A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

The quinoline-based allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are promising candidates for clinically useful antiviral agents. Studies using these compounds have highlighted the role of IN in both early and late stages of virus replication. However, dissecting the exact mechanism of action of the quinoline-based ALLINIs has been complicated by the multifunctional nature of these inhibitors because they both inhibit IN binding with its cofactor LEDGF/p75 and promote aberrant IN multimerization with similar potencies in vitro. Here we report design of small molecules that allowed us to probe the role of HIV-1 IN multimerization independently from IN-LEDGF/p75 interactions in infected cells. We altered the rigid quinoline moiety in ALLINIs and designed pyridine-based molecules with a rotatable single bond to allow these compounds to bridge between interacting IN subunits optimally and promote oligomerization. The most potent pyridine-based inhibitor, KF116, potently (EC₅₀ of 0.024 µM) blocked HIV-1 replication by inducing aberrant IN multimerization in virus particles, whereas it was not effective when added to target cells. Furthermore, KF116 inhibited the HIV-1 IN variant with the A128T substitution, which confers resistance to the majority of quinoline-based ALLINIs. A genome-wide HIV-1 integration site analysis demonstrated that addition of KF116 to target or producer cells did not affect LEDGF/p75-dependent HIV-1 integration in host chromosomes, indicating that this compound is not detectably inhibiting IN-LEDGF/p75 binding. These findings delineate the significance of correctly ordered IN structure for HIV-1 particle morphogenesis and demonstrate feasibility of exploiting IN multimerization as a therapeutic target. Furthermore, pyridine-based compounds present a novel class of multimerization selective IN inhibitors as investigational probes for HIV-1 molecular biology.     

Comparative 2D-DIGE Proteomic Analysis of Bovine Mammary Epithelial Cells during Lactation Reveals Protein Signatures for Lactation Persistency and Milk Yield

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.     

Anti-Inflammatory Activity of Odina wodier Roxb,an Indian Folk Remedy,through Inhibition of Toll-Like Receptor 4 Signaling Pathway

Inflammation is part of self-limiting non-specific immune response, which occurs during bodily injury. In some disorders the inflammatory process becomes continuous, leading to the development of chronic inflammatory diseases including cardiovascular diseases, diabetes, cancer etc. Several Indian tribes used the bark of Odina wodier (OWB) for treating inflammatory disorders. Thus, we have evaluated the immunotherapeutic potential of OWB methanol extract and its major constituent chlorogenic acid (CA), using three popular in vivo antiinflammatory models: Carrageenan- and Dextran-induced paw edema, Cotton pellet granuloma, and Acetic acid-induced vascular permeability. To elucidate the possible anti-inflammatory mechanism of action we determine the level of major inflammatory mediators (NO, iNOS, COX-2-dependent prostaglandin E2 or PGE2), and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-12). Further, we determine the toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88), c-Jun N-terminal kinases (JNK), nuclear factor kappa-B cells (NF-κB), and NF-kB inhibitor alpha (IK-Bα) by protein and mRNA expression, and Western blot analysis in drug treated LPS-induced murine macrophage model. Moreover, we determined the acute and sub-acute toxicity of OWB extract in BALB/c mice. Our study demonstrated a significant anti-inflammatory activity of OWB extract and CA along with the inhibition of TNF-α, IL-1β, IL-6 and IL-12 expressions. Further, the expression of TLR4, NF-κBp65, MyD88, iNOS and COX-2 molecules were reduced in drug-treated groups, but not in the LPS-stimulated untreated or control groups, Thus, our results collectively indicated that the OWB extract and CA can efficiently inhibit inflammation through the down regulation of TLR4/MyD88/NF-kB signaling pathway.     

‘Porosome’ discovered nearly 20 years ago provides molecular insights into the kiss‐and‐run mechanism of cell secretion

Secretion is a fundamental cellular process in living organisms, from yeast to cells in humans. Since the 1950s, it was believed that secretory vesicles completely merged with the cell plasma membrane during secretion. While this may occur, the observation of partially empty vesicles in cells following secretion suggests the presence of an additional mechanism that allows partial discharge of intra‐vesicular contents during secretion. This proposed mechanism requires the involvement of a plasma membrane structure called ‘porosome’, which serves to prevent the collapse of secretory vesicles, and to transiently fuse with the plasma membrane (Kiss‐and‐run), expel a portion of its contents and disengage. Porosomes are cup‐shaped supramolecular lipoprotein structures at the cell plasma membrane ranging in size from 15 nm in neurons and astrocytes to 100–180 nm in endocrine and exocrine cells. Neuronal porosomes are composed of nearly 40 proteins. In comparison, the 120 nm nuclear pore complex is composed of >500 protein molecules. Elucidation of the porosome structure, its chemical composition and functional reconstitution into artificial lipid membrane, and the molecular assembly of membrane‐associated t‐SNARE and v‐SNARE proteins in a ring or rosette complex resulting in the establishment of membrane continuity to form a fusion pore at the porosome base, has been demonstrated. Additionally, the molecular mechanism of secretory vesicle swelling, and its requirement for intra‐vesicular content release during cell secretion has also been elucidated. Collectively, these observations provide a molecular understanding of cell secretion, resulting in a paradigm shift in our understanding of the secretory process.     

Learning without Borders: A Review of the Implementation of Medical Error Reporting in Médecins Sans Frontières

Objective

To analyse the results from the first 3 years of implementation of a medical error reporting system in Médecins Sans Frontières-Operational Centre Amsterdam (MSF) programs.

Methodology

A medical error reporting policy was developed with input from frontline workers and introduced to the organisation in June 2010. The definition of medical error used was “the failure of a planned action to be completed as intended or the use of a wrong plan to achieve an aim.” All confirmed error reports were entered into a database without the use of personal identifiers.

Results

179 errors were reported from 38 projects in 18 countries over the period of June 2010 to May 2013. The rate of reporting was 31, 42, and 106 incidents/year for reporting year 1, 2 and 3 respectively. The majority of errors were categorized as dispensing errors (62 cases or 34.6%), errors or delays in diagnosis (24 cases or 13.4%) and inappropriate treatment (19 cases or 10.6%). The impact of the error was categorized as no harm (58, 32.4%), harm (70, 39.1%), death (42, 23.5%) and unknown in 9 (5.0%) reports. Disclosure to the patient took place in 34 cases (19.0%), did not take place in 46 (25.7%), was not applicable for 5 (2.8%) cases and not reported for 94 (52.5%). Remedial actions introduced at headquarters level included guideline revisions and changes to medical supply procedures. At field level improvements included increased training and supervision, adjustments in staffing levels, and adaptations to the organization of the pharmacy.

Conclusion

It was feasible to implement a voluntary reporting system for medical errors despite the complex contexts in which MSF intervenes. The reporting policy led to system changes that improved patient safety and accountability to patients. Challenges remain in achieving widespread acceptance of the policy as evidenced by the low reporting and disclosure rates.     

The A128T Resistance Mutation Reveals Aberrant Protein Multimerization as the Primary Mechanism of Action of Allosteric HIV-1 Integrase Inhibitors

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a very promising new class of anti-HIV-1 agents that exhibit a multimodal mechanism of action by allosterically modulating IN multimerization and interfering with IN-lens epithelium-derived growth factor (LEDGF)/p75 binding. Selection of viral strains under ALLINI pressure has revealed an A128T substitution in HIV-1 IN as a primary mechanism of resistance. Here, we elucidated the structural and mechanistic basis for this resistance. The A128T substitution did not affect the hydrogen bonding between ALLINI and IN that mimics the IN-LEDGF/p75 interaction but instead altered the positioning of the inhibitor at the IN dimer interface. Consequently, the A128T substitution had only a minor effect on the ALLINI IC₅₀ values for IN-LEDGF/p75 binding. Instead, ALLINIs markedly altered the multimerization of IN by promoting aberrant higher order WT (but not A128T) IN oligomers. Accordingly, WT IN catalytic activities and HIV-1 replication were potently inhibited by ALLINIs, whereas the A128T substitution in IN resulted in significant resistance to the inhibitors both in vitro and in cell culture assays. The differential multimerization of WT and A128T INs induced by ALLINIs correlated with the differences in infectivity of HIV-1 progeny virions. We conclude that ALLINIs primarily target IN multimerization rather than IN-LEDGF/p75 binding. Our findings provide the structural foundations for developing improved ALLINIs with increased potency and decreased potential to select for drug resistance.     

Effects of temperature on modulation of oxidative stress and antioxidant defenses in testes of tropical tasar silkworm Antheraea mylitta

High temperatures are known to cause physiological stress in organisms. This is often associated with enhanced generation of reactive oxygen species (ROS) leading to oxidative damage. The commercially important tropical tasar silkworm Antheraea mylitta has to endure high summer temperature before egg production on the onset of monsoon. In this study the status of pro-oxidants and antioxidants was studied in the testes of male pupae of tasar silkworm A. mylitta under thermal stress condition. Further, to find out the impact of temperature on physiological activity, oxygen consumption rate was measured. The result indicated higher level of thiobarbituric acid reactive substances (TBARS, as an index of lipid peroxidation) and total hydroperoxides in the male pupae exposed to high temperature (40±1 °C). Similarly, it was found that increased levels of antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), ascorbic acid (ASA) and low molecular thiols (L-SH) in testes are more prominent in high temperature rather than in moderate temperature (35±1 ^oC) suggesting the activation of physiological mechanism to scavenge the ROS produced during stress. Further more, the order of higher level of oxygen consumption rate was observed as high temperature (40±1°C) > moderate temperature (35±1°C) > control groups (28±1°C). Oxygen consumption rate was positively correlated with oxidative stress and antioxidant defence indices. We infer from these findings that the testes of A. mylitta pupae modulate testicular antioxidant responses to thermal stress.     

DNA damage by reactive species: Mechanisms, mutation and repair

DNA is continuously attacked by reactive species that can affect its structure and function severely. Structural modifications to DNA mainly arise from modifications in its bases that primarily occur due to their exposure to different reactive species. Apart from this, DNA strand break, inter- and intra-strand crosslinks and DNA-protein crosslinks can also affect the structure of DNA significantly. These structural modifications are involved in mutation, cancer and many other diseases. As it has the least oxidation potential among all the DNA bases, guanine is frequently attacked by reactive species, producing a plethora of lethal lesions. Fortunately, living cells are evolved with intelligent enzymes that continuously protect DNA from such damages. This review provides an overview of different guanine lesions formed due to reactions of guanine with different reactive species. Involvement of these lesions in inter- and intra-strand crosslinks, DNA-protein crosslinks and mutagenesis are discussed. How certain enzymes recognize and repair different guanine lesions in DNA are also presented.     

Water channels in platelet volume regulation

The regulation of platelet volume significantly affects its function. Because water is the major molecule in cells and its active transport via water channels called aquaporins (AQPs) have been implicated in cellular and organelle volume regulation, the presence of water channels in platelets and their potential role in platelet volume regulation was investigated. G-protein-mediated AQP regulation in secretory vesicle swelling has previously been reported in neurons and in pancreatic acinar cells. Mercuric chloride has been demonstrated to inhibit most AQPs except AQP6, which is stimulated by the compound. Exposure of platelets to HgCl(2)-induced swelling in a dose-dependent manner, suggesting the presence of AQP6 in platelets. Immunoblot analysis of platelet protein confirmed the presence of AQP6, and also of G(αo), G(αi-1) and G(αi-3) proteins. Results from this study demonstrate for the first time that in platelets AQP6 is involved in cell volume regulation via a G-protein-mediated pathway.