The Molecular Basis for Natural Competence in \u3ci\u3eAcinetobacter\u3c/i\u3e

Bacteria use Horizontal Gene Transfer (HGT) to acquire genetic material, leading to the development of novel traits, such as the spread and development of antibiotic resistance. Natural competence is one type of HGT accomplished by DNA uptake from the environment and incorporation into the genome. Bacteria from Acinetobacter are wildly distributed in the environment and are naturally competent. This propensity is a key factor to the steady increase in drug resistance in Acinetobacter, which is a cause for concern in human health as Acinetobacter is a major source of nosocomial infections. During natural competence, type IV pili (T4P) and related filaments are essential for DNA uptake. T4P are extracellular appendages composed of protein subunits (pilins) polymerized into helical fibers which can be extended into the extracellular space and retracted through depolymerization. T4P are required for diverse physiological processes, both retraction-dependent (HGT, twitching motility) and retraction-independent (biofilm formation, host cell adherence). While deletions of pilA (the primary pilin) and pilT (a cytosolic enzyme necessary for retraction) have been found to abrogate natural competence, the mechanism and DNA receptor remains unclear in Acinetobacter. In this thesis, we investigate the molecular basis for natural competence in Acinetobacter. Specific aim 1 is to identify extracellular DNA receptor(s) in Acinetobacter. To accomplish this, we proposed to identify DNA-receptors incorporated into T4P by directly measuring binding affinity of recombinantly-expressed pilin subunits to plasmid double-stranded DNA. Our data demonstrates that two pilin proteins, PilE1 and PilE2, of Acinetobacter baumannii are capable of binding plasmid DNA in vitro. Our other specific aim is to quantify the impact of allelic variation in pilA on DNA-uptake in A. baumannii. We measure natural competence using a common ΔpilA strain complemented with pilA genes from A. baumannii strains from distinct T4P subtypes to assess the impact of T4P pilA variation on natural competence. Our data shows an impact on natural competence from T4P subtype, which indicates that pilus subtype is one of the components influencing transformability in different strains of A.baumannii, potentially through T4P-subtype-depenent differences in pilus retraction. Our results provide a molecular description of HGT in Acinetobacter through natural competence. Advisor: Kurt Piepenbrin

Master of Science

thesisAccurate prediction of hurricane track and intensity is a challenging problem in numerical weather prediction (NWP). Evaluation of the performance of a forecast model is an important step in guiding model improvements. In this study, a statistical evaluation of track and intensity forecasts has been performed for the Navy Global Environmental Model (NAVGEM) during June 2014 to November 2014 for the Atlantic, East Pacific, and West Pacific basins. Results show that: 1) the averaged track errors of NAVGEM range from 100 km at day 1 to 460 km at day 5 and 2) the NAVGEM model has good skill in forecasting intensity trends, although the predicted intensifications lag the observed intensifications in many cases. Then, a notable recent hurricane, Hurricane Joaquin (2015) is used to evaluate the ability of NAVGEM analysis and forecasts to represent the atmospheric conditions in both the large-scale environment and the vortex core region of the hurricane. In order to do this, a series of high-resolution mesoscale numerical simulations of Hurricane Joaquin is performed with an advanced research version of the Weather Research and Forecasting (WRF or WRF ARW) model, and the outcomes are compared with NAVGEM largescale forecasts. Specifically, since there was considerable uncertainty in the Hurricane Joaquin NWP track forecast, five groups of sensitivity experiments with different cumulus, boundary layer, and microphysical schemes as well as different initial and boundary conditions and initial times in WRF simulations have been performed to investigate the large-scale environment and hurricane inner-core structures related to the best-track simulation of Joaquin. It is found that the midlevel steering flows and the thermal structure of the hurricane core region are crucial for track and intensity forecasts. A comparison between the NAVGEM forecasts and the WRF simulation during 1200 UTC 30 September 2015 to 0000 UTC 4 October 2015 shows that NAVGEM makes a fairly good track forecast with reasonable representation of hurricane environmental conditions at its resolution

A simple encoder scheme for distributed residual video coding.

Rate-Distortion (RD) performance of Distributed Video Coding (DVC) is considerably less than that of conventional predictive video coding. In order to reduce the performance gap, many methods and techniques have been proposed to improve the coding efficiency of DVC with increased system complexity, especially techniques employed at the encoder such as encoder mode decisions, optimal quantization, hash methods etc., no doubt increase the complexity of the encoder. However, low complexity encoder is a widely desired feature of DVC. In order to improve the coding efficiency while maintaining low complexity encoder, this paper focuses on Distributed Residual Video Coding (DRVC) architecture and proposes a simple encoder scheme. The main contributions of this paper are as follows: 1) propose a bit plane block based method combined with bit plane re-arrangement to improve the dependency between source and Side Information (SI), and meanwhile, to reduce the amount of data to be channel encoded 2) present a simple iterative dead-zone quantizer with 3 levels in order to adjust quantization from coarse to fine. The simulation results show that the proposed scheme outperforms DISCOVER scheme for low to medium motion video sequences in terms of RD performance, and maintains a low complexity encoder at the same time

Recognition of extracellular DNA by type IV pili promotes biofilm formation by \u3ci\u3eClostridioides difficile\u3c/i\u3e

Clostridioides difficile is a Gram-positive bacillus, which is a frequent cause of gastrointestinal infections triggered by the depletion of the gut microbiome. Because of the frequent recurrence of these infections after antibiotic treatment, mechanisms of C. difficile persistence and recurrence, including biofilm formation, are of increasing interest. Previously, our group and others found that type IV pili, filamentous helical appendages polymerized from protein subunits, promoted microcolony and biofilm formation in C. difficile. In Gram-negative bacteria, the ability of type IV pili to mediate bacterial self-association has been explained through interactions between the pili of adjacent cells, but type IV pili from several Gram-negative species are also required for natural competence through DNA uptake. Here, we report the ability of two C. difficile pilin subunits, PilJ and PilW, to bind to DNA in vitro, as well as the defects in biofilm formation in the pilJ and pilW gene-interruption mutants. Additionally, we have resolved the X-ray crystal structure of PilW, which we use to model possible structural mechanisms for the formation of C. difficile biofilm through interactions between type IV pili and the DNA of the extracellular matrix. Taken together, our results provide further insight into the relationship between type IV pilus function and biofilm formation in C. difficile and, more broadly, suggest that DNA recognition by type IV pili and related structures may have functional importance beyond DNA uptake for natural competence

Type IV Pilus-Mediated Inhibition of \u3ci\u3eAcinetobacter baumannii\u3c/i\u3e Biofilm Formation by Phenothiazine Compounds

Infections by pathogenic Acinetobacter species represent a significant burden on the health care system, despite their relative rarity, due to the difficulty of treating infections through oral antibiotics. Multidrug resistance is commonly observed in clinical Acinetobacter infections and multiple molecular mechanisms have been identified for this resistance, including multidrug efflux pumps, carbapenemase enzymes, and the formation of bacterial biofilm in persistent infections. Phenothiazine compounds have been identified as a potential inhibitor of type IV pilus production in multiple Gram-negative bacterial species. Here, we report the ability of two phenothiazines to inhibit type IV pilus-dependent surface (twitching) motility and biofilm formation in multiple Acinetobacter species. Biofilm formation was inhibited in both static and continuous flow models at micromolar concentrations without significant cytotoxicity, suggesting that type IV pilus biogenesis was the primary molecular target for these compounds. These results suggest that phenothiazines may be useful lead compounds for the development of biofilm dispersal agents against Gram-negative bacterial infections

Conformal Electrodeposition of Antimicrobial Hydrogels Formed by Self-Assembled Peptide Amphiphiles

The colonization of biomedical surfaces by bacterial biofilms is concerning because these microorganisms display higher antimicrobial resistance in biofilms than in liquid cultures. Developing antimicrobial coatings that can be easily applied to medically-relevant complex-shaped objects, such as implants and surgical instruments, is an important and challenging research direction. This work reports the preparation of antibacterial surfaces via the electrodeposition of a conformal hydrogel of self-assembling cationic peptide-amphiphiles (PAs). Hydrogels of three PAs are electrodeposited: C16K2, C16K3, and C18K2, where Cn is an alkyl chain of n methylene groups and Km is an oligopeptide of m lysines. The processing variables (electrodeposition time, potential, pH, salt concentration, agitation) enable fine control of film thickness, demonstrating the flexibility of the method and allowing to unravel the mechanisms underlying electrodeposition. The electrochemically prepared hydrogels inhibit the growth of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa in agar plates, and prevent the formation of biofilms of Acinetobacter baumannii and P. aeruginosa and the formation of A. baumannii colonies in solid media. C16K2 and C16K3 hydrogels outperform the antimicrobial activity of those of C18K2 while maintaining good compatibility with human cells

De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome.</p> <p>Methods</p> <p>The pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers.</p> <p>Results</p> <p>More than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients.</p> <p>Conclusions</p> <p>Our study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.</p

Cancer-Secreted miR-105 Destroys Vascular Endothelial Barriers to Promote Metastasis

SummaryCancer-secreted microRNAs (miRNAs) are emerging mediators of cancer-host crosstalk. Here we show that miR-105, which is characteristically expressed and secreted by metastatic breast cancer cells, is a potent regulator of migration through targeting the tight junction protein ZO-1. In endothelial monolayers, exosome-mediated transfer of cancer-secreted miR-105 efficiently destroys tight junctions and the integrity of these natural barriers against metastasis. Overexpression of miR-105 in nonmetastatic cancer cells induces metastasis and vascular permeability in distant organs, whereas inhibition of miR-105 in highly metastatic tumors alleviates these effects. miR-105 can be detected in the circulation at the premetastatic stage, and its levels in the blood and tumor are associated with ZO-1 expression and metastatic progression in early-stage breast cancer

Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats

Abstract Background Diseases caused by Toxoplasma gondii (T. gondii) have introduced serious threats to public health. There is an urgent need to develop a rapid detection method for T. gondii infection in cats, which are definitive hosts. Recombinant apical membrane antigen 1 (rAMA1) was produced in a prokaryotic expression system and used as the detection antigen. The aim of this study was to evaluate and optimize a reliable indirect enzyme-linked immunosorbent assay (iELISA) method based on rAMA1 for the detection of antibodies against T. gondii in cats. Results The rAMA1-iELISA method was developed and optimized by the chessboard titration method. There were no cross-reactions between T. gondii-positive cat serum and positive serum for other pathogens, indicating that rAMA1-iELISA could only detect T. gondii in most cases. The lowest detection limit of rAMA1-iELISA was 1:3200 (dilution of positive serum), and the CV of repeated tests within batches and between batches were confirmed to be less than 10%. The results of 247 cat serum samples detected by rAMA1-iELISA (kappa value = 0.622, p < 0.001) were in substantial agreement with commercial ELISA. The ROC curve analysis revealed the higher overall check accuracy of rAMA1-iELISA (sensitivity = 91.7%, specificity = 93.6%, AUC = 0.956, 95% CI 0.905 to 1.000) than GRA7-based iELISA (sensitivity = 91.7%, specificity = 85.5%, AUC = 0.936, 95% CI 0.892 to 0.980). Moreover, the positive rate of rAMA1-iELISA (6.5%, 16/247) was higher than that of GRA7-based iELISA (3.6%, 9/247) and that of commercial ELISA kit (4.9%, 12/247). Conclusion The iELISA method with good specificity, sensitivity, and reproducibility was established and can be used for large-scale detection of T. gondii infection in clinical cat samples