6) gene. However, no BNS cases have been reported in mainland China. In the present study, to the best of the authors' knowledge, the first patient with BNS was identified in China. A 39­year­old male was first diagnosed with hypogonadotropic hypogonadism. The proband additionally exhibited retinal degeneration and cerebellar dystrophy. Whole exome sequencing identified a compound heterozygous mutation in PNPLA6 (c.3386G>T+ c.3534G>C). The mutant amino acids were highly conserved and the mutations were predicted to be deleterious. This result further confirmed the role of PNPLA6 in BNS and suggested that whole exome sequencing may be applied for the diagnosis of complex syndromes, including BNS, prior to the observation of obvious symptoms.

Long non-coding RNA MEG3 induces cell apoptosis in esophageal cancer through endoplasmic reticulum stress.

Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes, such as cell growth, apoptosis and migration. Although downregulation of lncRNA MEG3 has been identified in several cancers, little is known about its role in esophageal squamous cell carcinoma (ESCC). The aim of the present study was to detect MEG3 expression in clinical ESCC tissues, investigate its biological functions and the endoplasmic reticulum (ER) stress-relative mechanism. MEG3 expression levels were detected by qRT-PCR in both tumor tissues and adjacent non-tumor tissues from 28 ESCC patients. PcDNA3.1-MEG3 recombinant plasmids were constructed and transfected to EC109 cells. Cell growth was analyzed by CCK-8 assay. Cell apoptosis was analyzed by fluorescence microscope and Annexin V/PI assay. The protein expression was determined by western blot analysis. The results showed that MEG3 decreased significantly in ESCC tissues relative to adjacent normal tissues. PcDNA3.1-MEG3 plasmids were successfully constructed and the expression level of MEG3 significantly increased after MEG3 transfection to EC109 cells. Ectopic expression of MEG3 inhibited EC109 cell proliferation and induced apoptosis in vitro. MEG3 overexpression increased the expression of ER stress­related proteins (GRP78, IRE1, PERK, ATF6, CHOP and cleaved­caspase-3). Our results first demonstrate that MEG3 is downregulated in ESCC tissues. MEG3 was able to inhibit cell growth and induced apoptosis in EC109 cells, most probably via activation of the ER stress pathway.

A dominant negative FGFR1 mutation identified in a Kallmann syndrome patient.

Kallmann syndrome (KS) is characterized by isolated hypogonadotropic hypogonadism (IHH) with anosmia. Fibroblast growth factor receptor 1 (FGFR1) is one of KS-associated genes, accounts for approximately 10% of total patients. FGFR1 mutations have also been identified in more severe craniosynostosis syndromes, and a subset of craniosynostosis syndromes-associated FGFR1 mutations show dominant negative effect. In this study, we identified a novel FGFR1 mutation (c.867G>A; p.W289X) in a KS patient. The p.W289X mutation leads premature termination, producing a truncated FGFR1 without the transmembrane and intracellular domains. Indeed, the W289X FGFR1 was secreted into culture medium. Further, W289X FGFR1 interfered with the function of wild type receptor to induce ERK1/2 phosphorylation. We therefore identified a dominant negative FGFR1 mutation in the KS patient, and this mutant FGFR1 may be used to decipher the physiological function of FGFR1.

The mechanism of adenosine-mediated activation of lncRNA MEG3 and its antitumor effects in human hepatoma cells.

Long non-coding RNA MEG3 is suggested to function as a tumor suppressor. However, the activation mechanism of MEG3 is still not well understood and data are not available on its role under adenosine-induced apoptosis. In this study, HepG2 cells were treated with adenosine or 5-Aza­cdR. Methylation status of MEG3 promoter was detected by methylation specific PCR (MSP) and MEG3 expression was determined by qRT-PCR. PcDNA3.1-MEG3 recombinant plasmid was constructed and transfected to hepatoma HepG2 and Huh7 cells. Cell growth, morphological changes, cell-cycle distribution and apoptosis were analyzed by MTT assay, fluorescence microscopy and flow cytometry. The mRNA and protein expression levels were detected by qRT-PCR and western blot analysis. MEG3 binding proteins were screened by the improved MS2 biotin tagged RNA affinity purification method. The co-expression network of MEG3 was generated by GO analysis and ILF3 was identified as MEG3 binding protein by RNA pulldown and western blot analysis. Both adenosine and 5-Aza-CdR increased MEG3 mRNA expression and the CpG island of MEG3 gene in HepG2 cells was typical hypermethylation. Ectopic expression of MEG3 inhibited hepatoma cell growth in a time-dependent manner, resulted in cell cycle arrest and induced apoptosis. Ectopic expression of MEG3 increased p53, caspase-3 mRNA and protein levels, decreased MDM2 and cyclin D1 mRNA and protein levels, as well as ILF3 protein expression in HepG2 cells. These findings are the first to identify that adenosine increases MEG3 expression by inhibition of DNA methylation and its antitumor effects is involved in MEG3 activation. ILF3 may participate in the anticancer regulation of MEG3 by interacting with MEG3.

Snapin interacts with G-protein coupled receptor PKR2.

Mutations in Prokineticin receptor 2 (PKR2), a G-protein-coupled receptor, have been identified in patients with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, characterized by delayed puberty and infertility. In this study, we performed yeast two-hybrid screening by using PKR2 C-terminus (amino acids 333-384) as a bait, and identified Snapin as a novel interaction partner for PKR2. The interaction of Snapin and PKR2 was confirmed in GST pull-down and co-immunoprecipitation studies. We further demonstrated that two α-helix domains in Snapin are required for the interaction. And the interactive motifs of PKR2 were mapped to YFK (343-345) and HWR (351-353), which shared a similar sequence of two aromatic amino acids followed by a basic amino acid. Disruption of Snapin-PKR2 interaction did not affect PKR2 signaling, but increased the ligand-induced degradation, implying a role of Snapin in the trafficking of PKR2.

Functional analysis of the distal region of the third intracellular loop of PROKR2.

Mutations in the G-protein-coupled receptor PROKR2 have been identified in patients with idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS) manifesting with delayed puberty and infertility. Recently, the homozygous mutation V274D was identified in a man displaying KS with an apparent reversal of hypogonadism. The affected amino acid, valine 274, is located at the junction region of the third intracellular loop (IL3) and the sixth transmembrane domain (TM6). In this study, we first studied the effect of V274D and related mutations (V274A, V274T, and V274R) on the signaling activity and cell surface expression of PROKR2. Our data indicate that a charged amino acid substitution at residue 274 of PROKR2 results in low cell surface expression and loss-of-function. Furthermore, we studied the effects of two clusters of basic amino acids located at the proximal region of Val274 on the cell surface expression and function of PROKR2. The deletion of RRK (270-272) resulted in undetectable cell surface expression, whereas RKR (264-266)-deleted PROKR2 was expressed normally on the cell surface but showed loss-of-function due to a deficiency in G-protein coupling. Our data indicate that the distal region of the IL3 of PROKR2 may differentially influence receptor trafficking and G-protein coupling.

[Construction and expression of the Echinococcus granulosus recombinant BCG-EgG1Y162].

OBJECTIVE: To construct and express Echinococcus granulosus recombinant bacille Calmette-Guerin (BCG) strain rBCG-EgG1Y162. METHODS: The encoding gene of the antigen EgG1Y162 of E. granulosus was recombined with E. coli-Mycobacterium shuttle expression plasmid vector pMV361 by genetic engineering technique, and transformed into E. coli for amplification. The recombinant plasmid rpMV-EgG1Y162 was identified by PCR, double digestion with restriction enzymes, and sequence analysis. The confirmed rpMV-EgG1Y162 was transformed into BCG strain via electroporation technique to construct the recombinant rBCG-EgG1Y162. After identification by PCR and double digestion with restriction enzymes, the recombinant strain was cultured for about 2 weeks. In order to induce the expression of target protein, the rBCG was placed in 45 degrees C for 30 min. SDS-PAGE and Western blotting were used to analyze the expressive protein. RESULTS: The product of recombinant plasmid rpMV-EgG1Y162 was approximately 360 bp by PCR amplification and double digestion with restriction enzymes, consistent with the expected fragment length. Sequencing results showed that the inserted sequence was correct. The rBCG-EgG1Y162 grew well and the identification of PCR and enzyme digestion revealed accuracy. The results of SDS-PAGE and Western blotting showed that the relative molecular weight (M(r)) of the protein was about 71 000. CONCLUSION: The E. granulosus rBCG-EgG1Y162 strain is constructed and expressed.

Components of the mitogen-activated protein kinase cascade are activated in hepatic cells by Echinococcus multilocularis metacestode.

AIM: To explore the effect of Echinococcus multilocularis (E. multilocularis) on the activation of mitogen-activated protein kinase (MAPK) signaling pathways and on liver cell proliferation. METHODS: Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen (PCNA) expression were measured in the liver of patients with alveolar echinococcosis (AE). MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase (ERK) kinase] and ribosomal S6 kinase (RSK) phosphorylation were detected in primary cultures of rat hepatocytes in contact in vitro with (1) E. multilocularis vesicle fluid (EmF), (2) E. multilocularis-conditioned medium (EmCM). RESULTS: In the liver of AE patients, ERK 1/2 and p38 MAPK were activated and PCNA expression was increased, especially in the vicinity of the metacestode. Upon exposure to EmF, p38, c-Jun N-terminal kinase (JNK) and ERK1/2 were also activated in hepatocytes in vitro, as well as MEK1/2 and RSK, in the absence of any toxic effect. Upon exposure to EmCM, only JNK was up-regulated. CONCLUSION: Previous studies have demonstrated an influence of the host on the MAPK cascade in E. multilocularis. Our data suggest that the reverse, i.e. parasite-derived signals efficiently acting on MAPK signaling pathways in host liver cells, is actually operating.

[In vitro cultivation of Echinococcus multilocularis metacestodes and observation of their growth].

OBJECTIVE: To observe the growth and development of Echinococcus multilocularis metacestodes under in vitro cultivation. METHODS: Hepatoma cell line was used for the cultivation. The number and morphology of the cysts were observed under light microscope. The parasite tissue was fixed and observed under electron microscope. RESULTS: During the first 21 days of cultivation, metacestodes in cyst-suspension derived cultures increased dramatically, and from the 22nd day on, the number of cysts remained as 6-7 times more than that of the 3rd-4th day of culture. Budding of new cysts was observed and the diameter of the cysts increased as time went on. On the 22nd day, larger cysts occupied 30%. Cysts were found with morphology between protoscolex and metacestode. CONCLUSION: An in vitro cultivation for the cysts of E. multilocularis has been established and basic feature of growth and development of the larvae observed.