Structurally Mapping Endogenous Heme in the CcmCDE Membrane Complex for Cytochrome c Biogenesis

Although many putative heme transporters have been discovered, it has been challenging to prove that these proteins are directly involved with heme trafficking in vivo and to identify their heme binding domains. The prokaryotic pathways for cytochrome c biogenesis, Systems I and II, transport heme from inside the cell to outside for stereochemical attachment to cytochrome c, making them excellent models to study heme trafficking. System I is composed of eight integral membrane proteins (CcmA–H) and is proposed to transport heme via CcmC to an external “WWD” domain for presentation to the membrane-tethered heme chaperone, CcmE. Herein, we develop a new cysteine/heme crosslinking approach to trap and map endogenous heme in CcmC (WWD domain) and CcmE (defining “2-vinyl” and “4-vinyl” pockets for heme). Crosslinking occurs when either of the two vinyl groups of heme localize near a thiol of an engineered cysteine residue. Double crosslinking, whereby both vinyls crosslink to two engineered cysteines, facilitated a more detailed structural mapping of the heme binding sites, including stereospecificity. Using heme crosslinking results, heme ligand identification, and genomic coevolution data, we model the structure of the CcmCDE complex, including the WWD heme binding domain. We conclude that CcmC trafficks heme via its WWD domain and propose the structural basis for stereochemical attachment of heme.     

Advances in thermochemical conversion of woody biomass to energy,fuels and chemicals

Biomass has been recognised as a promising resource for future energy and fuels. The biomass, originated from plants, is renewable and application of its derived energy and fuels is close to carbon-neutral by considering that the growing plants absorb CO₂ for photosynthesis. However, the complex physical structure and chemical composition of the biomass significantly hinder its conversion to gaseous and liquid fuels.This paper reviews recent advances in biomass thermochemical conversion technologies for energy, liquid fuels and chemicals. Combustion process produces heat or heat and power from the biomass through oxidation reactions; however, this is a mature technology and has been successfully applied in industry. Therefore, this review will focus on the remaining three thermochemical processes, namely biomass pyrolysis, biomass thermal liquefaction and biomass gasification. For biomass pyrolysis, biomass pretreatment and application of catalysts can simplify the bio-oil composition and retain high yield. In biomass liquefaction, application of appropriate solvents and catalysts improves the liquid product quality and yield. Gaseous product from biomass gasification is relatively simple and can be further processed for useful products. Dual fluidised bed (DFB) gasification technology using steam as gasification agent provides an opportunity for achieving high hydrogen content and CO₂ capture with application of appropriate catalytic bed materials. In addition, multi-staged gasification technology, and integrated biomass pyrolysis and gasification as well as gasification for poly-generation have attracted increasing attention.     

Synthesis and urease inhibitory potential of benzophenone sulfonamide hybrid in vitro and in silico

This study deals with the synthesis of benzophenone sulfonamides hybrids (1–31) and screening against urease enzyme in vitro. Studies showed that several synthetic compounds were found to have good urease enzyme inhibitory activity. Compounds 1 (N′-((4′-hydroxyphenyl)(phenyl)methylene)-4′′-nitrobenzenesulfonohydrazide), 2 (N′-((4′-hydroxyphenyl)(phenyl)methylene)-3′′-nitrobenzenesulfonohydrazide), 3 (N′-((4′-hydroxyphenyl)(phenyl)methylene)-4′′-methoxybenzenesulfonohydrazide), 4 (3′′,5′′-dichloro-2′′-hydroxy-N′-((4′-hydroxyphenyl)(phenyl)methylene)benzenesulfonohydrazide), 6 (2′′,4′′-dichloro-N′-((4′-hydroxyphenyl)(phenyl)methylene)benzenesulfonohydrazide), 8 (5-(dimethylamino)-N′-((4-hydroxyphenyl)(phenyl)methylene)naphthalene-1-sulfono hydrazide), 10 (2′′-chloro-N′-((4′-hydroxyphenyl)(phenyl)methylene)benzenesulfonohydrazide), 12 (N′-((4′-hydroxyphenyl)(phenyl)methylene)benzenesulfonohydrazide) have found to be potently active having an IC₅₀ value in the range of 3.90–17.99?µM. These compounds showed superior activity than standard acetohydroxamic acid (IC₅₀?=?29.20?±?1.01?µM). Moreover, in silico studies on most active compounds were also performed to understand the binding interaction of most active compounds with active sites of urease enzyme. Structures of all the synthetic compounds were elucidated by ¹H NMR, ¹³C NMR, EI-MS and FAB-MS spectroscopic techniques.     

Genetic characterization of antibiotic-resistant Staphylococcus aureus from milk in the North-West Province,South Africa

Food borne diseases are a major public health concern worldwide. Staphylococcus aureus is one of the potential food borne pathogens which causes nosocomial and community acquired infections. In the present study, 74 representative strains of S. aureus isolated and characterized in previous study from different milk samples were subjected to random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR to generate fingerprints to determine the genetic relationships of the isolated strains. A total of 20 RAPD patterns were generated and the number of amplified fragments obtained ranged from 0 to 8 with molecular weight ranging from 250 to 2000 bp. A dendrogram based on fingerprinting pattern grouped isolates into twelve major clusters (I–XII). In the case of ERIC-PCR 9 banding patterns were obtained with amplicons ranging from 1 to 8 and band sizes ranging from 250 to 2000 bp. A total of four major clusters (I–IV) were observed in the dendrogram based on ERIC fingerprints. The discrete banding patterns obtained both from ERIC-PCR and RAPD-PCR showed remarkably the genetic diversity of S. aureus. The findings of this study indicate that raw, bulk and pasteurized milk in the North-West Province was contaminated with toxigenic and multi-drug resistant S. aureus strains. This emphasizes the need to implement appropriate control measures to reduce contamination as well as the spread of virulent S. aureus strains to reduce the burden of disease in humans.     

Chemical fingerprint,acute oral toxicity and anti-inflammatory activity of the hydroalcoholic extract of leaves from Tocoyena formosa (Cham. & Schlecht.) K. Schum

Inflammation is a protective response of the organism against damaging agents, this process is considered beneficial, however in some situations, this response can be damage when exacerbated effect are present. This claim objective to evaluate the qualitative and quantitative chemical profile, acute toxic and anti-inflammatory effects of the hydroalcoholic extract of leaves from Tocoyena formosa (Cham. & Schlecht.) K. Schum. (HELTF). Quantitative and qualitative phytochemical analysis was performed by HPLC-DAD and colorimetric assay. The topical anti-inflammatory activity was determined in Croton oil-induced ear edema assay and systemic activity was performed in vascular permeability, paw edema induced by carrageenan and dextran. Phytochemical analysis of leaves from HELTF showed presence of tannin, flavonoid, saponins an other that confirmed by HPLC analysis. The extract did not cause significant with LD₅₀ greater than 5000 mg/kg and did not promote significate reduction in topical inflammatory process. However, HELTF demonstrate significant reduction of paw edema induced by carrageenan and dextran. The HELTF (200 mg/kg) reduced the protein/cell migration in the intradermal carrageenan-induced inflammation. Our results demonstrated that the first time the chemical profile and describe the effective action in systemic anti-inflammatory, antiedematogenic activity and low acute toxicity. This activity presents, supporting its traditional use. However, new studies are necessary for the detection and clarification of the possible mechanism of action.     

A novel multilocus sequence typing scheme identifying genetic diversity amongst Leishmania donovani isolates from a genetically homogeneous population in the Indian subcontinent

In the Indian subcontinent, infection with Leishmania donovani can cause fatal visceral leishmaniasis. Genetic variation in L. donovani is believed to occur rapidly from environmental changes and through selective drug pressures, thereby allowing continued disease occurrence in this region. All previous molecular markers that are commonly in use multilocus microsatellite typing and multilocus sequence typing, were monomorphic in L. donovani originating from the Indian subcontinent (with only a few exceptions) and hence are not suitable for this region. An multilocus sequence typing scheme consisting of a new set of seven housekeeping genes was developed in this study, based on recent findings from whole genome sequencing data. This new scheme was used to assess the genetic diversity amongst 22 autochthonous L. donovani isolates from Bangladesh. Nineteen additional isolates of the L. donovani complex (including sequences of L. donovani reference strain BPK282A1) from other countries were included for comparison. By using restriction fragment length polymorphism of the internal transcribed spacer 1 region (ITS1-RFLP) and ITS1 sequencing, all Bangladeshi isolates were confirmed to be L. donovani. Population genetic analyses of 41 isolates using the seven new MLST loci clearly separated L. donovani from Leishmania infantum. With this multilocus sequence typing scheme, seven genotypes were identified amongst Bangladeshi L. donovani isolates, and these isolates were found to be phylogenetically different compared with those from India, Nepal, Iraq and Africa. This novel multilocus sequence typing approach can detect intra- and inter-species variations within the L. donovani complex, but most importantly these molecular markers can be applied to resolve the phylogenetically very homogeneous L. donovani strains from the Indian subcontinent. Four of these markers were found suitable to differentiate strains originating from Bangladesh, with marker A2P being the most discriminative one.     

Dietary nitrate's effects on exercise performance in heart failure with reduced ejection fraction (HFrEF)

Heart failure with reduced ejection fraction (HFrEF) is a deadly and disabling disease. A key derangement contributing to impaired exercise performance in HFrEF is decreased nitric oxide (NO) bioavailability. Scientists recently discovered the inorganic nitrate pathway for increasing NO. This has advantages over organic nitrates and NO synthase production of NO. Small studies using beetroot juice as a source of inorganic nitrate demonstrate its power to improve exercise performance in HFrEF. A larger-scale trial is now underway to determine if inorganic nitrate may be a new arrow for physicians' quiver of HFrEF treatments.     

Mitochondrial and calcium perturbations in rat CNS neurons induce calpain-cleavage of Parkin: Phosphatase inhibition stabilizes pSer65Parkin reducing its calpain-cleavage

Mitochondrial impairment and calcium (Ca⁺⁺) dyshomeostasis are associated with Parkinson's disease (PD). When intracellular ATP levels are lowered, Ca⁺⁺-ATPase pumps are impaired causing cytoplasmic Ca⁺⁺ to be elevated and calpain activation. Little is known about the effect of calpain activation on Parkin integrity. To address this gap, we examined the effects of mitochondrial inhibitors [oligomycin (Oligo), antimycin and rotenone] on endogenous Parkin integrity in rat midbrain and cerebral cortical cultures. All drugs induced calpain-cleavage of Parkin to ~36.9/43.6 kDa fragments. In contrast, treatment with the proinflammatory prostaglandin J2 (PGJ2) and the proteasome inhibitor epoxomicin induced caspase-cleavage of Parkin to fragments of a different size, previously shown by others to be triggered by apoptosis. Calpain-cleaved Parkin was enriched in neuronal mitochondrial fractions. Pre-treatment with the phosphatase inhibitor okadaic acid prior to Oligo-treatment, stabilized full-length Parkin phosphorylated at Ser⁶⁵, and reduced calpain-cleavage of Parkin. Treatment with the Ca⁺⁺ ionophore A23187, which facilitates Ca⁺⁺ transport across the plasma membrane, mimicked the effect of Oligo by inducing calpain-cleavage of Parkin. Removing extracellular Ca⁺⁺ from the media prevented oligomycin- and ionophore-induced calpain-cleavage of Parkin. Computational analysis predicted that calpain-cleavage of Parkin liberates its UbL domain. The phosphagen cyclocreatine moderately mitigated Parkin cleavage by calpain. Moreover, the pituitary adenylate cyclase activating peptide (PACAP27), which stimulates cAMP production, prevented caspase but not calpain-cleavage of Parkin. Overall, our data support a link between Parkin phosphorylation and its cleavage by calpain. This mechanism reflects the impact of mitochondrial impairment and Ca⁺⁺-dyshomeostasis on Parkin integrity and could influence PD pathogenesis.