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1.  Regulation of transcription by eukaryotic-like serine-threonine kinases and phosphatases in Gram-positive bacterial pathogens  
Virulence  2015;5(8):863-885.
Bacterial eukaryotic-like serine threonine kinases (eSTKs) and serine threonine phosphatases (eSTPs) have emerged as important signaling elements that are indispensable for pathogenesis. Differing considerably from their histidine kinase counterparts, few eSTK genes are encoded within the average bacterial genome, and their targets are pleiotropic in nature instead of exclusive. The growing list of important eSTK/P substrates includes proteins involved in translation, cell division, peptidoglycan synthesis, antibiotic tolerance, resistance to innate immunity and control of virulence factors. Recently it has come to light that eSTK/Ps also directly modulate transcriptional machinery in many microbial pathogens. This novel form of regulation is now emerging as an additional means by which bacteria can alter their transcriptomes in response to host-specific environmental stimuli. Here we focus on the ability of eSTKs and eSTPs in Gram-positive bacterial pathogens to directly modulate transcription, the known mechanistic outcomes of these modifications, and their roles as an added layer of complexity in controlling targeted RNA synthesis to enhance virulence potential.
PMCID: PMC4601284  PMID: 25603430
bacteria; infection; phosphorylation; serine threonine kinase; serine threonine phosphatase; transcription
2.  Viewing Instructions Accompanying Action Observation Modulate Corticospinal Excitability 
Action observation interventions may have the potential to contribute to improved motor function in motor (re)learning settings by promoting functional activity and plasticity in the motor regions of the brain. Optimal methods for delivering such interventions, however, have yet to be established. This experiment investigated the effect on corticospinal excitability of manipulating the viewing instructions provided to participants (N = 21) prior to action observation. Specifically, motor evoked potential responses measured from the right hand muscles following single-pulse transcranial magnetic stimulation (TMS) to the left motor cortex were compared when participants were instructed to observe finger-thumb opposition movement sequences: (i) passively; (ii) with the intent to imitate the observed movement; or (iii) whilst simultaneously and actively imagining that they were performing the movement as they observed it. All three action observation viewing instructions facilitated corticospinal excitability to a greater extent than did observation of a static hand. In addition, the extent to which corticospinal excitability was facilitated was greater during combined observation and imagery, compared to passive observation. These findings have important implications for the design of action observation interventions in motor (re)learning settings, where instructions that encourage observers to simultaneously imagine themselves performing the observed movement may offer the current optimal method for improving motor function through action observation.
PMCID: PMC4740958  PMID: 26869901
combined observation and imagery; action observation; movement imagery; transcranial magnetic stimulation; motor (re)learning
3.  Progesterone treatment reduces neuroinflammation, oxidative stress and brain damage and improves long-term outcomes in a rat model of repeated mild traumatic brain injury 
Repeated mild traumatic brain injuries, such as concussions, may result in cumulative brain damage, neurodegeneration and other chronic neurological impairments. There are currently no clinically available treatment options known to prevent these consequences. However, growing evidence implicates neuroinflammation and oxidative stress in the pathogenesis of repetitive mild brain injuries; thus, these may represent potential therapeutic targets. Progesterone has been demonstrated to have potent anti-inflammatory and anti-oxidant properties after brain insult; therefore, here, we examined progesterone treatment in rats given repetitive mild brain injuries via the repeated mild fluid percussion injury model.
Male Long-Evans rats were assigned into four groups: sham injury + vehicle treatment, sham injury + progesterone treatment (8 mg/kg/day), repeated mild fluid percussion injuries + vehicle treatment, and repeated mild fluid percussion injuries + progesterone treatment. Rats were administered a total of three injuries, with each injury separated by 5 days. Treatment was initiated 1 h after the first injury, then administered daily for a total of 15 days. Rats underwent behavioural testing at 12-weeks post-treatment to assess cognition, motor function, anxiety and depression. Brains were then dissected for analysis of markers for neuroinflammation and oxidative stress. Ex vivo MRI was conducted in order to examine structural brain damage and white matter integrity.
Repeated mild fluid percussion injuries + progesterone treatment rats showed significantly reduced cognitive and sensorimotor deficits compared to their vehicle-treated counterparts at 12-weeks post-treatment. Progesterone treatment significantly attenuated markers of neuroinflammation and oxidative stress in rats given repeated mild fluid percussion injuries, with concomitant reductions in grey and white matter damage as indicated by MRI.
These findings implicate neuroinflammation and oxidative stress in the pathophysiological aftermath of mild brain injuries and suggest that progesterone may be a viable treatment option to mitigate these effects and their detrimental consequences.
PMCID: PMC4683966  PMID: 26683475
Concussion; Chronic traumatic encephalopathy; Animal model; DTI; MRI; Treatment; Microglia; Macrophages; Astrogliosis; Lipid peroxidation
4.  The four or more medicines (FOMM) support service: results from an evaluation of a new community pharmacy service aimed at over-65s 
Inappropriate prescribing and nonadherence have a significant impact on hospital admissions and patient quality of life. The English government has identified that community pharmacy could make a significant contribution to reducing nonadherence and improving the quality of prescribing, reducing both hospital admissions and medicines wastage. The objective of this study is to evaluate a community pharmacy service aimed at patients over the age of 65 years prescribed four or more medicines.
Patients were invited to participate in the service by the community pharmacy team. The pharmacist held regular consultations with the patient and discussed risk of falls, pain management, adherence and general health. They also reviewed the patient’s medication using STOPP/START criteria. Data were analysed for the first 6 months of participation in the service.
Key findings
Six hundred twenty patients were recruited with 441 (71.1%) completing the 6-month study period. Pharmacists made 142 recommendations to prescribers in 110 patients largely centred on potentially inappropriate prescribing of NSAIDs, PPIs or duplication of therapy. At follow-up, there was a significant decrease in the total number of falls (mean −0.116 (−0.217–−0.014)) experienced and a significant increase in medicine adherence (mean difference in Morisky Measure of Adherence Scale-8: 0.513 (0.337–0.689)) and quality of life. Cost per quality-adjusted life year estimates ranged from £11 885 to £32 466 depending on the assumptions made.
By focussing on patients over the age of 65 years with four or more medicines, community pharmacists can improve medicine adherence and patient quality of life.
PMCID: PMC4682463  PMID: 25847545
adherence; community pharmacy; falls; polypharmacy; quality of life
5.  Dietary Mannoheptulose Does Not Significantly Alter Daily Energy Expenditure in Adult Labrador Retrievers 
PLoS ONE  2015;10(12):e0143324.
Mannoheptulose (MH), a sugar found in avocados that inhibits glycolysis in vitro, has been preliminarily investigated as a novel food ingredient for dogs. This study aimed to determine the effects of dietary MH, delivered as an extract of un-ripened avocado, on energy expenditure (EE) in healthy adult Labrador Retriever dogs (total of 12 dogs, 26.99 ± 0.634 kg, 4.9 ± 0.2 y). The study was a double-blind, cross-over with each dog receiving both dietary treatments, control (CON) and MH (400 mg/kg of diet; 6 mg/kg BW), in random order. Resting and post-prandial (10 h) EE and respiratory quotient (RQ) were determined by indirect calorimetry (d 42). The following day, body composition was assessed using dual X-ray absorptiometry. Continuous activity monitoring was conducted using an Atical® accelerometer (d 43–47). A vastus lateralis muscle biopsy was obtained prior to the morning meal (d 49) and 4 h after consumption of their meal (d 56) to determine the protein content and phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK). Diet did not affect body weight, resting EE or skeletal muscle AMPK phosphorylation. Dogs fed MH had significantly lower post-prandial RQ (p = 0.02) and ratio of fat to lean body mass (p = 0.02). Physical activity during light time periods (but not dark) was lower in dogs fed MH (p < 0.05) during weekends, but not on weekdays. These results suggest that MH affects energy balance of adult dogs, but that these effects are not dose dependent and not due to physical activity.
PMCID: PMC4684352  PMID: 26656105
6.  Adherence to UK national guidance for discharge information: an audit in primary care 
Poor communication of clinical information between healthcare settings is associated with patient harm. In 2008, the UK National Prescribing Centre (NPC) issued guidance regarding the minimum information to be communicated upon hospital discharge. This study evaluates the extent of adherence to this guidance and identifies predictors of adherence.
This was an audit of discharge summaries received by medical practices in one UK primary care trust of patients hospitalized for 24 h or longer. Each discharge summary was scored against the applicable NPC criteria which were organized into: ‘patient, admission and discharge’, ‘medicine’ and ‘therapy change’ information.
Of 3444 discharge summaries audited, 2421 (70.3%) were from two teaching hospitals and 906 (26.3%) from three district hospitals. Unplanned admissions accounted for 2168 (63.0%) of the audit sample and 74.6% (2570) of discharge summaries were electronic. Mean (95% CI) adherence to the total NPC minimum dataset was 71.7% [70.2, 73.2]. Adherence to patient, admission and discharge information was 77.3% (95% CI 77.0, 77.7), 67.2% (95% CI 66.3, 68.2) for medicine information and 48.9% (95% CI 47.5, 50.3) for therapy change information. Allergy status, co-morbidities, medication history and rationale for therapy change were the most frequent omissions. Predictors of adherence included quality of the discharge template, electronic discharge summaries and smaller numbers of prescribed medicines.
Despite clear guidance regarding the content of discharge information, omissions are frequent. Adherence to the NPC minimum dataset might be improved by using comprehensive electronic discharge templates and implementation of effective medicines reconciliation at both sides of the health interface.
PMCID: PMC4256634  PMID: 25041244
care transition; discharge communication; discharge information; discharge summary; medicine reconciliation; NPC minimum dataset
7.  Gender-specific Regulatory Challenges to Product Approval: A Panel Discussion 
On May 13, 2014, a 1-hour panel discussion session titled “Gender-Specific Regulatory Challenges to Product Approval” was held during the Academic Emergency Medicine consensus conference, “Gender-Specific Research in Emergency Medicine: Investigate, Understand, and Translate How Gender Affects Patient Outcomes.” The session sought to bring together leaders in emergency medicine (EM) research, authors, and reviewers in EM research publications, as well as faculty, fellows, residents, and students engaged in research and clinical practice. A panel was convened involving a representative from the Office of Women’s Health of the U.S. Food and Drug Administration, two pharmaceutical executives, and a clinical EM researcher. The moderated discussion also involved audience members who contributed significantly to the dialogue. Historical background leading up to the session along with the main themes of the discussion are reproduced in this article. These revolve around sex- and gender-specific research, statistical analysis of sex and gender, clinical practice, financial costs associated with pharmaceutical development, adaptive design, and specific recommendations on the regulatory process as it affects the specialty of EM.
PMCID: PMC4311881  PMID: 25443664
8.  Gender Differences in Neurological Emergencies Part II: A Consensus Summary and Research Agenda on Traumatic Brain Injury 
Traumatic brain injury (TBI) is a major cause of death and disability worldwide. There is strong evidence that gender and sex play an important role across the spectrum of TBI, from pathophysiology to clinical care. In May 2014, Academic Emergency Medicine held a consensus conference “Gender-Specific Research in Emergency Care: Investigate, Understand, and Translate How Gender Affects Patient Outcomes.” A TBI working group was formed to explore what was known about the influence of sex and gender on TBI and to identify gaps for future research. The findings resulted in four major recommendations to guide the TBI research agenda.
PMCID: PMC4311997  PMID: 25420582
9.  Using the Self-Select Paradigm to Delineate the Nature of Speech Motor Programming 
The authors examined the involvement of 2 speech motor programming processes identified by S. T. Klapp (1995, 2003) during the articulation of utterances differing in syllable and sequence complexity. According to S. T. Klapp, 1 process, INT, resolves the demands of the programmed unit, whereas a second process, SEQ, oversees the serial order demands of longer sequences.
A modified reaction time paradigm was used to assess INT and SEQ demands. Specifically, syllable complexity was dependent on syllable structure, whereas sequence complexity involved either repeated or unique syllabi within an utterance.
INT execution was slowed when articulating single syllables in the form CCCV compared to simpler CV syllables. Planning unique syllables within a multisyllabic utterance rather than repetitions of the same syllable slowed INT but not SEQ.
The INT speech motor programming process, important for mental syllabary access, is sensitive to changes in both syllable structure and the number of unique syllables in an utterance.
PMCID: PMC4655590  PMID: 19474396
speech; motor response; memory; motor programming
10.  The stuff that motor chunks are made of: Spatial instead of motor representations? 
Experimental Brain Research  2015;234:353-366.
In order to determine how participants represent practiced, discrete keying sequences in the discrete sequence production task, we had 24 participants practice two six-key sequences on the basis of two pre-learned six-digit numbers. These sequences were carried out by fingers of the left (L) and right (R) hand with between-hand transitions always occurring between the second and third, and the fifth and sixth responses. This yielded the so-called LLRRRL and RRLLLR sequences. Early and late in practice, the keypad used for the right hand was briefly relocated from the front of the participants to 90° at their right side. The results indicate that after 600 practice trials, executing a keying sequence relies heavily on a spatial cross-hand representation in a trunk- or head-based reference frame that after about only 15 trials is fully adjusted to the changed hand location. The hand location effect was not found with the last sequence element. This is attributed to the application of explicit knowledge. The between-hand transitions appeared to induce initial segmentation in some of the participants, but this did not consolidate into a concatenation point of successive motor chunks.
PMCID: PMC4731443  PMID: 26487177
Cognitive models; Motor control; Sequence learning; Motor chunking; Spatial reference frames
11.  The role of clinically-relevant parameters on the cohesiveness of sclerosing foams in a biomimetic vein model 
We have recently reported on the development of a biomimetic vein model to measure the performance of sclerosing foams. In this study we employed the model to compare the commercially-available Varithena® (polidocanol injectable foam) 1 % varicose vein treatment (referred to as polidocanol endovenous microfoam, or PEM) with physician compounded foams (PCFs) made using different foam generation methods (Double Syringe System and Tessari methods) and different foam formulations [liquid to gas ratios of 1:3 or 1:7; gas mixtures composed of 100 % CO2, various CO2:O2 mixtures and room air (RA)]. PCFs produced using the DSS method had longer dwell times (DTs) (range 0.54–2.21 s/cm in the 4 mm diameter vein model) than those of the corresponding PCFs produced by the Tessari technique (range 0.29–0.94 s/cm). PEM had the longest DT indicating the best cohesive stability of any of the foams produced (2.92 s/cm). Other biomimetic model variables investigated included effect of vessel size, delayed injection and rate of plug formation (injection speed). When comparing the 4 and 10 mm vessel diameters, the DTs seen in the 10 mm vessel were higher than those observed for the 4 mm vessel, as the vein angle had been reduced to 5° to allow for foam plug formation. PCF foam performance was in the order RA > CO2:O2 (35:65) ≅ CO2:O2 (65:35) > CO2; PEM had a longer DT than all PCFs (22.10 s/cm) except that for RA made by DSS which was similar but more variable. The effect of delayed injection was also investigated and the DT for PEM remained the longest of all foams with the lowest percentage deviation with respect to the mean values, indicating a consistent foam performance. When considering rate of plug formation, PEM consistently produced the longest DTs and this was possible even at low plug expansion rates (mean 29.5 mm/s, minimum 20.9 mm/s). The developed vein model has therefore demonstrated that PEM consistently displays higher foam stability and cohesiveness when compared to PCFs, over a range of clinically-relevant operational variables.
Electronic supplementary material
The online version of this article (doi:10.1007/s10856-015-5587-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4598354  PMID: 26449448
12.  Case report: a diagnostically challenging conjunctival mass caused by the Epstein-Barr virus 
BMC Ophthalmology  2015;15:129.
We present a paediatric case of infectious mononucleosis in a 13-year old, manifesting with follicular conjunctivitis and a conjunctival mass in one eye with no evidence of leucocytosis on the blood count. The diagnosis was confirmed following surgical excision and biopsy. The case represented a diagnostic challenge due to its atypism and given the steady increase in the prevalence of EBV-related ocular diseases in the last years, this report can serve as an example to prompt earlier serological tests to identify the aetiology in similar cases. This is important because EBV can be treated with acyclovir early in the active viral phase.
PMCID: PMC4597445  PMID: 26447043
13.  The solution structures of native and patient monomeric human IgA1 reveal asymmetric extended structures: implications for function and IgAN disease 
Biochemical Journal  2015;471(Pt 2):167-185.
Detailed analytical ultracentrifugation and X-ray/neutron scattering data and a new atomistic modelling approach revealed asymmetric extended solution structures for human IgA1 that account for its receptor-binding function. IgA1 with different hinge O-galactosylation patterns showed similar structures.
Native IgA1, for which no crystal structure is known, contains an O-galactosylated 23-residue hinge region that joins its Fab and Fc regions. IgA nephropathy (IgAN) is a leading cause of chronic kidney disease in developed countries. Because IgA1 in IgAN often has a poorly O-galactosylated hinge region, the solution structures of monomeric IgA1 from a healthy subject and three IgAN patients with four different O-galactosylation levels were studied. Analytical ultracentrifugation showed that all four IgA1 samples were monomeric with similar sedimentation coefficients, s020,w. X-ray scattering showed that the radius of gyration (Rg) slightly increased with IgA1 concentration, indicating self-association, although their distance distribution curves, P(r), were unchanged with concentration. Neutron scattering indicated similar Rg values and P(r) curves, although IgA1 showed a propensity to aggregate in heavy water buffer. A new atomistic modelling procedure based on comparisons with 177000 conformationally-randomized IgA1 structures with the individual experimental scattering curves revealed similar extended Y-shaped solution structures for all four differentially-glycosylated IgA1 molecules. The final models indicated that the N-glycans at Asn263 were folded back against the Fc surface, the C-terminal tailpiece conformations were undefined and hinge O-galactosylation had little effect on the solution structure. The solution structures for full-length IgA1 showed extended hinges and the Fab and Fc regions were positioned asymmetrically to provide ample space for the functionally-important binding of two FcαR receptors to its Fc region. Whereas no link between O-galactosylation and the IgA1 solution structure was detected, an increase in IgA1 aggregation with reduced O-galactosylation may relate to IgAN.
PMCID: PMC4692083  PMID: 26268558
analytical ultracentrifugation; antibody; constrained modelling; human immunoglobulin A1 (IgA1); immunoglobulin A (IgA) nephropathy; neutron scattering; X-ray scattering
14.  Isolation and characterisation of transport-defective substrate-binding mutants of the tetracycline antiporter TetA(B) 
Biochimica et Biophysica Acta  2015;1848(10):2261-2270.
The tetracycline antiporter TetA(B) is a member of the Major Facilitator Superfamily which confers tetracycline resistance to cells by coupling the efflux of tetracycline to the influx of protons down their chemical potential gradient. Although it is a medically important transporter, its structure has yet to be determined. One possibility for why this has proven difficult is that the transporter may be conformationally heterogeneous in the purified state. To overcome this, we developed two strategies to rapidly identify TetA(B) mutants that were transport-defective and that could still bind tetracycline. Up to 9 amino acid residues could be deleted from the loop between transmembrane α-helices 6 and 7 with only a slight decrease in affinity of tetracycline binding as measured by isothermal titration calorimetry, although the mutant was transport-defective. Scanning mutagenesis where all the residues between 2 and 389 were mutated to either valine, alanine or glycine (VAG scan) identified 15 mutants that were significantly impaired in tetracycline transport. Of these mutants, 12 showed no evidence of tetracycline binding by isothermal titration calorimetry performed on the purified transporters. In contrast, the mutants G44V and G346V bound tetracycline 4–5 fold more weakly than TetA(B), with Kds of 28 μM and 36 μM, respectively, whereas the mutant R70G bound tetracycline 3-fold more strongly (Kd 2.1 μM). Systematic mutagenesis is thus an effective strategy for isolating transporter mutants that may be conformationally constrained and which represent attractive targets for crystallisation and structure determination.
Graphical abstract
•A rapid method was developed for the identification of transport-defective mutants of TetA(B).•ITC was used to determine the affinity of tetracycline binding to the mutants.•Fifteen transport-defective point mutations were identified.•Three mutants bound tetracycline whereas the remainder did not•The transport-defective mutants may facilitate crystallisation of TetA(B).
PMCID: PMC4579554  PMID: 26143388
Membrane protein; Transporter; Isothermal titration calorimetry; Conformational thermostabilisation
15.  Feeding butter with elevated content of trans-10, cis-12 conjugated linoleic acid to obese-prone rats impairs glucose and insulin tolerance 
We recently demonstrated that feeding a natural CLAt10,c12-enriched butter to lean female rats resulted in small, but significant increases in fasting glucose and insulin concentrations, and impaired insulin tolerance. Our goal was to extend these findings by utilizing the diabetes-prone female fatty Zucker rat. Rats were fed custom diets containing 45 % kcal of fat derived from control and CLAt10,c12-enriched butter for 8 weeks.
CLA t10,c12-enriched butter was prepared from milk collected from cows fed a high fermentable carbohydrate diet to create subacute rumen acidosis (SARA); control (non-SARA) butter was collected from cows fed a low grain diet. Female fatty Zucker rats (10 weeks old) were randomly assigned to one of four diet treatments: i) low fat (10 % kcal), ii) 45 % kcal lard, iii) 45 % kcal SARA butter, or iv) 45 % kcal non-SARA butter. A low fat fed lean Zucker group was used as a control group. After 8 weeks, i) glucose and insulin tolerance tests, ii) insulin signaling in muscle, adipose and liver, and iii) metabolic caging measurements were performed.
Glucose and insulin tolerance were significantly impaired in all fatty Zucker groups, but to the greatest extent in the LARD and SARA conditions. Insulin signaling (AKT phosphorylation) was impaired in muscle, visceral (perigonadal) adipose tissue and liver in fatty Zucker rats, but was generally similar across dietary groups. Physical activity, oxygen consumption, food intake and weight gain were also similar amongst the various fatty Zucker groups.
Increasing the consumption of a food naturally enriched with CLAt10,c12 significantly worsens glucose and insulin tolerance in a diabetes-prone rodent model. This outcome is not explained by changes in tissue insulin signaling, physical activity, energy expenditure, food intake or body mass.
PMCID: PMC4587826  PMID: 26415741
Conjugated linoleic acid; CLA (t10,c12); Glucose tolerance; Insulin tolerance; Insulin-stimulated glucose uptake; Female Zucker rats
16.  Social pairing of Seychelles warblers under reduced constraints: MHC, neutral heterozygosity, and age 
Behavioral Ecology  2015;27(1):295-303.
Lay Summary
We manipulated factors that normally constrain the social mate choice of female Seychelles warblers to test if, when less constrained, these females chose partners based on variation at specific immune genes that have been linked to mate choice in other species. We found that older and more generally genetically diverse males were more likely to end up paired with females, but contrary to our predictions, variation at the immune genes did not appear to influence social pairing.
The prevalence and significance of precopulatory mate choice remains keenly debated. The major histocompatibility complex (MHC) plays a key role in vertebrate adaptive immunity, and variation at the MHC influences individual survival. Although MHC-dependent mate choice has been documented in certain species, many other studies find no such pattern. This may be, at least in part, because in natural systems constraints may reduce the choices available to individuals and prevent full expression of underlying preferences. We used translocations to previously unoccupied islands to experimentally reduce constraints on female social mate choice in the Seychelles warbler (Acrocephalus sechellensis), a species in which patterns of MHC-dependent extrapair paternity (EPP), but not social mate choice, have been observed. We find no evidence of MHC-dependent social mate choice in the new populations. Instead, we find that older males and males with more microsatellite heterozygosity are more likely to have successfully paired. Our data cannot resolve whether these patterns in pairing were due to male–male competition or female choice. However, our research does suggest that female Seychelles warblers do not choose social mates using MHC class I to increase fitness. It may also indicate that the MHC-dependent EPP observed in the source population is probably due to mechanisms other than female precopulatory mate choice based on MHC cues.
PMCID: PMC4718175  PMID: 26792973
compatibility; extrapair paternity; good genes; MHC class I; sexual selection; translocation.
17.  Herd-level bovine tuberculosis risk factors: assessing the role of low-level badger population disturbance 
Scientific Reports  2015;5:13062.
Bovine TB (bTB) is endemic in Irish cattle and has eluded eradication despite considerable expenditure, amid debate over the relative roles of badgers and cattle in disease transmission. Using a comprehensive dataset from Northern Ireland (>10,000 km2; 29,513 cattle herds), we investigated interactions between host populations in one of the first large-scale risk factor analyses for new herd breakdowns to combine data on both species. Cattle risk factors (movements, international imports, bTB history, neighbours with bTB) were more strongly associated with herd risk than area-level measures of badger social group density, habitat suitability or persecution (sett disturbance). Highest risks were in areas of high badger social group density and high rates of persecution, potentially representing both responsive persecution of badgers in high cattle risk areas and effects of persecution on cattle bTB risk through badger social group disruption. Average badger persecution was associated with reduced cattle bTB risk (compared with high persecution areas), so persecution may contribute towards sustaining bTB hotspots; findings with important implications for existing and planned disease control programmes.
PMCID: PMC4642523  PMID: 26279310
18.  Urinary Concentrations of Environmental Phenols in Pregnant Women in a Pilot Study of the National Children’s Study 
Environmental research  2014;129:32-38.
Environmental phenols are a group of chemicals with widespread uses in consumer and personal care products, food and beverage processing, and in pesticides. We assessed exposure to benzophenone-3, bisphenol A (BPA), triclosan, methyl- and propyl parabens, and 2,4- and 2,5-dichlorophenol or their precursors in 506 pregnant women enrolled in the National Children’s Study (NCS) Vanguard Study. We measured the urinary concentrations of the target phenols by using online solid-phase extraction-isotope dilution high performance liquid chromatography-tandem mass spectrometry. NCS women results were compared to those of 524 similar-aged women in the National Health and Nutrition Examination Survey (NHANES) 2009–2010, and to 174 pregnant women in NHANES 2005–2010. In the NCS women, we found significant racial/ethnic differences (p<0.05) in regression adjusted mean concentrations of benzophenone-3, triclosan, 2,4- and 2,5-dichlorophenol, but not of BPA. Urinary 2,4- and 2,5-dichlorophenol concentrations were highly correlated (r=0.66, p<0.0001). Except for BPA and triclosan, adjusted mean concentrations were significantly different across the 7 study sites. Education was marginally significant for benzophenone-3, triclosan, propyl paraben, and 2,5-dichlorophenol. Urinary concentrations of target phenols in NCS pregnant women and U.S. women and pregnant women were similar. In NCS pregnant women, race/ethnicity and geographic location determined urinary concentrations of most phenols (except BPA), suggesting differential exposures. NCS Main Study protocols should collect urine biospecimens and information about exposures to environmental phenols.
PMCID: PMC4530794  PMID: 24529000
Environmental phenols; Biomonitoring; National Children’s Study; National Health and Nutrition Examination Survey; Pregnancy
19.  Mutation V111I in HIV-2 Reverse Transcriptase Increases the Fitness of the Nucleoside Analogue-Resistant K65R and Q151M Viruses 
Journal of Virology  2014;89(1):833-843.
Infection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with a combination of nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have described the development of HIV-1 resistance to NRTIs and identified mutations in the polymerase domain of RT. Recent studies have shown that mutations in the connection and RNase H domains of HIV-1 RT may also contribute to resistance. However, only limited information exists regarding the resistance of HIV-2 to NRTIs. In this study, therefore, we analyzed the polymerase, connection, and RNase H domains of RT in HIV-2 patients failing NRTI-containing therapies. Besides the key resistance mutations K65R, Q151M, and M184V, we identified a novel mutation, V111I, in the polymerase domain. This mutation was significantly associated with mutations K65R and Q151M. Sequencing of the connection and RNase H domains of the HIV-2 patients did not reveal any of the mutations that were reported to contribute to NRTI resistance in HIV-1. We show that V111I does not strongly affect drug susceptibility but increases the replication capacity of the K65R and Q151M viruses. Biochemical assays demonstrate that V111I restores the polymerization defects of the K65R and Q151M viruses but negatively affects the fidelity of the HIV-2 RT enzyme. Molecular dynamics simulations were performed to analyze the structural changes mediated by V111I. This showed that V111I changed the flexibility of the 110-to-115 loop region, which may affect deoxynucleoside triphosphate (dNTP) binding and polymerase activity.
IMPORTANCE Mutation V111I in the HIV-2 reverse transcriptase enzyme was identified in patients failing therapies containing nucleoside analogues. We show that the V111I change does not strongly affect the sensitivity of HIV-2 to nucleoside analogues but increases the fitness of viruses with drug resistance mutations K65R and Q151M.
PMCID: PMC4301157  PMID: 25355888
20.  Tuberculosis Biomarker Extraction and Isothermal Amplification in an Integrated Diagnostic Device 
PLoS ONE  2015;10(7):e0130260.
In this study, we integrated magnetic bead-based sample preparation and isothermal loop mediated amplification (LAMP) of TB in a single tube. Surrogate sputum samples produced by the Program for Appropriate Technology in Health containing inactivated TB bacteria were used to test the diagnostic. In order to test the sample preparation method, samples were lysed, and DNA was manually extracted and eluted into water in the tube. In a thermal cycler, LAMP amplified TB DNA from 103 TB cells/mL of sputum at 53.5 ± 3.3 minutes, 104 cells/mL at 46.3 ± 2.2 minutes, and 105 cells/mL at 41.6 ± 1.9 minutes. Negative control samples did not amplify. Next, sample preparation was combined with in-tubing isothermal LAMP amplification by replacing the water elution chamber with a LAMP reaction chamber. In this intermediate configuration, LAMP amplified 103 cells/mL at 74 ± 10 minutes, 104 cells/mL at 60 ± 9 minutes, and 105 TB cells/mL of sputum at 54 ± 9 minutes. Two of three negative controls did not amplify; one amplified at 100 minutes. In the semi-automated system, DNA was eluted directly into an isothermal reaction solution containing the faster OptiGene DNA polymerase. The low surrogate sputum concentration, 103 TB cells/mL, amplified at 52.8 ± 3.3 minutes, 104 cells/mL at 45.4 ± 11.3 minutes, and 105 cells/mL at 31.8 ± 2.9 minutes. TB negative samples amplified at 66.4 ± 7.4 minutes. This study demonstrated the feasibility of a single tube design for integrating sample preparation and isothermal amplification, which with further development could be useful for point-of-care applications, particularly in a low-resource setting.
PMCID: PMC4488445  PMID: 26132307
21.  Very Early Administration of Progesterone for Acute Traumatic Brain Injury 
The New England journal of medicine  2014;371(26):2457-2466.
Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Progesterone has been shown to improve neurologic outcome in multiple experimental models and two early-phase trials involving patients with TBI.
We conducted a double-blind, multicenter clinical trial in which patients with severe, moderate-to-severe, or moderate acute TBI (Glasgow Coma Scale score of 4 to 12, on a scale from 3 to 15, with lower scores indicating a lower level of consciousness) were randomly assigned to intravenous progesterone or placebo, with the study treatment initiated within 4 hours after injury and administered for a total of 96 hours. Efficacy was defined as an increase of 10 percentage points in the proportion of patients with a favorable outcome, as determined with the use of the stratified dichotomy of the Extended Glasgow Outcome Scale score at 6 months after injury. Secondary outcomes included mortality and the Disability Rating Scale score.
A total of 882 of the planned sample of 1140 patients underwent randomization before the trial was stopped for futility with respect to the primary outcome. The study groups were similar with regard to baseline characteristics; the median age of the patients was 35 years, 73.7% were men, 15.2% were black, and the mean Injury Severity Score was 24.4 (on a scale from 0 to 75, with higher scores indicating greater severity). The most frequent mechanism of injury was a motor vehicle accident. There was no significant difference between the progesterone group and the placebo group in the proportion of patients with a favorable outcome (relative benefit of progesterone, 0.95; 95% confidence interval [CI], 0.85 to 1.06; P = 0.35). Phlebitis or thrombophlebitis was more frequent in the progesterone group than in the placebo group (relative risk, 3.03; CI, 1.96 to 4.66). There were no significant differences in the other prespecified safety outcomes.
This clinical trial did not show a benefit of progesterone over placebo in the improvement of outcomes in patients with acute TBI. (Funded by the National Institute of Neurological Disorders and Stroke and others; PROTECT III number, NCT00822900.)
PMCID: PMC4303469  PMID: 25493974
22.  Optimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials 
Malaria Journal  2015;14:253.
The activity of several well-known anti-malarials, including chloroquine (CQ), is attributed to their ability to inhibit the formation of haemozoin (Hz) in the malaria parasite. The formation of inert Hz, or malaria pigment, from toxic haem acquired from the host red blood cell of the parasite during haemoglobin digestion represents a pathway essential for parasite survival. Inhibition of this critical pathway therefore remains a desirable target for novel anti-malarials. A recent publication described the results of a haem fractionation assay used to directly determine haemoglobin, free haem and Hz in Plasmodium falciparum inoculated with CQ. CQ was shown to cause a dose-dependent increase in cellular-free haem that was correlated with decreased parasite survival. The method provided valuable information but was limited due to its low throughput and high demand on parasite starting material. Here, this haem fractionation assay has been successfully adapted to a higher throughput method in 24-well plates, significantly reducing lead times and starting material volumes.
All major haem species in P. falciparum trophozoites, isolated through a series of cellular fractionation steps were determined spectrophotometrically in aqueous pyridine (5 % v/v, pH 7.5) as a low spin complex with haematin. Cell counts were determined using a haemocytometer and a rapid novel fluorescent flow cytometry method.
A higher throughput haem fractionation assay in 24-well plates, containing at most ten million trophozoites was validated against the original published method using CQ and its robustness was confirmed. It provided a minimum six-fold improvement in productivity and 24-fold reduction in starting material volume. The assay was successfully applied to amodiaquine (AQ), which was shown to inhibit Hz formation, while the antifolate pyrimethamine (PYR) and the mitochondrial electron transporter inhibitor atovaquone (Atov) demonstrated no increase in toxic cellular free haem.
This higher throughput cellular haem fractionation assay can easily be applied to novel anti-malarials with a significantly decreased lead time, providing a valuable tool with which to probe the mechanisms of action of both new and established anti-malarials.
PMCID: PMC4484700  PMID: 26099266
Malaria; Plasmodium falciparum; Haem; Haemozoin; β-haematin; Colorimetry; 24-well plate assay; Flow cytometry
23.  Identification of β-hematin inhibitors in the MMV Malaria Box 
The Malaria Box, assembled by the Medicines for Malaria Venture, is a set of 400 structurally diverse, commercially available compounds with demonstrated activity against blood-stage Plasmodium falciparum. The compounds are a representative subset of the 20,000 in vitro antimalarials identified from the high-throughput screening efforts of St. Jude Children's Research Hospital (TN, USA), Novartis and GlaxoSmithKline. In addition, a small set of active compounds from commercially available libraries was added to this group, but it has not previously been published. Elucidation of the biochemical pathways on which these compounds act is a major challenge; therefore, access to these compounds has been made available free of charge to the investigator community. Here, the Malaria Box compounds were tested for activity against the formation of β-hematin, a synthetic form of the heme detoxification biomineral, hemozoin. Further, the mechanism of action of these compounds within the malaria parasite was explored. Ten of the Malaria Box compounds demonstrated significant inhibition of β-hematin formation. In this assay, dose–response data revealed IC50 values ranging from 8.7 to 22.7 μM for these hits, each of which is more potent than chloroquine (a known inhibitor of hemozoin formation). The in vitro antimalarial activity of these ten hits was confirmed in cultures of the chloroquine sensitive D6 strain of the parasite resulting in IC50 values of 135–2165 nM, followed by testing in the multidrug resistant strain, C235. Cultures of P. falciparum (D6) were then examined for their heme distribution following treatment with nine of the commercially available confirmed compounds, seven of which disrupted the hemozoin pathway.
Graphical abstract
•Ten of 400 Malaria Box compounds were found to be potent β-hematin inhibitors.•We confirmed similar in vitro antimalarial activity to results from previous screens.•7 of the 9 commercially available hits were validated hemozoin inhibitors in culture.
PMCID: PMC4486462  PMID: 26150923
Antimalarial; Hemozoin; β-hematin; Biomineralization; Plasmodium falciparum; MMV Malaria Box
24.  Granulocyte-Macrophage Colony-Stimulating Factor Is Neuroprotective in Experimental Traumatic Brain Injury 
Journal of Neurotrauma  2014;31(10):976-983.
Traumatic brain injury (TBI) is an international health concern with a complex pathogenesis resulting in major long-term neurological, neurocognitive, and neuropsychiatric outcomes. Although neuroinflammation has been identified as an important pathophysiological process resulting from TBI, the function of specific inflammatory mediators in the aftermath of TBI remains poorly understood. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an inflammatory cytokine that has been reported to have neuroprotective effects in various animal models of neurodegenerative disease that share pathological similarities with TBI. The importance of GM-CSF in TBI has yet to be studied, however. We examined the role of GM-CSF in TBI by comparing the effects of a lateral fluid percussion (LFP) injury or sham injury in GM-CSF gene deficient (GM-CSF-/-) versus wild-type (WT) mice. After a 3-month recovery interval, mice were assessed using neuroimaging and behavioral outcomes. All mice given a LFP injury displayed significant brain atrophy and behavioral impairments compared with those given sham-injuries; however, this was significantly worse in the GM-CSF-/- mice compared with the WT mice. GM-CSF-/- mice given LFP injury also had reduced astrogliosis compared with their WT counterparts. These novel findings indicate that the inflammatory mediator, GM-CSF, may have significant protective properties in the chronic sequelae of experimental TBI and suggest that further research investigating GM-CSF and its potential benefits in the injured brain is warranted.
PMCID: PMC4012635  PMID: 24392832
animal studies; cytokine; inflammation; models of injury; MRI; traumatic brain injury
25.  SCT: a suite of programs for comparing atomistic models with small-angle scattering data 
Journal of Applied Crystallography  2015;48(Pt 3):953-961.
The SCT suite of tools for the atomistic modelling of X-ray and neutron scattering curves has resulted in 77 macromolecular structures to date. SCT is now made publicly available as open source, alongside an easier-to-use reimplementation of the same algorithms in Python.
Small-angle X-ray and neutron scattering techniques characterize proteins in solution and complement high-resolution structural studies. They are of particular utility when large proteins cannot be crystallized or when the structure is altered by solution conditions. Atomistic models of the averaged structure can be generated through constrained modelling, a technique in which known domain or subunit structures are combined with linker models to produce candidate global conformations. By randomizing the configuration adopted by the different elements of the model, thousands of candidate structures are produced. Next, theoretical scattering curves are generated for each model for trial-and-error fits to the experimental data. From these, a small family of best-fit models is identified. In order to facilitate both the computation of theoretical scattering curves from atomistic models and their comparison with experiment, the SCT suite of tools was developed. SCT also includes programs that provide sequence-based estimates of protein volume (either incorporating hydration or not) and add a hydration layer to models for X-ray scattering modelling. The original SCT software, written in Fortran, resulted in the first atomistic scattering structures to be deposited in the Protein Data Bank, and 77 structures for antibodies, complement proteins and anionic oligosaccharides were determined between 1998 and 2014. For the first time, this software is publicly available, alongside an easier-to-use reimplementation of the same algorithms in Python. Both versions of SCT have been released as open-source software under the Apache 2 license and are available for download from
PMCID: PMC4453981  PMID: 26089768
analytical ultracentrifugation; constrained modelling; hydration shells; neutron scattering; X-ray scattering

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