Coagulation abnormalities in Dengue fever infection: A systematic review and meta-analysis

BackgroundCoagulation mechanisms are reported to be affected in dengue illness and evidenced by prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT). The main aim of this systematic review and meta-analysis is to determine the magnitude of coagulation abnormalities among patients with dengue fever infection.MethodThis systematic review and meta-analysis were conducted per the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. The Joana Brigg’s Institute (JBI) critical appraisal checklist was used for quality appraisal. STATA version 11 software was used for meta-analysis. The magnitude of coagulation abnormalities among dengue fever patients was determined by using a random-effects model. Subgroup and sensitivity analysis were performed to investigate the possible source of heterogeneity. Egger weighted regression tests were used to check the presence of publication bias among the included articles.ResultForty-two studies with a total of 12,221 dengue fever patients were eligible for meta-analysis in this study. Of which 22, 15, and 26 studies were used to determine the magnitude of prolonged APTT, PT, and thrombocytopenia, respectively. The magnitude of prolonged APTT and PT among patients with dengue fever infection were 42.91% (95% CI: 30.95, 54.87) I² = 99.1% and 16.48% (95% CI: 10.95, 22.01) I² = 97.0%, respectively. Besides, the magnitude of thrombocytopenia among dengue fever patients was 70.29% (95% CI: 62.69, 77.89) I² = 99.3%. The magnitude of prolonged APTT in children and adults was 51.21% (95% CI: 24.54, 77.89) and 44.89% (95% CI: 28.32, 61.45), respectively. Similarly, the overall magnitude of prolonged PT in children and adults were 13.40% (95% CI: 6.09, 20.71) and 18.73% (95% CI: 7.49, 29.96), respectively.ConclusionThe result of this study showed that there is a high magnitude of prolonged APTT and PT in dengue fever patients. Therefore, screening and early correction of coagulation abnormalities may be helpful to reduce further complications in those patients.     

Neurotransmitter vesicle release from human model neurons (NT2) is sensitive to botulinum toxin A

Botulinum neurotoxins (BoNTs) internalize into nerve terminals and block the release of neurotransmitters into the synapse. BoNTs are widely used as a therapeutic agent for treatment of movement disorders and recently gained more attention as a biological weapon. Consequently, there is strong interest to develop a cell-based assay platform to screen the toxicity and bioactivity of the BoNTs. In this study, we present an in vitro screening assay for BoNT/A based on differentiated human embryonal carcinoma stem (NT2) cells. The human NT2 cells fully differentiated into mature neurons that display immunoreactivity to cytoskeletal markers (βIII-tubulin and MAP2) and presynaptic proteins (synapsin and synaptotagmin I). We showed that the human NT2 cells undergo a process of exo-endocytotic synaptic vesicle recycling upon depolarization with high K(+) buffer. By employing an antibody directed against light chain of BoNT/A, we detected internalized toxin as a punctate staining along the neurites of the NT2 neurons. Using well-established methods of synaptic vesicle exocytosis assay (luminal synaptotagmin I and FM1-43 imaging) we show that pre-incubation with BoNT/A resulted in a blockade of vesicle release from human NT2 neurons in a dose-dependent manner. Moreover, this blocking effect of BoNT/A was abolished by pre-adsorbing the toxin with neutralizing antibody. In a proof of principle, we demonstrate that our cell culture assay for vesicle release is sensitive to BoNT/A and the activity of BoNT/A can be blocked by specific neutralizing antibodies. Overall our data suggest that human NT2 neurons are suitable for large scale screening of botulinum bioactivity.     

Rapid Differentiation of Human Embryonal Carcinoma Stem Cells (NT2) into Neurons for Neurite Outgrowth Analysis

Human neurons derived from stem cells can be employed as in vitro models to predict the potential of neurochemicals affecting neurodevelopmental cellular processes including proliferation, migration, and differentiation. Here, we developed a model of differentiating human neurons from well characterized human embryonal carcinoma stem cells (NT2). NT2 cells were induced to differentiate into neuronal phenotypes after 2 weeks of treatment with retinoic acid in aggregate culture. Nestin positive progenitor cells migrate out of NT2 aggregates and differentiate into βIII-tubulin expressing neuronal cells. Culturing the NT2 cells for an additional 7–14 days resulted in increased percentage of βIII-tubulin expressing cells, elaborating a long neurite that positively stained for axonal marker (Tau) and presynaptic protein (synapsin). We then asked whether neurite outgrowth from NT2 cells is modulated by bioactive chemicals. Since the cAMP/PKA pathway has been widely investigated as a regulator of neurite outgrowth/regeneration in several experimental systems, we used chemical activators and inhibitors of cAMP/PKA pathway in the culture. The adenylyl cyclase activator, forskolin, and cell-permeable analog of cAMP, 8-Br-cAMP increased the percentage of neurite bearing cells and neurite extension. Application of the protein kinase A inhibitors, H-89 and Rp-cAMP, blocked neurite formation. Taken together, NT2 aggregates undergo migration, differentiation, and neurite elaboration and can be used as a model of differentiating human neurons to screen neurochemicals and to understand cellular mechanisms of human nerve cell development.     

Nitric oxide and cGMP signal transduction positively regulates the motility of human neuronal precursor (NT2) cells

Developmental studies in both vertebrates and invertebrates implicate an involvement of nitric oxide (NO) signaling in cell proliferation, neuronal motility, and synaptic maturation. However, it is unknown whether NO plays a role in the development of the human nervous system. We used a model of human neuronal precursor cells from a well-characterized teratocarcinoma cell line (NT2). The precursor cells proliferate during retinoic acid treatment as spherical aggregate culture that stains for nestin and βIII-tubulin. Cells migrate out of the aggregates to acquire fully differentiated neuronal phenotypes. The cells express neuronal nitric oxide synthase and soluble guanylyl cyclase (sGC), an enzyme that synthesizes cGMP upon activation by NO. The migration of the neuronal precursor cell is blocked by the use of nNOS, sGC, and protein kinase G (PKG) inhibitors. Inhibition of sGC can be rescued by a membrane permeable analog of cGMP. In gain of function experiments the application of a NO donor and cGMP analog facilitate cell migration. Our results from the differentiating NT2 model neurons point towards a vital role of the NO/cGMP/PKG signaling cascade as positive regulator of cell migration in the developing human brain.     

Non‐cultivated grass hosts of yellow dwarf viruses in Ethiopia and their epidemiological consequences on cultivated cereals

The yellow dwarf (YD) disease complex epidemics in cultivated cereals grown in a specific period of the year mainly depend on the presence of potential reservoir alternative hosts harbouring both the viruses and the vectors over the off‐season and serve as a source of inoculum in subsequent cropping season, further spread being supported by efficient aphid vectors. As such, an extensive and intensive exploration to generate base line information on the identity and prevalence of YD viruses [barley yellow dwarf virus (BYDV)‐PAV, BYDV‐MAV and BYDV‐SGV; cereal yellow dwarf virus (CYDV)‐RPV; and maize yellow dwarf virus (MYDV)‐RMV] on wild annual and perennial grasses and forage cereals alternative hosts was conducted consecutively during 2013–2015 main‐ and short‐rainy seasons in cereals growing belts of Ethiopia. Random sampling was employed to collect the samples that were tested by the tissue blot immunoassay (TBIA) to identify the YDVs associated with the hosts using a battery of virus‐specific polyclonal antibodies. Of 13,604 samples analysed, YDVs were detected in 392 (2.9%) samples, which consisted of various wild grasses, forage cereals and three cultivated crops. YDVs were identified from at least 26 grass species and forage cereals, some of them are new records, and some are previously documented hosts. To our knowledge, this is the first report of YDV infection of Andropogon abyssinicus (FresenR.Br. ex Fresen.) (BYDV‐PAV), Avena abyssinica Hochst (BYDV‐PAV), Bromus pectinatus Thunb. (BYDV‐PAV and BYDV‐MAV), Eragrostis tef (Zuccagni) Trotter (BYDV‐PAV), Eragrostis sp. (BYDV‐PAV), Hyparrhenia anthistrioides Stapf. (BYDV‐PAV), Panicum coloratum L. (BYDV‐PAV), Polypogon monspeliensis (L.) Desf. (BYDV‐PAV), Setaria pumila (Poir.) Roem & Schult (BYDV‐PAV, BYDV‐SGV and MYDV‐RMV), Setaria australiensis (Scribn. & Merrill) Vickery (BYDV‐PAV, BYDV‐MAV and CYDV‐RPV) and Snowdenia polystachya (Fresen.) Pilg (BYDV‐PAV, BYDV‐MAV, BYDV‐SGV, CYDV‐RPV and MYDV‐RMV).     

Anticancer and Apoptotic Activity in Cervical Adenocarcinoma HeLa Using Crude Extract of Ganoderma applanatum

Cancer is currently one of the foremost health challenges and a leading cause of death worldwide. Cervical cancer is caused by cofactors, including oral contraceptive use, smoking, multiparity, and HIV infection. One of the major and considerable etiologies is the persistent infection of the oncogenic human papilloma virus. G. applanatum is a valuable medicinal mushroom that has been widely used as a folk medicine for the treatment and prevention of various diseases. In this study, we obtained crude extract from G. applanatum mushroom with a subcritical water extraction method; cell viability assay was carried out and the crude extract showed an antiproliferative effect in HeLa cells with IC₅₀ of 1.55 ± 0.01 mg/mL; however, it did not show any sign of toxicity in HaCaT. Protein expression was detected by Western blot, stability of IκBα and downregulation of NFκB, IKKα, IKKβ, p-NFκB-65(Ser 536) and p-IKKα/β(Ser 176/180), suggesting loss of survival in a dose-dependent manner. RT-qPCR revealed RNA/mRNA expression; fold changes of gene expression in Apaf-1, caspase-3, cytochrome-c, caspase-9, Bax and Bak were increased, which implies apoptosis, and NFκB was decreased in a dose-dependent manner. DNA fragmentation was seen in the treatment groups as compared to the control group using gel electrophoresis. Identification and quantification of compounds were carried out by GC–MS and HPLC, respectively; 2(5H)furanone with IC₅₀ of 1.99 ± 0.01 μg/mL could be the responsible anticancer compound. In conclusion, these findings suggest the potential use of the crude extract of G. applanatum as a natural source with anticancer activity against cervical cancer.     

Production of thermotolerant entomopathogenic fungal conidia on millet grain

Thermotolerance of entomopathogenic (insect-killing) fungi should be seriously considered before industrialization. This work describes the feasibility of millet grain as a substrate for production of thermotolerant Beauveria bassiana (Bb) GHA and ERL1170 and Metarhizium anisopliae (Ma) ERL1171 and ERL1540 conidia. First, conidial suspensions of the Bb isolates, produced on millet grain in polyethylene bags, were exposed to five temperatures (43–47°C) at 15-min intervals for up to 120 min (experiment I). Agar-based quarter-strength (¼) Sabouraud dextrose agar supplemented with yeast extract (SDAY) and whey permeate media served as controls. Millet-grain-based culture was superior in producing the most thermotolerant Bb conidia, followed by whey permeate agar and ¼SDAY-based cultures. Secondly, to compare the thermotolerance of conidia produced at the same conditions, the Bb isolates were then produced on agar-based millet powder medium, with ¼SDAY and whey permeate agar media as controls, and the two Ma isolates were added (experiment II). They were then exposed to the same temperatures as above. More thermotolerant Bb and Ma conidia were produced on millet powder agar than on whey permeate agar and ¼SDAY overall. These results suggest that millet grain can be used as a substrate to produce thermotolerant conidia in a mass production system.     

Computer-aided molecular design of 1H-imidazole-2,4-diamine derivatives as potential inhibitors of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> DHFR enzyme

Design and discovery of new potential inhibitors of Plasmodium falciparum dihydrofolate reductase (PfDHFR), equally active against both the wild-type and mutant strains, is urgently needed. In this study, a computer-aided molecular design approach that involved ab initio molecular orbital and density functional theory calculations, along with molecular electrostatic potential analysis, and molecular docking studies was employed to design 15 1H-imidazole-2,4-diamine derivatives as potential inhibitors of PfDHFR enzyme. Visual inspection of the binding modes of the compounds demonstrated that they all interact, via H-bond interactions, with key amino acid residues (Asp54, Ileu/Leu164, Asn/Ser108 and Ile14) similar to those of WR99210 (3) in the active site of the enzymes used in the study. These interactions are known to be essential for enzyme inhibition. These compounds showed better or comparable binding affinities to that of the bound ligand (WR99210). In silico toxicity predictions, carried out using TOPKAT software, also indicated that the compounds are non-toxic.     

Diagnosis of latent tuberculosis infection in healthy young adults in a country with high tuberculosis burden and BCG vaccination at birth

ABSTRACT: BACKGROUND: One third of the world's population is thought to have latent tuberculosis infection (LTBI) with the potential for subsequent reactivation of disease. To better characterize this important population, studies comparing Tuberculin Skin Test (TST) and the new interferon-gamma release assays including QuantiFERON(R)-TB Gold In-Tube (QFT-GIT) have been conducted in different parts of the world, but most of these have been in countries with a low incidence of tuberculosis (TB). OBJECTIVE: To evaluate the use of QFT-GIT assay as compared with TST in the diagnosis of LTBI in a country with a high burden of TB and routine BCG vaccination at birth. METHODS: Healthy medical and paramedical male students at the Faculty of Medicine, Addis Ababa University, Ethiopia were enrolled into the study from December 2008 to February 2009. The TST and QFTG-IT assay were performed using standard methods. RESULTS: The mean age of the study participants was 20.9 years. From a total of 107 study participants, 46.7% (95%CI: 37.0% to 56.6%) had a positive TST result (TST greater than or equal to 10 mm), 43.9% (95%CI: 34.3% to 53.9%) had a positive QFT-GIT assay result and 44.9% (95%CI: 35.2% to 54.8%) had BCG scar. There was strong agreement between TST (TST greater than or equal to 10mm) and QFT-GIT assay (Kappa = 0.83, p value=0.000). CONCLUSION: The TST and QFT-GIT assay show similar efficacy for the diagnosis of LTBI in healthy young adults residing in Ethiopia, a country with high TB incidence.     

Genetic consequences of Pleistocene range shifts: contrast between the Arctic, the Alps and the East African mountains

In wide-ranging species, the genetic consequences of range shifts in response to climate change during the Pleistocene can be predicted to differ among different parts of the distribution area. We used amplified fragment length polymorphism data to compare the genetic structure of Arabis alpina, a widespread arctic-alpine and afro-alpine plant, in three distinct parts of its range: the North Atlantic region, which was recolonized after the last ice age, the European Alps, where range shifts were probably primarily altitudinal, and the high mountains of East Africa, where the contemporary mountain top populations result from range contraction. Genetic structure was inferred using clustering analyses and estimates of genetic diversity within and between populations. There was virtually no diversity in the vast North Atlantic region, which was probably recolonized from a single refugial population, possibly located between the Alps and the northern ice sheets. In the European mountains, genetic diversity was high and distinct genetic groups had a patchy and sometimes disjunct distribution. In the African mountains, genetic diversity was high, clearly structured and partially in accordance with a previous chloroplast phylogeography. The fragmented structure in the European and African mountains indicated that A. alpina disperses little among established populations. Occasional long-distance dispersal events were, however, suggested in all regions. The lack of genetic diversity in the north may be explained by leading-edge colonization by this pioneer plant in glacier forelands, closely following the retracting glaciers. Overall, the genetic structure observed corresponded to the expectations based on the environmental history of the different regions.