Biological And Computational Studies Of The Structure And Function Of Pul103, A Human Cytomegalovirus Tegument Protein

Human cytomegalovirus (HCMV) is an enveloped, single segment, double-stranded DNA virus. HCMV infection causes disease in immunocompromised (HIV patients, transplant recipients) and immunodeficient (fetuses, neonates) populations. Current treatments are effective but are either limited in use or can lead to organ damage and/or antiviral resistance, and no vaccines are available. Additional antiviral targets are needed. HCMV pUL103 is a potential antiviral target. pUL103 is a conserved herpesvirus protein present in the tegument, layer of proteins and RNA between the envelope and capsid of HCMV virions. pUL103 helps reorganize cellular secretory machinery (Golgi, endosomes) to form the cytoplasmic virion assembly compartment (cVAC) that is hypothesized to facilitate efficient virion assembly and egress. pUL103 is also important for cell-to-cell spread and virion maturation. The structure, function, and mechanisms of action of pUL103 are unknown. To identify amino acids important for pUL103 functions, I aligned the sequences of 14 pUL103 homologs using MUSCLE (Multiple Sequence Comparison by Log-Expectation). My data showed 12 conserved amino acids outside of the conserved herpesvirus domain. Within the N terminus there are 13 conserved residues, which shows the importance of this region and these residues in pUL103 function. To guide biological studies of pUL103, I used I-TASSER (Iterative Threading ASSEmbly Refinement) to predict structures of pUL103 and its homologs HSV-1 pUL7 (pUL7) and EBV BBRF2 (BBRF2) using their amino acid sequences. My data showed (a) their predicted secondary structures are similar, (b) the predicted structure of BBRF2 differs from pUL103 and pUL7, which are similar, and (c) the predicted pUL103 structure aligned with the non-catalytic LIM domain of human terminal uridylyl transferase 4 (TUT4), a nucleotidyl transferase domain lacking catalytic residues. This data suggests pUL103 may bind nucleic acids; experiments can be designed to test this hypothesis. To identify when pUL103 mechanistic studies should be performed, I determined when pUL103 is necessary for maximal cVAC abundance. During 120-hour infections, I showed pUL103 must be present between 72 and 84 hpi. Such studies might include analysis of virion assembly, intra- and extracellular viral titers, and deep temporal and spatial proteomics analysis of viral and cellular proteins during this process

Data-Driven Scratch Generation for Rigid-Body Models

The procedural scratch generation process for rigid-body models automates the process of locating and painting scratches on a texture map. A script computes points where a scratch is most likely to occur at each frame of an animation sequence based on whether the distance between two objects lies within a threshold. These points are then used to create a map of the scratch pattern to guide the blending of scratches onto the image. The tool may be useful to expedite the animated film production pipeline as well as predict areas of wear on machines before they are manufactured

Sn-modification of Pt7/alumina model catalysts: Suppression of carbon deposition and enhanced thermal stability.

An atomic layer deposition process is used to modify size-selected Pt7/alumina model catalysts by Sn addition, both before and after Pt7 cluster deposition. Surface science methods are used to probe the effects of Sn-modification on the electronic properties, reactivity, and morphology of the clusters. Sn addition, either before or after cluster deposition, is found to strongly affect the binding properties of a model alkene, ethylene, changing the number and type of binding sites, and suppressing decomposition leading to carbon deposition and poisoning of the catalyst. Density functional theory on a model system, Pt4Sn3/alumina, shows that the Sn and Pt atoms are mixed, forming alloy clusters with substantial electron transfer from Sn to Pt. The presence of Sn also makes all the thermally accessible structures closed shell, such that ethylene binds only by π-bonding to a single Pt atom. The Sn-modified catalysts are quite stable in repeated ethylene temperature programmed reaction experiments, suggesting that the presence of Sn also reduces the tendency of the sub-nano-clusters to undergo thermal sintering

Single-cell profiling of human megakaryocyte-erythroid progenitors identifies distinct megakaryocyte and erythroid differentiation pathways

Background Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by “bulk” assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined. Results To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlate the surface immunophenotype, transcriptional profile, and differentiation potential of individual MEP cells. Highly purified, single MEP cells were analyzed using index fluorescence-activated cell sorting and parallel targeted transcriptional profiling of the same cells was performed using a specifically designed panel of genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrate that immunophenotypic MEP comprise three distinct subpopulations: “Pre-MEP,” enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity; “E-MEP,” strongly biased towards erythroid differentiation; and “MK-MEP,” a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally defined MEP are a mixed population, as a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally primed to generate exclusively single-lineage output. Conclusions Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders, and erythromegakaryocytic leukemias

Searching for stochastic gravitational-wave background with the co-located LIGO interferometers

This paper presents techniques developed by the LIGO Scientific Collaboration to search for the stochastic gravitational-wave background using the co-located pair of LIGO interferometers at Hanford, WA. We use correlations between interferometers and environment monitoring instruments, as well as time-shifts between two interferometers (described here for the first time) to identify correlated noise from non-gravitational sources. We veto particularly noisy frequency bands and assess the level of residual non-gravitational coupling that exists in the surviving data.Comment: Proceedings paper from the 7th Edoardo Amaldi Conference on Gravitational Waves, held in Sydney, Australia from 8-14 July 2007. Accepted to J. Phys.: Conf. Se

Kinomic Profiling of Electromagnetic Navigational Bronchoscopy Specimens: A New Approach for Personalized Medicine

Purpose Researchers are currently seeking relevant lung cancer biomarkers in order to make informed decisions regarding therapeutic selection for patients in so-called “precision medicine.” However, there are challenges to obtaining adequate lung cancer tissue for molecular analyses. Furthermore, current molecular testing of tumors at the genomic or transcriptomic level are very indirect measures of biological response to a drug, particularly for small molecule inhibitors that target kinases. Kinase activity profiling is therefore theorized to be more reflective of in vivo biology than many current molecular analysis techniques. As a result, this study seeks to prove the feasibility of combining a novel minimally invasive biopsy technique that expands the number of lesions amenable for biopsy with subsequent ex vivo kinase activity analysis. Methods Eight patients with lung lesions of varying location and size were biopsied using the novel electromagnetic navigational bronchoscopy (ENB) technique. Basal kinase activity (kinomic) profiles and ex vivo interrogation of samples in combination with tyrosine kinase inhibitors erlotinib, crizotinib, and lapatinib were performed by PamStation 12 microarray analysis. Results Kinomic profiling qualitatively identified patient specific kinase activity profiles as well as patient and drug specific changes in kinase activity profiles following exposure to inhibitor. Thus, the study has verified the feasibility of ENB as a method for obtaining tissue in adequate quantities for kinomic analysis and has demonstrated the possible use of this tissue acquisition and analysis technique as a method for future study of lung cancer biomarkers. Conclusions We demonstrate the feasibility of using ENB-derived biopsies to perform kinase activity assessment in lung cancer patients

Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia

Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis

The clustering of galaxies in the SDSS-III Baryon Oscillation Spectroscopic Survey : measuring DA and H at z = 0.57 from the baryon acoustic peak in the Data Release 9 spectroscopic Galaxy sample

We present measurements of the angular diameter distance to and Hubble parameter at z = 0.57 from the measurement of the baryon acoustic peak in the correlation of galaxies from the Sloan Digital Sky Survey III Baryon Oscillation Spectroscopic Survey. Our analysis is based on a sample from Data Release 9 of 264 283 galaxies over 3275 square degrees in the redshift range 0.43 < z < 0.70. We use two different methods to provide robust measurement of the acoustic peak position across and along the line of sight in order to measure the cosmological distance scale. We find DA(0.57) = 1408 ± 45 Mpc and H(0.57) = 92.9 ± 7.8 km s−1 Mpc−1 for our fiducial value of the sound horizon. These results from the anisotropic fitting are fully consistent with the analysis of the spherically averaged acoustic peak position presented in Anderson et al. Our distance measurements are a close match to the predictions of the standard cosmological model featuring a cosmological constant and zero spatial curvature.Publisher PDFPeer reviewe

Changes in the Treatment Responses to Artesunate-Mefloquine on the Northwestern Border of Thailand during 13 Years of Continuous Deployment

Background: Artemisinin combination treatments (ACT) are recommended as first line treatment for falciparum malaria throughout the malaria affected world. We reviewed the efficacy of a 3-day regimen of mefloquine and artesunate regimen (MAS ), over a 13 year period of continuous deployment as first-line treatment in camps for displaced persons and in clinics for migrant population along the Thai-Myanmar border. Methods and Findings: 3,264 patients were enrolled in prospective treatment trials between 1995 and 2007 and treated with MAS. The proportion of patients with parasitaemia persisting on day-2 increased significantly from 4.5% before 2001 to 21.9% since 2002 (p&lt;0.001). Delayed parasite clearance was associated with increased risk of developing gametocytaemia (AOR = 2.29; 95% CI, 2.00-2.69, p = 0.002). Gametocytaemia on admission and carriage also increased over the years (p = 0.001, test for trend, for both). MAS efficacy has declined slightly but significantly (Hazards ratio 1.13; 95% CI, 1.07-1.19, p&lt;0.001), although efficacy in 2007 remained well within acceptable limits: 96.5% (95% CI, 91.0-98.7). The in vitro susceptibility of P. falciparum to artesunate increased significantly until 2002, but thereafter declined to levels close to those of 13 years ago (geometric mean in 2007: 4.2 nM/l; 95% CI, 3.2-5.5). The proportion of infections caused by parasites with increased pfmdr1 copy number rose from 30% (12/ 40) in 1996 to 53% (24/45) in 2006 (p = 0.012, test for trend). Conclusion: Artesunate-mefloquine remains a highly efficacious antimalarial treatment in this area despite 13 years of widespread intense deployment, but there is evidence of a modest increase in resistance. Of particular concern is the slowing of parasitological response to artesunate and the associated increase in gametocyte carriage. © 2009 Carrara et al