Recombinant Snail Sialic Acid Aldolase is Promiscuous towards Aliphatic Aldehydes.

Aldolases are enzymes that reversibly catalyze the cleavage of carbon-carbon bonds. Here we describe a recombinant sialic acid aldolase originating from the freshwater snail Biomphalaria glabrata (sNPL), and compare its substrate spectrum with a sialic acid aldolase originating from chicken (chNPL). In contrast to vertebrate animals which can synthesize, degrade, and incorporate sialic acids on glycoconjugate ubiquitously, snails (as all mollusks) cannot synthesize sialic acids endogenously, and therefore the biological function and substrate scope of sNPL ought to differ significantly from vertebrate sialic aldolases such as chNPL. sNPL was active towards a series of sialic acid derivatives but was in contrast to chNPL unable to catalyze the cleavage of N-acetylneuraminic acid into N-acetylmannosamine and pyruvate. Interestingly, chNPL and sNPL showed contrasting C4(R)/(S) diastereoselectivity towards the substrates d-mannose and d-galactose in the presence of pyruvate. In addition, sNPL was able to synthesize a series of 4-hydroxy-2-oxoates using the corresponding aliphatic aldehyde substrates in the presence of pyruvate, which could be not achieved by chNPL.

CB1 receptor antagonist rimonabant protects against chronic intermittent hypoxia-induced renal injury in rats.

BACKGROUND: Obstructive sleep apnoea (OSA) induced chronic kidney disease is mainly caused by chronic intermittent hypoxia (CIH). Our study investigate the mechanism underlying CIH-induced renal damage and whether the cannabinoid receptor 1 (CB1R) antagonist rimonabant (Ri) alleviates CIH-induced renal injury. METHODS: Male Sprague-Dawley rats were randomly divided into five groups: one normal control (NC) group, two chronic intermittent hypoxia (CIH) groups, and two CIH + Ri groups. Rats in the NC groups were exposed to room air, while the CIH groups were exposed to a CIH environment for 4 weeks (4w CIH group) and 6 weeks (6w CIH group), respectively. Additionally, rats in the CIH + Ri groups were administered 1.5 mg/kg/day Ri for 4 weeks (4w CIH + Ri group) and 6 weeks (6w CIH + Ri group), respectively. Following this, the rats were euthanized and kidneys were excised for downstream analysis. In the renal tissues, the morphological alterations were examined via haematoxylin eosin (HE) staining and periodic acid schiff (PAS) staining, CB1R, Fis1, Mfn1, and p66Shc expression was assessed through western blot and immunohistochemistry, and the mitochondrial ultrastructural changes in kidney sections were assessed by electron microscopy. RESULTS: CB1R expression in the 4w and 6w CIH groups was significantly elevated, and further increased with prolonged hypoxia; however, Ri prevented the increase in CIH-induced CB1R expression. Fis1 and p66Shc expression in the CIH groups were increased, but Mfn1 expression decreased. Ri decreased Fis1 and p66Shc expression and increased Mfn1 expression. Renal damage in the 4w or 6w CIH + Ri group was evidently improved compared with that in the 4w or 6w CIH group. CB1R expression was positively correlated with Fis1 and p66Shc and negatively correlated with Mfn1. Meanwhile, electron microscopy showed that the percentage of fragmented mitochondria in the tubular cells in each group was consistent with the trend of CB1R expression. CONCLUSION: CIH causes endocannabinoid disorders and induces abnormal mitochondrial dynamics, resulting in renal injury. Treatment with CB1R antagonists reduces CIH-induced renal damage by inhibiting dysregulated renal mitochondrial dynamics.

Posttranscriptional regulation of MMP-9 by HuR contributes to IL-1ß-induced pterygium fibroblast migration and invasion.

Inflammation is considered to be critical in the pterygium progression and recurrence. However, the underlying molecular mechanism is not well understood. Herein, we investigated the potential role of RNA binding protein human antigen R (HuR) responsible for the impact of inflammation on pterygium development. The expression of HuR and matrix metallopeptidase-9 (MMP-9) in pterygium and normal conjunctiva was detected with immunohistochemistry and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The influence of interleukin-1ß (IL-1ß) on HuR expression and cellular distribution was determined with western blot and immunofluorescence. The pterygium fibroblast (PTF) migration was determined with scratch wound healing assay and Transwell migration assay. MMP-9 production was determined with qRT-PCR and gelatin zymography. The interaction between HuR and MMP-9 was investigated with RNP immunoprecipitation (IP) followed by RT-PCR and messenger RNA (mRNA) stability analysis. HuR and MMP-9 expression are elevated in pterygium, especially progressive pterygium compared with normal conjunctiva. IL-1ß could increase the expression and nucleus-cytoplasm shuttle of HuR in cultured PTFs. HuR mediated the stimulatory effect of IL-1ß on PTF migration and MMP-9 production. HuR bound to MMP-9 mRNA and in turn increased it stability. Our results suggest that posttranscriptional regulation of MMP-9 via stabilizing mRNA by HuR might contribute to the stimulatory effect of inflammatory factor IL-1ß on pterygium progression. These findings shed light on the pathogenesis of pterygium and provide a promising target for adjuvant treatment of pterygium.

Quantitative proteomic analysis of human corneal epithelial cells infected with HSV-1.

HSV-1 infection in corneal epithelium initiates the process of herpes simplex keratitis. We investigated the dynamic change of the host proteins in corneal epithelial cells infected with HSV-1 to understand the virus-host interaction. iTRAQ coupled with LC-MS/MS was applied to quantitatively analyze the protein profiles in HSV-1 infected corneal epithelial cells at 6 and 24 h post-infection (hpi), and the results were validated by multiple reaction monitoring (MRM). We also performed bioinformatic analysis to investigate the potentially important signal pathways and protein interaction networks in the host response to HSV-1 infection. We identified 292 proteins were up-regulated and 168 proteins were down-regulated at 6 hpi, while 132 proteins were up-regulated and 89 proteins were down-regulated at 24 hpi, which were validated by MRM analysis. We found the most enriched GO terms were translational initiation, cytosol, poly(A) RNA binding, mRNA splicing via spliceosome and extracellular exosome for the dysregulated proteins. KEGG pathway analysis revealed significant changes in metabolism pathway characterized by decreased tricarboxylic acid cycle activity and increased glycolysis. Proteins interaction network analysis indicated several proteins including P4HB, ACLY, HSP90AA1 and EIF4A3, might be critical proteins in the host-virus response. Our study for the first time analyzed the protein profile of HSV-1 infected primary corneal epithelial cells by quantitative proteomics. These findings help to better understand the host-virus interaction and the pathogenesis of herpes simplex keratitis.

Recombinant human N-acetylneuraminate lyase as a tool to study clinically relevant mutant variants.

N-acetylneuraminic acid (sialic acid) is an abundantly found carbohydrate moiety covering the surface of all vertebrate cells and secreted glycoproteins. The human N-acetylneuraminate pyruvate lyase (NPL) interconverts sialic acid to N-acetylmannosamine and pyruvate, and mutations of the NPL gene were found to cause sialuria and impair the functionality of muscles. Here we report the soluble and functional expression of human NPL in Escherichia coli, which allowed us to study the biochemical properties of two clinically relevant NLP mutations (Asn45Asp and Arg63Cys). The Asn45Asp mutant variant was enzymatically active, but had lower expression levels and showed reduced stability when compared to the wild-type NPL variant. Expression trials of the Arg63Cys mutant did not yield any recombinant protein and consequently, no enzymatic activity was detected. The locations of these clinically relevant amino acid substitutions are also discussed by using a human NPL homology model.

Portal vein thrombosis in a noncirrhotic patient after hemihepatectomy: A case report and review of literature.

BACKGROUND: Portal vein thrombosis (PVT) is a condition caused by hemodynamic disorders. It may be noted in the portal vein system when there is an inflammatory stimulus in the abdominal cavity. However, PVT is rarely reported after hepatectomy. At present, related guidelines and major expert opinions tend to consider vitamin K antagonists or low-molecular weight heparin (LMWH) as the standard treatment. But based on research, direct oral anticoagulants may be more effective and safe for noncirrhotic PVT and are also beneficial by reducing the recurrence rate of PVT. CASE SUMMARY: A 51-year-old woman without any history of disease felt discomfort in her right upper abdomen for 20 d, with worsening for 7 d. Contrast-enhanced computed tomography (CECT) of the upper abdomen showed right liver intrahepatic cholangiocarcinoma with multiple intrahepatic metastases but not to the left liver. Therefore, she underwent right hepatic and caudate lobectomy. One week after surgery, the patient underwent a CECT scan, due to nausea, vomiting, and abdominal distension. Thrombosis in the left branch and main trunk of the portal vein and near the confluence of the splenic vein was found. After using LMWH for 22 d, CECT showed no filling defect in the portal vein system. CONCLUSION: Although PVT after hepatectomy is rare, it needs to be prevented during the perioperative period.

Cyanidin-3-O-glucoside Protects Lens Epithelial Cells against High Glucose-Induced Apoptosis and Prevents Cataract Formation via Suppressing NF-κB Activation and Cox-2 Expression.

Diabetic cataract is one of the most important causes of blindness worldwide. Cyanidin-3-O-glucoside (C3G) is found to exert beneficial effects on many diabetic complications. However, its effect on diabetic cataract is not well known. Herein, we investigated the effect of C3G on high glucose-induced lens epithelial cell (SRA01/04) apoptosis and cataract formation as well as the involved mechanisms. We found C3G (20 µM) could preserve cell viability in SRA01/04 cells exposed to high glucose (100 µM). Meanwhile, C3G inhibited SRA01/04 cell apoptosis and regulated the Bcl-2/Bax ratio. Additionally, C3G suppressed NF-κB activation and subsequent cyclooxygenases-2 (Cox-2) expression, which are associated with the protection against apoptosis. Moreover, C3G attenuated lens opacity and protein aggregation in lens culture exposed to high glucose. In conclusion, C3G protected against high glucose-induced SRA01/04 cell apoptosis and cataract formation, which indicated the potential protection of anthocyanins on diabetic cataract.

Transcriptional Profile and Integrative Analyses of Long Noncoding RNAs in Primary Human Corneal Epithelial Cells in Response to HSV-1 Infection.

PURPOSE: Long noncoding RNAs (lncRNAs) have been demonstrated to have important regulatory functions in diverse cellular processes; however, the role of lncRNAs in the pathogenesis of herpes simplex keratitis (HSK) remains poorly understood. METHODS: Primary human corneal epithelial cells (HCECs) were infected with herpes simplex virus-1 (HSV-1) and the total RNAs extracted from both the infected group and the mock-infected group subjected to microarray analysis to identify the differential expression of lncRNAs and mRNAs. We also performed bioinformatic analysis including gene ontology (GO) analysis, pathway analysis and co-expression network analysis. RESULTS: Compared with mock-infected group, the expression of thousands of lncRNAs and mRNAs were significantly changed, and the microarray results were validated by qRT-PCR. The most enriched GOs targeted by up-regulated transcripts were defense response, intrinsic component of plasma membrane and cytokine activity，and the most enriched GOs targeted by the down-regulated transcripts were cellular metabolic process, intracellular part and poly (A) RNA binding. Pathway analysis indicated that the most correlated pathways for up- and down-regulated transcripts were cytokine-cytokine receptor interaction and RNA transport, respectively. CONCLUSIONS: Our study identified the genome-wide profile of lncRNAs and mRNAs expression in primary corneal epithelial cells with HSV-1 infection. These transcriptomic data together with subsequent bioinformatic analysis will provide us with novel clue to the insight into molecular mechanism and potential therapeutic targets of HSK. Further studies are expected to verify the potentially functional genes and pathways and explore the critical lncRNAs. ABBREVIATIONS: Long noncoding RNAs: lncRNAs; herpes simplex virus-1: HSV-1; herpes simplex virus keratitis: HSK; human corneal epithelial cells: HCECs.

Airborne particulate matter impairs corneal epithelial cells migration via disturbing FAK/RhoA signaling pathway and cytoskeleton organization.

BACKGROUND: Cornea is the outmost structure of the eye and exposed directly to the air pollution. However, little is known about the effect of PM2.5 on corneal epithelium, which is critical for maintenance of cornea homeostasis and visual function. OBJECTIVE: We investigated the influence of PM2.5 exposure on corneal epithelial migration and the possible mechanisms involved in the process. METHODS: We observed wound healing in mouse model of cornea abrasion, evaluated the migration and mobility of cultured corneal epithelial cells with wound scratch assay and Transwell migration assay, detected the phosphorylation and interaction of FAK/paxillin with immunofluorescence and immunoprecipitation, and determined the RhoA activity and actin reorganization, in response to PM2.5 exposure. RESULTS: Exposure to PM2.5 remarkably inhibited corneal epithelial cell migration both in mouse model of corneal abrasion and in cell culture model. We found the phosphorylation and interaction of FAK/paxillin, RhoA activity as well as actin reorganization were suppressed by PM2.5 exposure. Moreover, formation of ROS might play a role in the action of PM2.5. CONCLUSIONS: PM2.5 exposure could result in delay of corneal epithelium wound healing by inhibiting cell migration, thus more attention should be paid to the potential risk of corneal infection and effort should be made to protect eyes against impairment induced by PM2.5.

Aberrant expression of genes and proteins in pterygium and their implications in the pathogenesis.

Pterygium is a common ocular surface disease induced by a variety of factors. The exact pathogenesis of pterygium remains unclear. Numbers of genes and proteins are discovered in pterygium and they function differently in the occurrence and development of this disease. We searched the Web of Science and PubMed throughout history for literatures about the subject. The keywords we used contain pterygium, gene, protein, angiogenesis, fibrosis, proliferation, inflammation, pathogenesis and therapy. In this review, we summarize the aberrant expression of a range of genes and proteins in pterygium compared with normal conjunctiva or cornea, including growth factors, matrix metalloproteinases and tissue inhibitors of metalloproteinases, interleukins, tumor suppressor genes, proliferation related proteins, apoptosis related proteins, cell adhesion molecules, extracellular matrix proteins, heat shock proteins and tight junction proteins. We illustrate their possible mechanisms in the pathogenesis of pterygium as well as the related intervention based on them for pterygium therapy.