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1.  The pmr Gene, Encoding a Ca2+-ATPase, Is Required for Calcium and Manganese Homeostasis and Normal Development of Hyphae and Conidia in Neurospora crassa 
Eukaryotic Cell  2012;11(11):1362-1370.
The pmr gene is predicted to encode a Ca2+-ATPase in the secretory pathway. We examined two strains of Neurospora crassa that lacked PMR: the Δpmr strain, in which pmr was completely deleted, and pmrRIP, in which the gene was extensively mutated. Both strains had identical, complex phenotypes. Compared to the wild type, these strains required high concentrations of calcium or manganese for optimal growth and had highly branched, slow-growing hyphae. They conidiated poorly, and the shape and size of the conidia were abnormal. Calcium accumulated in the Δpmr strains to only 20% of the wild-type level. High concentrations of MnCl2 (1 to 5 mM) in growth medium partially suppressed the morphological defects but did not alter the defect in calcium accumulation. The Δpmr Δnca-2 double mutant (nca-2 encodes a Ca2+-ATPase in the plasma membrane) accumulated 8-fold more calcium than the wild type, and the morphology of the hyphae was more similar to that of wild-type hyphae. Previous experiments failed to show a function for nca-1, which encodes a SERCA-type Ca2+-ATPase in the endoplasmic reticulum (B. J. Bowman, S. Abreu, E. Margolles-Clark, M. Draskovic, and E. J. Bowman, Eukaryot. Cell 10:654-661, 2011). The pmrRIP Δnca-1 double mutant accumulated small amounts of calcium, like the Δpmr strain, but exhibited even more extreme morphological defects. Thus, PMR can apparently replace NCA-1 in the endoplasmic reticulum, but NCA-1 cannot replace PMR. The morphological defects in the Δpmr strain are likely caused, in part, by insufficient concentrations of calcium and manganese in the Golgi compartment; however, PMR is also needed to accumulate normal levels of calcium in the whole cell.
PMCID: PMC3486030  PMID: 22983986
2.  Role of Four Calcium Transport Proteins, Encoded by nca-1, nca-2, nca-3, and cax, in Maintaining Intracellular Calcium Levels in Neurospora crassa ▿ 
Eukaryotic Cell  2011;10(5):654-661.
We have examined the distribution of calcium in Neurospora crassa and investigated the role of four predicted calcium transport proteins. The results of cell fractionation experiments showed 4% of cellular calcium in mitochondria, approximately 11% in a dense vacuolar fraction, 40% in an insoluble form that copurifies with microsomes, and 40% in a high-speed supernatant, presumably from large vacuoles that had broken. Strains lacking NCA-1, a SERCA-type Ca2+-ATPase, or NCA-3, a PMC-type Ca2+-ATPase, had no obvious defects in growth or distribution of calcium. A strain lacking NCA-2, which is also a PMC-type Ca2+-ATPase, grew slowly in normal medium and was unable to grow in high concentrations of calcium tolerated by the wild type. Furthermore, when grown in normal concentrations of calcium (0.68 mM), this strain accumulated 4- to 10-fold more calcium than other strains, elevated in all cell fractions. The data suggest that NCA-2 functions in the plasma membrane to pump calcium out of the cell. In this way, it resembles the PMC-type enzymes of animal cells, not the Pmc1p enzyme in Saccharomyces cerevisiae that resides in the vacuole. Strains lacking the cax gene, which encodes a Ca2+/H+ exchange protein in vacuolar membranes, accumulate very little calcium in the dense vacuolar fraction but have normal levels of calcium in other fractions. The cax knockout strain has no other observable phenotypes. These data suggest that “the vacuole” is heterogeneous and that the dense vacuolar fraction contains an organelle that is dependent upon the CAX transporter for accumulation of calcium, while other components of the vacuolar system have multiple calcium transporters.
PMCID: PMC3127652  PMID: 21335528
3.  Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa▿  
Eukaryotic Cell  2009;8(12):1845-1855.
We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca2+-ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca2+-ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca2+/H+ exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 μm from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.
PMCID: PMC2794220  PMID: 19801418
4.  Selective criteria for the microbiological examination of faecal specimens. 
Journal of Clinical Pathology  1992;45(9):838-839.
To assess the effectiveness of predetermined investigation criteria for the examination of faecal samples from inpatients, cultured stool specimens were prospectively examined for Salmonella spp, Shigella spp, Campylobacter spp and Clostridium difficile, and screened microscopically for intestinal parasites. Out of a total of 505 specimens, 421 (83%) fulfilled the criteria for examination for C difficile, 254 (50%) for Salmonella spp, Shigella spp, and Campylobacter spp, and 87 (17%) for intestinal parasites. Isolation rates for these organisms in those groups of patients where examination was indicated were 22.5% for C difficile and 9.1% for Salmonella spp, Shigella spp, and Campylobacter spp; the detection rate for parasites was 3.5%. In those patients where the criteria did not suggest investigation, the isolation or detection rates were 3.6% for C difficile, 0% for Salmonella spp, Shigella spp, and Campylobacter spp, and 1.7% for intestinal parasites, suggesting that the use of predetermined investigation criteria was effective.
PMCID: PMC495122  PMID: 1401225
5.  Identification and properties of an ATPase in vacuolar membranes of Neurospora crassa. 
Journal of Bacteriology  1982;151(3):1326-1337.
Using a vacuolar preparation virtually free of contamination by other organelles, we isolated vacuolar membranes and demonstrated that they contain an ATPase. Sucrose density gradient profiles of vacuolar membranes show a single peak of ATPase activity at a density of 1.11 g/cm3. Comparison of this enzyme with the two well-studied proton-pumping ATPases of Neurospora plasma membranes and mitochondria shows that it is clearly distinct. The vacuolar membrane ATPase is insensitive to the inhibitors oligomycin, azide, and vanadate, but sensitive to N,N'-dicyclohexylcarbodiimide (Ki = 2 microM). It has a pH optimum of 7.5, requires a divalent cation (Mg2+ or Mn2+) for activity, and is remarkably unaffected (+/- 20%) by a number of monovalent cations, anions, and buffers. In its substrate affinity (Km for ATP = 0.2 mM), substrate preference (ATP greater than GTP, ITP greater than UTP greater than CTP), and loss of activity with repeated 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid washes, the vacuolar membrane ATPase resembles the F1F0 type of ATPase found in mitochondria and differs from the integral membrane type of ATPase in plasma membranes.
PMCID: PMC220411  PMID: 6213602
7.  Strength of Axial Water Ligation in Substrate-Free Cytochrome P450s Is Isoform Dependent 
Biochemistry  2014;53(9):1428-1434.
The heme-containing cytochrome P450s exhibit isoform-dependent ferric spin equilibria in the resting state and differential substrate-dependent spin equilibria. The basis for these differences is not well understood. Here, magnetic circular dichroism (MCD) reveals significant differences in the resting low spin ligand field of CYPs 3A4, 2E1, 2C9, 125A1, and 51B1, which indicates differences in the strength of axial water ligation to the heme. The near-infrared bands that specifically correspond to charge-transfer porphyrin-to-metal transitions span a range of energies of nearly 2 kcal/mol. In addition, the experimentally determined MCD bands are not entirely in agreement with the expected MCD energies calculated from electron paramagnetic resonance parameters, thus emphasizing the need for the experimental data. MCD marker bands of the high spin heme between 500 and 680 nm were also measured and suggest only a narrow range of energies for this ensemble of high spin Cys(S–) → Fe3+ transitions among these isoforms. The differences in axial ligand energies between CYP isoforms of the low spin states likely contribute to the energetics of substrate-dependent spin state perturbation. However, these ligand field energies do not correlate with the fraction of high spin vs low spin in the resting state enzyme, suggestive of differences in water access to the heme or isoform-dependent differences in the substrate-free high spin states as well.
PMCID: PMC3985942  PMID: 24576089
8.  A Catalytic Enantiotopic-Group-Selective Suzuki Reaction for the Construction of Chiral Organoboronates 
Catalytic enantiotopic-group-selective cross-couplings of achiral geminal bis(pinacolboronates) provide a route for the construction of nonracemic chiral organoboronates. In the presence of a chiral monodentate taddol-derived phosphoramidite ligand, these reactions occur with high levels of asymmetric induction. Mechanistic experiments with chiral 10B-enriched geminal bis(boronates) suggest that the reaction occurs by a stereochemistry-determining transmetalation that occurs with inversion of configuration at carbon.
PMCID: PMC4021567  PMID: 24564423
9.  Investigating the introduction of porcine epidemic diarrhea virus into an Ohio swine operation 
Porcine Epidemic Diarrhea virus (PEDV) is a highly transmissible coronavirus that causes a severe enteric disease that is particularly deadly for neonatal piglets. Since its introduction to the United States in 2013, PEDV has spread quickly across the country and has caused significant financial losses to pork producers. With no fully licensed vaccines currently available in the United States, prevention and control of PEDV disease is heavily reliant on biosecurity measures. Despite proven, effective biosecurity practices, multiple sites and production stages, within and across designated production flows in an Ohio swine operation broke with confirmed PEDV in January 2014, leading the producer and attending veterinarian to investigate the route of introduction.
Case presentation
On January 12, 2014, several sows within a production flow were noted with signs of enteric illness. Within a few days, illness had spread to most of the sows in the facility and was confirmed by RT-PCR to be PEDV. Within a short time period, confirmed disease was present on multiple sites within and across breeding and post weaning production flows of the operation and mortality approached 100% in neonatal piglets. After an epidemiologic investigation, an outsourced, pelleted piglet diet was identified for assessment, and a bioassay, where naïve piglets were fed the suspected feed pellets, was initiated to test the pellets for infectious PEDV.
The epidemiological investigation provided strong evidence for contaminated feed as the source of the outbreak. In addition, feed pellets collected from unopened bags at the affected sites tested positive for PEDV using RT-PCR. However, the bioassay study was not able to show infectivity when feeding the suspected feed pellets to a small number of naïve piglets. The results highlight the critical need for surveillance of feed and feed components to further define transmission avenues in an effort to limit the spread of PEDV throughout the U.S. swine industry.
PMCID: PMC4334577
Feed; PEDV; Swine
10.  Absolute Oxygen R1e Imaging In Vivo with Pulse Electron Paramagnetic Resonance 
Tissue oxygen (O2) levels are among the most important and most quantifiable stimuli to which cells and tissues respond through inducible signaling pathways. Tumor O2 levels are major determinants of the response to cancer therapy. Developing more accurate measurements and images of tissue O2 partial pressure (pO2), assumes enormous practical, biological, and medical importance.
We present a fundamentally new technique to image pO2 in tumors and tissues with pulse electron paramagnetic resonance (EPR) imaging enabled by an injected, nontoxic, triaryl methyl (trityl) spin probe whose unpaired electron’s slow relaxation rates report the tissue pO2. Heretofore, virtually all in vivo EPR O2 imaging measures pO2 with the transverse electron spin relaxation rate, R2e, which is susceptible to the self-relaxation confounding O2 sensitivity.
We found that the trityl electron longitudinal relaxation rate, R1e, is an order of magnitude less sensitive to confounding self-relaxation. R1e imaging has greater accuracy and brings EPR O2 images to an absolute pO2 image, within uncertainties.
R1e imaging more accurately determines oxygenation of cancer and normal tissue in animal models than has been available. It will enable enhanced, rapid, noninvasive O2 images for understanding oxygen biology and the relationship of oxygenation patterns to therapy outcome in living animal systems.
PMCID: PMC4318240  PMID: 24006331
Oxygen; Imaging; EPR; Pulse; R1; Spin Lattice Relaxation; In Vivo; Tumor
11.  Randomized Clinical Trial To Evaluate the Immunogenicity of Quadrivalent Meningococcal Conjugate and Polysaccharide Vaccines in Adults in the United Kingdom 
Meningococcal conjugate vaccines are today successfully deployed in universal programs for children and adolescents in different geographic regions to control meningitis and septicemia. However, in adults, the advantages of these conjugates over the older polysaccharide vaccines are less clear. In this randomized clinical trial, we demonstrated that both conjugate and polysaccharide quadrivalent meningococcal vaccines elicit protective antibody responses in adults aged 18 to 70. (This study has been registered at under registration no. NCT00901940.)
PMCID: PMC4135917  PMID: 24964805
12.  Regulation Where Autophagy Intersects the Inflammasome 
Antioxidants & Redox Signaling  2014;20(3):495-506.
Significance: The autophagy and inflammasome pathways are ancient innate immune mechanisms for controlling invading pathogens that are linked by mutual regulation. In addition to controlling the metabolic homeostasis of the cell through nutrient recycling, the “self-eating” process of autophagy is also responsible for the degradation of damaged organelles, cells, and pathogens to protect the integrity of the organism. As a cytosolic pathogen recognition receptor (PRR) complex, the inflammasome both induces and is induced by autophagy through direct interactions with autophagy proteins or through the effects of secondary molecules, such as mitochondrial reactive oxygen species and mitochondrial DNA. Recent Advances: While the molecular mechanisms of inflammasome activation and regulation are largely unknown, much of the current knowledge has been established through investigation of the role of autophagy in innate immunity. Likewise, regulatory proteins in the NOD-like receptor family, which includes inflammasome PRRs, are able to stimulate autophagy in response to the presence of a pathogen. Critical Issues: Many of the newly uncovered links between autophagy and inflammasomes have raised new questions about the mechanisms controlling inflammasome function, which are highlighted in this review. Future Directions: Our basic understanding of the mutual regulation of inflammasomes and autophagy will be essential for designing therapeutics for chronic inflammatory diseases, especially those for which autophagy and inflammasome genes have already been linked. Antioxid. Redox Signal. 20, 495–506.
PMCID: PMC3894701  PMID: 23642014
13.  Using polymeric materials to generate an amplified response to molecular recognition events 
Nature materials  2007;7(1):52-56.
Clinical and field-portable diagnostic devices require the detection of atto- to zeptomoles of biological molecules rapidly, easily and at low cost, with stringent requirements in terms of robustness and reliability. Though a number of creative approaches to this difficult problem have been reported1–9, numerous unmet needs remain in the marketplace, particularly in resource-poor settings10–12. Using rational materials design, we investigated harnessing the amplification inherent in a radical chain polymerization reaction to detect molecular recognition. Polymerization-based amplification is shown to yield a macroscopically observable polymer, easily visible to the unaided eye, as a result of as few as ~1,000 recognition events (10 zeptomoles). Design and synthesis of a dual-functional macromolecule that is capable both of selective recognition and of initiating a polymerization reaction was central to obtaining high sensitivity and eliminating the need for any detection equipment. Herein, we detail the design criteria that were used and compare our findings with those obtained using enzymatic amplification. Most excitingly, this new approach is general in that it is readily adaptable to facile detection at very low levels of specific biological interactions of any kind.
PMCID: PMC4299824  PMID: 17965717
14.  Shaping the Future of Research: a perspective from junior scientists 
F1000Research  2015;3:291.
The landscape of scientific research and funding is in flux as a result of tight budgets, evolving models of both publishing and evaluation, and questions about training and workforce stability. As future leaders, junior scientists are uniquely poised to shape the culture and practice of science in response to these challenges. A group of postdocs in the Boston area who are invested in improving the scientific endeavor, planned a symposium held on October 2 nd and 3 rd, 2014, as a way to join the discussion about the future of US biomedical research. Here we present a report of the proceedings of participant-driven workshops and the organizers’ synthesis of the outcomes.
PMCID: PMC4304227  PMID: 25653845
biomedical research; funding; training; publishing
16.  Long-term follow-up of imatinib in pediatric Philadelphia chromosome-positive acute lymphoblastic leukemia: Children’s Oncology Group Study AALL0031 
Leukemia  2014;28(7):1467-1471.
We previously reported preliminary findings that post induction imatinib mesylate (340 mg/m2/day), in combination with intensive chemotherapy, resulted in outcomes similar to blood and marrow transplant (BMT) for pediatric patients with Philadelphia chromosome-positive (Ph + ) acute lymphoblastic leukemia (ALL). We now report 5-year outcomes of imatinib plus intensive chemotherapy in 91 children (1–21 years) with and without allogeneic BMT (N = 91). We explore the impacts of additional chromosomal abnormalities and minimal residual disease (MRD) by flow cytometry on outcomes. The 5-year disease-free survival was similar for Cohort 5 patients, treated with chemotherapy plus imatinib (70% ± 12%, n = 28), sibling donor BMT patients (65% ± 11%, n = 21) and unrelated donor BMT patients (59 ± 15%; P = 0.60, n = 13). Patients with additional cytogenetic abnormalities had worse outcomes (P = 0.05). End induction (pre-imatinib) MRD was not prognostic for Cohort 5 or allogeneic BMT patients, although limited by small numbers. The re-induction rate following relapse was similar to other higher-risk ALL groups. Longer-term follow-up confirms our initial observation of substantially good outcomes for children and adolescents with Ph + ALL treated with imatinib plus intensive chemotherapy with no advantage for allogeneic BMT.
PMCID: PMC4282929  PMID: 24441288
imatinib mesylate; Philadelphia chromosome; acute lymphoblastic leukemia; toxicity; event-free survival; blood and marrow transplantation
17.  Isolation of antiplasmodial anthraquinones from Kniphofia ensifolia, and synthesis and structure-activity relationships of related compounds 
Bioorganic & medicinal chemistry  2013;22(1):269-276.
Bioassay-guided separation of the South African plant Kniphofia ensifolia for antiplasmodial activity led to the isolation of two new anthraquinones, named kniphofiones A and B (3, 4), together with three known bioactive anthraquinone monomers (1, 2 and 5), and four known bisanthraquinones (6–9). The structures of the two new compounds were elucidated based on analyses of their 1D and 2D NMR spectra and mass spectrometric data. The dimeric compounds 6 and 7 displayed the strongest antiplasmodial activity among all the isolated compounds, with IC50 values of 0.4 ± 0.1 and 0.2 ± 0.1 μM, respectively. The two new compounds displayed modest activities, with IC50 values of 26 ± 4 and 9 ± 1 μM, respectively. Due to the synthetic accessibility of the new compounds and the increased activity shown by the dimeric compounds, a structure-activity relationship study was conducted. As a result, one analogue of kniphofione B (4), the caffeic acid derivative of aloe-emodin, was found to have the highest activity among all the aloe-emodin derivatives, with an IC50 value of 1.3 ± 0.2 μM.
PMCID: PMC3919637  PMID: 24326280
Anthraquinone; Antiplasmodial acitivity; Kniphofia ensifolia; Structure-activity relationship
18.  A Randomized Trial of Electro-acupuncture for Arthralgia related to Aromatase Inhibitor Use 
Arthralgia is a common and debilitating side effect experienced by breast cancer patients receiving aromatase inhibitors (AIs) and often results in premature drug discontinuation.
We conducted a randomized controlled trial of electro-acupuncture (EA) as compared to waitlist control (WLC) and sham acupuncture (SA) in postmenopausal women with breast cancer who self-reported arthralgia attributable to AIs. Acupuncturists performed ten EA/SA treatments over eight weeks using a manualized protocol with 2 Hz electro-stimulation delivered by a TENS unit. Acupuncturists administered SA using Streitberger (non-penetrating) needles at non-traditional acupuncture points without electro-stimulation. The primary endpoint was pain severity by Brief Pain Inventory (BPI) between EA and WLC at Week 8; durability of response at Week 12 and comparison of EA to SA were secondary aims.
Of the 67 randomly assigned patients, mean reduction in pain severity was greater in the EA group than in the WLC group at Week 8 (−2.2 vs. −0.2, p=0.0004) and at Week 12 (−2.4 vs. −0.2, p<0.0001). Pain-related interference measured by BPI also improved in the EA group compared to the WLC group at both Week 8 (−2.0 vs. 0.2, p=0.0006) and Week 12 (−2.1 vs. −0.1, p=0.0034). SA produced a magnitude of change in pain severity and pain-related interference at Week 8 (−2.3, −1.5 respectively) and Week 12 (−1.7, −1.3 respectively) similar to that of EA. Participants in both EA and SA groups reported few minor adverse events.
Compared to usual care, EA produced clinically important and durable improvement in arthralgia related to AIs in breast cancer patients, and SA had a similar effect. Both EA and SA were safe.
PMCID: PMC3972040  PMID: 24210070
Acupuncture; breast neoplasm; clinical trial; Aromatase inhibitors/*adverse effects; musculoskeletal; joint pain
19.  Statistical Learning Methods for Longitudinal High-dimensional Data 
Recent studies have collected high-dimensional data longitudinally. Examples include brain images collected during different scanning sessions and time-course gene expression data. Because of the additional information learned from the temporal changes of the selected features, such longitudinal high-dimensional data, when incorporated with appropriate statistical learning techniques, are able to more accurately predict disease status or responses to a therapeutic treatment. In this article, we review recently proposed statistical learning methods dealing with longitudinal high-dimensional data.
PMCID: PMC4181610  PMID: 25285184
High-dimensionality; Multiple times points; Prediction; Support vector machines; Shrinkage; Temporal effects
20.  The Professional Socialization of the Athletic Trainer Serving as a Preceptor 
Journal of Athletic Training  2014;49(1):75-82.
The role of the preceptor requires the athletic trainer to be versed in effective instructional techniques, supervisory skills, and communication skills beyond his or her competence as an athletic trainer, but many have not received formal training in educational techniques.
To gain a better understanding about the professional socialization process for the athletic trainer assuming the role of the preceptor.
Qualitative study.
Athletic training education programs.
Patients or Other Participants:
Twenty-four preceptors (11 men, 13 women; age = 32 ± 7 years, clinical experience = 9 ± 6 years, preceptor experience = 5 ± 3 years) employed in the collegiate (n = 12) or secondary school (n = 12) setting.
Data Collection and Analysis:
We gathered data using asynchronous, in-depth interviewing via QuestionPro. We analyzed data using a general inductive approach to uncover the dominant themes. Credibility was secured by using consistency and stakeholder checks and a peer review.
We identified 2 main themes by which preceptors develop in their roles as clinical instructors: formal processes and informal processes. The participants used observations, previous experiences or interactions with role models, and self-reflection and evaluation as informal socialization processes. Formal socialization processes included preceptor training/workshops, professional development, and formal teacher certification.
Athletic trainers who serve as preceptors learned their roles by a combination of informal and formal processes. Preceptor training sessions appeared to be effective in initially helping preceptors learn their responsibilities, whereby more informal processes seemed to help them refine their skills. Furthermore, one socialization strategy did not appear to dominate role learning; rather, a combination of several processes fostered an understanding.
PMCID: PMC3917300  PMID: 24377957
instructional techniques; mentoring; learning
21.  Two cases of intranasal naloxone self-administration in opioid overdose 
Overdose is a leading cause of death for former prisoners, exacting its greatest toll during the first 2 weeks post-release. Protective effects have been observed with training individuals at high risk of overdose and prescribing them naloxone, an opioid antagonist that reverses the effects of the opioid-induced respiratory depression that causes death.
We report two people with opiate use histories who self-administered intranasal naloxone to treat their own heroin overdoses following release from prison. Patient A is a 34-year-old male, who reported having experienced an overdose on heroin the day after he was released from incarceration. Patient B is a 29-year-old female, who reported an overdose on her first injection of heroin, 17 days post release from incarceration. Both patients self-administered the medication but were assisted at some point during the injury by a witness whom they had personally instructed in how to prepare and administer the medication. Neither patient experienced withdrawal symptoms following exposure to naloxone.
Self-administration of naloxone should not be a goal of overdose death prevention training. A safer, more reliable approach is to prescribe naloxone to at-risk patients and train and also equip members of their household and social or drug using networks in overdose prevention and response.
PMCID: PMC4019939  PMID: 24821348
overdose; naloxone; criminal justice
22.  Brain Imaging Analysis 
The increasing availability of brain imaging technologies has led to intense neuroscientific inquiry into the human brain. Studies often investigate brain function related to emotion, cognition, language, memory, and numerous other externally induced stimuli as well as resting-state brain function. Studies also use brain imaging in an attempt to determine the functional or structural basis for psychiatric or neurological disorders and, with respect to brain function, to further examine the responses of these disorders to treatment. Neuroimaging is a highly interdisciplinary field, and statistics plays a critical role in establishing rigorous methods to extract information and to quantify evidence for formal inferences. Neuroimaging data present numerous challenges for statistical analysis, including the vast amounts of data collected from each individual and the complex temporal and spatial dependence present. We briefly provide background on various types of neuroimaging data and analysis objectives that are commonly targeted in the field. We present a survey of existing methods targeting these objectives and identify particular areas offering opportunities for future statistical contribution.
PMCID: PMC4189192  PMID: 25309940
Neuroimaging; fMRI; DTI; connectivity; prediction; activation
23.  The linear ubiquitin assembly complex (LUBAC) is essential for NLRP3 inflammasome activation 
The Journal of Experimental Medicine  2014;211(7):1333-1347.
Independent of its known role in NF-κB transcription, the HOIL-1L containing LUBAC is required for assembly and activation of the NLRP3 inflammasome via linear ubiquitination of ASC.
Linear ubiquitination is a newly discovered posttranslational modification that is currently restricted to a small number of known protein substrates. The linear ubiquitination assembly complex (LUBAC), consisting of HOIL-1L, HOIP, and Sharpin, has been reported to activate NF-κB–mediated transcription in response to receptor signaling by ligating linear ubiquitin chains to Nemo and Rip1. Despite recent advances, the detailed roles of LUBAC in immune cells remain elusive. We demonstrate a novel HOIL-1L function as an essential regulator of the activation of the NLRP3/ASC inflammasome in primary bone marrow–derived macrophages (BMDMs) independently of NF-κB activation. Mechanistically, HOIL-1L is required for assembly of the NLRP3/ASC inflammasome and the linear ubiquitination of ASC, which we identify as a novel LUBAC substrate. Consequently, we find that HOIL-1L−/− mice have reduced IL-1β secretion in response to in vivo NLRP3 stimulation and survive lethal challenge with LPS. Together, these data demonstrate that linear ubiquitination is required for NLRP3 inflammasome activation, defining the molecular events of NLRP3 inflammasome activation and expanding the role of LUBAC as an innate immune regulator. Furthermore, our observation is clinically relevant because patients lacking HOIL-1L expression suffer from pyogenic bacterial immunodeficiency, providing a potential new therapeutic target for enhancing inflammation in immunodeficient patients.
PMCID: PMC4076580  PMID: 24958845
24.  Characteristics associated with willingness to participate in a randomized controlled behavioral clinical trial using home-based personal computers and a webcam 
Trials  2014;15(1):508.
Trials aimed at preventing cognitive decline through cognitive stimulation among those with normal cognition or mild cognitive impairment are of significant importance in delaying the onset of dementia and reducing dementia prevalence. One challenge in these prevention trials is sample recruitment bias. Those willing to volunteer for these trials could be socially active, in relatively good health, and have high educational levels and cognitive function. These participants’ characteristics could reduce the generalizability of study results and, more importantly, mask trial effects. We developed a randomized controlled trial to examine whether conversation-based cognitive stimulation delivered through personal computers, a webcam and the internet would have a positive effect on cognitive function among older adults with normal cognition or mild cognitive impairment. To examine the selectivity of samples, we conducted a mass mail-in survey distribution among community-dwelling older adults, assessing factors associated with a willingness to participate in the trial.
Two thousand mail-in surveys were distributed to retirement communities in order to collect data on demographics, the nature and frequency of social activities, personal computer use and additional health-related variables, and interest in the prevention study. We also asked for their contact information if they were interested in being contacted as potential participants in the trial.
Of 1,102 surveys returned (55.1% response rate), 983 surveys had complete data for all the variables of interest. Among them, 309 showed interest in the study and provided their contact information (operationally defined as the committed with interest group), 74 provided contact information without interest in the study (committed without interest group), 66 showed interest, but provided no contact information (interest only group), and 534 showed no interest and provided no contact information (no interest group). Compared with the no interest group, the committed with interest group were more likely to be personal computer users (odds ratio (OR) = 2.78), physically active (OR = 1.03) and had higher levels of loneliness (OR = 1.16).
Increasing potential participants’ familiarity with a personal computer and the internet before trial recruitment could increase participation rates and improve the generalizability of future studies of this type.
Trial registration
The trial was registered on 29 March 2012 at (ID number NCT01571427).
PMCID: PMC4307639  PMID: 25539637
Sample recruitment selection bias; Volunteer bias; Behavioral randomized controlled trial; PC; Internet; Webcam; Conversation-based social interaction; Cognitive function; Mild cognitive impairment
25.  Climate, not Aboriginal landscape burning, controlled the historical demography and distribution of fire-sensitive conifer populations across Australia 
Climate and fire are the key environmental factors that shape the distribution and demography of plant populations in Australia. Because of limited palaeoecological records in this arid continent, however, it is unclear as to which factor impacted vegetation more strongly, and what were the roles of fire regime changes owing to human activity and megafaunal extinction (since ca 50 kya). To address these questions, we analysed historical genetic, demographic and distributional changes in a widespread conifer species complex that paradoxically grows in fire-prone regions, yet is very sensitive to fire. Genetic demographic analysis showed that the arid populations experienced strong bottlenecks, consistent with range contractions during the Last Glacial Maximum (ca 20 kya) predicted by species distribution models. In southern temperate regions, the population sizes were estimated to have been mostly stable, followed by some expansion coinciding with climate amelioration at the end of the last glacial period. By contrast, in the flammable tropical savannahs, where fire risk is the highest, demographic analysis failed to detect significant population bottlenecks. Collectively, these results suggest that the impact of climate change overwhelmed any modifications to fire regimes by Aboriginal landscape burning and megafaunal extinction, a finding that probably also applies to other fire-prone vegetation across Australia.
PMCID: PMC3826224  PMID: 24174110
aboriginal fire management; conifer; climate change; fire; phylogeography; population demography

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