Model of a putative pore: the pentameric alpha-helical bundle of SARS coronavirus E protein in lipid bilayers

The coronavirus responsible for the severe acute respiratory syndrome contains a small envelope protein, E, with putative involvement in host apoptosis and virus morphogenesis. To perform these functions, it has been suggested that protein E can form a membrane destabilizing transmembrane (TM) hairpin, or homooligomerize to form a TM pore. Indeed, in a recent study we reported that the alpha-helical putative transmembrane domain of E protein (ETM) forms several SDS-resistant TM interactions: a dimer, a trimer, and two pentameric forms. Further, these interactions were found to be evolutionarily conserved. Herein, we have studied multiple isotopically labeled ETM peptides reconstituted in model lipid bilayers, using the orientational parameters derived from infrared dichroic data. We show that the topology of ETM is consistent with a regular TM alpha-helix. Further, the orientational parameters obtained unequivocally correspond to a homopentameric model, by comparison with previous predictions. We have independently confirmed that the full polypeptide of E protein can also aggregate as pentamers after expression in Escherichia coli. This interaction must be stabilized, at least partially, at the TM domain. The model we report for this pentameric alpha-helical bundle may explain some of the permabilizing properties of protein E, and should be the basis of mutagenesis efforts in future functional studies.     

The impact of administration of tranexamic acid in reducing the use of red blood cells and other blood products in cardiac surgery

Background

Cervicogenic headache (CEH) is a unilateral headache localised in the neck or occipital region, projecting to the frontal and temporal regions. Since the pathogenesis of this syndrome appears to have an anatomical basis in the cervical region, several surgical procedures aimed at reducing the nociceptive input on the cervical level, have been tested. We developed a sequence of various cervical radiofrequency neurotomies (facet joint denervations eventually followed by upper dorsal root ganglion neurotomies) that proved successful in a prospective pilot trial with 15 CEH patients. To further evaluate this sequential treatment program we conducted a randomised controlled trial

Methods

30 patients with cervicogenic headache according to the Sjaastad diagnostic criteria, were randomised. 15 patients received a sequence of radiofrequency treatments (cervical facet joint denervation, followed by cervical dorsal root ganglion lesions when necessary), and the other 15 patients underwent local injections with steroid and anaesthetic at the greater occipital nerve, followed by transcutaneous electrical nerve stimulation (TENS) when necessary. Visual analogue scores for pain, global perceived effects scores, quality of life scores were assessed at 8, 16, 24 and 48 weeks. Patients also kept a headache diary.

Results

There were no statistically significant differences between the two treatment groups at any time point in the trial.

Conclusion

We did not find evidence that radiofrequency treatment of cervical facet joints and upper dorsal root ganglions is a better treatment than the infiltration of the greater occipital nerve, followed by TENS for patients fulfilling the clinical criteria of cervicogenic headache.     

Structure of a novel phosphotyrosine-binding domain in Hakai that targets E-cadherin

Phosphotyrosine-binding domains, typified by the SH2 (Src homology 2) and PTB domains, are critical upstream components of signal transduction pathways. The E3 ubiquitin ligase Hakai targets tyrosine-phosphorylated E-cadherin via an uncharacterized domain. In this study, the crystal structure of Hakai (amino acids 106-206) revealed that it forms an atypical, zinc-coordinated homodimer by utilizing residues from the phosphotyrosine-binding domain of two Hakai monomers. Hakai dimerization allows the formation of a phosphotyrosine-binding pocket that recognizes specific phosphorylated tyrosines and flanking acidic amino acids of Src substrates, such as E-cadherin, cortactin and DOK1. NMR and mutational analysis identified the Hakai residues required for target binding within the binding pocket, now named the HYB domain. ZNF645 also possesses a HYB domain but demonstrates different target specificities. The HYB domain is structurally different from other phosphotyrosine-binding domains and is a potential drug target due to its novel structural features.     

Differential regulation of CD4+ T helper cell responses by curcumin in experimental autoimmune encephalomyelitis

Nutraceuticals and phytochemicals are important regulators of human health and diseases. Curcumin is a polyphenolic phytochemical isolated from the rhizome of the plant Curcuma longa (turmeric) that has been traditionally used for the treatment of inflammation and wound healing for centuries. Systematic analyses have shown that curcumin exerts its beneficial effects through antioxidant, antiproliferative and anti-inflammatory properties. We and others have shown earlier that curcumin ameliorates experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis. In this study, we show that C57BL/6 mice induced to develop EAE express elevated levels of interferon (IFN) γ and interleukin (IL)-17 in the central nervous system (CNS) and lymphoid organs that decreased significantly following in vivo treatment with curcumin. The EAE mice also showed elevated expression of IL-12 and IL-23 that decreased after treatment with curcumin. Ex vivo and in vitro treatment with curcumin resulted in a dose-dependent decrease in the secretion of IFNγ, IL-17, IL-12 and IL-23 in culture. The inhibition of EAE by curcumin was also associated with an up-regulation of IL-10, peroxisome proliferator activated receptor γ and CD4⁺CD25⁺Foxp3⁺ Treg cells in the CNS and lymphoid organs. These findings highlight that curcumin differentially regulates CD4⁺ T helper cell responses in EAE.     

Preliminary characterization of exopolysaccharides produced by a marine biofilm-forming bacterium Pseudoalteromonas ruthenica (SBT 033)

AIM: The major objective of the present study was the partial characterization of the exopolysaccharides (EPS) produced by a marine biofilm-forming bacterium Pseudoalteromonas ruthenica under shake culture conditions. METHODS AND RESULTS: EPS-producing bacterial cultures were isolated from the sea water collected from the vicinity of coastal electric power station. Zobell marine broth medium was used for growth of the cultures and the EPS produced was quantified using phenol sulfuric acid method. Chemical characterization of the EPS was carried out using Fourier transform infrared spectroscopy (FTIR), and capillary gas chromatography (GC). Further, viscosity and rheological properties of the purified EPS were studied. The FTIR spectrum revealed prominent peaks of various groups of OH and CH(3) bending. GC analysis showed the presence of eight individual sugars. Rheological studies of the aqueous EPS showed good shearing property. CONCLUSIONS: Pseudoalteromonas ruthenica isolated from marine environment produced copious amount of EPS under shake culture conditions. GC analysis of the EPS revealed the presence of eight individual sugars and the EPS had good shearing property. SIGNIFICANCE AND IMPACT OF THE STUDY: The EPS produced by P. ruthenica is pseudoplastic in nature and is stable at higher pH levels. These properties suggest that the EPS may have potential applications in the oil, textiles and food industries.     

Evidence for Hydroxyl Radical Scavenging Action of Nitric Oxide Donors in the Protection Against 1-Methyl-4-phenylpyridinium-induced Neurotoxicity in Rats

In the present study we provide evidence for hydroxyl radical (^•OH) scavenging action of nitric oxide (NO^•), and subsequent dopaminergic neuroprotection in a hemiparkinsonian rat model. Reactive oxygen species are strongly implicated in the nigrostriatal dopaminergic neurotoxicity caused by the parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP⁺). Since the role of this free radical as a neurotoxicant or neuroprotectant is debatable, we investigated the effects of some of the NO^• donors such as S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine hydrochloride (SIN-1), sodium nitroprusside (SNP) and nitroglycerin (NG) on in vitro ^•OH generation in a Fenton-like reaction involving ferrous citrate, as well as in MPP⁺-induced ^•OH production in the mitochondria. We also tested whether co-administration of NO^• donor and MPP⁺ could protect against MPP⁺-induced dopaminergic neurotoxicity in rats. While NG, SNAP and SIN-1 attenuated MPP⁺-induced ^•OH generation in the mitochondria, and in a Fenton-like reaction, SNP caused up to 18-fold increase in ^•OH production in the latter reaction. Striatal dopaminergic depletion following intranigral infusion of MPP⁺ in rats was significantly attenuated by NG, SNAP and SIN-1, but not by SNP. Solutions of NG, SNAP and SIN-1, exposed to air for 48 h to remove NO^•, when administered similarly failed to attenuate MPP⁺-induced neurotoxicity in vivo. Conversely, long-time air-exposed SNP solution when administered in rats intranigrally, caused a dose-dependent depletion of the striatal dopamine. These results confirm the involvement of ^•OH in the nigrostriatal degeneration caused by MPP⁺, indicate the ^•OH scavenging ability of NO^•, and demonstrate protection by NO^• donors against MPP⁺-induced dopaminergic neurotoxicity in rats.     

Substituting dietary linoleic acid with α-linolenic acid improves insulin sensitivity in sucrose fed rats

This study describes the effect of substituting dietary linoleic acid (18:2 n-6) with α-linolenic acid (18:3 n-3) on sucrose-induced insulin resistance (IR). Wistar NIN male weanling rats were fed casein based diet containing 22 energy percent (en%) fat with ～6, 9 and 7 en% saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) respectively for 3 months. IR was induced by replacing starch (ST) with sucrose (SU). Blends of groundnut, palmolein, and linseed oil in different proportions furnished the following levels of 18:3 n-3 (g/100 g diet) and 18:2 n-6/18:3 n-3 ratios respectively: ST-220 (0.014, 220), SU-220 (0.014, 220), SU-50 (0.06, 50), SU-10 (0.27, 10) and SU-2 (1.1, 2). The results showed IR in the sucrose fed group (SU-220) as evidenced by increase in fasting plasma insulin and area under the curve (AUC) of insulin in response to oral glucose load. In SU-220, the increase in adipocyte plasma membrane cholesterol/phospholipid ratio was associated with a decrease in fluidity, insulin stimulated glucose transport, antilipolytic effect of insulin and increase in basal and norepinephrine stimulated lipolysis in adipocytes. In SU-50, sucrose induced alterations in adipocyte lipolysis and antilipolysis were normalized. However, in SU-2, partial corrections in plasma insulin, AUC of insulin and adipocyte insulin stimulated glucose transport were observed. Further, plasma triglycerides and cholesterol decreased in SU-2. In diaphragm phospholipids, the observed dose dependent increase in long chain (LC) n-3 PUFA was associated with a decrease in LC-n-6 PUFA but insulin stimulated glucose transport increased only in SU-2. Thus, this study shows that the substitution of one-third of dietary 18:2 n-6 with 18:3 n-3 (SU-2) results in lowered blood lipid levels and increases peripheral insulin sensitivity, possibly due to the resulting high LCn-3 PUFA levels in target tissues of insulin action. These findings suggest a role for 18:3 n-3 in the prevention of insulin resistant states. The current recommendation to increase 18:3 n-3 intake for reducing cardiovascular risk may also be beneficial for preventing IR in humans.     

Molecular Epidemiology of Nontyphoidal Salmonella in Poultry and Poultry Products in India: Implications for Human Health

Human infections with non-typhoidal Salmonella (NTS) serovars are increasingly becoming a threat to human health globally. While all motile Salmonellae have zoonotic potential, Salmonella Enteritidis and Salmonella Typhimurium are most commonly associated with human disease, for which poultry are a major source. Despite the increasing number of human NTS infections, the epidemiology of NTS in poultry in India has not been fully understood. Hence, as a first step, we carried out epidemiological analysis to establish the incidence of NTS in poultry to evaluate the risk to human health. A total of 1215 samples (including poultry meat, tissues, egg and environmental samples) were collected from 154 commercial layer farms from southern India and screened for NTS. Following identification by cultural and biochemical methods, Salmonella isolates were further characterized by multiplex PCR, allele-specific PCR, enterobacterial repetitive intergenic consensus (ERIC) PCR and pulse field gel electrophoresis (PFGE). In the present study, 21/1215 (1.73 %) samples tested positive for NTS. We found 12/392 (3.06 %) of tissue samples, 7/460 (1.52 %) of poultry products, and 2/363 (0.55 %) of environmental samples tested positive for NTS. All the Salmonella isolates were resistant to oxytetracycline, which is routinely used as poultry feed additive. The multiplex PCR results allowed 16/21 isolates to be classified as S. Typhimurium, and five isolates as S. Enteritidis. Of the five S. Enteritidis isolates, four were identified as group D Salmonella by allele-specific PCR. All of the isolates produced different banding patterns in ERIC PCR. Of the thirteen macro restriction profiles (MRPs) obtained by PFGE, MRP 6 was predominant which included 6 (21 %) isolates. In conclusion, the findings of the study revealed higher incidence of contamination of NTS Salmonella in poultry tissue and animal protein sources used for poultry. The results of the study warrants further investigation on different type of animal feed sources, food market chains, processing plants, live bird markets etc., to evaluate the risk factors, transmission and effective control measures of human Salmonella infection from poultry products.     

Homology modeling and atomic level binding study of Leishmania MAPK with inhibitors

The current therapy for leishmaniasis is not sufficient and it has two severe drawbacks, host-toxicity and drug resistance. The substantial knowledge of parasite biology is not yet translating into novel drugs for leishmaniasis. Based on this observation, a 3D structural model of Leishmania mitogen-activated protein kinase (MAPK) homologue has been developed, for the first time, by homology modeling and molecular dynamics simulation techniques. The model provided clear insight in its structure features, i.e. ATP binding pocket, phosphorylation lip, and common docking site. Sequence-structure homology recognition identified Leishmania CRK3 (LCRK3) as a distant member of the MAPK superfamily. Multiple sequence alignment and 3D structure model provided the putative ATP binding pocket of Leishmania with respect to human ERK2 and LCRK3. This analysis was helpful in identifying the binding sites and molecular function of the Leishmania specific MAPK homologue. Molecular docking study was performed on this 3D structural model, using different classes of competitive ATP inhibitors of LCRK3, to check whether they exhibit affinity and could be identified as Leishmania MAPK specific inhibitors. It is well known that MAP kinases are extracellular signal regulated kinases ERK1 and ERK2, which are components of the Ras-MAPK signal transduction pathway which is complexed with HDAC4 protein, and their inhibition is of significant therapeutic interest in cancer biology. In order to understand the mechanism of action, docking of indirubin class of molecules to the active site of histone deacetylase 4 (HDAC4) protein is performed, and the binding affinity of the protein-ligand interaction was computed. The new structural insights obtained from this study are all consistent with the available experimental data, suggesting that the homology model of the Leishmania MAPK and its ligand interaction modes are reasonable. Further the comparative molecular electrostatic potential and cavity depth analysis of Leishmania MAPK and human ERK2 suggested several important differences in its ATP binding pocket. Such differences could be exploited in the future for designing Leishmania specific MAPK inhibitors.     

Manganese and cobalt recovery by surface display of metal binding peptide on various loops of OmpC in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In a cell-surface display (CSD) system, successful display of a protein or peptide is highly dependent on the anchoring motif and the position of the display in that anchoring motif. In this study, a recombinant bacterial CSD system for manganese (Mn) and cobalt (Co) recovery was developed by employing OmpC as an anchoring motif on three different external loops. A portion of Cap43 protein (TRSRSHTSEG)₃ was employed as a manganese and cobalt binding peptide (MCBP), which was fused with OmpC at three different external loops. The fusions were made at the loop 2 [fusion protein-2 (FP2)], loop 6 (FP6), and loop 8 (FP8) of OmpC, respectively. The efficacy of the three recombinant strains in the recovery of Mn and Co was evaluated by varying the concentration of the respective metal. Molecular modeling studies showed that the short trimeric repeats of peptide probably form a secondary structure with OmpC, thereby giving rise to a difference in metal recovery among the three recombinant strains. Among the three recombinant strains, FP6 showed increased metal recovery with both Mn and Co, at 1235.14 (1 mM) and 379.68 (0.2 mM) µmol/g dry cell weight (DCW), respectively.