A Chromosome Segment Substitution Library of Weedy Rice for Genetic Dissection of Complex Agronomic and Domestication Traits

Chromosome segment substitution lines (CSSLs) are a powerful alternative for locating quantitative trait loci (QTL), analyzing gene interactions, and providing starting materials for map-based cloning projects. We report the development and characterization of a CSSL library of a U.S. weedy rice accession ‘PSRR-1’ with genome-wide coverage in an adapted rice cultivar ‘Bengal’ background. The majority of the CSSLs carried a single defined weedy rice segment with an average introgression segment of 2.8 % of the donor genome. QTL mapping results for several agronomic and domestication traits from the CSSL population were compared with those obtained from two recombinant inbred line (RIL) populations involving the same weedy rice accession. There was congruence of major effect QTLs between both types of populations, but new and additional QTLs were detected in the CSSL population. Although, three major effect QTLs for plant height were detected on chromosomes 1, 4, and 8 in the CSSL population, the latter two escaped detection in both RIL populations. Since this was observed for many traits, epistasis may play a major role for the phenotypic variation observed in weedy rice. High levels of shattering and seed dormancy in weedy rice might result from an accumulation of many small effect QTLs. Several CSSLs with desirable agronomic traits (e.g. longer panicles, longer grains, and higher seed weight) identified in this study could be useful for rice breeding. Since weedy rice is a reservoir of genes for many weedy and agronomic attributes, the CSSL library will serve as a valuable resource to discover latent genetic diversity for improving crop productivity and understanding the plant domestication process through cloning and characterization of the underlying genes.     

Dynamic Proteome Response of Pseudomonas aeruginosa to Tobramycin Antibiotic Treatment

Genetically susceptible bacteria become antibiotic tolerant during chronic infections, and the mechanisms responsible are poorly understood. One factor that may contribute to differential sensitivity in vitro and in vivo is differences in the time-dependent tobramycin concentration profile experienced by the bacteria. Here, we examine the proteome response induced by subinhibitory concentrations of tobramycin in Pseudomonas aeruginosa cells grown under planktonic conditions. These efforts revealed increased levels of heat shock proteins and proteases were present at higher dosage treatments (0.5 and 1 μg/ml), while less dramatic at 0.1 μg/ml dosage. In contrast, many metabolic enzymes were significantly induced by lower dosages (0.1 and 0.5 μg/ml) but not at 1 μg/ml dosage. Time course proteome analysis further revealed that the increase of heat shock proteins and proteases was most rapid from 15 min to 60 min, and the increased levels sustained till 6 h (last time point tested). Heat shock protein IbpA exhibited the greatest induction by tobramycin, up to 90-fold. Nevertheless, deletion of ibpA did not enhance sensitivity to tobramycin. It seemed possible that the absence of sensitization could be due to redundant functioning of IbpA with other proteins that protect cells from tobramycin. Indeed, inactivation of two heat shock chaperones/proteases in addition to ibpA in double mutants (ibpA/clpB, ibpA/PA0779 and ibpA/hslV) did increase tobramycin sensitivity. Collectively, these results demonstrate the time- and concentration-dependent nature of the P. aeruginosa proteome response to tobramycin and that proteome modulation and protein redundancy are protective mechanisms to help bacteria resist antibiotic treatments.The opportunistic pathogen Pseudomonas aeruginosa is ubiquitous in the natural environment and causes human infections (1). P. aeruginosa can metabolize various carbon and nitrogen compounds and persists under nutrient-poor and hostile growth environments (2, 3). One example is P. aeruginosa pulmonary infection of cystic fibrosis (CF) patients. Despite stress induced by host defenses and high concentrations of antibiotics, P. aeruginosa cells are able to persistently colonize CF airways (4).The aminoglycoside tobramycin is a front-line drug currently used in the treatment of P. aeruginosa in CF and other diseases. It is supplied in the forms of inhaled solution (TOBI) and intravenous injection. The tobramycin concentrations in airways after 300-mg dosage TOBI inhalation can reach 1,000 μg per g of sputum (5, 6). This concentration is in the range of 10 to 1,000 times of the minimal inhibitory concentration (MIC) for P. aeruginosa clinical isolates tested ex vivo (6). However, even with such high tobramycin concentrations, chronic P. aeruginosa infections are rarely eradicated (6). This is true even when the infecting bacteria are antibiotic sensitive, as is the case early in disease (7).One possible reason for P. aeruginosa persistence in vivo could relate to the time dependence of local concentrations of tobramycin experienced by P. aeruginosa in CF patient airways. Many factors, including inflammatory responses, blood and lymphatic circulations, and air flow distribution (for inhaled antibiotics), can alter the local antibiotic concentrations. In addition, P. aeruginosa cells can form biofilms in CF lungs and other infection sites (8), and biofilm exopolysaccharide layers may slow the diffusion of tobramycin (9, 10). P. aeruginosa cells in the inner layers of biofilms may experience lower concentrations and more gradual increase of tobramycin levels than those in outer layers (10, 11). Furthermore, even if final tobramycin concentration levels inside the biofilm eventually grow to match the highest levels experienced elsewhere, bacteria in these inner regions have experienced a slower increase, during which time proteome levels could be altered to promote the “adapted resistant state” (12). Adaptive resistance can also be induced in planktonic (free-living) P. aeruginosa (13, 14), and conventional MIC assays are not designed to measure this.Once induced, the adaptive resistance confers bacteria higher resistance to antibiotic treatments (13, 14) and is associated with decreased clinical antibiotic treatment efficacy (15). Interestingly, the adaptive resistance is time dependent and reversible. Typical adaptive resistance was observed starting 1 h after antibiotic exposure, and the drug susceptibility was regained after 36 h intervals (14, 15). Thus, adaptive resistance mechanisms may contribute in part to the disparity of in vivo persistence and ex vivo susceptibility to antibiotics in MIC tests.As an initial step toward defining adaptive resistance mechanisms, we investigated the time- and concentration-dependence of P. aeruginosa proteome response to tobramycin in planktonic conditions. Since the most effective protective responses may operate before killing begins and the rate of change of drug levels is likely to depend on ambient conditions, we studied bacteria exposed to low, subinhibitory levels of tobramycin (0.1, 0.5, and 1.0 μg/ml) at a range of time points (15, 60, 120, and 360 min) after exposure. The candidate proteome marker of P. aeruginosa for tobramycin response, heat shock protein IbpA, was further investigated with genetic mutagenesis and MIC assays.     

Evaluation of a Minimally Invasive Cell Sampling Device Coupled with Assessment of Trefoil Factor 3 Expression for Diagnosing Barrett's Esophagus: A Multi-Center Case–Control Study

BackgroundBarrett''s esophagus (BE) is a commonly undiagnosed condition that predisposes to esophageal adenocarcinoma. Routine endoscopic screening for BE is not recommended because of the burden this would impose on the health care system. The objective of this study was to determine whether a novel approach using a minimally invasive cell sampling device, the Cytosponge, coupled with immunohistochemical staining for the biomarker Trefoil Factor 3 (TFF3), could be used to identify patients who warrant endoscopy to diagnose BE.ConclusionsThe Cytosponge-TFF3 test is safe and acceptable, and has accuracy comparable to other screening tests. This test may be a simple and inexpensive approach to identify patients with reflux symptoms who warrant endoscopy to diagnose BE.     

AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility

Automated docking of drug-like molecules into receptors is an essential tool in structure-based drug design. While modeling receptor flexibility is important for correctly predicting ligand binding, it still remains challenging. This work focuses on an approach in which receptor flexibility is modeled by explicitly specifying a set of receptor side-chains a-priori. The challenges of this approach include the: 1) exponential growth of the search space, demanding more efficient search methods; and 2) increased number of false positives, calling for scoring functions tailored for flexible receptor docking. We present AutoDockFR–AutoDock for Flexible Receptors (ADFR), a new docking engine based on the AutoDock4 scoring function, which addresses the aforementioned challenges with a new Genetic Algorithm (GA) and customized scoring function. We validate ADFR using the Astex Diverse Set, demonstrating an increase in efficiency and reliability of its GA over the one implemented in AutoDock4. We demonstrate greatly increased success rates when cross-docking ligands into apo receptors that require side-chain conformational changes for ligand binding. These cross-docking experiments are based on two datasets: 1) SEQ17 –a receptor diversity set containing 17 pairs of apo-holo structures; and 2) CDK2 –a ligand diversity set composed of one CDK2 apo structure and 52 known bound inhibitors. We show that, when cross-docking ligands into the apo conformation of the receptors with up to 14 flexible side-chains, ADFR reports more correctly cross-docked ligands than AutoDock Vina on both datasets with solutions found for 70.6% vs. 35.3% systems on SEQ17, and 76.9% vs. 61.5% on CDK2. ADFR also outperforms AutoDock Vina in number of top ranking solutions on both datasets. Furthermore, we show that correctly docked CDK2 complexes re-create on average 79.8% of all pairwise atomic interactions between the ligand and moving receptor atoms in the holo complexes. Finally, we show that down-weighting the receptor internal energy improves the ranking of correctly docked poses and that runtime for AutoDockFR scales linearly when side-chain flexibility is added.     

Treatment of W. bancrofti (Wb) in HIV/Wb Coinfections in South India

BackgroundThe disease course of human immunodeficiency virus (HIV) is often altered by existing or newly acquired coincident infections.Conclusions/SignificanceWe were unable to find a significant effect of W. bancrofti infection or its treatment on HIV clinical course or surrogate markers of HIV disease progression though we recognized that our study was limited by the smaller than predicted sample size and by the use of ART in half of the patients. Treatment of W. bancrofti coinfection in HIV positive subjects (as is usual in mass drug administration campaigns) did not represent an increased risk to the subjects, and should therefore be considered for PLWHA living in W. bancrofti endemic areas.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00344279","term_id":"NCT00344279"}}NCT00344279     

Prion Protein Does Not Confer Resistance to Hippocampus-Derived Zpl Cells against the Toxic Effects of Cu2+, Mn2+, Zn2+ and Co2+ Not Supporting a General Protective Role for PrP in Transition Metal Induced Toxicity

The interactions of transition metals with the prion protein (PrP) are well-documented and characterized, however, there is no consensus on their role in either the physiology of PrP or PrP-related neurodegenerative disorders. PrP has been reported to protect cells from the toxic stimuli of metals. By employing a cell viability assay, we examined the effects of various concentrations of Cu²⁺, Zn²⁺, Mn²⁺, and Co²⁺ on Zpl (Prnp ^-/-) and ZW (Prnp ^+/+) hippocampus-derived mouse neuronal cells. Prnp ^-/- Zpl cells were more sensitive to all four metals than PrP-expressing Zw cells. However, when we introduced PrP or only the empty vector into Zpl cells, we could not discern any protective effect associated with the presence of PrP. This observation was further corroborated when assessing the toxic effect of metals by propidium-iodide staining and fluorescence activated cell sorting analysis. Thus, our results on this mouse cell culture model do not seem to support a strong protective role for PrP against transition metal toxicity and also emphasize the necessity of extreme care when comparing cells derived from PrP knock-out and wild type mice.     

Tumor T1 Relaxation Time for Assessing Response to Bevacizumab Anti-Angiogenic Therapy in a Mouse Ovarian Cancer Model

Purpose

To assess whether T₁ relaxation time of tumors may be used to assess response to bevacizumab anti-angiogenic therapy. Procedures: 12 female nude mice bearing subcutaneous SKOV3ip1-LC ovarian tumors were administered bevacizumab (6.25ug/g, n=6) or PBS (control, n=6) therapy twice a week for two weeks. T₁ maps of tumors were generated before, two days, and 2 weeks after initiating therapy. Tumor weight was assessed by MR and at necropsy. Histology for microvessel density, proliferation, and apoptosis was performed.

Results

Bevacizumab treatment resulted in tumor growth inhibition (p<0.04, n=6), confirming therapeutic efficacy. Tumor T₁ relaxation times increased in bevacizumab treated mice 2 days and 2 weeks after initiating therapy (p<.05, n=6). Microvessel density decreased 59% and cell proliferation (Ki67+) decreased 50% in the bevacizumab treatment group (p<.001, n=6), but not apoptosis.

Conclusions

Findings suggest that increased tumor T₁ relaxation time is associated with response to bevacizumab therapy in ovarian cancer model and might serve as an early indicator of response.     

Naturally Occurring Genetic Variants in Human Chromogranin A (CHGA) Associated with Hypertension as well as Hypertensive Renal Disease

Chromogranin A (CHGA) plays a fundamental role in the biogenesis of catecholamine secretory granules. Changes in storage and release of CHGA in clinical and experimental hypertension prompted us to study whether genetic variation at the CHGA locus might contribute to alterations in autonomic function, and hence hypertension and its target organ consequences such as hypertensive renal disease (nephrosclerosis). Systematic polymorphism discovery across the human CHGA locus revealed both common and unusual variants in both the open reading frame and such regulatory regions as the proximal promoter and 30-UTR. In chromaffin cell-transfected CHGA 30-UTR and promoter/luciferase reporter plasmids, the functional consequences of the regulatory/non-coding allelic variants were documented. Variants in both the proximal promoter and the 30-UTR displayed statistical associations with hypertension. Genetic variation in the proximal CHGA promoter predicted glomerular filtration rate in healthy twins. However, for hypertensive renal damage, both end-stage renal disease and rate of progression of earlier disease were best predicted by variants in the 30-UTR. Finally, mechanistic studies were undertaken initiated by the clue that CHGA promoter variation predicted circulating endothelin-1. In cultured endothelial cells, CHGA triggered co-release of not only the vasoconstrictor and pro-fibrotic endothelin-1, but also the pro-coagulant von Willebrand Factor and the pro-angiogenic angiopoietin-2. These findings, coupled with stimulation of endothelin-1 release from glomerular capillary endothelial cells by CHGA, suggest a plausible mechanism whereby genetic variation at the CHGA locus eventuates in alterations in human renal function. These results document the consequences of genetic variation at the CHGA locus for cardiorenal disease and suggest mechanisms whereby such variation achieves functional effects.