Andrew P. Norgan

Multilevel Parallelization of AutoDock 4.2.

BackgroundVirtual (computational) screening is an increasingly important tool for drug discovery. AutoDock is a popular open-source application for performing molecular docking, the prediction of ligand-receptor interactions. AutoDock is a serial application, though several previous efforts have parallelized various aspects of the program. In this paper, we report on a multi-level parallelization of AutoDock 4.2 (mpAD4).ResultsUsing MPI and OpenMP, AutoDock 4.2 was parallelized for use on MPI-enabled systems and to multithread the execution of individual docking jobs. In addition, code was implemented to reduce input/output (I/O) traffic by reusing grid maps at each node from docking to docking. Performance of mpAD4 was examined on two multiprocessor computers.ConclusionsUsing MPI with OpenMP multithreading, mpAD4 scales with near linearity on the multiprocessor systems tested. In situations where I/O is limiting, reuse of grid maps reduces both system I/O and overall screening time. Multithreading of AutoDocks Lamarkian Genetic Algorithm with OpenMP increases the speed of execution of individual docking jobs, and when combined with MPI parallelization can significantly reduce the execution time of virtual screens. This work is significant in that mpAD4 speeds the execution of certain molecular docking workloads and allows the user to optimize the degree of system-level (MPI) and node-level (OpenMP) parallelization to best fit both workloads and computational resources.

The HECT Domain of the Ubiquitin Ligase Rsp5 Contributes to Substrate Recognition

Ubiquitin modification of endosomal membrane proteins is a signal for active inclusion into the Multivesicular Body (MVB) pathway, resulting in lysosomal degradation. However, the endosome represents a dynamic site of protein sorting with a majority of proteins destined for recycling, rather than MVB targeting. Substrate recognition by ubiquitin ligases is therefore highly regulated. We have investigated substrate recognition by the Nedd4 ortholog Rsp5 as a model for understanding ligase-substrate interactions. Rsp5 interacts directly with its substrate Cps1 via a novel interaction mode. Perturbation of this mode of interaction revealed a compensatory role for the Rsp5 adaptor Bsd2. These results highlight the ability of Rsp5 to interact with substrates via multiple modalities, suggesting additional mechanisms of regulating this interaction and relevant outcomes.

Relief of Autoinhibition Enhances Vta1 Activation of Vps4 via the Vps4 Stimulatory Element

Background: Vta1 promotes Vps4 ATPase activity and facilitates ESCRT-III stimulation of Vps4. Results: The Vta1 VSE (Vps4 stimulatory element) mediates ESCRT-III-enhanced activation of Vps4 and contributes to Vta1 function in vivo. Conclusion: ESCRT-III binding Vta1 relieves autoinhibition of the VSE to promote activation of Vps4. Significance: These studies identify a novel mechanism whereby ESCRT-III and Vta1 regulate Vps4 activity. The endosomal sorting complexes required for transport (ESCRTs) impact multiple cellular processes including multivesicular body sorting, abscission, and viral budding. The AAA-ATPase Vps4 is required for ESCRT function, and its full activity is dependent upon the co-factor Vta1. The Vta1 carboxyl-terminal Vta1 SBP1 Lip5 (VSL) domain stimulates Vps4 function by facilitating oligomerization of Vps4 into its active state. Here we report the identification of the Vps4 stimulatory element (VSE) within Vta1 that is required for additional stimulation of Vps4 activity in vitro and in vivo. VSE activity is autoinhibited in a manner dependent upon the unstructured linker region joining the amino-terminal microtubule interacting and trafficking domains and the carboxyl-terminal VSL domain. The VSE is also required for Vta1-mediated Vps4 stimulation by ESCRT-III subunits Vps60 and Did2. These results suggest that ESCRT-III binding to the Vta1 microtubule interacting and trafficking domains relieves linker region autoinhibition of the VSE to produce maximal activation of Vps4 during ESCRT function.

Vps4 stimulatory element of the cofactor Vta1 contacts the ATPase Vps4 α7 and α9 to stimulate ATP hydrolysis.

Background: ESCRT-III can enhance Vta1 stimulation of Vps4 ATPase activity via the Vps4 stimulatory element (VSE) of Vta1. Results: α7 and α9 of the Vps4 small AAA domain mediate VSE stimulation, contributing to Vps4 function in vivo. Conclusion: Vta1 contacts Vps4 α7 and α9 during ESCRT-III-enhanced stimulation of Vps4. Significance: These studies identify a novel mechanism of Vps4 stimulation. The endosomal sorting complexes required for transport (ESCRTs) function in a variety of membrane remodeling processes including multivesicular body sorting, abscission during cytokinesis, budding of enveloped viruses, and repair of the plasma membrane. Vps4 ATPase activity modulates ESCRT function and is itself modulated by its cofactor Vta1 and its substrate ESCRT-III. The carboxyl-terminal Vta1/SBP-1/Lip5 (VSL) domain of Vta1 binds to the Vps4 β-domain to promote Vps4 oligomerization-dependent ATP hydrolysis. Additionally, the Vps4 stimulatory element (VSE) of Vta1 contributes to enhancing Vps4 oligomer ATP hydrolysis. The VSE is also required for Vta1-dependent stimulation of Vps4 by ESCRT-III subunits. However, the manner by which the Vta1 VSE contributes to Vps4 activation is unknown. Existing structural data were used to generate a model of the Vta1 VSE in complex with Vps4. This model implicated residues within the small ATPase associated with various activities (AAA) domain, specifically α-helices 7 and 9, as relevant contact sites. Rational generation of Vps4 mutants defective for VSE-mediated stimulation, as well as intergenic compensatory mutations, support the validity of this model. These findings have uncovered the Vps4 surface responsible for coordinating ESCRT-III-stimulated Vta1 input during ESCRT function and identified a novel mechanism of Vps4 stimulation.

A method for reducing the sloughing of thick blood films for malaria diagnosis

BackgroundThe gold standard for malaria diagnosis is the examination of thick and thin blood films. Thick films contain 10 to 20 times more blood than thin films, correspondingly providing increased sensitivity for malaria screening. A potential complication of thick film preparations is sloughing of the blood droplet from the slide during staining or rinsing, resulting in the loss of sample. In this work, two methods for improving thick film slide adherence (‘scratch’ (SCM) and ‘acetone dip’ (ADM) methods) were compared to the ‘standard method’ (SM) of thick film preparation.MethodsStandardized blood droplets from 26 previously examined EDTA whole blood specimens (22 positive and four negative) were concurrently spread on glass slides using the SM, ADM, and SCM. For the SM and ADM prepared slides, the droplet was gently spread to an approximate 22 millimeters in diameter spot on the slide using the edge of a second glass slide. For the SCM, the droplet was spread by carefully grinding (or scratching) it into the slide with the point of a second glass slide. Slides were dried for one hour in a laminar flow hood. For the ADM, slides were dipped once in an acetone filled Coplin jar and allowed to air dry. All slides were then Giemsa-stained and examined in a blinded manner. Adherence was assessed by blinded reviewers.ResultsNo significant or severe defects were observed for slides prepared with the SCM. In contrast, 8 slides prepared by the ADM and 3 prepared using the SM displayed significant or severe defects. Thick films prepared by the three methods were microscopically indistinguishable and concordant results (positive or negative) were obtained for the three methods. Estimated parasitaemia of the blood samples ranged from 25 to 429,169 parasites/μL of blood.ConclusionsThe SCM is an inexpensive, rapid, and simple method that improves the adherence of thick blood films to standard glass slides without altering general slide preparation, microscopic appearance or interpretability. Using the SCM, thick films can be reliably examined less than two hours after sample receipt. This represents a significant diagnostic improvement over protocols requiring extended drying periods.

In-hospital outcomes associated with stent-assisted endovascular treatment of unruptured cerebral aneurysms in the USA

Background Adjunctive stenting has increasingly become an acceptable option for the endovascular treatment of unruptured aneurysms. The Nationwide Inpatient Sample (NIS) was used to compare US in-hospital outcomes related to coiling with and without adjunctive stenting for unruptured aneurysms. Methods Hospitalizations for coiling of unruptured cerebral aneurysms from 2004 to 2008 were identified in the NIS by extracting ICD-9-CM codes for the diagnosis of unruptured aneurysm (437.3) and intracranial stenting (00.65) with coiling (39.52, 39.79 or 39.72) of cerebral aneurysms. All patients with a diagnosis of subarachnoid hemorrhage (430) and/or intracerebral hemorrhage (431) were excluded. Mortality and discharge to a long-term facility were compared between stent and non-stent patient groups using multivariate regression analysis. Results Patients treated with stent-assisted coiling had an in-hospital mortality rate of 0.08–0.8% compared with a death rate of 0.5% (95% CI 0.3% to 0.7%) for patients who did not receive a stent during coiling (p=0.36). Patients in the stent group had a 3% rate of discharge to a care facility (95% CI 1.5% to 5.8%) compared with 5% (95% CI 4.5% to 5.6%) for those in the non-stent group (p=0.14). Patients treated with a stent had a similar likelihood of in-hospital mortality (adjusted OR, 2.12 (95% CI 0.32 to 7.11), p=0.34) and a lower likelihood of discharge to a long-term care facility (adjusted OR 0.59 (95% CI 0.24 to 1.16), p=0.16) compared with the non-stent group. Conclusions Adjunctive stenting adds little in-hospital risk to the endovascular treatment of cerebral aneurysms. However, the need for dual antiplatelet therapy may predispose to delayed hemorrhagic complications and discontinuation of dual antiplatelet therapy may lead to delayed thromboembolic complications.

How do I … manage the platelet transfusion–refractory patient?

Platelet transfusion–refractoriness is a challenging and expensive clinical scenario seen most often in patients with hematologic malignancies. Although the majority of platelet transfusion–refractory cases are due to nonimmune causes, a significant minority are caused by alloimmunization against Class I human leukocyte antigens (HLAs) or human platelet antigens (HPAs). Such platelet transfusion–refractory patients can be effectively managed with appropriate antigen‐negative products.

An Immunocompromised Child with Bloodstream Infection Caused by Two Escherichia coli Strains, One Harboring NDM-5 and the Other Harboring OXA-48-Like Carbapenemase

ABSTRACT We describe a 16-year-old neutropenic patient from the Middle East with bloodstream infection caused by two carbapenemase-producing Escherichia coli isolates that we characterized by whole-genome sequencing. While one displayed meropenem resistance and was blaNDM positive, the other demonstrated meropenem susceptibility yet harbored blaOXA181 (which encodes a blaOXA48-like enzyme). This report highlights the challenge of laboratory detection of blaOXA48-like enzymes and the clinical implications of genotypic resistance detection in carbapenemase-producing Enterobacteriaceae.

ESCRT-Independent Budding of HIV-1 Gag Virus-Like Particles from Saccharomyces cerevisiae Spheroplasts

Heterologous expression of HIV-1 Gag in a variety of host cells results in its packaging into virus-like particles (VLPs) that are subsequently released into the extracellular milieu. This phenomenon represents a useful tool for probing cellular factors required for viral budding and has contributed to the discovery of roles for ubiquitin ligases and the endosomal sorting complexes required for transport (ESCRTs) in viral budding. These factors are highly conserved throughout eukaryotes and have been studied extensively in the yeast Saccharomyces cerevisiae, a model eukaryote previously utilized as a host for the production of VLPs. We used heterologous expression of HIV Gag in yeast spheroplasts to examine the role of ESCRTs and associated factors (Rsp5, a HECT ubiquitin ligase of the Nedd4 family; Bro1, a homolog of Alix; and Vps4, the AAA-ATPase required for ESCRT function in all contexts/organisms investigated) in the generation of VLPs. Our data reveal: 1) characterized Gag-ESCRT interaction motifs (late domains) are not required for VLP budding, 2) loss of function alleles of the essential HECT ubiquitin ligase Rsp5 do not display defects in VLP formation, and 3) ESCRT function is not required for VLP formation from spheroplasts. These results suggest that the egress of HIV Gag from yeast cells is distinct from the most commonly described mode of exit from mammalian cells, instead mimicking ESCRT-independent VLP formation observed in a subset of mammalian cells. As such, budding of Gag from yeast cells appears to represent ESCRT-independent budding relevant to viral replication in at least some situations. Thus the myriad of genetic and biochemical tools available in the yeast system may be of utility in the study of this aspect of viral budding.

The use of cytapheresis in the treatment of infectious diseases

Cytapheresis (removal of cellular blood components) has been employed for treatment of infectious diseases since the 1960s. Techniques have included thrombocytapheresis (buffy coat apheresis) for loiasis, erythrocytapheresis for malaria and babesiosis, and leukocytapheresis for pertussis‐associated lymphocytosis. Published data on these applications is largely limited to case level data and small observational studies; as such, recommendations for or against the use of cytapheresis in the treatment of infections have been extrapolated from these limited (and at times flawed) data sets. Consequently, utilization of cytapheresis in many instances is not uniform between institutions, and typically occurs at the discretion of treating medical teams. This review revisits the existing literature on the use of cytapheresis in the treatment of four infections (loasis, malaria, babesiosis, and pertussis) and examines the rationale underlying current treatment recommendations concerning its use.