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1.  Secretory Competence in a Gateway Endocrine Cell Conferred by the Nuclear Receptor βFTZ-F1 Enables Stage-specific Ecdysone Responses Throughout Development in Drosophila 
Developmental biology  2013;385(2):253-262.
Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETH). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor βFTZ-F1. Selective RNA silencing of βftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in βftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted βFTZ-F1 expression. Moreover, premature βftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a “gateway cell”, whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed βFTZ-F1 expression late in the molt as steroids decline.
PMCID: PMC3900412  PMID: 24247008
Drosophila; ecdysis; ecdysis triggering hormone (ETH); Inka cells; βFTZ-F1; RNA-silencing; secretory competence
2.  Mannosylglycerate and Di-myo-Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress 
Applied and Environmental Microbiology  2014;80(14):4226-4233.
Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities. Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for P. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.
PMCID: PMC4068688  PMID: 24795373
3.  A Bio-Hybrid Tactile Sensor Incorporating Living Artificial Skin and an Impedance Sensing Array 
Sensors (Basel, Switzerland)  2014;14(12):23781-23802.
The development of a bio-hybrid tactile sensor array that incorporates a skin analogue comprised of alginate encapsulated fibroblasts is described. The electrical properties are modulated by mechanical stress induced during contact, and changes are detected by a ten-channel dual-electrode impedance sensing array. By continuously monitoring the impedance of the sensor array at a fixed frequency, whilst normal and tangential loads are applied to the skin surface, transient mechanotransduction has been observed. The results demonstrate the effectiveness and feasibility of the preliminary prototype bio-hybrid tactile sensor.
PMCID: PMC4299087  PMID: 25615726
bio-hybrid sensors; bioimpedance; haptics; tactile sensors; artificial touch; artificial skin; microfluidics
4.  Functional Phylogenetics Reveals Contributions of Pleiotropic Peptide Action to Ligand-Receptor Coevolution 
Scientific Reports  2014;4:6800.
The evolution of peptidergic signaling has been accompanied by a significant degree of ligand-receptor coevolution. Closely related clusters of peptide signaling molecules are observed to activate related groups of receptors, implying that genes encoding these ligands may orchestrate an array of functions, a phenomenon known as pleiotropy. Here we examine whether pleiotropic actions of peptide genes might influence ligand-receptor coevolution. Four test groups of neuropeptides characterized by conserved C-terminal amino acid sequence motifs and their cognate receptors were examined in the red flour beetle (Tribolium castaneum): 1) cardioacceleratory peptide 2b (CAPA); CAPAr, 2) pyrokinin/diapause hormone (PK1/DH); PKr-A, -B, 3) pyrokinin/pheromone biosynthesis activating hormone (PK2/PBAN); PKr-C, and 4) ecdysis triggering hormone (ETH); ETHr-b. Ligand-receptor specificities were established through heterologous expression of receptors in cell-based assays for 9 endogenous ligands. Based on ligand-receptor specificity analysis, we found positive pleiotropism exhibited by ETH on ETHR-b and CAPAr, whereas PK1/DH and CAPA are more highly selective for their respective authentic receptors than would be predicted by phylogenetic analysis. Disparities between evolutionary trees deduced from receptor sequences vs. functional ligand-receptor specificities lead to the conclusion that pleiotropy exhibited by peptide genes influences ligand-receptor coevolution.
PMCID: PMC4210869  PMID: 25348027
5.  Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway 
The extremely thermoacidophilic archaeon Metallosphaera sedula (optimum growth temperature, 73°C, pH 2.0) grows chemolithoautotrophically on metal sulfides or molecular hydrogen by employing the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle. This cycle adds two CO2 molecules to acetyl coenzyme A (acetyl-CoA) to generate 4HB, which is then rearranged and cleaved to form two acetyl-CoA molecules. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining one-third is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. Here, the final four enzymes of the 3HP/4HB cycle, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), and acetoacetyl-CoA β-ketothiolase (Msed_0656), were produced recombinantly in Escherichia coli, combined in vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO2 fixation and central metabolism were examined using a gas-intensive bioreactor system in which M. sedula was grown under autotrophic (CO2-limited) and heterotrophic conditions. Transcriptomic analysis revealed the importance of the 3HP/4HB pathway in supplying acetyl-CoA to anabolic pathways generating intermediates in M. sedula metabolism. The results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated posttranslationally by the protein acetyltransferase (Pat)/Sir2-dependent system. Taken together, this work confirms the final four steps of the 3HP/4HB pathway, thereby providing the framework for examining connections between CO2 fixation and central metabolism in M. sedula.
PMCID: PMC3993168  PMID: 24532060
6.  Complete Genome Sequence of Pelosinus sp. Strain UFO1 Assembled Using Single-Molecule Real-Time DNA Sequencing Technology 
Genome Announcements  2014;2(5):e00881-14.
Pelosinus species can reduce metals such as Fe(III), U(VI), and Cr(VI) and have been isolated from diverse geographical regions. Five draft genome sequences have been published. We report the complete genome sequence for Pelosinus sp. strain UFO1 using only PacBio DNA sequence data and without manual finishing.
PMCID: PMC4155594  PMID: 25189589
7.  Tobacco Dependence Treatment Teaching by Medical School Clerkship Preceptors: Survey Responses from more than 1,000 US Medical Students 
Preventive medicine  2013;57(2):81-86.
To determine factors associated with tobacco cessation counseling in medical school clerkships
Third-year medical students at 10 medical schools across the United States completed a 100-item survey, measuring the frequency with which they experienced their preceptors’ providing clinical teaching components: clear instruction, feedback, modeling behavior, setting clear objectives, and responding to questions about tobacco dependence counseling as well as frequency of use of tobacco prompts and office systems. Our primary dependent measure was student self-reported skill level for items of tobacco dependence treatment (e.g. “5As”).
Surveys were completed by 1213 students. For both family medicine and internal medicine clerkships, modeling and providing clear instruction on ways to provide tobacco counseling were reported most commonly. In contrast, providing feedback and clear objectives for tobacco dependence treatment lagged behind. Overall, students who reported preceptors’ provision of optimal clinical teaching components and office system prompts in both family medicine and internal medicine clerkships had higher self-reported skill (p<0.001) than students with no exposure or exposure during only one of the clerkships.
Future educational interventions intended to help students adopt effective tobacco dependence treatment techniques should be engineered to facilitate these critical precepting components.
PMCID: PMC3767283  PMID: 23623894
8.  Inflammation in rat pregnancy inhibits spiral artery remodeling leading to fetal growth restriction and features of preeclampsia 
Abnormal maternal inflammation leads to TNF-mediated fetal growth restriction and some features of preeclampsia that can be ameliorated with the nitric oxide mimetic nitroglycerin.
Fetal growth restriction (FGR) and preeclampsia (PE) are often associated with abnormal maternal inflammation, deficient spiral artery (SA) remodeling, and altered uteroplacental perfusion. Here, we provide evidence of a novel mechanistic link between abnormal maternal inflammation and the development of FGR with features of PE. Using a model in which pregnant rats are administered low-dose lipopolysaccharide (LPS) on gestational days 13.5–16.5, we show that abnormal inflammation resulted in FGR mediated by tumor necrosis factor-α (TNF). Inflammation was also associated with deficient trophoblast invasion and SA remodeling, as well as with altered uteroplacental hemodynamics and placental nitrosative stress. Moreover, inflammation increased maternal mean arterial pressure (MAP) and was associated with renal structural alterations and proteinuria characteristic of PE. Finally, transdermal administration of the nitric oxide (NO) mimetic glyceryl trinitrate prevented altered uteroplacental perfusion, LPS-induced inflammation, placental nitrosative stress, renal structural and functional alterations, increase in MAP, and FGR. These findings demonstrate that maternal inflammation can lead to severe pregnancy complications via a mechanism that involves increased maternal levels of TNF. Our study provides a rationale for the use of antiinflammatory agents or NO-mimetics in the treatment and/or prevention of inflammation-associated pregnancy complications.
PMCID: PMC3892976  PMID: 24395887
9.  Activities of Psilostachyin A and Cynaropicrin against Trypanosoma cruzi In Vitro and In Vivo 
Antimicrobial Agents and Chemotherapy  2013;57(11):5307-5314.
In vitro and in vivo activities against Trypanosoma cruzi were evaluated for two sesquiterpene lactones: psilostachyin A and cynaropicrin. Cynaropicrin had previously been shown to potently inhibit African trypanosomes in vivo, and psilostachyin A had been reported to show in vivo effects against T. cruzi, albeit in another test design. In vitro data showed that cynaropicrin was more effective than psilostachyin A. Ultrastructural alterations induced by cynaropicrin included shedding events, detachment of large portions of the plasma membrane, and vesicular bodies and large vacuoles containing membranous structures, suggestive of parasite autophagy. Acute toxicity studies showed that one of two mice died at a cynaropicrin dose of 400 mg/kg of body weight given intraperitoneally (i.p.). Although no major plasma biochemical alterations could be detected, histopathology demonstrated that the liver was the most affected organ in cynaropicrin-treated animals. Although cynaropicrin was as effective as benznidazole against trypomastigotes in vitro, the treatment (once or twice a day) of T. cruzi-infected mice (up to 50 mg/kg/day cynaropicrin) did not suppress parasitemia or protect against mortality induced by the Y and Colombiana strains. Psilostachyin A (0.5 to 50 mg/kg/day given once a day) was not effective in the acute model of T. cruzi infection (Y strain), reaching 100% animal mortality. Our data demonstrate that although it is very promising against African trypanosomes, cynaropicrin does not show efficacy compared to benznidazole in acute mouse models of T. cruzi infection.
PMCID: PMC3811247  PMID: 23939901
10.  Depressive Behavior and Coronary Artery Atherogenesis in Adult Female Cynomolgus Monkeys 
Psychosomatic medicine  2008;70(6):637-645.
To examine depressive behavior and early coronary artery atherogenesis in 36 socially housed female cynomolgus monkeys, an established model of atherogenesis and depression. Coronary heart disease (CHD) is caused by coronary artery atherosclerosis (CAA) and its sequelae which develop over a period of decades. Thus, in prospective studies of depression and CHD, CAA was likely present at baseline in most subjects who experienced cardiac events. Little is known about the relationship between depression and CAA.
The monkeys were free of atherosclerosis before being fed a diet containing moderate amounts of fat and cholesterol for 52 months. Depressed behavior and activity levels recorded in weekly 15-minute focal samples, telemetered 24-hour heart rate, plasma total (TPC) and high-density lipoprotein cholesterol (HDLC), luteal phase serum progesterone concentrations, basal cortisol, cortisol response to corticotrophin-releasing hormone (CRH), and CAA extent were assessed.
Time spent in depressed behavior over 4 years was significantly associated with early CAA (r = 73, p = .001), as were activity level, 24-hour heart rate, TPC, HDLC, cortisol response to CRH, and mean peak progesterone (all p ≤ 0.05). Depressed females had four times the CAA compared with nondepressed females.
Depression in primates is associated with perturbations in multiple CHD risk factors and accelerated early atherogenesis. These data are consistent with the hypotheses that depression and CAA both stem from a common mechanism and that depression may cause CAA.
PMCID: PMC3970186  PMID: 18596246
depression; CHD; coronary artery atherosclerosis; heart rate; ovarian function; women’s health; cholesterol
11.  Characterization of Ten Heterotetrameric NDP-Dependent Acyl-CoA Synthetases of the Hyperthermophilic Archaeon Pyrococcus furiosus 
Archaea  2014;2014:176863.
The hyperthermophilic archaeon Pyrococcus furiosus grows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of ATP. ACS1 and ACS2 were previously purified from P. furiosus and have α2β2 structures but the genome contains genes encoding three additional α-subunits. The ten possible combinations of α and β genes were expressed in E. coli and each resulted in stable and active α2β2 isoenzymes. The α-subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids. The β-subunit determined preference for adenine or guanine nucleotides. The GTP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GTP for GTP-dependent phosphoenolpyruvate carboxykinase and for other GTP-dependent processes. Transcriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both ATP and GTP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs of P. furiosus and other members of the Thermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea.
PMCID: PMC3942289  PMID: 24669200
12.  The Pheromone Biosynthesis Activating Neuropeptide (PBAN) Receptor of Heliothis virescens: Identification, Functional Expression, and Structure-Activity Relationships of Ligand Analogs 
Peptides  2007;29(2):268-275.
Pheromone biosynthesis activating neuropeptide (PBAN) promotes synthesis and release of sex pheromones in moths. We have identified and functionally expressed a PBAN receptor from Heliothis virescens (HevPBANR) and elucidated structure-activity relationships of PBAN analogs. Screening of a larval CNS cDNA library revealed three putative receptor subtypes and nucleotide sequence comparisons suggest they are produced through alternative splicing at the 3’-end. RT-PCR amplified preferentially HevPBANR-C from female pheromone glands. CHO cells expressing HevPBANR-C are highly sensitive to PBAN and related analogs, especially those sharing the C-terminal pentapeptide core, FXPRLamide (X = T, S or V). Alanine replacements in the C-terminal hexapeptide (YFTPRLamide) revealed the relative importance of each residue in the active core as follows, R5>L6>F2≫P4>T3≫Y1. This study provides a framework for the rational design of PBANR-specific agonists and/or antagonists that could be exploited for disruption of reproductive function in agriculturally important insect pests.
PMCID: PMC3900413  PMID: 18243415
13.  Optical gain in 1.3-μm electrically driven dilute nitride VCSOAs 
We report the observation of room-temperature optical gain at 1.3 μm in electrically driven dilute nitride vertical cavity semiconductor optical amplifiers. The gain is calculated with respect to injected power for samples with and without a confinement aperture. At lower injected powers, a gain of almost 10 dB is observed in both samples. At injection powers over 5 nW, the gain is observed to decrease. For nearly all investigated power levels, the sample with confinement aperture gives slightly higher gain.
PMCID: PMC3896757  PMID: 24417791
Dilute nitride; Optical injection; Gain; Vertical cavity semiconductor optical amplifiers
14.  Observation of the Fe—CN and Fe—CO Vibrations in the Active Site of [NiFe] Hydrogenase by Nuclear Resonance Vibrational Spectroscopy** 
Nuclear inelastic scattering of 57Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign Fe–CO and Fe–CN bending and stretching vibrations of the active site outside the spectral range of the Fe–S cluster normal modes.
PMCID: PMC3535562  PMID: 23136119
FeS clusters; hydrogenases; NiFe acvite site; spectroscopic methods; vibrational spectroscopy
15.  Risk of cardiovascular disease in HIV, Hepatitis C, or HIV/Hepatitis C patients compared to the general population 
Due to effective antiretroviral therapy HIV patients are living longer, and their risk of cardiovascular disease (CVD) is a growing concern. It is unknown whether co-infection with hepatitis C (HCV) changes an HIV person’s CVD risk, and how the risks compare to the general population. The objective of this study was to compare the Framingham Risk Score (FRS) and vascular age differences in persons with HIV, HCV or HIV/HCV disease to the general population.
HIV, HCV and HIV/HCV patients with clinic visits between 2004–2009 were sampled from medical clinics in Rochester, NY. Uninfected persons were randomly selected from the National Health and Nutrition Examination Survey (NHANES) and individually matched on sex, race, and age. We stratified by infection group and conducted separate multivariable linear regression between each infection group and the sex, race, and age matched participants from NHANES.
Rochester patients (HIV=239, HCV=167, HIV/HCV=182) were compared 3:1 to the NHANES participants. After controlling for weight, marital status, current pharmacotherapies, and the matching variables of sex, race and age, HIV/HCV patients had a 2% higher general FRS compared to the general population (p=0.03) and vascular age differences that were 4.1 years greater (p=.01). HCV patients had a 2.4% higher general FRS than the general population (p<.001), and vascular age differences that were 4.4 years greater (p<.001). CVD risk was elevated, but not significantly different between HIV patients and the general population.
CVD risk is elevated among HIV/HCV and HCV infected persons compared to the general population.
PMCID: PMC3533243  PMID: 23241046
HIV; Hepatitis C; HIV/HCV co-infection; cardiovascular disease risk; Framingham Risk Score
16.  Neuropeptide Action in Insects and Crustaceans* 
Physiological and biochemical zoology : PBZ  2010;83(5):10.1086/648470.
Physiological processes are regulated by a diverse array of neuropeptides that coordinate organ systems. The neuropeptides, many of which act through G protein–coupled receptors, affect the levels of cyclic nucleotides (cAMP and cGMP) and Ca2+ in target tissues. In this perspective, their roles in molting, osmoregulation, metabolite utilization, and cardiovascular function are highlighted. In decapod crustaceans, inhibitory neuropeptides (molt-inihibiting hormone and crustacean hyperglycemic hormone) suppress the molting gland through cAMP- and cGMP-mediated signaling. In insects, the complex movements during ecdysis are controlled by ecdysis-triggering hormone and a cascade of downstream neuropeptides. Adipokinetic/hypertrehalosemic/hyperprolinemic hormones mobilize energy stores in response to increased locomotory activity. Crustacean cardioacceleratory (cardioactive) peptide, proctolin, and FMRFamide-related peptides act on the heart, accessory pulsatile organs, and excurrent ostia to control hemolymph distribution to tissues. The osmoregulatory challenge of blood gorging in Rhodnius prolixus requires the coordinated release of serotonin and diuretic and antidiuretic hormones acting on the midgut and Malpighian tubules. These studies illustrate how multiple neuropeptides allow for flexibility in response to physiological challenges.
PMCID: PMC3844688  PMID: 20550437
17.  A 1.5 Å resolution X-ray structure of the catalytic module of Caldicellulosiruptor bescii family 3 pectate lyase 
Structural analysis of the catalytic module of Caldicellulosiruptor bescii family 3 pectate lyase shows that this new structure is very similar to the previously solved structure of a family 3 pectate lyase from Bacillus sp. strain KSM-P15.
A 1.5 Å resolution X-ray structure of the catalytic module of Caldicellulosi­ruptor bescii family 3 pectate lyase is reported (PDB entry 3t9g). The resulting structure was refined to an R factor of 0.143 and an R free of 0.178. Structural analysis shows that this new structure is very similar to the previously solved structure of a family 3 pectate lyase from Bacillus sp. strain KSM-P15 (PDB entry 1ee6), with a root-mean-square deviation of 0.93 Å and a sequence identity of 53%. This structural similarity is significant considering that C. bescii is a hyperthermophile and Bacillus sp. is a mesophile.
PMCID: PMC3232124  PMID: 22139151
pectate lyases; PL3; Caldicellulosiruptor bescii
18.  Short Communication: Risk of Elevated Total Cholesterol/High-Density Lipoprotein Cholesterol Ratio After Antiretroviral Therapy in HIV/Hepatitis C Virus Patients 
AIDS Research and Human Retroviruses  2012;28(12):1552-1556.
Dyslipidemia from highly active antiretroviral therapy (HAART) use has been reported to be less severe among persons with HIV and hepatitis C (HCV) compared to those with HIV monoinfection. However, the effect on lipoprotein ratios is less clear. The total cholesterol/high-density lipoprotein ratio (TC/HDL-C ratio) is a robust measure of cardiovascular disease (CVD) risk but has not been examined in the context of HIV/HCV-coinfected patients. We compared the TC/HDL-C ratio before HAART initiation and after at least 6 months on HAART between patients monoinfected with HIV and coinfected with HIV and HCV. Pre- and post-HAART TC, HDL-C, and non-HDL-C were also assessed. Although TC, HDL-C, and non-HDL-C significantly increased after HAART initiation in both HIV and HIV/HCV patients, the TC/HDL-C ratio did not. In addition, although the pre- and post-HAART TC, HDL-C, non-HDL-C, and TC/HDL-C ratio were significantly different between HIV and HIV/HCV patients, the magnitude in the change from pre- to post-HAART was not significantly different between infection groups. These results persisted after controlling for age, sex, race, current pharmacotherapy for lipoproteins, body mass index, and current CD4 cell count. The magnitude of change in the TC/HDL-C ratio after HAART initiation is not significantly different between HIV and HIV/HCV patients, suggesting subsequent CVD risk in HIV/HCV patients may be greater than currently appreciated.
PMCID: PMC3505066  PMID: 22380598
19.  The risk of bleeding with duloxetine treatment in patients who use nonsteroidal anti-inflammatory drugs (NSAIDs): analysis of placebo-controlled trials and post-marketing adverse event reports 
To assess the safety of duloxetine with regards to bleeding-related events in patients who concomitantly did, versus did not, use nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin.
Safety data from all placebo-controlled trials of duloxetine conducted between December 1993 and December 2010, and post-marketing reports from duloxetine-treated patients in the US Food and Drug Administration Adverse Event Reporting System (FAERS), were searched for bleeding-related treatment-emergent adverse events (TEAEs). The percentage of patients with bleeding-related TEAEs was summarized and compared between treatment groups in all the placebo-controlled studies. Differences between NSAID user and non-user subgroups from clinical trial data were analyzed by a logistic regression model that included therapy, NSAID use, and therapy-by-NSAID subgroup interaction. In addition, to determine if higher duloxetine doses are associated with an increased incidence of bleeding-related TEAEs, and whether the use of concomitant NSAIDs might influence the dose effect if one exists, placebo-controlled clinical trials with duloxetine fixed doses of 60 mg, 120 mg, and placebo were analyzed. Also, the incidence of bleeding-related TEAEs reported for duloxetine alone was compared with the incidence in patients treated with duloxetine and concomitant NSAIDs. Finally, the number of bleeding-related cases reported for duloxetine in the FAERS database was compared with the numbers reported for all other drugs.
Across duloxetine clinical trials, there was a significantly greater incidence of bleeding-related TEAEs in duloxetine- versus placebo-treated patients overall and also in those patients who did not take concomitant NSAIDS, but no significant difference was seen among those patients who did take concomitant NSAIDS. There was no significant difference in the incidence of bleeding-related TEAEs in the subset of patients treated with duloxetine 120 mg once daily versus those treated with 60 mg once daily regardless of concomitant NSAID use. The combination of duloxetine and NSAIDs was associated with a statistically significantly higher incidence of bleeding-related TEAEs compared with duloxetine alone. A similarly higher incidence of bleeding-related TEAEs was seen in patients treated with placebo and concomitant NSAIDs compared with placebo alone. Bleeding-related TEAEs reported in the FAERS database were disproportionally more frequent for duloxetine taken with NSAIDs compared with the full FAERS background, but there was no difference in the reporting of bleeding-related TEAEs when the cases reported for duloxetine taken with NSAIDs were compared against the cases reported for NSAIDs alone.
Concomitant use of NSAIDs was associated with a higher incidence of bleeding-related TEAEs in clinical trials regardless of whether patients were taking duloxetine or placebo; bleeding-related TEAEs did not appear to increase along with duloxetine dose regardless of NSAID use. In spontaneously reported post-marketing data, the combination of duloxetine and NSAID use was not associated with an increased reporting of bleeding-related events when compared to NSAID use alone.
PMCID: PMC3849082  PMID: 24348072
antidepressant; gastrointestinal bleeding; NSAID; aspirin
20.  Willingness of Parkinson's Disease Patients to Participate in Research Using Internet-Based Technology 
Telemedicine Journal and e-Health  2012;18(9):684-687.
Motor impairment and travel time have been shown to be important barriers to recruitment for Parkinson's disease (PD) clinical trials. This study determined whether use of Internet-based video communication for study visits would improve likelihood of participating in PD clinical trials. Subjects and Methods: University of Utah PD clinic patients were invited to complete a survey asking if they would be willing to participate in a hypothetical research study under four different scenarios. McNemar's test was used to test the hypothesis that remote assessments would improve willingness to participate. Results: Willingness to participate was 101/113 (87%) in the standard scenario. Willingness to participate was highest (93%; p=0.046) with most visits occurring via telemedicine at a local clinic, followed by some visits occurring via telemedicine at a local clinic (91%; p=0.157). Willingness to participate was lower with some (80%; p=0.008) or most (82%; p=0.071) visits occurring by home telemonitoring. Conclusions: Use of telemedicine may be an acceptable means to improve participation in clinical trials. This would need to be confirmed with the use of a larger-scale inquiry involving rural populations. Future research should assess subject or caregiver comfort and trainability with respect to computer-based technology in the home and systems barriers for wider implementation of telemedicine in neurology.
PMCID: PMC3491628  PMID: 22954069
Parkinson's disease; clinical trials; telemedicine; telemonitoring; neurodegenerative disease
21.  Regulation of Iron Metabolism by Pyrococcus furiosus 
Journal of Bacteriology  2013;195(10):2400-2407.
Iron is an essential element for the hyperthermophilic archaeon Pyrococcus furiosus, and many of its iron-containing enzymes have been characterized. How iron assimilation is regulated, however, is unknown. The genome sequence contains genes encoding two putative iron-responsive transcription factors, DtxR and Fur. Global transcriptional profiles of the dtxR deletion mutant (ΔDTXR) and the parent strain under iron-sufficient and iron-limited conditions indicated that DtxR represses the expression of the genes encoding two putative iron transporters, Ftr1 and FeoAB, under iron-sufficient conditions. Under iron limitation, DtxR represses expression of the gene encoding the iron-containing enzyme aldehyde ferredoxin oxidoreductase and a putative ABC-type transporter. Analysis of the dtxR gene sequence indicated an incorrectly predicted translation start site, and the corrected full-length DtxR protein, in contrast to the truncated version, specifically bound to the promoters of ftr1 and feoAB, confirming its role as a transcription regulator. Expression of the gene encoding Ftr1 was dramatically upregulated by iron limitation, but no phenotype was observed for the ΔFTR1 deletion mutant under iron-limited conditions. The intracellular iron concentrations of ΔFTR1 and the parent strain were similar, suggesting that under the conditions tested, Ftr1 is not an essential iron transporter despite its response to iron. In contrast to DtxR, the Fur protein appears not to be a functional regulator in P. furiosus, since it did not bind to the promoters of any of the iron-regulated genes and the deletion mutant (ΔFUR) revealed no transcriptional responses to iron availability. DtxR is therefore the key iron-responsive transcriptional regulator in P. furiosus.
PMCID: PMC3650548  PMID: 23504018
22.  Vitamin K Status in Chronic Kidney Disease 
Nutrients  2013;5(11):4390-4398.
The purpose of this review is to summarize the research to date on vitamin K status in chronic kidney disease (CKD). This review includes a summary of the data available on vitamin K status in patients across the spectrum of CKD as well as the link between vitamin K deficiency in CKD and bone dynamics, including mineralization and demineralization, as well as ectopic mineralization. It also describes two current clinical trials that are underway evaluating vitamin K treatment in CKD patients. These data may inform future clinical practice in this population.
PMCID: PMC3847737  PMID: 24212088
vitamin K; chronic kidney disease; renal insufficiency; vitamin K-dependent proteins
23.  Open questions: genomics and how far we haven’t come 
BMC Biology  2013;11:109.
PMCID: PMC4225822  PMID: 24228713
24.  Vertebral deformity arising from an accelerated “creep” mechanism 
European Spine Journal  2012;21(9):1684-1691.
Vertebral deformities often occur in patients who recall no trauma, and display no evident fracture on radiographs. We hypothesise that vertebral deformity can occur by a gradual creep mechanism which is accelerated following minor damage. “Creep” is continuous deformation under constant load.
Materials and methods
Forty-five thoracolumbar spine motion segments were tested from cadavers aged 42–92 years. Vertebral body areal BMD was measured using DXA. Specimens were compressed at 1 kN for 30 min, while creep in each vertebral body was measured using an optical MacReflex system. After 30 min recovery, each specimen was subjected to a controlled overload event which caused minor damage to one of its vertebrae. The creep test was then repeated.
Vertebral body creep was measurable in specimens with BMD <0.5 g/cm2. Creep was greater anteriorly than posteriorly (p < 0.001), so that vertebrae gradually developed a wedge deformity. Compressive overload reduced specimen height by 2.24 mm (STD 0.77 mm), and increased vertebral body creep by 800 % (anteriorly), 1,000 % (centrally) and 600 % (posteriorly). In 34 vertebrae with complete before-and-after data, anterior wedging occurring during the 1st creep test averaged 0.07° (STD 0.17°), and in the 2nd test (after minor damage) it averaged 0.79° (STD 1.03°). The increase was highly significant (P < 0.001). Vertebral body wedging during the 2nd creep test was proportional to the severity of damage, as quantified by specimen height loss during the overload event (r2 = 0.51, p < 0.001, n = 34).
Minor damage to an old vertebral body, even if it is barely discernible on radiographs, can accelerate creep to such an extent that it makes a substantial contribution to vertebral deformity.
PMCID: PMC3459129  PMID: 22447410
Vertebral deformity; Fracture; Creep; BMD; Cadaveric; Damage
25.  Increased Chondrocyte Apoptosis Is Associated with Progression of Osteoarthritis in Spontaneous Guinea Pig Models of the Disease 
Osteoarthritis (OA) is the most common joint disease characterised by degradation of articular cartilage and bone remodelling. For almost a decade chondrocyte apoptosis has been investigated as a possible mechanism of cartilage damage in OA, but its precise role in initiation and/or progression of OA remains to the determined. The aim of this study is to determine the role of chondrocyte apoptosis in spontaneous animal models of OA. Right tibias from six male Dunkin Hartley (DH) and Bristol Strain 2 (BS2) guinea pigs were collected at 10, 16, 24 and 30 weeks of age. Fresh-frozen sections of tibial epiphysis were microscopically scored for OA, and immunostained with caspase-3 and TUNEL for apoptotic chondrocytes. The DH strain had more pronounced cartilage damage than BS2, especially at 30 weeks. At this time point, the apoptotic chondrocytes were largely confined to the deep zone of articular cartilage (AC) with a greater percentage in the medial side of DH than BS2 (DH: 5.7%, 95% CI: 4.2–7.2), BS2: 4.8%, 95% CI: 3.8–5.8), p > 0.05). DH had a significant progression of chondrocyte death between 24 to 30 weeks during which time significant changes were observed in AC fibrillation, proteoglycan depletion and overall microscopic OA score. A strong correlation (p ≤ 0.01) was found between chondrocyte apoptosis and AC fibrillation (r = 0.3), cellularity (r = 0.4) and overall microscopic OA scores (r = 0.4). Overall, the rate of progression in OA and apoptosis over the study period was greater in the DH (versus BS2) and the medial AC (versus lateral). Chondrocyte apoptosis was higher at the later stage of OA development when the cartilage matrix was hypocellular and highly fibrillated, suggesting that chondrocyte apoptosis is a late event in OA.
PMCID: PMC3794750  PMID: 23994836
chondrocyte apoptosis; osteoarthritis; animal models; disease progression; caspase-3

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