Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia-specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms. Conditional mutants have been used to study essential genes of many microorganisms, so we screened a library of 4,184 ethyl methanesulfonate-mutagenized Chlamydia trachomatis isolates for temperature-sensitive (TS) mutants that developed normally at physiological temperature (37°C) but not at nonphysiological temperatures. Heat-sensitive TS mutants were identified at a high frequency, while cold-sensitive mutants were less common. Twelve TS mutants were mapped using a novel markerless recombination approach, PCR, and genome sequencing. TS alleles of genes that play essential roles in other bacteria and chlamydia-specific open reading frames (ORFs) of unknown function were identified. Temperature-shift assays determined that phenotypes of the mutants manifested at distinct points in the developmental cycle. Genome sequencing of a larger population of TS mutants also revealed that the screen had not reached saturation. In summary, we describe the first approach for studying essential chlamydial genes and broadly applicable strategies for genetic mapping in Chlamydia spp. and mutants that both define checkpoints and provide insights into the biology of the chlamydial developmental cycle. IMPORTANCE: Study of the pathogenesis of Chlamydia spp. has historically been hampered by a lack of genetic tools. Although there has been recent progress in chlamydial genetics, the existing approaches have limitations for the study of the genes that mediate growth of these organisms in cell culture. We used a genetic screen to identify conditional Chlamydia mutants and then mapped these alleles using a broadly applicable recombination strategy. Phenotypes of the mutants provide fundamental insights into unexplored areas of chlamydial pathogenesis and intracellular biology. Finally, the reagents and approaches we describe are powerful resources for the investigation of these organisms

U.S. Physician-Scientist Workforce in the 21st Century: Recommendations to Attract and Sustain the Pipeline

The U.S. physician-scientist (PS) workforce is invaluable to the nation's biomedical research effort. It is through biomedical research that certain diseases have been eliminated, cures for others have been discovered, and medical procedures and therapies that save lives have been developed. Yet, the U.S. PS workforce has both declined and aged over the last several years. The resulting decreased inflow and outflow to the PS pipeline renders the system vulnerable to collapsing suddenly as the senior workforce retires. In November 2015, the Alliance for Academic Internal Medicine hosted a consensus conference on the PS workforce to address issues impacting academic medical schools, with input from early-career PSs based on their individual experiences and concerns. One of the goals of the conference was to identify current impediments in attracting and supporting PSs and to develop a new set of recommendations for sustaining the PS workforce in 2016 and beyond. This Perspective reports on the opportunities and factors identified at the conference and presents five recommendations designed to increase entry into the PS pipeline and nine recommendations designed to decrease attrition from the PS workflow

Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection

The direct major histocompatibility complex (MHC) class I antigen presentation pathway ensures intracellular peptides are displayed at the cellular surface for recognition of infected or transformed cells by CD8⁺ cytotoxic T lymphocytes. Chlamydia spp. are obligate intracellular bacteria and, as such, should be targeted by CD8⁺ T cells. It is likely that Chlamydia spp. have evolved mechanisms to avoid the CD8⁺ killer T cell responses by interfering with MHC class I antigen presentation. Using a model system of self-peptide presentation which allows for posttranslational control of the model protein's stability, we tested the ability of various Chlamydia species to alter direct MHC class I antigen presentation. Infection of the JY lymphoblastoid cell line limited the accumulation of a model host protein and increased presentation of the model-protein-derived peptides. Enhanced self-peptide presentation was detected only when presentation was restricted to defective ribosomal products, or DRiPs, and total MHC class I levels remained unaltered. Skewed antigen presentation was dependent on a bacterial synthesized component, as evidenced by reversal of the observed phenotype upon preventing bacterial transcription, translation, and the inhibition of bacterial lipooligosaccharide synthesis. These data suggest that Chlamydia spp. have evolved to alter the host antigen presentation machinery to favor presentation of defective and rapidly degraded forms of self-antigen, possibly as a mechanism to diminish the presentation of peptides derived from bacterial proteins

ELEVATED PHENYLACETIC ACID LEVELS DO NOT CORRELATE WITH ADVERSE EVENTS IN PATIENTS WITH UREA CYCLE DISORDERS OR HEPATIC ENCEPHALOPATHY AND CAN BE PREDICTED BASED ON THE PLASMA PAA TO PAGN RATIO

Background Phenylacetic acid (PAA) is the active moiety in sodium phenylbutyrate (NaPBA) and glycerol phenylbutyrate (GPB, HPN-100), both are approved for treatment of urea cycle disorders (UCDs) - rare genetic disorders characterized by hyperammonemia. PAA is conjugated with glutamine in the liver to form phenylacetyleglutamine (PAGN), which is excreted in urine. PAA plasma levels ≥500 μg/dL have been reported to be associated with reversible neurological adverse events (AEs) in cancer patients receiving PAA intravenously. Therefore, we have investigated the relationship between PAA levels and neurological AEs in patients treated with these PAA pro-drugs as well as approaches to identifying patients most likely to experience high PAA levels. Methods The relationship between nervous system AEs, PAA levels and the ratio of plasma PAA to PAGN were examined in 4683 blood samples taken serially from: [1] healthy adults [2], UCD patients ≥2 months of age, and [3] patients with cirrhosis and hepatic encephalopathy (HE). The plasma ratio of PAA to PAGN was analyzed with respect to its utility in identifying patients at risk of high PAA values. Results Only 0.2% (11) of 4683 samples exceeded 500 ug/ml. There was no relationship between neurological AEs and PAA levels in UCD or HE patients, but transient AEs including headache and nausea that correlated with PAA levels were observed in healthy adults. Irrespective of population, a curvilinear relationship was observed between PAA levels and the plasma PAA:PAGN ratio, and a ratio > 2.5 (both in μg/mL) in a random blood draw identified patients at risk for PAA levels > 500 μg/ml. Conclusions The presence of a relationship between PAA levels and reversible AEs in healthy adults but not in UCD or HE patients may reflect intrinsic differences among the populations and/or metabolic adaptation with continued dosing. The plasma PAA:PAGN ratio is a functional measure of the rate of PAA metabolism and represents a useful dosing biomarker

Laboratory investigations of the interaction between benzene and bare silicate grain surfaces

Experimental results on the thermal desorption of benzene (C6H6) from amorphous silica (SiO2) are presented. The amorphous SiO2 substrate was imaged using atomic force microscopy (AFM), revealing a surface morphology reminiscent of that of interplanetary dust particles (IDPs). Temperature programmed desorption (TPD) experiments were conducted for a wide range of C6H6 exposures, yielding information on both C6H6-SiO2 interactions and the C6H6-C6H6 interactions present in the bulk C6H6 ice. The low coverage experiments reveal complicated desorption behaviour that results both from porosity and roughness in the SiO2 substrate, and repulsive interactions between C6H6 molecules. Kinetic parameters were obtained through a combination of direct analysis of the TPD traces and kinetic modelling, demonstrating the coverage dependence of both desorption energy and pre-exponential factor. Experiments were also performed whereby the pores were blocked by pre-exposure of the SiO2 to water vapour. C6H6 was observed to be adsorbed preferentially on the SiO2 film not covered by H2O at the temperature at which these experiments were performed. This observation means that intermolecular repulsion likely becomes important at smaller C6H6 exposures on grains with a H2O mantle. Kinetic modelling of C6H6 multilayer desorption yields kinetic parameters in good agreement with previous studies, with the SiO2 having little impact on the desorption beyond the first few layers.Comment: 23 pages, including 6 figures and 1 table ; Submitted to MNRA

1965: Abilene Christian College Bible Lectures - Full Text

LIFT UP YOUR EYES” Being the Abilene Christian College Annual Bible Lectures 1965 Price: $3.95 Published by ABILENE CHRISTIAN COLLEGE STUDENTS EXCHANGE ACC Station Abilene. Texa

Nanoparticle Orientation to Control RNA Loading and Ligand Display on Extracellular Vesicles for Cancer Regression

Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft

Psychologists’ Authenticity: Implications for Work in Professional and Therapeutic Settings

Derek J. Burks, PhD, is a licensed psychologist and postdoctoral psychology fellow at the Pacific Northwest Mental Illness Research, Education, and Clinical Center (MIRECC), located within the Veterans Affairs Medical Center in Portland, Oregon. He obtained his PhD in counseling psychology from the University of Oklahoma. His current research focuses on health disparities, posttraumatic stress disorder among sexual-minority U.S. military veterans, and culturally appropriate mental health treatment for American Indians.Yeshttps://us.sagepub.com/en-us/nam/manuscript-submission-guideline

The Chlamydia psittaci Genome: A Comparative Analysis of Intracellular Pathogens

Chlamydiaceae are a family of obligate intracellular pathogens causing a wide range of diseases in animals and humans, and facing unique evolutionary constraints not encountered by free-living prokaryotes. To investigate genomic aspects of infection, virulence and host preference we have sequenced Chlamydia psittaci, the pathogenic agent of ornithosis.A comparison of the genome of the avian Chlamydia psittaci isolate 6BC with the genomes of other chlamydial species, C. trachomatis, C. muridarum, C. pneumoniae, C. abortus, C. felis and C. caviae, revealed a high level of sequence conservation and synteny across taxa, with the major exception of the human pathogen C. trachomatis. Important differences manifest in the polymorphic membrane protein family specific for the Chlamydiae and in the highly variable chlamydial plasticity zone. We identified a number of psittaci-specific polymorphic membrane proteins of the G family that may be related to differences in host-range and/or virulence as compared to closely related Chlamydiaceae. We calculated non-synonymous to synonymous substitution rate ratios for pairs of orthologous genes to identify putative targets of adaptive evolution and predicted type III secreted effector proteins.This study is the first detailed analysis of the Chlamydia psittaci genome sequence. It provides insights in the genome architecture of C. psittaci and proposes a number of novel candidate genes mostly of yet unknown function that may be important for pathogen-host interactions