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1.  Flagellin induces antibody responses through a TLR5- and inflammasome-independent pathway1 
Flagellin is a potent immunogen that activates the innate immune system via TLR5 and Naip5/6, and generates strong T and B cell responses. The adaptor protein MyD88 is critical for signaling by TLR5, as well as IL-1 and IL-18 receptors, major downstream mediators of the Naip5/6 Nlrc4-inflammasome. Herein we define roles of known flagellin receptors and MyD88 in antibody responses generated towards flagellin. We used mice genetically deficient in flagellin recognition pathways to characterize innate immune components that regulate isotype specific antibody responses. Using purified flagellin from Salmonella, we dissected the contribution of innate flagellin recognition pathways to promote antibody responses towards flagellin and co-administered ovalbumin in C57BL/6 mice. We demonstrate IgG2c responses towards flagellin were TLR5- and inflammasome-dependent; IgG1 was the dominant isotype and partially TLR5- and inflammasome-dependent. Our data indicates a substantial flagellin-specific IgG1 response was induced through a TLR5-, inflammasome-, and MyD88-independent pathway. IgA anti-FliC responses were TLR5- & MyD88-dependent and caspase-1-independent. Unlike C57BL/6 mice, flagellin immunized A/J mice induced co-dominant IgG1 and IgG2a responses. Furthermore, MyD88-independent flagellin-induced antibody responses were even more pronounced in A/J MyD88−/− mice, and IgA anti-FliC responses were suppressed by MyD88. Flagellin also worked as an adjuvant toward co-administered ovalbumin, but it only promoted IgG1 anti-OVA responses. Our results demonstrate that a novel pathway for flagellin recognition contributes to antibody production. Characterization of this pathway will be useful for understanding immunity to flagellin and the rationale design of flagellin-based vaccines.
doi:10.4049/jimmunol.1301893
PMCID: PMC3925749  PMID: 24442437
2.  Diet Is Critical for Prolonged Glycemic Control after Short-Term Insulin Treatment in High-Fat Diet-Induced Type 2 Diabetic Male Mice 
PLoS ONE  2015;10(1):e0117556.
Background
Clinical studies suggest that short-term insulin treatment in new-onset type 2 diabetes (T2DM) can promote prolonged glycemic control. The purpose of this study was to establish an animal model to examine such a “legacy” effect of early insulin therapy (EIT) in long-term glycemic control in new-onset T2DM. The objective of the study was to investigate the role of diet following onset of diabetes in the favorable outcomes of EIT.
Methodology
As such, C57BL6/J male mice were fed a high-fat diet (HFD) for 21 weeks to induce diabetes and then received 4 weeks of daily insulin glargine or sham subcutaneous injections. Subsequently, mice were either kept on the HFD or switched to a low-fat diet (LFD) for 4 additional weeks.
Principal Findings
Mice fed a HFD gained significant fat mass and displayed increased leptin levels, increasing insulin resistance (poor HOMA-IR) and worse glucose tolerance test (GTT) performance in comparison to mice fed a LFD, as expected. Insulin-treated diabetic mice but maintained on the HFD demonstrated even greater weight gain and insulin resistance compared to sham-treated mice. However, insulin-treated mice switched to the LFD exhibited a better HOMA-IR compared to those mice left on a HFD. Further, between the insulin-treated and sham control mice, in spite of similar HOMA-IR values, the insulin-treated mice switched to a LFD following insulin therapy did demonstrate significantly better HOMA-B% values than sham control and insulin-treated HFD mice.
Conclusion/Interpretation
Early insulin treatment in HFD-induced T2DM in C57BL6/J mice was only beneficial in animals that were switched to a LFD after insulin treatment which may explain why a similar legacy effect in humans is achieved clinically in only a portion of cases studied, emphasizing a vital role for diet adherence in diabetes control.
doi:10.1371/journal.pone.0117556
PMCID: PMC4310595  PMID: 25633992
3.  Increased Wnt5a mRNA Expression in Advanced Atherosclerotic Lesions, and Oxidized LDL Treated Human Monocyte-Derived Macrophages 
Objective
Wnt5a is a secreted glycoprotein highly present in atherosclerotic lesions. Uptake of oxidized-low density lipoprotein (ox-LDL) by monocytes/macrophages plays a critical role in atherosclerosis. The objective of this study was to determine if Wnt5a mRNA expression correlates with the severity of atherosclerotic lesions, and if, ox-LDL can induce Wnt5a mRNA in macrophages.
Methods
Wnt5a mRNA in tissue sections from carotid arteries of patients undergoing endarterectomy was quantified via RT-PCR and correlated with plaque severity. Human monocyte-derived macrophages and differentiated THP-1 cells, a human monocytic cell line, were treated with ox-LDL or native-LDL. Subsequently, Wnt5a transcripts were quantified by RT-PCR.
Results
Regions of the arteries with more severe plaques had detectable and significant levels of Wnt5a mRNA, while regions of the arteries containing less vulnerable plaques had low or non-detectable Wnt5a. Ox-LDL, but not native-LDL, induced Wnt5a mRNA in both human monocyte-derived macrophages and differentiated THP-1 cells.
Conclusion
Our results demonstrate that the expression of Wnt5a correlates with the severity of atherosclerotic lesions, and that ox-LDL induces Wnt5a mRNA expression in human macrophages. These findings are consistent with the hypothesis that Wnt5a plays a critical role in atherosclerosis progression and that a source of Wnt5a is ox-LDL stimulated macrophages.
PMCID: PMC4270053  PMID: 25530821
Cardiovascular disease; atherosclerosis; inflammation; low density lipoprotein; Wnt5a
4.  Acute and Chronic Mood and Apathy Outcomes from a Randomized Study of Unilateral STN and GPi DBS 
PLoS ONE  2014;9(12):e114140.
Objective
To study mood and behavioral effects of unilateral and staged bilateral subthalamic nucleus (STN) and globus pallidus internus (GPi) deep brain stimulation (DBS) for Parkinson's disease (PD).
Background
There are numerous reports of mood changes following DBS, however, most have focused on bilateral simultaneous STN implants with rapid and aggressive post-operative medication reduction.
Methods
A standardized evaluation was applied to a subset of patients undergoing STN and GPi DBS and who were also enrolled in the NIH COMPARE study. The Unified Parkinson Disease Rating Scale (UPDRS III), the Hamilton depression (HAM-D) and anxiety rating scales (HAM-A), the Yale-Brown obsessive-compulsive rating scale (YBOCS), the Apathy Scale (AS), and the Young mania rating scale (YMRS) were used. The scales were repeated at acute and chronic intervals. A post-operative strategy of non-aggressive medication reduction was employed.
Results
Thirty patients were randomized and underwent unilateral DBS (16 STN, 14 GPi). There were no baseline differences. The GPi group had a higher mean dopaminergic dosage at 1-year, however the between group difference in changes from baseline to 1-year was not significant. There were no differences between groups in mood and motor outcomes. When combining STN and GPi groups, the HAM-A scores worsened at 2-months, 4-months, 6-months and 1-year when compared with baseline; the HAM-D and YMRS scores worsened at 4-months, 6-months and 1-year; and the UPDRS Motor scores improved at 4-months and 1-year. Psychiatric diagnoses (DSM-IV) did not change. No between group differences were observed in the cohort of bilateral cases.
Conclusions
There were few changes in mood and behavior with STN or GPi DBS. The approach of staging STN or GPi DBS without aggressive medication reduction could be a viable option for managing PD surgical candidates. A study of bilateral DBS and of medication reduction will be required to better understand risks and benefits of a bilateral approach.
doi:10.1371/journal.pone.0114140
PMCID: PMC4254912  PMID: 25469706
5.  Illness Representations of Lung Cancer, Lung Cancer Worry, and Perceptions of Risk by Smoking Status 
We examined perceived risk, worry, and illness representations of lung cancer by smoking status using data from the 2005 Health Information National Trends Survey (n=1,765). Perceived lung cancer risk was rated “very high” more frequently by current (15.2%) than former (1.9%) and never (1.6%) smokers. Current smokers more frequently reported worry about lung cancer (18.4%) than former (3.1%) and never smokers (1.8%). Confusion about lung cancer prevention was higher among current (55.2%) than former (41.3%) or never smokers (38.2%). Agreement that lung cancer is caused by a person’s behavior was higher among never (86.1%) and former (82.6%) than current smokers (75.4%). In multivariable models, never (OR=.07) and former smokers (OR=.16) were less likely than current smokers to perceive their lung cancer risk as high. Never smokers (OR=.21) were significantly less likely than current smokers to report worrying about lung cancer, while former and current smokers did not differ.
doi:10.1007/s13187-011-0247-6
PMCID: PMC4251766  PMID: 21688184
Smoking; Lung cancer worry; Risk perceptions
6.  Preliminary Validation of the Yale Food Addiction Scale for Children 
Eating behaviors  2013;14(4):10.1016/j.eatbeh.2013.07.002.
Introduction
Evidence is growing that an addictive process may play a role in problematic eating behavior. The majority of research on this topic has examined the concept of “food addiction” solely in adult samples. If certain foods have addictive potential, children may be impacted as much as (or more) than adults due to psychological and neurobiological vulnerabilities at younger developmental stages. In the current study, we developed a measure of food addiction in children that reflects the diagnostic indicators of addiction.
Materials and Methods
The content and reading level of the Yale Food Addiction Scale (YFAS) was altered to be appropriate for children (YFAS-C). The YFAS-C and other eating-related measures were administered to study participants to examine the validity and reliability of the YFAS-C.
Participants
75 children recruited from the community ranging from lean to obese.
Results
The validation of the YFAC-C provides preliminary support for its convergent validity with like constructs and incremental validity in predicting body mass index. Internal consistency was adequate given the small number of items on the scale.
Discussion
The YFAS-C appears to be a helpful tool for identifying addictive-like eating in children.
doi:10.1016/j.eatbeh.2013.07.002
PMCID: PMC3817415  PMID: 24183146
Obesity; Addiction; Children; Eating; Food
7.  Elevated IgE and atopy in patients treated for early onset ADA-SCID 
The Journal of allergy and clinical immunology  2013;132(6):10.1016/j.jaci.2013.05.040.
doi:10.1016/j.jaci.2013.05.040
PMCID: PMC3844080  PMID: 23895897
Early-onset ADA-SCID; primary immunodeficiency; atopy; IgE; T cell signaling; Th2 cytokines
8.  Diminished allergic disease in patients with STAT3 mutations reveals a role for STAT3 signaling in mast cell degranulation 
The Journal of allergy and clinical immunology  2013;132(6):10.1016/j.jaci.2013.08.045.
Background
Severe atopic conditions associated with elevated serum IgE are heterogeneous with few known causes. Nearly every patient with autosomal-dominant hyper-IgE syndrome (AD-HIES) due to signal transducer and activator of transcription 3 (STAT3) mutations has a history of eczematous dermatitis and elevated IgE; however, clinical atopy has never been systematically studied.
Objective
Understanding of genetic determinants of allergic disease may lead to novel therapies in controlling allergic disease.
Methods
We conducted clinical evaluation of the rates of food allergies and anaphylaxis in patients with AD-HIES, a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis, and healthy volunteers with no history of atopy. Morphine skin prick testing, ImmunoCAP assays for allergen-specific IgE, and basophil activation were measured. A model of systemic anaphylaxis was studied in transgenic mice carrying an AD-HIES mutation. STAT3 was silenced in LAD2 and primary human mast cells to study the role of STAT3 in signaling and degranulation after IgE cross-linking.
Results
Food allergies and anaphylaxis were markedly diminished in patients with AD-HIES compared with a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis. Morphine skin prick testing and basophil activation were diminished in patients with AD-HIES, whereas mice carrying an AD-HIES mutation were hyporesponsive to systemic anaphylaxis models. Rapid mast cell STAT3 serine727 phosphorylation was noted after IgE cross-linking, and inhibition of STAT3 signaling in mast cells lead to impaired FcεRI-mediated proximal and distal signaling, as well as reduced degranulation.
Conclusion
This study serves as an example for how mutations in specific atopic pathways can lead to discrete allergic phenotypes, encompassing increased risk of some phenotypes but a relative protection from others.
doi:10.1016/j.jaci.2013.08.045
PMCID: PMC3881191  PMID: 24184145
Autosomal dominant hyper-IgE syndrome; Job syndrome; signal transducer and activator of transcription 3; mast cell; degranulation
10.  Women who are motivated to eat and discount the future are more obese 
Obesity (Silver Spring, Md.)  2013;22(6):1394-1399.
Objective
Food reinforcement and delay discounting (DD) predict Body Mass Index (BMI), but there is no research studying whether these variables interact to improve prediction of BMI.
Design and Methods
BMI, the relative reinforcing value of high (PMAXHED) and low (PMAXLED) energy dense food, and DD for $10 and $100 future rewards (DD10, DD100) were measured in 199 adult females.
Results
PMAXHED (p = 0.017), DD10 (p = 0.003) and DD100 (p = 0.003) were independent predictors of BMI. The interaction of PMAXLED X DD10 (p = 0.033) and DD100 (p = 0.039), and PMAXHED X DD10 (p = 0.041) and DD100 (p = 0.045) increased the variance accounted for predicting BMI beyond the base model controlling for age, education, minority status, disinhibition and dietary restraint. Based on the regression model, BMI differed by about 2 BMI units for low versus high food reinforcement, by about 3 BMI units for low versus high DD, and by about 4 BMI units for those high in PMAXHED but low in DD versus high in PMAXHED and high in DD.
Conclusions
Reducing DD may help prevent obesity and improve treatment of obesity in those who are high in food reinforcement.
doi:10.1002/oby.20661
PMCID: PMC4007365  PMID: 24311480
Obesity; food reinforcement; delay discounting; reinforcement pathology
11.  Genome Sequencing of an Extended Series of NDM-Producing Klebsiella pneumoniae Isolates from Neonatal Infections in a Nepali Hospital Characterizes the Extent of Community- versus Hospital-Associated Transmission in an Endemic Setting 
Antimicrobial Agents and Chemotherapy  2014;58(12):7347-7357.
NDM-producing Klebsiella pneumoniae strains represent major clinical and infection control challenges, particularly in resource-limited settings with high rates of antimicrobial resistance. Determining whether transmission occurs at a gene, plasmid, or bacterial strain level and within hospital and/or the community has implications for monitoring and controlling spread. Whole-genome sequencing (WGS) is the highest-resolution typing method available for transmission epidemiology. We sequenced carbapenem-resistant K. pneumoniae isolates from 26 individuals involved in several infection case clusters in a Nepali neonatal unit and 68 other clinical Gram-negative isolates from a similar time frame, using Illumina and PacBio technologies. Within-outbreak chromosomal and closed-plasmid structures were generated and used as data set-specific references. Three temporally separated case clusters were caused by a single NDM K. pneumoniae strain with a conserved set of four plasmids, one being a 304,526-bp plasmid carrying blaNDM-1. The plasmids contained a large number of antimicrobial/heavy metal resistance and plasmid maintenance genes, which may have explained their persistence. No obvious environmental/human reservoir was found. There was no evidence of transmission of outbreak plasmids to other Gram-negative clinical isolates, although blaNDM variants were present in other isolates in different genetic contexts. WGS can effectively define complex antimicrobial resistance epidemiology. Wider sampling frames are required to contextualize outbreaks. Infection control may be effective in terminating outbreaks caused by particular strains, even in areas with widespread resistance, although this study could not demonstrate evidence supporting specific interventions. Larger, detailed studies are needed to characterize resistance genes, vectors, and host strains involved in disease, to enable effective intervention.
doi:10.1128/AAC.03900-14
PMCID: PMC4249533  PMID: 25267672
12.  Percutaneous Management of RetroFlex 3 Balloon Rupture and Separation of the Edwards Sapien Delivery System 
Texas Heart Institute Journal  2014;41(6):641-644.
We report the case of an 85-year-old woman with severe aortic stenosis who underwent transcatheter aortic valve replacement with use of the Edwards Sapien® valve system. The procedure was complicated by rupture of the valve-deployment balloon, with separation and retention of the nose cone of the RetroFlex 3® delivery system in the iliac artery. Our endovascular retrieval of the equipment was successful, and we achieved access-site hemostasis by deploying a covered stent. To our knowledge, this is the first report of the endovascular retrieval of a malfunctioning delivery system during transcatheter aortic valve replacement.
doi:10.14503/THIJ-13-3447
PMCID: PMC4251339  PMID: 25593532
Aged, 80 and over; cardiac catheterization/adverse effects; endovascular procedures/adverse effects/instrumentation; equipment failure; heart valve prosthesis implantation/adverse effects; iliac artery/injuries; treatment outcome
13.  Atrophy and Other Potential Factors Affecting Long Term Deep Brain Stimulation Response: A Case Series 
PLoS ONE  2014;9(10):e111561.
Objective
To describe three DBS cases which presented with new side effects or loss of benefit from stimulation after long-term follow-up and to discuss the potential contributing factors.
Methods
A University of Florida (UF) database (INFORM) search was performed, identifying three patients, two Parkinson's disease (PD) and one Essential Tremor (ET), with an unexpected change in long-term programming thresholds as compared to initial evaluation. Clinical follow-up, programming, imaging studies, and lead measurements were reviewed. The UF Institutional Review Board (IRB) approved this study.
Results
A substantial increase in the 3rd ventricular width (120%), Evans index (6%), ventricular index (5%), and cella media index (17%) was uncovered. A change in thresholds across lead contacts with a decrease in current densities as well as a relative lateral change of lead location was also observed. Hardware-related complications, lead migration, and impedance variability were not identified.
Conclusions
Potential factors contributing to long-term side effects should be examined during a DBS troubleshooting assessment. Clinicians should be aware that in DBS therapy there is delivery of electricity to a changing brain, and atrophy may possibly affect DBS programming settings as part of long-term follow-up.
doi:10.1371/journal.pone.0111561
PMCID: PMC4216112  PMID: 25360599
14.  Competitive Fitness Assays Indicate that the E138A Substitution in HIV-1 Reverse Transcriptase Decreases In Vitro Susceptibility to Emtricitabine 
We characterized the relative fitness of multiple nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-resistant HIV-1 variants in the presence of etravirine (ETV), rilpivirine (RPV), and/or the nucleoside RT inhibitor emtricitabine (FTC) by simultaneous competitive culture and 454 deep sequencing. The E138A substitution, typically associated with decreased virologic responses to ETV- and RPV-containing regimens, confers a clear fitness advantage to the virus in the presence of FTC and decreases FTC susceptibility 4.7-fold.
doi:10.1128/AAC.02114-13
PMCID: PMC4023788  PMID: 24419343
15.  Structure-based Programming of Lymph Node Targeting in Molecular Vaccines 
Nature  2014;507(7493):519-522.
In cancer patients, visual identification of sentinel lymph nodes (LNs) is achieved by the injection of dyes that bind avidly to endogenous albumin, targeting these compounds to LNs where they are efficiently filtered by resident phagocytes1,2. Here we translate this “albumin hitchhiking” approach to molecular vaccines, via the synthesis of amphiphiles (amph-vaccines) comprised of an antigen or adjuvant cargo linked to a lipophilic albumin-binding tail by a solubility-promoting polar polymer chain. Structurally-optimized CpG-DNA/peptide amph-vaccines exhibited dramatic increases in LN accumulation and decreased systemic dissemination relative to their parent compounds, leading to 30-fold increases in T-cell priming and enhanced anti-tumor efficacy while greatly reducing systemic toxicity. Amph-vaccines provide a simple, broadly-applicable strategy to simultaneously increase the potency and safety of subunit vaccines.
doi:10.1038/nature12978
PMCID: PMC4069155  PMID: 24531764
16.  SOCIAL TOLERANCE IN A DESPOTIC PRIMATE: CO-FEEDING BETWEEN CONSORTSHIP PARTNERS IN RHESUS MACAQUES 
Food-sharing among non-kin— one of the most fascinating cooperative behaviors in humans— is not widespread in non-human primates. Over the past few years, a large body of work has investigated the contexts in which primates cooperate and share food with unrelated individuals. This work has successfully demonstrated that species-specific differences in temperament constrain the extent to which food-sharing emerges in experimental situations, with despotic species being less likely to share food than tolerant ones. However, little experimental work has examined the contexts that promote food-sharing and cooperation within a species. Here, we examine whether one salient reproductive context—the consortship dyad— can allow the necessary social tolerance for co-feeding to emerge in an extremely despotic species, the rhesus macaque (Macaca mulatta). We gave naturally formed male-female rhesus macaque pairs access to a monopolizable food site in the free-ranging population at Cayo Santiago, Puerto Rico. Using this method, we were able to show that tolerated co-feeding between unrelated adults can take place in this despotic species. Specifically, our results show that consort pairs co-fed at the experimental food site more than non-consort control pairs, leading females to obtain more food in this context. These results suggest that co-feeding is possible even in the most despotic of primate species, but perhaps only in contexts that specifically promote the necessary social tolerance. Researchers might profit from exploring whether other kinds of within-species contexts could also generate cooperative behaviors.
doi:10.1002/ajpa.22043
PMCID: PMC4167600  PMID: 22415860
Social tolerance; food-sharing; sexual consortships; non-human primates
17.  In Vitro Characterization of a Sustained-Release Formulation for Enfuvirtide 
Although approved by the U.S. Food and Drug Administration, enfuvirtide is rarely used in combination antiretroviral therapies (cART) to treat HIV-1 infection, primarily because of its intense dosing schedule that requires twice-daily subcutaneous injection. Here, we describe the development of enfuvirtide-loaded, degradable poly(lactic-co-glycolic) acid microparticles that provide linear in vitro release of the drug over an 18-day period. This sustained-release formulation could make enfuvirtide more attractive for use in cART.
doi:10.1128/AAC.02440-13
PMCID: PMC3957899  PMID: 24366751
18.  Cerebral Venous Infarction: A Potentially Avoidable Complication of Deep Brain Stimulation Surgery 
Object
Despite numerous reports on the morbidity and mortality of deep brain stimulation (DBS), cerebral venous infarction has rarely been reported. We present four cases of venous infarct secondary to DBS surgery.
Methods
The diagnosis of venous infarction was based on: 1) delayed onset of new neurologic deficits on post-operative day 1 or 2, and 2) significant edema surrounding the superficial aspect of the implanted lead, with or without subcortical hemorrhage on CT scan.
Results
Four cases (0.8%/lead, 1.3%/patient) of symptomatic cerebral venous infarction were identified out of 500 DBS lead implantation procedures between July 2002 and August 2009. All four patients had Parkinson’s disease (PD). Their DBS leads were implanted in the subthalamic nucleus (STN) (n=2), and the internal globus pallidus (GPi) (n=2). Retrospective review of the targeting confirmed that the planned trajectory passed within 3mm of a cortical vein in two cases for which contrast-enhanced pre-operative MRI was available. In the other two cases, contrasted targeting images were not obtained preoperatively.
Conclusion
Cerebral venous infarction is a potentially avoidable, but serious complication. To minimize its incidence, we propose the use of high resolution, contrast-enhanced, T1 weighted MR images to delineate cerebral venous anatomy, along with careful stereotactic planning of the lead trajectory to avoid injury to venous structures.
doi:10.1111/ner.12052
PMCID: PMC3772976  PMID: 23738501
Deep Brain Stimulation; Venous Infarction; Complication; Adverse Event; Hemorrhage
19.  The Structural Basis for Divergence of Substrate Specificity and Biological Function within HAD Phosphatases in Lipopolysaccharide and Sialic Acid Biosynthesis 
Biochemistry  2013;52(32):5372-5386.
The haloacid dehalogenase enzyme superfamily (HADSF) is largely composed of phosphatases, which, relative to members of other phosphatase families, have been particularly successful at adaptation to novel biological functions. Herein, we examine the structural basis for divergence of function in two bacterial homologs: 2-keto-3-deoxy-D-manno-octulosonate 8-phosphate phosphohydrolase (KDO8P phosphatase, KDO8PP) and 2-keto-3-deoxy-9-O-phosphonononic acid phosphohydrolase (KDN9P phosphatase, KDN9PP). KDO8PP and KDN9PP catalyze the final step of KDO and KDN synthesis, respectively, prior to transfer to CMP to form the activated sugar nucleotide. KDO8PP and KDN9PP orthologs derived from an evolutionarily diverse collection of bacterial species were subjected to steady-state kinetic analysis to determine their specificities towards catalyzed KDO8P and KDN9P hydrolysis. Although each enzyme was more active with its biological substrate, the degree of selectivity (as defined by the ratio of kcat/Km for KDO8P vs. KDN9P) varied significantly. High-resolution X-ray structure determination of Haemophilus influenzae KDO8PP bound to KDO/VO3− and Bacteriodes thetaiotaomicron KDN9PP bound to KDN/VO3− revealed the substrate-binding residues. Structures of the KDO8PP and KDN9PP orthologs were also determined to reveal the differences in active site structure that underlies the variation in substrate preference. Bioinformatic analysis was carried out to define the sequence divergence among KDN9PP and KDO8PP orthologs. The KDN9PP orthologs were found to exist as single domain proteins or fused with the pathway nucleotidyl transferases; fusion of KDO8PP with the transferase is rare. The KDO8PP and KDN9PP orthologs share a stringently conserved Arg residue, which forms a salt bridge with the substrate carboxylate group. The split of the KDN9PP lineage from the KDO8PP orthologs is easily tracked by the acquisition of a Glu/Lys pair that supports KDN9P binding. Moreover, independently evolved lineages of KDO8PP orthologs exist, separated by diffuse active-site sequence boundaries. We infer high tolerance of the KDO8PP catalytic platform to amino acid replacements that, in turn, influence substrate specificity changes and thereby facilitate divergence of biological function.
doi:10.1021/bi400659k
PMCID: PMC3966652  PMID: 23848398
2-keto-3-deoxyoctulosonic acid (KDO); 2-keto-3-deoxynononic acid (KDN); phosphohydrolases; enzyme evolution; ortholog boundaries
20.  Human colonic crypts in culture: segregation of immunochemical markers in normal versus adenoma-derived 
In order to advance a culture model of human colonic neoplasia, we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomas. Crypts were maintained in three-dimensional Matrigel culture with a simple, serum-free, low Ca2+ (0.15 mM) medium. Intact colonic crypts from normal human mucosa were viably maintained for 3–5 days with preservation of the in situ crypt-like architecture, presenting a distinct base and apex. Abnormal structures from adenoma tissue could be maintained through multiple passages (up to months), with expanding buds/tubules. Immunohistochemical markers for intestinal stem cells (Lgr5), growth (Ki67), differentiation (E-cadherin, cytokeratin 20 (CK20) and mucin 2 (MUC2)) and epithelial turnover (Bax, cleaved Caspase-3), paralleled the changes in function. The epithelial cells in normal crypts followed the physiological sequence of progression from proliferation to differentiation to dissolution in a spatially and temporally appropriate manner. Lgr5 expression was seen in a few basal cells of freshly isolated crypts, but was not detected after 1–3 days in culture. After 24 h in culture, crypts from normal colonic tissue continued to show strong Ki67 and MUC2 expression at the crypt base, with a gradual decrease over time such that by days 3–4 Ki67 was not expressed. The differentiation marker CK20 increased over the same period, eventually becoming intense throughout the whole crypt. In adenoma-derived structures, expression of markers for all stages of progression persisted for the entire time in culture. Lgr5 showed expression in a few select cells after months in culture. Ki67 and MUC2 were largely associated with the proliferative budding regions while CK20 was localized to the parent structure. This ex vivo culture model of normal and adenomatous crypts provides a readily accessible tool to help understand the growth and differentiation process in human colonic epithelium.
doi:10.1038/labinvest.2013.145
PMCID: PMC4108175  PMID: 24365748
adenoma; apoptosis; chemoprevention; colon crypt culture; cytokeratin 20; Lgr5; mucin 2
21.  Predictors of eHealth Usage: Insights on The Digital Divide From the Health Information National Trends Survey 2012 
Background
Recent eHealth developments have elevated the importance of assessing the extent to which technology has empowered patients and improved health, particularly among the most vulnerable populations. With noted disparities across racial and social groups in chronic health outcomes, such as cancer, obesity, and diabetes, it is essential that researchers examine any differences in the implementation, uptake, and impact of eHealth strategies across groups that bear a disproportionate burden of disease.
Objective
The goal was to examine eHealth use by sociodemographic factors, such as race/ethnicity, socioeconomic status (SES), age, and sex.
Methods
We drew data from National Cancer Institute’s 2012 Health Information National Trends Survey (HINTS) (N=3959) which is publicly available online. We estimated multivariable logistic regression models to assess sociodemographic predictors of eHealth use among adult Internet users (N=2358) across 3 health communication domains (health care, health information–seeking, and user-generated content/sharing).
Results
Among online adults, we saw no evidence of a digital use divide by race/ethnicity. However, there were significant differences in use by SES, particularly for health care and health information–seeking items. Patients with lower levels of education had significantly lower odds of going online to look for a health care provider (high school or less: OR 0.50, 95% CI 0.33-0.76) using email or the Internet to communicate with a doctor (high school or less: OR 0.46, 95% CI 0.29-0.72), tracking their personal health information online (high school or less: OR 0.53, 95% CI 0.32-0.84), using a website to help track diet, weight, and physical activity (high school or less: OR 0.64, 95% CI 0.42-0.98; some college: OR 0.67, 95% CI 0.49-0.93), or downloading health information to a mobile device (some college: OR 0.54, 95% CI 0.33-0.89). Being female was a consistent predictor of eHealth use across health care and user-generated content/sharing domains, whereas age was primarily influential for health information–seeking.
Conclusions
This study illustrates that lower SES, older, and male online US adults were less likely to engage in a number of eHealth activities compared to their counterparts. Future studies should assess issues of health literacy and eHealth literacy and their influence on eHealth engagement across social groups. Clinical care and public health communication efforts attempting to leverage Web 2.0 and 3.0 platforms should acknowledge differential eHealth usage to better address communication inequalities and persistent disparities in health.
doi:10.2196/jmir.3117
PMCID: PMC4129114  PMID: 25048379
health communication; communication barriers; Internet; consumer health information
22.  Deep Brain Stimulation for Tremor Associated with Underlying Ataxia Syndromes: A Case Series and Discussion of Issues 
Background
Deep brain stimulation (DBS) has been utilized to treat various symptoms in patients suffering from movement disorders such as Parkinson's disease, dystonia, and essential tremor. Though ataxia syndromes have not been formally or frequently addressed with DBS, there are patients with ataxia and associated medication refractory tremor or dystonia who may potentially benefit from therapy.
Methods
A retrospective database review was performed, searching for cases of ataxia where tremor and/or dystonia were addressed by utilizing DBS at the University of Florida Center for Movement Disorders and Neurorestoration between 2008 and 2011. Five patients were found who had DBS implantation to address either medication refractory tremor or dystonia. The patient's underlying diagnoses included spinocerebellar ataxia type 2 (SCA2), fragile X associated tremor ataxia syndrome (FXTAS), a case of idiopathic ataxia (ataxia not otherwise specified [NOS]), spinocerebellar ataxia type 17 (SCA17), and a senataxin mutation (SETX).
Results
DBS improved medication refractory tremor in the SCA2 and the ataxia NOS patients. The outcome for the FXTAS patient was poor. DBS improved dystonia in the SCA17 and SETX patients, although dystonia did not improve in the lower extremities of the SCA17 patient. All patients reported a transient gait dysfunction postoperatively, and there were no reports of improvement in ataxia-related symptoms.
Discussion
DBS may be an option to treat tremor, inclusive of dystonic tremor in patients with underlying ataxia; however, gait and other symptoms may possibly be worsened.
doi:10.7916/D8542KQ5
PMCID: PMC4101398  PMID: 25120941
Tremor; SCA2; SCA17; fragile X syndrome; myoclonic dystonia; deep brain stimulation; unilateral
24.  Food reinforcement, dietary disinhibition and weight gain in non-obese adults 
Obesity (Silver Spring, Md.)  2013;22(1):254-259.
Objective
Food reinforcement is cross-sectionally related to body mass index and energy intake in adults, and prospectively predicts weight gain in children, but there has not been any research studying food reinforcement as a predictor of adult weight gain.
Design and Methods
This study examined whether the relative reinforcing value of food versus sedentary activities, as measured on a progressive ratio schedule, predicts 12 month weight gain. Dietary disinhibition and dietary restraint were also examined as potential moderators of this relationship, in a sample of 115 non-obese (Body Mass Index< 30) adults.
Results
In a hierarchical regression controlling for baseline age and weight, dietary hunger, income, sex and minority status, food reinforcement significantly increased the variance from 6.3% to 11.7% (p = 0.01) and predicted weight gain (p = 0.01). Dietary disinhibition moderated this relationship (p = 0.02) and increased the variance an additional 4.7% (p = 0.02), such that individuals with high food reinforcement had greater weight gain if they were also high in disinhibition.
Conclusions
These results suggest that food reinforcement is a significant contributor to weight change over time, and food reinforcement may have the biggest effect on those who are most responsive to food cues.
doi:10.1002/oby.20392
PMCID: PMC3815500  PMID: 23512958
25.  Peripheral vascular complications during TAVR: Management and potential role of chronic steroid use; A case report 
Purpose
To report a case of a major vascular complication during TAVR and the endovascular management thereof. Additionally, we discuss a possible correlation with long-term steroid use.
Case Report
A 79 year old woman with a history of critical aortic stenosis underwent elective transcatheter aortic valve replacement (TAVR). Her procedure was complicated by rupture of her right iliac artery, life threatening retroperitoneal hemorrhage, and thrombus extending into the distal right lower extremity. This case was emergently managed by stent placement, thrombectomy, and tPA via a percutaneous approach.
Conclusions
Peripheral vascular complications are common during percutaneous TAVR, and chronic steroid use may predispose patients. Endovascular management is often possible and may potentially save valuable time in emergent situations.
doi:10.1177/1531003513491985
PMCID: PMC4005891  PMID: 23793293
TAVR; TAVI; Peripheral; Endovascular

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