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1.  Characterization of saltern based Streptomyces sp. and statistical media optimization for its improved antibacterial activity 
A moderately halotolerant Streptomyces strain, designated JAJ13 was characterized and a culture medium was statistically optimized to improve its antibacterial activity. Based on the phenotypic and molecular characteristics, strain JAJ13 was identified as a moderately halotolerant Streptomyces sp. JAJ13. Novelty of the strain JAJ13 in production of antibacterial compound was assessed by sequence analysis of KSα gene and LC-MS analysis of the active compound. Optimization of the culture medium for antibacterial compound production by the strain JAJ13 was performed with statistical methodology based on experimental designs. Initially, a starch based basal production medium was selected out of eight different production media screened for antibacterial compound production by Streptomyces sp. JAJ13. Plackett-Burman design was employed to screen the influential media components affecting the antibacterial compound production. Subsequently, statistical optimization of selected medium components was performed by employing the response surface methodology (RSM) with Box-Behnken design. The optimum initial level of CuSO4.5H2O, (NH4)2SO4 and K2HPO4 for the highest antibacterial activity was determined to be at 4.45 mg, 1.96 g, and 1.15 g in 1 L of distilled H2O, respectively. PBD and RSM guided design of experiments resulted in a maximum antibacterial activity of 23.37 ± 2.08 mm, which is a 78.8% increase in comparison with that obtained in the unoptimized medium. This study points the success of statistical model in developing an optimized production media for enhanced antibacterial compound production by Streptomyces sp. JAJ13.
PMCID: PMC4301002  PMID: 25653640
Plackett-Burman design; response surface methodology; Box-Behnken design; solar saltern; Streptomyces; antibiotics
2.  Epidemiology of nontuberculous mycobacteria among patients with cystic fibrosis in Scandinavia 
Journal of Cystic Fibrosis  2015;14(1):46-52.
Nontuberculous mycobacteria (NTM) are an emerging threat to cystic fibrosis (CF) patients but their epidemiology is not well described.
In this retrospective observational study we identified all Scandinavian CF patients with a positive NTM culture from airway secretions from 2000 to the end of 2012 and used national CF databases to describe microbiological and clinical characteristics.
During the 13-year period 157 (11%) CF patients were culture positive for NTM at least once. Mycobacterium abscessus complex (MABSC) (45%) and Mycobacterium avium complex (MAC) (32%) were the predominant species with geographical differences in distribution. Younger patients were more prone to MABSC (p < 0.01). Despite treatment, less than one-third of MABSC patients with repeated positive cultures cleared their infection and a quarter had a lung transplant or died.
NTM are significant CF pathogens and are becoming more prevalent in Scandinavia. MABSC and MAC appear to target distinct patient groups. Having multiple positive cultures despite treatment conveys a poor outcome.
PMCID: PMC4298356  PMID: 25178871
Mycobacterium abscessus; Mycobacterium avium; Prevalence; Susceptibility
3.  Characterization of Antibiotic Producing Rare Actinomycete Nonomuraea sp. JAJ18 Derived from an Indian Coastal Solar Saltern 
The Scientific World Journal  2014;2014:456070.
Rare actinomycete genera are accepted as a promising source of novel metabolites having pharmaceutical importance. One such genus of rare actinomycete is Nonomuraea. The present study was aimed at characterizing the antibiotic producing Nonomuraea strain JAJ18 which was previously isolated from coastal solar saltern. Strain JAJ18 was recognized as a member of genus Nonomuraea based on its almost complete 16S rRNA gene sequence and phenotypic characteristics. The strain JAJ18 was found to be closely related to Nonomuraea maheshkhaliensis 16-5-14T (98.90%), Nonomuraea candida HMC10T (98.58%), and Nonomuraea jabiensis A4036T (98.43%). From cell-free culture broth of strain JAJ18, an antibiotic was extracted and purified by silica column chromatography. The obtained antibiotic was found to be active against a range of Gram-positive and Gram-negative bacteria including drug-resistant Staphylococcus, with minimal inhibitory concentration (MIC) ranging from 0.5 to 16.0 µg mL−1. The structural characteristics of antibiotic were determined by FTIR and NMR spectroscopy. The antibiotic was identified to be an aliphatic rich compound with significant dissimilarity to known antibiotics reported from members of the genus, Nonomuraea. As the trends to discover novel metabolites from Nonomuraea are vibrant, further studies are needed to understand the structural and biotechnological significance of antibiotic compound produced by Nonomuraea sp. JAJ18.
PMCID: PMC4281464  PMID: 25587565
4.  Smorgasbord or symphony? Assessing public health nutrition policies across 30 European countries using a novel framework 
BMC Public Health  2014;14(1):1195.
Countries across Europe have introduced a wide variety of policies to improve nutrition. However, the sheer diversity of interventions represents a potentially bewildering smorgasbord.
We aimed to map existing public health nutrition policies, and examine their perceived effectiveness, in order to inform future evidence-based diet strategies.
We created a public health nutrition policy database for 30 European countries . National nutrition policies were classified and assigned using the marketing "4Ps" approach Product (reformulation, elimination, new healthier products); Price (taxes, subsidies); Promotion (advertising, food labelling, health education) and Place (schools, workplaces, etc.).
We interviewed 71 senior policy-makers, public health nutrition policy experts and academics from 14 of the 30 countries, eliciting their views on diverse current and possible nutrition strategies.
Product Voluntary reformulation of foods is widespread but has variable and often modest impact. Twelve countries regulate maximum salt content in specific foods.
Denmark, Austria, Iceland and Switzerland have effective trans fats bans.
Price EU School Fruit Scheme subsidies are almost universal, but with variable implementation.
Taxes are uncommon. However, Finland, France, Hungary and Latvia have implemented ‘sugar taxes’ on sugary foods and sugar-sweetened beverages. Finland, Hungary and Portugal also tax salty products.
Promotion Dialogue, recommendations, nutrition guidelines, labelling, information and education campaigns are widespread. Restrictions on marketing to children are widespread but mostly voluntary.
Place Interventions reducing the availability of unhealthy foods were most commonly found in schools and workplace canteens.
Interviewees generally considered mandatory reformulation more effective than voluntary, and regulation and fiscal interventions much more effective than information strategies, but also politically more challenging.
Public health nutrition policies in Europe appear diverse, dynamic, complex and bewildering. The "4Ps" framework potentially offers a structured and comprehensive categorisation.
Encouragingly, the majority of European countries are engaged in activities intended to increase consumption of healthy food and decrease the intake of "junk" food and sugary drinks. Leading countries include Finland, Norway, Iceland, Denmark, Hungary, Portugal and perhaps the UK. However, all countries fall short of optimal activities. More needs to be done across Europe to implement the most potentially powerful fiscal and regulatory nutrition policies.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2458-14-1195) contains supplementary material, which is available to authorized users.
PMCID: PMC4251675  PMID: 25413832
Public health nutrition; Public health policy; Europe; Food policy mapping; Qualitative
5.  Fathering of Dizygotic Twins and Risk of Prostate Cancer: Nationwide, Population-Based Case-Control Study 
PLoS ONE  2014;9(10):e110506.
An association between male fertility and risk of prostate cancer has been suggested, possibly through lower androgen levels in subfertile men. We evaluated male fertility in relation to risk of prostate cancer by assessing the frequency of fathering of dizygotic twins, a marker of high fertility, among cases of prostate cancer and controls.
We performed a case-control study in Prostate Cancer data Base Sweden (PCBaSe), a nationwide, population-based cohort. PCBaSe was linked to the Swedish twin register for information on zygosity for same-sex twins and to other nationwide health care registers and demographic databases for information on socioeconomic factors, comorbidity, and tumor characteristics for 96 301 prostate cancer cases and 378 583 matched controls. To account for the influence of in vitro fertilization on dizygotic twinning, analyses were restricted to men who had fathered children before 1991, when in vitro fertilization was still uncommon in Sweden.
1 112 cases and 4 538 controls had fathered dizygotic twins. Men with dizygotic twins had no increased risk of prostate cancer compared to fathers of singletons; neither for total prostate cancer odds ratio (OR) 0.95(95% CI 0.89–1.02), nor for any risk category, OR 0.97 (95% CI 0.84–1.12) for low-risk disease, and OR 1.04 (95% CI 0.90–1.22) for metastatic disease.
The lack of association between fathering of dizygotic twins and prostate cancer risk give no support for an association between male fertility and prostate cancer risk.
PMCID: PMC4206421  PMID: 25337702
6.  Comprehensive geriatric assessment for older adults admitted to hospital 
Comprehensive geriatric assessment (CGA) is a multidimensional, interdisciplinary diagnostic process to determine the medical, psychological and functional capabilities of a frail elderly person in order to develop a co-ordinated and integrated plan for treatment and long-term follow up.
We sought to evaluate the effectiveness of CGA in hospital for older adults admitted as an emergency.
Search methods
We searched the Cochrane Effective Practice and Organisation of Care (EPOC) Group Register, the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library), the Database of Abstracts of Reviews of Effects (DARE), MEDLINE, EMBASE, CINAHL and AARP Ageline, and handsearched high-yield journals.
Selection criteria
We searched for randomised controlled trials comparing CGA (whether by mobile teams or in designated wards) to usual care.
Data collection and analysis
Two review authors initially assessed eligibility and trial quality and extracted published data.
Main results
Twenty-two trials evaluating 10,315 participants in six countries were identified. Patients in receipt of CGA were more likely to be alive and in their own homes at up to six months (OR 1.25, 95% CI 1.11 to 1.42, P = 0.0002) and at the end of scheduled follow up (median 12 months) (OR 1.16, 95% CI 1.05 to 1.28, P = 0.003) when compared to general medical care. In addition, patients were less likely to be institutionalised (OR 0.79, 95% CI 0.69 to 0.88, P < 0.0001). They were less likely to suffer death or deterioration (OR 0.76, 95% CI 0.64 to 0.90, P = 0.001), and were more likely to experience improved cognition in the CGA group (OR 1.11, 95% CI 0.20 to 2.01, P = 0.02). Subgroup interaction in the primary outcomes suggests that the effects of CGA are primarily the result of CGA wards.
Authors’ conclusions
Comprehensive geriatric assessment increases a patient’s likelihood of being alive and in their own home at up to 12 months.
PMCID: PMC4164377  PMID: 21735403
*Frail Elderly; *Hospitalization; *Outcome and Process Assessment (Health Care); Comprehensive Health Care [*methods]; Emergencies; Geriatric Assessment [*methods]; Independent Living; Mortality; Aged; Humans
7.  Successive Nonstatistical and Statistical Approaches for the Improved Antibiotic Activity of Rare Actinomycete Nonomuraea sp. JAJ18 
BioMed Research International  2014;2014:906097.
The selection and optimization of nutritional constituents as well as their levels for the improved production of antibiotic by Nonomuraea sp. JAJ18 were carried out using combination of both nonstatistical one-factor-at-a-time (OFAT) method and statistical response surface methodology (RSM). Using OFAT method, starch and (NH4)2SO4 were identified as suitable carbon and nitrogen sources, respectively. Subsequently, starch, NaCl, and MgSO4·7H2O were recognized as the most significant media components with confidence level of above 95% using the Plackett-Burman design. The levels of the three media components were further optimized using RSM employed with Box-Behnken design. Accordingly, a second-order polynomial regression model was fitted into the experimental data. By analyzing the response surface plots as well as using numerical optimization method, the optimal levels for starch, NaCl, and MgSO4·7H2O were determined as 15.6 g/L, 0.8 g/L, and 1.98 g/L, respectively. With the optimized medium, 15.5% increase was observed in antibiotic activity of JAJ18. Results further support the use of RSM for media optimization. To the best of our knowledge, this is the first report of statistical media optimization for antibiotic production in rare actinomycete Nonomuraea species, which will be useful for the development of Nonomuraea cultivation process for efficient antibiotic production on a large scale.
PMCID: PMC4168032  PMID: 25276828
8.  Prevalence of Anti-Peptidylarginine Deiminase Type 4 Antibodies in Rheumatoid Arthritis and Unaffected First-Degree Relatives in Indigenous North American Populations 
The Journal of rheumatology  2013;40(9):1523-1528.
The objective of this study was to determine whether anti-peptidylarginine deiminase type 4 (PAD4) antibodies were present in first-degree relatives of rheumatoid arthritis (RA) patients in two indigenous North American populations with high prevalence of RA.
Participants were recruited from two indigenous populations in Canada and the United States, including RA patients (probands), their unaffected first-degree relatives, and healthy unrelated controls. Sera were tested for the presence of anti-PAD4 antibodies, anti-cyclic citrullinated peptide (CCP) antibodies, and rheumatoid factor (RF). HLA-DRB1 subtyping was performed and participants were classified according to number of shared epitope alleles present.
Antibodies to PAD4 were detected in 24 of 82 (29.3%) probands; 2 of 147 (1.4%) relatives; and no controls (p <0.0001). Anti-CCP was present in 39/144 (27.1%) of the relatives, and there was no overlap between positivity for anti-CCP and PAD4 in the relatives. In RA patients, anti-PAD4 antibodies were associated with disease duration (p=0.0082) and anti-CCP antibodies (p=0.008), but not smoking or shared epitope alleles.
Despite a significant prevalence of anti-CCP in first-degree relatives, anti-PAD4 antibodies were almost exclusively found in established RA. The prevalence of anti-PAD4 antibodies in RA is similar to the prevalence described in other populations and these autoantibodies are associated with disease duration and anti-CCP in RA.
PMCID: PMC3969032  PMID: 23908443
Arthritis; Rheumatoid; Autoantibodies; peptidylarginine deiminase
9.  Impact of the 2008 Economic and Financial Crisis on Child Health: A Systematic Review 
The aim of this study was to provide an overview of studies in which the impact of the 2008 economic crisis on child health was reported. Structured searches of PubMed, and ISI Web of Knowledge, were conducted. Quantitative and qualitative studies reporting health outcomes on children, published since 2007 and related to the 2008 economic crisis were included. Two reviewers independently assessed studies for inclusion. Data were synthesised as a narrative review. Five hundred and six titles and abstracts were reviewed, from which 22 studies were included. The risk of bias for quantitative studies was mixed while qualitative studies showed low risk of bias. An excess of 28,000–50,000 infant deaths in 2009 was estimated in sub-Saharan African countries, and increased infant mortality in Greece was reported. Increased price of foods was related to worsening nutrition habits in disadvantaged families worldwide. An increase in violence against children was reported in the U.S., and inequalities in health-related quality of life appeared in some countries. Most studies suggest that the economic crisis has harmed children’s health, and disproportionately affected the most vulnerable groups. There is an urgent need for further studies to monitor the child health effects of the global recession and to inform appropriate public policy responses.
PMCID: PMC4078594  PMID: 25019121
adolescent; child health; economic and financial crisis; inequalities
10.  Implementation of Ultrasonic Sensing for High Resolution Measurement of Binary Gas Mixture Fractions 
Sensors (Basel, Switzerland)  2014;14(6):11260-11276.
We describe an ultrasonic instrument for continuous real-time analysis of the fractional mixture of a binary gas system. The instrument is particularly well suited to measurement of leaks of a high molecular weight gas into a system that is nominally composed of a single gas. Sensitivity < 5 × 10−5 is demonstrated to leaks of octaflouropropane (C3F8) coolant into nitrogen during a long duration (18 month) continuous study. The sensitivity of the described measurement system is shown to depend on the difference in molecular masses of the two gases in the mixture. The impact of temperature and pressure variances on the accuracy of the measurement is analysed. Practical considerations for the implementation and deployment of long term, in situ ultrasonic leak detection systems are also described. Although development of the described systems was motivated by the requirements of an evaporative fluorocarbon cooling system, the instrument is applicable to the detection of leaks of many other gases and to processes requiring continuous knowledge of particular binary gas mixture fractions.
PMCID: PMC4118389  PMID: 24961217
ultrasonic; binary gas analysis; leak detection
11.  Unexplored hypersaline habitats are sources of novel actinomycetes 
PMCID: PMC4034035  PMID: 24904555
actinomycetes; hypersaline environments; antibiotic; Streptomyces; drug discovery
12.  Predicting outcomes after blunt chest wall trauma: development and external validation of a new prognostic model 
Critical Care  2014;18(3):R98.
Blunt chest wall trauma accounts for over 15% of all trauma admissions to Emergency Departments worldwide. Reported mortality rates vary between 4 and 60%. Management of this patient group is challenging as a result of the delayed on-set of complications. The aim of this study was to develop and validate a prognostic model that can be used to assist in the management of blunt chest wall trauma.
There were two distinct phases to the overall study; the development and the validation phases. In the first study phase, the prognostic model was developed through the retrospective analysis of all blunt chest wall trauma patients (n = 274) presenting to the Emergency Department of a regional trauma centre in Wales (2009 to 2011). Multivariable logistic regression was used to develop the model and identify the significant predictors for the development of complications. The model’s accuracy and predictive capabilities were assessed. In the second study phase, external validation of the model was completed in a multi-centre prospective study (n = 237) in 2012. The model’s accuracy and predictive capabilities were re-assessed for the validation sample. A risk score was developed for use in the clinical setting.
Significant predictors of the development of complications were age, number of rib fractures, chronic lung disease, use of pre-injury anticoagulants and oxygen saturation levels. The final model demonstrated an excellent c-index of 0.96 (95% confidence intervals: 0.93 to 0.98).
In our two phase study, we have developed and validated a prognostic model that can be used to assist in the management of blunt chest wall trauma patients. The final risk score provides the clinician with the probability of the development of complications for each individual patient.
PMCID: PMC4095687  PMID: 24887537
13.  Exo70E2 is essential for exocyst subunit recruitment and EXPO formation in both plants and animals 
Molecular Biology of the Cell  2014;25(3):412-426.
Exocyst-positive organelle (EXPO) is a double-membrane organelle mediating unconventional protein secretion in plants. The Arabidopsis exocyst subunit AtExo70E2 is essential for the recruitment of other exocyst subunits to EXPO and plays a key role in EXPO formation in both plant and animal cells.
In contrast to a single copy of Exo70 in yeast and mammals, the Arabidopsis genome contains 23 paralogues of Exo70 (AtExo70). Using AtExo70E2 and its GFP fusion as probes, we recently identified a novel double-membrane organelle termed exocyst-positive organelle (EXPO) that mediates an unconventional protein secretion in plant cells. Here we further demonstrate that AtExo70E2 is essential for exocyst subunit recruitment and for EXPO formation in both plants and animals. By performing transient expression in Arabidopsis protoplasts, we established that a number of exocyst subunits (especially the members of the Sec family) are unable to be recruited to EXPO in the absence of AtExo70E2. The paralogue AtExo70A1 is unable to substitute for AtExo70E2 in this regard. Fluorescence resonance energy transfer assay and bimolecular fluorescence complementation analyses confirm the interaction between AtExo70E2 and Sec6 and Sec10. AtExo70E2, but not its yeast counterpart, is also capable of inducing EXPO formation in an animal cell line (HEK293A cells). Electron microscopy confirms the presence of double-membraned, EXPO-like structures in HEK293A cells expressing AtExo70E2. Inversely, neither human nor yeast Exo70 homologues cause the formation of EXPO in Arabidopsis protoplasts. These results point to a specific and crucial role for AtExo70E2 in EXPO formation.
PMCID: PMC3907280  PMID: 24307681
14.  The histone chaperones Vps75 and Nap1 form ring-like, tetrameric structures in solution 
Nucleic Acids Research  2014;42(9):6038-6051.
NAP-1 fold histone chaperones play an important role in escorting histones to and from sites of nucleosome assembly and disassembly. The two NAP-1 fold histone chaperones in budding yeast, Vps75 and Nap1, have previously been crystalized in a characteristic homodimeric conformation. In this study, a combination of small angle X-ray scattering, multi angle light scattering and pulsed electron–electron double resonance approaches were used to show that both Vps75 and Nap1 adopt ring-shaped tetrameric conformations in solution. This suggests that the formation of homotetramers is a common feature of NAP-1 fold histone chaperones. The tetramerisation of NAP-1 fold histone chaperones may act to shield acidic surfaces in the absence of histone cargo thus providing a ‘self-chaperoning’ type mechanism.
PMCID: PMC4027167  PMID: 24688059
15.  AZD3514: a small molecule that modulates androgen receptor signaling and function in vitro and in vivo 
Molecular cancer therapeutics  2013;12(9):1715-1727.
Continued androgen receptor (AR) expression and signaling is a key driver in castration resistant prostate cancer (CRPC) after classical androgen ablation therapies have failed, and therefore remains a target for the treatment of progressive disease. Here we describe the biological characterization of AZD3514, an orally bioavailable drug that inhibits androgen-dependent and–independent AR signaling. AZD3514 modulates AR signaling through two distinct mechanisms, an inhibition of ligand driven nuclear translocation of AR and a down-regulation of receptor levels, both of which were observed in vitro and in vivo. AZD3514 inhibited testosterone-driven seminal vesicle development in juvenile male rats and the growth of androgen-dependent Dunning R3327H prostate tumors in adult rats. Furthermore, this class of compound demonstrated anti-tumor activity in the HID28 mouse model of CRPC in vivo. AZD3514 is currently in Phase I clinical evaluation.
PMCID: PMC3769207  PMID: 23861347
16.  Phosphorylation of Sli15 by Ipl1 Is Important for Proper CPC Localization and Chromosome Stability in Saccharomyces cerevisiae 
PLoS ONE  2014;9(2):e89399.
The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A) or to acidic residues to mimic constitutive phosphorylation (sli15-20D). Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the ‘tension checkpoint’ – the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism.
PMCID: PMC3928436  PMID: 24558497
17.  Expression of Vesicular Glutamate Transporters Type 1 and 2 in Sensory and Autonomic Neurons Innervating the Mouse Colorectum 
The Journal of comparative neurology  2011;519(16):3346-3366.
Vesicular glutamate transporters (VGLUTs) have been extensively studied in various neuronal systems, but their expression in visceral sensory and autonomic neurons remains to be analyzed in detail. Here we studied VGLUTs type 1 and 2 (VGLUT1 and VGLUT2, respectively) in neurons innervating the mouse colorectum. Lumbosacral and thoracolumbar dorsal root ganglion (DRG), lumbar sympathetic chain (LSC), and major pelvic ganglion (MPG) neurons innervating the colorectum of BALB/C mice were retrogradely traced with Fast Blue, dissected, and processed for immunohistochemistry. Tissue from additional naïve mice was included. Previously characterized antibodies against VGLUT1, VGLUT2, and calcitonin gene-related peptide (CGRP) were used. Riboprobe in situ hybridization, using probes against VGLUT1 and VGLUT2, was also performed. Most colorectal DRG neurons expressed VGLUT2 and often colocalized with CGRP. A smaller percentage of neurons expressed VGLUT1. VGLUT2-immunoreactive (IR) neurons in the MPG were rare. Abundant VGLUT2-IR nerves were detected in all layers of the colorectum; VGLUT1-IR nerves were sparse. A subpopulation of myenteric plexus neurons expressed VGLUT2 protein and mRNA, but VGLUT1 mRNA was undetectable. In conclusion, we show 1) that most colorectal DRG neurons express VGLUT2, and to a lesser extent, VGLUT1; 2) abundance of VGLUT2-IR fibers innervating colorectum; and 3) a subpopulation of myenteric plexus neurons expressing VGLUT2. Altogether, our data suggests a role for VGLUT2 in colorectal glutamatergic neurotransmission, potentially influencing colorectal sensitivity and motility.
PMCID: PMC3919020  PMID: 21800314
colon; DRG; glutamate; neuropeptides; pain; pelvic nerve
19.  Megalencephaly Syndromes: Exome Pipeline Strategies for Detecting Low-Level Mosaic Mutations 
PLoS ONE  2014;9(1):e86940.
Two megalencephaly (MEG) syndromes, megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyriapolydactyly-hydrocephalus (MPPH), have recently been defined on the basis of physical and neuroimaging features. Subsequently, exome sequencing of ten MEG cases identified de-novo postzygotic mutations in PIK3CA which cause MCAP and de-novo mutations in AKT and PIK3R2 which cause MPPH. Here we present findings from exome sequencing three unrelated megalencephaly patients which identified a causal PIK3CA mutation in two cases and a causal PIK3R2 mutation in the third case. However, our patient with the PIK3R2 mutation which is considered to cause MPPH has a marked bifrontal band heterotopia which is a feature of MCAP. Furthermore, one of our patients with a PIK3CA mutation lacks syndactyly/polydactyly which is a characteristic of MCAP. These findings suggest that the overlap between MCAP and MPPH may be greater than the available studies suggest. In addition, the PIK3CA mutation in one of our patients could not be detected using standard exome analysis because the mutation was observed at a low frequency consistent with somatic mosaicism. We have therefore investigated several alternative methods of exome analysis and demonstrate that alteration of the initial allele frequency spectrum (AFS), used as a prior for variant calling in samtools, had the greatest power to detect variants with low mutant allele frequencies in our 3 MEG exomes and in simulated data. We therefore recommend non-default settings of the AFS in combination with stringent quality control when searching for causal mutation(s) that could have low levels of mutant reads due to post-zygotic mutation.
PMCID: PMC3908952  PMID: 24497998
20.  Bisubstrate UDP–peptide conjugates as human O-GlcNAc transferase inhibitors 
Biochemical Journal  2014;457(Pt 3):497-502.
Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, eliminating the GlcNAc pyranoside ring. Goblin1 co-crystallizes with OGT, revealing an ordered C3 linker and retained substrate-binding modes, and binds the enzyme with micromolar affinity, inhibiting glycosyltransfer on to protein and peptide substrates.
Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, inhibiting glycosyltransfer on to protein and peptide substrates.
PMCID: PMC3927924  PMID: 24256146
bisubstrate analogue inhibitor; glycosyltransferase; O-GlcNAc; rational drug design; DIPEA, N,N-di-isopropylethylamine; DMF, dimethylformamide; goblin, OGT bisubstrate-linked inhibitor; h, human; HRMS, high-resolution MS; MP, p-methoxyphenyl; OGA, O-GlcNAc hydrolase; OGT, O-GlcNAc:polypeptidyl transferase; TAB1, TGF (transforming growth factor)-β-activated kinase-binding protein 1
21.  Formulation and Statistical Optimization of Culture Medium for Improved Production of Antimicrobial Compound by Streptomyces sp. JAJ06 
Streptomyces sp. JAJ06 is a seawater-dependent antibiotic producer, previously isolated and characterised from an Indian coastal solar saltern. This paper reports replacement of seawater with a defined salt formulation in production medium and subsequent statistical media optimization to ensure consistent as well as improved antibiotic production by Streptomyces sp. JAJ06. This strain was observed to be proficient to produce antibiotic compound with incorporation of chemically defined sodium-chloride-based salt formulation instead of seawater into the production medium. Plackett-Burman design experiment was applied, and three media constituents, starch, KBr, and CaCO3, were recognised to have significant effect on the antibiotic production of Streptomyces JAJ06 at their individual levels. Subsequently, Response surface methodology with Box-Behnken design was employed to optimize these influencing medium constituents for the improved antibiotic production of Streptomyces sp. JAJ06. A total of 17 experiments were conducted towards the construction of a quadratic model and a second-order polynomial equation. Optimum levels of medium constituents were obtained by analysis of the model and numerical optimization method. When the strain JAJ06 was cultivated in the optimized medium, the antibiotic activity was increased to 173.3 U/mL, 26.8% increase as compared to the original (136.7 U/mL). This study found a useful way to cultivate Streptomyces sp. JAJ06 for enhanced production of antibiotic compound.
PMCID: PMC3885193  PMID: 24454383
22.  Atrazine biodegradation efficiency, metabolite detection, and trzD gene expression by enrichment bacterial cultures from agricultural soil 
Atrazine is a selective herbicide used in agricultural fields to control the emergence of broadleaf and grassy weeds. The persistence of this herbicide is influenced by the metabolic action of habituated native microorganisms. This study provides information on the occurrence of atrazine mineralizing bacterial strains with faster metabolizing ability. The enrichment cultures were tested for the biodegradation of atrazine by high-performance liquid chromatography (HPLC) and mass spectrometry. Nine cultures JS01.Deg01 to JS09.Deg01 were identified as the degrader of atrazine in the enrichment culture. The three isolates JS04.Deg01, JS07.Deg01, and JS08.Deg01 were identified as efficient atrazine metabolizers. Isolates JS04.Deg01 and JS07.Deg01 produced hydroxyatrazine (HA) N-isopropylammelide and cyanuric acid by dealkylation reaction. The isolate JS08.Deg01 generated deethylatrazine (DEA), deisopropylatrazine (DIA), and cyanuric acid by N-dealkylation in the upper degradation pathway and later it incorporated cyanuric acid in their biomass by the lower degradation pathway. The optimum pH for degrading atrazine by JS08.Deg01 was 7.0 and 16S rDNA phylogenetic typing identified it as Enterobacter cloacae strain JS08.Deg01. The highest atrazine mineralization was observed in case of isolate JS08.Deg01, where an ample amount of trzD mRNA was quantified at 72 h of incubation with atrazine. Atrazine bioremediating isolate E. cloacae strain JS08.Deg01 could be the better environmental remediator of agricultural soils and the crop fields contaminated with atrazine could be the source of the efficient biodegrading microbial strains for the environmental cleanup process.
PMCID: PMC3863374  PMID: 24302716
Atrazine; Biodegradation; Enterobacter cloacae; Deethylatrazine; Cyanuric acid; trzD gene
23.  Design and Analysis of Bar-seq Experiments 
G3: Genes|Genomes|Genetics  2013;4(1):11-18.
High-throughput quantitative DNA sequencing enables the parallel phenotyping of pools of thousands of mutants. However, the appropriate analytical methods and experimental design that maximize the efficiency of these methods while maintaining statistical power are currently unknown. Here, we have used Bar-seq analysis of the Saccharomyces cerevisiae yeast deletion library to systematically test the effect of experimental design parameters and sequence read depth on experimental results. We present computational methods that efficiently and accurately estimate effect sizes and their statistical significance by adapting existing methods for RNA-seq analysis. Using simulated variation of experimental designs, we found that biological replicates are critical for statistical analysis of Bar-seq data, whereas technical replicates are of less value. By subsampling sequence reads, we found that when using four-fold biological replication, 6 million reads per condition achieved 96% power to detect a two-fold change (or more) at a 5% false discovery rate. Our guidelines for experimental design and computational analysis enables the study of the yeast deletion collection in up to 30 different conditions in a single sequencing lane. These findings are relevant to a variety of pooled genetic screening methods that use high-throughput quantitative DNA sequencing, including Tn-seq.
PMCID: PMC3887526  PMID: 24192834
yeast; Bar-seq; galactose; functional genomics; Sacchromyces cerevisiae
24.  Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis 
Open Biology  2013;3(9):130022.
The fungal cell possesses an essential carbohydrate cell wall. The outer layer, mannan, is formed by mannoproteins carrying highly mannosylated O- and N-linked glycans. Yeast mannan biosynthesis is initiated by a Golgi-located complex (M-Pol I) of two GT-62 mannosyltransferases, Mnn9p and Van1p, that are conserved in fungal pathogens. Saccharomyces cerevisiae and Candida albicans mnn9 knockouts show an aberrant cell wall and increased antibiotic sensitivity, suggesting the enzyme is a potential drug target. Here, we present the structure of ScMnn9 in complex with GDP and Mn2+, defining the fold and catalytic machinery of the GT-62 family. Compared with distantly related GT-78/GT-15 enzymes, ScMnn9 carries an unusual extension. Using a novel enzyme assay and site-directed mutagenesis, we identify conserved amino acids essential for ScMnn9 ‘priming’ α-1,6-mannosyltransferase activity. Strikingly, both the presence of the ScMnn9 protein and its product, but not ScMnn9 catalytic activity, are required to activate subsequent ScVan1 processive α-1,6-mannosyltransferase activity in the M-Pol I complex. These results reveal the molecular basis of mannan synthesis and will aid development of inhibitors targeting this process.
PMCID: PMC3787745  PMID: 24026536
cell wall; glycobiology; glycosyltransferase; mannan; M-Pol I; protein crystallography
25.  A Longitudinal Study of the Impact of Social Deprivation and Disease Severity on Employment Status in the UK Cystic Fibrosis Population 
PLoS ONE  2013;8(8):e73322.
People with Cystic Fibrosis (CF) in the UK and elsewhere are increasingly surviving into adulthood, yet there is little research on the employment consequences of having CF. We investigated, for the first time in a UK-wide cohort, longitudinal employment status, and its association with deprivation, disease severity, and time in hospital.
We did a longitudinal registry study of adults with CF in the UK aged 20 to 40 (3458 people with 15,572 observations between 1996 and 2010), using mixed effects models.
Around 50% of adults with CF were in employment. Male sex, higher lung function and body mass index, and less time in hospital were associated with improved employment chances. All other things being equal, being in the most deprived quintile was associated with a reduction of employment prevalence of 17.6 percentage points compared to the prevalence in the least deprived quintile. Having poor lung function was associated with a reduced employment prevalence of 7.2 percentage points compared to the prevalence for people with relatively good lung function. Acting synergistically, deprivation modifies the effect of lung function on employment chances – poor lung function in the least deprived group was associated with a 3 percentage point reduction in employment chances, while poor lung function in the most deprived quintile was associated with a 7.7 point reduction in employment chances.
Greater deprivation, disease severity, and time in hospital are all associated with employment chances in adults with CF. Furthermore, our analysis suggests that deprivation amplifies the harmful association of disease severity on employment. Future studies should focus on understanding and mitigating the barriers to employment faced by people with CF.
PMCID: PMC3751887  PMID: 24009747

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