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1.  Mapping the Pareto Optimal Design Space for a Functionally Deimmunized Biotherapeutic Candidate 
PLoS Computational Biology  2015;11(1):e1003988.
The immunogenicity of biotherapeutics can bottleneck development pipelines and poses a barrier to widespread clinical application. As a result, there is a growing need for improved deimmunization technologies. We have recently described algorithms that simultaneously optimize proteins for both reduced T cell epitope content and high-level function. In silico analysis of this dual objective design space reveals that there is no single global optimum with respect to protein deimmunization. Instead, mutagenic epitope deletion yields a spectrum of designs that exhibit tradeoffs between immunogenic potential and molecular function. The leading edge of this design space is the Pareto frontier, i.e. the undominated variants for which no other single design exhibits better performance in both criteria. Here, the Pareto frontier of a therapeutic enzyme has been designed, constructed, and evaluated experimentally. Various measures of protein performance were found to map a functional sequence space that correlated well with computational predictions. These results represent the first systematic and rigorous assessment of the functional penalty that must be paid for pursuing progressively more deimmunized biotherapeutic candidates. Given this capacity to rapidly assess and design for tradeoffs between protein immunogenicity and functionality, these algorithms may prove useful in augmenting, accelerating, and de-risking experimental deimmunization efforts.
Author Summary
Protein therapeutics have created a revolution in disease therapy, providing improved outcomes for prevalent illnesses and conditions while at the same time yielding treatments for diseases that were previously intractable. However, this powerful class of drugs is subject to their own unique challenges and risk factors. In particular, the biological origins of therapeutic proteins predispose them towards eliciting a detrimental immune response from the patient's own body. Therefore, fully capitalizing on the medicinal reservoir of natural and engineered proteins will require efficient, effective, and broadly applicable deimmunization technologies. We have developed deimmunization algorithms that simultaneously optimize therapeutic candidates for both low immunogenicity and high-level activity and stability. Here, we combine computational modeling and experimental analysis to show that the process of protein deimmunization manifests inherent tradeoffs between immunogenic potential and biomolecular function. Our experimental results demonstrate that dual objective optimization allows us to assess and design for these tradeoffs, thereby enabling facile construction of deimmunized variants that span a broad range of immunogenicity and functionality performance parameters. Thus, we can rapidly map the design space for deimmunized drug candidates, and we can use this information to guide selection of engineered proteins that are most likely to meet performance benchmarks for a given clinical application.
PMCID: PMC4288714  PMID: 25568954
2.  Multifrequency bioelectrical impedance analysis and bioimpedance spectroscopy for monitoring fluid and body cell mass changes after gastric bypass surgery✩ 
Background & aims
There is a growing need for clinically applicable body composition assessment tools for extremely obese individuals. The objective of this research was to evaluate several bio impedance techniques for monitoring changes in fluid, particularly intracellular water (reflecting body cell mass) after bariatric surgery.
Fifteen extremely obese women (body mass index: 48.9 ±7.0 kg/m2; age: 48 ±9 years) were assessed before (baseline; T1), and approximately 6 weeks after gastric bypass surgery (T2) by several multifrequency bioelectrical impedance analysis approaches (MFBIA; QuadScan 4000), a bioimpedance spectroscopy device (BIS; Hydra 4200), and multiple dilution.
BIS provided intracellular water estimates that were comparable to criterion, based on mean comparisons, at both time points (T1: criterion: 24.2 ±3.1 L, BIS: 24.0 ± 3.7 L; T2: criterion: 20.6 ± 3.7 L, BIS: 19.7 ± 3.2 L). MFBIA (with Deurenberg equations) provided comparable measures to criterion only at T2 (criterion: 20.3 ± 3.7 L, MFBIA: 20.6 ± 2.7 L). Both MFBIA (with QuadScan proprietary equations) and BIS produced estimates of intracellular water change that were comparable to dilution. There was substantial variability in individual volume measures.
Although MFBIA and BIS hold promise as convenient techniques for assessing fluid changes, individual variability in measurements makes them impractical for assessment of extremely obese patients in the clinical setting.
PMCID: PMC4284052  PMID: 18676066
Bioimpedance; Multiple frequency; Obesity; Intracellular water
3.  Changes in Gastrointestinal Hormones and Leptin After Roux-en-Y Gastric Bypass Surgery 
Roux-en-Y gastric bypass (RYGB) imparts long-term weight loss, the mechanisms for which are not well understood. Changes in leptin and gastrointestinal (GI) hormones, including glucagon-like peptide 1 (GLP-1), peptide YY (PYY), and ghrelin, may contribute to the relative success of RYGB compared with conventional weight loss methods. This study evaluated changes in GI hormones and leptin post-RYGB. The study also evaluated whether GI hormones differed after a short-term dose of protein or fat.
GLP-1, PYY, ghrelin, and leptin were assessed in 16 women before RYGB and up to 1 year after RYGB. Plasma was collected before and at several times after a short-term equicaloric dose of protein or fat.
GLP-1 area under the curve (AUC) increased at week 6 and 1 year in the fat beverage (FAT-BEV) group compared with baseline. PYY AUC remained elevated at 1 year in the FAT-BEV group. Ghrelin AUC decreased at week 2, week 6, and 1 year in the protein beverage (PRO-BEV) group compared with baseline. Ghrelin AUC was lower in the PRO-BEV group compared with the FAT-BEV group at week 6. Fasted leptin decreased at all visits in both groups and was lower in the FAT-BEV group compared with the PRO-BEV group at 1 year.
Changes from baseline were evident for all GI hormones and leptin; some differences were evident soon after surgery (ghrelin, leptin), whereas others were maintained long term (GLP-1, PYY, ghrelin, leptin). In response to a short-term stimulus, protein suppressed ghrelin and fat potently stimulated GLP-1 and PYY. Future work in this area is warranted.
PMCID: PMC4284080  PMID: 21378246
obesity; ghrelin; peptide YY; glucagon-like peptide 1; leptin; gastric bypass; gastrointestinal hormones
4.  Prostate laser vaporization is safe and effective in elderly men 
Urology Annals  2015;7(1):36-40.
There are few data on the safety and efficacy of laser photoselective vaporization (LVP) in elderly men. We compared the safety and efficacy of LVP for the treatment of symptomatic benign prostatic hyperplasia (BPH) in men ≥75 years, who we defined as elderly, to those <75 years.
Materials and Methods:
Safety and efficacy outcomes in elderly men undergoing LVP for lower urinary tract symptoms secondary to BPH from 2005 to 2012 were compared with men <75 years. Differences between-groups in demographics, perioperative outcomes, complications, and postoperative changes in International Prostate Symptom Score (I-PSS) were calculated.
Of 202 patients, 49 (24%) were elderly (range: 75-95 years) and 153 (76%) were <75 years. Preoperatively, elderly men were more likely to have heart disease (35% vs. 20%, P = 0.03), gross hematuria (6.1% vs. 0.7%, P = 0.05), urinary retention (57% vs. 41%, P = 0.07), and take anti-coagulants (61% vs. 35%, P = 0.002). Elderly men had a longer median length of stay (1 day vs. 0 day, P = 0.001). There were no significant between-group differences in transfusion frequency (4.4% vs. 0.7%, P = 0.14) or Clavien III complications (2% vs. 2.6%, P = 1.0). One month postsurgery, elderly patients reported smaller median decreases in I-PSS (5.5 vs. 9, P = 0.02) and urinary bother (1 point vs. 2, P = 0.03) compared with preoperative values. At till 9 months follow-up, there were no significant between-group differences in median I-PSS or urinary bother scores.
Despite a higher prevalence of preoperative comorbidity and urinary retention, elderly LVP patients experienced perioperative safety and shorter term efficacy outcomes comparable to younger men.
PMCID: PMC4310114  PMID: 25657541
Benign prostatic hyperplasia; complications; elderly; lower urinary tract symptoms; outcomes
5.  Structure-Activity Relationships of Retro-dihydrochalcones Isolated from Tacca sp 
Journal of natural products  2013;76(12):2189-2194.
Several biologically active compounds have been identified from Tacca species, including glycosides, diarylheptanoids, saponins, withanolides, and the taccalonolide class of microtubule stabilizers. More recently, two cytotoxic retro-dihydrochalcones named evelynin A (7) and taccabulin A (6) were isolated and their biological activities characterized, including the finding that taccabulin has microtubule destabilizing effects. Here we describe the identification and characterization of five new retro-chalcones, named taccabulins B – E (1–4) and evelynin B (5) from Tacca sp. extracts. Their structures were determined using 1D and 2D NMR as well as mass spectroscopic data and modeled into the colchicine binding site of tubulin. The antiproliferative and microtubule effects of each compound were determined experimentally and found to be well correlated with modeling studies. The isolation and biological characterization of several retro-dihydrochalcones facilitated preliminary structure-activity relationships for this compound class concerning its antiproliferative and microtubule depolymerizing activities.
PMCID: PMC4012383  PMID: 24303844
6.  Cks Confers Specificity to Phosphorylation-Dependent Cdk Signaling Pathways 
Nature structural & molecular biology  2013;20(12):1407-1414.
Cks is an evolutionarily conserved protein that regulates cyclin-dependent kinase (Cdk) activity. Clarifying the underlying mechanisms and cellular contexts of Cks function is critical, as Cks is essential for proper cell growth, and its overexpression has been linked to cancer. We observe that budding yeast Cks associates with select phosphorylated sequences in cell cycle regulatory proteins. We characterize the molecular interactions responsible for this specificity and demonstrate that Cks enhances Cdk activity in response to specific priming phosphosites. Identification of the binding consensus sequence allows us to identify putative Cks-directed Cdk substrates and binding partners. We characterize novel Cks binding sites in the mitotic regulator Wee1 and discover a novel role for Cks in regulating Cdk activity at mitotic entry. Together our results portray Cks as a multifunctional phosphoadaptor that serves as a specificity factor for Cdk activity.
PMCID: PMC4242096  PMID: 24186063
7.  Learning Sequence Determinants of Protein:protein Interaction Specificity with Sparse Graphical Models 
In studying the strength and specificity of interaction between members of two protein families, key questions center on which pairs of possible partners actually interact, how well they interact, and why they interact while others do not. The advent of large-scale experimental studies of interactions between members of a target family and a diverse set of possible interaction partners offers the opportunity to address these questions. We develop here a method, DgSpi (Data-driven Graphical models of Specificity in Protein:protein Interactions), for learning and using graphical models that explicitly represent the amino acid basis for interaction specificity (why) and extend earlier classification-oriented approaches (which) to predict the ΔG of binding (how well). We demonstrate the effectiveness of our approach in analyzing and predicting interactions between a set of 82 PDZ recognition modules, against a panel of 217 possible peptide partners, based on data from MacBeath and colleagues. Our predicted ΔG values are highly predictive of the experimentally measured ones, reaching correlation coefficients of 0.69 in 10-fold cross-validation and 0.63 in leave-one-PDZ-out cross-validation. Furthermore, the model serves as a compact representation of amino acid constraints underlying the interactions, enabling protein-level ΔG predictions to be naturally understood in terms of residue-level constraints. Finally, as a generative model, DgSpi readily enables the design of new interacting partners, and we demonstrate that designed ligands are novel and diverse.
PMCID: PMC4235964  PMID: 25414914
8.  Fecal Indicator Bacteria Contamination of Fomites and Household Demand for Surface Disinfection Products: A Case Study from Peru 
Surface-mediated disease transmission is understudied in developing countries, particularly in light of the evidence that surface concentrations of fecal bacteria typically exceed concentrations in developed countries by 10- to 100-fold. In this study, we examined fecal indicator bacterial contamination of dinner plates at 21 households in four peri-urban communities in the Peruvian Amazon. We also used surveys to estimate household use of and demand for surface disinfectants at 280 households. Despite detecting total coliform, enterococci, and Escherichia coli on 86%, 43%, and 24% of plates sampled, respectively, less than one-third of households were regularly using bleach to disinfect surfaces. Among non-users of bleach, only 3.2% of respondents reported a new demand for bleach, defined as a high likelihood of using bleach within the next year. This study highlights the potential for marketing approaches to increase use of and demand for surface disinfectants to improve domestic hygiene.
PMCID: PMC3820328  PMID: 24019431
9.  Gene and Protein Sequence Optimization for High-Level Production of Fully Active and Aglycosylated Lysostaphin in Pichia pastoris 
Lysostaphin represents a promising therapeutic agent for the treatment of staphylococcal infections, in particular those of methicillin-resistant Staphylococcus aureus (MRSA). However, conventional expression systems for the enzyme suffer from various limitations, and there remains a need for an efficient and cost-effective production process to facilitate clinical translation and the development of nonmedical applications. While Pichia pastoris is widely used for high-level production of recombinant proteins, there are two major barriers to the production of lysostaphin in this industrially relevant host: lack of expression from the wild-type lysostaphin gene and aberrant glycosylation of the wild-type protein sequence. The first barrier can be overcome with a synthetic gene incorporating improved codon usage and balanced A+T/G+C content, and the second barrier can be overcome by disrupting an N-linked glycosylation sequon using a broadened choice of mutations that yield aglyscosylated and fully active lysostaphin. The optimized lysostaphin variants could be produced at approximately 500 mg/liter in a small-scale bioreactor, and 50% of that material could be recovered at high purity with a simple 2-step purification. It is anticipated that this novel high-level expression system will bring down one of the major barriers to future development of biomedical, veterinary, and research applications of lysostaphin and its engineered variants.
PMCID: PMC3993296  PMID: 24561590
10.  Binding Mode Characterization of 6α- and 6β-N-Heterocyclic Substituted Naltrexamine Derivatives via Docking in Opioid Receptor Crystal Structures and Site-directed Mutagenesis Studies: Application of the “Message–Address” Concept in Development of Mu Opioid Receptor Selective Antagonists 
Bioorganic & medicinal chemistry  2013;21(21):10.1016/j.bmc.2013.08.042.
Highly selective opioid receptor antagonists are essential pharmacological probes in opioid receptor structural characterization and opioid agonist functional studies. Currently, there is no highly selective, non-peptidyl and reversible mu opioid receptor antagonist available. Among a series of naltrexamine derivatives that have been designed and synthesized, two compounds, NAP and NAQ, were previously identified as novel leads for this purpose based on their in vitro and in vivo pharmacological profiles. Both compounds displayed high binding affinity and selectivity to the mu opioid receptor. To further study the interaction of these two ligands with the three opioid receptors, the recently released opioid receptor crystal structures were employed in docking studies to further test our original hypothesis that the ligands recognize a unique “address” domain in the mu opioid receptor involving Trp318 that facilitates their selectivity. These modeling results were supported by site-directed mutagenesis studies on the mu opioid receptor, where the mutants Y210A and W318A confirmed the role of the latter in binding. Such work not only enriched the “message-address” concept, also facilitated our next generation ligand design and development.
PMCID: PMC3831368  PMID: 24055076
Mu opioid receptor; selective antagonists; crystal structures; automated docking; site-directed mutagenesis; GPCR
11.  Comparing Bacterial Community Composition of Healthy and Dark Spot-Affected Siderastrea siderea in Florida and the Caribbean 
PLoS ONE  2014;9(10):e108767.
Coral disease is one of the major causes of reef degradation. Dark Spot Syndrome (DSS) was described in the early 1990's as brown or purple amorphous areas of tissue on a coral and has since become one of the most prevalent diseases reported on Caribbean reefs. It has been identified in a number of coral species, but there is debate as to whether it is in fact the same disease in different corals. Further, it is questioned whether these macroscopic signs are in fact diagnostic of an infectious disease at all. The most commonly affected species in the Caribbean is the massive starlet coral Siderastrea siderea. We sampled this species in two locations, Dry Tortugas National Park and Virgin Islands National Park. Tissue biopsies were collected from both healthy colonies and those with dark spot lesions. Microbial-community DNA was extracted from coral samples (mucus, tissue, and skeleton), amplified using bacterial-specific primers, and applied to PhyloChip G3 microarrays to examine the bacterial diversity associated with this coral. Samples were also screened for the presence of a fungal ribotype that has recently been implicated as a causative agent of DSS in another coral species, but the amplifications were unsuccessful. S. siderea samples did not cluster consistently based on health state (i.e., normal versus dark spot). Various bacteria, including Cyanobacteria and Vibrios, were observed to have increased relative abundance in the discolored tissue, but the patterns were not consistent across all DSS samples. Overall, our findings do not support the hypothesis that DSS in S. siderea is linked to a bacterial pathogen or pathogens. This dataset provides the most comprehensive overview to date of the bacterial community associated with the scleractinian coral S. siderea.
PMCID: PMC4188562  PMID: 25289937
12.  Smarter vaccine design will circumvent regulatory T cell-mediated evasion in chronic HIV and HCV infection 
Despite years of research, vaccines against HIV and HCV are not yet available, due largely to effective viral immunoevasive mechanisms. A novel escape mechanism observed in viruses that cause chronic infection is suppression of viral-specific effector CD4+ and CD8+ T cells by stimulating regulatory T cells (Tregs) educated on host sequences during tolerance induction. Viral class II MHC epitopes that share a T cell receptor (TCR)-face with host epitopes may activate Tregs capable of suppressing protective responses. We designed an immunoinformatic algorithm, JanusMatrix, to identify such epitopes and discovered that among human-host viruses, chronic viruses appear more human-like than viruses that cause acute infection. Furthermore, an HCV epitope that activates Tregs in chronically infected patients, but not clearers, shares a TCR-face with numerous human sequences. To boost weak CD4+ T cell responses associated with persistent infection, vaccines for HIV and HCV must circumvent potential Treg activation that can handicap efficacy. Epitope-driven approaches to vaccine design that involve careful consideration of the T cell subsets primed during immunization will advance HIV and HCV vaccine development.
PMCID: PMC4186478  PMID: 25339942
HIV; HCV; T cell epitope; immunoinformatics; vaccines; cross-reactivity; regulatory T cells; MHC class II
13.  The Burden of Urinary Incontinence and Urinary Bother Among Elderly Prostate Cancer Survivors 
European urology  2013;64(4):672-679.
Data describing urinary health in elderly, community-dwelling prostate cancer (PCa) survivors are limited.
To elucidate the prevalence of lower urinary tract symptoms, urinary bother, and incontinence in elderly PCa survivors compared with peers without PCa.
Design, setting, and participants
A cross-sectional analysis of 5990 participants in the Osteoporotic Fractures in Men Research Group, a cohort study of community-dwelling men ≥65 yr.
Outcome measurements and statistical analysis
We characterized urinary health using self-reported urinary incontinence and the American Urological Association Symptom Index (AUA-SI). We compared urinary health measures according to type of PCa treatment in men with PCa and men without PCa using multivariate log-binomial regression to generate prevalence ratios (PRs).
Results and limitations
At baseline, 706 men (12%) reported a history of PCa, with a median time since diagnosis of 6.3 yr. Of these men, 426 (60%) reported urinary incontinence. In adjusted analyses, observation (PR: 1.92; 95% confidence interval [CI], 1.15–3.21; p = 0.01), surgery (PR: 4.68; 95% CI, 4.11–5.32; p < 0.0001), radiation therapy (PR: 1.64; 95% CI, 1.20– 2.23; p = 0.002), and androgen-deprivation therapy (ADT) (PR: 2.01; 95% CI, 1.35–2.99; p = 0.0006) were each associated with daily incontinence. Daily incontinence risk increased with time since diagnosis independently of age. Observation (PR: 1.33; 95% CI, 1.00–1.78; p = 0.05), surgery (PR: 1.25; 95% CI, 1.10–1.42; p = 0.0008), and ADT (PR: 1.50; 95% CI, 1.26–1.79; p < 0.0001) were associated with increased AUA-SI bother scores. Cancer stage and use of adjuvant or salvage therapies were not available for analysis.
Compared with their peers without PCa, elderly PCa survivors had a two-fold to five-fold greater prevalence of urinary incontinence, which rose with increasing survivorship duration. Observation, surgery, and ADT were each associated with increased urinary bother. These data suggest a substantially greater burden of urinary health problems among elderly PCa survivors than previously recognized.
PMCID: PMC3938018  PMID: 23587870
Aging male; Elderly; Epidemiology; Incontinence; Lower urinary tract symptoms; Prostate cancer; Prostate cancer treatment; Urinary bother
14.  Unclassified Mucinous Renal Cell Carcinoma: A Rare Histopathological Entity 
Korean Journal of Urology  2014;55(10):690-692.
Renal cell carcinoma (RCC) with mucin production is extremely rare. We present the case of a previously healthy 76-year-old woman who underwent a robotic-assisted laparoscopic right nephrectomy for a 5-cm heterogeneously enhancing right renal mass. Pathology revealed mucin-producing epithelial RCC. We discuss the presentation and pathological features of this case and comment on its definitive treatment.
PMCID: PMC4198770  PMID: 25324954
Mucin; Renal cell carcinoma
15.  Phlorotannins from Alaskan Seaweed Inhibit Carbolytic Enzyme Activity 
Marine Drugs  2014;12(10):5277-5294.
Global incidence of type 2 diabetes has escalated over the past few decades, necessitating a continued search for natural sources of enzyme inhibitors to offset postprandial hyperglycemia. The objective of this study was to evaluate coastal Alaskan seaweed inhibition of α-glucosidase and α-amylase, two carbolytic enzymes involved in serum glucose regulation. Of the six species initially screened, the brown seaweeds Fucus distichus and Alaria marginata possessed the strongest inhibitory effects. F. distichus fractions were potent mixed-mode inhibitors of α-glucosidase and α-amylase, with IC50 values of 0.89 and 13.9 μg/mL, respectively; significantly more efficacious than the pharmaceutical acarbose (IC50 of 112.0 and 137.8 μg/mL, respectively). The activity of F. distichus fractions was associated with phlorotannin oligomers. Normal-phase liquid chromatography-mass spectrometry (NPLC-MS) was employed to characterize individual oligomers. Accurate masses and fragmentation patterns confirmed the presence of fucophloroethol structures with degrees of polymerization from 3 to 18 monomer units. These findings suggest that coastal Alaskan seaweeds are sources of α-glucosidase and α-amylase inhibitory phlorotannins, and thus have potential to limit the release of sugar from carbohydrates and thus alleviate postprandial hyperglycemia.
PMCID: PMC4210899  PMID: 25341030
Alaska; seaweed; diabetes; glucosidase; hyperglycemia; amylase; type 2 diabetes mellitus; phlorotannin; polyphenol; ethnopharmacology
16.  How to Deal with Low-Resolution Target Structures: Using SAR, Ensemble Docking, Hydropathic Analysis, and 3D-QSAR to Definitively Map the αβ-Tubulin Colchicine Site 
Journal of medicinal chemistry  2013;56(18):10.1021/jm400954h.
αβ-tubulin colchicine site inhibitors (CSIs) from four scaffolds that we previously tested for antiproliferative activity were modeled to better understand their effect on microtubules. Docking models, constructed by exploiting the SAR of a pyrrole subset and HINT scoring, guided ensemble docking of all 59 compounds. This conformation set and two variants having progressively less structure knowledge were subjected to CoMFA, CoMFA+HINT, and CoMSIA 3D-QSAR analyses. The CoMFA+HINT model (docked alignment) showed the best statistics: leave-one-out q2 of 0.616, r2 of 0.949 and r2pred (internal test set) of 0.755. An external (tested in other laboratories) collection of 24 CSIs from eight scaffolds were evaluated with the 3D-QSAR models, which correctly ranked their activity trends in 7/8 scaffolds for CoMFA+HINT (8/8 for CoMFA). The combination of SAR, ensemble docking, hydropathic analysis and 3D-QSAR provides an atomic-scale colchicine site model more consistent with a target structure resolution much higher than the ~3.6 Å available for αβ-tubulin.
PMCID: PMC3831284  PMID: 23961916
tubulin; 3D-QSAR; microtubule depolymerization; colchicine site inhibitor; HINT; ensemble docking and scoring
17.  Vitamin E and the Risk of Prostate Cancer: Updated Results of The Selenium and Vitamin E Cancer Prevention Trial (SELECT) 
JAMA  2011;306(14):1549-1556.
The initial report of the Selenium and Vitamin E Cancer Prevention Trial (SELECT) found no reduction in risk of prostate cancer with either selenium or vitamin E supplements but a non-statistically significant increase in prostate cancer risk with vitamin E. Longer follow-up and more prostate cancer events provide further insight into the relationship of vitamin E and prostate cancer.
To determine the long-term effect of vitamin E and selenium on risk of prostate cancer in relatively healthy men.
Design, Setting and Participants
SELECT randomized 35,533 men from 427 study sites in the United States, Canada and Puerto Rico in a double-blind manner between August 22, 2001 and June 24, 2004. Eligible men were 50 years or older (African Americans) or 55 years or older (all others) with a PSA ≤4.0 ng/mL and a digital rectal examination not suspicious for prostate cancer. Included in the analysis are 34,887 men randomly assigned to one of four treatment groups: selenium (n=8752), vitamin E (n=8737), both agents (n=8702), or placebo (n=8696). Data reflect the final data collected by the study sites on their participants through July 5, 2011.
Oral selenium (200 μg/day from L-selenomethionine) with matched vitamin E placebo, vitamin E (400 IU/d of all rac-α-tocopheryl acetate) with matched selenium placebo, both agents, or both matched placebos for a planned follow-up of a minimum of 7 and maximum of 12 years.
Main Outcome Measures
Prostate cancer incidence.
This report includes 54,464 additional person-years of follow-up since the primary report. Hazard ratios (99% confidence intervals [CI]) and numbers of prostate cancers were 1.17(99% CI 1.004-1.36, p=.008, n=620) for vitamin E, 1.09 (99% CI 0.93-1.27, p=.18, n=575) for selenium, 1.05 (99%CI 0.89-1.22, p=.46, n=555) for selenium + vitamin E vs. 1.00 (n=529) for placebo.The absolute increase in risk compared with placebo for vitamin E, selenium and the combination were 1.6, 0.9 and 0.4 cases of prostate cancer per 1,000 person-years.
Dietary supplementation with Vitamin E significantly increases the risk of prostate cancer among healthy men.
Trial registration identifier: NCT00006392
PMCID: PMC4169010  PMID: 21990298
18.  A Randomized Pilot Trial of Dietary Modification for the Chemoprevention of Non-invasive Bladder Cancer: The Dietary Intervention in Bladder Cancer Study (DIBS) 
Cancer prevention research (Philadelphia, Pa.)  2013;6(9):10.1158/1940-6207.CAPR-13-0050.
Epidemiological data suggest robust associations of high vegetable intake with decreased risks of bladder cancer incidence and mortality, but translational prevention studies have yet to be performed. We designed and tested a novel intervention to increase vegetable intake in patients with non-invasive bladder cancer. We randomized 48 patients aged 50 to 80 years with biopsy-proven non-invasive (Ta, T1, or carcinoma in situ) urothelial cell carcinoma to telephone- and Skype-based dietary counseling or a control condition that provided print materials only. The intervention behavioral goals promoted 7 daily vegetable servings, with at least 2 of these as cruciferous vegetables. Outcome variables were self-reported diet and plasma carotenoid and 24-hour urinary isothiocyanate (ITC) concentrations. We used 2-sample t-tests to assess between-group differences at 6-month follow-up. After 6 months, intervention patients had higher daily intakes of vegetable juice (p=0.02), total vegetables (p=0.02), and cruciferous vegetables (p=0.07); lower daily intakes of energy (p=0.007), (p=0.002) and energy from fat (p=0.06); and higher plasma alpha-carotene concentrations (p=0.03). Self-reported cruciferous vegetable intake correlated with urinary ITC concentrations at baseline (p<0.001) and at 6 months (p=0.03). Although urinary ITC concentrations increased in the intervention group and decreased in the control group, these changes did not attain between-group significance (p=0.32). In patients with non-invasive bladder cancer, our novel intervention induced diet changes associated with protective effects against bladder cancer. These data demonstrate the feasibility of implementing therapeutic dietary modifications to prevent recurrent and progressive bladder cancer.
PMCID: PMC3857028  PMID: 23867158
19.  Utility of a clinical practice guideline in treatment of chylothorax in the post-operative congenital heart patient 
The Annals of thoracic surgery  2013;96(3):10.1016/j.athoracsur.2013.05.058.
Chylothorax following congenital heart surgery is a common complication with associated morbidities, but consensus treatment guidelines are lacking. Variability exists in the duration of medical treatment and timing for surgical intervention.
Following institution of a clinical practice guideline for management of post-operative chylothorax at a single center, pediatric cardiothoracic intensive care unit (ICU) in June 2010, we retrospectively analyzed two cohorts of patients: those with chylothorax from 1/2008-5/2010 (early cohort; n = 118) and from 6/2010-8/2011 (late cohort; n = 45). Data collected included demographics, cardiac surgical procedure, treatments for chylothorax, bloodstream infections, hospital mortality, length of hospitalization, duration of mechanical ventilation, and device utilization.
There were no demographic differences between the cohorts. No differences were found in octreotide use or surgical treatments for chylothorax. Significant differences were found in median times to chylothorax diagnosis (9 in early cohort versus 6 days in late cohort, p = 0.004), ICU length of stay (18 vs. 9 days, p = 0.01), hospital length of stay (30 vs. 23 days, p = 0.005), and total durations of mechanical ventilation (11 vs. 5 days, p = 0.02), chest tube use (20 vs. 14 days, p = 0.01), central venous line use (27 vs. 15 days, p = 0.001), and NPO status (9.5 vs. 6 days, p = 0.04).
Institution of a clinical practice guideline for treatment of chylothorax following congenital heart surgery was associated with earlier diagnosis, reduced hospital length of stay, mechanical ventilation, and device utilization for these patients.
PMCID: PMC3886283  PMID: 23915583
chyle; pleural effusion; congenital heart disease; practice guidelines
20.  Divergent Antibody Subclass and Specificity Profiles but Not Protective HLA-B Alleles Are Associated with Variable Antibody Effector Function among HIV-1 Controllers 
Journal of Virology  2014;88(5):2799-2809.
Understanding the coordination between humoral and cellular immune responses may be the key to developing protective vaccines, and because genetic studies of long-term HIV-1 nonprogressors have associated specific HLA-B alleles with spontaneous control of viral replication, this subject group presents an opportunity to investigate relationships between arms of the adaptive immune system. Given evidence suggesting that cellular immunity may play a role in viral suppression, we sought to determine whether and how the humoral immune response might vary among controllers. Significantly, Fc-mediated antibody effector functions have likewise been associated with durable viral control. In this study, we compared the effector function and biophysical features of HIV-specific antibodies in a cohort of controllers with and without protective HLA-B alleles in order to investigate whether there was evidence for multiple paths to HIV-1 control, or whether cellular and humoral arms of immunity might exhibit coordinated profiles. However, with the exception of IgG2 antibodies to gp41, HLA status was not associated with divergent humoral responses. This finding did not result from uniform antibody responses across subjects, as controllers could be regrouped according to strong differences in their HIV-specific antibody subclass specificity profiles. These divergent antibody profiles were further associated with significant differences in nonneutralizing antibody effector function, with levels of HIV-specific IgG1 acting as the major distinguishing factor. Thus, while HLA background among controllers was associated with minimal differences in humoral function, antibody subclass and specificity profiles were associated with divergent effector function, suggesting that these features could be used to make functional predictions. Because these nonneutralizing antibody activities have been associated with spontaneous viral control, reduced viral load, and nonprogression in infected subjects and protection in vaccinated subjects, understanding the specific features of IgGs with potentiated effector function may be critical to vaccine and therapeutic antibody development.
IMPORTANCE In this study, we investigated whether the humoral and cellular arms of adaptive immunity exhibit coordinated or compensatory activity by studying the antibody response among HIV-1 controllers with different genetic backgrounds.
PMCID: PMC3958053  PMID: 24352471
21.  PP2ARts1 is a master regulator of pathways that control cell size 
The Journal of Cell Biology  2014;204(3):359-376.
PP2ARts1 controls diverse pathways that influence cell size and may link cell cycle entry to cell growth via the transcription factor Ace2.
Cell size checkpoints ensure that passage through G1 and mitosis occurs only when sufficient growth has occurred. The mechanisms by which these checkpoints work are largely unknown. PP2A associated with the Rts1 regulatory subunit (PP2ARts1) is required for cell size control in budding yeast, but the relevant targets are unknown. In this paper, we used quantitative proteome-wide mass spectrometry to identify proteins controlled by PP2ARts1. This revealed that PP2ARts1 controls the two key checkpoint pathways thought to regulate the cell cycle in response to cell growth. To investigate the role of PP2ARts1 in these pathways, we focused on the Ace2 transcription factor, which is thought to delay cell cycle entry by repressing transcription of the G1 cyclin CLN3. Diverse experiments suggest that PP2ARts1 promotes cell cycle entry by inhibiting the repressor functions of Ace2. We hypothesize that control of Ace2 by PP2ARts1 plays a role in mechanisms that link G1 cyclin accumulation to cell growth.
PMCID: PMC3912523  PMID: 24493588
22.  Nutritional Control of Antibiotic Resistance via an Interface between the Phosphotransferase System and a Two-Component Signaling System 
Enterococci are ubiquitous inhabitants of the gastrointestinal (GI) tract. However, antibiotic-resistant enterococci are also major causes of hospital-acquired infections. Enterococci are intrinsically resistant to cephalosporins, enabling growth to abnormally high densities in the GI tract in patients during cephalosporin therapy, thereby promoting dissemination to other sites where they cause infection. Despite its importance, many questions about the underlying basis for cephalosporin resistance remain. A specific two-component signaling system, composed of the CroS sensor kinase and its cognate response regulator (CroR), is required for cephalosporin resistance in Enterococcus faecalis, but little is known about the factors that control this signaling system to modulate resistance. To explore the signaling network in which CroR participates to influence cephalosporin resistance, we employed a protein fragment complementation assay to detect protein-protein interactions in E. faecalis cells, revealing a previously unknown association of CroR with the HPr protein of the phosphotransferase system (PTS) responsible for carbohydrate uptake and catabolite control of gene expression. Genetic and physiological analyses indicate that association with HPr restricts the ability of CroR to promote cephalosporin resistance and gene expression in a nutrient-dependent manner. Mutational analysis suggests that the interface used by HPr to associate with CroR is distinct from the interface used to associate with other cellular partners. Our results define a physical and functional connection between a critical nutrient-responsive signaling system (the PTS) and a two-component signaling system that drives antibiotic resistance in E. faecalis, and they suggest a general strategy by which bacteria can integrate their nutritional status with diverse environmental stimuli.
PMCID: PMC3910890  PMID: 24277024
23.  Laparoscopic Appendectomy Is Safer Than Open Appendectomy in an Elderly Population 
Background and Objectives:
This study describes perioperative patient safety outcomes comparing laparoscopic appendectomy with open appendectomy in the elderly population (defined as age ≥65 years) during the diffusion of laparoscopic appendectomy into widespread clinical practice.
We performed a cross-sectional analysis of patients undergoing open or laparoscopic appendectomy in the US Nationwide Inpatient Sample, a 20% sample of inpatient discharges from 1056 hospitals, from 1998 to 2009, and used weighted sampling to estimate national trends. Multivariate logistic regression modeling was used to examine the association of laparoscopy with perioperative outcomes.
Patients who met the inclusion criteria totaled 257 484. Of these, 87 209 (34%) underwent laparoscopic appendectomy. These patients were younger (P < .001); had lower Charlson comorbidity scores (P < .001); were more likely to be white (P < .001), to be privately insured (P = .005), and to undergo surgery in urban hospitals (P < .001); and were less likely to have appendiceal rupture (P < .001). Laparoscopic appendectomy was associated with a decreased length of stay (4.44 days vs 7.86 days, P < .001), fewer total patient safety indicator events (1.8% vs 3.5%, P < .001), and a decreased mortality rate (0.9% vs 2.8%, P < .001). On multivariate analyses, we observed a 32% (odds ratio, 0.68) decreased probability of patient safety events occurring in laparoscopic appendectomy cases versus open appendectomy cases as measured by patient safety indicators.
The data suggest that laparoscopic appendectomy is associated with improved clinical outcomes in the elderly and that diffusion of laparoscopic appendectomy is not associated with adverse patient safety events in this population.
PMCID: PMC4208904  PMID: 25392668
Appendectomy; Patient safety indicator; Surgery in elderly
24.  Cricket antennae shorten when bending (Acheta domesticus L.) 
Insect antennae are important mechanosensory and chemosensory organs. Insect appendages, such as antennae, are encased in a cuticular exoskeleton and are thought to bend only between segments or subsegments where the cuticle is thinner, more flexible, or bent into a fold. There is a growing appreciation of the dominating influence of folds in the mechanical behavior of a structure, and the bending of cricket antennae was considered in this context. Antennae will bend or deflect in response to forces, and the resulting bending behavior will affect the sensory input of the antennae. In some cricket antennae, such as in those of Acheta domesticus, there are a large number (>100) of subsegments (flagellomeres) that vary in their length. We evaluated whether these antennae bend only at the joints between flagellomeres, which has always been assumed but not tested. In addition we questioned whether an antenna undergoes a length change as it bends, which would result from some patterns of joint deformation. Measurements using light microscopy and SEM were conducted on both male and female adult crickets (Acheta domesticus) with bending in four different directions: dorsal, ventral, medial, and lateral. Bending occurred only at the joints between flagellomeres, and antennae shortened a comparable amount during bending, regardless of sex or bending direction. The cuticular folds separating antennal flagellomeres are not very deep, and therefore as an antenna bends, the convex side (in tension) does not have a lot of slack cuticle to “unfold” and does not lengthen during bending. Simultaneously on the other side of the antenna, on the concave side in compression, there is an increasing overlap in the folded cuticle of the joints during bending. Antennal shortening during bending would prevent stretching of antennal nerves and may promote hemolymph exchange between the antenna and head.
PMCID: PMC4071997  PMID: 25018734
cricket; antenna; bending; cuticle; insect; Acheta domesticus
25.  Finasteride Reduces the Risk of Incident Clinical Benign Prostatic Hyperplasia 
European urology  2012;62(2):234-241.
Despite the high prevalence of clinical benign prostatic hyperplasia (BPH) among older men, there remains a notable absence of studies focused on BPH prevention.
To determine if finasteride prevents incident clinical BPH in healthy older men.
Design, setting, and participants
Data for this study are from the Prostate Cancer Prevention Trial. After excluding those with a history of BPH diagnosis or treatment, or an International Prostate Symptom Score (IPSS) ≥8 at study entry, 9253 men were available for analysis.
Outcome measurements and statistical analysis
The primary outcome was incident clinical BPH, defined as the initiation of medical treatment, surgery, or sustained, clinically significant urinary symptoms (IPSS >14). Finasteride efficacy was estimated using Cox proportional regression models to generate hazards ratios (HRs).
Results and limitations
Mean length of follow-up was 5.3 yr. The rate of clinical BPH was 19 per 1000 person-years in the placebo arm and 11 per 1000 person-years in the finasteride arm (p < 0.001). In a covariate-adjusted model, finasteride reduced the risk of incident clinical BPH by 40% (HR: 0.60; 95% confidence interval, 0.51–0.69; p < 0.001). The effect of finasteride on incident clinical BPH was attenuated in men with a body mass index ≥30 kg/m2 (pinteraction = 0.04) but otherwise did not differ significantly by physical activity, age, race, current diabetes, or current smoking. The post hoc nature of the analysis is a potential study limitation.
Finasteride substantially reduces the risk of incident clinical BPH in healthy older men. These results should be considered in formulating recommendations for the use of finasteride to prevent prostate diseases in asymptomatic older men.
PMCID: PMC4059403  PMID: 22459892
Prostatic hyperplasia; Finasteride; 5α-reductase inhibitor; Prevention and control; Prostatic neoplasm

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