Coupling of Calcium and Substrate Binding through Loop Alignment in the Outer-Membrane Transporter BtuB

In Gram-negative bacteria, TonB-dependent outer-membrane transporters bind large, scarce organometallic substrates with high affinity preceding active transport. The cobalamin transporter BtuB requires the additional binding of two Ca²⁺ ions before substrate binding can occur, but the underlying molecular mechanism is unknown. Using the crystallographic structures available for different bound states of BtuB, we have carried out extended molecular dynamics simulations of multiple functional states of BtuB to address the role of Ca²⁺ in substrate recruitment. We find that Ca²⁺ binding both stabilizes and repositions key extracellular loops of BtuB, optimizing interactions with the substrate. Interestingly, replacement by Mg²⁺ abolishes this effect, in accordance with experiments. Using a set of new force-field parameters developed for cyanocobalamin, we also simulated the substrate-bound form of BtuB, where we observed interactions not seen in the crystal structure between the substrate and loops previously found to be important for binding and transport. Based on our results, we suggest that the large size of cobalamin compared to other TonB-dependent transporter substrates explains the requirement of Ca²⁺ binding for high-affinity substrate recruitment in BtuB.     

Bio-shielding <Emphasis Type="Italic">In Situ</Emphasis> Forming Gels (BSIFG) Loaded With Lipospheres for Depot Injection of Quetiapine Fumarate: <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Evaluation

Quetiapine fumarate (QF), an anti-schizophrenic drug, suffers from rapid elimination and poor bioavailability due to extensive first-pass effect. Intramuscularly (IM) injected lipospheres were designed to enhance the drug’s bioavailability and extend its release. A central composite design was applied to optimize the liposphere preparation by a melt dispersion technique using Compritol® 888 ATO or glyceryl tristearate as lipid component and polyvinyl alcohol as surfactant. Lipospheres were evaluated for their particle size, entrapment efficiency, and in vitro release. The optimized QF lipospheres were prepared using a Compritol® 888 ATO fraction of 18.88% in the drug/lipid mixture under a stirring rate of 3979 rpm. The optimized lipospheres were loaded into a thermoresponsive in situ forming gel (TRIFG) and a liquid crystalline in situ forming gel (LCIFG) to prevent in vivo degradation by lipases. The loaded gels were re-evaluated for their in vitro release and injectability. Bioavailability of QF from liposphere suspension and bio-shielding in situ gels loaded with QF lipospheres were assessed in rabbits compared to drug suspension. Results revealed that the AUC_0–72 obtained from the liposphere-loaded TRIFG was ～3-fold higher than that obtained from the aqueous drug suspension indicating the bio-shielding effect of Poloxamer® 407 gel to inhibit the biodegradation of the lipospheres prolonging the residence of the drug in the muscle for higher absorption. Our results propose that bio-shielding in situ Poloxamer® 407 gels loaded with lipospheres is promising for the development of IM depot injection of drugs having extensive first-pass metabolism and rapid elimination.     

Molecular determinants of phospholipid synergy in blood clotting

Many regulatory processes in biology involve reversible association of proteins with membranes. Clotting proteins bind to phosphatidylserine (PS) on cell surfaces, but a clear picture of this interaction has yet to emerge. We present a novel explanation for membrane binding by GLA domains of clotting proteins, supported by biochemical studies, solid-state NMR analyses, and molecular dynamics simulations. The model invokes a single "phospho-L-serine-specific" interaction and multiple "phosphate-specific" interactions. In the latter, the phosphates in phospholipids interact with tightly bound Ca(2+) in GLA domains. We show that phospholipids with any headgroup other than choline strongly synergize with PS to enhance factor X activation. We propose that phosphatidylcholine and sphingomyelin (the major external phospholipids of healthy cells) are anticoagulant primarily because their bulky choline headgroups sterically hinder access to their phosphates. Following cell damage or activation, exposed PS and phosphatidylethanolamine collaborate to bind GLA domains by providing phospho-L-serine-specific and phosphate-specific interactions, respectively.     

Molecular diagnosis of Helicobacter pylori antibiotic resistance in the Taif region,Saudi Arabia

Success in eradication of Helicobacter pylori is declining globally because H. pylori has developed resistance against most of the antibiotics proposed for eradication regimens, mainly through point mutations. The present study included 200 patients with dyspepsia attending Taif Hospital. Gastric biopsies were obtained during gastroscopy and subjected to rapid urease testing. Molecular methods were used to confirm diagnoses of H. pylori infection and to identify resistance gene variants of four antibiotics; namely, clarithromycin, metronidazole, fluoroquinolones and tetracycline (23S rRNA, gyrA, rdxA and 16S rRNA respectively). Of all investigated patients, Molecular diagnoses were made in 143 of all investigated patients; thus, the prevalence was .5%. The overall rate of resistance to clarithromycin among the H. pylori‐positive patients was high (39.9%) and the rate of resistance significantly greater (48.2%) among the secondary resistance group, secondary resistance being defined as resistance as a result of previous exposure to the relevant antibiotic. The rate of resistance to fluoroquinolones was considered moderate; the difference in rate of resistance between the primary and secondary resistance groups (8.4% and 9.5%, respectively) was not significant Also, there was a low prevalence of both primary and the secondary tetracycline resistance in the study cohort. In contrast, the prevalence of metronidazole resistance was considered high with no significant difference between the two resistance groups. H. pylori showed an increased prevalence of resistance to all four of the commonly used therapeutic agents. Thus, eradication therapy should be based on the regional results of susceptibility testing. Moreover, treatment tailored according to individually determined H. pylori susceptibility may be a reasonable future goal.     

Stabilization and solidification of hydrocarbon‐contaminated soils in concrete

This article describes an experimental program developed to investigate the potential for using hydrocarbon‐contaminated soils as a fine aggregate replacement in concrete. Five different contaminated soil types with a total petroleum hydrocarbon content of less than 1% were investigated. For each soil type, three concrete mixtures were obtained by replacing sand with contaminated soils (10, 20, and 40% replacement ratio). The resulting concrete was tested for setting times, compression strength, flexural strength, durability, and teachability of benzene to water.

The results indicate that the addition of hydrocarbon‐contaminated soil adversely affects the strength of concrete. The strength reduction at each soil replacement level depends on contamination concentration, contaminant type, and soil type. The durability of the tested concrete is comparable to normal concrete. For all five soils at a 40% replacement ratio, the leachability of benzene was nondetectable after 24 h and after 10 d. After testing the leachability of artificially contaminated soils (0.5 and 3% neat benzene contamination) for 24 h, it was found that the leaching of benzene increases with the percentage of contamination. However, the fraction of benzene that leached was about 95% lower than the values for loose soils.     

PyContact: Rapid,Customizable, and Visual Analysis of Noncovalent Interactions in MD Simulations

Molecular dynamics (MD) simulations have become ubiquitous in all areas of life sciences. The size and model complexity of MD simulations are rapidly growing along with increasing computing power and improved algorithms. This growth has led to the production of a large amount of simulation data that need to be filtered for relevant information to address specific biomedical and biochemical questions. One of the most relevant molecular properties that can be investigated by all-atom MD simulations is the time-dependent evolution of the complex noncovalent interaction networks governing such fundamental aspects as molecular recognition, binding strength, and mechanical and structural stability. Extracting, evaluating, and visualizing noncovalent interactions is a key task in the daily work of structural biologists. We have developed PyContact, an easy-to-use, highly flexible, and intuitive graphical user interface-based application, designed to provide a toolkit to investigate biomolecular interactions in MD trajectories. PyContact is designed to facilitate this task by enabling identification of relevant noncovalent interactions in a comprehensible manner. The implementation of PyContact as a standalone application enables rapid analysis and data visualization without any additional programming requirements, and also preserves full in-program customization and extension capabilities for advanced users. The statistical analysis representation is interactively combined with full mapping of the results on the molecular system through the synergistic connection between PyContact and VMD. We showcase the capabilities and scientific significance of PyContact by analyzing and visualizing in great detail the noncovalent interactions underlying the ion permeation pathway of the human P2X₃ receptor. As a second application, we examine the protein-protein interaction network of the mechanically ultrastable cohesin-dockering complex.     

Imiquimod‐induced apoptosis of melanoma cells is mediated by ER stress‐dependent Noxa induction and enhanced by NF‐κB inhibition

Melanoma is characterized by dysregulated intracellular signalling pathways including an impairment of the cell death machinery, ultimately resulting in melanoma resistance, survival and progression. This explains the tumour's extraordinary resistance to the standard treatment. Imiquimod is a topical immune response modifier (imidazoquinoline) with both antiviral and antitumour activities. The mechanism by which imiquimod triggers the apoptosis of melanoma cells has now been carefully elucidated. Imiquimod‐induced apoptosis is associated with the activation of apoptosis signalling regulating kinase1/c‐Jun‐N‐terminal kinase/p38 pathways and the induction of endoplasmic stress characterized by the activation of the protein kinase RNA‐like endoplasmic reticulum kinase signalling pathway, increase in intracellular Ca²⁺ release, degradation of calpain and subsequent cleavage of caspase‐4. Moreover, imiquimod triggers the activation of NF‐κB and the expression of the inhibitor of apoptosis proteins (IAPs) such as, X‐linked IAP (XIAP) together with the accumulation of reactive oxygen species (ROS). Also, imiquimod triggers mitochondrial dysregulation characterized by the loss of mitochondrial membrane potential (Δψm), the increase in cytochrome c release, and cleavage of caspase‐9, caspase‐3 and poly(ADP‐ribose) polymerase (PARP). Inhibitors of specific pathways, permit the elucidation of possible mechanisms of imiquimod‐induced apoptosis. They demonstrate that inhibition of NF‐kB by the inhibitor of nuclear factor kappa‐B kinase (IKK) inhibitor Bay 11‐782 or knockdown of XIAP induces melanoma apoptosis in cells exposed to imiquimod. These findings support the use of either IKK inhibitors or IAP antagonists as adjuvant therapies to improve the effectiveness topical imiquimod in the treatment of melanoma.