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1.  Intergenomic Comparisons Highlight Modularity of the Denitrification Pathway and Underpin the Importance of Community Structure for N2O Emissions 
PLoS ONE  2014;9(12):e114118.
Nitrous oxide (N2O) is a potent greenhouse gas and the predominant ozone depleting substance. The only enzyme known to reduce N2O is the nitrous oxide reductase, encoded by the nosZ gene, which is present among bacteria and archaea capable of either complete denitrification or only N2O reduction to di-nitrogen gas. To determine whether the occurrence of nosZ, being a proxy for the trait N2O reduction, differed among taxonomic groups, preferred habitats or organisms having either NirK or NirS nitrite reductases encoded by the nirK and nirS genes, respectively, 652 microbial genomes across 18 phyla were compared. Furthermore, the association of different co-occurrence patterns with enzymes reducing nitric oxide to N2O encoded by nor genes was examined. We observed that co-occurrence patterns of denitrification genes were not randomly distributed across taxa, as specific patterns were found to be more dominant or absent than expected within different taxonomic groups. The nosZ gene had a significantly higher frequency of co-occurrence with nirS than with nirK and the presence or absence of a nor gene largely explained this pattern, as nirS almost always co-occurred with nor. This suggests that nirS type denitrifiers are more likely to be capable of complete denitrification and thus contribute less to N2O emissions than nirK type denitrifiers under favorable environmental conditions. Comparative phylogenetic analysis indicated a greater degree of shared evolutionary history between nosZ and nirS. However 30% of the organisms with nosZ did not possess either nir gene, with several of these also lacking nor, suggesting a potentially important role in N2O reduction. Co-occurrence patterns were also non-randomly distributed amongst preferred habitat categories, with several habitats showing significant differences in the frequencies of nirS and nirK type denitrifiers. These results demonstrate that the denitrification pathway is highly modular, thus underpinning the importance of community structure for N2O emissions.
PMCID: PMC4250227  PMID: 25436772
2.  Dissecting the organ specificity of insecticide resistance candidate genes in Anopheles gambiae: known and novel candidate genes 
BMC Genomics  2014;15(1):1018.
The elevated expression of enzymes with insecticide metabolism activity can lead to high levels of insecticide resistance in the malaria vector, Anopheles gambiae. In this study, adult female mosquitoes from an insecticide susceptible and resistant strain were dissected into four different body parts. RNA from each of these samples was used in microarray analysis to determine the enrichment patterns of the key detoxification gene families within the mosquito and to identify additional candidate insecticide resistance genes that may have been overlooked in previous experiments on whole organisms.
A general enrichment in the transcription of genes from the four major detoxification gene families (carboxylesterases, glutathione transferases, UDP glucornyltransferases and cytochrome P450s) was observed in the midgut and malpighian tubules. Yet the subset of P450 genes that have previously been implicated in insecticide resistance in An gambiae, show a surprisingly varied profile of tissue enrichment, confirmed by qPCR and, for three candidates, by immunostaining. A stringent selection process was used to define a list of 105 genes that are significantly (p ≤0.001) over expressed in body parts from the resistant versus susceptible strain. Over half of these, including all the cytochrome P450s on this list, were identified in previous whole organism comparisons between the strains, but several new candidates were detected, notably from comparisons of the transcriptomes from dissected abdomen integuments.
The use of RNA extracted from the whole organism to identify candidate insecticide resistance genes has a risk of missing candidates if key genes responsible for the phenotype have restricted expression within the body and/or are over expression only in certain tissues. However, as transcription of genes implicated in metabolic resistance to insecticides is not enriched in any one single organ, comparison of the transcriptome of individual dissected body parts cannot be recommended as a preferred means to identify new candidate insecticide resistant genes. Instead the rich data set on in vivo sites of transcription should be consulted when designing follow up qPCR validation steps, or for screening known candidates in field populations.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1018) contains supplementary material, which is available to authorized users.
PMCID: PMC4256904  PMID: 25421852
Detoxification; Insecticide resistance; Microarray; Mosquito; Transcriptome
3.  Casein Kinase Iδ Mutations in Familial Migraine and Advanced Sleep Phase 
Science translational medicine  2013;5(183):183ra56-11.
Migraine is a common disabling disorder with a significant genetic component, characterized by severe headache and often accompanied by nausea, vomiting, and light sensitivity. We identified two families, each with a distinct missense mutation in the gene encoding casein kinase Iδ (CKIδ), in which the mutation cosegregated with both the presence of migraine and advanced sleep phase. The resulting alterations (T44A and H46R) occurred in the conserved catalytic domain of CKIδ, where they caused reduced enzyme activity. Mice engineered to carry the CKIδ-T44A allele were more sensitive to pain after treatment with the migraine trigger nitroglycerin. CKIδ-T44A mice also exhibited a reduced threshold for cortical spreading depression (believed to be the physiological analog of migraine aura) and greater arterial dilation during cortical spreading depression. Astrocytes from CKIδ-T44A mice showed increased spontaneous and evoked calcium signaling. These genetic, cellular, physiological, and behavioral analyses suggest that decreases in CKIδ activity can contribute to the pathogenesis of migraine.
PMCID: PMC4220792  PMID: 23636092
4.  Clinical trials registries are under-utilized in the conduct of systematic reviews: a cross-sectional analysis 
Systematic Reviews  2014;3:126.
Publication bias is a major threat to the validity of systematic reviews. Searches of clinical trials registries can help to identify unpublished trials, though little is known about how often these resources are utilized. We assessed the usage and results of registry searches reported in systematic reviews published in major general medical journals.
This cross-sectional analysis includes data from systematic reviews assessing medical interventions which were published in one of six major general medical journals between July 2012 and June 2013. Two authors independently examined each published systematic review and all available supplementary materials to determine whether at least one clinical trials registry was searched.
Of the 117 included systematic reviews, 41 (35%) reported searching a trials registry. Of the 29 reviews which also provided detailed registry search results, 15 (52%) identified at least one completed trial and 18 (62%) identified at least one ongoing trial.
Clinical trials registry searches are not routinely included in systematic reviews published in major medical journals. Routine examination of registry databases may allow a more accurate characterization of publication and outcome reporting biases and improve the validity of estimated effects of medical treatments.
PMCID: PMC4217330  PMID: 25348628; Systematic review; Trials registry; Publication bias
5.  The Tachykinin Peptide Neurokinin B Binds Copper Forming an Unusual [CuII(NKB)2] Complex and Inhibits Copper Uptake into 1321N1 Astrocytoma Cells 
ACS Chemical Neuroscience  2013;4(10):1371-1381.
Neurokinin B (NKB) is a member of the tachykinin family of neuropeptides that have neuroinflammatory, neuroimmunological, and neuroprotective functions. In a neuroprotective role, tachykinins can help protect cells against the neurotoxic processes observed in Alzheimer’s disease. A change in copper homeostasis is a clear feature of Alzheimer’s disease, and the dysregulation may be a contributory factor in toxicity. Copper has recently been shown to interact with neurokinin A and neuropeptide γ and can lead to generation of reactive oxygen species and peptide degradation, which suggests that copper may have a place in tachykinin function and potentially misfunction. To explore this, we have utilized a range of spectroscopic techniques to show that NKB, but not substance P, can bind CuII in an unusual [CuII(NKB)2] neutral complex that utilizes two N-terminal amine and two imidazole nitrogen ligands (from each molecule of NKB) and the binding substantially alters the structure of the peptide. Using 1321N1 astrocytoma cells, we show that copper can enter the cells and subsequently open plasma membrane calcium channels but when bound to neurokinin B copper ion uptake is inhibited. This data suggests a novel role for neurokinin B in protecting cells against copper-induced calcium changes and implicates the peptide in synaptic copper homeostasis.
PMCID: PMC3798990  PMID: 23875773
Neurokinin B; tachykinin; copper; calcium; neurodegeneration; substance P; Fura2; EPR
6.  Surface-Induced Dissociation Shows Potential to be More Informative than Collision-Induced Dissociation for Structural Studies of Large Systems 
The ability to preserve non-covalent, macromolecular assemblies intact in the gas-phase has paved the way for mass spectrometry to characterize ions of increasing size and become a powerful tool in the field of structural biology. Tandem mass spectrometry experiments have the potential to expand the capabilities of this technique through the gas-phase dissociation of macromolecular complexes, but collisions with small gas atoms currently provide very limited fragmentation. One alternative for dissociating large ions is to collide them into a surface, a more massive target. Here, we demonstrate the ability and benefit of fragmenting large protein complexes and inorganic salt clusters by surface-induced dissociation (SID). SID provides more extensive fragmentation of these systems and shows promise as an activation method for ions of increasing size.
PMCID: PMC4186222  PMID: 18598898
7.  Hepatic Lesions with Secondary Syphilis in an HIV-Infected Patient 
Case Reports in Medicine  2014;2014:604794.
Syphilis among HIV-infected patients continues to be a public health concern, especially in men who have sex with men. The clinical manifestations of syphilis are protean; syphilitic hepatitis is an unusual complication that can occur at any stage of the disease. We report a case of an HIV-infected male who presented with systemic symptoms and liver lesions highly suggestive of lymphoma and was proven to have syphilitic hepatitis by liver biopsy. Our case reinforces the importance of recognizing syphilis as a possible cause of unexplained abnormal liver enzymes and/or hepatic lesions in HIV-infected patients.
PMCID: PMC4202275  PMID: 25349616
8.  Increased Pyrethroid Resistance in Malaria Vectors and Decreased Bed Net Effectiveness, Burkina Faso 
Emerging Infectious Diseases  2014;20(10):1691-1696.
This new resistance will have serious effects on malaria control.
Malaria control is dependent on insecticides. Increases in prevalence of insecticide resistance in malaria vectors across Africa are well-documented. However, few attempts have been made to quantify the strength of this resistance and link it to the effectiveness of control tools. Using quantitative bioassays, we show that in Burkina Faso pyrethroid resistance in Anopheles gambiae mosquitoes has increased in intensity in recent years and now exceeds 1,000-fold. In laboratory assays, this level of resistance renders insecticides used to impregnate bed nets ineffective. Thus, the level of personal and community protection afforded by long-lasting insecticide-treated net campaigns will probably be reduced. Standardized methods are needed to quantify resistance levels in malaria vectors and link these levels to failure of vector control methods.
PMCID: PMC4193182  PMID: 25279965
malaria; parasites; pyrethroid resistance; Anopheles gambiae; mosquitoes; vector control; bed nets; bed net effectiveness; Burkina Faso
9.  Cognitive behavioural therapy versus other psychosocial treatments for schizophrenia 
Cognitive behavioural therapy (CBT) is now a recommended treatment for people with schizophrenia. This approach helps to link the person’s distress and problem behaviours to underlying patterns of thinking.
To review the effects of CBT for people with schizophrenia when compared with other psychological therapies.
Search methods
We searched the Cochrane Schizophrenia Group Trials Register (March 2010) which is based on regular searches of CINAHL, EMBASE, MEDLINE and PsycINFO. We inspected all references of the selected articles for further relevant trials, and, where appropriate, contacted authors.
Selection criteria
All relevant randomised controlled trials (RCTs) of CBT for people with schizophrenia-like illnesses.
Data collection and analysis
Studies were reliably selected and assessed for methodological quality. Two review authors, working independently, extracted data. We analysed dichotomous data on an intention-to-treat basis and continuous data with 65% completion rate are presented. Where possible, for dichotomous outcomes, we estimated a risk ratio (RR) with the 95% confidence interval (CI) along with the number needed to treat/harm.
Main results
Thirty one papers described 20 trials. Trials were often small and of limited quality. When CBT was compared with other psychosocial therapies, no difference was found for outcomes relevant to adverse effect/events (2 RCTs, n = 202, RR death 0.57 CI 0.12 to 2.60). Relapse was not reduced over any time period (5 RCTs, n = 183, RR long-term 0.91 CI 0.63 to 1.32) nor was rehospitalisation (5 RCTs, n = 294, RR in longer term 0.86 CI 0.62 to 1.21). Various global mental state measures failed to show difference (4 RCTs, n = 244, RR no important change in mental state 0.84 CI 0.64 to 1.09). More specific measures of mental state failed to show differential effects on positive or negative symptoms of schizophrenia but there may be some longer term effect for affective symptoms (2 RCTs, n = 105, mean difference (MD) Beck Depression Inventory (BDI) −6.21 CI −10.81 to −1.61). Few trials report on social functioning or quality of life. Findings do not convincingly favour either of the interventions (2 RCTs, n = 103, MD Social Functioning Scale(SFS) 1.32 CI −4.90 to 7.54; n = 37, MD EuroQOL −1.86 CI −19.20 to 15.48). For the outcome of leaving the study early, we found no significant advantage when CBT was compared with either non-active control therapies (4 RCTs, n = 433, RR 0.88 CI 0.63 to 1.23) or active therapies (6 RCTs, n = 339, RR 0.75 CI 0.40 to 1.43)
Authors’ conclusions
Trial-based evidence suggests no clear and convincing advantage for cognitive behavioural therapy over other - and sometime much less sophisticated - therapies for people with schizophrenia.
PMCID: PMC4163968  PMID: 22513966
Cognitive Therapy [*methods]; Schizophrenia [*therapy]; Adult; Humans; Middle Aged
10.  Structure of Dengue Virus: Implications for Flavivirus Organization, Maturation, and Fusion 
Cell  2002;108(5):717-725.
The first structure of a flavivirus has been determined by using a comThe first structure of a flavivirus has been determined by using a combination of cryoelectron microscopy and fitting of the known structure of glycoprotein E into the electron density map. The virus core, within a lipid bilayer, has a less-ordered structure than the external, icosahedral scaffold of 90 glycoprotein E dimers. The three E monomers per icosahedral asymmetric unit do not have quasiequivalent symmetric environments. Difference maps indicate the location of the small membrane protein M relative to the overlaying scaffold of E dimers. The structure suggests that flaviviruses, and by analogy also alphaviruses, employ a fusion mechanism in which the distal β barrels of domain II of the glycoprotein E are inserted into the cellular membrane.
PMCID: PMC4152842  PMID: 11893341
11.  Molecular characterization of DDT resistance in Anopheles gambiae from Benin 
Parasites & Vectors  2014;7(1):409.
Insecticide resistance in the mosquito vector is the one of the main obstacles against effective malaria control. In order to implement insecticide resistance management strategies, it is important to understand the genetic factors involved. In this context, we investigated the molecular basis of DDT resistance in the main malaria vector from Benin.
Anopheles gambiae mosquitoes were collected from four sites across Benin and identified to species/molecular form. Mosquitoes from Cotonou (M-form), Tori-Bossito (S-form) and Bohicon (S-form) were exposed to DDT 4% at a range of exposure times (30 min to 300 min). Another batch of mosquitoes from Cotonou and Malanville were exposed to DDT for 1 hour and the survivors 48 hours post exposure were used to quantify metabolic gene expression. Quantitative PCR assays were used to quantify mRNA levels of metabolic enzymes: GSTE2, GSTD3, CYP6P3 and CYP6M2. Expression (fold-change) was calculated using the ∆∆Ct method and compared to susceptible strains. Detection of target-site mutations (L1014F, L1014S and N1575Y) was performed using allelic discrimination TaqMan assays.
DDT resistance was extremely high in all populations, regardless of molecular form, with no observed mortality after 300 min exposure. In both DDT-survivors and non-exposed mosquitoes, GSTE2 and GSTD3 were over-expressed in the M form at 4.4-fold and 3.5-fold in Cotonou and 1.5-fold and 2.5-fold in Malanville respectively, when compared to the susceptible strain. The CYP6M2 and CYP6P3 were over-expressed at 4.6-fold and 3.8-fold in Cotonou and 1.2-fold and 2.5-fold in Malanville respectively. In contrast, no differences in GSTE2 and CYP6M2 were observed between S form mosquitoes from Tori-Bossito and Bohicon compared to susceptible strain. The 1014 F allele was fixed in the S-form and at high frequency in the M-form (0.7-0.914). The frequency of 1575Y allele was 0.29-0.36 in the S-form and nil in the M-form. The 1014S allele was detected in the S form of An. gambiae in a 1014 F/1014S heterozygous specimen.
Our results show that the kdr 1014 F, 1014S and 1575Y alleles are widespread in Benin and the expression of two candidate metabolic markers (GSTE2 and CYP6M2) are over-expressed specifically in the M-form.
PMCID: PMC4164740  PMID: 25175167
An. gambiae; Insecticide resistance; qPCR; Kdr mutation; Vector control; Metabolic enzymes
12.  A randomized, controlled trial comparing the efficacy and safety of aqueous subcutaneous progesterone with vaginal progesterone for luteal phase support of in vitro fertilization 
Human Reproduction (Oxford, England)  2014;29(10):2212-2220.
Is the ongoing pregnancy rate with a new aqueous formulation of subcutaneous progesterone (Prolutex®) non-inferior to vaginal progesterone (Endometrin®) when used for luteal phase support of in vitro fertilization?
In the per-protocol (PP) population, the ongoing pregnancy rates per oocyte retrieval at 12 weeks of gestation were comparable between Prolutex and Endometrin (41.6 versus 44.4%), with a difference between groups of −2.8% (95% confidence interval (CI) −9.7, 4.2), consistent with the non-inferiority of subcutaneous progesterone for luteal phase support.
Luteal phase support has been clearly demonstrated to improve pregnancy rates in women undergoing in vitro fertilization (IVF). Because of the increased risk of ovarian hyperstimulation syndrome associated with the use of hCG, progesterone has become the treatment of choice for luteal phase support.
This prospective, open-label, randomized, controlled, parallel-group, multicentre, two-arm, non-inferiority study was performed at eight fertility clinics. A total of 800 women, aged 18–42 years, with a BMI of ≤30 kg/m2, with <3 prior completed assisted reproductive technology (ART) cycles, exhibiting baseline (Days 2–3) FSH of ≤15 IU/L and undergoing IVF at 8 centres (seven private, one academic) in the USA, were enrolled from January 2009 through June 2011.
In total, 800 women undergoing IVF were randomized after retrieval of at least three oocytes to an aqueous preparation of progesterone administered subcutaneously (25 mg daily) or vaginal progesterone (100 mg bid daily). Randomization was performed to enrol 100 patients at each site using a randomization list that was generated with Statistical Analysis Software (SAS®). If a viable pregnancy occurred, progesterone treatment was continued up to 12 weeks of gestation.
Using a PP analysis, which included all patients who received an embryo transfer (Prolutex = 392; Endometrin = 390), the ongoing pregnancy rate per retrieval for subcutaneous versus vaginal progesterone was 41.6 versus 44.4%, with a difference between groups of −2.8% (95% CI −9.7, 4.2), consistent with the non-inferiority of subcutaneous progesterone for luteal phase support. In addition, rates of initial positive β-hCG (56.4% subcutaneous versus 59.0% vaginal; 95% CI −9.5, 4.3), clinical intrauterine pregnancy with fetal cardiac activity (42.6 versus 46.4%; 95% CI −10.8, 3.2), implantation defined as number of gestational sacs divided by number of embryos transferred (33.2 versus 35.1%; 95% CI −7.6, 4.0), live birth (41.1 versus 43.1%; 95% CI −8.9, 4.9) and take-home baby (41.1 versus 42.6%; 95% CI −8.4, 5.4) were comparable. Both formulations were well-tolerated, with no difference in serious adverse events. Analysis with the intention-to-treat population also demonstrated no difference for any outcomes between the treatment groups.
The conclusions are limited to the progesterone dosing regimen studied and duration of treatment for the patient population examined in this study.
Subcutaneous progesterone represents a novel option for luteal phase support in women undergoing IVF who for personal reasons prefer not to use a vaginal preparation or who wish to avoid the side effects of vaginal or i.m. routes of administration.
The study was funded by Institut Biochimique SA (IBSA). CAJ, BC, ST and CJ are employees of IBSA. FH currently consults for IBSA.
PMCID: PMC4164149  PMID: 25100106
progesterone; luteal phase support; in vitro fertilization; RCT
13.  Loss in microbial diversity affects nitrogen cycling in soil 
The ISME Journal  2013;7(8):1609-1619.
Microbial communities have a central role in ecosystem processes by driving the Earth's biogeochemical cycles. However, the importance of microbial diversity for ecosystem functioning is still debated. Here, we experimentally manipulated the soil microbial community using a dilution approach to analyze the functional consequences of diversity loss. A trait-centered approach was embraced using the denitrifiers as model guild due to their role in nitrogen cycling, a major ecosystem service. How various diversity metrics related to richness, eveness and phylogenetic diversity of the soil denitrifier community were affected by the removal experiment was assessed by 454 sequencing. As expected, the diversity metrics indicated a decrease in diversity in the 1/103 and 1/105 dilution treatments compared with the undiluted one. However, the extent of dilution and the corresponding reduction in diversity were not commensurate, as a dilution of five orders of magnitude resulted in a 75% decrease in estimated richness. This reduction in denitrifier diversity resulted in a significantly lower potential denitrification activity in soil of up to 4–5 folds. Addition of wheat residues significantly increased differences in potential denitrification between diversity levels, indicating that the resource level can influence the shape of the microbial diversity–functioning relationship. This study shows that microbial diversity loss can alter terrestrial ecosystem processes, which suggests that the importance of functional redundancy in soil microbial communities has been overstated.
PMCID: PMC3721106  PMID: 23466702
biodiversity; ecosystem functioning; denitrification; functional redundancy; nitrogen cycling; soil
15.  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.
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.
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).
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.
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.
PMCID: PMC4082164  PMID: 24946780
Anopheles arabiensis; Genes; Microarrays; Permethrin; Resistance; Transcription
16.  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 
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.
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.
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.
PMCID: PMC3646974  PMID: 23474109
Prostate carcinoma; Ki-67 Antigen/Analysis; Prognosis; Metastasis; Biomarkers
17.  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.
PMCID: PMC3658935  PMID: 23563879
18.  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.
PMCID: PMC3742305  PMID: 23517755
Exoribonuclease; XRN; XRN1/PACMAN; XRN4; XRN2/RAT1; RNA decay; Small RNA; Transcriptional surveillance
19.  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.
PMCID: PMC3646915  PMID: 23416558
chaperone; α-crystallin domain; protein flexibility; subunit exchange; hydrogen deuterium exchange; mass spectrometry of protein complexes
20.  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.
PMCID: PMC3894417  PMID: 24259577
21.  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.
PMCID: PMC4023308  PMID: 24868514
Left atrium calcification; Heart of stone; Atrial calcification; Dystrophic cardiac calcification; Renal failure
22.  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.
PMCID: PMC3968121  PMID: 24675720
23.  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.
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.
PMCID: PMC3964821  PMID: 24668172
electron cryotomography; correlative microscopy; chemotaxis; Rhodobacter sphaeroides; Vibrio cholerae; E. coli
24.  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.
PMCID: PMC3752004  PMID: 23432621
Fragment-based drug discovery; Molecular dynamics; MM/PBSA; N5-CAIR mutase; PurE
25.  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.
PMCID: PMC3964364  PMID: 22309720
sleep deprivation; stress; stressors; mood

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