The correlation between exaggerated fluid in lumbar facet joints and degenerative spondylolisthesis: prospective study of 52 patients

Magnetic resonance imaging (MRI) is often used to evaluate low back pain; however, MRI in the supine position does not always reveal degenerative spondylolisthesis. The existence of a linear correlation between increased fluid in the facet joints seen on the supine axial T2 MRI of the lumbosacral spine and lumbar instability seen on standing lateral flexion-extension lumbosacral radiographs has recently been reported. The objective of this prospective study was to determine the incidence of increased fluid in the lumbar facet joints seen on the supine axial T2 MRI, and to evaluate the correlation of this finding with radiographic evidence of lumbar instability

Long-acting formulations for the treatment of latent tuberculous infection: opportunities and challenges

Long-acting/extended-release drug formulations have proved very successful in diverse areas of medicine, including contraception, psychiatry and, most recently, human immunodeficiency virus (HIV) disease. Though challenging, application of this technology to anti-tuberculosis treatment could have substantial impact. The duration of treatment required for all forms of tuberculosis (TB) put existing regimens at risk of failure because of early discontinuations and treatment loss to follow-up. Long-acting injections, for example, administered every month, could improve patient adherence and treatment outcomes. We review the state of the science for potential long-acting formulations of existing tuberculosis drugs, and propose a target product profile for new formulations to treat latent tuberculous infection (LTBI). The physicochemical properties of some anti-tuberculosis drugs make them unsuitable for long-acting formulation, but there are promising candidates that have been identified through modeling and simulation, as well as other novel agents and formulations in preclinical testing. An efficacious long-acting treatment for LTBI, particularly for those co-infected with HIV, and if coupled with a biomarker to target those at highest risk for disease progression, would be an important tool to accelerate progress towards TB elimination

Analyses of HIV-1 integrase sequences prior to South African national HIV-treatment program and available of integrase inhibitors in Cape Town, South Africa

HIV-Integrase (IN) has proven to be a viable target for highly specific HIV-1 therapy. We aimed to characterize the HIV-1 IN gene in a South African context and identify resistance-associated mutations (RAMs) against available first and second generation Integrase strand-transfer inhibitors (InSTIs). We performed genetic analyses on 91 treatment-naïve HIV-1 infected patients, as well as 314 treatmentnaive South African HIV-1 IN-sequences, downloaded from Los Alamos HIV Sequence Database. Genotypic analyses revealed the absence of major RAMs in the cohort collected before the broad availability of combination antiretroviral therapy (cART) and INSTI in South Africa, however, occurred at a rate of 2.85% (9/314) in database derived sequences. RAMs were present at IN-positions 66, 92, 143, 147 and 148, all of which may confer resistance to Raltegravir (RAL) and Elvitegravir (EVG), but are unlikely to affect second-generation Dolutegravir (DTG), except mutations in the Q148 pathway. Furthermore, protein modeling showed, naturally occurring polymorphisms impact the stability of the intasome-complex and therefore may contribute to an overall potency against InSTIs. Our data suggest the prevalence of InSTI RAMs, against InSTIs, is low in South Africa, but natural polymorphisms and subtype-specific differences may influence the effect of individual treatment regimens

Phospholipase C-eta enzymes as putative protein kinase C and Ca2+ signalling components in neuronal and neuroendocrine tissues

Phosphoinositol-specific phospholipase C enzymes (PLCs) are central to inositol lipid signalling pathways, facilitating intracellular Ca2+ release and protein kinase C activation. A sixth class of phosphoinositol-specific PLC with a novel domain structure, PLC-eta (PLCeta) has recently been discovered in mammals. Recent research, reviewed here, shows that this class consists of two enzymes, PLCeta1 and PLCeta2. Both enzymes hydrolyze phosphatidylinositol 4,5-bisphosphate and are more sensitive to Ca2+ than other PLC isozymes and are likely to mediate G-protein-coupled receptor (GPCR) signalling pathways. Both enzymes are expressed in neuron-enriched regions, being abundant in the brain. We demonstrate that they are also expressed in neuroendocrine cell lines. PLCeta enzymes therefore represent novel proteins influencing intracellular Ca2+ dynamics and protein kinase C activation in the brain and neuroendocrine systems as putative mediation of GPCR regulation

Evolution of the mammalian lysozyme gene family

<p>Abstract</p> <p>Background</p> <p>Lysozyme <it>c </it>(chicken-type lysozyme) has an important role in host defense, and has been extensively studied as a model in molecular biology, enzymology, protein chemistry, and crystallography. Traditionally, lysozyme <it>c </it>has been considered to be part of a small family that includes genes for two other proteins, lactalbumin, which is found only in mammals, and calcium-binding lysozyme, which is found in only a few species of birds and mammals. More recently, additional testes-expressed members of this family have been identified in human and mouse, suggesting that the mammalian lysozyme gene family is larger than previously known.</p> <p>Results</p> <p>Here we characterize the extent and diversity of the lysozyme gene family in the genomes of phylogenetically diverse mammals, and show that this family contains at least eight different genes that likely duplicated prior to the diversification of extant mammals. These duplicated genes have largely been maintained, both in intron-exon structure and in genomic context, throughout mammalian evolution.</p> <p>Conclusions</p> <p>The mammalian lysozyme gene family is much larger than previously appreciated and consists of at least eight distinct genes scattered around the genome. Since the lysozyme <it>c </it>and lactalbumin proteins have acquired very different functions during evolution, it is likely that many of the other members of the lysozyme-like family will also have diverse and unexpected biological properties.</p

Comparison of metal-dependent catalysis by HIV-1 and ASV integrase proteins using a new and rapid, moderate throughput assay for joining activity in solution

<p>Abstract</p> <p>Background</p> <p>HIV-1 integrase (IN) is an attractive target for the development of drugs to treat AIDS, and inhibitors of this viral enzyme are already in the clinic. Nevertheless, there is a continuing need to devise new approaches to block the activity of this viral protein because of the emergence of resistant strains. To facilitate the biochemical analysis of wild-type IN and its derivatives, and to measure the potency of prospective inhibitory compounds, a rapid, moderate throughput solution assay was developed for IN-catalyzed joining of viral and target DNAs, based on the detection of a fluorescent tag.</p> <p>Results</p> <p>A detailed, step-by-step description of the new joining assay is provided. The reactions are run in solution, the products captured on streptavidin beads, and activity is measured by release of a fluorescent tag. The procedure can be scaled up for the analysis of numerous samples, and is substantially more rapid and sensitive than the standard radioactive gel methods. The new assay is validated and its utility demonstrated via a detailed comparison of the Mg⁺⁺- and Mn⁺⁺-dependent activities of the IN proteins from human immunodeficiency virus type 1 (HIV-1) and the avian sarcoma virus (ASV). The results confirm that ASV IN is considerably more active than HIV-1 IN, but with both enzymes the initial rates of joining, and the product yields, are higher in the presence of Mn⁺⁺than Mg⁺⁺. Although the pH optima for these two enzymes are similar with Mn⁺⁺, they differ significantly in the presence of Mg⁺⁺, which is likely due to differences in the molecular environment of the binding region of this physiologically relevant divalent cation. This interpretation is strengthened by the observation that a compound that can inhibit HIV-1 IN in the presence of either metal cofactors is only effective against ASV in the presence of Mn⁺⁺.</p> <p>Conclusion</p> <p>A simplified, assay for measuring the joining activity of retroviral IN in solution is described, which offers several advantages over previous methods and the standard radioactive gel analyses. Based on comparisons of signal to background ratios, the assay is 10–30 times more sensitive than gel analysis, allows more rapid and accurate biochemical analyses of IN catalytic activity, and moderate throughput screening of inhibitory compounds. The assay is validated, and its utility demonstrated in a comparison of the metal-dependent activities of HIV-1 and ASV IN proteins.</p

Bunyaviridae RNA Polymerases (L-Protein) Have an N-Terminal, Influenza-Like Endonuclease Domain, Essential for Viral Cap-Dependent Transcription

Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase (L-protein) to transcribe viral mRNAs. It was recently shown that the cap-snatching endonuclease of influenza virus resides in a discrete N-terminal domain of the PA polymerase subunit. Here we structurally and functionally characterize a similar endonuclease in La Crosse orthobunyavirus (LACV) L-protein. We expressed N-terminal fragments of the LACV L-protein and found that residues 1-180 have metal binding and divalent cation dependent nuclease activity analogous to that of influenza virus endonuclease. The 2.2 Å resolution X-ray crystal structure of the domain confirms that LACV and influenza endonucleases have similar overall folds and identical two metal binding active sites. The in vitro activity of the LACV endonuclease could be abolished by point mutations in the active site or by binding 2,4-dioxo-4-phenylbutanoic acid (DPBA), a known influenza virus endonuclease inhibitor. A crystal structure with bound DPBA shows the inhibitor chelating two active site manganese ions. The essential role of this endonuclease in cap-dependent transcription was demonstrated by the loss of transcriptional activity in a RNP reconstitution system in cells upon making the same point mutations in the context of the full-length LACV L-protein. Using structure based sequence alignments we show that a similar endonuclease almost certainly exists at the N-terminus of L-proteins or PA polymerase subunits of essentially all known negative strand and cap-snatching segmented RNA viruses including arenaviruses (2 segments), bunyaviruses (3 segments), tenuiviruses (4–6 segments), and orthomyxoviruses (6–8 segments). This correspondence, together with the well-known mapping of the conserved polymerase motifs to the central regions of the L-protein and influenza PB1 subunit, suggests that L-proteins might be architecturally, and functionally equivalent to a concatemer of the three orthomyxovirus polymerase subunits in the order PA-PB1-PB2. Furthermore, our structure of a known influenza endonuclease inhibitor bound to LACV endonuclease suggests that compounds targeting a potentially broad spectrum of segmented RNA viruses, several of which are serious or emerging human, animal and plant pathogens, could be developed using structure-based optimisation

Unprocessed Viral DNA Could Be the Primary Target of the HIV-1 Integrase Inhibitor Raltegravir

Integration of HIV DNA into host chromosome requires a 3′-processing (3′-P) and a strand transfer (ST) reactions catalyzed by virus integrase (IN). Raltegravir (RAL), commonly used in AIDS therapy, belongs to the family of IN ST inhibitors (INSTIs) acting on IN-viral DNA complexes (intasomes). However, studies show that RAL fails to bind IN alone, but nothing has been reported on the behaviour of RAL toward free viral DNA. Here, we assessed whether free viral DNA could be a primary target for RAL, assuming that the DNA molecule is a receptor for a huge number of pharmacological agents. Optical spectroscopy, molecular dynamics and free energy calculations, showed that RAL is a tight binder of both processed and unprocessed LTR (long terminal repeat) ends. Complex formation involved mainly van der Waals forces and was enthalpy driven. Dissociation constants (Kds) revealed that RAL affinity for unbound LTRs was stronger than for bound LTRs. Moreover, Kd value for binding of RAL to LTRs and IC50 value (half concentration for inhibition) were in same range, suggesting that RAL binding to DNA and ST inhibition are correlated events. Accommodation of RAL into terminal base-pairs of unprocessed LTR is facilitated by an extensive end fraying that lowers the RAL binding energy barrier. The RAL binding entails a weak damping of fraying and correlatively of 3′-P inhibition. Noteworthy, present calculated RAL structures bound to free viral DNA resemble those found in RAL-intasome crystals, especially concerning the contacts between the fluorobenzyl group and the conserved 5′C4pA33′ step. We propose that RAL inhibits IN, in binding first unprocessed DNA. Similarly to anticancer drug poisons acting on topoisomerases, its interaction with DNA does not alter the cut, but blocks the subsequent joining reaction. We also speculate that INSTIs having viral DNA rather IN as main target could induce less resistance