Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: The Fab is directed against an intermediate in the helix-coil dynamics of the enzyme

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a γ turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme. The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme reveal the structural basis of its preference for binding to DNA polymerases of the Thermus species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies remarkably high relative mobility between these two domains.     

Cannabis and Δ-tetrahydrocannabinol (THC) for weight loss?

Obesity is one of the highest preventable causes of morbidity and mortality in the developed world [1]. It has been well known for a long time that exposure to cannabis produces an increase of appetite (a phenomenon referred to as the ‘munchies’). This phenomenon led to an exploration of the role of the endocannabinoid system in the regulation of obesity and associated metabolic syndrome. This effort subsequently led to the development of a successful therapeutic approach for obesity that consisted of blocking the cannabinoid CB₁ receptors using ligands such as Rimonabant in order to produce weight loss and improve metabolic profile [2]. Despite being efficacious, Rimonabant was associated with increased rates of depression and anxiety and therefore removed from the market. We recently discovered that the prevalence of obesity is paradoxically much lower in cannabis users as compared to non-users and that this difference is not accounted for by tobacco smoking status and is still present after adjusting for variables such as sex and age. Here, we propose that this effect is directly related to exposure to the Δ⁹-tetrahydrocannabinol (THC) present in cannabis smoke. We therefore propose the seemingly paradoxical hypothesis that THC or a THC/cannabidiol combination drug may produce weight loss and may be a useful therapeutic for the treatment of obesity and its complications.     

Locus assignment of two alpha-globin structural mutants from the Caribbean basin: alpha Fort de France (alpha 45 Arg) and alpha Spanish Town (alpha 27 Val)

Two alpha-globin structural mutants were mapped to their encoding loci by in vitro translation of hybrid-selected alpha 1- and alpha 2-globin mRNA. The more highly expressed mutant, alpha Spanish Town (alpha 27Val), is encoded at the alpha 2 locus and the less expressed mutant, alpha Fort de France (alpha 45Arg), is encoded at the alpha 1 locus. These results further define the distribution of alpha-globin structural mutations within the alpha-globin gene cluster and substantiate the dominant role of the alpha 2-globin locus in alpha- globin expression.     

Modeling of the bryostatins to the phorbol ester pharmacophore on protein kinase C

The bryostatins are macrocyclic lactones that represent an additional structural class of potent activators of protein kinase C. These marine animal biosynthetic products are of unusual interest because they induce only a subset of the biological responses induced by the phorbol esters. We have now determined the binding affinities of naturally occurring and semisynthetic bryostatins for protein kinase C by competition analysis with [26-3H]bryostatin 4 as the radioactive ligand. Esterification of the hydroxyl group at C26 caused dramatic loss of activity as did inversion of the asymmetric center at this position. In contrast, neither of the ester groups at C7 and C20 had a major influence on activity. Computer modeling of the phorbol esters, related diterpenes, and indole alkaloids suggested that the C20, C9, and C4 oxygens of phorbol represented critical elements of the phorbol ester pharmacophore. The C26 oxygen of the bryostatins, together with the C1 and C19 oxygens, gave an excellent spatial correlation with this model, with a root-mean-square deviation of 0.16 A (compared to 0.10-0.35 A among phorbol-related diterpenes). The extension of the phorbol ester pharmacophore model to the bryostatins and its agreement with the structure-activity relations for the bryostatin class of compounds provide additional support for the validity of the model.     

The strength of combined cytogenetic and mate-pair sequencing techniques illustrated by a germline chromothripsis rearrangement involving FOXP2

Next-generation mate-pair sequencing (MPS) has revealed that many constitutional complex chromosomal rearrangements (CCRs) are associated with local shattering of chromosomal regions (chromothripsis). Although MPS promises to identify the molecular basis of the abnormal phenotypes associated with many CCRs, none of the reported mate-pair sequenced complex rearrangements have been simultaneously studied with state-of-the art molecular cytogenetic techniques. Here, we studied chromothripsis-associated CCR involving chromosomes 2, 5 and 7, associated with global developmental and psychomotor delay and severe speech disorder. We identified three truncated genes: CDH12, DGKB and FOXP2, confirming the role of FOXP2 in severe speech disorder, and suggestive roles of CDH12 and/or DGKB for the global developmental and psychomotor delay. Our study confirmes the power of MPS for detecting breakpoints and truncated genes at near nucleotide resolution in chromothripsis. However, only by combining MPS data with conventional G-banding and extensive fluorescence in situ hybridizations could we delineate the precise structure of the derivative chromosomes.     

Community Health Workers and Stand-Alone or Integrated Case Management of Malaria: A Systematic Literature Review

A systematic literature review was conducted to assess the effectiveness of strategies to improve community case management (CCM) of malaria. Forty-three studies were included; most (38) reported indicators of community health worker (CHW) performance, 14 reported on malaria CCM integrated with other child health interventions, 16 reported on health system capacity, and 13 reported on referral. The CHWs are able to provide good quality malaria care, including performing procedures such as rapid diagnostic tests. Appropriate training, clear guidelines, and regular supportive supervision are important facilitating factors. Crucial to sustainable success of CHW programs is strengthening health system capacity to support commodity supply, supervision, and appropriate treatment of referred cases. The little evidence available on referral from community to health facility level suggests that this is an area that needs priority attention. The studies of integrated CCM suggest that additional tasks do not reduce the quality of malaria CCM provided sufficient training and supervision is maintained.     

Is Venous Foot Pump Effective In Prevention of Thromboembolic Disease After Joint Arthroplasty: A Meta-Analysis

The goal of this meta-analysis was to evaluate the efficacy of venous foot pumps in prevention of venous thromboembolism following joint arthroplasty. Using different databases, we found 13 prospective clinical trials published meeting our inclusion criteria. In total, 1514 patients were included in the final analysis. Venous foot pump devices are effective in prevention of venous thromboembolic disease after total hip and knee arthroplasty compared to chemoprophylaxis. This was especially significant in prevention of major deep vein thrombosis and pulmonary emboli rate. The use of mechanical devices like venous calf or foot pump, either alone or in combination with less potent chemical prophylaxis, on the other hand can reduce the rate of venous thromboembolism and complications of potent chemoprophylaxis like wound hematoma.     

Advances in small molecules promoting neurotrophic function

Nerve growth factor (NGF) and other members of the neurotrophin family are critical for the survival and differentiation of neurons and have been implicated in the pathophysiology of numerous disease states. Although the therapeutic potential of neurotrophins has generated much excitement over the past decade, inconvenient pharmacokinetics and adverse side-effect profiles have limited the clinical usefulness of neurotrophic factors themselves. Compounds that mimic neurotrophin signaling and overcome the pharmacokinetic and side-effect barriers may have greater therapeutic potential. Here, we review the progress to date of clinical trials with direct neurotrophin modulators and describe alternative strategies to target (modulate) neurotrophin production and/or their signal transduction pathways. Particular emphasis is placed on small molecules that are able to modulate neurotrophin function in diseases of the nervous system. These alternative strategies show promise in preclinical studies, with some advancing into clinical development. Moreover, the recognition that clinically effective therapeutics, such as antidepressants and immunophilin ligands, can modulate neurotrophin function suggests that the concept of small molecule therapeutics that promote neurotrophic function may still be viable.     

Revision Knee Arthroplasty for Bone Loss: Choosing the Right Degree of Constraint

Revision total knee arthroplasty (TKA) in the setting of bone deficiency requires varied levels of constraint to restore knee stability. However, the outcomes between different levels remain controversial. Clinical outcomes for 183 AORI Type I knees, 168 Type II knees and 124 Type III knees utilizing posterior stabilized (PS), unlinked constrained (UC) or hinged prostheses were evaluated with standardized clinical assessment tools and radiographic results over an average of 7.4 years. PS yielded superior knee scores in AORI Type I patients (P < 0.05), UC in Type II and III aseptic patients (P < 0.05), and a hinge was preferred in septic Type II or III knees (P < 0.05). Revision TKA conducted with increased constraint appears effective in the setting of increased bone deficiency.