Publications by authors named "Pamela Brown"

137 Publications

d-canavanine affects peptidoglycan structure, morphogenesis and fitness in Rhizobiales.

Environ Microbiol 2021 Apr 8. Epub 2021 Apr 8.

Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden.

The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for maintenance of cell shape and survival. Many bacteria alter their PG chemistry as a strategy to adapt their cell wall to external challenges. Therefore, identifying these environmental cues is important to better understand the interplay between microbes and their habitat. Here, we used the soil bacterium Pseudomonas putida to uncover cell wall modulators from plant extracts and found canavanine (CAN), a non-proteinogenic amino acid. We demonstrated that cell wall chemical editing by CAN is licensed by P. putida BSAR, a broad-spectrum racemase which catalyses production of dl-CAN from l-CAN, which is produced by many legumes. Importantly, d-CAN diffuses to the extracellular milieu thereby having a potential impact on other organisms inhabiting the same niche. Our results show that d-CAN alters dramatically the PG structure of Rhizobiales (e.g., Agrobacterium tumefaciens, Sinorhizobium meliloti), impairing PG crosslinkage and cell division. Using A. tumefaciens, we demonstrated that the detrimental effect of d-CAN is suppressed by a single amino acid substitution in the cell division PG transpeptidase penicillin binding protein 3a. Collectively, this work highlights the role of amino acid racemization in cell wall chemical editing and fitness.
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http://dx.doi.org/10.1111/1462-2920.15513DOI Listing
April 2021

Influence of Lipophilicity on the Antibacterial Activity of Polymyxin Derivatives and on Their Ability to Act as Potentiators of Rifampicin.

ACS Infect Dis 2021 04 10;7(4):894-905. Epub 2021 Mar 10.

Cantab Anti-Infectives Ltd., BioPark, Broadwater Road, Welwyn Garden City AL7 3AX, United Kingdom.

Novel polymyxin derivatives are often classified either as having direct activity against Gram-negative pathogens or as compounds inactive in their own right, which through permeabilization of the outer membrane act as potentiators of other antibiotics. Here, we report the systematic investigation of the influence of lipophilicity on microbiological activity (including against strains with reduced susceptibility to polymyxins), potentiation of rifampicin, and toxicity within a series of next-generation polymyxin nonapeptides. We demonstrate that the lipophilicity at the N-terminus and amino acids 6 and 7 in the cyclic peptide core is interchangeable and that the activity, ability to potentiate, and cytotoxicity all appear to be primarily driven by overall lipophilicity. Our work also suggests that the characterization of a polymyxin molecule as either a direct acting compound or a potentiator is more of a continuum that is strongly influenced by lipophilicity rather than as a result of fundamentally different modes-of-action.
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http://dx.doi.org/10.1021/acsinfecdis.0c00917DOI Listing
April 2021

A comparison of in vivo viral targeting systems identifies adeno-associated virus serotype 9 (AAV9) as an effective vector for genetic manipulation of Leydig cells in adult mice.

Andrology 2021 01 23;9(1):460-473. Epub 2020 Oct 23.

MRC Centre for Reproductive Health, University of Edinburgh, The Queen's Medical Research Institute, Edinburgh, UK.

Background: Despite the increasing popularity of deliverable transgenics, a robust and fully validated method for targeting Leydig cells, capable of delivering long-term transgene expression, is yet to be defined.

Objectives: We compared three viral vector systems in terms of their cell targeting specificity, longevity of gene expression and impact on targeted cell types when delivered to the interstitial compartment of the mouse testis.

Materials & Methods: We delivered lentiviral, adenoviral and adeno-associated (AAV) viral particles to the interstitial compartment of adult mouse testis. Immunolocalization and stereology were performed to characterize ability of vectors to target and deliver transgenes to Leydig cells.

Results: Viral vectors utilized in this study were found to specifically target Leydig cells when delivered interstitially. Transgene expression in lentiviral-targeted Leydig cells was detected for 7 days post-injection before Leydig cells underwent apoptosis. Adenoviral-delivered transgene expression was detected for 10 days post-injection with no evidence of targeted cell apoptosis. We found serotype differences in AAV injected testis with AAV serotype 9 targeting a significant proportion of Leydig cells. Targeting efficiency increased to an average of 59.63% (and a maximum of 80%) of Leydig cells with the addition of neuraminidase during injection. In AAV injected testis sections, transgene expression was detectable for up to 50 days post-injection.

Discussion & Conclusion: Lentivirus, Adenovirus and Adeno-Associated virus delivery to the testis resulted in key variances in targeting efficiency of Leydig cells and in longevity of transgene expression, but identified AAV9 + Neuraminidase as an efficient vector system for transgene delivery and long-term expression. Simple viral delivery procedures and the commercial availability of viral vectors suggests AAV9 + Neuraminidase will be of significant utility to researchers investigating the genetics underpinning Leydig cell function and holds promise to inform the development of novel therapeutics for the treatment of male reproductive disorders.
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http://dx.doi.org/10.1111/andr.12915DOI Listing
January 2021

Direct modifications of the cyclic peptide Polymyxin B leading to analogues with enhanced in vitro antibacterial activity.

Bioorg Med Chem Lett 2020 06 30;30(11):127163. Epub 2020 Mar 30.

Cantab Anti-Infectives Ltd, Welwyn Garden City AL7 3AX, UK; Spero Therapeutics, Inc, 675 Massachusetts Avenue, 14th Floor, Cambridge, MA 02139 USA.

Synthetic modifications have been made directly to the cyclic peptide core of polymyxin B, enabling the further understanding of structure activity relationships of this antimicrobial peptide. Such modified polymyxins are also substrates for enzymic hydrolysis, enabling the synthesis of a variety of semi-synthetic analogues, resulting in compounds with increased in vitro antibacterial activity.
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http://dx.doi.org/10.1016/j.bmcl.2020.127163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215238PMC
June 2020

Applicability of the ACE-III and RBANS Cognitive Tests for the Detection of Alcohol-Related Brain Damage.

Front Psychol 2019 28;10:2636. Epub 2019 Nov 28.

Institute of Health & Wellbeing, University of Glasgow, Glasgow, United Kingdom.

Background And Aims: Recent investigations have highlighted the value of neuropsychological testing for the assessment and screening of Alcohol-Related Brain Damage (ARBD). The aim of the present study was to evaluate the suitability of the Addenbrooke's Cognitive Examination (ACE-III) and the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) for this purpose.

Methods: Comparing 28 participants with ARBD (11 with Korsakoff's Syndrome and 17 with the umbrella "ARBD" diagnosis) and 30 alcohol-dependent participants without ARBD (ALs) we calculated Area Under the Curve (AUC) statistics, sensitivity and specificity values, base-rate adjusted predictive values, and likelihood ratios for both tests.

Results: High levels of screening accuracy were found for the total scores of both the ACE-III ( = 0.823, 95% CIs [0.714, 0.932], = 0.056; optimal cut-off ≤86: sensitivity = 82%, specificity = 73%) and RBANS ( = 0.846, 95% CIs [0.746, 0.947], = 0.052; optimal cut-off ≤83: sensitivity = 89%, specificity = 67%) at multiple cut-off points. Removing participants with a history of polysubstance from the samples (10 ALs and 1 ARBD) improved the diagnostic capabilities of the RBANS substantially ( = 0.915, 95% CIs [0.831, 0.999], = 0.043; optimal cut-off ≤85: sensitivity = 98%, specificity = 80%), while only minor improvements to the ACE-III's accuracy were observed ( = 0.854, 95% CIs [0.744, 0.963], = 0.056; optimal cut-off ≤88: sensitivity = 85%, specificity = 75%).

Conclusion: Overall, both the ACE-III and RBANS are suitable tools for ARBD screening within an alcohol-dependent population, though the RBANS is the superior of the two. Clinicians using these tools for ARBD screening should be cautious of false-positive outcomes and should therefore combine them with other assessment methods (e.g., neuroimaging, clinical observations) and more detailed neuropsychological testing before reaching diagnostic decisions.
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http://dx.doi.org/10.3389/fpsyg.2019.02636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892773PMC
November 2019

Design of Next Generation Polymyxins with Lower Toxicity: The Discovery of SPR206.

ACS Infect Dis 2019 10 24;5(10):1645-1656. Epub 2019 Sep 24.

Cantab Anti-Infectives Ltd. , BioPark, Broadwater Road , Welwyn Garden City , Hertfordshire AL7 3AX , United Kingdom.

Polymyxins are an important class of antibiotics for the treatment of bacterial infections due to multidrug resistant Gram-negative pathogens. However, their clinical utility is limited by nephrotoxicity. Here, we report a series of promising next generation polymyxin nonapeptides identified on the basis of our understanding of the relationship of structure with activity, cytotoxicity, and kidney compartment accumulation. We demonstrate that nonapeptides with an amine-containing N-terminal moiety of specific regio- and stereochemistry possess superior activity, together with lower cytotoxicity compared to polymyxin B. We further demonstrate that compounds with a β-branched aminobutyrate N-terminus with an aryl substituent offer a promising combination of low cytotoxicity and kidney exposure, leading to low toxicity in the mouse. From this series, SPR206 has been selected as a development candidate.
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http://dx.doi.org/10.1021/acsinfecdis.9b00217DOI Listing
October 2019

Isolation and Characterization T4- and T7-Like Phages that Infect the Bacterial Plant Pathogen .

Viruses 2019 06 7;11(6). Epub 2019 Jun 7.

Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA.

In the rhizosphere, bacteria-phage interactions are likely to have important impacts on the ecology of microbial communities and microbe-plant interactions. To better understand the dynamics of Agrobacteria-phage interactions, we have isolated diverse bacteriophages which infect the bacterial plant pathogen, . Here, we complete the genomic characterization of phages Atu_ph04 and Atu_ph08. Atu_ph04-a T4-like phage belonging to the family-was isolated from waste water and has a 143,349 bp genome that encodes 223 predicted open reading frames (ORFs). Based on phylogenetic analysis and whole-genome alignments, Atu_ph04 is a member of a newly described T4 superfamily that contains other -infecting phages. Atu_ph08, a member of the T7-like family, was isolated from waste water, has a 59,034 bp genome, and encodes 75 ORFs. Based on phylogenetic analysis and whole-genome alignments, Atu_ph08 may form a new T7 superfamily which includes phage PCB5 and phage POI1126. Atu_ph08 is predicted to have lysogenic activity, as we found evidence of an integrase and several transcriptional repressors with similarity to proteins in transducing phage P22. Together, this data suggests that phages are diverse in morphology, genomic content, and lifestyle.
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http://dx.doi.org/10.3390/v11060528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6630229PMC
June 2019

Higher functionality of bacterial plasmid DNA in water after peracetic acid disinfection compared with chlorination.

Sci Total Environ 2019 Oct 11;685:419-427. Epub 2019 May 11.

Department of Civil & Environmental Engineering, University of Missouri, Columbia, MO 65211, United States. Electronic address:

Peracetic acid (PAA) is an emerging disinfectant with a low disinfection by-product formation potential, but how PAA destroys gene function after killing bacteria remains to be studied. Bacterial plasmid DNA is a mobile genetic element that often harbors undesirable genes encoding antibiotic resistance and virulence factors. Even though PAA efficiently kills bacteria, bacterial plasmids and other mobile genetic elements might still be intact and functional after PAA disinfection, posing potential public health and environmental risks. This study evaluated the impact of PAA disinfection on the functionality of plasmid DNA in vivo and compared the results with those from chlorination. We delivered a plasmid DNA harboring two antibiotic resistance genes to Escherichia coli TOP10 to form an antibiotic-resistant bacterium (ARB). The planktonic ARB was treated with PAA and chlorine to find the minimum doses inhibiting the regrowth of the strain. PAA and chlorine stopped the regrowth at 8 ± 1 mg PAA·L and 20 ± 9 mg Cl·L, respectively. The functionality of the plasmid DNA after PAA and chlorine disinfection was then determined at higher doses in vivo. Neither PAA nor chlorine completely destroyed the plasmid DNA. However, chlorine was more efficient than PAA in eliminating the plasmid DNA. PAA at 25 mg PAA·L reduced the transforming activity of the plasmid DNA by less than 0.3 log units, whereas chlorine at 25 mg Cl·L reduced the transforming activity by approximately 1.7 log units. Chlorine had a more pronounced impact on the functionality of the plasmid DNA because it oxidizes or destroys bacterial components including plasmid DNA faster than PAA. In addition, environmental scanning electron microscopy shows that chlorination desiccated the cells resulting in the flat cellular structure and possibly more complete loss of plasmid DNA, whereas PAA disinfection had a less impact on cell structure and morphology. This study demonstrates that more plasmid DNA remains functional in water after PAA disinfection than after chlorination. These functional genetic elements could be acquired by other microorganisms via horizontal gene transfer to pose potential public health and environmental risks.
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http://dx.doi.org/10.1016/j.scitotenv.2019.05.074DOI Listing
October 2019

An Essential Regulator of Bacterial Division Links FtsZ to Cell Wall Synthase Activation.

Curr Biol 2019 05 25;29(9):1460-1470.e4. Epub 2019 Apr 25.

Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address:

Bacterial growth and division require insertion of new peptidoglycan (PG) into the existing cell wall by PG synthase enzymes. Emerging evidence suggests that many PG synthases require activation to function; however, it is unclear how activation of division-specific PG synthases occurs. The FtsZ cytoskeleton has been implicated as a regulator of PG synthesis during division, but the mechanisms through which it acts are unknown. Here, we show that FzlA, an FtsZ-binding protein and essential regulator of constriction in Caulobacter crescentus, helps link FtsZ to PG synthesis to promote division. We find that hyperactive mutants of the PG synthases FtsW and FtsI specifically render fzlA, but not other division genes, non-essential. However, FzlA is still required to maintain proper constriction rate and efficiency in a hyperactive PG synthase background. Intriguingly, loss of fzlA in the presence of hyperactivated FtsWI causes cells to rotate about the division plane during constriction and sensitizes cells to cell-wall-specific antibiotics. We demonstrate that FzlA-dependent signaling to division-specific PG synthesis is conserved in another α-proteobacterium, Agrobacterium tumefaciens. These data establish that FzlA helps link FtsZ to cell wall remodeling and is required for signaling to both activate and spatially orient PG synthesis during division. Overall, our findings support the paradigm that activation of SEDS-PBP PG synthases is a broadly conserved requirement for bacterial morphogenesis.
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http://dx.doi.org/10.1016/j.cub.2019.03.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504580PMC
May 2019

Androgen receptor expression is required to ensure development of adult Leydig cells and to prevent development of steroidogenic cells with adrenal characteristics in the mouse testis.

BMC Dev Biol 2019 04 17;19(1). Epub 2019 Apr 17.

MRC Centre for Reproductive Health, University of Edinburgh, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK.

Background: The interstitium of the mouse testis contains Leydig cells and a small number of steroidogenic cells with adrenal characteristics which may be derived from the fetal adrenal during development or may be a normal subset of the developing fetal Leydig cells. Currently it is not known what regulates development and/or proliferation of this sub-population of steroidogenic cells in the mouse testis. Androgen receptors (AR) are essential for normal testicular function and in this study we have examined the role of the AR in regulating interstitial cell development.

Results: Using a mouse model which lacks gonadotropins and AR (hpg.ARKO), stimulation of luteinising hormone receptors in vivo with human chorionic gonadotropin (hCG) caused a marked increase in adrenal cell transcripts/protein in a group of testicular interstitial cells. hCG also induced testicular transcripts associated with basic steroidogenic function in these mice but had no effect on adult Leydig cell-specific transcript levels. In hpg mice with functional AR, treatment with hCG induced Leydig cell-specific function and had no effect on adrenal transcript levels. Examination of mice with cell-specific AR deletion and knockdown of AR in a mouse Leydig cell line suggests that AR in the Leydig cells are likely to regulate these effects.

Conclusions: This study shows that in the mouse the androgen receptor is required both to prevent development of testicular cells with adrenal characteristics and to ensure development of an adult Leydig cell phenotype.
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http://dx.doi.org/10.1186/s12861-019-0189-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472051PMC
April 2019

1,25-Dihydroxyvitamin D Restrains CD4 T Cell Priming Ability of CD11c Dendritic Cells by Upregulating Expression of CD31.

Front Immunol 2019 28;10:600. Epub 2019 Mar 28.

MRC Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom.

Dendritic cells (DC) are specialized sentinel cells that bridge the innate and adaptive immune response and play a crucial role in shaping the adaptive immune response. Vitamin D, a known epidemiological risk factor for the development of several autoimmune diseases, influences the development of dendritic cells. Consequently, vitamin D metabolites are frequently used in protocols to develop therapeutic dendritic cell therapies for autoimmune diseases. However, the mechanisms by which vitamin D modulates DC function remain poorly understood. We investigated the effects of vitamin D on murine CD11c bone marrow derived DC (BMDC) function by analyzing global gene expression in CD11c BMDC generated in the presence (VitD-CD11cBMDC) or absence (Veh-CD11cBMDC) of the active vitamin D metabolite, 1,25-dihydroxyvitamin D (1,25(OH)D). Seven genes were significantly increased in expression in both immature and LPS-matured VitD-CD11cBMDC, one of which was CD31, a member of the immunoglobulin superfamily. Gene knockdown of CD31 enhanced the ability of VitD-CD11cBMDC to prime naïve CD4 T cells ; conversely, increased expression of CD31 on vehicle treated CD11cBMDC restrained their T cell priming abilities. Time-lapse imaging of BMDC and CD4 T cells during priming revealed that CD31 reduced the BMDC-T cell interaction time. Finally, we confirmed a similar effect of 1,25(OH)D on human CD34 cell-derived CD11cDC, whereby DC generated in the presence of 1,25(OH)D had increased CD31 expression. In summary, we show that both mouse and human DC generated in the presence of 1,25(OH)D upregulate CD31 expression, resulting in a reduced ability to prime CD4 T cells by impairing a stable cell-cell contact.
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http://dx.doi.org/10.3389/fimmu.2019.00600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6447667PMC
August 2020

Agrobacterium tumefaciens divisome proteins regulate the transition from polar growth to cell division.

Mol Microbiol 2019 04 4;111(4):1074-1092. Epub 2019 Mar 4.

Division of Biological Sciences, University of Missouri, Columbia, MO, 65203, USA.

The mechanisms that restrict peptidoglycan biosynthesis to the pole during elongation and re-direct peptidoglycan biosynthesis to mid-cell during cell division in polar-growing Alphaproteobacteria are largely unknown. Here, we explore the role of early division proteins of Agrobacterium tumefaciens including three FtsZ homologs, FtsA and FtsW in the transition from polar growth to mid-cell growth and ultimately cell division. Although two of the three FtsZ homologs localize to mid-cell, exhibit GTPase activity and form co-polymers, only one, FtsZ , is required for cell division. We find that FtsZ is required not only for constriction and cell separation, but also for initiation of peptidoglycan synthesis at mid-cell and cessation of polar peptidoglycan biosynthesis. Depletion of FtsZ in A. tumefaciens causes a striking phenotype: cells are extensively branched and accumulate growth active poles through tip splitting events. When cell division is blocked at a later stage by depletion of FtsA or FtsW, polar growth is terminated and ectopic growth poles emerge from mid-cell. Overall, this work suggests that A. tumefaciens FtsZ makes distinct contributions to the regulation of polar growth and cell division.
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http://dx.doi.org/10.1111/mmi.14212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482847PMC
April 2019

Inhibition of regrowth of planktonic and biofilm bacteria after peracetic acid disinfection.

Water Res 2019 02 24;149:640-649. Epub 2018 Oct 24.

Department of Civil & Environmental Engineering, University of Missouri, Columbia, MO, 65211, United States. Electronic address:

Peracetic acid (PAA) is a promising alternative to chlorine for disinfection; however, bacterial regrowth after PAA disinfection is poorly understood. This study compared the regrowth of bacteria (Gram-negative Pseudomonas aeruginosa PAO1 and Gram-positive Bacillus sp.) after disinfection with PAA or free chlorine. In the absence of organic matter, PAA and free chlorine prevented the regrowth of planktonic cells of P. aeruginosa PAO1 at C·t (= disinfectant concentration × contact time) doses of (28.5 ± 9.8) mg PAA·min·L and (22.5 ± 10.6) mg Cl·min·L, respectively, suggesting that they had comparable efficiencies in preventing the regrowth of planktonic bacteria. For comparison, the minimum C·t doses of PAA and free chlorine to prevent the regrowth of P. aeruginosa PAO1 biofilm cells in the absence of organic matter were (14,000 ± 1,732) mg PAA·min·L and (6,500 ± 2,291) mg Cl·min·L, respectively. PAA was less effective than free chlorine in killing bacteria within biofilms in the absence of organic matter most likely because PAA reacts with biofilm matrix constituents slower than free chlorine. In the presence of organic matter, although the bactericidal efficiencies of both disinfectants significantly decreased, PAA was less affected due to its slower reaction with organic matter and/or slower self-decomposition. For instance, in a dilute Lysogeny broth-Miller, the minimum concentrations of PAA and free chlorine to prevent the regrowth of planktonic P. aeruginosa PAO1 were 20 mg PAA·L and 300 mg Cl·L, respectively. While both disinfectants are strong oxidants disrupting cell membrane, environmental scanning electron microscopy (ESEM) revealed that PAA made holes in the center of the cells, whereas free chlorine desiccated the cells. Overall, this study shows that PAA is a powerful disinfectant to prevent bacterial regrowth even in the presence of organic matter.
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http://dx.doi.org/10.1016/j.watres.2018.10.062DOI Listing
February 2019

Towards cardiac MRI based risk stratification in idiopathic dilated cardiomyopathy.

Heart 2019 02 30;105(4):270-275. Epub 2018 Oct 30.

Cardiac MRI Department, North West Heart Centre, Manchester University Foundation Trust-Wythenshawe Site, Manchester, UK.

Sudden cardiac death (SCD) secondary to arrhythmia remains a risk in those with dilated cardiomyopathy (DCM), an implantable cardiac defibrillator (ICD) is an effective strategy to prevent SCD. Current guidelines recommend selection for ICD based on ejection fraction (EF) less than 35%, however, most SCD occurs in those with EF>35%. Although meta-analysis has demonstrated a survival benefit for primary prevention ICD in DCM, no randomised trial has shown a significant reduction in overall mortality including the most recent 'Danish Study to Assess the Efficacy of ICDs in Patients With Non-Ischemic Systolic Heat Failure on Mortality' study. Clearly, a more sophisticated selection strategy is required. Cardiac MRI (CMR) is an ideal non-invasive imaging technique which allows calculation of EF as well as tissue characterisation with gadolinium contrast, parametric mapping and feature tracking. Late gadolinium enhancement detects mid-wall fibrosis in approximately 30% of those with DCM, three meta-analyses have demonstrated an association between fibrosis in DCM and SCD, and those without fibrosis are at low risk of SCD. T1 mapping and extracellular volume (ECV) calculation are methods of demonstrating diffuse fibrosis in the myocardium. Raised ECV and native T1 have been associated with worse outcomes but the relationship to SCD has not been well studied. Undoubtedly, more research is required but CMR has several tools which offer incremental value above EF to improve risk stratification and consequent outcomes and resource utilisation in those with DCM.
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http://dx.doi.org/10.1136/heartjnl-2018-313767DOI Listing
February 2019

DMRT1 repression using a novel approach to genetic manipulation induces testicular dysgenesis in human fetal gonads.

Hum Reprod 2018 11;33(11):2107-2121

MRC Centre for Reproductive Health, The Queen's Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh, Scotland, UK.

Study Question: Does loss of DMRT1 in human fetal testis alter testicular development and result in testicular dysgenesis?

Summary Answer: DMRT1 repression in human fetal testis alters the expression of key testicular and ovarian determining genes, and leads to focal testicular dysgenesis.

What Is Known Already: Testicular dysgenesis syndrome (TDS) is associated with common testicular disorders in young men, but its etiology is unknown. DMRT1 has been shown to play a role in the regulation of sex differentiation in the vertebrate gonad. Downregulation of DMRT1 in male mice results in trans-differentiation of Sertoli cells into granulosa (FOXL2+) cells resulting in an ovarian gonadal phenotype.

Study Design, Size, Duration: To determine the effect of DMRT1 repression on human fetal testes, we developed a novel system for genetic manipulation, which utilizes a Lentivral delivered miRNA during short-term in vitro culture (2 weeks). A long-term (4-6 weeks) ex vivo xenograft model was used to determine the subsequent effects of DMRT1 repression on testicular development and maintenance. We included first and second-trimester testis tissue (8-20 weeks gestation; n = 12) in the study.

Participants/materials, Setting, Methods: Human fetal testes were cultured in vitro and exposed to either of two DMRT1 miRNAs (miR536, miR641), or to scrambled control miRNA, for 24 h. This was followed by a further 14 days of culture (n = 3-4), or xenografting (n = 5) into immunocompromised mice for 4-6 weeks. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence and quantitative RT-PCR. Endpoints included histological evaluation of seminiferous cord integrity, mRNA expression of testicular, ovarian and germ cell genes, and assessment of cell number and protein expression for proliferation, apoptosis and pluripotency factors. Statistical analysis was performed using a linear mixed effect model.

Main Results And The Role Of Chance: DMRT1 repression (miR536/miR641) resulted in a loss of DMRT1 protein expression in a sub-population of Sertoli cells of first trimester (8-11 weeks gestation) human fetal testis; however, this did not affect the completion of seminiferous cord formation or morphological appearance. In second-trimester testis (12-20 weeks gestation), DMRT1 repression (miR536/miR641) resulted in disruption of seminiferous cords with absence of DMRT1 protein expression in Sertoli (SOX9+) cells. No differences in proliferation (Ki67+) were observed and apoptotic cells (CC3+) were rare. Expression of the Sertoli cell associated gene, SOX8, was significantly reduced (miR536, 34% reduction, P = 0.031; miR641 36% reduction, P = 0.026), whilst SOX9 expression was unaffected. Changes in expression of AMH (miR536, 100% increase, P = 0.033), CYP26B1 (miR641, 38% reduction, P = 0.05) and PTGDS (miR642, 30% reduction, P = 0.0076) were also observed. Amongst granulosa cell associated genes, there was a significant downregulation in R-spondin 1 expression (miR536, 76% reduction, P < 0.0001; miR641, 49% reduction, P = 0.046); however, there were no changes in expression of the granulosa cell marker, FOXL2. Analysis of germ cell associated genes demonstrated a significant increase in the expression of the pluripotency gene OCT4 (miR536, 233%, P < 0.001). We used the xenograft system to investigate the longer-term effects of seminiferous cord disruption via DMRT1 repression. As was evident in vitro for second-trimester samples, DMRT1 repression resulted in focal testicular dysgenesis similar to that described in adults with TDS. These dysgenetic areas were devoid of germ cells, whilst expression of FOXL2 within the dysgenetic areas, indicated trans-differentiation from a male (Sertoli cell) to female (granulosa cell) phenotype.

Limitations, Reasons For Caution: Human fetal testis tissue is a limited resource; however, we were able to demonstrate significant effects of DMRT1 repression on the expression of germ and somatic cell genes, in addition to the induction of focal testicular dysgenesis, using these limited samples. In vitro culture may not reflect all aspects of human fetal testis development and function; however, the concurrent use of the xenograft model which represents a more physiological system supports the validity of the in vitro findings.

Wider Implications Of The Findings: Our findings have important implications for understanding the role of DMRT1 in human testis development and in the origin of testicular dysgenesis. In addition, we provide validation of a novel system that can be used to determine the effects of repression of genes that have been implicated in gonadal development and associated human reproductive disorders.

Study Funding/competing Interest(s): This project was funded by a Wellcome Trust Intermediate Clinical Fellowship (Grant No. 098522) awarded to RTM. LBS was supported by MRC Programme Grant MR/N002970/1. RAA was supported by MRC Programme Grant G1100357/1. RMS was supported by MRC Programme Grant G33253. This work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. The funding bodies had no input into the conduct of the research or the production of this manuscript. The authors have declared no conflicts of interest.
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http://dx.doi.org/10.1093/humrep/dey289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195803PMC
November 2018

Larger Than Life: Isolation and Genomic Characterization of a Jumbo Phage That Infects the Bacterial Plant Pathogen, .

Front Microbiol 2018 14;9:1861. Epub 2018 Aug 14.

Division of Biological Sciences, University of Missouri, Columbia, MO, United States.

is a plant pathogen that causes crown gall disease, leading to the damage of agriculturally-important crops. As part of an effort to discover new phages that can potentially be used as biocontrol agents to prevent crown gall disease, we isolated and characterized phage Atu_ph07 from Sawyer Creek in Springfield, MO, using the virulent strain C58 as a host. After surveying its host range, we found that Atu_ph07 exclusively infects . Time-lapse microscopy of cells subjected to infection at a multiplicity of infection (MOI) of 10 with Atu_ph07 reveals that lysis occurs within 3 h. Transmission electron microscopy (TEM) of virions shows that Atu_ph07 has a typical morphology with an icosahedral head, long tail, and tail fibers. The sequenced genome of Atu_ph07 is 490 kbp, defining it as a jumbo phage. The Atu_ph07 genome contains 714 open reading frames (ORFs), including 390 ORFs with no discernable homologs in other lineages (ORFans), 214 predicted conserved hypothetical proteins with no assigned function, and 110 predicted proteins with a functional annotation based on similarity to conserved proteins. The proteins with predicted functional annotations share sequence similarity with proteins from bacteriophages and bacteria. The functionally annotated genes are predicted to encode DNA replication proteins, structural proteins, lysis proteins, proteins involved in nucleotide metabolism, and tRNAs. Characterization of the gene products reveals that Atu_ph07 encodes homologs of 16 T4 core proteins and is closely related to Rak2-like phages. Using ESI-MS/MS, the majority of predicted structural proteins could be experimentally confirmed and 112 additional virion-associated proteins were identified. The genomic characterization of Atu_ph07 suggests that this phage is lytic and the dynamics of Atu_ph07 interaction with its host indicate that this phage may be suitable for inclusion in a phage cocktail to be used as a biocontrol agent.
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http://dx.doi.org/10.3389/fmicb.2018.01861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102473PMC
August 2018

An aryl-homoserine lactone quorum-sensing signal produced by a dimorphic prosthecate bacterium.

Proc Natl Acad Sci U S A 2018 07 2;115(29):7587-7592. Epub 2018 Jul 2.

Integrative Microbiology Research Centre, South China Agricultural University, 510642 Guangzhou, People's Republic of China;

Many species of produce acyl-homoserine lactone (AHL) compounds as quorum-sensing (QS) signals for cell density-dependent gene regulation. Most known AHL synthases, LuxI-type enzymes, produce fatty AHLs, and the fatty acid moiety is derived from an acyl-acyl carrier protein (ACP) intermediate in fatty acid biosynthesis. Recently, a class of LuxI homologs has been shown to use CoA-linked aromatic or amino acid substrates for AHL synthesis. By using an informatics approach, we found the CoA class of LuxI homologs exists primarily in α-Proteobacteria. The genome of , a dimorphic prosthecate bacterium, possesses a like AHL synthase gene that we predicted to encode a CoA-utilizing enzyme. We show the LuxI homolog catalyzes synthesis of phenylacetyl-homoserine lactone (PA-HSL). Our experiments show obtains phenylacetate from its environment and uses a CoA ligase to produce the phenylacetyl-CoA substrate for the LuxI homolog. By using an AHL degrading enzyme, we showed that PA-HSL controls aggregation, biofilm formation, and pigment production in These findings advance a limited understanding of the CoA-dependent AHL synthases. We describe how to identify putative members of the class, we describe a signal synthesized by using an environmental aromatic acid, and we identify phenotypes controlled by the aryl-HSL.
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http://dx.doi.org/10.1073/pnas.1808351115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055194PMC
July 2018

Cell Wall Biogenesis During Elongation and Division in the Plant Pathogen Agrobacterium tumefaciens.

Curr Top Microbiol Immunol 2018;418:87-110

Division of Biological Sciences, University of Missouri, Columbia, MO, USA.

A great diversity of bacterial cell shapes can be found in nature, suggesting that cell wall biogenesis is regulated both spatially and temporally. Although Agrobacterium tumefaciens has a rod-shaped morphology, the mechanisms underlying cell growth are strikingly different than other well-studied rod-shaped bacteria including Escherichia coli. Technological advances, such as the ability to deplete essential genes and the development of fluorescent D-amino acids, have enabled recent advances in our understanding of cell wall biogenesis during cell elongation and division of A. tumefaciens. In this review, we address how the field has evolved over the years by providing a historical overview of cell elongation and division in rod-shaped bacteria. Next, we summarize the current understanding of cell growth and cell division processes in A. tumefaciens. Finally, we highlight the need for further research to answer key questions related to the regulation of cell wall biogenesis in A. tumefaciens.
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http://dx.doi.org/10.1007/82_2018_92DOI Listing
June 2019

A novel whole-cell biosensor of Pseudomonas aeruginosa to monitor the expression of quorum sensing genes.

Appl Microbiol Biotechnol 2018 Jul 5;102(14):6023-6038. Epub 2018 May 5.

Department of Civil & Environmental Engineering, University of Missouri, Columbia, MO, USA.

A novel whole-cell biosensor was developed to noninvasively and simultaneously monitor the in situ genetic activities of the four quorum sensing (QS) networks in Pseudomonas aeruginosa PAO1, including the las, rhl, pqs, and iqs systems. P. aeruginosa PAO1 is a model bacterium for studies of biofilm and pathogenesis while both processes are closely controlled by the QS systems. This biosensor worked well by selectively monitoring the expression of one representative gene from each network. In the biosensor, the promoter regions of lasI, rhlI, pqsA, and ambB (QS genes) controlled the fluorescent reporter genes of Turbo YFP, mTag BFP2, mNEON Green, and E2-Orange, respectively. The biosensor was successful in monitoring the impact of an important environmental factor, salt stress, on the genetic regulation of QS networks. High salt concentrations (≥ 20 g·L) significantly downregulated rhlI, pqsA, and ambB after the biosensor was incubated for 17 h to 18 h at 37 °C, resulting in slow bacterial growth.
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http://dx.doi.org/10.1007/s00253-018-9044-zDOI Listing
July 2018

Absence of the Min System Does Not Cause Major Cell Division Defects in .

Front Microbiol 2018 9;9:681. Epub 2018 Apr 9.

Division of Biological Sciences, University of Missouri, Columbia, MO, United States.

In , the essential FtsZ protein is located at the growth pole before shifting to the mid-cell right before division. Loss of FtsZ causes a halt in cell separation and lysis of cells. To understand how FtsZ polymerization is regulated to properly localize the FtsZ ring at the mid-cell, we have conducted a systematic characterization of the Min system in . Our findings indicate that the Min system is not required for cell survival. Yet, we find that the deletion of either or results in a broad cell size distribution, including an increase in the proportion of short and long cells. We observe that the site of constriction is misplaced in the or deletion strains allowing for short cells to arise from sites of constriction near the cell poles. Remarkably, the short cells are viable and contain DNA. In order to observe chromosome replication and segregation in these strains, YFP-ParB is used as a proxy to track the origin of replication as cells elongate and divide. In the absence of the Min proteins, duplication and segregation of the origin of replication is frequently delayed. Taken together, our data suggest that the Min system contributes to the proper regulation of FtsZ placement and subsequent cell division. Furthermore, the failure to precisely place FtsZ rings at mid-cell in the mutants impacts other cell cycle features including chromosome segregation.
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http://dx.doi.org/10.3389/fmicb.2018.00681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5900048PMC
April 2018

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth.

J Vis Exp 2017 11 24(129). Epub 2017 Nov 24.

Division of Biological Sciences, University of Missouri;

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.
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http://dx.doi.org/10.3791/56497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755469PMC
November 2017

Thermodynamic properties of an emerging chemical disinfectant, peracetic acid.

Sci Total Environ 2018 Apr 27;621:948-959. Epub 2017 Nov 27.

Department of Civil & Environmental Engineering, University of Missouri, Columbia, MO 65211, United States. Electronic address:

Peracetic acid (PAA or CHCOOOH) is an emerging disinfectant with a low potential to form carcinogenic disinfection by-products (DBPs). Basic thermodynamic properties of PAA are, however, absent or inconsistently reported in the literature. This review aimed to summarize important thermodynamic properties of PAA, including standard Gibbs energy of formation and oxidation-reduction (redox) potential. The standard Gibbs energies of formation of CHCOOOH, CHCOOOH, CHCOOOH, and CHCOOO are -299.41kJ·mol, -283.02kJ·mol, -276.10kJ·mol, and -252.60kJ·mol, respectively. The standard redox potentials of PAA are 1.748V and 1.005V vs. standard hydrogen electrode (SHE) at pH 0 and pH 14, respectively. Under biochemical standard state conditions (pH 7, 25°C, 101,325Pa), PAA has a redox potential of 1.385V vs. SHE, higher than many disinfectants. Finally, the environmental implications of the thermodynamic properties of PAA were systematically discussed. Those properties can be used to predict the physicochemical and biological behavior of aquatic systems exposed to PAA.
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http://dx.doi.org/10.1016/j.scitotenv.2017.10.195DOI Listing
April 2018

Expression of a Peptidoglycan Hydrolase from Lytic Bacteriophages Atu_ph02 and Atu_ph03 Triggers Lysis of Agrobacterium tumefaciens.

Appl Environ Microbiol 2017 Dec 16;83(23). Epub 2017 Nov 16.

Division of Biological Sciences, University of Missouri, Columbia, Missouri, USA

To provide food security, innovative approaches to preventing plant disease are currently being explored. Here, we demonstrate that lytic bacteriophages and phage lysis proteins are effective at triggering lysis of the phytopathogen Phages Atu_ph02 and Atu_ph03 were isolated from wastewater and induced lysis of C58-derived strains of The coinoculation of with phages on potato discs limited tumor formation. The genomes of Atu_ph02 and Atu_ph03 are nearly identical and are ∼42% identical to those of T7 supercluster phages. attempts to find a canonical lysis cassette were unsuccessful; however, we found a putative hage eptidoglycan ydrolase (PPH), which contains a C-terminal transmembrane domain. Remarkably, the endogenous expression of in the absence of additional phage genes causes a block in cell division and subsequent lysis of cells. When the presumed active site of the -acetylmuramidase domain carries an inactivating mutation, PPH expression causes extensive cell branching due to a block in cell division but does not trigger rapid cell lysis. In contrast, the mutation of positively charged residues at the extreme C terminus of PPH causes more rapid cell lysis. Together, these results suggest that PPH causes a block in cell division and triggers cell lysis through two distinct activities. Finally, the potent killing activity of this single lysis protein can be modulated, suggesting that it could be engineered to be an effective enzybiotic. The characterization of bacteriophages such as Atu_ph02 and Atu_ph03, which infect plant pathogens such as , may be the basis of new biocontrol strategies. First, cocktails of diverse bacteriophages could be used as a preventative measure to limit plant diseases caused by bacteria; a bacterial pathogen is unlikely to simultaneously develop resistances to multiple bacteriophage species. The specificity of bacteriophage treatment for the host is an asset in complex communities, such as in orchards where it would be detrimental to harm the symbiotic bacteria in the environment. Second, bacteriophages are potential sources of enzymes that efficiently lyse bacterial cells. These phage proteins may have a broad specificity, but since proteins do not replicate as phages do, their effect is highly localized, providing an alternative to traditional antibiotic treatments. Thus, studies of lytic bacteriophages that infect may provide insights for designing preventative strategies against bacterial pathogens.
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http://dx.doi.org/10.1128/AEM.01498-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691410PMC
December 2017

Targeting lysyl oxidase reduces peritoneal fibrosis.

PLoS One 2017 11;12(8):e0183013. Epub 2017 Aug 11.

MRC/University of Edinburgh Centre for Reproductive Health, Edinburgh Medical School, Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh, United Kingdom.

Background: Abdominal surgery and disease cause persistent abdominal adhesions, pelvic pain, infertility and occasionally, bowel obstruction. Current treatments are ineffective and the aetiology is unclear, although excessive collagen deposition is a consistent feature. Lysyl oxidase (Lox) is a key enzyme required for crosslinking and deposition of insoluble collagen, so we investigated whether targeting Lox might be an approach to reduce abdominal adhesions.

Methods: Female C57Bl/6 mice were treated intraperitoneally with multiwalled carbon nanotubes (NT) to induce fibrosis, together with chemical (ß-aminoproprionitrile-BAPN) or miRNA Lox inhibitors, progesterone or dexamethasone. Fibrotic lesions on the diaphragm, and expression of fibrosis-related genes in abdominal wall peritoneal mesothelial cells (PMC) were measured. Effects of BAPN and dexamethasone on collagen fibre alignment were observed by TEM. Isolated PMC were cultured with interleukin-1 alpha (IL-1α) and progesterone to determine effects on Lox mRNA in vitro.

Results: NT-induced fibrosis and collagen deposition on the diaphragm was ameliorated by BAPN, Lox miRNA, or steroids. BAPN and dexamethasone disrupted collagen fibres. NT increased PMC Lox, Col1a1, Col3a1 and Bmp1 mRNA, which was inhibited by steroids. Progesterone significantly inhibited IL-1α induced Lox expression by PMC in vitro.

Conclusion: Our results provide proof-of-concept that targeting peritoneal Lox could be an effective approach in ameliorating fibrosis and adhesion development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183013PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553776PMC
October 2017

Absence of the Polar Organizing Protein PopZ Results in Reduced and Asymmetric Cell Division in Agrobacterium tumefaciens.

J Bacteriol 2017 09 8;199(17). Epub 2017 Aug 8.

Division of Biological Sciences, University of Missouri, Columbia, Missouri, USA

is a rod-shaped bacterium that grows by polar insertion of new peptidoglycan during cell elongation. As the cell cycle progresses, peptidoglycan synthesis at the pole ceases prior to insertion of new peptidoglycan at midcell to enable cell division. The homolog of the polar organelle development protein PopZ has been identified as a growth pole marker and a candidate polar growth-promoting factor. Here, we characterize the function of PopZ in cell growth and division of Consistent with previous observations, we observe that PopZ localizes specifically to the growth pole in wild-type cells. Despite the striking localization pattern of PopZ, we find the absence of the protein does not impair polar elongation or cause major changes in the peptidoglycan composition. Instead, we observe an atypical cell length distribution, including minicells, elongated cells, and cells with ectopic poles. Most minicells lack DNA, suggesting a defect in chromosome segregation. Furthermore, the canonical cell division proteins FtsZ and FtsA are misplaced, leading to asymmetric sites of cell constriction. Together, these data suggest that PopZ plays an important role in the regulation of chromosome segregation and cell division. is a bacterial plant pathogen and a natural genetic engineer. However, very little is known about the spatial and temporal regulation of cell wall biogenesis that leads to polar growth in this bacterium. Understanding the molecular basis of growth may allow for the development of innovations to prevent disease or to promote growth during biotechnology applications. Finally, since many closely related plant and animal pathogens exhibit polar growth, discoveries in may be broadly applicable for devising antimicrobial strategies.
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http://dx.doi.org/10.1128/JB.00101-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553032PMC
September 2017

CDC's DELTA FOCUS Program: Identifying Promising Primary Prevention Strategies for Intimate Partner Violence.

J Womens Health (Larchmt) 2017 01;26(1):9-12

Centers for Disease Control and Prevention , Atlanta, Georgia .

According to 2011 data, nearly one in four women and one in seven men in the United States experience severe physical violence by an intimate partner, creating a public health burden requiring population-level solutions. To prevent intimate partner violence (IPV) before it occurs, the CDC developed Domestic Violence Prevention Enhancements and Leadership Through Alliances, Focusing on Outcomes for Communities United with States to identify promising community- and societal-level prevention strategies to prevent IPV. The program funds 10 state domestic violence coalitions for 5 years to implement and evaluate programs and policies to prevent IPV by influencing the environments and conditions in which people live, work, and play. The program evaluation goals are to promote IPV prevention by identifying promising prevention strategies and describing those strategies using case studies, thereby creating a foundation for building practice-based evidence with a health equity approach.
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http://dx.doi.org/10.1089/jwh.2016.6251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808986PMC
January 2017

Development of new polymyxin derivatives for multi-drug resistant Gram-negative infections.

J Antibiot (Tokyo) 2017 Apr 11;70(4):386-394. Epub 2017 Jan 11.

Cantab Anti-infectives Ltd., BioPark Hertfordshire, Welwyn Garden City, Hertfordshire, UK.

Over the last decade, there has been a resurgence of interest in polymyxins owing to the rapid rise in multi-drug resistant Gram-negative bacteria against which polymyxins offer a last-resort treatment. Although having excellent antibacterial activity, the clinical utility of polymyxins is limited by toxicity, especially renal toxicity. There is much interest therefore in developing polymyxin analogues with an improved therapeutic index. This review describes recent work aimed at improving the activity and/or reducing the toxicity of polymyxins. Consideration to providing activity against emerging strains with reduced susceptibility to polymyxins is also made.
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http://dx.doi.org/10.1038/ja.2016.146DOI Listing
April 2017

How can Primary Care Physicians Best Support Contraceptive Decision Making? A Qualitative Study Exploring the Perspectives of Baltimore Latinas.

Womens Health Issues 2017 Mar - Apr;27(2):158-166. Epub 2016 Nov 4.

Department of Family & Community Medicine, University of Maryland School of Medicine, Baltimore, Maryland.

Objectives: U.S. Latinas experience disproportionately high rates of unintended pregnancy and low rates of consistent contraception use. Not well known are Latinas' perspectives about how primary care physicians (PCPs) might facilitate or deter contraceptive decision making. The theory of planned behavior has been used previously to explain contraceptive behaviors. This study used the theory of planned behavior as a guide to help describe Latinas' perspectives regarding specific factors that influence their contraceptive decision making and to describe their perspectives about the role of PCPs in the decision making.

Study Design And Methods: We conducted focus groups (n = 3) and interviews (n = 8) of Latinas ages 15 to 24 years, recruited from urban primary care sites in Baltimore, Maryland. Concepts from the theory of planned behavior were used to develop a coding scheme and guide identification of themes.

Results: Sixteen Latinas participated; all were immigrants.

Themes: The desire to avoid unintended pregnancy is dominant and, not surprisingly, is the main driver of contraceptive intentions. The role of PCPs in contraceptive decision making is to build strong patient relationships through heightened communication and trust. PCPs should develop trust and foster communication by using a shared decision-making approach in contraceptive counseling. Religious norms rarely operate as barriers to contraceptive use, yet positive reinforcement from family, friends, and schools is viewed as supportive.

Conclusions And Implications: For this group of young, immigrant Latinas, there is a pervasive desire for effective communication and trusting relationships with PCPs. Findings suggest that providers can facilitate contraceptive decision making for this population by using a shared decision-making approach to contraceptive counseling.
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http://dx.doi.org/10.1016/j.whi.2016.09.015DOI Listing
December 2017

Building the bacterial cell wall at the pole.

Curr Opin Microbiol 2016 12 6;34:53-59. Epub 2016 Aug 6.

Division of Biological Sciences, 423 Tucker Hall, 612 Hitt St., University of Missouri, Columbia, MO 65211, USA. Electronic address:

Polar growth is the predominant mode of cell wall extension in the Actinobacteria and the alphaproteobacterial clade Rhizobiales. The observation of polar elongation in taxonomically diverse bacteria suggests that polar growth may have evolved independently. Indeed, the regulatory mechanisms governing the assembly of cell wall biosynthesis machinery at the pole are distinct in the Actinobacteria and Rhizobiales. Here we highlight recent advances in our understanding of polar growth mechanisms in bacteria, with an emphasis on Streptomyces and Agrobacterium. This review illustrates that common themes are emerging in the regulation of polar growth in diverse bacteria. Emerging themes include the use of landmark proteins to direct growth to the pole and coordination of polar growth with cell-cycle progression.
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http://dx.doi.org/10.1016/j.mib.2016.07.021DOI Listing
December 2016