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1.  Genetic basis of pyrethroid resistance in a population of Anopheles arabiensis, the primary malaria vector in Lower Moshi, north-eastern Tanzania 
Parasites & Vectors  2014;7:274.
Background
Pyrethroid resistance has been slower to emerge in Anopheles arabiensis than in An. gambiae s.s and An. funestus and, consequently, studies are only just beginning to unravel the genes involved. Permethrin resistance in An. arabiensis in Lower Moshi, Tanzania has been linked to elevated levels of both P450 monooxygenases and β-esterases. We have conducted a gene expression study to identify specific genes linked with metabolic resistance in the Lower Moshi An. arabiensis population.
Methods
Microarray experiments employing an An. gambiae whole genome expression chip were performed on An. arabiensis, using interwoven loop designs. Permethrin-exposed survivors were compared to three separate unexposed mosquitoes from the same or a nearby population. A subsection of detoxification genes were chosen for subsequent quantitative real-time PCR (qRT-PCR).
Results
Microarray analysis revealed significant over expression of 87 probes and under expression of 85 probes (in pairwise comparisons between permethrin survivors and unexposed sympatric and allopatric samples from Dar es Salaam (controls). For qRT-PCR we targeted over expressed ABC transporter genes (ABC ‘2060’), a glutathione-S-transferase, P450s and esterases. Design of efficient, specific primers was successful for ABC ‘2060’and two P450s (CYP6P3, CYP6M2). For the CYP4G16 gene, we used the primers that were previously used in a microarray study of An. arabiensis from Zanzibar islands. Over expression of CYP4G16 and ABC ‘2060’ was detected though with contrasting patterns in pairwise comparisons between survivors and controls. CYP4G16 was only up regulated in survivors, whereas ABC ‘2060’ was similar in survivors and controls but over expressed in Lower Moshi samples compared to the Dar es Salaam samples. Increased transcription of CYP4G16 and ABC ‘2060’ are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis.
Conclusions
Increased transcription of a P450 (CYP4G16) and an ABC transporter (ABC 2060) are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis. Our study provides replication of CYP4G16 as a candidate gene for pyrethroid resistance in An. arabiensis, although its role may not be in detoxification, and requires further investigation.
doi:10.1186/1756-3305-7-274
PMCID: PMC4082164  PMID: 24946780
Anopheles arabiensis; Genes; Microarrays; Permethrin; Resistance; Transcription
2.  Ki-67 is an Independent Predictor of Metastasis and Cause-Specific Mortality for Prostate Cancer Patients Treated on Radiation Therapy Oncology Group (RTOG) 94-08 
Purpose
The association of Ki-67 staining index (Ki67-SI) with overall survival (OS), disease-specific mortality (DSM), distant metastasis (DM), and biochemical failure (BF) was examined in men with favorable-to-intermediate risk prostate cancer receiving radiotherapy (RT) alone or with short-term androgen deprivation (ADT) in Radiation Therapy Oncology Group (RTOG) 94-08.
Methods and Materials
468 patients (23.6%) on RTOG 94-08 had sufficient tissue for Ki67-SI analysis. Median follow-up was 7.9 years. Ki67-SI was determined by immunohistochemistry and quantified manually and by image analysis. Correlative analysis versus clinical outcome was performed using the third quartile (≥Q3) cut-point. A proportional hazards multivariable analysis (MVA) dichotomized covariates in accordance with trial stratification and randomization criteria.
Results
In MVAs adjusted for all treatment covariates, high Ki67-SI (≥Q3) was correlated with increased DSM (HR 2.48, p=0.03), DM (HR 3.5, p=0.002) and BF (HR 3.55, p<0.0001). MVA revealed similar Ki67-associated hazard ratios in each separate treatment arm for DSM, DM and BF, these only reached significance for DM in the RT alone arm and for BF in both arms. Ki67-SI was not a significant predictor of intraprostatic recurrence assessed by rebiopsy at 2 years post-treatment. Patients with a high or low Ki67-SI appeared to experience similar relative benefit from the addition of ADT to radiation.
Conclusions
High Ki67-SI independently predicts for increased disease specific mortality, distant metastasis and protocol biochemical failure in primarily intermediate risk prostate cancer patients treated with radiation therapy with or without androgen deprivation therapy on RTOG 9408, but does not predict for local recurrence nor for increased relative benefit from ADT. This and prior studies lend support for use of Ki67-SI as a stratification factor in future trials.
doi:10.1016/j.ijrobp.2013.01.016
PMCID: PMC3646974  PMID: 23474109
Prostate carcinoma; Ki-67 Antigen/Analysis; Prognosis; Metastasis; Biomarkers
3.  Diagnostic and Management Approach to Common Sleep Disorders During Pregnancy 
The significance for maternal and fetal health of gestational Obstructive Sleep Apnea, Primary Insomnia, Restless Legs Syndrome, & Narcolepsy are summarized. The pathophysiology, signs, symptoms, and basic Sleep Medicine concepts that assist the obstetrician in suspecting these four conditions are described. Where appropriate, initial management options are also outlined. Referral guidelines to a Sleep Medicine specialist are included when further diagnostic, severity assessment, and management suggestions are needed.
doi:10.1097/GRF.0b013e31828f2717
PMCID: PMC3658935  PMID: 23563879
4.  XRN 5’→3’ exoribonucleases: Structure, mechanisms and functions 
Biochimica et biophysica acta  2013;1829(0):590-603.
The XRN family of 5’→3’ exoribonucleases is critical for ensuring the fidelity of cellular RNA turnover in eukaryotes. Highly conserved across species, the family is typically represented by one cytoplasmic enzyme (XRN1/PACMAN or XRN4) and one or more nuclear enzymes (XRN2/RAT1 and XRN3). Cytoplasmic and/or nuclear XRNs have proven to be essential in all organisms tested, and deficiencies can have severe developmental phenotypes, demonstrating that XRNs are indispensable in fungi, plants and animals. XRNs degrade diverse RNA substrates during general RNA decay and function in specialized processes integral to RNA metabolism, such as nonsense-mediated decay (NMD), gene silencing, rRNA maturation, and transcription termination. Here, we review current knowledge of XRNs, highlighting recent work of high impact and future potential. One example is the breakthrough in our understanding of how XRN1 processively degrades 5’ monophosphorylated RNA, revealed by its crystal structure and mutational analysis. The expanding knowledge of XRN substrates and interacting partners is outlined and the functions of XRNs are interpreted at the organismal level using available mutant phenotypes. Finally, three case studies are discussed in more detail to underscore a few of the most exciting areas of research on XRN function: XRN4 involvement in small RNA-associated processes in plants, the roles of XRN1/PACMAN in Drosophila development, and the function of human XRN2 in nuclear transcriptional quality control. This article is part of a Special Issue entitled: RNA Decay Mechanisms.
doi:10.1016/j.bbagrm.2013.03.005
PMCID: PMC3742305  PMID: 23517755
Exoribonuclease; XRN; XRN1/PACMAN; XRN4; XRN2/RAT1; RNA decay; Small RNA; Transcriptional surveillance
5.  An unusual dimeric small heat shock protein provides insight into the mechanism of this class of chaperones 
Journal of molecular biology  2013;425(10):1683-1696.
Small heat shock proteins (sHSPs) are virtually ubiquitous stress proteins that are also found in many normal tissues and accumulate in diseases of protein folding. They generally act as ATP-independent chaperones to bind and stabilize denaturing proteins that can be later reactivated by ATP-dependent Hsp70/DnaK, but the mechanism of substrate capture by sHSPs remains poorly understood. A majority of sHSPs form large oligomers, a property that has been linked to their effective chaperone action. We describe AtHsp18.5 from Arabidopsis thaliana, demonstrating it is dimeric and exhibits robust chaperone activity, adding support to the model that suboligomeric sHSP forms are a substrate binding species. Notably, like oligomeric sHSPs, when bound to substrate AtHsp18.5 assembles into large complexes, indicating reformation of sHSP oligomeric contacts are not required for assembly of sHSP-substrate complexes. Monomers of AtHsp18.5 freely exchange between dimers, but fail to coassemble in vitro with dodecameric plant cytosolic sHSPs, suggesting AtHsp18.5 does not interact by coassembly with these other sHSPs in vivo. Data from controlled proteolysis and hydrogen-deuterium exchange coupled with mass spectrometry show that the N- and C-termini of AtHsp18.5 are highly accessible and lack stable secondary structure, most likely a requirement for substrate interaction. Chaperone activity of a series of AtHsp18.5 truncation mutants confirm that the N-terminal arm is required for substrate protection and that different substrates interact differently with the N-terminal arm. In total, these data imply that the core α-crystallin domain of the sHSPs is a platform for flexible arms that capture substrates to maintain their solubility.
doi:10.1016/j.jmb.2013.02.011
PMCID: PMC3646915  PMID: 23416558
chaperone; α-crystallin domain; protein flexibility; subunit exchange; hydrogen deuterium exchange; mass spectrometry of protein complexes
6.  Functional Subpopulations of V3 Interneurons in the Mature Mouse Spinal Cord 
The Journal of Neuroscience  2013;33(47):18553-18565.
V3 interneurons (INs) are a major group of excitatory commissural interneurons in the spinal cord, and they are essential for producing a stable and robust locomotor rhythm. V3 INs are generated from the ventral-most progenitor domain, p3, but migrate dorsally and laterally during postmitotic development. At birth, they are located in distinctive clusters in the ventral horn and deep dorsal horn. To assess the heterogeneity of this genetically identified group of spinal INs, we combined patch-clamp recording and anatomical tracing with cluster analysis. We examined electrophysiological and morphological properties of mature V3 INs identified by their expression of tdTomato fluorescent proteins in Sim1Cre/+; Rosafloxstop26TdTom mice. We identified two V3 subpopulations with distinct intrinsic properties and spatial distribution patterns. Ventral V3 INs, primarily located in lamina VIII, possess a few branching processes and were capable of generating rapid tonic firing spikes. By contrast, dorsal V3 INs exhibited a more complex morphology and relatively slow average spike frequency with strong adaptation, and they also displayed large sag voltages and post-inhibitory rebound potentials. Our data suggested that hyperpolarization-activated cation channel currents and T-type calcium channel currents may account for some of the membrane properties of V3 INs. Finally, we observed that ventral and dorsal V3 INs were active in different ways during running and swimming, indicating that ventral V3 INs may act as premotor neurons and dorsal V3 INs as relay neurons mediating sensory inputs. Together, we detected two physiologically and topographically distinct subgroups of V3 INs, each likely playing different roles in locomotor activities.
doi:10.1523/JNEUROSCI.2005-13.2013
PMCID: PMC3894417  PMID: 24259577
7.  Atrium of stone: A case of confined left atrial calcification without hemodynamic compromise 
Dystrophic cardiac calcification is often associated with conditions causing systemic inflammation and when present, is usually extensive, often encompassing multiple cardiac chambers and valves. We present an unusual case of dystrophic left atrial calcification in the setting of end stage renal disease on hemodialysis diagnosed by echocardiography and computed tomography. Significant calcium deposition is confined within the walls of the left atrium with no involvement of the mitral valve, and no hemodynamic effects.
doi:10.12998/wjcc.v2.i5.142
PMCID: PMC4023308  PMID: 24868514
Left atrium calcification; Heart of stone; Atrial calcification; Dystrophic cardiac calcification; Renal failure
8.  Psychological and Behavioral Changes during Confinement in a 520-Day Simulated Interplanetary Mission to Mars 
PLoS ONE  2014;9(3):e93298.
Behavioral health risks are among the most serious and difficult to mitigate risks of confinement in space craft during long-duration space exploration missions. We report on behavioral and psychological reactions of a multinational crew of 6 healthy males confined in a 550 m3 chamber for 520 days during the first Earth-based, high-fidelity simulated mission to Mars. Rest-activity of crewmembers was objectively measured throughout the mission with wrist-worn actigraphs. Once weekly throughout the mission crewmembers completed the Beck Depression Inventory-II (BDI-II), Profile of Moods State short form (POMS), conflict questionnaire, the Psychomotor Vigilance Test (PVT-B), and series of visual analogue scales on stress and fatigue. We observed substantial inter-individual differences in the behavioral responses of crewmembers to the prolonged mission confinement and isolation. The crewmember with the highest average POMS total mood disturbance score throughout the mission also reported symptoms of depression in 93% of mission weeks, which reached mild-to-moderate levels in >10% of mission weeks. Conflicts with mission control were reported five times more often than conflicts among crewmembers. Two crewmembers who had the highest ratings of stress and physical exhaustion accounted for 85% of the perceived conflicts. One of them developed a persistent sleep onset insomnia with ratings of poor sleep quality, which resulted in chronic partial sleep deprivation, elevated ratings of daytime tiredness, and frequent deficits in behavioral alertness. Sleep-wake timing was altered in two other crewmembers, beginning in the first few months of the mission and persisting throughout. Two crewmembers showed neither behavioral disturbances nor reports of psychological distress during the 17-month period of mission confinement. These results highlight the importance of identifying behavioral, psychological, and biological markers of characteristics that predispose prospective crewmembers to both effective and ineffective behavioral reactions during the confinement of prolonged spaceflight, to inform crew selection, training, and individualized countermeasures.
doi:10.1371/journal.pone.0093298
PMCID: PMC3968121  PMID: 24675720
9.  Structure of bacterial cytoplasmic chemoreceptor arrays and implications for chemotactic signaling 
eLife  2014;3:e02151.
Most motile bacteria sense and respond to their environment through a transmembrane chemoreceptor array whose structure and function have been well-studied, but many species also contain an additional cluster of chemoreceptors in their cytoplasm. Although the cytoplasmic cluster is essential for normal chemotaxis in some organisms, its structure and function remain unknown. Here we use electron cryotomography to image the cytoplasmic chemoreceptor cluster in Rhodobacter sphaeroides and Vibrio cholerae. We show that just like transmembrane arrays, cytoplasmic clusters contain trimers-of-receptor-dimers organized in 12-nm hexagonal arrays. In contrast to transmembrane arrays, however, cytoplasmic clusters comprise two CheA/CheW baseplates sandwiching two opposed receptor arrays. We further show that cytoplasmic fragments of normally transmembrane E. coli chemoreceptors form similar sandwiched structures in the presence of molecular crowding agents. Together these results suggest that the 12-nm hexagonal architecture is fundamentally important and that sandwiching and crowding can replace the stabilizing effect of the membrane.
DOI: http://dx.doi.org/10.7554/eLife.02151.001
eLife digest
Many bacteria swim through water by rotating tiny hair-like structures called flagella. In E. coli, if all the flagella on the surface of a bacterium rotate in a counterclockwise fashion, then it will swim in a particular direction, but if the flagella all rotate in an clockwise fashion, then the bacterium will stop swimming and start to tumble.
Bacteria use a combination of swimming and tumbling in order to move towards or away from certain chemicals. For example, a bacterium is able to move towards a source of nutrients because it is constantly evaluating its environment and will swim forward for longer periods of time when it recognizes the concentration of the nutrient is increasing. And if it senses that the nutrient concentration is decreasing, it will tumble in an effort to move in a different direction.
Many bacteria, such as E. coli, rely on proteins in their cell membrane called chemoreceptors to sense specific chemicals and then send signals that tell the flagella how to rotate. These transmembrane receptors and their role in chemotaxis—that is, movement towards or away from specific chemicals in the environment—have been widely studied. However, other bacteria also have chemoreceptors in the cytoplasm inside the bacterial cell, and much less is known about these.
Now, Briegel et al. have examined the cytoplasmic chemoreceptors of two unrelated bacteria, R. sphaeroides and V. cholera, and found that the cytoplasmic chemoreceptors arrange themselves in hexagonal arrays, similar to the way that transmembrane chemoreceptors are arranged. However, the cytoplasmic chemoreceptors arrange themselves in a two-layer sandwich-like structure, whereas the transmembrane chemoreceptors are arranged in just one layer. The next step is to understand how chemical binding causes these arrays to send their signals to the motor. A complete understanding of this signaling system may ultimately allow scientists to re-engineer it to draw bacteria to targets of medical or environmental interest, such as cancer cells or contaminated soils.
DOI: http://dx.doi.org/10.7554/eLife.02151.002
doi:10.7554/eLife.02151
PMCID: PMC3964821  PMID: 24668172
electron cryotomography; correlative microscopy; chemotaxis; Rhodobacter sphaeroides; Vibrio cholerae; E. coli
10.  Fragment-based drug discovery using a multi-domain, parallel MD-MM/PBSA screening protocol 
We have developed a rigorous computational screening protocol to identify novel fragment-like inhibitors of N5-CAIR mutase (PurE), a key enzyme involved in de novo purine synthesis that represents a novel target for the design of antibacterial agents. This computational screening protocol utilizes molecular docking, graphics processing unit (GPU)-accelerated molecular dynamics and Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) free energy estimations to investigate the binding modes and energies of fragments in the active sites of PurE. PurE is a functional octamer comprised of identical subunits. The octameric structure, with its eight active sites, provided a distinct advantage in these studies because, for a given simulation length, we were able to place eight separate fragment compounds in the active sites to increase the throughput of the MM/PBSA analysis. To validate this protocol, we have screened an in-house fragment library consisting of 352 compounds. The theoretical results were then compared with the results of two experimental fragment screens, Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) binding analyses. In these validation studies, the protocol was able to effectively identify the competitive binders that had been independently identified by experimental testing, suggesting the potential utility of this method for the identification of novel fragments for future development as PurE inhibitors.
doi:10.1021/ci300502h
PMCID: PMC3752004  PMID: 23432621
Fragment-based drug discovery; Molecular dynamics; MM/PBSA; N5-CAIR mutase; PurE
11.  Sleep Deprivation and Stressors: Evidence for Elevated Negative Affect in Response to Mild Stressors When Sleep Deprived 
Emotion (Washington, D.C.)  2012;12(5):1015-1020.
Stress often co-occurs with inadequate sleep duration, and both are believed to impact mood and emotion. It is not yet known whether inadequate sleep simply increases the intensity of subsequent stress responses or interacts with stressors in more complicated ways. To address this issue, we investigated the effects of one night of total sleep deprivation on subjective stress and mood in response to low-stress and high-stress cognitive testing conditions in healthy adult volunteers in two separate experiments (total N = 53). Sleep was manipulated in a controlled, laboratory setting and stressor intensity was manipulated by changing difficulty of cognitive tasks, time pressure, and feedback about performance. Sleep-deprived participants reported greater subjective stress, anxiety, and anger than rested controls following exposure to the low-stressor condition, but not in response to the high-stressor condition, which elevated negative mood and stress about equally for both sleep conditions. These results suggest that sleep deprivation lowers the psychological threshold for the perception of stress from cognitive demands but does not selectively increase the magnitude of negative affect in response to high-stress performance demands.
doi:10.1037/a0026871
PMCID: PMC3964364  PMID: 22309720
sleep deprivation; stress; stressors; mood
12.  Total Synthesis of the Antitumor Antibiotic (±)-Streptonigrin: First- and Second-Generation Routes for de Novo Pyridine Formation Using Ring-Closing Metathesis 
The Journal of Organic Chemistry  2013;78(24):12338-12350.
The total synthesis of (±)-streptonigrin, a potent tetracyclic aminoquinoline-5,8-dione antitumor antibiotic that reached phase II clinical trials in the 1970s, is described. Two routes to construct a key pentasubstituted pyridine fragment are depicted, both relying on ring-closing metathesis but differing in the substitution and complexity of the precursor to cyclization. Both routes are short and high yielding, with the second-generation approach ultimately furnishing (±)-streptonigrin in 14 linear steps and 11% overall yield from inexpensive ethyl glyoxalate. This synthesis will allow for the design and creation of druglike late-stage natural product analogues to address pharmacological limitations. Furthermore, assessment of a number of chiral ligands in a challenging asymmetric Suzuki–Miyaura cross-coupling reaction has enabled enantioenriched (up to 42% ee) synthetic streptonigrin intermediates to be prepared for the first time.
doi:10.1021/jo402388f
PMCID: PMC3964827  PMID: 24328139
13.  CYP6 P450 Enzymes and ACE-1 Duplication Produce Extreme and Multiple Insecticide Resistance in the Malaria Mosquito Anopheles gambiae 
PLoS Genetics  2014;10(3):e1004236.
Malaria control relies heavily on pyrethroid insecticides, to which susceptibility is declining in Anopheles mosquitoes. To combat pyrethroid resistance, application of alternative insecticides is advocated for indoor residual spraying (IRS), and carbamates are increasingly important. Emergence of a very strong carbamate resistance phenotype in Anopheles gambiae from Tiassalé, Côte d'Ivoire, West Africa, is therefore a potentially major operational challenge, particularly because these malaria vectors now exhibit resistance to multiple insecticide classes. We investigated the genetic basis of resistance to the most commonly-applied carbamate, bendiocarb, in An. gambiae from Tiassalé. Geographically-replicated whole genome microarray experiments identified elevated P450 enzyme expression as associated with bendiocarb resistance, most notably genes from the CYP6 subfamily. P450s were further implicated in resistance phenotypes by induction of significantly elevated mortality to bendiocarb by the synergist piperonyl butoxide (PBO), which also enhanced the action of pyrethroids and an organophosphate. CYP6P3 and especially CYP6M2 produced bendiocarb resistance via transgenic expression in Drosophila in addition to pyrethroid resistance for both genes, and DDT resistance for CYP6M2 expression. CYP6M2 can thus cause resistance to three distinct classes of insecticide although the biochemical mechanism for carbamates is unclear because, in contrast to CYP6P3, recombinant CYP6M2 did not metabolise bendiocarb in vitro. Strongly bendiocarb resistant mosquitoes also displayed elevated expression of the acetylcholinesterase ACE-1 gene, arising at least in part from gene duplication, which confers a survival advantage to carriers of additional copies of resistant ACE-1 G119S alleles. Our results are alarming for vector-based malaria control. Extreme carbamate resistance in Tiassalé An. gambiae results from coupling of over-expressed target site allelic variants with heightened CYP6 P450 expression, which also provides resistance across contrasting insecticides. Mosquito populations displaying such a diverse basis of extreme and cross-resistance are likely to be unresponsive to standard insecticide resistance management practices.
Author Summary
Malaria control depends heavily on only four classes of insecticide to which Anopheles mosquitoes are increasingly resistant. It is important to manage insecticide application carefully to minimise increases in resistance, for example by using different compounds in combination or rotation. Recently, mosquitoes resistant to all available insecticides have been found in Tiassalé, West Africa, which could be problematic for resistance management, particularly if common genetic mechanisms are responsible (‘cross-resistance’). Tiassalé mosquitoes also exhibit extreme levels of resistance to the two most important classes, pyrethroids and carbamates. We investigated the genetic basis of extreme carbamate resistance and cross-resistance in Tiassalé, and the applicability of results in an additional population from Togo. We find that specific P450 enzymes are involved in both extreme and cross-resistance, including one, CYP6M2, which can cause resistance to three insecticide classes. However, amplification of a mutated version of the gene which codes for acetycholinesterase, the target site of both the carbamate and organophosphate insecticides, also plays an important role. Mechanisms involved in both extreme resistance and cross resistance are likely to be very resilient to insecticide management practices, and represent an alarming scenario for mosquito-targeted malaria control.
doi:10.1371/journal.pgen.1004236
PMCID: PMC3961184  PMID: 24651294
14.  ChIP-Seq and RNA-Seq Reveal an AmrZ-Mediated Mechanism for Cyclic di-GMP Synthesis and Biofilm Development by Pseudomonas aeruginosa 
PLoS Pathogens  2014;10(3):e1003984.
The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ΔamrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ΔamrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ΔamrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon, and characterized AmrZ as an activator or repressor at each binding site. We identified an AmrZ-repressed DGC-encoding gene (PA4843) from this cohort, which we named AmrZ dependent cyclase A (adcA). PAO1 overexpressing adcA accumulates 29-fold more c-di-GMP than the wild type strain, confirming the cyclase activity of AdcA. In biofilm reactors, a ΔamrZ ΔadcA double mutant formed smaller microcolonies than the single ΔamrZ mutant, indicating adcA is responsible for the hyper biofilm phenotype of the ΔamrZ mutant. This study combined the techniques of ChIP-Seq and RNA-Seq to define the comprehensive regulon of a bifunctional transcriptional regulator. Moreover, we identified a c-di-GMP mediated mechanism for AmrZ regulation of biofilm formation and chronicity.
Author Summary
Pathogenic bacteria such as Pseudomonas aeruginosa utilize a wide variety of systems to sense and respond to the changing conditions during an infection. When a stress is sensed, signals are transmitted to impact expression of many genes that allow the bacterium to adapt to the changing conditions. AmrZ is a protein that regulates production of several virulence-associated gene products, though we predicted that its role in virulence was more expansive than previously described. Transcription factors such as AmrZ often affect the expression of a gene by binding and promoting or inhibiting expression of the target gene. Two global techniques were utilized to determine where AmrZ binds in the genome, and what effect AmrZ has once bound. This approach revealed that AmrZ represses the production of a signaling molecule called cyclic diguanylate, which is known to induce the formation of difficult to treat communities of bacteria called biofilms. This study also identified many novel targets of AmrZ to promote future studies of this regulator. Collectively, these data can be utilized to develop treatments to inhibit biofilm formation during devastating chronic infections.
doi:10.1371/journal.ppat.1003984
PMCID: PMC3946381  PMID: 24603766
15.  Does surgery improve prognosis in patients with small-cell lung carcinoma? 
A best evidence topic was written according to a structured protocol, asking ‘does surgery improve prognosis in patients with small-cell lung carcinoma (SCLC)?’ One hundred and thirteen papers were identified, of which the nine papers best able to answer the question were selected and the details of each tabulated. The prohibitive attitude of clinicians toward surgery in SCLC has prevailed since the 1960s, informed by a prospective randomized trial in which 144 patients were assigned to surgical treatment or to radical radiotherapy. Surgery conferred no survival benefit when compared with radical radiotherapy as assessed at 6 monthly intervals up to 10 years post-treatment. Patients with metastatic disease were excluded; however, diagnostic advances subsequent to these trials justify a re-evaluation of the issue, given the greater degree of accuracy with which sub-groups of patients who might benefit from surgery can now be defined. Only one further prospective, randomized trial features in the literature. This study also discerned that no survival benefit was accrued by adding surgery to chemotherapy. However, this study only included patients who responded to an initial course of chemotherapy and also excluded patients with peripheral nodules only. Subsequent investigators have asserted the value of surgery in SCLC. A retrospective case–control study found that surgery significantly improved median survival in patients with stage I disease when compared with patients undergoing medical therapy. One British centre reported survival rates of 52% at 5 years amongst patients undergoing resection and nodal dissection for stage II–IIIA disease. In a retrospective analysis of the Norwegian cancer database, 5-year survival for patients with stage I undergoing surgery was 44.9%, as opposed to 11.3% amongst those treated medically. This finding was echoed in the analysis of the surveillance epidemiology and end results database in the USA, which found improved median survival amongst patients undergoing surgery for limited SCLC. Prospective studies of carefully selected patients have documented good median survival in patients whose tumour was completely resected. We conclude that surgery for early-stage SCLC improves prognosis as part of a multi-modality approach. This echoes the advice of the 2011 national institute of health and clinical excellence guidelines regarding surgery in SCLC.
doi:10.1093/icvts/ivs475
PMCID: PMC3568800  PMID: 23169878
Small cell lung cancer; Surgery
16.  A Course-Based Research Experience: How Benefits Change with Increased Investment in Instructional Time 
Shaffer, Christopher D. | Alvarez, Consuelo J. | Bednarski, April E. | Dunbar, David | Goodman, Anya L. | Reinke, Catherine | Rosenwald, Anne G. | Wolyniak, Michael J. | Bailey, Cheryl | Barnard, Daron | Bazinet, Christopher | Beach, Dale L. | Bedard, James E. J. | Bhalla, Satish | Braverman, John | Burg, Martin | Chandrasekaran, Vidya | Chung, Hui-Min | Clase, Kari | DeJong, Randall J. | DiAngelo, Justin R. | Du, Chunguang | Eckdahl, Todd T. | Eisler, Heather | Emerson, Julia A. | Frary, Amy | Frohlich, Donald | Gosser, Yuying | Govind, Shubha | Haberman, Adam | Hark, Amy T. | Hauser, Charles | Hoogewerf, Arlene | Hoopes, Laura L. M. | Howell, Carina E. | Johnson, Diana | Jones, Christopher J. | Kadlec, Lisa | Kaehler, Marian | Silver Key, S. Catherine | Kleinschmit, Adam | Kokan, Nighat P. | Kopp, Olga | Kuleck, Gary | Leatherman, Judith | Lopilato, Jane | MacKinnon, Christy | Martinez-Cruzado, Juan Carlos | McNeil, Gerard | Mel, Stephanie | Mistry, Hemlata | Nagengast, Alexis | Overvoorde, Paul | Paetkau, Don W. | Parrish, Susan | Peterson, Celeste N. | Preuss, Mary | Reed, Laura K. | Revie, Dennis | Robic, Srebrenka | Roecklein-Canfield, Jennifer | Rubin, Michael R. | Saville, Kenneth | Schroeder, Stephanie | Sharif, Karim | Shaw, Mary | Skuse, Gary | Smith, Christopher D. | Smith, Mary A. | Smith, Sheryl T. | Spana, Eric | Spratt, Mary | Sreenivasan, Aparna | Stamm, Joyce | Szauter, Paul | Thompson, Jeffrey S. | Wawersik, Matthew | Youngblom, James | Zhou, Leming | Mardis, Elaine R. | Buhler, Jeremy | Leung, Wilson | Lopatto, David | Elgin, Sarah C. R. | O'Dowd, Diane K.
CBE Life Sciences Education  2014;13(1):111-130.
While course-based research in genomics can generate both knowledge gains and a greater appreciation for how science is done, a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. Nonetheless, this is a very cost-effective way to reach larger numbers of students.
There is widespread agreement that science, technology, engineering, and mathematics programs should provide undergraduates with research experience. Practical issues and limited resources, however, make this a challenge. We have developed a bioinformatics project that provides a course-based research experience for students at a diverse group of schools and offers the opportunity to tailor this experience to local curriculum and institution-specific student needs. We assessed both attitude and knowledge gains, looking for insights into how students respond given this wide range of curricular and institutional variables. While different approaches all appear to result in learning gains, we find that a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. An alumni survey revealed that time spent on a research project is also a significant factor in the value former students assign to the experience one or more years later. We conclude: 1) implementation of a bioinformatics project within the biology curriculum provides a mechanism for successfully engaging large numbers of students in undergraduate research; 2) benefits to students are achievable at a wide variety of academic institutions; and 3) successful implementation of course-based research experiences requires significant investment of instructional time for students to gain full benefit.
doi:10.1187/cbe-13-08-0152
PMCID: PMC3940452  PMID: 24591510
17.  The unaccounted yet abundant nitrous oxide-reducing microbial community: a potential nitrous oxide sink 
The ISME Journal  2012;7(2):417-426.
Nitrous oxide (N2O) is a major radiative forcing and stratospheric ozone-depleting gas emitted from terrestrial and aquatic ecosystems. It can be transformed to nitrogen gas (N2) by bacteria and archaea harboring the N2O reductase (N2OR), which is the only known N2O sink in the biosphere. Despite its crucial role in mitigating N2O emissions, knowledge of the N2OR in the environment remains limited. Here, we report a comprehensive phylogenetic analysis of the nosZ gene coding the N2OR in genomes retrieved from public databases. The resulting phylogeny revealed two distinct clades of nosZ, with one unaccounted for in studies investigating N2O-reducing communities. Examination of N2OR structural elements not considered in the phylogeny revealed that the two clades differ in their signal peptides, indicating differences in the translocation pathway of the N2OR across the membrane. Sequencing of environmental clones of the previously undetected nosZ lineage in various environments showed that it is widespread and diverse. Using quantitative PCR, we demonstrate that this clade was most often at least as abundant as the other, thereby more than doubling the known extent of the overall N2O-reducing community in the environment. Furthermore, we observed that the relative abundance of nosZ from either clade varied among habitat types and environmental conditions. Our results indicate a physiological dichotomy in the diversity of N2O-reducing microorganisms, which might be of importance for understanding the relationship between the diversity of N2O-reducing microorganisms and N2O reduction in different ecosystems.
doi:10.1038/ismej.2012.125
PMCID: PMC3554408  PMID: 23151640
denitrification; functional gene diversity; nitrous oxide reductase; protein translocation pathway
18.  Different Requirements for Scavenger Receptor Class B Type I in Hepatitis C Virus Cell-Free versus Cell-to-Cell Transmission 
Journal of Virology  2013;87(15):8282-8293.
Hepatitis C virus (HCV) is believed to initially infect the liver through the basolateral side of hepatocytes, where it engages attachment factors and the coreceptors CD81 and scavenger receptor class B type I (SR-BI). Active transport toward the apical side brings the virus in close proximity of additional entry factors, the tight junction molecules claudin-1 and occludin. HCV is also thought to propagate via cell-to-cell spread, which allows highly efficient virion delivery to neighboring cells. In this study, we compared an adapted HCV genome, clone 2, characterized by superior cell-to cell spread, to its parental genome, J6/JFH-1, with the goal of elucidating the molecular mechanisms of HCV cell-to-cell transmission. We show that CD81 levels on the donor cells influence the efficiency of cell-to-cell spread and CD81 transfer between neighboring cells correlates with the capacity of target cells to become infected. Spread of J6/JFH-1 was blocked by anti-SR-BI antibody or in cells knocked down for SR-BI, suggesting a direct role for this receptor in HCV cell-to-cell transmission. In contrast, clone 2 displayed a significantly reduced dependence on SR-BI for lateral spread. Mutations in E1 and E2 responsible for the enhanced cell-to-cell spread phenotype of clone 2 rendered cell-free virus more susceptible to antibody-mediated neutralization. Our results indicate that although HCV can lose SR-BI dependence for cell-to-cell spread, vulnerability to neutralizing antibodies may limit this evolutionary option in vivo. Combination therapies targeting both the HCV glycoproteins and SR-BI may therefore hold promise for effective control of HCV dissemination.
doi:10.1128/JVI.01102-13
PMCID: PMC3719822  PMID: 23698298
19.  Randomized Trials in Emergency Medicine Journals, 2008-2011 
Study Objective
Knowledge of current areas of activity in emergency medicine research may improve collaboration among investigators and may help inform decisions about future research priorities. Randomized controlled trials are a key component of research activity and an essential tool for improving care. We investigated the characteristics of randomized trials recently published in emergency medicine journals.
Methods
This was a retrospective analysis of randomized trials published in the five highest-impact emergency medicine journals. Pubmed was searched for reports of randomized trials involving human subjects indexed to MEDLINE between January 1, 2008 and December 31, 2011. Included trials were classified with respect to study topic, funding source, presence of age-related inclusion criteria, and country of origin.
Results
163 published studies were included for analysis. Pain management was the most commonly studied topic (N=28, 17%) followed by orthopedics (N = 24, 15%), cardiovascular disease (N=13, 8%), and pre-hospital medicine (N=13, 8%). Less than half of studies received extramural funding support. Children were specifically examined in 22 (13%) of trials; only 5 trials (3%) specifically examined patients age 60 or older.
Conclusions
Emergency medicine journals publish randomized trials addressing a wide range of clinical topics. Randomized trials focusing on geriatric patients are not commonly published in these journals.
doi:10.1016/j.ajem.2012.06.003
PMCID: PMC3494801  PMID: 22867836
Emergency medicine; randomized controlled trials; geriatrics
20.  The dynamics of pyrethroid resistance in Anopheles arabiensis from Zanzibar and an assessment of the underlying genetic basis 
Parasites & Vectors  2013;6:343.
Background
The emergence of pyrethroid resistance in the malaria vector, Anopheles arabiensis, threatens to undermine the considerable gains made towards eliminating malaria on Zanzibar. Previously, resistance was restricted to the island of Pemba while mosquitoes from Unguja, the larger of the two islands of Zanzibar, were susceptible. Here, we characterised the mechanism(s) responsible for resistance on Zanzibar using a combination of gene expression and target-site mutation assays.
Methods
WHO resistance bioassays were conducted using 1-5d old adult Anopheles gambiae s.l. collected between 2011 and 2013 across the archipelago. Synergist assays with the P450 inhibitor piperonyl-butoxide were performed in 2013. Members of the An. gambiae complex were PCR-identified and screened for target-site mutations (kdr and Ace-1). Gene expression in pyrethroid resistant An. arabiensis from Pemba was analysed using whole-genome microarrays.
Results
Pyrethroid resistance is now present across the entire Zanzibar archipelago. Survival to the pyrethroid lambda-cyhalothrin in bioassays conducted in 2013 was 23.5-54.3% on Unguja and 32.9-81.7% on Pemba. We present evidence that resistance is mediated, in part at least, by elevated P450 monoxygenases. Whole-genome microarray scans showed that the most enriched gene terms in resistant An. arabiensis from Pemba were associated with P450 activity and synergist assays with PBO completely restored susceptibility to pyrethroids in both islands. CYP4G16 was the most consistently over-expressed gene in resistant mosquitoes compared with two susceptible strains from Unguja and Dar es Salaam. Expression of this P450 is enriched in the abdomen and it is thought to play a role in hydrocarbon synthesis. Microarray and qPCR detected several additional genes putatively involved in this pathway enriched in the Pemba pyrethroid resistant population and we hypothesise that resistance may be, in part, related to alterations in the structure of the mosquito cuticle. None of the kdr target-site mutations, associated with pyrethroid/DDT resistance in An. gambiae elsewhere in Africa, were found on the islands.
Conclusion
The consequences of this resistance phenotype are discussed in relation to future vector control strategies on Zanzibar to support the ongoing malaria elimination efforts on the islands.
doi:10.1186/1756-3305-6-343
PMCID: PMC3895773  PMID: 24314005
Anopheles arabiensis; Zanzibar; Pyrethroid resistance; P450s; Gene expression
21.  Predicting Return of Fear Following Exposure Therapy With an Implicit Measure of Attitudes 
Behaviour research and therapy  2012;50(12):767-774.
We sought to advance understanding of the processes underlying the efficacy of exposure therapy and particularly the phenomenon of return of fear (ROF) following treatment by drawing on a social psychological view of phobias as attitudes. Specifically, a dual process theory of attitude-related behavior predicts that a positive response to exposure therapy may reflect change in either the automatic (the attitude representation itself) or controlled (skills and confidence at coping with the fear) responses to the phobic stimulus, or both. However, if the attitude representation remains negative following treatment, ROF should be more likely. We tested this hypothesis in a clinical sample of individuals with public speaking phobia using a single-session exposure therapy protocol previously shown to be efficacious but also associated with some ROF. Consistent with predictions, a post-treatment implicit measure of attitudes toward public speaking (the Personalized Implicit Association Test [PIAT]) predicted ROF at 1-month follow-up. These results suggest that change in the automatically activated attitude toward the phobic stimulus is an important goal of exposure therapy and that an implicit measure like the PIAT can provide a useful measure of such change by which to gauge the adequacy of exposure treatment and predict its long-term efficacy.
doi:10.1016/j.brat.2012.08.007
PMCID: PMC3498531  PMID: 23085186
Exposure; Return of fear; Social phobia; Fear of public speaking; Implicit measures; Attitudes
22.  Metabolomics of forage plants: a review 
Annals of Botany  2012;110(6):1281-1290.
Background
Forage plant breeding is under increasing pressure to deliver new cultivars with improved yield, quality and persistence to the pastoral industry. New innovations in DNA sequencing technologies mean that quantitative trait loci analysis and marker-assisted selection approaches are becoming faster and cheaper, and are increasingly used in the breeding process with the aim to speed it up and improve its precision. High-throughput phenotyping is currently a major bottle neck and emerging technologies such as metabolomics are being developed to bridge the gap between genotype and phenotype; metabolomics studies on forages are reviewed in this article.
Scope
Major challenges for pasture production arise from the reduced availability of resources, mainly water, nitrogen and phosphorus, and metabolomics studies on metabolic responses to these abiotic stresses in Lolium perenne and Lotus species will be discussed here. Many forage plants can be associated with symbiotic microorganisms such as legumes with nitrogen fixing rhizobia, grasses and legumes with phosphorus-solubilizing arbuscular mycorrhizal fungi, and cool temperate grasses with fungal anti-herbivorous alkaloid-producing Neotyphodium endophytes and metabolomics studies have shown that these associations can significantly affect the metabolic composition of forage plants. The combination of genetics and metabolomics, also known as genetical metabolomics can be a powerful tool to identify genetic regions related to specific metabolites or metabolic profiles, but this approach has not been widely adopted for forages yet, and we argue here that more studies are needed to improve our chances of success in forage breeding.
Conclusions
Metabolomics combined with other ‘-omics’ technologies and genome sequencing can be invaluable tools for large-scale geno- and phenotyping of breeding populations, although the implementation of these approaches in forage breeding programmes still lags behind. The majority of studies using metabolomics approaches have been performed with model species or cereals and findings from these studies are not easily translated to forage species. To be most effective these approaches should be accompanied by whole-plant physiology and proof of concept (modelling) studies. Wider considerations of possible consequences of novel traits on the fitness of new cultivars and symbiotic associations need also to be taken into account.
doi:10.1093/aob/mcs023
PMCID: PMC3478039  PMID: 22351485
Metabolomics; forage plants; Lolium perenne; drought stress, nutrient stress; Neotyphodium spp. endophytes; Medicago sativa; Lotus spp.; arbuscular mycorrhizal fungi; rhizobia; metabolic profiling; genetical metabolomics
23.  Genetic Basis of Human Circadian Rhythm Disorders 
Experimental neurology  2012;243:28-33.
Circadian rhythm disorders constitute a group of phenotypes that usually present as altered sleep-wake schedules. Until a human genetics approach was applied to investigate these traits, the genetic components regulating human circadian rhythm and sleep behaviors remained mysterious. Steady advances in the last decade have dramatically improved our understanding of the genes involved in circadian rhythmicity and sleep regulation. Finding these genes presents new opportunities to use a wide range of approaches, including in vitro molecular studies and in vivo animal modeling, to elevate our understanding of how sleep and circadian rhythms are regulated and maintained. Ultimately, this knowledge will reveal how circadian and sleep disruption contribute to various ailments and shed light on how best to maintain and recover good health.
doi:10.1016/j.expneurol.2012.07.012
PMCID: PMC3514403  PMID: 22849821
Circadian rhythm; Circadian rhythm sleep disorders; Advanced sleep phase; Delayed sleep phase; Advanced sleep phase disorder
24.  Contrasting selected reproductive challenges of today with those of antiquity—the past is prologue 
The Ulster Medical Journal  2013;82(3):150-156.
Viewing human history through a medical lens provides a renewed appreciation for today’s vexing reproductive challenges, as some modern dilemmas are actually continuations of similar challenges experienced long ago. Certainly there are many examples of assisted fertility therapy that were entirely theoretical only a generation ago, but have become commonplace in modern practice and society. In particular posthumous birth and infertility have, over time, been the focus of compelling social interest, occasionally even impacting national security and dynastic succession. While the concepts have remained static, the tools available to extend and improve reproductive success have changed radically. Appropriately regarded as confidential and private, an individual’s reproductive details are typically impervious to formal study. Yet, archival sources including ancient literature and formal court records can occasionally provide evidence of otherwise deeply personal concerns of a different era. Our assessment finds the issues, worries, and desires of patients of antiquity to align closely with contemporary reproductive challenges. Because children and family have always been central to the human experience, the consequences of reproduction (or the lack thereof) can make substantial imprints upon the cultural, economic, and political landscape—irrespective of civilization or century. In this article, selected motifs are described in a broad historical context to illustrate how challenges of human reproduction have remained essentially unchanged, despite a vast accumulation of knowledge made possible by gains in reproductive science and technology.
Plus ça change, plus c’est la même chose.
-Jean-Baptiste Alphonse Karr (1808–1890)
PMCID: PMC3913404  PMID: 24505149
25.  AmrZ Modulates Pseudomonas aeruginosa Biofilm Architecture by Directly Repressing Transcription of the psl Operon 
Journal of Bacteriology  2013;195(8):1637-1644.
Pseudomonas aeruginosa strains recovered from chronic pulmonary infections in cystic fibrosis patients are frequently mucoid. Such strains express elevated levels of alginate but reduced levels of the aggregative polysaccharide Psl; however, the mechanistic basis for this regulation is not completely understood. Elevated pslA expression was observed in an amrZ null mutant and in strains expressing a DNA-binding-deficient AmrZ. AmrZ is a transcription factor that positively regulates twitching motility and alginate synthesis, two phenotypes involved in P. aeruginosa biofilm development. AmrZ bound directly to the pslA promoter in vitro, and molecular analyses indicate that AmrZ represses psl expression by binding to a site overlapping the promoter. Altered expression of amrZ in nonmucoid strains impacted biofilm structure and architecture, as structured microcolonies were observed with low AmrZ production and flat biofilms with amrZ overexpression. These biofilm phenotypes correlated with Psl levels, since we observed elevated Psl production in amrZ mutants and lower Psl production in amrZ-overexpressing strains. These observations support the hypothesis that AmrZ is a multifunctional regulator mediating transition of P. aeruginosa biofilm infections from colonizing to chronic biofilms through repression of the psl operon while activating the algD operon.
doi:10.1128/JB.02190-12
PMCID: PMC3624555  PMID: 23354748

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