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1.  Direct, Catalytic and Regioselective Synthesis of 2-Alkyl, Aryl, and Alkenyl-Substituted N-Heterocycles from N-Oxides 
Organic letters  2014;16(3):864-867.
A one-step transformation of heterocyclic N-oxides to 2-alkyl, aryl, and alkenyl-substituted N-heterocycles is described. The success of this broad-scope methodology hinges on the combination of copper catalysis and activation by lithium fluoride or magnesium chloride. The utility of this method for the late-stage modification of complex N-heterocycles is exemplified by facile syntheses of new structural analogs of several antimalarial, antimicrobial and fungicidal agents.
PMCID: PMC4134923  PMID: 24410049
2.  Why not lie? Costs enforce honesty in an experimental signalling game 
Communication depends on reliability. Yet, the existence of stable honest signalling presents an evolutionary puzzle. Why should animals signal honestly in the face of a conflict of interest? While students of animal signalling have offered several theoretical answers to this puzzle, the most widely studied model, commonly called the ‘handicap principle’, postulates that the costs of signals stabilize honesty. This model is the motivating force behind an enormous research enterprise that explores signal costs—whether they are physiological, immunological, neural, developmental or caloric. While there can be no question that many signals are costly, we lack definitive experimental evidence demonstrating that costs stabilize honesty. This study presents a laboratory signalling game using blue jays (Cyanocitta cristata) that provides, to our knowledge, the first experimental evidence showing honesty persists when costs are high and disappears when costs are low.
PMCID: PMC3843836  PMID: 24225460
communication; honesty; signal costs; handicap principle; game theory
3.  Propensity Score Estimation in the Presence of Length-biased Sampling: A Nonparametric Adjustment Approach 
Stat  2014;3(1):83-94.
The pervasive use of prevalent cohort studies on disease duration increasingly calls for an appropriate methodology to account for the biases that invariably accompany samples formed by such data. It is well-known, for example, that subjects with shorter lifetime are less likely to be present in such studies. Moreover, certain covariate values could be preferentially selected into the sample, being linked to the long-term survivors. The existing methodology for estimating the propensity score using data collected on prevalent cases requires the correct conditional survival/hazard function given the treatment and covariates. This requirement can be alleviated if the disease under study has stationary incidence, the so-called stationarity assumption. We propose a nonparametric adjustment technique based on a weighted estimating equation for estimating the propensity score which does not require modeling the conditional survival/hazard function when the stationarity assumption holds. The estimator’s large-sample properties are established and its small-sample behavior is studied via simulation. The estimated propensity score is utilized to estimate the survival curves.
PMCID: PMC4142657  PMID: 25170178
Propensity score; Length-biased sampling; Causal inference; Survival curve
4.  Two-year seizure reduction in adults with medically intractable partial onset epilepsy treated with responsive neurostimulation: Final results of the RNS System Pivotal trial 
Epilepsia  2014;55(3):432-441.
To demonstrate the safety and effectiveness of responsive stimulation at the seizure focus as an adjunctive therapy to reduce the frequency of seizures in adults with medically intractable partial onset seizures arising from one or two seizure foci.
Randomized multicenter double-blinded controlled trial of responsive focal cortical stimulation (RNS System). Subjects with medically intractable partial onset seizures from one or two foci were implanted, and 1 month postimplant were randomized 1:1 to active or sham stimulation. After the fifth postimplant month, all subjects received responsive stimulation in an open label period (OLP) to complete 2 years of postimplant follow-up.
All 191 subjects were randomized. The percent change in seizures at the end of the blinded period was −37.9% in the active and −17.3% in the sham stimulation group (p = 0.012, Generalized Estimating Equations). The median percent reduction in seizures in the OLP was 44% at 1 year and 53% at 2 years, which represents a progressive and significant improvement with time (p < 0.0001). The serious adverse event rate was not different between subjects receiving active and sham stimulation. Adverse events were consistent with the known risks of an implanted medical device, seizures, and of other epilepsy treatments. There were no adverse effects on neuropsychological function or mood.
Responsive stimulation to the seizure focus reduced the frequency of partial-onset seizures acutely, showed improving seizure reduction over time, was well tolerated, and was acceptably safe. The RNS System provides an additional treatment option for patients with medically intractable partial-onset seizures.
PMCID: PMC4233950  PMID: 24621228
Cortical stimulation; Partial seizures; Focal seizures; Responsive stimulation; Neurostimulator
5.  Subunit composition of the human cytoplasmic dynein-2 complex 
Journal of Cell Science  2014;127(21):4774-4787.
Cytoplasmic dynein-2 is the motor for retrograde intraflagellar transport (IFT), and mutations in dynein-2 are known to cause skeletal ciliopathies. Here, we define for the first time the composition of the human cytoplasmic dynein-2 complex. We show that the proteins encoded by the ciliopathy genes WDR34 and WDR60 are bona fide dynein-2 intermediate chains and are both required for dynein-2 function. In addition, we identify TCTEX1D2 as a unique dynein-2 light chain that is itself required for cilia function. We define several subunits common to both dynein-1 and dynein-2, including TCTEX-1 (also known as DYNLT1) and TCTEX-3 (also known as DYNLT3), roadblock-1 (also known as DYNLRB1) and roadblock-2 (also known as DYNLRB2), and LC8-1 and LC8-2 light chains (DYNLL1 and DYNLL2, respectively). We also find that NudCD3 associates with dynein-2 as it does with dynein-1. By contrast, the common dynein-1 regulators dynactin, LIS1 (also known as PAFAH1B1) and BICD2 are not found in association with dynein-2. These data explain why mutations in either WDR34 or WDR60 cause disease, as well as identifying TCTEX1D2 as a candidate ciliopathy gene.
PMCID: PMC4215718  PMID: 25205765
Microtubule motor; Dynein; Cilia; Intraflagellar transport
6.  Molecular signatures of antibody responses derived from a systems biological study of 5 human vaccines 
Nature immunology  2013;15(2):195-204.
Many vaccines induce protective immunity via antibodies. Recent studies have used systems biological approaches to determine signatures that predict vaccine immunity in humans, but whether there is a ‘universal signature’ that can predict antibody responses to any vaccine, is unknown. Here we performed systems analyses of immune responses to the meningococcal polysaccharide and conjugate vaccines in healthy adults, in the broader context of our previous studies with the yellow fever and two influenza vaccines. To achieve this, we performed a large-scale network integration of public human blood transcriptomes, and systems-scale databases in specific biological contexts, and deduced a set of blood transcription modules. These modules revealed distinct transcriptional signatures of antibody responses to different classes of vaccines providing key insights into primary viral, protein recall and anti-polysaccharide responses. These results illuminate the early transcriptional programs orchestrating vaccine immunity in humans, and demonstrate the power of integrative network modeling.
PMCID: PMC3946932  PMID: 24336226
7.  Tonic Inhibition of Accumbal Spiny Neurons by Extrasynaptic α4βδ GABAA Receptors Modulates the Actions of Psychostimulants 
The Journal of Neuroscience  2014;34(3):823-838.
Within the nucleus accumbens (NAc), synaptic GABAA receptors (GABAARs) mediate phasic inhibition of medium spiny neurons (MSNs) and influence behavioral responses to cocaine. We demonstrate that both dopamine D1- and D2-receptor-expressing MSNs (D-MSNs) additionally harbor extrasynaptic GABAARs incorporating α4, β, and δ subunits that mediate tonic inhibition, thereby influencing neuronal excitability. Both the selective δ-GABAAR agonist THIP and DS2, a selective positive allosteric modulator, greatly increased the tonic current of all MSNs from wild-type (WT), but not from δ−/− or α4−/− mice. Coupling dopamine and tonic inhibition, the acute activation of D1 receptors (by a selective agonist or indirectly by amphetamine) greatly enhanced tonic inhibition in D1-MSNs but not D2-MSNs. In contrast, prolonged D2 receptor activation modestly reduced the tonic conductance of D2-MSNs. Behaviorally, WT and constitutive α4−/− mice did not differ in their expression of cocaine-conditioned place preference (CPP). Importantly, however, mice with the α4 deletion specific to D1-expressing neurons (α4D1−/−) showed increased CPP. Furthermore, THIP administered systemically or directly into the NAc of WT, but not α4−/− or α4D1−/− mice, blocked cocaine enhancement of CPP. In comparison, α4D2−/− mice exhibited normal CPP, but no cocaine enhancement. In conclusion, dopamine modulation of GABAergic tonic inhibition of D1- and D2-MSNs provides an intrinsic mechanism to differentially affect their excitability in response to psychostimulants and thereby influence their ability to potentiate conditioned reward. Therefore, α4βδ GABAARs may represent a viable target for the development of novel therapeutics to better understand and influence addictive behaviors.
PMCID: PMC3891962  PMID: 24431441
addiction; GABAA receptors; gaboxadol; nucleus accumbens; THIP
8.  Tracking a changing environment: optimal sampling, adaptive memory and overnight effects 
Behavioural processes  2011;89(2):86-94.
Foraging in a variable environment presents a classic problem of decision making with incomplete information. Animals must track the changing environment, remember the best options and make choices accordingly. While several experimental studies have explored the idea that sampling behavior reflects the amount of environmental change, we take the next logical step in asking how change influences memory. We explore the hypothesis that memory length should be tied to the ecological relevance and the value of the information learned, and that environmental change is a key determinant of the value of memory. We use a dynamic programming model to confirm our predictions and then test memory length in a factorial experiment. In our experimental situation we manipulate rates of change in a simple foraging task for blue jays over a 36 hour period. After jays experienced an experimentally determined change regime, we tested them at a range of retention intervals, from 1 to 72 hours. Manipulated rates of change influenced learning and sampling rates: subjects sampled more and learned more quickly in the high change condition. Tests of retention revealed significant interactions between retention interval and the experienced rate of change. We observed a striking and surprising difference between the high and low change treatments at the 24 hour retention interval. In agreement with earlier work we find that a circadian retention interval is special, but we find that the extent of this ‘specialness’ depends on the subject’s prior experience of environmental change. Specifically, experienced rates of change seem to influence how subjects balance recent information against past experience in a way that interacts with the passage of time.
PMCID: PMC4070854  PMID: 22024660
tracking; sampling; memory; environmental change; foraging; blue jays
9.  Models of Interinstitutional Partnerships between Research Intensive Universities and Minority Serving Institutions (MSI) across the Clinical Translational Science Award (CTSA) Consortium 
Health disparities are an immense challenge to American society. Clinical and Translational Science Awards (CTSAs) housed within the National Center for Advancing Translational Science (NCATS) are designed to accelerate the translation of experimental findings into clinically meaningful practices and bring new therapies to the doorsteps of all patients. Research Centers at Minority Institutions (RCMI) program at the National Institute on Minority Health and Health Disparities (NIMHD) are designed to build capacity for biomedical research and training at minority serving institutions. The CTSA created a mechanism fostering formal collaborations between research intensive universities and minority serving institutions (MSI) supported by the RCMI program. These consortium-level collaborations activate unique translational research approaches to reduce health disparities with credence to each academic institutions history and unique characteristics. Five formal partnerships between research intensive universities and MSI have formed as a result of the CTSA and RCMI programs. These partnerships present a multifocal approach; shifting cultural change and consciousness toward addressing health disparities, and training the next generation of minority scientists. This collaborative model is based on the respective strengths and contributions of the partnering institutions, allowing bidirectional interchange and leveraging NIH and institutional investments providing measurable benchmarks toward the elimination of health disparities.
PMCID: PMC4038384  PMID: 24119157
translational research; consortium; partnerships; minority serving institutions
Nature communications  2013;4:10.1038/ncomms3816.
Alcohol-dependence is a common, complex and debilitating disorder with genetic and environmental influences. Here we show that alcohol consumption increases following mutations to the γ-aminobutyric acidA receptor (GABAAR) β1 subunit gene (Gabrb1). Using N-ethyl-N-nitrosourea mutagenesis on an alcohol-averse background (F1 BALB/cAnN × C3H/HeH), we develop a mouse model exhibiting strong heritable preference for ethanol resulting from a dominant mutation (L285R) in Gabrb1. The mutation causes spontaneous GABA ion channel opening and increases GABA sensitivity of recombinant GABAARs, coupled to increased tonic currents in the nucleus accumbens, a region long-associated with alcohol reward. Mutant mice work harder to obtain ethanol, and are more sensitive to alcohol intoxication. Another spontaneous mutation (P228H) in Gabrb1 also causes high ethanol consumption accompanied by spontaneous GABA ion channel opening and increased accumbal tonic current. Our results provide a new and important link between GABAAR function and increased alcohol consumption that could underlie some forms of alcohol abuse.
PMCID: PMC3843143  PMID: 24281383
11.  Opposing microtubule motors control motility, morphology and cargo segregation during ER-to-Golgi transport 
Biology Open  2014;3(5):307-313.
We recently demonstrated that dynein and kinesin motors drive multiple aspects of endosomal function in mammalian cells. These functions include driving motility, maintaining morphology (notably through providing longitudinal tension to support vesicle fission), and driving cargo sorting. Microtubule motors drive bidirectional motility during traffic between the endoplasmic reticulum (ER) and Golgi. Here, we have examined the role of microtubule motors in transport carrier motility, morphology, and domain organization during ER-to-Golgi transport. We show that, consistent with our findings for endosomal dynamics, microtubule motor function during ER-to-Golgi transport of secretory cargo is required for motility, morphology, and cargo sorting within vesicular tubular carriers en route to the Golgi. Our data are consistent with previous findings that defined roles for dynein-1, kinesin-1 (KIF5B) and kinesin-2 in this trafficking step. Our high resolution tracking data identify some intriguing aspects. Depletion of kinesin-1 reduces the number of motile structures seen, which is in line with other findings relating to the role of kinesin-1 in ER export. However, those transport carriers that were produced had a much greater run length suggesting that this motor can act as a brake on anterograde motility. Kinesin-2 depletion did not significantly reduce the number of motile transport carriers but did cause a similar increase in run length. These data suggest that kinesins act as negative regulators of ER-to-Golgi transport. Depletion of dynein not only reduced the number of motile carriers formed but also caused tubulation of carriers similar to that seen for sorting nexin-coated early endosomes. Our data indicated that the previously observed anterograde–retrograde polarity of transport carriers in transit to the Golgi from the ER is maintained by microtubule motor function.
PMCID: PMC4021352  PMID: 24705013
Microtubule motor; Dynein; Kinesin; Endoplasmic reticulum; Golgi; Secretory cargo trafficking
12.  A Comparison of Supervised Classification Methods for the Prediction of Substrate Type Using Multibeam Acoustic and Legacy Grain-Size Data 
PLoS ONE  2014;9(4):e93950.
Detailed seabed substrate maps are increasingly in demand for effective planning and management of marine ecosystems and resources. It has become common to use remotely sensed multibeam echosounder data in the form of bathymetry and acoustic backscatter in conjunction with ground-truth sampling data to inform the mapping of seabed substrates. Whilst, until recently, such data sets have typically been classified by expert interpretation, it is now obvious that more objective, faster and repeatable methods of seabed classification are required. This study compares the performances of a range of supervised classification techniques for predicting substrate type from multibeam echosounder data. The study area is located in the North Sea, off the north-east coast of England. A total of 258 ground-truth samples were classified into four substrate classes. Multibeam bathymetry and backscatter data, and a range of secondary features derived from these datasets were used in this study. Six supervised classification techniques were tested: Classification Trees, Support Vector Machines, k-Nearest Neighbour, Neural Networks, Random Forest and Naive Bayes. Each classifier was trained multiple times using different input features, including i) the two primary features of bathymetry and backscatter, ii) a subset of the features chosen by a feature selection process and iii) all of the input features. The predictive performances of the models were validated using a separate test set of ground-truth samples. The statistical significance of model performances relative to a simple baseline model (Nearest Neighbour predictions on bathymetry and backscatter) were tested to assess the benefits of using more sophisticated approaches. The best performing models were tree based methods and Naive Bayes which achieved accuracies of around 0.8 and kappa coefficients of up to 0.5 on the test set. The models that used all input features didn't generally perform well, highlighting the need for some means of feature selection.
PMCID: PMC3974812  PMID: 24699553
13.  The Endoplasmic Reticulum Coat Protein II Transport Machinery Coordinates Cellular Lipid Secretion and Cholesterol Biosynthesis*  
The Journal of Biological Chemistry  2013;289(7):4244-4261.
Background: Sar1 mediates the onward transport of ER cargo.
Results: Sar1B promotes VLDL secretion, whereas Sar1A antagonizes this activity, and a deficit of both reduces cholesterol biosynthesis.
Conclusion: Sar1B independently of and through its lipoprotein secretion function promotes the expression of genes regulating cholesterol biosynthesis.
Significance: Sar1B-mediated transport activities contribute to both the functional integrity of the ER membrane and blood cholesterol levels.
Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions.
PMCID: PMC3924288  PMID: 24338480
Apolipoproteins; Cholesterol Regulation; Endoplasmic Reticulum (ER); Lipoprotein Secretion; Transcriptomics
14.  A role for endoplasmic reticulum exit sites in foot-and-mouth disease virus infection 
The Journal of General Virology  2013;94(Pt 12):2636-2646.
Picornaviruses replicate their genomes in association with cellular membranes. While enteroviruses are believed to utilize membranes of the early secretory pathway, the origin of the membranes used by foot-and-mouth disease virus (FMDV) for replication are unknown. Secretory-vesicle traffic through the early secretory pathway is mediated by the sequential acquisition of two distinct membrane coat complexes, COPII and COPI, and requires the coordinated actions of Sar1, Arf1 and Rab proteins. Sar1 is essential for generating COPII vesicles at endoplasmic reticulum (ER) exit sites (ERESs), while Arf1 and Rab1 are required for subsequent vesicle transport by COPI vesicles. In the present study, we have provided evidence that FMDV requires pre-Golgi membranes of the early secretory pathway for infection. Small interfering RNA depletion of Sar1 or expression of a dominant-negative (DN) mutant of Sar1a inhibited FMDV infection. In contrast, a dominant-active mutant of Sar1a, which allowed COPII vesicle formation but inhibited the secretory pathway by stabilizing COPII coats, caused major disruption to the ER–Golgi intermediate compartment (ERGIC) but did not inhibit infection. Treatment of cells with brefeldin A, or expression of DN mutants of Arf1 and Rab1a, disrupted the Golgi and enhanced FMDV infection. These results show that reagents that block the early secretory pathway at ERESs have an inhibitory effect on FMDV infection, while reagents that block the early secretory pathway immediately after ER exit but before the ERGIC and Golgi make infection more favourable. Together, these observations argue for a role for Sar1 in FMDV infection and that initial virus replication takes place on membranes that are formed at ERESs.
PMCID: PMC3836498  PMID: 23963534
15.  Adjunctive perampanel for refractory partial-onset seizures 
French, Jacqueline A. | Krauss, Gregory L. | Biton, Victor | Squillacote, David | Yang, Haichen | Laurenza, Antonio | Kumar, Dinesh | Rogawski, Michael A. | Campanille, Verónica | Floridia, Jorge | Ilari, Rita | Consalvo, Damián E. | Thomson, Alfredo | Sfaello, Ignacio | Pociecha, Juan | Nieto, Flavia | Firstenfeld, Alfredo | Zuin, Daniel | Mesri, Jacobo | Silva, Walter | Nofal, Pedro | Cristalli, Diana | Clement, Jean-Francois | Hwang, Paul | McLachlan, Richard | Pillay, Neelan | Lasso, Jorge | Peralta, Balduin Lawson | Hernandez, Marcelo Leiva | Tenhamm, Eugenio | Barroso, Noe | Milian, Albino Contreras | Morales, Sarug Reyes | Rodríguez, Ildefonso | Jain, Sanjay | Wilfong, Angus | Kalra, Arun | Renfroe, Ben | Moore, David | Flamini, J. | Klapper, Jack | Szaflarski, Jerzy | Teasley, Jean | Halford, Jonathan | Ferreira, Jose | Goodpasture, Hewitt | Sperling, Michael | Klein, Pavel | Ayala, Ricardo | Brower, Richard | Leroy, Robert | Davis, Ronald | Elterman, Roy | Enlow, Thomas | Sosa, Veronica | Puri, Vinay | Miller, John | Shneker, Bassel | Bautista, Ramon | Chung, Steve | Lesch, David | Steiner, David | Webb, Randall | Armstrong, Robert | Biton, Victor | Krauss, Gregory | Mitchell, Wendy | Hogan, Robert | Knowlton, Robert | Wheless, James | Aung-Din, Ronald | Ali, Imran | Brown, Lawrence | Gruenthal, Michael | Kankirawatana, Pongkiat | Charlet, Michael | Conry, Joan | Owen, Meriem Bensalem | Yerby, Mark | Herrman, Craig | Fessler, Albert | Modur, Pradeep | Miranda, Fernando | King-Stephens, David
Neurology  2012;79(6):589-596.
To assess efficacy and safety of once-daily 8 or 12 mg perampanel, a noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor antagonist, when added to concomitant antiepileptic drugs (AEDs) in the treatment of drug-resistant partial-onset seizures.
This was a multicenter, double-blind, placebo-controlled trial ( identifier: NCT00699972). Patients (≥12 years, with ongoing seizures despite 1–3 AEDs) were randomized (1:1:1) to once-daily perampanel 8 mg, 12 mg, or placebo. Following baseline (6 weeks), patients entered a 19-week double-blind phase: 6-week titration (2 mg/week increments to target dose) followed by a 13-week maintenance period. Percent change in seizure frequency was the primary endpoint; 50% responder rate was the primary endpoint for EU registration.
Of 388 patients randomized and treated, 387 provided seizure frequency data. Using this intent-to-treat population over the double-blind phase, the median percent change in seizure frequency was −21.0%, −26.3%, and −34.5% for placebo and perampanel 8 and 12 mg, respectively (p = 0.0261 and p = 0.0158 for 8 and 12 mg vs placebo, respectively). Fifty percent responder rates during the maintenance period were 26.4%, 37.6%, and 36.1%, respectively, for placebo, perampanel 8 mg, and perampanel 12 mg; these differences were not statistically significant for 8 mg (p = 0.0760) or 12 mg (p = 0.0914). Sixty-eight (17.5%) patients discontinued, including 40 (10.3%) for adverse events. Most frequent treatment-emergent adverse events were dizziness, somnolence, irritability, headache, fall, and ataxia.
This trial demonstrated that once-daily, adjunctive perampanel at doses of 8 or 12 mg improved seizure control in patients with uncontrolled partial-onset seizures. Doses of perampanel 8 and 12 mg were safe, and tolerability was acceptable.
Classification of evidence:
This study provides Class I evidence that once-daily 8 and 12 mg doses of adjunctive perampanel are effective in patients with uncontrolled partial-onset seizures.
PMCID: PMC3413761  PMID: 22843280
16.  Reductions in intestinal Clostridiales precede the development of nosocomial Clostridium difficile infection 
Microbiome  2013;1:18.
Antimicrobial use is thought to suppress the intestinal microbiota, thereby impairing colonization resistance and allowing Clostridium difficile to infect the gut. Additional risk factors such as proton-pump inhibitors may also alter the intestinal microbiota and predispose patients to Clostridium difficile infection (CDI). This comparative metagenomic study investigates the relationship between epidemiologic exposures, intestinal bacterial populations and subsequent development of CDI in hospitalized patients. We performed a nested case–control study including 25 CDI cases and 25 matched controls. Fecal specimens collected prior to disease onset were evaluated by 16S rRNA gene amplification and pyrosequencing to determine the composition of the intestinal microbiota during the at-risk period.
The diversity of the intestinal microbiota was significantly reduced prior to an episode of CDI. Sequences corresponding to the phylum Bacteroidetes and to the families Bacteroidaceae and Clostridiales Incertae Sedis XI were depleted in CDI patients compared to controls, whereas sequences corresponding to the family Enterococcaceae were enriched. In multivariable analyses, cephalosporin and fluoroquinolone use, as well as a decrease in the abundance of Clostridiales Incertae Sedis XI were significantly and independently associated with CDI development.
This study shows that a reduction in the abundance of a specific bacterial family - Clostridiales Incertae Sedis XI - is associated with risk of nosocomial CDI and may represent a target for novel strategies to prevent this life-threatening infection.
PMCID: PMC3971611  PMID: 24450844
Intestinal microbiota; Clostridium difficile infection; 16S rRNA gene sequencing; Clostridiales Incertae Sedis XI
17.  Acquired inducible antimicrobial resistance in Gram-positive bacteria 
Future microbiology  2012;7:959-978.
A major contributor to the emergence of antibiotic resistance in Gram-positive bacterial pathogens is the expansion of acquired, inducible genetic elements. Although acquired, inducible antibiotic resistance is not new, the interest in its molecular basis has been accelerated by the widening distribution and often ‘silent’ spread of the elements responsible, the diagnostic challenges of such resistance and the mounting limitations of available agents to treat Gram-positive infections. Acquired, inducible antibiotic resistance elements belong to the accessory genome of a species and are horizontally acquired by transformation/recombination or through the transfer of mobile DNA elements. The two key, but mechanistically very different, induction mechanisms are: ribosome-sensed induction, characteristic of the macrolide–lincosamide–streptogramin B antibiotics and tetracycline resistance, leading to ribosomal modifications or efflux pump activation; and resistance by cell surface-associated sensing of β-lactams (e.g., oxacillin), glycopeptides (e.g., vancomycin) and the polypeptide bacitracin, leading to drug inactivation or resistance due to cell wall alterations.
PMCID: PMC3464494  PMID: 22913355
antimicrobial resistance; Gram-positive bacteria; inducible resistance; mobile genetic elements; ribosomal stalling; transposon
18.  Microtubule motors mediate endosomal sorting by maintaining functional domain organization 
Journal of Cell Science  2013;126(11):2493-2501.
Many microtubule motors have been shown to couple to endosomal membranes. These motors include dynein in addition to many different kinesin family members. Sorting nexins (SNXs) are central to the organization and function of endosomes. These proteins can actively shape endosomal membranes and couple directly or indirectly to the minus-end microtubule motor dynein. Motor proteins acting on endosomes drive their motility, dictate their morphology and affect cargo segregation. We have used well-characterized members of the SNX family to elucidate motor coupling using high-resolution light microscopy coupled with depletion of specific microtubule motors. Endosomal domains labelled with SNX1, SNX4 and SNX8 couple to discrete combinations of dynein and kinesin motors. These specific combinations govern the structure and motility of each SNX-coated membrane in addition to the segregation of distinct functional endosomal subdomains. Taken together, our data show that these key features of endosome dynamics are governed by the same set of opposing microtubule motors. Thus, microtubule motors help to define the mosaic layout of endosomes that underpins cargo sorting.
PMCID: PMC3679488  PMID: 23549789
Microtubule motor; Endosome; Cargo sorting; Tubulation
19.  Description and Nomenclature of Neisseria meningitidis Capsule Locus 
Emerging Infectious Diseases  2013;19(4):566-573.
Pathogenic Neisseria meningitidis isolates contain a polysaccharide capsule that is the main virulence determinant for this bacterium. Thirteen capsular polysaccharides have been described, and nuclear magnetic resonance spectroscopy has enabled determination of the structure of capsular polysaccharides responsible for serogroup specificity. Molecular mechanisms involved in N. meningitidis capsule biosynthesis have also been identified, and genes involved in this process and in cell surface translocation are clustered at a single chromosomal locus termed cps. The use of multiple names for some of the genes involved in capsule synthesis, combined with the need for rapid diagnosis of serogroups commonly associated with invasive meningococcal disease, prompted a requirement for a consistent approach to the nomenclature of capsule genes. In this report, a comprehensive description of all N. meningitidis serogroups is provided, along with a proposed nomenclature, which was presented at the 2012 XVIIIth International Pathogenic Neisseria Conference.
PMCID: PMC3647402  PMID: 23628376
Neisseria meningitidis; capsule; serogroup; bacteria; nomenclature
20.  A role for Tctex-1 (DYNLT1) in controlling primary cilium length 
European journal of cell biology  2011;90(10):865-871.
The microtubule motor complex cytoplasmic dynein is known to be involved in multiple processes including endomembrane organization and trafficking, mitosis, and microtubule organization. The majority of studies of cytoplasmic dynein have focussed on the form of the motor that is built around the dynein-1 heavy chain. A second isoform, dynein heavy chain-2, and its specifically associated light intermediate chain, LIC3 (D2LIC), are known to be involved in the formation and function of primary cilia. We have used RNAi in human epithelial cells to define the cytoplasmic dynein subunits that function with dynein heavy chain 2 in primary cilia. We identify the dynein light chain Tctex-1 as a key modulator of cilia length control; depletion of Tctex-1 results in longer cilia as defined by both acetylated tubulin labelling of the axoneme and Rab8a labelling of the cilia membrane. Suppression of dynein heavy chain-2 causes concomitant loss of Tctex-1 and this correlates with an increase in cilia length. Compared to individual depletions, double siRNA depletion of DHC2 and Tctex-1 causes an even greater increase in cilia length. Our data show that Tctex-1 is a key regulator of cilia length and most likely functions as part of dynein-2.
PMCID: PMC3566477  PMID: 21700358
dynein; cilia
Nature  2012;482(7386):474-475.
Cells package proteins into vesicles for secretion to the extracellular milieu. A study shows that an enzyme modifies the packaging machinery to encapsulate unusually large proteins such as collagen.
PMCID: PMC3566552  PMID: 22358830
22.  Attachment and Invasion of Neisseria meningitidis to Host Cells Is Related to Surface Hydrophobicity, Bacterial Cell Size and Capsule 
PLoS ONE  2013;8(2):e55798.
We compared exemplar strains from two hypervirulent clonal complexes, strain NMB-CDC from ST-8/11 cc and strain MC58 from ST-32/269 cc, in host cell attachment and invasion. Strain NMB-CDC attached to and invaded host cells at a significantly greater frequency than strain MC58. Type IV pili retained the primary role for initial attachment to host cells for both isolates regardless of pilin class and glycosylation pattern. In strain MC58, the serogroup B capsule was the major inhibitory determinant affecting both bacterial attachment to and invasion of host cells. Removal of terminal sialylation of lipooligosaccharide (LOS) in the presence of capsule did not influence rates of attachment or invasion for strain MC58. However, removal of either serogroup B capsule or LOS sialylation in strain NMB-CDC increased bacterial attachment to host cells to the same extent. Although the level of inhibition of attachment by capsule was different between these strains, the regulation of the capsule synthesis locus by the two-component response regulator MisR, and the level of surface capsule determined by flow cytometry were not significantly different. However, the diplococci of strain NMB-CDC were shown to have a 1.89-fold greater surface area than strain MC58 by flow cytometry. It was proposed that the increase in surface area without changing the amount of anchored glycolipid capsule in the outer membrane would result in a sparser capsule and increase surface hydrophobicity. Strain NMB-CDC was shown to be more hydrophobic than strain MC58 using hydrophobicity interaction chromatography and microbial adhesion-to-solvents assays. In conclusion, improved levels of adherence of strain NMB-CDC to cell lines was associated with increased bacterial cell surface and surface hydrophobicity. This study shows that there is diversity in bacterial cell surface area and surface hydrophobicity within N. meningitidis which influence steps in meningococcal pathogenesis.
PMCID: PMC3566031  PMID: 23405216
23.  Early stages of retinal development depend on Sec13 function 
Biology Open  2013;2(3):256-266.
ER-to-Golgi transport of proteins destined for the extracellular space or intracellular compartments depends on the COPII vesicle coat and is constitutive in all translationally active cells. Nevertheless, there is emerging evidence that this process is regulated on a cell- and tissue-specific basis, which means that components of the COPII coat will be of differential importance to certain cell types. The COPII coat consists of an inner layer, Sec23/24 and an outer shell, Sec13/31. We have shown previously that knock-down of Sec13 results in concomitant loss of Sec31. In zebrafish and cultured human cells this leads to impaired trafficking of large cargo, namely procollagens, and is causative for defects in craniofacial and gut development. It is now widely accepted that the outer COPII coat is key to the architecture and stability of ER export vesicles containing large, unusual cargo proteins. Here, we investigate zebrafish eye development following Sec13 depletion. We find that photoreceptors degenerate or fail to develop from the onset. Impaired collagen trafficking from the retinal pigment epithelium and defects in overall retinal lamination also seen in Sec13-depleted zebrafish might have been caused by increased apoptosis and reduced topical proliferation in the retina. Our data show that the outer layer of the COPII coat is also necessary for the transport of large amounts of cargo proteins, in this case rhodopsin, rather than just large cargo as previously thought.
PMCID: PMC3603407  PMID: 23519012
COPII; Rhodopsin; ER export; Zebrafish; Eye
24.  Deletion of the gabra2 Gene Results in Hypersensitivity to the Acute Effects of Ethanol but Does Not Alter Ethanol Self Administration 
PLoS ONE  2012;7(10):e47135.
Human genetic studies have suggested that polymorphisms of the GABRA2 gene encoding the GABAA α2-subunit are associated with ethanol dependence. Variations in this gene also convey sensitivity to the subjective effects of ethanol, indicating a role in mediating ethanol-related behaviours. We therefore investigated the consequences of deleting the α2-subunit on the ataxic and rewarding properties of ethanol in mice. Ataxic and sedative effects of ethanol were explored in GABAA α2-subunit wildtype (WT) and knockout (KO) mice using a Rotarod apparatus, wire hang and the duration of loss of righting reflex. Following training, KO mice showed shorter latencies to fall than WT littermates under ethanol (2 g/kg i.p.) in both Rotarod and wire hang tests. After administration of ethanol (3.5 g/kg i.p.), KO mice took longer to regain the righting reflex than WT mice. To ensure the acute effects are not due to the gabra2 deletion affecting pharmacokinetics, blood ethanol concentrations were measured at 20 minute intervals after acute administration (2 g/kg i.p.), and did not differ between genotypes. To investigate ethanol’s rewarding properties, WT and KO mice were trained to lever press to receive increasing concentrations of ethanol on an FR4 schedule of reinforcement. Both WT and KO mice self-administered ethanol at similar rates, with no differences in the numbers of reinforcers earned. These data indicate a protective role for α2-subunits, against the acute sedative and ataxic effects of ethanol. However, no change was observed in ethanol self administration, suggesting the rewarding effects of ethanol remain unchanged.
PMCID: PMC3480382  PMID: 23115637
25.  Transmission Clustering Drives the Onward Spread of the HIV Epidemic Among Men Who Have Sex With Men in Quebec 
The Journal of Infectious Diseases  2011;204(7):1115-1119.
Phylodynamic analysis and epidemiologic data identified 3 patterns of spread of primary human immunodeficiency virus type 1 infection (PHI) among men who have sex with men (2001–2009): 420 unique PHIs, 102 small clusters (2–4 PHIs per cluster, n = 280), and 46 large clusters (5–31 PHIs per cluster, n = 450). Large clusters disproportionately increased from 25.2% of PHIs in 2005 to 39.1% in 2009 (χ2 = 33.9, P < .001). Scalar expansion of large clusters over 11 months (interquartile range, 3.5–25.5 months) correlated with cluster membership size (r2 = 0.174, F = 4.424, P = .047). PHI cohort data revealed variations in social networks and risk behaviors among the 3 groups, suggesting the need for tailored prevention measures.
PMCID: PMC3164430  PMID: 21881127

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