Antiport Mechanism for Cl/H in ClC-ec1 from Normal-Mode Analysis

ClC chloride channels and transporters play major roles in cellular excitability, epithelial salt transport, volume, pH, and blood pressure regulation. One family member, ClC-ec1 from Escherichia coli, has been structurally resolved crystallographically and subjected to intensive mutagenetic, crystallographic, and electrophysiological studies. It functions as a Cl⁻/H⁺ antiporter, not a Cl⁻ channel; however, the molecular mechanism for Cl⁻/H⁺ exchange is largely unknown. Using all-atom normal-mode analysis to explore possible mechanisms for this antiport, we propose that Cl⁻/H⁺ exchange involves a conformational cycle of alternating exposure of Cl⁻ and H⁺ binding sites of both ClC pores to the two sides of the membrane. Both pores switch simultaneously from facing outward to facing inward, reminiscent of the standard alternating-access mechanism, which may have direct implications for eukaryotic Cl⁻/H⁺ transporters and Cl⁻ channels.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Structure based virtual screening of ligands to identify cysteinyl leukotriene receptor 1 antagonist

Montelukast and Zafirlukast are known leukotriene receptor antagonists prescribed in asthma treatment. However, these fall short as mono therapy and are frequently used in combination with inhaled glucocorticosteroids with or without long acting beta 2 agonists. Therefore, it is of interest to apply ligand and structure based virtual screening strategies to identify compounds akin to lead compounds Montelukast and Zafirlukast. Hence, compounds with structures having 95% similarity to these compounds were retrieved from NCBI׳s PubChem database. Compounds similar to lead were grouped and docked at the antagonist binding site of cysteinyl leukotriene receptor 1. This exercise identified compounds UNII 70RV86E50Q (Pub Cid 71587778) and Sure CN 9587085 (Pub Cid 19793614) with higher predicted binding compared to Montelukast and Zafirlukast. It is shown that the compound Sure CN 9587085 showed appreciable ligand receptor interaction compared to UNII 70RV86E50Q. Thus, the compound Sure CN 9587085 is selected as a potent antagonist to cysteinyl leukotriene receptor 1 for further consideration in vitro and in vivo validation.     

The open state gating mechanism of gramicidin a requires relative opposed monomer rotation and simultaneous lateral displacement

The gating mechanism of the open state of the gramicidin A (gA) channel is studied by using a new Monte Carlo Normal Mode Following (MC-NMF) technique, one applicable even without a target structure. The results demonstrate that the lowest-frequency normal mode (NM) at approximately 6.5 cm(-1) is the crucial mode that initiates dissociation. Perturbing the gA dimer in either direction along this NM leads to opposed, nearly rigid-body rotations of the gA monomers around the central pore axis. Tracking this NM by using the eigenvector-following technique reveals the channel's gating mechanism: dissociation via relative opposed monomer rotation and simultaneous lateral displacement. System evolution along the lowest-frequency eigenvector shows that the large-amplitude motions required for gating (dissociation) are not simple relative rigid-body motions of the monomers. Gating involves coupling intermonomer hydrogen bond breaking, backbone realignment, and relative monomer tilt with complex side chain reorganization at the intermonomer junction.     

Conformational changes in the selectivity filter of the open-state KcsA channel: an energy minimization study

Potassium channels switch between closed and open conformations and selectively conduct K⁺ ions. There are at least two gates. The TM2 bundle at the intracellular site is the primary gate of KcsA, and rearrangements at the selectivity filter (SF) act as the second gate. The SF blocks ion flow via an inactivation process similar to C-type inactivation of voltage-gated K⁺ channels. We recently generated the open-state conformation of the KcsA channel. We found no major, possibly inactivating, structural changes in the SF associated with this massive inner-pore rearrangement, which suggests that the gates might act independently. Here we energy-minimize the open state of wild-type and mutant KcsA, validating in silico structures of energy-minimized SFs by comparison with crystallographic structures, and use these data to gain insight into how mutation, ion depletion, and K⁺ to Na⁺ substitution influence SF conformation. Both E71 or D80 protonations/mutations and the presence/absence of protein-buried water molecule(s) modify the H-bonding network stabilizing the P-loops, spawning numerous SF conformations. We find that the inactivated state corresponds to conformations with a partially unoccupied or an entirely empty SF. These structures, involving modifications in all four P-loops, are stabilized by H-bonds between amide H and carbonyl O atoms from adjacent P-loops, which block ion passage. The inner portions of the P-loops are more rigid than the outer parts. Changes are localized to the outer binding sites, with innermost site S4 persisting in the inactivated state. Strong binding by Na⁺ locally contracts the SF around Na⁺, releasing ligands that do not participate in Na⁺ coordination, and occluding the permeation pathway. K⁺ selectivity primarily appears to arise from the inability of the SF to completely dehydrate Na⁺ ions due to basic structural differences between liquid water and the “quasi-liquid” SF matrix.     

Identification of structural DNA variations in human cell cultures after long-term passage

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8–12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.     

Weekly vs. tri-weekly cisplatin based chemoradiation in carcinoma cervix: a prospective randomized study of toxicity and compliance

BackgroundAddition of chemotherapy to radiation has improved 5-year survival by 6%. However, the optimal dose and schedule of concurrent cisplatin is not well defined, though widely accepted practice is the weekly schedule of 40 mg/m² for 5 weeks. Repeated admissions for weekly cisplatin drain the limited resources in high volume centres. We intended to study the compliance and toxicity of two cisplatin schedules in our patients diagnosed with carcinoma cervix.Materials and methodsBetween 2007–2011, 212 patients, histologically proven squamous cell carcinoma with stages IIB to IIIB were randomized into two arms. All patients were planned for external beam radiotherapy 45 Gy/25 frs over 5 weeks followed by Intracavitary or Interstitial brachytherapy to a total BED dose of 75–85 Gy. Single agent cisplatin given concomitantly, was scheduled weekly (40 mg/m²/cycle, 5 cycles) in an arm A and three weekly (100 mg/m²/cycle, 2 cycles) in an arm B. Toxicity and compliance were evaluated weekly according to the RTOG guidelines. Analysis of the compiled data was done using SSPS version 20.ResultsOf the evaluable 212, 109 patients received weekly cisplatin chemotherapy and 103 patients received three weekly cisplatin. The most common acute toxicity observed was grade I–II leucopoenia. The upper and lower gastrointestinal reactions were high in three weekly arms, which was statistically significant (57% and 42.7%, p < 0.05). Proctitis was observed in 10% of patients in both of the arms and only two patients had Gr1 Cystitis after 6 months of treatment.ConclusionsTri-weekly cisplatin based concurrent chemoradiation can be adopted in high volume centres with manageable haematological and gastrointestinal acute toxicities.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

Open-state conformation of the KcsA K+ channel: Monte Carlo normal mode following simulations

Potassium channels fluctuate between closed and open states. The detailed mechanism of the conformational changes opening the intracellular pore in the K+ channel from Streptomyces lividans (KcsA) is unknown. Applying Monte Carlo normal mode following, we find that gating involves rotation and unwinding of the TM2 bundle, lateral movement of the TM2 helices away from the channel axis, and disappearance of the TM2 bundle. The open-state conformation of KcsA exhibits a very wide inner vestibule, with a radius approximately 5-7 A and inner helices bent at the A98-G99 hinge. Computed conformational changes demonstrate that spin labeling and X-ray experiments illuminate different stages in gating: transition begins with clockwise rotation of the TM2 helices ending at a final state with the TM2 bend hinged near residues A98-G99. The concordance between the computational and experimental results provides atomic-level insights into the structural rearrangements of the channel's inner pore.     

Genetic diversity and relationships in mulberry (genusMorus) as revealed by RAPD and ISSR marker assays

Background  

The genus Morus, known as mulberry, is a dioecious and cross-pollinating plant that is the sole food for the domesticated silkworm, Bombyx mori. Traditional methods using morphological traits for classification are largely unsuccessful in establishing the diversity and relationships among different mulberry species because of environmental influence on traits of interest. As a more robust alternative, PCR based marker assays including RAPD and ISSR were employed to study the genetic diversity and interrelationships among twelve domesticated and three wild mulberry species.