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1.  Comparative promoter region analysis powered by CORG 
BMC Genomics  2005;6:24.
Promoters are key players in gene regulation. They receive signals from various sources (e.g. cell surface receptors) and control the level of transcription initiation, which largely determines gene expression. In vertebrates, transcription start sites and surrounding regulatory elements are often poorly defined. To support promoter analysis, we present CORG , a framework for studying upstream regions including untranslated exons (5' UTR).
The automated annotation of promoter regions integrates information of two kinds. First, statistically significant cross-species conservation within upstream regions of orthologous genes is detected. Pairwise as well as multiple sequence comparisons are computed. Second, binding site descriptions (position-weight matrices) are employed to predict conserved regulatory elements with a novel approach. Assembled EST sequences and verified transcription start sites are incorporated to distinguish exonic from other sequences.
As of now, we have included 5 species in our analysis pipeline (man, mouse, rat, fugu and zebrafish). We characterized promoter regions of 16,127 groups of orthologous genes. All data are presented in an intuitive way via our web site. Users are free to export data for single genes or access larger data sets via our DAS server . The benefits of our framework are exemplarily shown in the context of phylogenetic profiling of transcription factor binding sites and detection of microRNAs close to transcription start sites of our gene set.
The CORG platform is a versatile tool to support analyses of gene regulation in vertebrate promoter regions. Applications for CORG cover a broad range from studying evolution of DNA binding sites and promoter constitution to the discovery of new regulatory sequence elements (e.g. microRNAs and binding sites).
PMCID: PMC555765  PMID: 15723697
2.  Rapid profiling of RSV antibody repertoires from the memory B cells of naturally infected adult donors 
Science immunology  2016;1(6):eaaj1879.
Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in young children and the elderly. There are currently no licensed RSV vaccines, and passive prophylaxis with the monoclonal antibody palivizumab is restricted to high-risk infants in part due to its modest efficacy. Although it is widely agreed that an effective RSV vaccine will require the induction of a potent neutralizing antibody response against the RSV fusion (F) glycoprotein, little is known about the specificities and functional activities of RSV F-specific antibodies induced by natural infection. Here, we have comprehensively profiled the human antibody response to RSV F by isolating and characterizing 364 RSV F-specific monoclonal antibodies from the memory B cells of three healthy adult donors. In all donors, the antibody response to RSV F is comprised of a broad diversity of clones that target several antigenic sites. Nearly half of the most potent antibodies target a previously undefined site of vulnerability near the apex of the prefusion conformation of RSV F (preF), providing strong support for the development of RSV vaccine candidates that preserve the membrane-distal hemisphere of the preF protein. Additionally, the antibodies targeting this new site display convergent sequence features, thus providing a future means to rapidly detect the presence of these antibodies in human vaccine samples. Many of the antibodies that bind preF-specific surfaces are over 100 times more potent than palivizumab, and several cross-neutralize human metapneumovirus (HMPV). Taken together, the results have implications for the design and evaluation of RSV vaccine candidates and offer new options for passive prophylaxis.
One Sentence Summary
High-throughput profiling of anti-RSV F antibody repertoires reveals new opportunities for vaccine design and passive therapy.
PMCID: PMC5244814
3.  Identification of wood-boring beetles (Cerambycidae and Buprestidae) intercepted in trade-associated solid wood packaging material using DNA barcoding and morphology 
Scientific Reports  2017;7:40316.
Global trade facilitates the inadvertent movement of insect pests and subsequent establishment of populations outside their native ranges. Despite phytosanitary measures, nonnative insects arrive at United States (U.S.) ports of entry as larvae in solid wood packaging material (SWPM). Identification of wood-boring larval insects is important for pest risk analysis and management, but is difficult beyond family level due to highly conserved morphology. Therefore, we integrated DNA barcoding and rearing of larvae to identify wood-boring insects in SWPM. From 2012 to 2015, we obtained larvae of 338 longhorned beetles (Cerambycidae) and 38 metallic wood boring beetles (Buprestidae) intercepted in SWPM associated with imported products at six U.S. ports. We identified 265 specimens to species or genus using DNA barcodes. Ninety-three larvae were reared to adults and identified morphologically. No conflict was found between the two approaches, which together identified 275 cerambycids (23 genera) and 16 buprestids (4 genera). Our integrated approach confirmed novel DNA barcodes for seven species (10 specimens) of woodborers not in public databases. This study demonstrates the utility of DNA barcoding as a tool for regulatory agencies. We provide important documentation of potential beetle pests that may cross country borders through the SWPM pathway.
PMCID: PMC5238391  PMID: 28091577
4.  Lipid droplets in Leukocytes: organelles linked to inflammatory responses 
Experimental cell research  2015;340(2):193-197.
Studies on lipid droplets (LDs) in leukocytes have attracted attention due to their association with human diseases. In these cells, LDs are rapidly formed in response to inflammatory stimuli or allergic/inflammatory diseases including infections with parasites and bacteria. Leukocyte LDs are linked to the regulation of immune responses by compartmentalization of several proteins and lipids involved in the control and biosynthesis of inflammatory mediators (eicosanoids). In this mini review, we summarize current knowledge on the composition, structure and function of leukocyte LDs, organelles now considered as structural markers of inflammation.
PMCID: PMC4744558  PMID: 26515551
lipid bodies; inflammation; eicosanoids; immune responses; cell activation; electron microscopy
5.  Calcium/Vitamin D (CaD) Supplementation, Serum 25(OH) Vitamin D Concentrations, and Cholesterol Profiles in the Women’s Health Initiative CaD Randomized Trial 
Menopause (New York, N.Y.)  2014;21(8):823-833.
The objective of this study was to evaluate whether increased serum 25OHD3 concentrations, in response to calcium plus vitamin D (CaD) supplementation, are associated with improved lipids in postmenopausal women.
The parent trial was a double-blinded, randomized, placebo-controlled, parallel group trial designed to test the effects of CaD supplementation (1,000 mg of elemental calcium plus 400 IU of vitamin D3 daily) versus placebo in postmenopausal women. Women were enrolled between 1993 and 1998 from the general community including multiple sites in the U.S. This cohort included 300 White, 200 African American, and 100 Hispanic participants randomly selected from the WHI CaD trial. Serum 25OHD3 and lipids (fasting plasma triglycerides [TG], high density lipoprotein cholesterol [HDL-C], and calculated low density lipoprotein cholesterol [LDL-C]) were assessed prior to CaD randomization and again post-randomization.
There was a 38% increase in mean serum 25OHD3 concentrations after two years (95% CI 1.29–1.47, p< 0.001) for women randomized to CaD (24.3ng/mL post randomization mean) compared with placebo (18.2 ng/mL). Women randomized to CaD had a 4.46 mg/dL mean decrease in LDL-C (p=0.03). Higher concentrations of 25OHD3, were associated with higher HDL-C (p=0.003) along with lower LDL-C and TG levels (p=0.02 and p<0.001, respectively).
Supplemental CaD significantly increased concentrations of 25OHD3 and decreased LDL-C. Women with higher 25OHD3 had more favorable lipid profiles, including increased HDL-C as well as lower LDL-C and TG. These results support the hypothesis that higher concentrations of 25OHD3, in response to CaD supplementation, are associated with improved LDL-C.
PMCID: PMC5234488  PMID: 24594863
Vitamin D; Cholesterol; Coronary Artery Disease; Menopause; Low Density Lipoprotein Cholesterol
6.  Protection against Plasmodium falciparum malaria by PfSPZ Vaccine 
JCI Insight  null;2(1):e89154.
BACKGROUND: A radiation-attenuated Plasmodium falciparum (Pf) sporozoite (SPZ) malaria vaccine, PfSPZ Vaccine, protected 6 of 6 subjects (100%) against homologous Pf (same strain as in the vaccine) controlled human malaria infection (CHMI) 3 weeks after 5 doses administered intravenously. The next step was to assess protective efficacy against heterologous Pf (different from Pf in the vaccine), after fewer doses, and at 24 weeks.
METHODS: The trial assessed tolerability, safety, immunogenicity, and protective efficacy of direct venous inoculation (DVI) of 3 or 5 doses of PfSPZ Vaccine in non-immune subjects.
RESULTS: Three weeks after final immunization, 5 doses of 2.7 × 105 PfSPZ protected 12 of 13 recipients (92.3% [95% CI: 48.0, 99.8]) against homologous CHMI and 4 of 5 (80.0% [10.4, 99.5]) against heterologous CHMI; 3 doses of 4.5 × 105 PfSPZ protected 13 of 15 (86.7% [35.9, 98.3]) against homologous CHMI. Twenty-four weeks after final immunization, the 5-dose regimen protected 7 of 10 (70.0% [17.3, 93.3]) against homologous and 1 of 10 (10.0% [–35.8, 45.6]) against heterologous CHMI; the 3-dose regimen protected 8 of 14 (57.1% [21.5, 76.6]) against homologous CHMI. All 22 controls developed Pf parasitemia. PfSPZ Vaccine was well tolerated, safe, and easy to administer. No antibody or T cell responses correlated with protection.
CONCLUSIONS: We have demonstrated for the first time to our knowledge that PfSPZ Vaccine can protect against a 3-week heterologous CHMI in a limited group of malaria-naive adult subjects. A 3-dose regimen protected against both 3-week and 24-week homologous CHMI (87% and 57%, respectively) in this population. These results provide a foundation for developing an optimized immunization regimen for preventing malaria.
FUNDING: Support was provided through the US Army Medical Research and Development Command, Military Infectious Diseases Research Program, and the Naval Medical Research Center’s Advanced Medical Development Program.
PfSPZ Vaccine provides high level protection against controlled human malaria infection in malaria-naïve subjects challenged by homologous and heterologous strains of Plasmodium falciparum.
PMCID: PMC5214067  PMID: 28097230
8.  Age-related macular degeneration associated polymorphism rs10490924 in ARMS2 results in deficiency of a complement activator 
Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The polymorphism rs10490924 in the ARMS2 gene is highly associated with AMD and linked to an indel mutation (del443ins54), the latter inducing mRNA instability. At present, the function of the ARMS2 protein, the exact cellular sources in the retina and the biological consequences of the rs10490924 polymorphism are unclear.
Recombinant ARMS2 was expressed in Pichia pastoris, and protein functions were studied regarding cell surface binding and complement activation in human serum using fluoresence-activated cell sorting (FACS) as well as laser scanning microscopy (LSM). Biolayer interferometry defined protein interactions. Furthermore, endogenous ARMS2 gene expression was studied in human blood derived monocytes and in human induced pluripotent stem cell-derived microglia (iPSdM) by PCR and LSM. The ARMS2 protein was localized in human genotyped retinal sections and in purified monocytes derived from AMD patients without the ARMS2 risk variant by LSM. ARMS2 expression in monocytes under oxidative stress was determined by Western blot analysis.
Here, we demonstrate for the first time that ARMS2 functions as surface complement regulator. Recombinant ARMS2 binds to human apoptotic and necrotic cells and initiates complement activation by recruiting the complement activator properdin. ARMS2-properdin complexes augment C3b surface opsonization for phagocytosis. We also demonstrate for the first time expression of ARMS2 in human monocytes especially under oxidative stress and in microglia cells of the human retina. The ARMS2 protein is absent in monocytes and also in microglia cells, derived from patients homozygous for the ARMS2 AMD risk variant (rs10490924).
ARMS2 is likely involved in complement-mediated clearance of cellular debris. As AMD patients present with accumulated proteins and lipids on Bruch’s membrane, ARMS2 protein deficiency due to the genetic risk variant might be involved in drusen formation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12974-016-0776-3) contains supplementary material, which is available to authorized users.
PMCID: PMC5234120  PMID: 28086806
9.  Detoxification of Mitochondrial Oxidants and Apoptotic Signaling Are Facilitated by Thioredoxin-2 and Peroxiredoxin-3 during Hyperoxic Injury 
PLoS ONE  2017;12(1):e0168777.
Mitochondria play a fundamental role in the regulation of cell death during accumulation of oxidants. High concentrations of atmospheric oxygen (hyperoxia), used clinically to treat tissue hypoxia in premature newborns, is known to elicit oxidative stress and mitochondrial injury to pulmonary epithelial cells. A consequence of oxidative stress in mitochondria is the accumulation of peroxides which are detoxified by the dedicated mitochondrial thioredoxin system. This system is comprised of the oxidoreductase activities of peroxiredoxin-3 (Prx3), thioredoxin-2 (Trx2), and thioredoxin reductase-2 (TrxR2). The goal of this study was to understand the role of the mitochondrial thioredoxin system and mitochondrial injuries during hyperoxic exposure. Flow analysis of the redox-sensitive, mitochondrial-specific fluorophore, MitoSOX, indicated increased levels of mitochondrial oxidant formation in human adenocarcinoma cells cultured in 95% oxygen. Increased expression of Trx2 and TrxR2 in response to hyperoxia were not attributable to changes in mitochondrial mass, suggesting that hyperoxic upregulation of mitochondrial thioredoxins prevents accumulation of oxidized Prx3. Mitochondrial oxidoreductase activities were modulated through pharmacological inhibition of TrxR2 with auranofin and genetically through shRNA knockdown of Trx2 and Prx3. Diminished Trx2 and Prx3 expression was associated with accumulation of mitochondrial superoxide; however, only shRNA knockdown of Trx2 increased susceptibility to hyperoxic cell death and increased phosphorylation of apoptosis signal-regulating kinase-1 (ASK1). In conclusion, the mitochondrial thioredoxin system regulates hyperoxic-mediated death of pulmonary epithelial cells through detoxification of oxidants and regulation of redox-dependent apoptotic signaling.
PMCID: PMC5207683  PMID: 28045936
10.  Eosinophil ETosis-derived DNA traps: their presence in secretions and their functional attributes 
Activated human eosinophils, as well as neutrophils, can release extracellular chromatin to form DNA traps through cytolytic extracellular trap cell death (ETosis). Although formations of neutrophil DNA traps are recognized in various inflammatory conditions, neither the presence of ETosis-derived eosinophil DNA traps in human allergic diseases nor the characteristics of these DNA traps have been studied.
We investigated the presence of ETosis-derived DNA traps in eosinophil-rich sinus and ear secretions and the functional attributes of ETosis DNA traps.
Eosinophil-rich secretions obtained from patients with eosinophilic chronic rhinosinusitis (ECRS) and eosinophilic otitis media (EOM) were studied microscopically. In vitro studies of ETosis and DNA trap formation used blood-derived eosinophils and neutrophils, and binding capacities of DNA traps used labeled bacteria and fluorescent microbeads. Stabilities of DNA traps were evaluated by fluorescence microscopy.
Abundant nuclear histone H1-bearing DNA traps had formed in vivo in the eosinophilic secretions and contributed to their increased viscosity. In vitro, following brief shear flow, eosinophil ETosis-elicited DNA traps assembled to form stable aggregates. Eosinophil DNA traps entrapped bacteria and fungi and by hydrophobic interactions microbeads. In comparison with neutrophil-derived DNA traps, eosinophil DNA traps ultrastructurally exhibited thicker fibers with globular structures and were less susceptible to leukocyte-derived proteolytic degradation, likely due to the lesser protease activities of eosinophils.
In human allergic diseases, the local cytolysis of eosinophils not only releases free eosinophil granules but also generates nuclear-derived DNA traps that are major extracellular structural components within eosinophil-rich secretions.
PMCID: PMC4674385  PMID: 26070883
eosinophilic chronic rhinosinusitis; eosinophils; ETosis; extracellular DNA traps; neutrophils
11.  Zinc Oxide Nanoparticle Suspensions and Layer-By-Layer Coatings Inhibit Staphylococcal Growth 
Despite a decade of engineering and process improvements, bacterial infection remains the primary threat to implanted medical devices. Zinc oxide nanoparticles (ZnO-NPs) have demonstrated antimicrobial properties. Their microbial selectivity, stability, ease of production, and low cost make them attractive alternatives to silver NPs or antimicrobial peptides. Here we sought to (1) determine the relative efficacy of ZnO-NPs on planktonic growth of medically relevant pathogens; (2) establish the role of bacterial surface chemistry on ZnO-NP effectiveness; (3) evaluate NP shape as a factor in the dose-response; and (4) evaluate layer-by-layer (LBL) ZnO-NP surface coatings on biofilm growth. ZnO-NPs inhibited bacterial growth in a shape-dependent manner not previously seen or predicted. Pyramid shape particles were the most effective and contrary to previous work, larger particles were more effective than smaller particles. Differential susceptibility of pathogens may be related to their surface hydrophobicity. LBL coatings of ZnO-NP reduced staphylococcal biofilm burden by >95%.
Graphical Abstract
Despite a decade of engineering and process improvements, bacterial colonization and infection remain the primary threats to implanted medical devices. Zinc oxide nanoparticles (ZnO-NPs) are attractive alternatives to silver NPs or antimicrobial peptides for device coatings to prevent infection. Here we address several questions regarding ZnO-NPs and their interactions with bacteria in effort to translate this material toward antibacterial medical device coatings. Using a Layer-by-layer technique we demonstrate that ZnO-NP coatings reduce staphylococcal biofilm burden by >95%.
PMCID: PMC4728027  PMID: 26515755
Zinc oxide; nanoparticles; layer-by-layer; biofilm; staphylococcus
12.  Using incentives to improve resource utilization: a quasi-experimental evaluation of an ICU quality improvement program 
Critical care medicine  2016;44(1):162-170.
Healthcare systems strive to provide quality care at lower cost. Arterial blood gas testing (ABGs), chest radiographs (CXRs), and red blood cell transfusions (RBCs) provide an important example of opportunities to reduce excess resource utilization within the ICU. We describe the effect of a multifaceted quality improvement program designed to decrease avoidable ABGs, CXRs, and RBCs utilization on utilization of these resources and patient outcomes.
Prospective pre-post cohort study
Seven ICUs in an academic healthcare system
All adult ICU patients admitted to study ICUs during consecutive baseline (n=7,357), intervention (n=7,553), and follow up (n=7,657) years between September 2010 and August 2013.
A multifaceted quality improvement program including provider education, audit and feedback, and unit-based provider financial incentives targeting ABG, CXR, and RBC utilization.
Measurements and Main Results
The primary outcome was the number of orders for ABGs, CXRs, and RBCs per patient. Compared to the baseline period, unadjusted ABG, CXR, and RBC utilization in the intervention period was reduced by 42%, 26%, and 17%, respectively (p<0.01). After adjusting for potentially relevant patient factors, the intervention was associated with 128 fewer ABGs, 73 fewer CXRs, and 16 fewer RBCs per 100 patients (p<0.01). This effect was durable during the follow up year. This reduction yielded an approximate net savings of $1.5 M in direct costs over the intervention and follow-up years after accounting for the direct costs of the program. Unadjusted hospital mortality decreased from 7% in the baseline period to 5.2% in the intervention period (p<0.01). This reduction remained significant after adjusting for patient factors (OR= 0.43, P<0.01).
Implementation of a multifaceted quality improvement program including financial incentives was associated with significant improvements in resource utilization. Our findings provide evidence supporting the safety, effectiveness, and sustainability of incentive-based quality improvement interventions.
PMCID: PMC4684419  PMID: 26496444
Quality of Health Care; Outcome and Process Assessment; Evidence-based Practice; Intensive Care Unit; Organizational Efficiency; Physician Incentive Plans
13.  High-Temperature-Induced Defects in Tomato (Solanum lycopersicum) Anther and Pollen Development Are Associated with Reduced Expression of B-Class Floral Patterning Genes 
PLoS ONE  2016;11(12):e0167614.
Sexual reproduction is a critical process in the life-cycle of plants and very sensitive to environmental perturbations. To better understand the effect of high temperature on plant reproduction, we cultivated tomato (Solanum lycopersicum) plants in continuous mild heat. Under this condition we observed a simultaneous reduction in pollen viability and appearance of anthers with pistil-like structures, while in a more thermotolerant genotype, both traits were improved. Ectopic expression of two pistil-specific genes, TRANSMITTING TISSUE SPECIFIC and TOMATO AGAMOUS LIKE11, in the anthers confirmed that the anthers had gained partial pistil identity. Concomitantly, expression of the B-class genes TOMATO APETALA3, TOMATO MADS BOX GENE6 (TM6) and LePISTILLATA was reduced in anthers under continuous mild heat. Plants in which TM6 was partially silenced reacted hypersensitively to temperature elevation with regard to the frequency of pistilloid anthers, pollen viability and pollen quantity. Taken together, these results suggest that high-temperature-induced down-regulation of tomato B-class genes contributes to anther deformations and reduced male fertility. Improving our understanding of how temperature perturbs the molecular mechanisms of anther and pollen development will be important in the view of maintaining agricultural output under current climate changes.
PMCID: PMC5147909  PMID: 27936079
14.  The Yersinia pestis Effector YopM Inhibits Pyrin Inflammasome Activation 
PLoS Pathogens  2016;12(12):e1006035.
Type III secretion systems (T3SS) are central virulence factors for many pathogenic Gram-negative bacteria, and secreted T3SS effectors can block key aspects of host cell signaling. To counter this, innate immune responses can also sense some T3SS components to initiate anti-bacterial mechanisms. The Yersinia pestis T3SS is particularly effective and sophisticated in manipulating the production of pro-inflammatory cytokines IL-1β and IL-18, which are typically processed into their mature forms by active caspase-1 following inflammasome formation. Some effectors, like Y. pestis YopM, may block inflammasome activation. Here we show that YopM prevents Y. pestis induced activation of the Pyrin inflammasome induced by the RhoA-inhibiting effector YopE, which is a GTPase activating protein. YopM blocks YopE-induced Pyrin-mediated caspase-1 dependent IL-1β/IL-18 production and cell death. We also detected YopM in a complex with Pyrin and kinases RSK1 and PKN1, putative negative regulators of Pyrin. In contrast to wild-type mice, Pyrin deficient mice were also highly susceptible to an attenuated Y. pestis strain lacking YopM, emphasizing the importance of inhibition of Pyrin in vivo. A complex interplay between the Y. pestis T3SS and IL-1β/IL-18 production is evident, involving at least four inflammasome pathways. The secreted effector YopJ triggers caspase-8- dependent IL-1β activation, even when YopM is present. Additionally, the presence of the T3SS needle/translocon activates NLRP3 and NLRC4-dependent IL-1β generation, which is blocked by YopK, but not by YopM. Taken together, the data suggest YopM specificity for obstructing the Pyrin pathway, as the effector does not appear to block Y. pestis-induced NLRP3, NLRC4 or caspase-8 dependent caspase-1 processing. Thus, we identify Y. pestis YopM as a microbial inhibitor of the Pyrin inflammasome. The fact that so many of the Y. pestis T3SS components are participating in regulation of IL-1β/IL-18 release suggests that these effects are essential for maximal control of innate immunity during plague.
Author Summary
Many pathogenic Gram-negative bacteria express type III secretion systems (T3SS) that translocate bacterial proteins into host cells with the potential of altering normal cell processes. Yersinia pestis, the causative agent of plague, harbors a T3SS which is particularly effective in suppressing innate immunity and release of pro-inflammatory cytokines IL-1β and IL-18, potent triggers of anti-bacterial responses. These cytokines are produced via processing by active caspase-1 in inflammasome complexes. Pyrin is an inflammasome component that recognizes alterations in certain host cell signals. Here we show that the T3SS effector protein YopM inhibits effector YopE-mediated Pyrin-induced caspase-1 activation, IL-1β, IL-18 and cell death triggered by Y. pestis. We also found that blocking the Pyrin pathway is important for disease development in a mouse model of bubonic plague. Thus, YopM is a microbial molecule blocking Pyrin inflammasomes.
PMCID: PMC5135138  PMID: 27911947
15.  Laboratory Measures as Proxies for Primary Care Encounters: Implications for Quantifying Clinical Retention Among HIV-Infected Adults in North America 
American Journal of Epidemiology  2015;182(11):952-960.
Because of limitations in the availability of data on primary care encounters, patient retention in human immunodeficiency virus (HIV) care is often estimated using laboratory measurement dates as proxies for clinical encounters, leading to possible outcome misclassification. This study included 83,041 HIV-infected adults from 14 clinical cohorts in the North American AIDS Cohort Collaboration on Research and Design (NA-ACCORD) who had ≥1 HIV primary care encounters during 2000–2010, contributing 468,816 person-years of follow-up. Encounter-based retention (REB) was defined as ≥2 encounters in a calendar year, ≥90 days apart. Laboratory-based retention (RLB) was defined similarly, using the dates of CD4-positive cell counts or HIV-1 RNA measurements. Percentage of agreement and the κ statistic were used to characterize agreement between RLB and REB. Logistic regression with generalized estimating equations and stabilized inverse-probability-of-selection weights was used to elucidate temporal trends and the discriminatory power of RLB as a predictor of REB, accounting for age, sex, race/ethnicity, primary HIV risk factor, and cohort site as potential confounders. Both REB and RLB increased from 2000 to 2010 (from 67% to 78% and from 65% to 77%, respectively), though REB was higher than RLB throughout (P < 0.01). RLB agreed well with REB (80%–86% agreement; κ = 0.55–0.62, P < 0.01) and had a strong, imperfect ability to discriminate between persons retained and not retained in care by REB (C statistic: C = 0.81, P < 0.05). As a proxy for REB, RLB had a sensitivity and specificity of 84% and 77%, respectively, with misclassification error of 18%.
PMCID: PMC4655744  PMID: 26578717
clinical encounters; clinical retention; HIV; laboratory measurements; measurement error; misclassification; proxies
16.  An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35 
Applied and Environmental Microbiology  2016;82(12):3481-3492.
Streptomyces iranensis HM 35 is an alternative rapamycin producer to Streptomyces rapamycinicus. Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation in S. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer from Escherichia coli to Streptomyces, and the failure of this step has frustrated its application to Streptomyces iranensis HM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca2+ ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions in S. iranensis HM 35. Among them were rapK, which generates the starter unit for rapamycin biosynthesis, and hutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants of Streptomyces species that are genetically difficult to manipulate.
IMPORTANCE The developed protocol of conjugational plasmid transfer from Escherichia coli to Streptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable, Streptomyces species.
PMCID: PMC4959151  PMID: 27037115
17.  Efficacy and Safety of Abiraterone Acetate in Elderly (≥75 Years) Chemotherapy-Naïve Patients with Metastatic Castration-Resistant Prostate Cancer 
The Journal of urology  2015;194(5):1277-1284.
Metastatic castration-resistant prostate cancer primarily affects elderly men. In this post hoc analysis we investigated the safety and efficacy of abiraterone acetate in elderly (≥75 years) and younger (<75 years) patient subgroups at the prespecified interim analysis (55% of total overall survival [OS] events) for the COU-AA-302 trial.
Materials and Methods
Patients were stratified and randomized 1:1 to abiraterone acetate 1,000 mg plus prednisone/prednisolone 5 mg bid (abiraterone-prednisone) vs placebo plus prednisone/prednisolone 5 mg bid (prednisone alone). Co-primary end points were radiographic progression-free survival (rPFS) and OS. Median time to event and hazard ratio (HR) were estimated using Kaplan-Meier method and Cox model, respectively.
Elderly patients (n=350) treated with abiraterone-prednisone had significant improvements in OS and rPFS vs prednisone alone (HR=0.71 [95% CI 0.53–0.96] and HR=0.63 [95% CI 0.48–0.83], respectively), similar to younger patients (n=738, HR=0.81 [95% CI 0.63–1.03] and HR=0.49 [95% CI 0.40–0.59], respectively). All secondary end points favored the abiraterone-prednisone arm for both age subgroups. Specific adverse events with abiraterone-prednisone were similar between age subgroups. Elderly patients in both treatment arms had higher rates of fluid retention and cardiac disorders than younger patients, although rates of dose reduction or treatment interruptions due to adverse events were low in both age subgroups.
Abiraterone acetate demonstrated clinical benefit and was well tolerated in both elderly and younger men with chemotherapy-naïve metastatic castration-resistant prostate cancer, thus supporting it as a treatment option for elderly patients who may not tolerate other therapies with greater toxicity.
PMCID: PMC5129174  PMID: 26151676
prostatic neoplasms; aged; abiraterone acetate; safety; treatment outcome
18.  Filamin C-related myopathies: pathology and mechanisms 
Acta neuropathologica  2012;125(1):33-46.
The term filaminopathy was introduced after a truncating mutation in the dimerization domain of filamin C (FLNc) was shown to be responsible for a devastating muscle disease. Subsequently, the same mutation was found in patients from diverse ethnical origins, indicating that this specific alteration is a mutational hot spot. Patients initially present with proximal muscle weakness, while distal and respiratory muscles become affected with disease progression. Muscle biopsies of these patients show typical signs of myofibrillar myopathy, including disintegration of myofibrils and aggregation of several proteins into distinct intracellular deposits. Highly similar phenotypes were observed in patients with other mutations in Ig-like domains of FLNc that result in expression of a noxious protein. Biochemical and biophysical studies showed that the mutated domains acquire an abnormal structure causing decreased stability and eventually becoming a seed for abnormal aggregation with other proteins. The disease usually presents only after the fourth decade of life possibly as a result of ageing-related impairments in the machinery that is responsible for disposal of damaged proteins. This is confirmed by mutations in components of this machinery that cause a highly similar phenotype. Transfection studies of cultured muscle cells reflect the events observed in patient muscles and, therefore, may provide a helpful model for testing future dedicated therapeutic strategies. More recently, FLNC mutations were also found in families with a distal myopathy phenotype, caused either by mutations in the actin-binding domain of FLNc that result in increased actin-binding and non-specific myopathic abnormalities without myofibrillar myopathy pathology, or a nonsense mutation in the rod domain that leads to RNA instability, haploinsufficiency with decreased expression levels of FLNc in the muscle fibers and myofibrillar abnormalities, but not to the formation of desmin-positive protein aggregates required for the diagnosis of myofibrillar myopathy.
PMCID: PMC5127197  PMID: 23109048
Filamin C; Filaminopathy; Myofibrillar myopathy; Distal myopathy; Limb-girdle muscular dystrophy; Pathomechanism
19.  Phylogenetic distribution of plant snoRNA families 
BMC Genomics  2016;17:969.
Small nucleolar RNAs (snoRNAs) are one of the most ancient families amongst non-protein-coding RNAs. They are ubiquitous in Archaea and Eukarya but absent in bacteria. Their main function is to target chemical modifications of ribosomal RNAs. They fall into two classes, box C/D snoRNAs and box H/ACA snoRNAs, which are clearly distinguished by conserved sequence motifs and the type of chemical modification that they govern. Similarly to microRNAs, snoRNAs appear in distinct families of homologs that affect homologous targets. In animals, snoRNAs and their evolution have been studied in much detail. In plants, however, their evolution has attracted comparably little attention.
In order to chart the phylogenetic distribution of individual snoRNA families in plants, we applied a sophisticated approach for identifying homologs of known plant snoRNAs across the plant kingdom. In response to the relatively fast evolution of snoRNAs, information on conserved sequence boxes, target sequences, and secondary structure is combined to identify additional snoRNAs. We identified 296 families of snoRNAs in 24 species and traced their evolution throughout the plant kingdom. Many of the plant snoRNA families comprise paralogs. We also found that targets are well-conserved for most snoRNA families.
The sequence conservation of snoRNAs is sufficient to establish homologies between phyla. The degree of this conservation tapers off, however, between land plants and algae. Plant snoRNAs are frequently organized in highly conserved spatial clusters. As a resource for further investigations we provide carefully curated and annotated alignments for each snoRNA family under investigation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-3301-2) contains supplementary material, which is available to authorized users.
PMCID: PMC5122169  PMID: 27881081
snoRNAs; Evolution; Small RNAs; snoRNA targets
20.  Changes of bivalent chromatin coincide with increased expression of developmental genes in cancer 
Scientific Reports  2016;6:37393.
Bivalent (poised or paused) chromatin comprises activating and repressing histone modifications at the same location. This combination of epigenetic marks at promoter or enhancer regions keeps genes expressed at low levels but poised for rapid activation. Typically, DNA at bivalent promoters is only lowly methylated in normal cells, but frequently shows elevated methylation levels in cancer samples. Here, we developed a universal classifier built from chromatin data that can identify cancer samples solely from hypermethylation of bivalent chromatin. Tested on over 7,000 DNA methylation data sets from several cancer types, it reaches an AUC of 0.92. Although higher levels of DNA methylation are often associated with transcriptional silencing, counter-intuitive positive statistical dependencies between DNA methylation and expression levels have been recently reported for two cancer types. Here, we re-analyze combined expression and DNA methylation data sets, comprising over 5,000 samples, and demonstrate that the conjunction of hypermethylation of bivalent chromatin and up-regulation of the corresponding genes is a general phenomenon in cancer. This up-regulation affects many developmental genes and transcription factors, including dozens of homeobox genes and other genes implicated in cancer. Thus, we reason that the disturbance of bivalent chromatin may be intimately linked to tumorigenesis.
PMCID: PMC5120258  PMID: 27876760
21.  Optimization of Multi-Stage Dynamic Treatment Regimes Utilizing Accumulated Data 
Statistics in medicine  2015;34(26):3424-3443.
In medical therapies involving multiple stages, a physician’s choice of a subject’s treatment at each stage depends on the subject’s history of previous treatments and outcomes. The sequence of decisions is known as a dynamic treatment regime, or treatment policy. We consider dynamic treatment regimes in settings where each subject’s final outcome can be defined as the sum of longitudinally observed values, each corresponding to a stage of the regime. Q-learning, which is a backward induction method, is used to first optimize the last stage treatment, then sequentially optimize each previous stage treatment until the first stage treatment is optimized. During this process, model-based expectations of outcomes of late stages are used in the optimization of earlier stages. When the outcome models are misspecified, bias can accumulate from stage to stage and become severe, especially when the number of treatment stages is large. We demonstrate that a modification of standard Q-learning can help reduce the accumulated bias. We provide a computational algorithm, estimators, and closed-form variance formulas. Simulation studies show that the modified Q-learning method has a higher probability of identifying the optimal treatment regime even in settings with mis-specified models for outcomes. It is applied to identify optimal treatment regimes in a study for advanced prostate cancer, and to estimate and compare the final mean rewards of all the possible discrete two-stage treatment sequences.
PMCID: PMC4596799  PMID: 26095711
Backward induction; Multi-stage treatment; Optimal treatment sequence; Q-learning; Treatment decision making
22.  Proteogenomic analyses indicate bacterial methylotrophy and archaeal heterotrophy are prevalent below the grass root zone 
PeerJ  2016;4:e2687.
Annually, half of all plant-derived carbon is added to soil where it is microbially respired to CO2. However, understanding of the microbiology of this process is limited because most culture-independent methods cannot link metabolic processes to the organisms present, and this link to causative agents is necessary to predict the results of perturbations on the system. We collected soil samples at two sub-root depths (10–20 cm and 30–40 cm) before and after a rainfall-driven nutrient perturbation event in a Northern California grassland that experiences a Mediterranean climate. From ten samples, we reconstructed 198 metagenome-assembled genomes that represent all major phylotypes. We also quantified 6,835 proteins and 175 metabolites and showed that after the rain event the concentrations of many sugars and amino acids approach zero at the base of the soil profile. Unexpectedly, the genomes of novel members of the Gemmatimonadetes and Candidate Phylum Rokubacteria phyla encode pathways for methylotrophy. We infer that these abundant organisms contribute substantially to carbon turnover in the soil, given that methylotrophy proteins were among the most abundant proteins in the proteome. Previously undescribed Bathyarchaeota and Thermoplasmatales archaea are abundant in deeper soil horizons and are inferred to contribute appreciably to aromatic amino acid degradation. Many of the other bacteria appear to breakdown other components of plant biomass, as evidenced by the prevalence of various sugar and amino acid transporters and corresponding hydrolyzing machinery in the proteome. Overall, our work provides organism-resolved insight into the spatial distribution of bacteria and archaea whose activities combine to degrade plant-derived organics, limiting the transport of methanol, amino acids and sugars into underlying weathered rock. The new insights into the soil carbon cycle during an intense period of carbon turnover, including biogeochemical roles to previously little known soil microbes, were made possible via the combination of metagenomics, proteomics, and metabolomics.
PMCID: PMC5103831  PMID: 27843720
Genome-resolved metagenomics; Methanol dehydrogenase; Soil bacteria; Soil archaea; Proteomics; Metabolomics
24.  Vorinostat Combined with High-Dose Gemcitabine, Busulfan, and Melphalan with Autologous Stem Cell Transplantation in Patients with Refractory Lymphomas 
More active high-dose regimens are needed for refractory/poor-risk relapsed lymphomas. We previously developed a regimen of infusional gemcitabine/busulfan/melphalan, exploiting the synergistic interaction. Its encouraging activity in refractory lymphomas led us to further enhance its use as a platform for epigenetic modulation. We previously observed increased cytotoxicity in refractory lymphoma cell lines when the histone deacetylase inhibitor vorinostat was added to gemcitabine/busulfan/melphalan, which prompted us to clinically study this four-drug combination. Patients ages 12 to 65 with refractory diffuse large B cell lymphoma (DLCL), Hodgkin (HL), or T lymphoma were eligible. Vorinostat was given at 200 mg/day to 1000 mg/day (days −8 to −3). Gemcitabine was infused continuously at 10 mg/m2/minute over 4.5 hours (days −8 and −3). Busulfan dosing targeted 4000 μM-minute/day (days −8 to −5). Melphalan was infused at 60 mg/m2/day (days −3 and −2). Patients with CD20+ tumors received rituximab (375 mg/m2, days +1 and +8). We enrolled 78 patients: 52 DLCL, 20 HL, and 6 T lymphoma; median age 44 years (range, 15 to 65); median 3 prior chemotherapy lines (range, 2 to 7); and 48% of patients had positron emission tomography–positive tumors at high-dose chemotherapy (29% unresponsive). The vorinostat dose was safely escalated up to 1000 mg/day, with no treatment-related deaths. Toxicities included mucositis and dermatitis. Neutrophils and platelets engrafted promptly. At median follow-up of 25 (range, 16 to 41) months, event-free and overall survival were 61.5% and 73%, respectively (DLCL) and 45% and 80%, respectively (HL). In conclusion, vorinostat/gemcitabine/busulfan/melphalan is safe and highly active in refractory/poor-risk relapsed lymphomas, warranting further evaluation.
PMCID: PMC4781754  PMID: 26071868
Vorinostat; Phase 1 trial; High-dose chemotherapy; Autologous stem-cell; transplantation; Lymphoma
25.  Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria vaccines 
Vaccine  2015;33(52):7452-7461.
Sanaria Inc. has developed methods to manufacture, purify and cryopreserve aseptic Plasmodium falciparum (Pf) sporozoites (SPZ), and is using this platform technology to develop an injectable PfSPZ-based vaccine that provides high-grade, durable protection against infection with Pf malaria. Several candidate vaccines are being developed and tested, including PfSPZ Vaccine, in which the PfSPZ are attenuated by irradiation, PfSPZ-CVac, in which fully infectious PfSPZ are attenuated in vivo by concomitant administration of an anti-malarial drug, and PfSPZ-GA1, in which the PfSPZ are attenuated by gene knockout. Forty-three research groups in 15 countries, organized as the International PfSPZ Consortium (I-PfSPZ-C), are collaborating to advance this program by providing intellectual, clinical, and financial support. Fourteen clinical trials of these products have been completed in the USA, Europe and Africa, two are underway and at least 12 more are planned for 2015–2016 in the US (four trials), Germany (2 trials), Tanzania, Kenya, Mali, Burkina Faso, Ghana and Equatorial Guinea. Sanaria anticipates application to license a first generation product as early as late 2017, initially to protect adults, and a year later to protect all persons >6 months of age for at least six months. Improved vaccine candidates will be advanced as needed until the following requirements have been met: long-term protection against natural transmission, excellent safety and tolerability, and operational feasibility for population-wide administration. Here we describe the three most developed whole PfSPZ vaccine candidates, associated clinical trials, initial plans for licensure and deployment, and long-term objectives for a final product suitable for mass administration to achieve regional malaria elimination and eventual global eradication.
PMCID: PMC5077156  PMID: 26469720
Malaria vaccine; Plasmodium falciparum; Sporozoite; PfSPZ Vaccine; PfSPZ-CVac; PfSPZ Challenge

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