Penicillin-binding protein 1B from Escherichia coli contains a membrane association site in addition to its transmembrane anchor

A working structural model of penicillin-binding protein 1B (PBP 1B) from Escherichia coli derived from previous data consists of a highly charged amino-terminal cytoplasmic tail, a 23-amino-acid hydrophobic transmembrane anchor, and a 758-amino-acid periplasmic domain. Using an engineered thrombin cleavage site, we have investigated the solubility properties of the periplasmic domain of PBP 1B. Twelve amino acids, comprised of the consensus thrombin cleavage site (LVPR↓GS) and flanking glycine residues, were inserted into PBP 1B just past its putative transmembrane segment. To aid in purification, a hexahistidine tag was also inserted at its amino terminus, and the engineered protein (PBP 1B-GT/H6) was purified and characterized.A working structural model of penicillin-binding protein 1B (PBP 1B) from Escherichia coli derived from previous data consists of a highly charged amino-terminal cytoplasmic tail, a 23-amino-acid hydrophobic transmembrane anchor, and a 758-amino-acid periplasmic domain. Using an engineered thrombin cleavage site, we have investigated the solubility properties of the periplasmic domain of PBP 1B. Twelve amino acids, comprised of the consensus thrombin cleavage site (LVPR↓GS) and flanking glycine residues, were inserted into PBP 1B just past its putative transmembrane segment. To aid in purification, a hexahistidine tag was also inserted at its amino terminus, and the engineered protein (PBP 1B-GT/H6) was purified and characterized

Comparison of explicit and mean-field models of cytoskeletal filaments with crosslinking motors

In cells, cytoskeletal filament networks are responsible for cell movement, growth, and division. Filaments in the cytoskeleton are driven and organized by crosslinking molecular motors. In reconstituted cytoskeletal systems, motor activity is responsible for far-from-equilibrium phenomena such as active stress, self-organized flow, and spontaneous nematic defect generation. How microscopic interactions between motors and filaments lead to larger-scale dynamics remains incompletely understood. To build from motor-filament interactions to predict bulk behavior of cytoskeletal systems, more computationally efficient techniques for modeling motor-filament interactions are needed. Here we derive a coarse-graining hierarchy of explicit and continuum models for crosslinking motors that bind to and walk on filament pairs. We compare the steady-state motor distribution and motor-induced filament motion for the different models and analyze their computational cost. All three models agree well in the limit of fast motor binding kinetics. Evolving a truncated moment expansion of motor density speeds the computation by $10^3$--$10^6$ compared to the explicit or continuous-density simulations, suggesting an approach for more efficient simulation of large networks. These tools facilitate further study of motor-filament networks on micrometer to millimeter length scales.Comment: 54 pages, 7 figures, 1 tabl

Evaluation of the use of sludge containing plutonium as a soil conditioner for food crops

An experiment was conducted to assess the potential hazard associated with the use of sludge containing plutonium as a soil conditioner for food crops. Conditions were chosen that would maximize exposure to the $sup 239$Pu in the sludge through resuspension and in plant content and thus approximated the maximum potential hazards due to the inhalation and ingestion pathways. The estimated 50-year radiation doses to the pulmonary region of the lung, bone, and liver based on the results of the inhalation experiment are 6 x 10$sup -4$ rem, 1.2 x 10$sup -3$ rem, and 0.55 x 10$sup -4$ rem, respectively. Similarly, the 50- year radiation doses attributable to ingestion of the sludge-grown vegetables were 2.2 x 10$sup -5$ rem to the bone and 1.5 x 10$sup -5$ rem to the liver. Thus, the inhalation pathway is the more critical of the two. The maximum permissible annual doses to the lungs, bone, and the liver for a member of the general public are 1.5, 3.0, and 1.5 rem, respectively. Thus, the maximum credible 50-year lung, bone, and liver dose commitments associated with the use of the $sup 239$Pu-contaminated sludge as a soil conditioner are approximately 4.0 x 10$sup -2$ percent of the annual maximum permissible dose. Under more realistic exposure circumstances, one might expect less drying of the sludge, less resuspension of dust and flying dirt before and during rototilling, and a much smaller sludge vegetable consumption rate. The conservative assumptions made in this analysis tend to assure that actual radiation doses would be even less than those calculated. (auth

Novel Human Rhinoviruses and Exacerbation of Asthma in Children1

To determine links between human rhinoviruses (HRV) and asthma, we used data from a case–control study, March 2003–February 2004, among children with asthma. Molecular characterization identified several likely new HRVs and showed that association with asthma exacerbations was largely driven by HRV-A and a phylogenetically distinct clade of 8 strains, genogroup C

Hypoxia-inducible factor-1 (HIF-1) pathway activation by quercetin in human lens epithelial cells

Quercetin is a dietary bioflavonoid which has been shown to inhibit lens opacification in a number of models of cataract. The objectives of this study were to determine gene expression changes in human lens epithelial cells in response to quercetin and to investigate in detail the mechanisms underlying the responses. FHL-124 cells were treated with quercetin (10 µM) and changes in gene expression were measured by microarray. It was found that 65% of the genes with increased expression were regulated by the hypoxia-inducible factor-1 (HIF-1) pathway. Quercetin (10 and 30 µM) induced a time-dependent increase in HIF-1a protein levels. Quercetin (30 µM) was also responsible for a rapid and long-lasting translocation of HIF-1a from the cytoplasm to the nucleus. Activation of HIF-1 signaling by quercetin was confirmed by qRT–PCR which showed upregulation of the HIF-1 regulated genes EPO, VEGF, PGK1 and BNIP3. Analysis of medium taken from FHL-124 cells showed a sustained dose-dependent increase in VEGF secretion following quercetin treatment. The quercetin-induced increase and nuclear translocation of HIF-1a was reversed by addition of excess iron (100 µM). These results demonstrate that quercetin activates the HIF-1 signaling pathway in human lens epithelial cells

Mechanisms of chromosome biorientation and bipolar spindle assembly analyzed by computational modeling

The essential functions required for mitotic spindle assembly and chromosome biorientation and segregation are not fully understood, despite extensive study. To illuminate the combinations of ingredients most important to align and segregate chromosomes and simultaneously assemble a bipolar spindle, we developed a computational model of fission-yeast mitosis. Robust chromosome biorientation requires progressive restriction of attachment geometry, destabilization of misaligned attachments, and attachment force dependence. Large spindle length fluctuations can occur when the kinetochore-microtubule attachment lifetime is long. The primary spindle force generators are kinesin-5 motors and crosslinkers in early mitosis, while interkinetochore stretch becomes important after biorientation. The same mechanisms that contribute to persistent biorientation lead to segregation of chromosomes to the poles after anaphase onset. This model therefore provides a framework to interrogate key requirements for robust chromosome biorientation, spindle length regulation, and force generation in the spindle.</p

Genetics, recombination and clinical features of human rhinovirus species C (HRV-C) infections; interactions of HRV-C with other respiratory viruses

To estimate the frequency, molecular epidemiological and clinical associations of infection with the newly described species C variants of human rhinoviruses (HRV), 3243 diagnostic respiratory samples referred for diagnostic testing in Edinburgh were screened using a VP4-encoding region-based selective polymerase chain reaction (PCR) for HRV-C along with parallel PCR testing for 13 other respiratory viruses. HRV-C was the third most frequently detected behind respiratory syncytial virus (RSV) and adenovirus, with 141 infection episodes detected among 1885 subjects over 13 months (7.5%). Infections predominantly targeted the very young (median age 6–12 months; 80% of infections in those &#60;2 years), occurred throughout the year but with peak incidence in early winter months. HRV-C was detected significantly more frequently among subjects with lower (LRT) and upper respiratory tract (URT) disease than controls without respiratory symptoms; HRV-C mono-infections were the second most frequently detected virus (behind RSV) in both disease presentations (6.9% and 7.8% of all cases respectively). HRV variants were classified by VP4/VP2 sequencing into 39 genotypically defined types, increasing the current total worldwide to 60. Through sequence comparisons of the 5′untranslated region (5′UTR), the majority grouped with species A (n = 96; 68%, described as HRV-Ca), the remainder forming a phylogenetically distinct 5′UTR group (HRV-Cc). Multiple and bidirectional recombination events between HRV-Ca and HRV-Cc variants and with HRV species A represents the most parsimonious explanation for their interspersed phylogeny relationships in the VP4/VP2-encoding region. No difference in age distribution, seasonality or disease associations was identified between HRV-Ca and HRV-Cc variants. HRV-C-infected subjects showed markedly reduced detection frequencies of RSV and other respiratory viruses, providing evidence for a major interfering effect of HRV-C on susceptibility to other respiratory virus infections. HRV-C's disease associations, its prevalence and evidence for interfering effects on other respiratory viruses mandates incorporation of rhinoviruses into future diagnostic virology screening

Longitudinal molecular microbial analysis of influenza-like illness in New York City, may 2009 through may 2010

<p>Abstract</p> <p>Background</p> <p>We performed a longitudinal study of viral etiology in samples collected in New York City during May 2009 to May 2010 from outpatients with fever or respiratory disease symptoms in the context of a pilot respiratory virus surveillance system.</p> <p>Methods</p> <p>Samples were assessed for the presence of 13 viruses, including influenza A virus, by MassTag PCR.</p> <p>Results</p> <p>At least one virus was detected in 52% of 940 samples analyzed, with 3% showing co-infections. The most frequently detected agents were rhinoviruses and influenza A, all representing the 2009 pandemic H1N1 strain. The incidence of influenza H1N1-positive samples was highest in late spring 2009, followed by a decline in summer and early fall, when rhinovirus infections became predominant before H1N1 reemerged in winter. Our study also identified a focal outbreak of enterovirus 68 in the early fall of 2009.</p> <p>Conclusion</p> <p>MassTag multiplex PCR affords opportunities to track the epidemiology of infectious diseases and may guide clinicians and public health practitioners in influenza-like illness and outbreak management. Nonetheless, a substantial proportion of influenza-like illness remains unexplained underscoring the need for additional platforms.</p