Deciphering the DNA code for the function of the Drosophila polydactyl zinc finger protein Suppressor of Hairy-wing.

Polydactyl zinc finger (ZF) proteins have prominent roles in gene regulation and often execute multiple regulatory functions. To understand how these proteins perform varied regulation, we studiedDrosophila Suppressor of Hairy-wing [Su(Hw)], an exemplar multifunctional polydactyl ZF protein. We identified separation-of-function (SOF) alleles that encode proteins disrupted in a single ZF that retain one of the Su(Hw) regulatory activities. Through extended in vitro analyses of the Su(Hw) ZF domain, we show that clusters of ZFs bind individual modules within a compound DNA consensus sequence. Through in vivo analysis of SOF mutants, we find that Su(Hw) genomic sites separate into sequence subclasses comprised of combinations of modules, with subclasses enriched for different chromatin features. These data suggest a Su(Hw) code, wherein DNA binding dictates its cofactor recruitment and regulatory output. We propose that similar DNA codes might be used to confer multiple regulatory functions of other polydactyl ZF proteins.

Design, Synthesis and Microbiological Evaluation of Novel Compounds as Potential Staphylococcus aureus Phenylalanine tRNA Synthetase Inhibitors.

AS THE RESISTANCE of Staphylococcus aureus to antibiotics represents a major threat to global health, anti-infectives with novel mechanisms must be developed. Novel compounds were generated as potential phenylalanine tRNA synthetase (PheRS) inhibitors based on the published homology model of S. aureus PheRS to aid the design process using Molecular Operating Environment (MOE) software. PheRS was selected as it is structurally unique enzyme among the aminoacyl-tRNA synthetases (aaRS), it is considerably different from human cytosolic and human mitochondrial aaRS and it is essential and conserved across bacterial species. The designed compounds were synthesized according to different clear schemes. The compounds were confirmed by 1H NMR, 13C NMR, HRMS and/or microanalysis, and they were microbiologically evaluated.

Discovery and Analysis of Natural-Product Compounds Inhibiting Protein Synthesis in Pseudomonas aeruginosa.

Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation (A/T) protein synthesis system composed of phenylalanyl-tRNA synthetases (PheRS), ribosomes, and ribosomal factors from Pseudomonas aeruginosa This system has been used for high-throughput screening of a natural-compound library. Assays were developed for each component of the system to ascertain the specific target of inhibitory compounds. In high-throughput screens, 13 compounds were identified that inhibit protein synthesis with 50% inhibitory concentrations ranging from 0.3 to >80 µM. MICs were determined for the compounds against the growth of a panel of pathogenic organisms, including Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, Moraxella catarrhalis, P. aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae Three of the compounds were observed to have broad-spectrum activity and inhibited a hypersensitive strain of P. aeruginosa with MICs of 8 to 16 µg/ml. The molecular target of each of the three compounds was determined to be PheRS. One compound was found to be bacteriostatic, and one compound was bactericidal against both Gram-positive and Gram-negative pathogens. The third compound was observed to be bacteriostatic against Gram-positive and bactericidal against Gram-negative bacteria. All three compounds were competitive with the substrate ATP; however, one compound was competitive, one was uncompetitive, and one noncompetitive with the amino acid substrate. Macromolecular synthesis assays confirm the compounds inhibit protein synthesis. The compounds were shown to be more than 25,000-fold less active than the control staurosporine in cytotoxicity MTT testing in human cell lines.

The origin of the Haitian cholera outbreak strain.

BACKGROUND: Although cholera has been present in Latin America since 1991, it had not been epidemic in Haiti for at least 100 years. Recently, however, there has been a severe outbreak of cholera in Haiti. METHODS: We used third-generation single-molecule real-time DNA sequencing to determine the genome sequences of 2 clinical Vibrio cholerae isolates from the current outbreak in Haiti, 1 strain that caused cholera in Latin America in 1991, and 2 strains isolated in South Asia in 2002 and 2008. Using primary sequence data, we compared the genomes of these 5 strains and a set of previously obtained partial genomic sequences of 23 diverse strains of V. cholerae to assess the likely origin of the cholera outbreak in Haiti. RESULTS: Both single-nucleotide variations and the presence and structure of hypervariable chromosomal elements indicate that there is a close relationship between the Haitian isolates and variant V. cholerae El Tor O1 strains isolated in Bangladesh in 2002 and 2008. In contrast, analysis of genomic variation of the Haitian isolates reveals a more distant relationship with circulating South American isolates. CONCLUSIONS: The Haitian epidemic is probably the result of the introduction, through human activity, of a V. cholerae strain from a distant geographic source. (Funded by the National Institute of Allergy and Infectious Diseases and the Howard Hughes Medical Institute.).

Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany.

BACKGROUND: A large outbreak of diarrhea and the hemolytic-uremic syndrome caused by an unusual serotype of Shiga-toxin-producing Escherichia coli (O104:H4) began in Germany in May 2011. As of July 22, a large number of cases of diarrhea caused by Shiga-toxin-producing E. coli have been reported--3167 without the hemolytic-uremic syndrome (16 deaths) and 908 with the hemolytic-uremic syndrome (34 deaths)--indicating that this strain is notably more virulent than most of the Shiga-toxin-producing E. coli strains. Preliminary genetic characterization of the outbreak strain suggested that, unlike most of these strains, it should be classified within the enteroaggregative pathotype of E. coli. METHODS: We used third-generation, single-molecule, real-time DNA sequencing to determine the complete genome sequence of the German outbreak strain, as well as the genome sequences of seven diarrhea-associated enteroaggregative E. coli serotype O104:H4 strains from Africa and four enteroaggregative E. coli reference strains belonging to other serotypes. Genomewide comparisons were performed with the use of these enteroaggregative E. coli genomes, as well as those of 40 previously sequenced E. coli isolates. RESULTS: The enteroaggregative E. coli O104:H4 strains are closely related and form a distinct clade among E. coli and enteroaggregative E. coli strains. However, the genome of the German outbreak strain can be distinguished from those of other O104:H4 strains because it contains a prophage encoding Shiga toxin 2 and a distinct set of additional virulence and antibiotic-resistance factors. CONCLUSIONS: Our findings suggest that horizontal genetic exchange allowed for the emergence of the highly virulent Shiga-toxin-producing enteroaggregative E. coli O104:H4 strain that caused the German outbreak. More broadly, these findings highlight the way in which the plasticity of bacterial genomes facilitates the emergence of new pathogens.

Cell envelope phospholipid composition of Burkholderia multivorans.

Burkholderia multivorans causes opportunistic pulmonary infections in cystic fibrosis and immunocompromised patients. The purpose of the present study was to determine the nature of the phospholipids and their fatty acid constituents comprising the cell envelope membranes of strains isolated from three disparate sources. A conventional method for obtaining the readily extractable lipids fraction from bacteria was employed to obtain membrane lipids for thin-layer chromatographic and gas chromatography-mass spectrophotometric analyses. Major fatty acid components of the B. multivorans readily extractable lipid fractions included C(16:0) (palmitic acid), C(16:1) (palmitoleic acid), and C(18:1) (oleic acid), while C(14:0) (myristic acid), ΔC(17:0) (methylene hexadecanoic acid), C(18:0) (stearic acid), and ΔC(19:0) (methylene octadecanoic acid) were present in lesser amounts. Fatty acid composition differed quantitatively among strains with regard to C(16:0), C(16:1), ΔC(17:0), C(18:1), and ΔC(19:0) with the unsaturated:saturated fatty acid ratios being significantly less in a cystic fibrosis type strain than either environmental or chronic granulomatous disease strains. Phospholipids identified in all B. multivorans strains included lyso-phosphatidylethanolamine, phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol in similar ratios. These data support the conclusion that the cell envelope phospholipid profiles of disparate B. multivorans strains are similar, while their respective fatty acyl substituent profiles differ quantitatively under identical cultivation conditions.

A rationally designed antimicrobial peptide from structural and functional insights of Clostridioides difficile translation initiation factor 1.

A significant increase of hospital-acquired bacterial infections during the COVID-19 pandemic has become an urgent medical problem. Clostridioides difficile is an urgent antibiotic-resistant bacterial pathogen and a leading causative agent of nosocomial infections. The increasing recurrence of C. difficile infection and antibiotic resistance in C. difficile has led to an unmet need for the discovery of new compounds distinctly different from present antimicrobials, while antimicrobial peptides as promising alternatives to conventional antibiotics have attracted growing interest recently. Protein synthesis is an essential metabolic process in all bacteria and a validated antibiotic target. Initiation factor 1 from C. difficile (Cd-IF1) is the smallest of the three initiation factors that acts to establish the 30S initiation complex to initiate translation during protein biosynthesis. Here, we report the solution nuclear magnetic resonance (NMR) structure of Cd-IF1 which adopts a typical ß-barrel fold and consists of a five-stranded ß-sheet and one short α-helix arranged in the sequential order ß1-ß2-ß3-α1-ß4-ß5. The interaction of Cd-IF1 with the 30S ribosomal subunit was studied by NMR titration for the construction of a structural model of Cd-IF1 binding with the 30S subunit. The short α-helix in IF1 was found to be critical for IF1 ribosomal binding. A peptide derived from this α-helix was tested and displayed a high ability to inhibit the growth of C. difficile and other bacterial strains. These results provide a clue for the rational design of new antimicrobials.IMPORTANCEBacterial infections continue to represent a major worldwide health hazard due to the emergence of drug-resistant strains. Clostridioides difficile is a common nosocomial pathogen and the causative agent in many infections resulting in an increase in morbidity and mortality. Bacterial protein synthesis is an essential metabolic process and an important target for antibiotic development; however, the precise structural mechanism underlying the process in C. difficile remains unknown. This study reports the solution structure of C. difficile translation initiation factor 1 (IF1) and its interaction with the 30S ribosomal subunit. A short α-helix in IF1 structure was identified as critically important for ribosomal binding and function in regulating the translation initiation, which allowed a rational design of a new peptide. The peptide demonstrated a high ability to inhibit bacterial growth with broad-spectrum antibacterial activity. This study provides a new clue for the rational design of new antimicrobials against bacterial infections.

Polygenic and directional regulatory evolution across pathways in Saccharomyces.

The search to understand how genomes innovate in response to selection dominates the field of evolutionary biology. Powerful molecular evolution approaches have been developed to test individual loci for signatures of selection. In many cases, however, an organism's response to changes in selective pressure may be mediated by multiple genes, whose products function together in a cellular process or pathway. Here we assess the prevalence of polygenic evolution in pathways in the yeasts Saccharomyces cerevisiae and S. bayanus. We first established short-read sequencing methods to detect cis-regulatory variation in a diploid hybrid between the species. We then tested for the scenario in which selective pressure in one species to increase or decrease the activity of a pathway has driven the accumulation of cis-regulatory variants that act in the same direction on gene expression. Application of this test revealed a variety of yeast pathways with evidence for directional regulatory evolution. In parallel, we also used population genomic sequencing data to compare protein and cis-regulatory variation within and between species. We identified pathways with evidence for divergence within S. cerevisiae, and we detected signatures of positive selection between S. cerevisiae and S. bayanus. Our results point to polygenic, pathway-level change as a common evolutionary mechanism among yeasts. We suggest that pathway analyses, including our test for directional regulatory evolution, will prove to be a relevant and powerful strategy in many evolutionary genomic applications.

Triclosan-induced modification of unsaturated fatty acid metabolism and growth in Pseudomonas aeruginosa PAO1.

Triclosan is a broad-spectrum antimicrobial agent having low toxicity which facilitates its incorporation into numerous personal and health care products. Although triclosan acts against a wide range of gram-positive and gram-negative bacteria by affecting fatty acid biosynthesis, it is ineffective against the opportunistic pathogen Pseudomonas aeruginosa. Wild-type strain P. aeruginosa PAO1 was used as a model system to determine the effects of triclosan on fatty acid metabolism in resistant microorganisms. This was accomplished by cultivating P. aeruginosa PAO1 cultures in the presence of different concentrations of triclosan, monitoring growth rates turbidimetrically, and harvesting in stationary phase. Readily extractable lipids (RELs) were obtained from freeze-dried cells after washing and analyzed using gas chromatography coupled with mass spectrometry. Resultant data demonstrated that triclosan caused dose-dependent increases in the amounts of trans-C(16:1) and trans-C(18:1) fatty acids, with concomitant decreases in their respective cyclopropyl analogs. Triclosan did not affect the relative concentrations of saturated, cis unsaturated, or the overall ratios of combined C(16) to C(18) fatty acid species. The readily extractable lipid fractions contained triclosan proportional to triclosan concentrations in the growth media. The presence or absence of triclosan in either liquid or solid media did not affect the antimicrobial susceptibilities of P. aeruginosa PAO1 to a battery of unrelated antimicrobials. Triclosan decreased growth rate in a dose-dependent manner at soluble concentrations. Incorporation of triclosan into the REL fraction was accompanied by increased levels of trans unsaturated fatty acids, decreased levels of cyclopropyl fatty acids, and decrease in growth rate. These alterations may contribute to triclosan resistance in P. aeruginosa PAO1.

Novel low abundance and transient RNAs in yeast revealed by tiling microarrays and ultra high-throughput sequencing are not conserved across closely related yeast species.

A complete description of the transcriptome of an organism is crucial for a comprehensive understanding of how it functions and how its transcriptional networks are controlled, and may provide insights into the organism's evolution. Despite the status of Saccharomyces cerevisiae as arguably the most well-studied model eukaryote, we still do not have a full catalog or understanding of all its genes. In order to interrogate the transcriptome of S. cerevisiae for low abundance or rapidly turned over transcripts, we deleted elements of the RNA degradation machinery with the goal of preferentially increasing the relative abundance of such transcripts. We then used high-resolution tiling microarrays and ultra high-throughput sequencing (UHTS) to identify, map, and validate unannotated transcripts that are more abundant in the RNA degradation mutants relative to wild-type cells. We identified 365 currently unannotated transcripts, the majority presumably representing low abundance or short-lived RNAs, of which 185 are previously unknown and unique to this study. It is likely that many of these are cryptic unstable transcripts (CUTs), which are rapidly degraded and whose function(s) within the cell are still unclear, while others may be novel functional transcripts. Of the 185 transcripts we identified as novel to our study, greater than 80 percent come from regions of the genome that have lower conservation scores amongst closely related yeast species than 85 percent of the verified ORFs in S. cerevisiae. Such regions of the genome have typically been less well-studied, and by definition transcripts from these regions will distinguish S. cerevisiae from these closely related species.

Structure of PolC reveals unique DNA binding and fidelity determinants.

PolC is the polymerase responsible for genome duplication in many Gram-positive bacteria and represents an attractive target for antibacterial development. We have determined the 2.4-A resolution crystal structure of Geobacillus kaustophilus PolC in a ternary complex with DNA and dGTP. The structure reveals nascent base pair interactions that lead to highly accurate nucleotide incorporation. A unique beta-strand motif in the PolC thumb domain contacts the minor groove, allowing replication errors to be sensed up to 8 nt upstream of the active site. PolC exhibits the potential for large-scale conformational flexibility, which could encompass the catalytic residues. The structure suggests a mechanism by which the active site can communicate with the rest of the replisome to trigger proofreading after nucleotide misincorporation, leading to an integrated model for controlling the dynamic switch between replicative and repair polymerases. This ternary complex of a cellular replicative polymerase affords insights into polymerase fidelity, evolution, and structural diversity.

Evaluation of the microbial flora found in woodwind and brass instruments and their potential to transmit diseases.

Previous studies of dental devices (toothbrushes, dentures, and protective athletic mouthguards) have demonstrated microbial contamination of these devices and possible transmission of infectious diseases to the users. Since woodwind and brass instruments come into intimate contact with the musician's oral cavity and often are passed from student to student without sanitization, the question arises as to whether these instruments are contaminated and can transmit microbial diseases. The purpose of this study was to determine if woodwind and brass instruments and/or their cases harbor opportunistic, pathogenic, or allergenic microorganisms that can be transmitted to the musician. The internal components of woodwind and brass instruments harbored opportunistic, pathogenic, and/or allergenic microorganisms. The highest concentrations of microorganisms were found consistently at the mouthpiece end, but there was evidence of contamination throughout the instruments and their cases. The close proximity of contaminated mouthpieces to the oral cavity could facilitate local and systemic dissemination of the resident opportunistic, pathogenic, and/or allergenic microorganisms. General dentists should determine whether patients play a brass or woodwind instrument and be aware of the possible impact of this activity on the oral cavity and the entire body.

Evaluation of cleansing methods for previously worn prostheses.

STATEMENT OF PROBLEM: Although there are many product claims that address the issue of denture sanitization, controlled scientific studies on previously worn dentures have not been performed. PURPOSE: The purpose of this study was to evaluate procedures directed at sanitizing previously worn contaminated dentures from two regions of the United States. MATERIALS AND METHODS: This study examined 51 previously worn dentures from two regions. An established method of denture retrieval, sectioning, and culturing was used, including isolation of anaerobes. Evaluation of microbial contamination posttreatment was used to determine the effects of soaking dentures in Polident (US and European formulations) for varying periods of times/temperatures, microwaving dentures with varying temperatures, sonicating dentures, and immersing the dentures while using a vacuum. A combination of analysis of variance (ANOVA) and general linear model (GLM) of the SPSS was used to analyze the data with P < .05 being considered statistically significant when using a two-tailed test. RESULTS: While all Polident treatments were found to significantly reduce microorganism loads in dentures, extended soaking (8 hours) and 65 degrees C (5 minutes) were the most effective. Microwaving was slightly more effective than either sonication or vacuum. Regardless the treatment, dentures underwent sanitization rather than sterilization. CONCLUSIONS: Denture-borne microorganisms can be significantly reduced by using a Polident solution for 8 hours at room temperature or for 5 minutes at 65 degrees C. Microwaving, sonication, and use of a vacuum were less effective. ClLINICAL IMPLICATIONS The importance of daily use of Polident solution for 8 hours or for 5 minutes at 65 degrees C to sanitize worn prostheses must be stressed.

Medical Food Assessment Using a Smartphone App With Continuous Glucose Monitoring Sensors: Proof-of-Concept Study.

BACKGROUND: Novel wearable biosensors, ubiquitous smartphone ownership, and telemedicine are converging to enable new paradigms of clinical research. A new generation of continuous glucose monitoring (CGM) devices provides access to clinical-grade measurement of interstitial glucose levels. Adoption of these sensors has become widespread for the management of type 1 diabetes and is accelerating in type 2 diabetes. In parallel, individuals are adopting health-related smartphone-based apps to monitor and manage care. OBJECTIVE: We conducted a proof-of-concept study to investigate the potential of collecting robust, annotated, real-time clinical study measures of glucose levels without clinic visits. METHODS: Self-administered meal-tolerance tests were conducted to assess the impact of a proprietary synbiotic medical food on glucose control in a 6-week, double-blind, placebo-controlled, 2×2 cross-over pilot study (n=6). The primary endpoint was incremental glucose measured using Abbott Freestyle Libre CGM devices associated with a smartphone app that provided a visual diet log. RESULTS: All subjects completed the study and mastered CGM device usage. Over 40 days, 3000 data points on average per subject were collected across three sensors. No adverse events were recorded, and subjects reported general satisfaction with sensor management, the study product, and the smartphone app, with an average self-reported satisfaction score of 8.25/10. Despite a lack of sufficient power to achieve statistical significance, we demonstrated that we can detect meaningful changes in the postprandial glucose response in real-world settings, pointing to the merits of larger studies in the future. CONCLUSIONS: We have shown that CGM devices can provide a comprehensive picture of glucose control without clinic visits. CGM device usage in conjunction with our custom smartphone app can lower the participation burden for subjects while reducing study costs, and allows for robust integration of multiple valuable data types with glucose levels remotely. TRIAL REGISTRATION: ClinicalTrials.gov NCT04424888; http://clinicaltrials.gov/ct2/show/NCT04424888.

Rational Design of an Antimicrobial Peptide Based on Structural Insight into the Interaction of Pseudomonas aeruginosa Initiation Factor 1 with Its Cognate 30S Ribosomal Subunit.

Bacterial infections continue to represent a major worldwide health hazard following the emergence of drug-resistant pathogenic strains. Pseudomonas aeruginosa is an opportunistic pathogen causing nosocomial infections with increased morbidity and mortality. The increasing antibiotic resistance in P. aeruginosa has led to an unmet need for discovery of new antibiotic candidates. Bacterial protein synthesis is an essential metabolic process and a validated target for antibiotic development; however, the precise structural mechanism in P. aeruginosa remains unknown. In this work, the interaction of P. aeruginosa initiation factor 1 (IF1) with the 30S ribosomal subunit was studied by NMR, which enabled us to construct a structure of IF1-bound 30S complex. A short α-helix in IF1 was found to be critical for IF1 ribosomal binding and function. A peptide derived from this α-helix was tested and displayed a high ability to inhibit bacterial growth. These results provide a clue for rational design of new antimicrobials.

Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments.

BACKGROUND: High-throughput sequencing technologies, such as the Illumina Genome Analyzer, are powerful new tools for investigating a wide range of biological and medical questions. Statistical and computational methods are key for drawing meaningful and accurate conclusions from the massive and complex datasets generated by the sequencers. We provide a detailed evaluation of statistical methods for normalization and differential expression (DE) analysis of Illumina transcriptome sequencing (mRNA-Seq) data. RESULTS: We compare statistical methods for detecting genes that are significantly DE between two types of biological samples and find that there are substantial differences in how the test statistics handle low-count genes. We evaluate how DE results are affected by features of the sequencing platform, such as, varying gene lengths, base-calling calibration method (with and without phi X control lane), and flow-cell/library preparation effects. We investigate the impact of the read count normalization method on DE results and show that the standard approach of scaling by total lane counts (e.g., RPKM) can bias estimates of DE. We propose more general quantile-based normalization procedures and demonstrate an improvement in DE detection. CONCLUSIONS: Our results have significant practical and methodological implications for the design and analysis of mRNA-Seq experiments. They highlight the importance of appropriate statistical methods for normalization and DE inference, to account for features of the sequencing platform that could impact the accuracy of results. They also reveal the need for further research in the development of statistical and computational methods for mRNA-Seq.

Discovery and analysis of 4H-pyridopyrimidines, a class of selective bacterial protein synthesis inhibitors.

Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation protein synthesis system composed of phenyl-tRNA synthetases, ribosomes, and ribosomal factors from Escherichia coli. This system, utilizing purified components, has been used for high-throughput screening of a small-molecule chemical library. We have identified a series of compounds that inhibit protein synthesis with 50% inhibitory concentrations (IC(50)s) ranging from 3 to 14 µM. This series of compounds all contained the same central scaffold composed of tetrahydropyrido[4,3-d]pyrimidin-4-ol (e.g., 4H-pyridopyrimidine). All analogs contained an ortho pyridine ring attached to the central scaffold in the 2 position and either a five- or a six-member ring tethered to the 6-methylene nitrogen atom of the central scaffold. These compounds inhibited the growth of E. coli, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, with MICs ranging from 0.25 to 32 µg/ml. Macromolecular synthesis (MMS) assays with E. coli and S. aureus confirmed that antibacterial activity resulted from specific inhibition of protein synthesis. Assays were developed for the steps performed by each component of the system in order to ascertain the target of the compounds, and the ribosome was found to be the site of inhibition.

Evaluation of microbial flora found in previously worn prostheses from the Northeast and Southwest regions of the United States.

STATEMENT OF PROBLEM: Denture-induced stomatitis is a recognized clinical challenge. The responsible microorganisms have not been delineated and may differ among regions of the United States. PURPOSE: The purpose of this study was to identify the microorganisms found in dentures from 2 geographic regions. MATERIAL AND METHODS: Previously worn dentures from 51 available subjects living in the Southwest (41) and Northeast (10) were aseptically retrieved in sterile plastic bags. A posterior piece of the mandibular denture was removed and sampled on appropriate media under anaerobic conditions. The remaining denture material was divided into 7 equal pieces. Each piece was touched to appropriate aerobic media and incubated at 37 degrees C. Bacteria and yeasts were identified using standard clinical laboratory procedures. Data were analyzed by using descriptive statistics. Denture fragments were further analyzed by scanning electron microscopy (SEM). RESULTS: A total of 916 isolates were carried to final speciation. Of these, 711 were aerobic bacteria, 67 were anaerobic bacteria, 125 were yeasts, and 13 were amoebae. Microorganisms were found on the denture surfaces and interstices (denture pores). Most subjects wore their dentures for extended periods without sanitization. SEM analyses confirmed substantial porosity of the denture material with microbial penetration and biofilm formation within the pores. CONCLUSIONS: A wide range of potentially pathogenic microorganisms was found in dentures. There were also regional differences in the microbial flora.

Lysyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Identification of Inhibitory Compounds.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections and has highly developed systems for acquiring resistance against numerous antibiotics. The gene (lysS) encoding P. aeruginosa lysyl-tRNA synthetase (LysRS) was cloned and overexpressed, and the resulting protein was purified to 98% homogeneity. LysRS was kinetically evaluated, and the Km values for the interaction with lysine, adenosine triphosphate (ATP), and tRNALys were determined to be 45.5, 627, and 3.3 µM, respectively. The kcatobs values were calculated to be 13, 22.8, and 0.35 s-1, resulting in kcatobs/KM values of 0.29, 0.036, and 0.11 s-1µM-1, respectively. Using scintillation proximity assay technology, natural product and synthetic compound libraries were screened to identify inhibitors of function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified with inhibitory activity against LysRS. The IC50 values were 17, 30, and 27 µM for each compound, respectively. The minimum inhibitory concentrations were determined against a panel of clinically important pathogens. All three compounds were observed to inhibit the growth of gram-positive organisms with a bacteriostatic mode of action. However, two compounds (BT06F11 and BT08F04) were bactericidal against cultures of gram-negative bacteria. When tested against human cell cultures, BT06F11 was not toxic at any concentration tested, and BM01D09 was toxic only at elevated levels. However, BT08F04 displayed a CC50 of 61 µg/mL. In studies of the mechanism of inhibition, BM01D09 inhibited LysRS activity by competing with ATP for binding, and BT08F04 was competitive with ATP and uncompetitive with the amino acid. BT06F11 inhibited LysRS activity by a mechanism other than substrate competition.

Two Forms of Tyrosyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Discovery of Inhibitory Compounds.

Pseudomonas aeruginosa is a multidrug-resistant (MDR) pathogen and a causative agent of both nosocomial and community-acquired infections. The genes (tyrS and tyrZ) encoding both forms of P. aeruginosa tyrosyl-tRNA synthetase (TyrRS-S and TyrRS-Z) were cloned and the resulting proteins purified. TyrRS-S and TyrRS-Z were kinetically evaluated and the Km values for interaction with Tyr, ATP, and tRNATyr were 172, 204, and 1.5 µM and 29, 496, and 1.9 µM, respectively. The kcatobs values for interaction with Tyr, ATP, and tRNATyr were calculated to be 3.8, 1.0, and 0.2 s-1 and 3.1, 3.8, and 1.9 s-1, respectively. Using scintillation proximity assay (SPA) technology, a druglike 2000-compound library was screened to identify inhibitors of the enzymes. Four compounds (BCD37H06, BCD38C11, BCD49D09, and BCD54B04) were identified with inhibitory activity against TyrRS-S. BCD38C11 also inhibited TyrRS-Z. The IC50 values for BCD37H06, BCD38C11, BCD49D09, and BCD54B04 against TyrRS-S were 24, 71, 65, and 50 µM, respectively, while the IC50 value for BCD38C11 against TyrRS-Z was 241 µM. Minimum inhibitory concentrations (MICs) were determined against a panel of clinically important pathogens. All four compounds were observed to inhibit the growth of cultures of both Gram-positive and Gram-negative bacteria organisms with a bacteriostatic mode of action. When tested against human cell cultures, none of the compounds were toxic at concentrations up to 400 µg/mL. In mechanism of inhibition studies, BCD38C11 and BCD49D09 selectively inhibited TyrRS activity by competing with ATP for binding. BCD37H06 and BCD54B04 inhibited TyrRS activity by a mechanism other than substrate competition.