Conversations with a Killer Bacterium: Identifying new targets of Quorum Sensing sRNAs in Vibrio cholera

A presentation in the Undergraduate Research Kaleidoscope: Library East Commons Performance Space, February 9, 2009Runtime: 06:42 minutes (Edited)Runtime: 06:43 minutes (Original)Bacteria communicate with each other by releasing signal molecules into the environment that travel between cells. When the molecules accumulate, surface receptors detect the signals and trigger pathways that alter gene expression on a population-wide scale. This process, called quorum sensing (QS), allows bacterial groups to behave like multicellular organisms. In the human pathogen Vibrio cholerae, QS synchronizes the production of multiple small RNAs (sRNAs) that do not encode proteins, but instead bind to and alter messenger RNA targets, including one that controls essential virulence traits. We used a new computer algorithm, TargetRNA, to verify the known sRNA-controlled targets and also to predict several new candidates. I will genetically engineer a fusion of each candidate to a bioluminescent “reporter” gene, visually monitor responses to QS signals, and define the mechanism of each sRNA/mRNA binding interaction. These studies will clarify how chemical information is processed and converted into behavioral changes in this deadly microbe.Brian K. Hamme

CERAMIDE-ENRICHED EXTRACELLULAR VESICLES: A ROLE IN ENHANCING AMYLOID-BETA NEUROTOXICITY AND MITOCHONDRIAL DAMAGE IN ALZHEIMER’S DISEASE

Alzheimer’s disease (AD) is an age-dependent, progressive, neurodegenerative disorder that is characterized clinically by the impairment of cognitive functions concomitant with behavioral and personality changes. AD is associated with distinct pathological hallmarks, namely, intracellular neurofibrillary tangles comprised of hyperphosphorylated tau protein, extracellular amyloid beta (Aβ) plaques, and marked brain atrophy. Besides their main role as the core component of amyloid plaques, oligomeric Aβ have been shown to be neurotoxic. The exact mechanism of Aβ neurotoxicity is yet to be elucidated. Recently, a pathogenic function of small extracellular vesicles- also known as exosomes- has been proposed, suggesting that exosomes can transfer pathogens between cells. One such pathogen that exploits this pathway is Aβ in Alzheimer’s disease, however, it is not known yet whether this Aβ/exosomes association would affect the neuronal toxicity of Aβ. Exosomes are nano-sized lipid vesicles that are formed by inward budding of late endosomes to form multi vesicular bodies (MVB) which fuse to the plasma membrane and release exosomes to the extracellular space. Exosomes serve as a means of intercellular communication due to their ability in carrying cargoes including microRNA (miRNA), messenger RNA (mRNA), proteins, and other biomolecules. There are several established pathways for exosomes biogenesis, one of which is triggered by the sphingolipid ceramide. Ceramide is a key molecule in sphingolipids metabolism and it is involved in several cellular processes such as proliferation, senescence and apoptosis. It has also been reported that ceramide levels are elevated in AD patients brain specimens. Exploiting the fact that exosomes can cross the blood brain barrier we therefore used serum derived exosomes to study the biophysical and biochemical characteristics of Alzheimer’s disease mouse model (5xFAD) and AD patients’ exosomes compared to wild type and healthy individuals. We found that serum from 5xFAD mice and AD patients contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide, particularly C16:0, C18:0, C20:0, 22:0, C24:0, and C24:1 ceramide species. This subpopulation (termed astrosomes) was shown to associated with Aβ and are prone to aggregation as confirmed by nanoparticle tracking and cluster analyses. To study the functional characteristics of these Aβ-associated astrosomes, we used Neuro-2a (N2a) cells, human iPS cell-derived neurons, and mouse primary cultured neurons as in vitro tissue culture models. When taken up by neurons, Aβ-associated astrosomes were specifically transported to mitochondria where they induced mitochondria clustering, evident by elevation of expression of the fission protein dynamin related protein1 (Drp1). Aβ-associated astrosomes, but not wild type or healthy control human exosomes, mediated binding of Aβ to voltage-dependent anion channel 1 (VDAC1), a gate keeper protein in the outer mitochondrial membrane that is involved in regulating passage of metabolites, nucleotides, and ions; it plays a crucial role in regulating apoptosis. This Aβ/VDAC1 interaction leads to caspase activation and subsequently apoptosis. Interestingly, removing the ceramide-enriched astrosomes from the exosome pool using lipid-mediated affinity chromatography (LIMAC) mitigated that toxic effect on neurons. These results were replicated using brain derived exosomes. To investigate the in vivo significance of our in vitro results, we stereotaxically injected wild type mice (two weeks old) with 5xFAD or wild type brain derived exosomes (nine months old). We found that within two days, the injected exosomes were specifically taken up by neurons and transported to mitochondria. Consistent with our in vitro data using Aβ-associated astrosomes, the exosomes isolated from 5xFAD brain, but not those from wild type brain, induced complex formation of Aβ with VDAC1 and activation of caspase 3. To test that our observations hold true in physiological conditions, we generated a novel astrosome reporter mouse model. This was accomplished by crossing of Aldh1l1-Cre/ERT with floxed CD63-GFP and 5xFAD mice (5XFAD xAldh1l1-Cre/ERTxCD63-GFPfl/fl) which allows us to track astrosome uptake and their subsequent effects. As seen with the injected exosomes, we found that endogenous GFP-labeled astrosomes are taken up by neurons where they shuttle Aβ and induce mitotoxicity. In conclusion, our data show that association of Aβ to astrosomes in critical for Aβ neurotoxicity. Therefore, we discovered a novel mechanism by which Aβ induces AD neuropathology as well as potential pharmacological target

Contrawound toroidal helical antenna modeling using the FDTD method

The contrawound toroidal helical antenna (CTHA) is a patented antenna developed at the Center for Industrial Research Applications (CIRA) of West Virginia University. In this study, a computer numerical model was built to simulate the behavior of printed circuit board versions of the CTHA. The numerical model was built upon the finite difference time domain method (FDTD). The results were compared to results from physical antennas, obtained from the CIRA anechoic chamber located in the WVU hanger, to verify the validity of the results of the numerical model. The results from both the numerical and experimental models were also used to reach an understanding of the general performance of the PCB-CTHA and its behavior under different feed configurations.;In order to understand the behavior of the antenna under different feed configurations, a feed study was conducted to check for all the possible feeds of a PCB-CTHA. These were found to be thirteen different feeds and were named feeds 1 through 13. Thorough numerical and experimental analysis was done on all thirteen feeds for both understanding the behavior of the antenna under these different feeds, and also comparing the predictions of the numerical model to the data obtained from the anechoic chamber.;It was found that the reactance curves can be predicted to an accuracy of 3% or better by the developed numerical model. The far field gains, on the other hand, are far from being reliably predicted by the model. The study also found that significant differences in the behavior of the PCB-CTHA occur when the feed configuration is changed, and three different feed configurations were recommended for future consideration, feeds 7, 12, and 13

Characterizing a novel direct target of the quorum-sensing controlled small RNAs in V. cholerae

n/aUndergraduat

Electron-deficient chiral lactic acid-based hypervalent iodine reagents

Novel electron-deficient chiral hypervalent iodine reagents were prepared in good overall yields. The reactivity and stereoselectivity of these reagents in oxidative rearrangements of alkenes to α-aryl ketones were investigated. The results show that the new reagents have good reactivity and generate products with high enantiomeric excess

Laparoscopic esophagomyotomy in children: is routine fundoplication necessary?

Purpose Laparoscopic esophagomyotomy (LE) with or without fundoplication has been described as a modality for the treatment of achalasia in children. Our aim is to evaluate the safety and efficacy of LE without fundoplication in the management of achalasia in children.Patients and methods A retrospective review of the medical record was carried out on patients with achalasia at our institution from January 2006 to March 2011. Eight children were diagnosed with esophageal achalasia and all of them underwent LE without fundoplication. These patients were reviewed to evaluate intraoperative and postoperative complications, as well as outcome.Results Eight children with achalasia had LE without fundoplication successfully completed. There were four boys and four girls ranging in age between 1 and 13 years (mean 4.3 years). None of the patients had received therapy before LE. The mean operating time was 44 min. The mean follow-up was 40.3 months (range 6–62 months). There were no intraoperative or postoperative esophageal perforations or complications. The mean length of hospital stay was 2.7 days. None of the patients required redo esophagomyotomy or esophageal dilatation. Dysphagia symptoms improved uniformly and gastroesophageal reflux symptoms were not evident in follow-up.Conclusion This technique of LE without fundoplication in the treatment of achalasia is safe, effective, and yields excellent cosmetic results. The routine addition of fundoplication to LE for the treatment of achalasia in our series appears to be unnecessary. Yet, a multicenter study with a large group of patients should be carried out. Keywords: achalasia, fundoplication for achalasia, laparoscopic esophagomyotom

Laparoscopically assisted anorectal pull-through for rectovestibular fistula

Purpose Laparoscopically assisted anorectal pull-through (LAARP) has been  described as an alternative to posterior sagittal anorectoplasty for the surgical  treatment of rectourethral fistula in boys. The aim of the present study was to evaluate the feasibility, safety, efficacy, and advantages of LAARP in the repair of  rectovestibular fistula (RVF) in girls.Patients and methods From January 2010 to January 2015, we conducted a  prospective collection of data of our patients with RVF who were treated with LAARP, regarding demographics, VACTERL (vertebral, anal, cardiac, tracheoesophageal, renal, limb malformations) screening, perioperative measurements, complications, and outcome. Anorectal function of these patients was evaluated using Kelly’s clinical score.Results Sixteen girls with RVF underwent LAARP at our hospital. For all these girls, umbilical colostomy had been performed at the time of their birth. The mean age at LAARP operation was 3 months (range = 2–5 months). Mean operative time was 99 min. Mean hospital stay was 3.2 days. There were no intraoperative complications.  All the patients had their colostomy reversed. No patient had a stricture at the anorectal anastomosis. The mean follow-up time was 35.7 months (range = 6–60  months). The cosmetic appearance was satisfactory. Seven patients, who were older than 3 years, achieved continence and had regular bowel movements with good  Kelly’s clinical score of 6. For the remaining nine patients, the longest follow-up was 3 years, and therefore continence could not be evaluated.Conclusion LAARP for the repair of RVF in girls is feasible, safe, and efficient. Long-term follow-up, which would remain unavailable for several years, is necessary.Keywords: anorectal malformation, imperforated anus, laparoscopically assisted anorectoplasty, rectovestibular fistul

Laparoscopic repair of Morgagni diaphragmatic hernia in infants and children: do we need to resect the hernia sac?

Purpose Removal of the hernia sac for Morgagni diaphragmatic hernia repair in infants and children is a controversial issue. In our series, we elected not to excise the sac in all the cases.Methods Nine children with retrosternal (Morgagni) hernias underwent primary laparoscopic repair without excision of the hernia sac, and we analyzed our results, complications, and outcome.Results Between January 2007 and March 2011, nine children, comprising five boys and four girls, with Morgagni hernia underwent repair laparoscopically at our hospital. The mean age of the children was 15.1 months (range, 3–38 months), mean operative time was 50.5 min, and mean hospital stay was 3.6 days. There were no intraoperative or postoperative complications. The mean follow-up time was 32.8 months (range, 6–54 months). There were no recurrences. All patients had complete obliteration of the residual cavity.Conclusion Laparoscopic closure of the defect by suturing the posterior rim of the hernia to the full thickness of the anterior abdominal wall without excision of the hernia sac is safe and effective in repairing Morgagni hernia without any risk accruing from leaving the hernia sac intact.Keywords: laparoscopic Morgagni hernia repair, Morgagni diaphragmatic hernia, retrosternal diaphragmatic herni

Silencing the nosocomial pathogen Serratia marcescens by glyceryl trinitrate

Background: Quorum sensing is a cell-to-cell communication system in bacteria that controls the production of virulence factors. Serratia marcescens is a causative agent of hospital-acquired infections that shows high resistance to antibiotics. This makes the treatment of these infections difficult. Quorum sensing regulates the production of virulence factors of S. marcescens such as prodigiosin, protease, swimming and swarming motilities and formation of biofilms. Inhibition of quorum sensing may be an alternative to antibiotic treatment to avoid emergence of resistance.Objectives: Testing the ability of glyceryl trinitrate to inhibit quorum sensing and virulence factors of Serratia marcescens.Methods: The inhibiting activities of sub-inhibitory concentration of glyceryl trinitrate against the quorum-sensing regulated violacein pigment in Chromobacterium violaceum CV026 was performed to evaluate the anti-quorum sensing effect of glyceryl trinitrate. The anti-virulence activity was assessed against prodigiosin, protease, biofilm formation in addition to swimming and swarming motilities.Results: Glyceryl trinitrate at at a concentration of 0.25 mg/ml produced significant inhibitory effects against violacein (67.01%), prodigiosin (82.67%), protease, swimming (36.72%) and swarming (79.31%) motilities and biofilm formation (87.83%).Conclusion: Glyceryl trinitrate is a quorum sensing and virulence inhibitor that may be useful in treatment of nosocomial infections caused by Serratia marcescens.Keywords: Serratia marcescens, quorum sensing, virulence, glyceryl trinitrate