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Crystal structures of human CtBP in complex with substrate MTOB reveal active site features useful for inhibitor design

Wed, 01/21/2015 - 3:22pm

The oncogenic corepressors C-terminal Binding Protein (CtBP) 1 and 2 harbor regulatory d-isomer specific 2-hydroxyacid dehydrogenase (d2-HDH) domains. 4-Methylthio 2-oxobutyric acid (MTOB) exhibits substrate inhibition and can interfere with CtBP oncogenic activity in cell culture and mice. Crystal structures of human CtBP1 and CtBP2 in complex with MTOB and NAD(+) revealed two key features: a conserved tryptophan that likely contributes to substrate specificity and a hydrophilic cavity that links MTOB with an NAD(+) phosphate. Neither feature is present in other d2-HDH enzymes. These structures thus offer key opportunities for the development of highly selective anti-neoplastic CtBP inhibitors. Elsevier B.V. All rights reserved.

HIV-1 protease-substrate coevolution in nelfinavir resistance

Wed, 01/21/2015 - 3:22pm

Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. IMPORTANCE: Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations.

Drug Resistance Mutations Alter Dynamics of Inhibitor-Bound HIV-1 Protease

Wed, 01/21/2015 - 3:22pm

Under the selective pressure of therapy, HIV-1 protease mutants resistant to inhibitors evolve to confer drug resistance. Such mutations can impact both the dynamics and structures of the bound and unbound forms of the enzyme. Flap+ is a multidrug-resistant variant of HIV-1 protease with a combination of primary and secondary resistance mutations (L10I, G48V, I54V, V82A) and a strikingly altered thermodynamic profile for darunavir (DRV) binding relative to the wild-type protease. We elucidated the impact of these mutations on protein dynamics in the DRV-bound state using molecular dynamics simulations and NMR relaxation experiments. Both methods concur in that the conformational ensemble and dynamics of protease are impacted by the drug resistance mutations in Flap+ variant. Surprisingly this change in ensemble dynamics is different from that observed in the unliganded form of the same variant (Cai, Y. et al. J. Chem. Theory Comput. 2012, 8, 3452-3462). Our comparative analysis of both inhibitor-free and bound states presents a comprehensive picture of the altered dynamics in drug-resistant mutant HIV-1 protease and underlies the importance of incorporating dynamic analysis of the whole system, including the unliganded state, into revealing drug resistance mechanisms.

Drug resistance conferred by mutations outside the active site through alterations in the dynamic and structural ensemble of HIV-1 protease

Wed, 01/21/2015 - 3:22pm

HIV-1 protease inhibitors are part of the highly active antiretroviral therapy effectively used in the treatment of HIV infection and AIDS. Darunavir (DRV) is the most potent of these inhibitors, soliciting drug resistance only when a complex combination of mutations occur both inside and outside the protease active site. With few exceptions, the role of mutations outside the active site in conferring resistance remains largely elusive. Through a series of DRV-protease complex crystal structures, inhibition assays, and molecular dynamics simulations, we find that single and double site mutations outside the active site often associated with DRV resistance alter the structure and dynamic ensemble of HIV-1 protease active site. These alterations correlate with the observed inhibitor binding affinities for the mutants, and suggest a network hypothesis on how the effect of distal mutations are propagated to pivotal residues at the active site and may contribute to conferring drug resistance.

Structural basis and distal effects of Gag substrate coevolution in drug resistance to HIV-1 protease

Wed, 01/21/2015 - 3:22pm

Drug resistance mutations in response to HIV-1 protease inhibitors are selected not only in the drug target but elsewhere in the viral genome, especially at the protease cleavage sites in the precursor protein Gag. To understand the molecular basis of this protease-substrate coevolution, we solved the crystal structures of drug resistant I50V/A71V HIV-1 protease with p1-p6 substrates bearing coevolved mutations. Analyses of the protease-substrate interactions reveal that compensatory coevolved mutations in the substrate do not restore interactions lost due to protease mutations, but instead establish other interactions that are not restricted to the site of mutation. Mutation of a substrate residue has distal effects on other residues' interactions as well, including through the induction of a conformational change in the protease. Additionally, molecular dynamics simulations suggest that restoration of active site dynamics is an additional constraint in the selection of coevolved mutations. Hence, protease-substrate coevolution permits mutational, structural, and dynamic changes via molecular mechanisms that involve distal effects contributing to drug resistance.

Structural analysis of asunaprevir resistance in HCV NS3/4A protease

Wed, 01/21/2015 - 3:22pm

Asunaprevir (ASV), an isoquinoline-based competitive inhibitor targeting the hepatitis C virus (HCV) NS3/4A protease, is very potent in vivo. However, the potency is significantly compromised by the drug resistance mutations R155K and D168A. In this study three crystal structures of ASV and an analogue were determined to analyze the structural basis of drug resistance susceptibility. These structures revealed that ASV makes extensive contacts with Arg155 outside the substrate envelope. Arg155 in turn is stabilized by Asp168, and thus when either residue is mutated, the enzyme's interaction with ASV's P2* isoquinoline is disrupted. Adding a P1-P3 macrocycle to ASV enhances the inhibitor's resistance barrier, likely due to poising the inhibitor to its bound conformation. Macrocyclic inhibitors with P2* extension moieties avoiding interaction with the protease S2 residues including Arg155 must be chosen for future design of more robust protease inhibitors.

Acute stroke imaging: recent updates

Thu, 01/15/2015 - 1:43pm

Acute ischemic stroke imaging is one of the leading causes of death and disability worldwide. Neuroimaging plays a crucial role in early diagnosis and yields essential information regarding tissue integrity, a factor that remains a key therapeutic determinant. Given the widespread public health implications of stroke and central role of neuroimaging in overall management, acute stroke imaging remains a heavily debated, extensively researched, and rapidly evolving subject. There has been recent debate in the scientific community due to divided opinions on the use of CT perfusion and access-related limitations of MRI. In this paper we review and summarize recent updates relevant to acute stroke imaging and propose an imaging paradigm based on the recently available evidence.

Control of neutrophil inflammation at mucosal surfaces by secreted epithelial products

Thu, 01/15/2015 - 1:43pm

The human intestine is a large and delicately balanced organ, responsible for efficiently absorbing nutrients and selectively eliminating disease-causing pathogens. The gut architecture consists of a single layer of epithelial cells that forms a barrier against the food antigens and resident microbiota within the lumen. This barrier is augmented by a thick layer of mucus on the luminal side and an underlying lamina propria containing a resident population of immune cells. Attempted breaches of the intestinal barrier by pathogenic bacteria result in the rapid induction of a coordinated innate immune response that includes release of antimicrobial peptides, activation of pattern recognition receptors, and recruitment of various immune cells. In recent years, the role of epithelial cells in initiating this immune response has been increasingly appreciated. In particular, epithelial cells are responsible for the release of a variety of factors that attract neutrophils, the body's trained bacterial killers. In this review we will highlight recent research that details a new understanding of how epithelial cells directionally secrete specific compounds at distinct stages of the inflammatory response in order to coordinate the immune response to intestinal microbes. In addition to their importance during the response to infection, evidence suggests that dysregulation of these pathways may contribute to pathologic inflammation during inflammatory bowel disease. Therefore, a continued understanding of the mechanisms by which epithelial cells control neutrophil migration into the intestine will have tremendous benefits in both the understanding of biological processes and the identification of potential therapeutic targets.

Evaluation and management of patients with isolated neutropenia

Thu, 01/15/2015 - 1:43pm

Neutropenia, defined as an absolute neutrophil count (ANC) < 1.5 x 10(9)/L, encompasses a wide range of diagnoses, from normal variants to life-threatening acquired and congenital disorders. This review addresses the diagnosis and management of isolated neutropenia, not multiple cytopenias due to splenomegaly, bone marrow replacement, or myelosuppression by chemotherapy or radiation. Laboratory evaluation generally includes repeat complete blood cell counts (CBCs) with differentials and bone marrow examination with cytogenetics. Neutrophil antibody testing may be useful but only in the context of clinical and bone marrow findings. The discovery of genes responsible for congenital neutropenias now permits genetic diagnosis in many cases. Management of severe chronic neutropenia includes commonsense precautions to avoid infection, aggressive treatment of bacterial or fungal infections, and administration of granulocyte colony-stimulating factor (G-CSF). Patients with severe chronic neutropenia, particularly those who respond poorly to G-CSF, have a risk of eventually developing myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) and require monitoring for this complication, which also can occur without G-CSF therapy. Patients with cyclic, idiopathic, and autoimmune neutropenia have virtually no risk of evolving to MDS or AML. Hematopoietic stem cell transplantation is a curative therapy for congenital neutropenia with MDS/AML or with cytogenetic abnormalities indicating impending conversion.

hnRNP A1 and secondary structure coordinate alternative splicing of Mag

Thu, 01/15/2015 - 1:43pm

Myelin-associated glycoprotein (MAG) is a major component of myelin in the vertebrate central nervous system. MAG is present in the periaxonal region of the myelin structure, where it interacts with neuronal proteins to inhibit axon outgrowth and protect neurons from degeneration. Two alternatively spliced isoforms of Mag mRNA have been identified. The mRNA encoding the shorter isoform, known as S-MAG, contains a termination codon in exon 12, while the mRNA encoding the longer isoform, known as L-MAG, skips exon 12 and produces a protein with a longer C-terminal region. L-MAG is required in the central nervous system. How inclusion of Mag exon 12 is regulated is not clear. In a previous study, we showed that heteronuclear ribonucleoprotein A1 (hnRNP A1) contributes to Mag exon 12 skipping. Here, we show that hnRNP A1 interacts with an element that overlaps the 5' splice site of Mag exon 12. The element has a reduced ability to interact with the U1 snRNP compared with a mutant that improves the splice site consensus. An evolutionarily conserved secondary structure is present surrounding the element. The structure modulates interaction with both hnRNP A1 and U1. Analysis of splice isoforms produced from a series of reporter constructs demonstrates that the hnRNP A1-binding site and the secondary structure both contribute to exclusion of Mag exon 12.

Seven propositions of the science of improvement: exploring foundations

Thu, 01/15/2015 - 1:43pm

CONTEXT: The phrase "Science of Improvement" or "Improvement Science" is commonly used today by a range of people and professions to mean different things, creating confusion to those trying to learn about improvement. In this article, we briefly define the concepts of improvement and science, and review the history of the consideration of "improvement" as a science.

METHODS: We trace key concepts and ideas in improvement to their philosophical and theoretical foundation with a focus on Deming's System of Profound Knowledge. We suggest that Deming's system has a firm association with many contemporary and historic philosophic and scientific debates and concepts. With reference to these debates and concepts, we identify 7 propositions that provide the scientific and philosophical foundation for the science of improvement.

FINDINGS: A standard view of the science of improvement does not presently exist that is grounded in the philosophical and theoretical basis of the field. The 7 propositions outlined here demonstrate the value of examining the underpinnings of improvement. This is needed to both advance the field and minimize confusion about what the phrase "science of improvement" represents. We argue that advanced scientists of improvement are those who like Deming and Shewhart can integrate ideas, concepts, and models between scientific disciplines for the purpose of developing more robust improvement models, tools, and techniques with a focus on application and problem solving in real world contexts.

CONCLUSIONS: The epistemological foundations and theoretical basis of the science of improvement and its reasoning methods need to be critically examined to ensure its continued development and relevance. If improvement efforts and projects in health care are to be characterized under the canon of science, then health care professionals engaged in quality improvement work would benefit from a standard set of core principles, a standard lexicon, and an understanding of the evolution of the science of improvement.

Data citation and the author byline: who's line is it anyway

Thu, 01/15/2015 - 1:43pm

A recent article by Rohlfing & Poline has started a dialog regarding the use of "Institutional Authorship" in the authorship list of manuscripts that utilize the resources of some consortia and organizations.

Vertebral artery ostial stenosis: prevalence by digital subtraction angiography, MR angiography, and CT angiography

Thu, 01/15/2015 - 1:43pm

BACKGROUND AND PURPOSE: (1) To determine the prevalence of vertebral arterial ostial stenosis (VOS) as determined by the "gold standard" of digital subtraction angiography (DSA). (2) To learn the correlation between vertebral ostial stenosis and study indication. (3) To determine the ability of contrast-enhanced magnetic resonance angiography (CE MRA) and computed tomographic angiography (CTA) to reflect the true prevalence of vertebral ostial stenosis as determined by DSA.

METHODS: Three hundred and twenty-nine patients who underwent DSA had recorded evaluation of 443 vertebral artery origins. Cases were categorized by patient age and study indication. Similar numbers of CTA and MRA studies were assessed.

RESULTS: The prevalence of VOS in the study population was 5.4%. VOS was not observed in patients under 40 years of age, and was seen in 12.5% of patients over 70 years. CE MRA demonstrated decreased signal at the vertebral origins consistent with stenosis in 20% of patients. CTA estimated VOS at .8%, and yielded 7.3% of studies, which were nondiagnostic for VOS.

CONCLUSION: The prevalence of VOS as determined by DSA is low and increases with patient age and correlates with factors such as anterior infarct (18.4%), posterior infarct (33.3%), carotid atherosclerosis (30.8%), and vertebrobasilar insufficiency (33%). Patients being evaluated for reasons less closely correlated with atherosclerotic disease, such as arteriovenous malformation (AVM) or hemorrhage showed a lower prevalence of VA stenosis (brain aneurysm or AVM 5/121, 4.1%, brain hemorrhage 5/153, 3.3%). Routine clinical MRA significantly overestimates VOS prevalence, and findings suggest that CTA underestimates the degree and prevalence of VOS.

DNA Replication Timing

Thu, 01/15/2015 - 1:42pm

Patterns of replication within eukaryotic genomes correlate with gene expression, chromatin structure, and genome evolution. Recent advances in genome-scale mapping of replication kinetics have allowed these correlations to be explored in many species, cell types, and growth conditions, and these large data sets have allowed quantitative and computational analyses. One striking new correlation to emerge from these analyses is between replication timing and the three-dimensional structure of chromosomes. This correlation, which is significantly stronger than with any single histone modification or chromosome-binding protein, suggests that replication timing is controlled at the level of chromosomal domains. This conclusion dove tails with parallel work on the heterogeneity of origin firing and the competition between origins for limiting activators to suggest a model in which the stochastic probability of individual origin firing is modulated by chromosomal domain structure to produce patterns of replication. Whether these patterns have inherent biological functions or simply reflect higher-order genome structure is an open question.

Five top stories in anatomic pathology: stories from the faculty at UMass Medical School and UMass Memorial Medical Center, Worcester, Massachusetts

Thu, 01/15/2015 - 1:42pm

This month's issue of the Archives of Pathology & Laboratory Medicine introduces a new series of special sections featuring relevant and contemporary topics in Anatomic Pathology and Cytopathology, herein referred to as “Five Top Stories.”

Clinical implications of current developments in genitourinary pathology

Thu, 01/15/2015 - 1:42pm

CONTEXT: Several developments in genitourinary pathology are likely to change our understanding and management of some genitourinary cancers considerably.

OBJECTIVE: To review 5 stories in genitourinary pathology: (1) fusion in the ETS (E26) gene family in prostatic adenocarcinoma; (2) insulin-like growth factor II messenger RNA-binding protein 3 (IMP3), an important prognostic biomarker for kidney and bladder cancers; (3) translocation renal cell carcinoma; (4) UroVysion fluorescence in situ hybridization test in urine cytology for detection of bladder cancer; and (5) the use of triple immunostaining for diagnosis of prostate cancer.

DATA SOURCES: Literature review and authors' personal experiences.

CONCLUSIONS: Many scientific findings have contributed recently to the understanding of the natural pathogenesis and progression of genitourinary cancers. This translational research helps in diagnosing, predicting, and potentially, treating genitourinary cancers.

Five top stories in cytopathology

Thu, 01/15/2015 - 1:42pm

CONTEXT: Cytology relies heavily on morphology to make diagnoses, and morphologic criteria have not changed much in recent years. The field is being shaped predominantly by new techniques for imaging and for acquiring and processing samples, advances in molecular diagnosis and therapeutics, and regulatory issues.

OBJECTIVE: To review the importance of classical morphology in the future of cytopathology, to identify areas in which cytology is expanding or contracting in its scope, and to identify factors that are shaping the field.

DATA SOURCES: Literature review.

CONCLUSIONS: Five stories paint a picture in which classical cytomorphology will continue to have essential importance, both for diagnosis and for improving our understanding of cancer biology. New endoscopy and imaging techniques are replacing surgical biopsies with cytology samples. New molecularly targeted therapies offer a chance for cytology to play a major role, but they pose new challenges. New molecular tests have the potential to synergize with, but not replace, morphologic interpretation of thyroid fine-needle aspirations. Ultrasound-guided fine-needle aspiration performed by cytopathologists is opening a new field of "interventional cytopathology" with unique value. For the productive evolution of the field, it will be important for cytopathologists to play an active role in clinical trials that document the ability of cytology to achieve cost-effective health care outcomes.

Adaptive beta-cell proliferation increases early in high-fat feeding in mice, concurrent with metabolic changes, with induction of islet cyclin D2 expression

Thu, 01/15/2015 - 1:42pm

Type 2 diabetes (T2D) is caused by relative insulin deficiency, due in part to reduced beta-cell mass (11, 62). Therapies aimed at expanding beta-cell mass may be useful to treat T2D (14). Although feeding rodents a high-fat diet (HFD) for an extended period (3-6 mo) increases beta-cell mass by inducing beta-cell proliferation (16, 20, 53, 54), evidence suggests that adult human beta-cells may not meaningfully proliferate in response to obesity. The timing and identity of the earliest initiators of the rodent compensatory growth response, possible therapeutic targets to drive proliferation in refractory human beta-cells, are not known. To develop a model to identify early drivers of beta-cell proliferation, we studied mice during the first week of HFD exposure, determining the onset of proliferation in the context of diet-related physiological changes. Within the first week of HFD, mice consumed more kilocalories, gained weight and fat mass, and developed hyperglycemia, hyperinsulinemia, and glucose intolerance due to impaired insulin secretion. The beta-cell proliferative response also began within the first week of HFD feeding. Intriguingly, beta-cell proliferation increased before insulin resistance was detected. Cyclin D2 protein expression was increased in islets by day 7, suggesting it may be an early effector driving compensatory beta-cell proliferation in mice. This study defines the time frame and physiology to identify novel upstream regulatory signals driving mouse beta-cell mass expansion, in order to explore their efficacy, or reasons for inefficacy, in initiating human beta-cell proliferation.

Ablation of the X-linked retinitis pigmentosa 2 (Rp2) gene in mice results in opsin mislocalization and photoreceptor degeneration

Thu, 01/15/2015 - 1:42pm

PURPOSE: Mutations in the RP2 gene are associated with 10% to 15% of X-linked retinitis pigmentosa (XLRP), a debilitating disorder characterized by the degeneration of retinal rod and cone photoreceptors. The molecular mechanism of pathogenesis of photoreceptor degeneration in XLRP-RP2 has not been elucidated, and no treatment is currently available. This study was undertaken to investigate the pathogenesis of RP2-associated retinal degeneration.

METHODS: We introduced loxP sites that flank exon 2, a mutational hotspot in XLRP-RP2, in the mouse Rp2 gene. We then produced Rp2-null allele using transgenic mice that expressed Cre-recombinase under control of the ubiquitous CAG promoter. Electroretinography (ERG), histology, light microscopy, transmission electron microscopy, and immunofluorescence microscopy were performed to ascertain the effect of ablation of Rp2 on photoreceptor development, function, and protein trafficking.

RESULTS: Although no gross abnormalities were detected in the Rp2(null) mice, photopic (cone) and scotopic (rod) function as measured by ERG showed a gradual decline starting as early as 1 month of age. We also detected slow progressive degeneration of the photoreceptor membrane discs in the mutant retina. These defects were associated with mislocalization of cone opsins to the nuclear and synaptic layers and reduced rhodopsin content in the outer segment of mutant retina prior to the onset of photoreceptor degeneration.

CONCLUSIONS: Our studies suggest that RP2 contributes to the maintenance of photoreceptor function and that cone opsin mislocalization represents an early step in XLRP caused by RP2 mutations. The Rp2(null) mice should serve as a useful preclinical model for testing gene- and cell-based therapies.