A Unique Group I Intron in Coxiella Burnetii is a Natural Splice Mutant

Cbu.L1917, a group I intron present in the 23S rRNA gene of Coxiella burnetii, possesses a unique 3\u27-terminal adenine in place of a conserved guanine. Here, we show that, unlike all other group I introns, Cbu.L1917 utilizes a different cofactor for each splicing step and has a decreased self-splicing rate in vitro

Pentamidine inhibits Coxiella burnetii growth and 23S rRNA intron splicing in vitro

Coxiella burnetii is the bacterial agent of Q fever in humans. Acute Q fever generally manifests as a flu-like illness and is typically self-resolving. In contrast, chronic Q fever usually presents with endocarditis and is often life-threatening without appropriate antimicrobial therapy. Unfortunately, available options for the successful treatment of chronic Q fever are both limited and protracted (\u3e18 months). Pentamidine, an RNA splice inhibitor used to treat fungal and protozoal infections, was shown to reduce intracellular growth of Coxiella by ca. 73% at a concentration of 1 microM (ca. 0.6 microg/mL) compared with untreated controls, with no detectable toxic effects on host cells. Bacterial targets of pentamidine include Cbu.L1917 and Cbu.L1951, two group I introns that disrupt the 23S rRNA gene of Coxiella, as demonstrated by the drug\u27s ability to inhibit intron RNA splicing in vitro. Since both introns are highly conserved amongst all eight genotypes of the pathogen, pentamidine is predicted to be efficacious against numerous strains of C. burnetii. To our knowledge, this is the first report describing antibacterial activity for this antifungal/antiprotozoal agent

A DNA-Binding Peroxiredoxin of Coxiella Burnetii is Involved in Countering Oxidative Stress During Exponential-Phase Growth

Coxiella burnetii is a Gram-negative, obligate intracellular bacterial pathogen that resides within the harsh, acidic confines of a lysosome-like compartment of the host cell that is termed a parasitophorous vacuole. In this study, we characterized a thiol-specific peroxidase of C. burnetii that belongs to the atypical 2-cysteine subfamily of peroxiredoxins, commonly referred to as bacterioferritin comigratory proteins (BCPs). Coxiella BCP was initially identified as a potential DNA-binding protein by two-dimensional Southwestern (SW) blots of the pathogen\u27s proteome, probed with biotinylated C. burnetii genomic DNA. Confirmation of the identity of the DNA-binding protein as BCP (CBU_0963) was established by matrix-assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). Recombinant Coxiella BCP (rBCP) was generated, and its DNA binding was demonstrated by two independent methods, including SW blotting and electrophoretic mobility shift assays (EMSAs). rBCP also demonstrated peroxidase activity in vitro that required thioredoxin-thioredoxin reductase (Trx-TrxR). Both the DNA-binding and peroxidase activities of rBCP were lost upon heat denaturation (100 degrees C, 10 min). Functional expression of Coxiella bcp was demonstrated by trans-complementation of an Escherichia coli bcp mutant, as evidenced by the strain\u27s ability to grow in an oxidative-stress growth medium containing tert-butyl hydroperoxide to levels that were indistinguishable from, or significantly greater than, those observed with its wild-type parental strain and significantly greater than bcp mutant levels (P \u3c 0.05). rBCP was also found to protect supercoiled plasmid DNA from oxidative damage (i.e., nicking) in vitro. Maximal expression of the bcp gene coincided with the pathogen\u27s early (day 2 to 3) exponential-growth phase in an experiment involving synchronized infection of an epithelial (Vero) host cell line. Taken as a whole, the results show that Coxiella BCP binds DNA and likely serves to detoxify endogenous hydroperoxide byproducts of Coxiella\u27s metabolism during intracellular replication

Nivel de adherencia al tratamiento y calidad de vida relacionada a la salud en pacientes con VIH/sida, de un hospital MINSA nivel II-1 de Ferreñafe – Lambayeque, durante agosto – diciembre, 2017

Se ha observado que pacientes infectados por el virus de inmunodeficiencia humana y síndrome de inmunodeficiencia adquirida (VIH/SIDA) que no toman en cuenta las indicaciones del médico para su tratamiento, pueden generar una propagación constante y continua, dando paso a un problema de salud más grave a nivel mundial. Por ello se realizó una investigación no experimental - descriptiva, con el propósito de determinar los niveles de adherencia al tratamiento y calidad de vida relacionada a la salud según sexo y grado de instrucción, además de describir las dimensiones sociodemográficas en pacientes con VIH/SIDA, de un Hospital MINSA nivel II-1 de Ferreñafe - Lambayeque, durante agosto a diciembre del 2017. Se utilizó los cuestionarios CEAT-VIH para hallar los niveles de adherencia al tratamiento y MOS-SF 30 para determinar los niveles de calidad de vida relacionada a la salud, estimando su validez y confiabilidad en una población similar, siendo válidos y fiables. Además, en cuanto la variable sexo, se obtuvo que los varones tienen una adecuada adherencia al tratamiento, sin embargo para calidad de vida relacionada a la salud ambos sexos se encontraron en un nivel bajo. Por ende la población estudiada presenta baja adherencia al tratamiento y calidad de vida relacionada a la salud. Con respecto a los aspectos sociodemográficos, el 53% es de sexo masculino, el 62% es soltera, siendo la vía sexual (96%) la principal forma de transmisión del virus del VIH. Además el 75% lleva un tiempo mayor a 12 meses de tratamiento en el servicio de TARGA.Tesi

Identification of Novel Small RNAs and Characterization of the 6S RNA of \u3ci\u3eCoxiella burnetii\u3c/i\u3e

Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV) and a dormant small cell variant (SCV). As such, the bacterium undoubtedly employs complex modes of regulating its lifecycle, metabolism and pathogenesis. Small RNAs (sRNAs) have been shown to play important regulatory roles in controlling metabolism and virulence in several pathogenic bacteria. We hypothesize that sRNAs are involved in regulating growth and development of C. burnetii and its infection of host cells. To address the hypothesis and identify potential sRNAs, we subjected total RNA isolated from Coxiella cultured axenically and in Vero host cells to deep-sequencing. Using this approach, we identified fifteen novel C. burnetii sRNAs (CbSRs). Fourteen CbSRs were validated by Northern blotting. Most CbSRs showed differential expression, with increased levels in LCVs. Eight CbSRs were upregulated ($2-fold) during intracellular growth as compared to growth in axenic medium. Along with the fifteen sRNAs, we also identified three sRNAs that have been previously described from other bacteria, including RNase P RNA, tmRNA and 6S RNA. The 6S regulatory sRNA of C. burnetii was found to accumulate over log phase-growth with a maximum level attained in the SCV stage. The 6S RNA-encoding gene (ssrS) was mapped to the 59 UTR of ygfA; a highly conserved linkage in eubacteria. The predicted secondary structure of the 6S RNA possesses three highly conserved domains found in 6S RNAs of other eubacteria. We also demonstrate that Coxiella’s 6S RNA interacts with RNA polymerase (RNAP) in a specific manner. Finally, transcript levels of 6S RNA were found to be at much higher levels when Coxiella was grown in host cells relative to axenic culture, indicating a potential role in regulating the bacterium’s intracellular stress response by interacting with RNAP during transcription

Streamlined preparation of genomic DNA in agarose plugs for pulsed-field gel electrophoresis

Genome analysis using pulsed-field gel electrophoresis (PFGE) has been used in applications ranging from typing bacterial strains to radiobiology to cancer research. While methods for running PFGE have been significantly improved since its invention, the method for preparing chromosomal DNA itself has remained essentially unchanged. This limits the applicability of PFGE, especially when analyses require many samples. We have streamlined sample preparation for routine applications of PFGE through the use of deep-well 48-well plates. Besides saving time, our protocol has the added advantage of reducing the volume of expensive reagents. Our improved protocol enables us to reduce throughput time and simplify the procedure, facilitating wider application of PFGE-based analyses in the laboratory

Ready or Not, Here They Come: Acting Interns’ Experience and Perceived Competency Performing Basic Medical Procedures

OBJECTIVE: To assess acting interns’ (AI’s) experience with and perceived level of competency performing 6 basic medical procedures. DESIGN: Fourth-year medical students at the University of Cincinnati (UCCOM) are required to complete 2 AI rotations in Internal Medicine. All AIs in 2003–2004 (n = 150) and 2004–2005 (n = 151) were asked to complete a survey about whether during each of their rotations they had performed and felt competent performing the following procedures: phlebotomy, intravenous (IV) catheter insertion, arterial blood gas (ABG), nasogastric (NG) tube insertion, lumbar puncture (LP), and Foley catheter insertion. RESULTS: Four hundred sixty-seven of 601 possible surveys (across both years and both rotations) were completed (78% response rate). During both rotations, relatively few students performed the procedures, ranging from 9% for Foley catheter insertion (24/208) to 50% for both ABG and NG tube insertion (130/259). The two procedures most often performed were ABG (range 46–50%) and NG tube insertion (range 42–50%). Feelings of competency varied from 12% (LP) to 82% (Foley catheter). Except for LP, if students performed a procedure at least once, they reported feeling more competent (range 85% for ABG to 96% for Foley catheter insertion). Among the students who performed LP during a rotation, many still did not feel competent performing LPs: 23 (74%) in rotation 1 and 20 (40%) in rotation 2. CONCLUSION: Many fourth-year students at UCCOM do not perform basic procedures during their acting internship rotations. Procedural performance correlates with feelings of competency. Lumbar puncture competency may be too ambitious a goal for medical students

Are Canadian General Internal Medicine training program graduates well prepared for their future careers?

BACKGROUND: At a time of increased need and demand for general internists in Canada, the attractiveness of generalist careers (including general internal medicine, GIM) has been falling as evidenced by the low number of residents choosing this specialty. One hypothesis for the lack of interest in a generalist career is lack of comfort with the skills needed to practice after training, and the mismatch between the tertiary care, inpatient training environment and "real life". This project was designed to determine perceived effectiveness of training for 10 years of graduates of Canadian GIM programs to assist in the development of curriculum and objectives for general internists that will meet the needs of graduates and ultimately society. METHODS: Mailed survey designed to explore perceived importance of training for and preparation for various aspects of Canadian GIM practice. After extensive piloting of the survey, including a pilot survey of two universities to improve the questionnaire, all graduates of the 16 universities over the previous ten years were surveyed. RESULTS: Gaps (difference between importance and preparation) were demonstrated in many of the CanMEDS 2000/2005(® )competencies. Medical problems of pregnancy, perioperative care, pain management, chronic care, ambulatory care and community GIM rotations were the medical expert areas with the largest gaps. Exposure to procedural skills was perceived to be lacking. Some procedural skills valued as important for current GIM trainees and performed frequently (example ambulatory ECG interpretation) had low preparation ratings by trainees. Other areas of perceived discrepancy between training and practice included: manager role (set up of an office), health advocate (counseling for prevention, for example smoking cessation), and professional (end of life issues, ethics). CONCLUSION: Graduates of Canadian GIM training programs over the last ten years have identified perceived gaps between training and important areas for practice. They have identified competencies that should be emphasized in Canadian GIM programs. Ongoing review of graduate's perceptions of training programs as it applies to their current practice is important to ensure ongoing appropriateness of training programs. This information will be used to strengthen GIM training programs in Canada