7, 8-dihydroxyflavone Ameliorates Cholinergic Dysfunction, Inflammation, Oxidative Stress, and Apoptosis in a Rat Model of Vascular Dementia.

Vascular dementia (VD) is a degenerative cerebrovascular disorder associated with progressive cognitive decline. Previous reports have shown that 7,8-dihydroxyflavone (7,8-DHF), a well-known TrkB agonist, effectively ameliorates cognitive deficits in several disease models. Therefore, this study investigated the protective effects of 7,8-DHF against 2-VO-induced VD. VD was established in rats using the permanent bilateral carotid arteries occlusion (two-vessel occlusion, 2-VO) model. 7,8-DHF (5, 10, and 20 mg/kg) and Donepezil (10 mg/kg) were administered for 4 weeks. Memory function was assessed by the novel objective recognition task (NOR) and Morris water maze (MWM) tests. Inflammatory (TNF-α, IL-1ß, and NF-kß), oxidative stress, and apoptotic (BAX, BCL-2, caspase-3) markers, along with the activity of choline acetylcholinesterase (AChE) was assessed. p-AKT, p-CREB, BDNF, and neurotransmitter (NT) (GLU, GABA, and ACh) levels were also analyzed in the hippocampus of 2-VO rats. Our results show that 7,8-DHF effectively improved memory performance and cholinergic dysfunction in 2-VO model rats. Furthermore, 7,8-DHF treatment also increased p-AKT, p-CREB, and BDNF levels, suppressed oxidative, inflammatory, and apoptotic markers, and restored altered NT levels in the hippocampus. These findings imply that 7, 8-DHF may act via multiple mechanisms and as such serve as a promising neuroprotective agent in the context of VD.

Utility of the WHO dengue guidelines in pediatric immunological studies.

Dengue is a significant health problem due to the high burden of critical infections during outbreaks. In 1997, the World Health Organization (WHO) classified dengue as dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). It was revised in 2009 (updated in 2015), and the new guidelines recommended classifying patients as dengue without warning signs (DNS), dengue with warning signs (DWS), and severe dengue (SD). Although the utility of the revised 2009 classification for clinical studies is accepted, for immunological studies it needs to be clarified. We determined the usefulness of the 2009 classification for pediatric studies that analyze the circulating interleukin (IL)-6 and IL-8, two inflammatory cytokines. Plasma levels of IL-6 and IL-8 were evaluated in the acute and convalescent phases by flow cytometry in children with dengue classified using the 1997 and 2009 WHO guidelines. The plasma levels of IL-6 and IL-8 were elevated during the acute and decreased during convalescence, and both cytokines served as a good marker of acute dengue illness compared to convalescence. There were no differences in the plasma level of the evaluated cytokines among children with different clinical severity with any classification, except for the IL-8, which was higher in DWS than DNS. Based on the levels of IL-8, the 2009 classification identified DWS plus SD (hospital-treated children) compared to the DNS group [area under the curve (AUC): 0.7, p = 0.028]. These results support the utility of the revised 2009 (updated in 2015) classification in studies of immune markers in pediatric dengue.

Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action.

Methionyl-tRNA formyltransferase (Fmt)-mediated formylation of Met-tRNAfMet to fMet-tRNAfMet is crucial for efficient initiation of translation in bacteria and the eukaryotic organelles. Folate dehydrogenase-cyclohydrolase (FolD), a bifunctional enzyme, carries out conversion of 5,10-methylene tetrahydrofolate (5,10-CH2-THF) to 10-formyl-THF (10-CHO-THF), a metabolite utilized by Fmt as a formyl group donor. In this study, using in vivo and in vitro approaches, we show that 10-CHO-DHF may also be utilized by Fmt as an alternative substrate (formyl group donor) to formylate Met-tRNAfMet. Dihydrofolate (DHF) formed as a by-product in the in vitro assay was verified by LC-MS/MS analysis. FolD-deficient mutants and Fmt over-expressing strains were more sensitive to trimethoprim (TMP) than the ∆fmt strain, suggesting that the domino effect of TMP leads to inhibition of protein synthesis and strain growth. Antifolate treatment to Escherichia coli showed a decrease in the reduced folate species (THF, 5,10-CH2-THF, 5-CH3-THF, 5,10-CH+-THF and 5-CHO-THF) and increase in the oxidized folate species (folic acid and DHF). In cells, 10-CHO-DHF and 10-CHO-folic acid were enriched in the stationary phase. This suggests that 10-CHO-DHF is a bioactive metabolite in the folate pathway for generating other folate intermediates and fMet-tRNAfMet.

Association Between Soluble Notch Ligand Delta-like Ligand 1 and Bleeding Complications in Patients With Dengue Fever Infection.

Bleeding associated with endothelial damage is a key feature of severe dengue fever. In the current study, we investigated whether Notch ligands were associated with bleeding in 115 patients with confirmed dengue infection in Vietnam. Soluble Notch ligands were determined by means of enzyme-linked immunosorbent assay. Seventeen of 115 patients (14.8%) experienced bleeding manifestations. High soluble delta-like ligand 1 (sDLL1) plasma levels was associated with bleeding (median, 15 674 vs 7117 pg/mL; P < .001). Receiver operating characteristic (ROC) curve analysis demonstrated that sDLL1 had the best test performance (area under the ROC curve, 0.852), with 88% sensitivity and 84% specificity. The combination with alanine aminotransferase and aspartate aminotransferase slightly increased sDLL1 performance. sDLL1 may be useful to guide clinical management of patients with patients in endemic settings.

Clinicopathological alteration of symptoms with serotype among dengue infected pediatric patients.

Dengue fever is a self-limiting, acute febrile illness caused by an arbovirus. This infection may be asymptomatic or symptomatic with its potential life-threatening form as DHF/DSS. Severe dengue cases occur typically in children due to overproduction of proinflammatory and anti-inflammatory cytokines (called cytokines storm) as well as increased microvascular permeability in them. This study aimed to find circulating dengue serotype and their clinicopathological association among pediatric patients admitted to tertiary care hospitals in Kolkata, India. Overall, 210 patients were approached, among them, 170 dengue suspected children admitted to three tertiary care hospitals were included in this study. Dengue samples were screened for the presence of dengue NS1 antigen and IgM antibodies by enzyme-linked immunosorbent assay. Viral RNA was extracted from NS1 seropositive serum samples and subjected to molecular serotyping by semi-nested reverse-transcription polymerase chain reaction. All patients were followed up for clinical manifestations and biochemical parameters associated with dengue. Cocirculation of all four serotypes was observed and DENV2 was the major circulating strain. Physiological classification of associated clinical symptoms was done as per WHO guideline and represented as a percentage variable. A multivariate logistic regression approach was used for making a regression model including dengue-associated clinical symptoms with dengue positivity or negativity as dependent variables. Thrombocytopenia was observed in 69% of patients and the commonest bleeding manifestation was petechia. Liver function profiles of infected patients were observed during follow-up and represented using a box plot. A significant change in trends of dengue-associated clinical manifestations and differential expression of liver functional profile with different phases of transition of dengue fever was observed in this study population.

Identification of stage-related and severity-related biomarkers and exploration of immune landscape for Dengue by comprehensive analyses.

BACKGROUND: At present, there are still no specific therapeutic drugs and appropriate vaccines for Dengue. Therefore, it is important to explore distinct clinical diagnostic indicators. METHODS: In this study, we combined differentially expressed genes (DEGs) analysis, weighted co-expression network analysis (WGCNA) and Receiver Operator Characteristic Curve (ROC) to screen a stable and robust biomarker with diagnosis value for Dengue patients. CIBERSORT was used to evaluate immune landscape of Dengue patients. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene set enrichment analysis (GSEA) were applied to explore potential functions of hub genes. RESULTS: CD38 and Plasma cells have excellent Area Under the Curve (AUC) in distinguishing clinical stages for Dengue patients, and activated memory CD4+ T cells and Monocytes have good AUC for this function. ZNF595 has acceptable AUC in discriminating dengue hemorrhagic fever (DHF) from dengue fever (DF) in whole acute stages. Analyzing any serotype, we can obtain consistent results. Negative inhibition of viral replication based on GO, KEGG and GSEA analysis results, up-regulated autophagy genes and the impairing immune system are potential reasons resulting in DHF. CONCLUSIONS: CD38, Plasma cells, activated memory CD4+ T cells and Monocytes can be used to distinguish clinical stages for dengue patients, and ZNF595 can be used to discriminate DHF from DF, regardless of serotypes.

A hospital based cross-sectional study on platelet parameters in adult patients with dengue, its serological subgroups, and controls.

Our objective was to analyze if there was a significant relationship between platelet parameters (PLT, MPV, PDW, P-LCR, PCT) among dengue, its serological subgroups and controls. Serologically proven adult patients with dengue {(n = 238) (NS1 positive = 218, IgM positive = 14, NS1 & IgM positive = 6)} and age- and gender-matched controls (n = 254) were included. The MPV, PDW and P-LCR were significantly higher, and PLT and PCT were significantly lower in cases compared with controls. Cases as well as controls showed a positive correlation between PLT and PCT, both parameters individually showed negative correlation with MPV, PDW, P-LCR. MPV, PDW and P-LCR showed positive correlation with each other. The results were similar in the serological subgroups. Comparison of our results with other studies points toward an overall hyperdestructive etiology for thrombocytopenia in dengue. There were two subgroups of cases based on the severity of thrombocytopenia. The mean/median value of all the platelet parameters was lesser in the ≤20k group than the >20k group, except for PDW, which was high although not statistically significant. Suppression of megakaryopoiesis with concomitant immune destruction of platelets in severe dengue could explain low MPV and P-LCR with a high PDW in view of the presence of microthrombocytes as a result of immune destruction. Although an overall hyperdestructive mechanism contributes to thrombocytopenia in dengue, regular monitoring of the platelet indices could reflect the status of megakaryopoiesis and thrombokinetic axis, thus aiding easy determination of pathophysiology and treatment.

Alginate Degradation: Insights Obtained through Characterization of a Thermophilic Exolytic Alginate Lyase.

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. Herein, we describe a thermostable alginate lyase that belongs to polysaccharide lyase family 17 (PL17) and was derived from an Arctic Mid-Ocean Ridge (AMOR) metagenomics data set. This enzyme, AMOR_PL17A, is a thermostable exolytic oligoalginate lyase (EC 4.2.2.26), which can degrade alginate, poly-ß-d-mannuronate, and poly-α-l-guluronate within a broad range of pHs, temperatures, and salinity conditions. Site-directed mutagenesis showed that tyrosine Y251, previously suggested to act as a catalytic acid, indeed is essential for catalysis, whereas mutation of tyrosine Y446, previously proposed to act as a catalytic base, did not affect enzyme activity. The observed reaction products are protonated and deprotonated forms of the 4,5-unsaturated uronic acid monomer, Δ, two hydrates of DEH (4-deoxy-l-erythro-5-hexulosuronate), which are formed after ring opening, and, finally, two epimers of a 5-member hemiketal called 4-deoxy-d-manno-hexulofuranosidonate (DHF), formed through intramolecular cyclization of hydrated DEH. The detection and nuclear magnetic resonance (NMR) assignment of these hemiketals refine our current understanding of alginate degradation.IMPORTANCE The potential markets for seaweed-derived products and seaweed processing technologies are growing, yet commercial enzyme cocktails for complete conversion of seaweed to fermentable sugars are not available. Such an enzyme cocktail would require the catalytic properties of a variety of different enzymes, where fucoidanases, laminarinases, and cellulases together with endo- and exo-acting alginate lyases would be the key enzymes. Here, we present an exo-acting alginate lyase that efficiently produces monomeric sugars from alginate. Since it is only the second characterized exo-acting alginate lyase capable of degrading alginate at a high industrially relevant temperature (≥60°C), this enzyme may be of great biotechnological and industrial interest. In addition, in-depth NMR-based structural elucidation revealed previously undescribed rearrangement products of the unsaturated monomeric sugars generated from exo-acting lyases. The insight provided by the NMR assignment of these products facilitates future assessment of product formation by alginate lyases.

A novel anti-NS2BNS3pro antibody-based indirect ELISA test for the diagnosis of dengue virus infections.

Dengue virus reportedly circulates as four genetically distinct serotypes for which there is no widely accepted vaccine or drug at present. Morbidity and mortality caused by this virus are alarming for the possible increased threat to human health. A suitable diagnostic test is the prerequisite for designing and developing control measures. But, the tests being employed at present possess one or the other drawback for this disease diagnosis. During the dengue virus infections, NS2B is essential for the stability and catalytic activity of the NS3 protease. N-terminal 185 amino acids of NS3 protease domain along with hydrophilic portion of NS2B (NS2BNS3pro) is being used to screen dengue inhibitors but not for diagnosis until now. In the present study, we have used purified NS2BNS3pro as an antigen to trap anti-NS2BNS3pro antibodies of the clinical samples. Antibodies were detected successfully in both Western blot analysis and enzyme-linked immunosorbent assay (ELISA) tests. In ELISA, antibodies were detected in both primary and secondary infections of all serotypes. Interestingly, 17 samples declared as other febrile infections by NS1 and IgM/IgG tests were found to be positive in present test, which were further confirmed by reverse-transcription polymerase chain reaction. In silico studies suggested the absence of conserved epitopes between NS2BNS3pro and the counterpart in JEV, Zika, and CHIKV, indicating less possibility of crossreaction, which was in turn confirmed by using synthetic peptides representing the above epitopes. Statistical analysis with 76% specificity, 87% sensitivity, and 95% concordance also supported the present test as a suitable test for large scale diagnosis of dengue virus infections.

Modeling the Relationship Between Antibody-Dependent Enhancement and Disease Severity in Secondary Dengue Infection.

Sequential infections with different dengue serotypes (DENV-1, 4) significantly increase the risk of a severe disease outcome (fever, shock, and hemorrhagic disorders). Two hypotheses have been proposed to explain the severity of the disease: (1) antibody-dependent enhancement (ADE) and (2) original T cell antigenic sin. In this work, we explored the first hypothesis through mathematical modeling. The proposed model reproduces the dynamic of susceptible and infected target cells and dengue virus in scenarios of infection-neutralizing and infection-enhancing antibody competition induced by two distinct serotypes of the dengue virus during secondary infection. The enhancement and neutralization functions are derived from basic concepts of chemical reactions and used to mimic binding to the virus by two distinct populations of antibodies. The analytic study of the model showed the existence of two equilibriums: a disease-free equilibrium and an endemic one. Using the concept of the basic reproduction number [Formula: see text], we performed the asymptotic stability analysis for the two equilibriums. To measure the severity of the disease, we considered the maximum value of infected cells as well as the time when this maximum is reached. We observed that it corresponds to the time when the maximum enhancing activity for the infection occurs. This critical time was calculated from the model to be a few days after the occurrence of the infection, which corresponds to what is observed in the literature. Finally, using as output [Formula: see text], we were able to rank the contribution of each parameter of the model. In particular, we highlighted that the cross-reactive antibody responses may be responsible for the disease enhancement during secondary heterologous dengue infection.

The TrkB agonist, 7,8-dihydroxyflavone, impairs fracture healing in mice.

OBJECTIVES: To study the effects of the selective TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF), on fracture healing in mice and on an osteoprogenitor cell line, Kusa4b10, in vitro. METHODS: Mice received unilateral closed mid-shaft tibial fractures and treated for two weeks with vehicle or 5 mg/kg/day DHF and euthanised at 28 days post-fracture. Calluses were analysed by micro-computed tomography (µCT) and three-point bending biomechanical test. Kusa4b10 cells were cultured with 50nM of 7,8-DHF or vehicle for 3-, 7-, 14-days for RT-PCR, and 21 days for mineralization. RESULTS: µCT found 7,8-DHF calluses had decreased tissue volume (p=0.042), mean polar moment of inertia (p = 0.004), and mean cross-sectional area (p=0.042) compared to controls. At 28 days biomechanical analyses showed 7,8-DHF treatment decreased peak force (p=0.011) and stiffness per unit area (p=0.012). 7,8-DHF treatment did not change Kusa4b10 gene expression of Runx2 and alkaline phosphatase at all time points, nor mineralization. CONCLUSIONS: 7,8-DHF treatment had a negative impact on fracture healing at 28 days post-fracture via an unknown mechanism. 7,8-DHF may have had a central role in impairing fracture healing.

Dengue virus: epidemiology, biology, and disease aetiology.

Dengue is a vector-borne viral disease caused by the flavivirus dengue virus (DENV). Approximately 400 million cases and 22 000 deaths occur due to dengue worldwide each year. It has been reported in more than 100 countries in tropical and subtropical regions. A positive-stranded enveloped RNA virus (DENV) is principally transmitted by Aedes mosquitoes. It has four antigenically distinct serotypes, DENV-1 to DENV-4, with different genotypes and three structural proteins and seven non-structural proteins. Clinical symptoms of dengue range from mild fever to severe dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), with thrombocytopenia, leucopenia, and increased vascular permeability. Although primary infection causes activation of immune responses against DENV serotypes, the severity of the disease is enhanced via heterotypic infection by various serotypes as well as antibody-dependent enhancement (ADE). The first licensed DENV vaccine was tetravalent CYD Denvaxia, but it has not been approved in all countries. The lack of a suitable animal model, a proper mechanistic study in pathogenesis, and ADE are the main hindrances in vaccine development. This review summarizes the current knowledge on DENV epidemiology, biology, and disease aetiology in the context of prevention and protection from dengue virus disease.

7,8-Dihydroxiflavone Protects Adult Rat Axotomized Retinal Ganglion Cells through MAPK/ERK and PI3K/AKT Activation.

We analyze the 7,8-dihydroxyflavone (DHF)/TrkB signaling activation of two main intracellular pathways, mitogen-activated protein kinase (MAPK)/ERK and phosphatidylinositol 3 kinase (PI3K)/AKT, in the neuroprotection of axotomized retinal ganglion cells (RGCs). METHODS: Adult albino Sprague-Dawley rats received left intraorbital optic nerve transection (IONT) and were divided in two groups. One group received daily intraperitoneal DHF (5 mg/kg) and another vehicle (1%DMSO in 0.9%NaCl) from one day before IONT until processing. Additional intact rats were employed as control (n = 4). At 1, 3 or 7 days (d) after IONT, phosphorylated (p)AKT, p-MAPK, and non-phosphorylated AKT and MAPK expression levels were analyzed in the retina by Western blotting (n = 4/group). Radial sections were also immunodetected for the above-mentioned proteins, and for Brn3a and vimentin to identify RGCs and Müller cells (MCs), respectively (n = 3/group). RESULTS: IONT induced increased levels of p-MAPK and MAPK at 3d in DHF- or vehicle-treated retinas and at 7d in DHF-treated retinas. IONT induced a fast decrease in AKT in retinas treated with DHF or vehicle, with higher levels of phosphorylation in DHF-treated retinas at 7d. In intact retinas and vehicle-treated groups, no p-MAPK or MAPK expression in RGCs was observed. In DHF- treated retinas p-MAPK and MAPK were expressed in the ganglion cell layer and in the RGC nuclei 3 and 7d after IONT. AKT was observed in intact and axotomized RGCs, but the signal intensity of p-AKT was stronger in DHF-treated retinas. Finally, MCs expressed higher quantities of both MAPK and AKT at 3d in both DHF- and vehicle-treated retinas, and at 7d the phosphorylation of p-MAPK was higher in DHF-treated groups. CONCLUSIONS: Phosphorylation and increased levels of AKT and MAPK through MCs and RGCs in retinas after DHF-treatment may be responsible for the increased and long-lasting RGC protection afforded by DHF after IONT.

Infection severity across scales in multi-strain immuno-epidemiological Dengue model structured by host antibody level.

Infection by distinct Dengue virus serotypes and host immunity are intricately linked. In particular, certain levels of cross-reactive antibodies in the host may actually enhance infection severity leading to Dengue hemorrhagic fever (DHF). The coupled immunological and epidemiological dynamics of Dengue calls for a multi-scale modeling approach. In this work, we formulate a within-host model which mechanistically recapitulates characteristics of antibody dependent enhancement in Dengue infection. The within-host scale is then linked to epidemiological spread by a vector-host partial differential equation model structured by host antibody level. The coupling allows for dynamic population-wide antibody levels to be tracked through primary and secondary infections by distinct Dengue strains, along with waning of cross-protective immunity after primary infection. Analysis of both the within-host and between-host systems are conducted. Stability results in the epidemic model are formulated via basic and invasion reproduction numbers as a function of immunological variables. Additionally, we develop numerical methods in order to simulate the multi-scale model and assess the influence of parameters on disease spread and DHF prevalence in the population.

A case report of dengue hemorrhagic fever complicated with diabetic ketoacidosis in a child: challenges in clinical management.

BACKGROUND: Diabetic ketoacidosis (DKA) is a common presentation of type 1 diabetes mellitus (T1DM) precipitated by various bacterial and viral infections. Dengue infection is no exception for this and can be a precipitating factor for DKA. The presentation of DKA with dengue haemorrhagic fever (DHF) has been reported in adults. However, it is very rarely observed in children. CASE PRESENTATION: We present the case of a paediatric patient who was previously healthy and subsequently, developed polyuria (above 3 ml/kg/hour), irritability and high blood glucose (724 mg/dl) during the critical phase of DHF. DKA was diagnosed with DHF and managed successfully with insulin and intravenous fluids. He recovered without complications and discharged home with follow up being arranged at the endocrinology clinic. CONCLUSIONS: When both DHF and DKA present together in a patient, meticulous monitoring of glycaemic control as well as fluid management is required to reduce the potential risk for severe complications of both conditions. Since there are no similar paediatric case reported in the literature, this case report might inspire paediatricians to anticipate the possibility of DKA in children with DHF.

Osteopontin - a biomarker of disease, but also of stage stratification of the functional myocardial contractile deficit by chronic ischaemic heart disease.

The study analyses the significance of the plasmatic values of the OPN dosed to 91 people suffering from diastolic cardiac dysfunction with preserved ejection fraction, thus revealing significant growths of its level compared to the normal value. Despite being a clinical research, its conclusions are a breakthrough, differing from the results of other studies published in the relevant medical literature. We can make this assertion because this study analyses the clinical information given by the circulating values of the OPN, based on experimental models (animals), or on patients with congestive heart failure, which can be identified with the existence of a low systolic flow. The results of our study allow us to assert that the plasmatic values of this glycoprotein lead to its acceptance in the medical practice as a new biomarker that provides indicators regarding the stratification of risk with the patients suffering from heart failure of the diastolic dysfunction type, but whose systolic flow is preserved.

7,8-Dihydroxyflavone ameliorates high-glucose induced diabetic apoptosis in human retinal pigment epithelial cells by activating TrkB.

BACKGROUND: In diabetic retinopathy, prolonged high-level blood glucose induced significant impairments among various retinal tissues, including retinal pigment epithelial (RPE) cells. In an in vitro model of human RPE cells, we evaluated whether 7,8-Dihydroxyflavone (DHF) may effectively prevent high glucose-induced diabetic apoptosis among human RPE cells. METHOD: ARPE-19 cells, a Human RPE cell line, were treated with d-glucose (50 mM) to induce apoptosis in vitro. Prior to glucose, ARPE-19 cells were pre-incubated with various concentrations of DHF. The effect of DHF on d-glucose-induced apoptosis was examined by TUNEL assay, in a concentration-dependent manner. The biological effects of DHF on Caspase-9 (Casp-9) and TrkB signaling pathways in d-glucose-injured ARPE-19 cells were evaluated by qRT-PCR and western blot (WB) assays. A TrkB antagonist, K252a, was also applied in DHF and d-glucose treated ARPE-19 cells. Possible effect of K252a blocking TrkB signaling pathway, thus reversing DHF-modulated apoptosis prevention was also examined by TUNEL and WB assays. RESULTS: DHF ameliorated d-glucose-induced diabetic apoptosis in ARPE-19 cells. Apoptotic factor Casp-9, at both mRNA and protein levels, were drastically inhibited by DHF in d-glucose-injured ARPE-19 cells. Also, DHF activated TrkB signaling pathway through phosphorylation. K252a dramatically reversed the preventive effect of DHF on d-glucose-induced apoptosis in ARPE-19 cells. Further investigation showed that K252a functioned through de-activating or de-phosphorylating TrkB signaling pathway. CONCLUSION: This work demonstrates that DHF, through activation of TrkB signaling pathway, has a preventive function in d-glucose-induced apoptosis in PRE cells in diabetic retinopathy.

Utilisation of 10-formyldihydrofolate as substrate by dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) tranformylase/IMP cyclohydrolase (PurH) in Escherichia coli.

Dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/IMP cyclohydrolase (PurH) play key roles in maintaining folate pools in cells, and are targets of antimicrobial and anticancer drugs. While the activities of bacterial DHFR and PurH on their classical substrates (DHF and 10-CHO-THF, respectively) are known, their activities and kinetic properties of utilisation of 10-CHO-DHF are unknown. We have determined the kinetic properties (kcat/Km) of conversion of 10-CHO-DHF to 10-CHO-THF by DHFR, and to DHF by PurH. We show that DHFR utilises 10-CHO-DHF about one third as efficiently as it utilises DHF. The 10-CHO-DHF is also utilised (as a formyl group donor) by PurH albeit slightly less efficiently than 10-CHO-THF. The utilisation of 10-CHO-DHF by DHFR is ~50 fold more efficient than its utilisation by PurH. A folate deficient Escherichia coli (∆pabA) grows well when supplemented with adenine, glycine, thymine and methionine, the metabolites that arise from the one-carbon metabolic pathway. Notably, when the ∆pabA strain harboured a folate transporter, it grew in the presence of 10-CHO-DHF alone, suggesting that it (10-CHO-DHF) can enter one-carbon metabolic pathway to provide the required metabolites. Thus, our studies reveal that both DHFR and PurH could utilise 10-CHO-DHF for folate homeostasis in E. coli.

Sphingolipid signaling modulates trans-endothelial cell permeability in dengue virus infected HMEC-1 cells.

Dengue has emerged as a major mosquito-borne disease in the tropics and subtropics. In severe dengue, enhanced microvascular endothelial permeability leads to plasma leakage. Direct dengue virus (DENV) infection in human microvascular endothelial cells (HMEC-1) can enhance trans-endothelial leakage. Using a microarray-based analysis, we identified modulation of key endothelial cell signaling pathways in DENV-infected HMEC-1 cells. One among them was the sphingolipid pathway that regulates vascular barrier function. Sphingosine-1-phosphate receptor 2 (S1PR2) and S1PR5 showed significant up-regulation in the microarray data. In DENV-infected cells, the kinetics of S1PR2 transcript expression and enhanced in vitro trans-endothelial permeability showed a correlation. We also observed an internalization and cytoplasmic translocation of VE-Cadherin, a component of adherens junctions (AJ), upon infection indicating AJ disassembly. Further, inhibition of S1PR2 signaling by a specific pharmacological inhibitor prevented translocation of VE-Cadherin, thus helping AJ maintenance, and abrogated DENV-induced trans-endothelial leakage. Our results show that sphingolipid signaling, especially that involving S1PR2, plays a critical role in vascular leakage in dengue.

Serum fatty acids and progression from dengue fever to dengue haemorrhagic fever/dengue shock syndrome.

PUFA might modulate inflammatory responses involved in the development of severe dengue. We aimed to examine whether serum PUFA concentrations in patients diagnosed with dengue fever (DF) were related to the risk of progression to dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). A secondary aim was to assess correlations between fatty acids (FA) and inflammatory biomarkers in patients with DF. We conducted a prospective case-control study nested within a cohort of patients who were diagnosed with DF and followed during the acute episode. We compared the distribution of individual FA (% of total FA) at onset of fever between 109 cases who progressed to DHF/DSS and 235 DF non-progressing controls using unconditional logistic regression. We estimated correlations between baseline FA and cytokine concentrations and compared FA concentrations between the acute episode and >1 year post-convalescence in a subgroup. DHA was positively related to progression to DHF/DSS (multivariable adjusted OR (AOR) for DHA in quintile 5 v. 1=5·34, 95 % CI 2·03, 14·1; P trend=0·007). Dihomo-γ-linolenic acid (DGLA) was inversely associated with progression (AOR for quintile 5 v. 1=0·30, 95 % CI 0·13, 0·69; P trend=0·007). Pentadecanoic acid concentrations were inversely related to DHF/DSS. Correlations of PUFA with cytokines at baseline were low. PUFA were lower during the acute episode than in a disease-free period. In conclusion, serum DHA in patients with DF predicts higher odds of progression to DHF/DSS whereas DGLA and pentadecanoic acid predict lower odds.