Human Parechovirus Genotypes -10, -13 and -15 in Pakistani Children with Acute Dehydrating Gastroenteritis

Human parechoviruses are known to cause asymptomatic to severe clinical illness predominantly respiratory and gastroenetric infections. Despite their global prevalence, epidemiological studies have not been performed in Pakistan. In this study, we retrospectively analyzed 110 fecal specimen and found 26 (24%) positive for viral RNA with HPeV-10 (n = 3, 23%), HPeV-13 (n = 4, 31%) and HPeV-15 (n = 6, 46%) genotypes. Clinical features of patients with different HPeV genotypes were compared. All HPeV positive children were aged ≤4 years (mean 13.92 months). The male-to-female ratio was 1: 1.17 (46.2 vs 53.8%) with significant association (p = .031) to HPeV infectivity. HPeV-10 and -13 were found during summer while HPeV-15 was only detected during late winter season. Disease symptoms were more severe in children infected with HPeV-10 and -13 as compared to HPeV-15. Fever and vomiting were observed in 100% cases of HPeV-10 and -13 while only 17% patients of HPeV-15 had these complaints. Phylogenetic analyses showed that HPeV-10, -13 and -15 strains found in this study have 9–13%, 16.8% and 21.8% nucleotide divergence respectively from the prototype strains and were clustered to distinct genetic lineages. This is the first report of HPeV-15 infection in humans although first identified in rhesus macaques. The arginine-glycine-aspartic acid (RGD) motif present at the C-terminal of VP1 responsible for the viral attachment to cellular integrins was not found in all of these strains. In conclusion, these findings enhance our knowledge related to the epidemiology and genetic diversity of the HPeV in Pakistan and support the need for continued laboratory based surveillance programs especially in infants and neonatal clinical settings. Further, the parechovirus pathogenesis, cross-species transmission and disease reservoirs must be ascertained to adopt better prevention measures.     

Confidence Intervals for the Mean of a Poisson Distribution: A Review

The paper provides a comprehensive review of methodology for setting confidence intervals for the parameter of a Poisson distribution. The results are illustrated by a numerical example.     

One-pot conjugated linoleic acid production from castor oil by Rhizopus oryzae lipase and resting cells of Lactobacillus plantarum

Conjugated linoleic acid (CLA) has attracted as novel type of fatty acids having unusual health-promoting properties such as anticarcinogenic and antiobesitic effects. The present work employed castor oil as substrate for one-pot production of CLA using washed cells of Lactobacillus plantarum (L. plantarum) and lipases as catalysts. Among the screened lipases, the lipase Rhizopus oryzae (ROL) greatly assisted resting cells to produce CLA. Mass spectral analysis of the product showed that two major isomers of CLA were produced in the reaction mixture i.e. cis-9, trans-11 56.55% and trans-10, cis-12 43.45%. Optimum factors for CLA synthesis were found as substrate concentration (8 mg/mL), pH (6.5), washed cell concentration (12% w/v), and incubation time of 20 h. Hence, the combination of ROL with L. plantarum offers one pot production of CLA selectively using castor oil as a cost-effective substrate.     

Negative emotional biases in late chronotypes

Increasing evidence suggests evening chronotypes are at increased risk for developing depression. Here, we examined if, similar to acutely depressed patients, evening chronotype individuals display biases in emotional face recognition. Two hundred and twenty-six individuals completed an online survey including measures of sleep quality, depression/anxiety and chronotype followed by a simple emotion recognition task presenting male and female faces morphed in 10 steps between 0 (neutral) and 100% sad or happy. Evening chronotype was associated with increased recognition of sad facial expressions independently of sleep quality, mood, age and gender. The current results extend previous work indicating that negative biases in emotional processing are present in evening chronotypes and may have important implications for the prevention and treatment of depression in these vulnerable individuals.     

Design, synthesis and pharmacological evaluation of novel tetrasubstituted thiophene analogues as anti-inflammatory agents

A new series of tetrasubstituted thiophene analogues (4a-4f, 5a-5f and 8a-8i) were designed incorporating the pharmacophoric features of COX-1 (as in fenamates), 5-LOX and the p38 MAP kinase inhibitors. The designed series was synthesized by nucleophilic addition of aryl/aroylisothiocyanate and enamine (2) yielding the addition product l-(alpha-Carbomethoxy-beta-aminothiocrotonoyl)-aryl/aroyl amines (3/7); which on reaction with substituted phenacyl bromides gave the targeted tetrasubstituted thiophene esters (4a-4f / 8a-8i). The tetrasubstituted thiophenes esters (4a-4f ) on hydrolysis with one equivalent of potassium hydroxide solution in methanol at room temperature gave corresponding acids (5a-5f ). All the targeted compounds were evaluated for their anti-inflammatory activity in carrageenin-induced rat hind paw oedema model at the doses of 10, 20 and 40 mg/kg body weight using standard drugs mefanamic acid and ibuprofen. The compounds (4c, 4e, 4f, 5f, 8a- 8i) which gave reasonable protection to the inflamed paw, eliciting good or moderate comparable anti-inflammatory activity were selected for investigating their analgesic activity using acetic acid induced writhing response test in albino mice at 10 mg/kg dose using standard drug ibuprofen and in order to arrive at possible mechanism of their anti-inflammatory activity, in vitro antioxidant nitric oxide radical scavenging assay at the concentrations of 5, 10, 15, 20, 25, 30 and 35 microg/mL were performed using standard drug ascorbic acid.     

A modeled structure for amidase-03 from Bacillus anthracis

Homology models of amidase-03 from Bacillus anthracis were constructed using Modeller (9v2). Modeller constructs protein models using an automated approach for comparative protein structure modeling by the satisfaction of spatial restraints. A template structure of Listeria monocytogenes bacteriophage PSA endolysin PlyPSA (PDB ID: 1XOV) was selected from protein databank (PDB) using BLASTp with BLOSUM62 sequence alignment scoring matrix. We generated five models using the Modeller default routine in which initial coordinates are randomized and evaluated by pseudo-energy parameters. The protein models were validated using PROCHECK and energy minimized using the steepest descent method in GROMACS 3.2 (flexible SPC water model in cubic box of size 1 Å instead of rigid SPC model). We used G43a1 force field in GROMACS for energy calculations and the generated structure was subsequently analyzed using the VMD software for stereo-chemistry, atomic clash and misfolding. A detailed analysis of the amidase-03 model structure from Bacillus anthracis will provide insight to the molecular design of suitable inhibitors as drug candidates.     

The essential role of centrosomal NDE1 in human cerebral cortex neurogenesis

We investigated three families whose offspring had extreme microcephaly at birth and profound mental retardation. Brain scans and postmortem data showed that affected individuals had brains less than 10% of expected size (≤10 standard deviation) and that in addition to a massive reduction in neuron production they displayed partially deficient cortical lamination (microlissencephaly). Other body systems were apparently unaffected and overall growth was normal. We found two distinct homozygous mutations of NDE1, c.83+1G>T (p.Ala29GlnfsX114) in a Turkish family and c.684_685del (p.Pro229TrpfsX85) in two families of Pakistani origin. Using patient cells, we found that c.83+1G>T led to the use of a novel splice site and to a frameshift after NDE1 exon 2. Transfection of tagged NDE1 constructs showed that the c.684_685del mutation resulted in a NDE1 that was unable to localize to the centrosome. By staining a patient-derived cell line that carried the c.83+1G>T mutation, we found that this endogeneously expressed mutated protein equally failed to localize to the centrosome. By examining human and mouse embryonic brains, we determined that NDE1 is highly expressed in neuroepithelial cells of the developing cerebral cortex, particularly at the centrosome. We show that NDE1 accumulates on the mitotic spindle of apical neural precursors in early neurogenesis. Thus, NDE1 deficiency causes both a severe failure of neurogenesis and a deficiency in cortical lamination. Our data further highlight the importance of the centrosome in multiple aspects of neurodevelopment.     

Aromaticity of azines through dyotropic double hydrogen transfer reaction

The thermodynamic stabilities and IR spectra of the three water clusters (H₂O)_20, (H₂O)_54,, and (H₂O)₁₀₀ are studied by quantum-chemical computations. After full optimization of the (H₂O)_20,54,100 structures using the hybrid density functional B3LYP together with the 6-31+G(d,p) basis set, the electronic energies, zero-point energies, internal energies, enthalpies, entropies, and Gibbs free energies of the water clusters at 298 K are investigated. The OH stretching vibrational IR spectra of (H₂O)_20,54,100 are simulated and split into sub-spectra for different H-bond groups depending on the conformations of the hydrogen bonds. From the computed spectra the different spectroscopic fingerprint features of water molecules in different H-bond conformations in the water clusters are inferred.     

Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K _m and k _cat), thermodynamic stability (T _m and ΔH _m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.