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Many research articles on various helical gearbox stages are accessible in reputable journals. The majorities of earlier research, despite the fact that triple step helical gearboxes have several benefits, were confined on single step and double step helical gears. Lately, most researching experts who work in this field have started paying close attention to the importances of three stage helical gearboxes. Throughout this research, authors seek to carry out an exhaustive evaluation of the current triple step helical transmission articles for examining the most widely used survey designs, approaches, tools & techniques. The above investigation expansively analysed 315 journal articles on helical gearbox various stages and then used content analysis procedure with an inductive method of research for carrying out an organized review of 45 literatures on three step helix transmission box that appeared in different scientific publications over the preceding two decades. Apart from this; the study presents a broad assessment of currently utilized materials, several future possibilities and problems in employing different materials for various parts of gearbox and its casing. This important research finding indicates there’s an enormous potentiality towards dominating the area of research as well as continuing more chances for exploring growth of three stage helical gearboxes, which provides novel career opportunities for both experts and industrial professionals.

In Electric Spark Machining, choice of manufacturing constraints is a necessary feature. This Electrical Spark Machining is an advanced process in all manufacturing processes. For the manufacturing of sophisticated parts, which require accurate machining that time Electric Discharge Machining used. A super alloy, heat-treated tool steel, ceramics, heat resistance steels etc. which are tough to machining. For this, we use Electric Discharge Machining which also known as Electric Spark Machining. Incoloy alloy set in the group of super stainless steels. The main element of this Incoloy alloy is nickel-chromium-iron with other extra copper, molybdenum metal. High solidity at high peak temperature and super resist of erosion is the main feature of Incoloy alloy. Vast application of Incoloy 800 is like Heat exchangers, Carburizing equipments, Heating elements and nuclear steam generator tubing. In our research, we use hollow, flat, convex and concave electrode profile for machining of Incoloy 800.MRR and TWR are the parameters we analyze after the experiment.

Abstract Background This study was designed to determine if a natural mineral supplement, sierrasil, alone and in combination with a cat's claw extract (Uncaria guianensis), vincaria, has therapeutic potential in mild to moderate osteoarthritis of the knee. Methods Patients (n = 107) with mild to moderate osteoarthritis of the knee were randomly assigned to one of 4 groups; high dose sierrasil (3 g/day), low dose sierrasil (2 g/day), low dose sierrasil (2 g/day) + cat's claw extract (100 mg/day) or placebo, administered for 8 weeks. Treatment was double blinded. Primary efficacy variables were WOMAC scores (A, B, C and total). Visual analog score (VAS) for pain, consumption of rescue medication (paracetamol), and tolerability were secondary variables. Safety measures included vital signs and laboratory-based assays. Results Ninety-one of the 107 patients successfully completed the protocol. All four groups showed improvement in WOMAC and VAS scores after 8 weeks (p < 0.001), in all 3 groups receiving sierrasil the magnitude of benefits were greater vs. placebo (WOMAC Total 38–43% vs. 27%) but this was not statistically significant. In reference to baseline values sierrasil treated groups had a considerably faster onset of benefits. Placebo-treated individuals failed to show significant benefits at 4 weeks (11% reduction in total WOMAC). In contrast, after 1 or 2 weeks of therapy all the sierrasil groups displayed significant reductions in WOMAC scores (p < 0.05) and at week 4 displayed a 38–43% improvement. VAS was significantly improved at 4 weeks in all groups (p < 0.001) but was significantly greater in all sierrasil groups compared to placebo (p < 0.05). Rescue medication use was 28-23% lower in the herbomineral combination and high dose sierrasil groups although not statistically different from placebo (P = 0.101 and P = 0.193, respectively). Tolerability was good for all groups, no serious adverse events were noted and safety parameters remained unchanged. Conclusion The natural mineral supplement, sierrasil alone and in combination with a cat's claw extract, improved joint health and function within 1–2 weeks of treatment but significant benefits over placebo were not sustained, possibly due to rescue medication masking. Sierrasil may offer an alternative therapy in subjects with joint pain and dysfunction.

Antibiotic-resistant bacteria originating from hospitals are ultimately discharged to municipal wastewater treatment plants (WWTP), which may serve as important reservoirs for the spread of antibiotic resistant genes. This study traced and quantified the presence of a rare but clinically relevant antimicrobial resistance gene; Klebsiella pneumoniae carbapenamase (KPC)-and the viable organisms (KPCO) which carried this gene in hospital, non-hospital wastewater discharges, various compartments within a municipal WWTP, receiving water and sediment samples. High concentration of the gene, bla KPC harbored in viable and multispecies KPCO was detected in the hospital wastewater and in the forepart stages of the WWTP, but was not detected in the final effluent following UV disinfection. KPCO were not detected in multiple non-hospital sources of wastewater discharges tested. The treatment train used in the sampled WWTP was found to help remove and reduce KPCO load. Using whole-genome sequencing, a KPC-producing Klebsiella oxytoca strain identical to strains seen in the patients and hospital environment was isolated from the downstream receiving water on one sampling event. KPCO were also found to persist in the biosolids throughout the WWTP, but were not detected in the processed compost-products made from WWTP-biosolids. This study systematically demonstrates dissemination of KPCO from hospital point source to environment via municipal WWTP. Understanding hospitals as the origin and source of spread of some of the most clinically urgent antimicrobial-resistant organisms may help direct interventions that target rate at which antibiotic resistant bacteria evolve and spread via enhancement of wastewater treatment and mitigation of dissemination at source., (Copyright © 2022. Published by Elsevier Ltd.)

Background: The neuropeptide oxytocin regulates mammalian social behavior. Disruptions in oxytocin signaling are a feature of many psychopathologies. One commonly studied biomarker for oxytocin involvement in psychiatric diseases is DNA methylation at the oxytocin receptor gene (OXTR). Such studies focus on DNA methylation in two regions of OXTR, exon 3 and a region termed MT2 which overlaps exon 1 and intron 1. However, the relative contribution of exon 3 and MT2 in regulating OXTR gene expression in the brain is currently unknown., Results: Here, we use the prairie vole as a translational animal model to investigate genetic, epigenetic, and environmental factors affecting Oxtr gene expression in a region of the brain that has been shown to drive Oxtr related behavior in the vole, the nucleus accumbens. We show that the genetic structure of Oxtr in prairie voles resembles human OXTR. We then studied the effects of early life experience on DNA methylation in two regions of a CpG island surrounding the Oxtr promoter: MT2 and exon 3. We show that early nurture in the form of parental care results in DNA hypomethylation of Oxtr in both MT2 and exon 3, but only DNA methylation in MT2 is associated with Oxtr gene expression. Network analyses indicate that CpG sites in the 3' portion of MT2 are most highly associated with Oxtr gene expression. We also identify two novel SNPs in exon 3 of Oxtr in prairie voles and a novel alternative transcript originating from the third intron of the gene. Expression of the novel alternative transcript is associated with genotype at SNP KLW2., Conclusions: These results identify putative regulatory features of Oxtr in prairie voles which inform future studies examining OXTR in human social behaviors and disorders. These studies indicate that in prairie voles, DNA methylation in MT2, particularly in the 3' portion, is more predictive of Oxtr gene expression than DNA methylation in exon 3. Similarly, in human temporal cortex, we find that DNA methylation in the 3' portion of MT2 is associated with OXTR expression. Together, these results suggest that among the CpG sites studied, DNA methylation of MT2 may be the most reliable indicator of OXTR gene expression. We also identify novel features of prairie vole Oxtr, including SNPs and an alternative transcript, which further develop the prairie vole as a translational model for studies of OXTR.

Antimicrobial resistance has been recognized as a threat to human health. The role of hospital sinks acting as a reservoir for some of the most concerning antibiotic resistant organisms, carbapenemase producing Enterobacterales (CPE) is evident but not well understood. Strategies to prevent establishment, interventions to eliminate these reservoirs and factors which drive persistence of CPE are not well established. We use a uniquely designed sink lab to transplant CPE colonized hospital sink plumbing with an aim to understand CPE dynamics in a controlled setting, notably exploiting both molecular and culture techniques. After ex situ installation the CPE population in the sink plumbing drop from previously detectable to undetectable levels. The addition of nutrients is followed by a quick rebound in CPE detection in the sinks after as many as 37 days. We did not however detect a significant shift in microbial community structure or the overall resistance gene carriage in longitudinal samples from a subset of these transplanted sinks using whole shotgun metagenomic sequencing. Comparing nutrient types in a benchtop culture study model, protein rich nutrients appear to be the most supportive for CPE growth and biofilm formation ability. The role of nutrients exposure is determining factor for maintaining a high bioburden of CPE in the sink drains and P-traps. Therefore, limiting nutrient disposal into sinks has reasonable potential with regard to decreasing the CPE wastewater burden, especially in hospitals seeking to control an environmental reservoir., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: AJM has conflict of interest as Consultant for Antimicrobial Resistance (AMR) Services, Inc. SMK, HIP, KEB, & JC have conflict of interest as having conducted independent research contracted by Antimicrobial Resistance Services, Inc. None of the work presented in this manuscript was funded by Antimicrobial Resistance Services, Inc. All authors contributed significantly to the work and will transfer all copyright ownership of the manuscript in the event the work is accepted for publication. The manuscript has not been previously published nor is not being considered for publication elsewhere. A portion of this work was presented at the American Society of Microbiology Annual Meeting (ASM Microbe) in New Orleans, LA in 2017 as an oral presentation., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Anti-virulence approaches in the treatment of Pseudomonas aeruginosa (PA)-induced infections have shown clinical potential in multiple in vitro and in vivo studies. However, development of these compounds is limited by several factors, including the lack of molecules capable of penetrating the membrane of gram-negative organisms. Here, we report the identification of novel structurally diverse compounds that inhibit PqsR and LasR-based signaling and diminish virulence factor production and biofilm growth in two clinically relevant strains of P. aeruginosa. It is the first report where potential anti-virulent agents were evaluated for inhibition of several virulence factors of PA. Finally, co-treatment with these inhibitors significantly reduced the production of virulence factors induced by the presence of sub-inhibitory levels of ciprofloxacin. Further, we have analyzed the drug-likeness profile of designed compounds using quantitative estimates of drug-likeness (QED) and confirmed their potential as hit molecules for further development., (Published by Elsevier Masson SAS.)

Background: Mechanisms linking herpes simplex virus type 2 (HSV-2) with human immunodeficiency virus (HIV) are not fully defined. We tested the hypothesis that HSV-2 and HIV dual infection is associated with cervicovaginal inflammation and/or vaginal dysbiosis., Methods: Genital tract samples were obtained weekly over a 12-week period from 30 women seropositive (+) for HIV and HSV-2 and 15 women each who were seropositive for one or seronegative (-) for both viruses. Immune mediators, antimicrobial activity, and microbial composition and diversity were compared., Results: Significant differences in the concentrations of interferon-γ (P = .002), tumor necrosis factor-α (P = .03), human beta defensin 1 (P = .001), secretory leukocyte protease inhibitor (P = .01), and lysozyme (P = .03) were observed across the 4 groups (Kruskal-Wallis). There were also significant differences in vaginal microbial alpha diversity (Simpson index) (P = .0046). Specifically, when comparing HIV-1+/HSV-2+ to HIV-1-/HSV-2- women, a decrease in Lactobacillus crispatus and increase in diverse anaerobes was observed. The number of genital HSV outbreaks was greater in HIV+ versus HIV- women (39 versus 12) (P = .04), but there were no significant differences when comparing outbreak to non-outbreak visits., Conclusions: Increased microbial diversity and cervicovaginal inflammation in HIV and HSV-2 dually infected women may adversely impact genital health and, in the absence of antiretroviral therapy, facilitate HIV shedding., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Glucose hydrogen breath testing is a noninvasive test for small intestine bacterial overgrowth (SIBO). A positive glucose hydrogen breath test is common in children from low-income countries and has been found to be associated with malnutrition as measured by stunted growth. The microbiome associated with positive breath testing is relatively unstudied., Methods: We performed 16 S V4 rDNA microbiome analysis on the stool of 90 Bangladeshi children aged 2 years from an impoverished neighborhood who were tested at the same time for SIBO by glucose hydrogen breath testing. Data were analyzed by linear discriminant analysis effect size with SIBO as the outcome. Any selected genera were tested individually by Wilcoxon's rank-sum test to ensure that linear discriminant analysis effect size results were not outlier-skewed., Results: Linear discriminant analysis effect size analysis identified Lactobacillus (linear discriminate analysis score, 4.59; P = .03) as over-represented in 15 out of the 90 children who were SIBO positive., Conclusions: These results suggest that glucose hydrogen breath test positivity in children from low-income settings may be due to an upper intestinal Lactobacillus bloom, potentially explaining the association of SIBO with the gut damage and inflammation that leads to malnutrition.

Objective: Evidence supports an inverse association between vitamin D and bacterial vaginosis (BV) during pregnancy. Furthermore, both the vaginal microbiome and vitamin D status correlate with pregnancy outcome. Women of African ancestry are more likely to experience BV, to be vitamin D deficient, and to have certain pregnancy complications. We investigated the association between vitamin D status and the vaginal microbiome., Study Design: Subjects were assigned to a treatment (4400 IU) or a control group (400 IU vitamin D daily), sampled three times during pregnancy, and vaginal 16S rRNA gene taxonomic profiles and plasma 25-hydroxyvitamin D [25(OH)D] concentrations were examined., Result: Gestational age and ethnicity were significantly associated with the microbiome. Megasphaera correlated negatively (p = 0.0187) with 25(OH)D among women of African ancestry. Among controls, women of European ancestry exhibited a positive correlation between plasma 25(OH)D and L. crispatus abundance., Conclusion: Certain vaginal bacteria are associated with plasma 25(OH)D concentration.

The microbiome of the female reproductive tract has implications for women's reproductive health. We examined the vaginal microbiome in two cohorts of women who experienced normal term births: a cross-sectionally sampled cohort of 613 pregnant and 1,969 non-pregnant women, focusing on 300 pregnant and 300 non-pregnant women of African, Hispanic or European ancestry case-matched for race, gestational age and household income; and a longitudinally sampled cohort of 90 pregnant women of African or non-African ancestry. In these women, the vaginal microbiome shifted during pregnancy toward Lactobacillus-dominated profiles at the expense of taxa often associated with vaginal dysbiosis. The shifts occurred early in pregnancy, followed predictable patterns, were associated with simplification of the metabolic capacity of the microbiome and were significant only in women of African or Hispanic ancestry. Both genomic and environmental factors are likely contributors to these trends, with socioeconomic status as a likely environmental influence.

The incidence of preterm birth exceeds 10% worldwide. There are significant disparities in the frequency of preterm birth among populations within countries, and women of African ancestry disproportionately bear the burden of risk in the United States. In the present study, we report a community resource that includes 'omics' data from approximately 12,000 samples as part of the integrative Human Microbiome Project. Longitudinal analyses of 16S ribosomal RNA, metagenomic, metatranscriptomic and cytokine profiles from 45 preterm and 90 term birth controls identified harbingers of preterm birth in this cohort of women predominantly of African ancestry. Women who delivered preterm exhibited significantly lower vaginal levels of Lactobacillus crispatus and higher levels of BVAB1, Sneathia amnii, TM7-H1, a group of Prevotella species and nine additional taxa. The first representative genomes of BVAB1 and TM7-H1 are described. Preterm-birth-associated taxa were correlated with proinflammatory cytokines in vaginal fluid. These findings highlight new opportunities for assessment of the risk of preterm birth.

Several emerging pathogens have arisen as a result of selection pressures exerted by modern health care. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intraspecies transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, bla KPC , between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10- and 5-year periods, respectively. There were 32 bla KPC -positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from 8 patients and 11 sink drains, with evidence for seven separate bla KPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subsp. quasipneumoniae ( n  = 23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then possible transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique bla KPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases., (Copyright © 2019 Mathers et al.)

As the spread of antimicrobial resistance (AMR) genes becomes an increasing global threat, improved understanding of mobile genetic elements which contribute to the spread of antimicrobial resistance genes, becomes more critical. We created transconjugants from the mating of three chromosomally isogenic Klebsiella pneumoniae carbapenemase (bla KPC ) positive Citrobacter freundii isolates with a laboratory strain of Escherichia coli and evaluated the movement of small cryptic plasmids (SCPs), p3223 and p1916, when larger bla KPC -plasmids were transferred. In all of the 143 transconjugants, multiple plasmids, both large and small, transferred with each mating. When two bla KPC- plasmids were present in the host, frequently (87%; 98/113) both would be transferred during mating. p3223 is found in a wide range of bacterial hosts that harbor AMR genes; p1916 has been identified in only a limited number of publicly available sequences to date. From our evaluation, there is still much to learn about SCPs, and the high rate of co-transfer of multiple plasmids from real-world carbapenemase-producing Enterobacteriales., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Preterm premature rupture of membranes (PPROM) is the leading identifiable cause of preterm birth, a complication that is more common in African Americans. Attempts to identify genetic loci associated with preterm birth using genome-wide association studies (GWAS) have only been successful with large numbers of cases and controls, and there has yet to be a convincing genetic association to explain racial/ethnic disparities. Indeed, the search for ancestry-specific variants associated with preterm birth has led to the conclusion that spontaneous preterm birth could be the consequence of multiple rare variants. The hypothesis that preterm birth is due to rare genetic variants that would go undetected in standard GWAS has been explored in the present study. The detection and validation of these rare variants present challenges because of the low allele frequency. However, some success in the identification of fetal loci/genes associated with preterm birth using whole genome sequencing and whole exome sequencing (WES) has recently been reported. While encouraging, this is currently an expensive technology, and methods to leverage the sequencing data to quickly identify and cost-effectively validate variants are needed., Methods: We developed a WES data analysis strategy based on neonatal genomic DNA from PPROM cases and term controls that was unencumbered by preselection of candidate genes, and capable of identifying variants in African Americans worthy of focused evaluation to establish statistically significant associations., Results: We describe this approach and the identification of damaging nonsense variants of African ancestry in the DEFB1 and MBL2 genes that encode anti-microbial proteins that presumably defend the fetal membranes from infectious agents. Our approach also enabled us to rule out a likely contribution of a predicted damaging nonsense variant in the METTL7B gene., Conclusions: Our findings support the notion that multiple rare population-specific variants in the fetal genome contribute to preterm birth associated with PPROM.

Background: Twin studies have revealed a significant contribution of the fetal genome to risk of preterm birth. Preterm premature rupture of membranes (PPROM) is the leading identifiable cause of preterm delivery. Infection and inflammation of the fetal membranes is commonly found associated with PPROM., Methods: We carried out whole exome sequencing (WES) of genomic DNA from neonates born of African-American mothers whose pregnancies were complicated by PPROM (76) or were normal term pregnancies (N = 43) to identify mutations in 35 candidate genes involved in innate immunity and host defenses against microbes. Targeted genotyping of mutations in the candidates discovered by WES was conducted on an additional 188 PPROM cases and 175 controls., Results: We identified rare heterozygous nonsense and frameshift mutations in several of the candidate genes, including CARD6, CARD8, DEFB1, FUT2, MBL2, NLP10, NLRP12, and NOD2. We discovered that some mutations (CARD6, DEFB1, FUT2, MBL2, NLRP10, NOD2) were present only in PPROM cases., Conclusions: We conclude that rare damaging mutations in innate immunity and host defense genes, the majority being heterozygous, are more frequent in neonates born of pregnancies complicated by PPROM. These findings suggest that the risk of preterm birth in African-Americans may be conferred by mutations in multiple genes encoding proteins involved in dampening the innate immune response or protecting the host against microbial infection and microbial products., (© 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Preterm premature rupture of membranes (PPROM) is the leading identifiable cause of preterm birth with ~ 40% of preterm births being associated with PPROM and occurs in 1% - 2% of all pregnancies. We hypothesized that multiple rare variants in fetal genes involved in extracellular matrix synthesis would associate with PPROM, based on the assumption that impaired elaboration of matrix proteins would reduce fetal membrane tensile strength, predisposing to unscheduled rupture. We performed whole exome sequencing (WES) on neonatal DNA derived from pregnancies complicated by PPROM (49 cases) and healthy term deliveries (20 controls) to identify candidate mutations/variants. Genotyping for selected variants from the WES study was carried out on an additional 188 PPROM cases and 175 controls. All mothers were self-reported African Americans, and a panel of ancestry informative markers was used to control for genetic ancestry in all genetic association tests. In support of the primary hypothesis, a statistically significant genetic burden (all samples combined, SKAT-O p-value = 0.0225) of damaging/potentially damaging rare variants was identified in the genes of interest-fibrillar collagen genes, which contribute to fetal membrane strength and integrity. These findings suggest that the fetal contribution to PPROM is polygenic, and driven by an increased burden of rare variants that may also contribute to the disparities in rates of preterm birth among African Americans.

Background: Recent advances in next-generation sequencing have revolutionized genomic research. 16S rRNA amplicon sequencing using paired-end sequencing on the MiSeq platform from Illumina, Inc., is being used to characterize the composition and dynamics of extremely complex/diverse microbial communities. For this analysis on the Illumina platform, merging and quality filtering of paired-end reads are essential first steps in data analysis to ensure the accuracy and reliability of downstream analysis., Results: We have developed the Merging and Filtering Tool (MeFiT) to combine these pre-processing steps into one simple, intuitive pipeline. MeFiT invokes CASPER (context-aware scheme for paired-end reads) for merging paired-end reads and provides users the option to quality filter the reads using the traditional average Q-score metric or using a maximum expected error cut-off threshold., Conclusions: MeFiT provides an open-source solution that permits users to merge and filter paired end illumina reads. The tool has been implemented in python and the source-code is freely available at https://github.com/nisheth/MeFiT .

C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24μM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50=0.18μM) and 3-chloro- (IC50=0.17μM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Species-specific antimicrobial therapy has the potential to combat the increasing threat of antibiotic resistance and alteration of the human microbiome. We therefore set out to demonstrate the beginning of a pathogen-selective drug discovery method using the periodontal pathogen Porphyromonas gingivalis as a model. Through our knowledge of metabolic networks and essential genes we identified a "druggable" essential target, meso-diaminopimelate dehydrogenase, which is found in a limited number of species. We adopted a high-throughput virtual screen method on the ZINC chemical library to select a group of potential small-molecule inhibitors. Meso-diaminopimelate dehydrogenase from P. gingivalis was first expressed and purified in Escherichia coli then characterized for enzymatic inhibitor screening studies. Several inhibitors with similar structural scaffolds containing a sulfonamide core and aromatic substituents showed dose-dependent inhibition. These compounds were further assayed showing reasonable whole-cell activity and the inhibition mechanism was determined. We conclude that the establishment of this target and screening strategy provides a model for the future development of new antimicrobials.

Objective: The oral cavity represents an initial entry way for oral and gut indigenous colonization. Skin-to-skin (STS) care, in which the mother holds the diaper clad naked preterm (PT) infant between her breasts, is associated with improved digestive function, decreased stress, and improved survival. This study evaluated the development of oral microbial colonization repertoires and health characteristics in PT infants with or without STS exposure., Methods: Saliva from 42 PT infants (<32 weeks of gestation at birth) was collected prospectively at 1 month and/or at discharge. High-throughput 16S rRNA sequencing identified microbial diversity and prevalence of bacterial signatures correlated with clinical STS or non-STS care., Results: Corrected for gestational age (CGA) at sampling, bacterial taxa demonstrated increased Streptococcus as a signature of oral repertoire maturation. STS was associated with increased Streptococcus (p < 0.024), while non-STS was associated with greater Corynebacterium (p < 0.023) and Pseudomonas (p < 0.019) in infants ≤ 32 weeks CGA. In infants > 32 weeks CGA, Neisseria and Acinetobacter were more prevalent, 50 vs. 16.7% and 40 vs. 0%, respectively. STS care was associated with shorter hospitalization (p < 0.039)., Conclusion: STS care during earlier gestation was associated with a distinct microbial pattern and an accelerated pace of oral microbial repertoire maturity., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Human cytomegalovirus (CMV) causes significant disease in immunocompromised patients and serious birth defects if acquired in utero. Available CMV antivirals target the viral DNA polymerase, have significant toxicities, and suffer from resistance. New drugs targeting different pathways would be beneficial. The anthraquinone emodin is proposed to inhibit herpes simplex virus by blocking the viral nuclease. Emodin and related anthraquinones are also reported to inhibit CMV. In the present study, emodin reduced CMV infectious yield with an EC50 of 4.9μM but was cytotoxic at concentrations only twofold higher. Related anthraquinones acid blue 40 and alizarin violet R inhibited CMV at only high concentrations (238-265μM) that were also cytotoxic. However, atanyl blue PRL inhibited infectious yield of CMV with an EC50 of 6.3μM, significantly below its 50% cytotoxic concentration of 216μM. Atanyl blue PRL reduced CMV infectivity and inhibited spread. When added up to 1h after infection, it dramatically reduced CMV immediate early protein expression and blocked viral DNA synthesis. However, it had no antiviral activity when added 24h after infection. Interestingly, atanyl blue PRL inhibited nuclease activities of purified CMV UL98 protein with IC50 of 4.5 and 9.3μM. These results indicate that atanyl blue PRL targets very early post-entry events in CMV replication and suggest it may act through inhibition of UL98, making it a novel CMV inhibitor. This compound may provide valuable insights into molecular events that occur at the earliest times post-infection and serve as a lead structure for antiviral development., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Characterizing the nature of interaction between proteins that have not been experimentally cocrystallized requires a computational docking approach that can successfully predict the spatial conformation adopted in the complex. In this work, the Hydropathic INTeractions (HINT) force field model was used for scoring docked models in a data set of 30 high-resolution crystallographically characterized "dry" protein-protein complexes and was shown to reliably identify native-like models. However, most current protein-protein docking algorithms fail to explicitly account for water molecules involved in bridging interactions that mediate and stabilize the association of the protein partners, so we used HINT to illuminate the physical and chemical properties of bridging waters and account for their energetic stabilizing contributions. The HINT water Relevance metric identified the "truly" bridging waters at the 30 protein-protein interfaces and we utilized them in "solvated" docking by manually inserting them into the input files for the rigid body ZDOCK program. By accounting for these interfacial waters, a statistically significant improvement of ∼24% in the average hit-count within the top-10 predictions the protein-protein dataset was seen, compared to standard "dry" docking. The results also show scoring improvement, with medium and high accuracy models ranking much better than incorrect ones. These improvements can be attributed to the physical presence of water molecules that alter surface properties and better represent native shape and hydropathic complementarity between interacting partners, with concomitantly more accurate native-like structure predictions., (© 2013 Wiley Periodicals, Inc.)

Members of the RSK family of kinases constitute attractive targets for drug design, but a lack of structural information regarding the mechanism of selective inhibitors impedes progress in this field. The crystal structure of the N-terminal kinase domain (residues 45-346) of mouse RSK2, or RSK2(NTKD), has recently been described in complex with one of only two known selective inhibitors, a rare naturally occurring flavonol glycoside, kaempferol 3-O-(3'',4''-di-O-acetyl-α-L-rhamnopyranoside), known as SL0101. Based on this structure, it was hypothesized that quercitrin (quercetin 3-O-α-L-rhamnopyranoside), a related but ubiquitous and inexpensive compound, might also act as an RSK inhibitor. Here, it is demonstrated that quercitrin binds to RSK2(NTKD) with a dissociation constant (K(d)) of 5.8 µM as determined by isothermal titration calorimetry, and a crystal structure of the binary complex at 1.8 Å resolution is reported. The crystal structure reveals a very similar mode of binding to that recently reported for SL0101. Closer inspection shows a number of small but significant differences that explain the slightly higher K(d) for quercitrin compared with SL0101. It is also shown that quercitrin can effectively substitute for SL0101 in a biological assay, in which it significantly suppresses the contractile force in rabbit pulmonary artery smooth muscle in response to Ca(2+).

In our effort to develop selective sphingosine kinase-2 (SphK2) inhibitors as pharmacological tools, a thiazolidine-2,4-dione analogue, 3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145), was synthesized and biologically characterized. Biochemical assay results indicate that K145 is a selective SphK2 inhibitor. Molecular modeling studies also support this notion. In vitro studies using human leukemia U937 cells demonstrated that K145 accumulates in U937 cells, suppresses the S1P level, and inhibits SphK2. K145 also exhibited inhibitory effects on the growth of U937 cells as well as apoptotic effects in U937 cells, and that these effects may be through the inhibition of down-stream ERK and Akt signaling pathways. K145 also significantly inhibited the growth of U937 tumors in nude mice by both intraperitoneal and oral administration, thus demonstrating its in vivo efficacy as a potential lead anticancer agent. The antitumor activity of K145 was also confirmed in a syngeneic mouse model by implanting murine breast cancer JC cells in BALB/c mice. Collectively, these results strongly encourage further optimization of K145 as a novel lead compound for development of more potent and selective SphK2 inhibitors.