Acardiac anomaly spectrum

BACKGROUND: Acardiac anomaly spectrum is a rare congenital malformation found in monozygotic twin pregnancy. Besides the absence of heart, the condition is associated with variable grades of developmental disruption. Thus, no two cases are similar. METHODS: This case report is based on physical examination and autopsy findings. RESULTS: The twin had acardia and partial development of head and face. There was complete absence of upper extremities. CONCLUSIONS: The twin reversed arterial perfusion (TRAP) theory is the most accepted etiology of the disorder. Normally, the cephalic pole is the most severely affected, being most distal to the retrograde perfusion. In acardia, partial development of head, face, and brain is usually associated with the development of the upper extremities. However, in the present case, there was extensive cephalic development in the absence of upper extremity development.     

Human carbonic anhydrase I: Effect of specific site mutations on its function

Carbonic anhydrase I (CAI) is one out of ten CA isoenzymes that have been identified in humans. X-ray crystallographic and inhibitor complex studies of human carbonic anhydrase I (HCAI) and related studies in other CA isoenzymes identified several residues, in particular Thr199, GlulO6, Tyr7, Glull7, His l07, with likely involvement in the catalytic activity of HCAI. To further study the role of these residues, we undertook, site-directed mutagenesis of HCAI. Using a polymerase chain reaction based strategy and altered oligonucleotide primers, we modified a cloned wild type hCAI gene so as to produce mutant genes encoding proteins with single amino acid substitutions. Thrl99Val, Thrl99Cys, Thr199Ser, GlulO6Ile, Glul06Gln, Tyr7Trp, Glu.117Gln, and His 107Val mutations were thus generated and the activity of each measured by ester hydrolysis. Overproduction of the Glu117Gln and HisI07Val mutant proteins inEscherichia coli resulted in a large proportion of the enzyme forming aggregates probably due to folding defect. The mutations Thr199Val, GlulO6Ile and GlulO6Gln gave soluble protein with drastically reduced enzyme activity, while the Tyr7Trp mutation had only marginal effect on the activity, thus s.uggesting important roles for Thr199 and Glu lO6 but not for Tyr7 in the catalytic function of HCAI.     

In Vitro Culture of Functionally Active Buffalo Hepatocytes Isolated by Using a Simplified Manual Perfusion Method

Background

In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.

Results

Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3±0.66)×10⁷ cells per gram of liver tissue with a viability of 82.3±3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.

Conclusion

We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes for studying gene expression, regulation, hepatic genomics and proteomics in farm animals.     

Computational Identification of Post Translational Modification Regulated RNA Binding Protein Motifs

RNA and its associated RNA binding proteins (RBPs) mitigate a diverse array of cellular functions and phenotypes. The interactions between RNA and RBPs are implicated in many roles of biochemical processing by the cell such as localization, protein translation, and RNA stability. Recent discoveries of novel mechanisms that are of significant evolutionary advantage between RBPs and RNA include the interaction of the RBP with the 3’ and 5’ untranslated region (UTR) of target mRNA. These mechanisms are shown to function through interaction of a trans-factor (RBP) and a cis-regulatory element (3’ or 5’ UTR) by the binding of a RBP to a regulatory-consensus nucleic acid motif region that is conserved throughout evolution. Through signal transduction, regulatory RBPs are able to temporarily dissociate from their target sites on mRNAs and induce translation, typically through a post-translational modification (PTM). These small, regulatory motifs located in the UTR of mRNAs are subject to a loss-of-function due to single polymorphisms or other mutations that disrupt the motif and inhibit the ability to associate into the complex with RBPs. The identification of a consensus motif for a given RBP is difficult, time consuming, and requires a significant degree of experimentation to identify each motif-containing gene on a genomic scale. We have developed a computational algorithm to analyze high-throughput genomic arrays that contain differential binding induced by a PTM for a RBP of interest–RBP-PTM Target Scan (RPTS). We demonstrate the ability of this application to accurately predict a PTM-specific binding motif to an RBP that has no antibody capable of distinguishing the PTM of interest, negating the use of in-vitro exonuclease digestion techniques.     

Treatment of W. bancrofti (Wb) in HIV/Wb Coinfections in South India

BackgroundThe disease course of human immunodeficiency virus (HIV) is often altered by existing or newly acquired coincident infections.Conclusions/SignificanceWe were unable to find a significant effect of W. bancrofti infection or its treatment on HIV clinical course or surrogate markers of HIV disease progression though we recognized that our study was limited by the smaller than predicted sample size and by the use of ART in half of the patients. Treatment of W. bancrofti coinfection in HIV positive subjects (as is usual in mass drug administration campaigns) did not represent an increased risk to the subjects, and should therefore be considered for PLWHA living in W. bancrofti endemic areas.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00344279","term_id":"NCT00344279"}}NCT00344279     

Reduced Model Captures Mg-RNA Interaction Free Energy of Riboswitches

The stability of RNA tertiary structures depends heavily on Mg²⁺. The Mg²⁺-RNA interaction free energy that stabilizes an RNA structure can be computed experimentally through fluorescence-based assays that measure Γ₂₊, the number of excess Mg²⁺ associated with an RNA molecule. Previous explicit-solvent simulations predict that the majority of excess Mg²⁺ ions interact closely and strongly with the RNA, unlike monovalent ions such as K⁺, suggesting that an explicit treatment of Mg²⁺ is important for capturing RNA dynamics. Here we present a reduced model that accurately reproduces the thermodynamics of Mg²⁺-RNA interactions. This model is able to characterize long-timescale RNA dynamics coupled to Mg²⁺ through the explicit representation of Mg²⁺ ions. KCl is described by Debye-Hückel screening and a Manning condensation parameter, which represents condensed K⁺ and models its competition with condensed Mg²⁺. The model contains one fitted parameter, the number of condensed K⁺ ions in the absence of Mg²⁺. Values of Γ₂₊ computed from molecular dynamics simulations using the model show excellent agreement with both experimental data on the adenine riboswitch and previous explicit-solvent simulations of the SAM-I riboswitch. This agreement confirms the thermodynamic accuracy of the model via the direct relation of Γ₂₊ to the Mg²⁺-RNA interaction free energy, and provides further support for the predictions from explicit-solvent calculations. This reduced model will be useful for future studies of the interplay between Mg²⁺ and RNA dynamics.     

Chromatin-bound PCNA as S-phase marker in mononuclear blood cells of patients with acute lymphoblastic leukaemia or multiple myeloma

Objectives: Proliferating cell nuclear antigen (PCNA) has often been used as a marker to aid assessment of tumour growth fraction. This paper addresses the question of whether it can be used as an S‐phase marker, when the non‐chromatin‐bound form of the protein is removed by pepsin treatment. Materials and methods: Cytofluorometric measurements were carried out after immunofluorescence staining of PCNA and counterstaining of DNA. S‐phase fraction was determined with the help of windows on PCNA versus DNA scattergrams, or mathematically from DNA histograms. Results: S‐phase fractions obtained using the two methods correlated well, but did not always agree, exact discrepancies depending on the mathematical model used for histogram analysis. Conclusions: Determination of S‐phase fractions with the help of PCNA immunofluorescence staining is possible, and probably more reliable than calculation of S‐fractions from DNA histograms. It thus offers an alternative to assays involving BrdU labelling in vivo.     

Complete sequence and organisation of the <Emphasis Type="Italic">Jatropha curcas</Emphasis> (Euphorbiaceae) chloroplast genome

Jatropha curcas is an important non-edible oil seed tree species and is considered a promising source of biodiesel. The complete nucleotide sequence of J. curcas chloroplast genome (cpDNA) was determined by pyrosequencing and gaps filled by Sanger sequencing. The cpDNA is a circular molecule of 163,856 bp in length and codes for 110 distinct genes (78 protein coding, four rRNA and 28 distinct tRNA). Genome organisation and arrangement are similar to the reported angiosperm chloroplast genome. However, in Jatropha, the infA and the rps16 genes are non-functional. The inverted repeat (IR) boundary is within the rpl2 gene, and the 13 nucleotides at the ends of the two duplicate genes are different. Repeat analysis suggests the presence of 72 repeat regions (>30 bp) apart from the IR; of these, 48 were direct and 24 were palindromic repeats. Phylogenetic analysis of 81 protein coding chloroplast genes from 65 taxa by maximum parsimony, maximum likelihood and minimum evolution analyses at 100 bootstraps provide strong support for the placement of inaperturate crotonoids of which Jatropha is a member as sister to articulated crotonoids of which Manihot is a member.     

Ethanol fermentation of mahula (<Emphasis Type="Italic">Madhuca latifolia</Emphasis>) flowers using free and immobilized bacteria <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> MTCC 92

Mahula (Madhuca latifolia L.) is a deciduous tree commonly found in the tropical rain forests of Asian and Australian continent. Corolla, the edible part of its flowers, is rich in fermentable sugar (37 ± 0.23%; on dry weight basis). Batch fermentation of mahula flowers was carried out using Zymomonas mobilis MTCC 92 free cells and cells immobilized in calcium alginate matrix. The ethanol productions were 122.9 ± 0.972 and 134.6 ± 0.104 g/kg flowers on dry weight basis using free and immobilized cells, respectively, after 96 h of fermentation, which showed that cells entrapped in calcium alginate matrix yielded 8.7% more ethanol than free cells. Further, the immobilized cells were physiologically active up to three more cycles of fermentation producing 132.7 ± 0.095, 130.5 ± 0.09 and 128.7 ± 0.056 g ethanol per kg flower in first, second and third cycle, respectively.