Differential expression of microRNA, miR-150 and enhancer of zeste homolog 2 (EZH2) in peripheral blood cells as early prognostic markers of severe forms of dengue.

BACKGROUND: Dengue presents a wide clinical spectrum. Most patients recover following a self-limiting non-severe clinical course. A small proportion of patients progress to severe disease, mostly characterized by plasma leakage with or without hemorrhage. Early symptoms of severe dengue (SD) are similar to those of non-severe dengue fever (DF). Severe symptoms manifest after 3-5 days of fever, which can be life threatening due to lack of proper medications and inability to distinguish severe cases during the early stages. Early prediction of SD in patients with no warning signs who may later develop severe infection is very important for proper disease management to alleviate related complications and mortality. microRNA are small non-coding RNA molecules that regulate post-transcriptional gene expression. Due to the remarkable stability and the role of microRNA in gene expression, altered expression of microRNA was evaluated to explore clinically relevant prognostic markers of severe dengue. METHODS: The relative expression of microRNA hsa-let-7e (let-7e), hsa-miR-30b-5p (miR-30b), hsa-miR-30e-3p (miR-30e), hsa-miR-33a (miR-33a), and hsa-miR-150-5p (miR-150) and several putative target genes in peripheral blood cells (PBC) collected from 20 DF and 20 SD positive patients within 4 days from fever onset was evaluated by quantitative reverse transcription PCR (qRT-PCR). RESULTS: miR-150 showed significant (P < 0.01) up regulation in PBC of SD patients compared to DF patients during the acute phase of infection. Expression of enhancer of zeste homolog 2 (EZH2) was significantly (P < 0.01) down regulated indicating that genes involved in epigenetic regulation are also differentially expressed in SD patients during the early stage of infection. CONCLUSIONS: Differential expression of microRNA miR-150 and the putative target gene EZH2 may serve as reliable biomarkers of disease severity during early stages of dengue infection.

Evaluating suitable internal control genes for transcriptional studies in heat-stressed mammary explants of buffaloes.

It is now a well-accepted notion that each new experimental design requires proper evaluation of internal control genes (ICGs) for accurate normalization of expression data. In riverine buffaloes, till date no appropriate ICG has been reported for studying transcriptional response under any of the physiological stressful condition. The objective here was to test 16 well-known reference genes from different functional categories that could serve as suitable ICG during heat stress studies in buffalo mammary tissue. Briefly, the mammary explants were exposed to 45°C for 1 h and subsequently allowed to recover at 37°C for different time points (2-24 h). Three software programs, geNorm, Normfinder and BestKeeper, were used to measure gene transcript stability. RPL22 was excluded because of weak amplification and unacceptable PCR efficiency. Except GAPDH, all other genes showed expression stability within the acceptable range (<1.5). RPL4, B2M, RPS23 and EEF1A1 genes were found to be most stably expressed while GAPDH and ACTB showed least stability. The BestKeeper analysis identified high correlation for RPL4 (r=0.953) and EEF1A1 (r=0.914) with BestKeeper index. Based on the present findings, it could be suggested that geometric average of RPL4, B2M, RPS23 and EEF1A1 would provide accurate normalization to transcriptional data of buffalo mammary explant in response to heat stress.

A Computational Study of Carbazole Alkaloids from Murraya koenigii as Potential SARS-CoV-2 Main Protease Inhibitors.

Despite COVID-19 vaccination, immune escape of new SARS-CoV-2 variants has created an urgent priority to identify additional antiviral drugs. Targeting main protease (Mpro) expressed by SARS-CoV-2 is a therapeutic strategy for drug development due to its prominent role in viral replication cycle. Leaves of Murraya koenigii are used in various traditional medicinal applications and this plant is known as a rich source of carbazole alkaloids. Thus, this computational study was designed to investigate the inhibitory potential of carbazole alkaloids from Murraya koenigii against Mpro. Molecular docking was initially used to determine the binding affinity and molecular interactions of carbazole alkaloids and the reference inhibitor (3WL) in the active site of SARS-CoV-2 Mpro (PDB ID: 6M2N).The top scoring compounds were further assessed for protein structure flexibility, physicochemical properties and drug-likeness, pharmacokinetic and toxicity (ADME/T) properties, antiviral activity, and pharmacophore modeling. Five carbazole alkaloids (koenigicine, mukonicine, o-methylmurrayamine A, koenine, and girinimbine) displayed a unique binding mechanism that shielded the catalytic dyad of Mpro with stronger binding affinities and molecular interactions than 3WL. Furthermore, the compounds with high affinity displayed favorable physicochemical and ADME/T properties that satisfied the criteria for oral bioavailability and druggability. The pharmacophore modeling study shows shared pharmacophoric features of those compounds for their biological interaction with Mpro. During the molecular dynamics simulation, the top docking complexes demonstrated precise stability except koenigicine. Therefore, mukonicine, o-methylmurrayamine A, koenine, and girinimbine may have the potential to restrict SARS-CoV-2 replication by inactivating the Mpro catalytic activity.

Expression of Nitric Oxide Synthase and Nitric Oxide Levels in Peripheral Blood Cells and Oxidized Low-Density Lipoprotein Levels in Saliva as Early Markers of Severe Dengue.

BACKGROUND: Severe dengue (SD), experienced by only a fraction of dengue patients, can be lethal. Due to the lack of early markers that can predict the evolution of SD, all dengue patients have to be monitored under hospital care. We discovered early oxidative stress markers of SD to identify patients who can benefit from early intervention before the symptoms appear. METHODS: The expression of inducible nitric oxide synthase (iNOS) in peripheral blood cells (PBC), nitric oxide (NO), and oxidized low-density lipoprotein (oxLDL) levels in plasma and saliva collected at early stages of dengue infection from 20 nonsevere dengue fever (DF) patients and 20 patients who later developed SD were analyzed in a retrospective nested case-control study. RESULTS: The expression of iNOS is significantly (P < 0.05) lower in patients who developed SD than in DF patients at admission within 4 days from fever onset. Median plasma NO concentration within 4 days from fever onset is also significantly (P < 0.05) lower in patients who developed SD (17.9 ± 1.6 µmol/L) than DF (23.0 ± 2.1 µmol/L). Median oxLDL levels in plasma within 3 days from fever onset is significantly (P < 0.05) lower in patients who developed SD (509.4 ± 224.1 ng/mL) than DF (740.0 ± 300.0 ng/mL). Median salivary oxLDL levels are also significantly (P < 0.05) lower in patients who developed SD (0.8 ± 0.5 ng/mL) than DF (3.6 ± 2.6 ng/mL) within 4 days from fever onset. CONCLUSIONS: These findings suggest that the expression of iNOS (73% sensitivity, 86% specificity) and plasma NO (96% sensitivity, 61% specificity at 22.3 µmol/L; P < 0.05) may serve as early markers of SD within 3 days from fever onset. Salivary oxLDL levels may serve as early noninvasive markers of SD with a sensitivity and specificity, respectively, of 57% and 91% at 0.9 ng/mL; 76% and 55% at 2.3 ng/mL; and 100% and 50% at 4.6 ng/mL (P < 0.05) within 4 days from fever onset.

Effect of chain length and saturation of the fatty acids in dietary triglycerides on lipid metabolism in Wistar rats.

We investigated the effect of the chain length and the degree of saturation of fatty acids in dietary triglycerides on serum lipid profiles and hepatic lipid metabolism in Wistar rats. Fat component of the basal diet (soybean oil) was replaced with fats with fatty acids of different chain lengths and saturation and the serum lipids were monitored for 150 days. Principal component (PC) analysis of serum lipid components was related to chain length and saturation. The combined effect of chain length and saturation on PC 1 scores was evaluated by multiple regression analysis. The results indicated that average chain length of the fatty acids of triglycerides has a higher influence on the quality of serum lipid parameters than the average degree of saturation. Expression of selected genes responsible for lipid metabolism showed similar trends in medium chain saturated and long chain polyunsaturated diet groups. PRACTICAL APPLICATIONS: Dietary lipids contain a wide range of saturated and unsaturated fatty acids with different chain lengths. Overall contribution of these different fatty acids decides the health effects of the lipids in the diet. Present study shows that the fats with medium chains and higher degree of saturation and fats with long chains and higher degree of unsaturation (lower degree of saturation) affect serum lipid parameters and expression of hepatic genes involved in the lipid metabolism in a similar manner. Such information is important for physicians to plan dietary schemes to improve the nutritional health and manage the noncommunicable diseases.

Soybean oil and coconut oil enhance the absorption of chlorogenic acid in humans.

The effect of a mainly polyunsaturated oil (soybean oil) and a mainly medium chain triglyceride oil (coconut oil) on the absorption of the phenolic antioxidant chlorogenic acid (5-caffeoylquinic acid) was investigated using 90 healthy volunteers. Serum concentrations and the absorbed percentages of chlorogenic acid of volunteers who received chlorogenic acid without oils (0.006 ± 0.001 mg/ml, 5.7 ± 0.2%), chlorogenic acid with soybean oil (0.012 ± 0.001 mg/ml, 11.8 ± 1.3%), and chlorogenic acid with coconut oil (0.067 ± 0.014 mg/ml, 65.6 ± 18.1%) were significantly different from each other (p < .05). There was a strong positive correlation between the increase in serum and plasma antioxidant capacity and the absorption of chlorogenic acid. The major fatty acid of each of soybean oil and coconut oil also improved the permeability of chlorogenic acid in Caco-2 cell monolayers. The results suggest that the tested edible oils may improve the nutritional value of chlorogenic acid-containing foods by improving the absorption of chlorogenic acid. PRACTICAL APPLICATIONS: Small polar antioxidants such as phenolic acids and flavonoids are poorly absorbed through the intestinal epithelium. Chlorogenic acid was used in the present study as a model for small polar phenolic antioxidants. According to the present study, soybean oil with mainly polyunsaturated fatty acids and coconut oil with mainly medium chain fatty acids improve the absorption of these antioxidants. These findings suggest that proper planning of diets or food supplements containing phenolic antioxidants with medium chain or polyunsaturated fatty acid-rich edible oils may enhance the nutritional benefits expected from phenolic antioxidants.

Antioxidant and Nutritional Properties of Domestic and Commercial Coconut Milk Preparations.

The aqueous extract of scraped coconut kernel is known as coconut milk. Coconut milk preparations are also commercially available in the form of desiccated powders or liquids. While these various coconut milk preparations are heavily used in cooking in the Asian countries as a major source of dietary fat, limited studies have been conducted on their chemical and nutritional composition. In this study, we have determined the chemical composition and nutritional effects of both domestic preparations of coconut milk and the commercially available counterparts. The results indicate that the phenolic compounds of all coconut milk preparations provide protection against oxidative damage on lipids and inhibit oxidative damage of both proteins and DNA. The lipid profiles are not significantly affected by the consumption of the three coconut milk preparations despite their different fat contents.

Effect of three edible oils on the intestinal absorption of caffeic acid: An in vivo and in vitro study.

Polyphenolic antioxidants are mainly absorbed through passive paracellular permeation regulated by tight junctions. Some fatty acids are known to modulate tight junctions. Fatty acids resulting from the digestion of edible oils may improve the absorption of polyphenolic antioxidants. Therefore, we explored the effect of three edible oils on the intestinal absorption of caffeic acid. Rats were fed with soybean oil and caffeic acid dissolved in distilled water. Caffeic acid contents in the plasma collected up to 1 hr were quantified. The experiment was repeated with coconut oil and olive oil. Component fatty acids of the oils were individually tested in vitro for their effect on permeability of caffeic acid using Caco-2 cell monolayers. Highest absorption of caffeic acid was observed in animals fed with coconut oil. In vitro transport percentages of caffeic acid in 2.5 mmol/L solutions of fatty acids were 22.01±0.12 (lauric), 15.30 ± 0.25 (myristic acid), 13.59 ± 0.35 (linoleic acid), 3.70 ± 0.09 (oleic acid) and 0.10-2.0 (all other fatty acids). Lauric acid and myristic acid are the two major fatty acids present in coconut oil. Therefore, these fatty acids may contribute to the higher absorption of caffeic acid in the presence of coconut oil.

Prevention of blood-borne HIV transmission using a decentralized approach in Shaba, Zaire.

OBJECTIVE: To prevent blood transfusion-acquired HIV infection with a decentralized approach to HIV screening of blood donors, using an instrument-free rapid test. SETTING: Shaba province, Zaire (496,877 km2). METHODS: The programme consisted of training health-care workers, distribution of a rapid HIV-antibody test (DuPont's HIVCHEK) for screening of all blood donations, and quality control of testing by a regional reference centre. RESULTS: Over a 2-year period, 11,940 rapid tests were distributed to 37 hospitals, covering 75% of all hospital beds outside the copper mine's health system in Shaba. Eighty-five per cent of the tests were used to screen blood donors (5.4% positive test rate) and 13% to test patients (39.7% positive test rate). At least 265 cases of HIV-positive blood donation were prevented, at an estimated cost of 137-279 ECU per case. Only 26% of initially positive specimens reached the central laboratory for supplemental testing, and sterile transfusion equipment and blood-grouping reagents were frequently unavailable. The lack of transport and communications and a deteriorating health system were major constraints. CONCLUSIONS: District hospitals in Africa are often long distances from major cities, difficult to reach for most of the year, and perform a small number of transfusions. In this context a classical centralized regional blood bank may not be a feasible option to ensure safe blood transfusions. However, safe blood transfusion can be achieved with a decentralized approach using a rapid test, provided that minimum standards of health-care services are available.

PIP: This program aimed at preventing blood transfusion-acquired HIV infection with a decentralized approach to HIV screening of blood donors using an instrument free raid test was initiated in Shaba province in Zaire (496,877 sq. km and included training of health care workers, distribution of a rapid HIV-antibody test (DuPont's HIVCHEK) for screening of all blood donations, and quality control of testing by a regional reference center. Over a 2-year period, 11,940 rapid tests were distributed to 37 hospitals, covering 75% of all hospital beds outside the copper mine's health system in Shaba. 85% of the tests were used to screen blood donors (5.4% positive test rate) and 13% to test patients (39.7% positive test rate). At least 265 cases of HIV-positive blood donation were prevented at an estimated cost of 137-279 ECU per case. Only 26% of initially positive specimens reached the central laboratory for supplemental testing, and sterile transfusion equipment and blood-grouping reagents were frequently unavailable. The lack of transport and communications and a deteriorating health system were major constraints. District hospitals in Africa are often long distances from major cities, difficult to reach for most of the year, and perform a small number of transfusions. In this context, a classical centralized regional blood bank may not be a feasible option to ensure safe blood transfusions. However, safe blood transfusion can be achieved with a decentralized approach using a rapid test, provided that minimum standards of health care services, are available.

Use of a rapid test and an ELISA for HIV antibody screening of pooled serum samples in Lubumbashi, Zaire.

Pools with a size of 3 and 5 were prepared by mixing one HIV confirmed HIV-1 seropositive serum with either 2 or 4 HIV seronegative sera at the Regional HIV Laboratory in Lubumbashi, Zaire. These pools were assessed in a blind fashion by ELISA (Vironostika anti-HTLV-III microELISA system, Organon Technika). Similarly constituted pools of 3 samples were assayed by a rapid test with visual reading (HIVCHEK 1 + 2, Dupont de Nemours). With the HIVCHEK, pooling was achieved on the test device itself by dropping consecutively 3 different serum samples on the devices's membrane. After the last serum was soaked in, wash fluid and conjugate were added. Results of the pooling experiments were compared with testing sera individually. The ELISA results from pools and from individual tested samples matched completely if, and only if, the final dilution of individual samples in the reagent medium was the same as recommended by the manufacturer for testing of individual samples. With the HIVCHEK a sensitivity of 99-100% was obtained with pooled sera. Both approaches seemed sensitive enough to enable their use for screening of blood donors and patient management, but a prospective study to validate these preliminary results is necessary.

Interactions of Rh(III)-dihydrido-bis(phosphine) complexes with semicarbazones.

Interaction of cis,trans,cis-[Rh(H)2(PR3)2(acetone)2]PF6 complexes (R = aryl or R3 = Ph2Me, Ph2Et) under H2 with E-semicarbazones gives the Rh(III)-dihydrido-bis(phosphine)-semicarbazone species cis,trans-[Rh(H)2(PR3)2{R'(R' ')C=N-N(H)CONH2}]PF6, where R' and R' ' are Ph, Et, or Me. The complexes are generally characterized by elemental analysis, 31P{1H} NMR, 1H NMR, and IR spectroscopies, and MS. X-ray analysis of three PPh3 complexes reveals chelation of E-semicarbazones by the imine-N atom and the carbonyl-O atom. In contrast, the corresponding reaction of [Rh(H)2(PPhMe2)2(acetone)2]PF6 with acetophenone semicarbazone gives the ortho-metalated-semicarbazone species cis-[RhH(PPhMe2)2{o-C6H4(Me)C=N-N(H)CONH2}]PF6. The X-ray structure of E-propiophenone semicarbazone is also reported. Rhodium-catalyzed, homogeneous hydrogenation of semicarbazones was not observed even at 40 atm H2.

A simplified and less expensive strategy for confirming anti HIV-1 screening results in a diagnostic laboratory in Lubumbashi, Zaire.

The conventional algorithm for HIV testing based on the confirmation of all positive anti-HIV screening reactions by Western blot (WB) is too expensive for developing countries. We investigated the validity of confirming positive screening assay reactions by a second screening test, limiting the use of the supplemental assay to the discrepant test results (algorithm 3), or screening all sera with 2 different assays and retesting all discrepant results by a supplemental assay (algorithm 4) on a panel of 519 sera in a regional reference laboratory in Lubumbashi, Zaire. Combining the Vironostika anti-HTLV-III ELISA with HIV Chek 1 + 2 or Clonatec Rapid HIV 1/2 Ab on all samples and retesting the discrepant results in WB or a line immunoassay (INNO-LIA) (algorithm 4), yielded a sensitivity of 100% and specificities of 98.4% and 99.0% respectively, at costs of 7.3 US $ and 9.3 US $ per test, respectively, for a 40% prevalence of HIV antibody positive samples. The conventional algorithm scored a sensitivity of 97.1% and a specificity of 100% for 11.3 US $ per test. The testing strategy of combining HIV Chek 1 + 2 and Clonatec Rapid HIV 1/2 Ab, an interesting option for small isolated centra, had a 96.6% sensitivity, but yielded only a slightly better specificity of 99.0%, as compared to 97.8% for HIV Chek alone. The price of combining the two simple assays using algorithm 3 was 6.8 US $ per test, using algorithm 4 was 10.6 US $. HIV testing strategies based on ELISA and a simple HIV test are a valuable alternative for reference laboratories faced with a high prevalence of HIV positive samples.

Imine hydrolysis and role of a rhodium(I)-imine-amine complex in homogeneous H2-hydrogenation of the imine and a rare example of inequivalent NH2 protons.

A Rh-catalyzed, homogeneous hydrogenation of the imine, PhCH(2)N=CHPh, is shown to involve a Rh-imine-amine species that subsequently activates H(2), the amine (benzylamine) being formed via a Rh-catalyzed hydrolysis of the imine by adventitious water. The imine-amine complex, cis-(Rh[P(p-tolyl)(3)](2)(PhCH(2)N=CHPh)(NH(2)CH(2)Ph))PF(6) (2b), is structurally characterized, and the solution (1)H NMR data reveal inequivalent NH(2) protons.