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1.  An extract of the medicinal plant Artemisia annua modulates production of inflammatory markers in activated neutrophils 
Purpose
To investigate the ability of a commercial extract from the medicinal plant Artemisia annua to modulate production of the cytokine, tumor necrosis factor-alpha (TNF-α), and the cyclooxygenase (COX) inflammatory marker, prostaglandin E2 (PGE2) in activated neutrophils.
Methods
Neutrophils were harvested from rat whole blood and cultured in the presence of plant extract or control samples. Neutrophils, except unactivated control cells, were activated with 10 μg/mL lipopolysaccharide (LPS). The cells were cultured with a range of different concentrations of the A. annua extracts (400–1 μg/mL) and artemisinin (200 and 100 μg/mL) and the supernatants were then tested by enzyme-linked immunosorbent assay (ELISA) for the concentrations of TNF-α and PGE2. Each sample was assayed in triplicate. Positive controls with an inhibitor were assayed in triplicate: chloroquine 2.58 and 5.16 μg/mL for TNF-α, and ibuprofen 400 μg/mL for PGE2. An unsupplemented group was also assessed in triplicate as a baseline control.
Results
Neutrophils were stimulated to an inflammatory state by the addition of LPS. A. annua extract significantly inhibited TNF-α production by activated neutrophils in a dose-dependent manner. There was complete inhibition by the A. annua extract at 200, 100, and 50 μg/mL (all P≤0.0003). At A. annua extract concentrations of 25, 10, and 5 μg/mL, TNF-α production was inhibited by 89% (P<0.0001), 54% (P=0.0002), and 38% (P=0.0014), respectively. A. annua 1 μg/mL did not significantly inhibit TNF-α production (8.8%; P>0.05). Concentrations of 400, 200, and 100 μg/mL A. annua extract significantly inhibited PGE2 production by 87% (P=0.0128), 91% (P=0.0017), and 93% (P=0.0114), respectively.
Conclusion
An extract of A. annua was shown to be a potent inhibitor of TNF-α and a strong inhibitor of PGE2 production in activated neutrophils at the concentrations tested. Further studies are warranted with this promising plant extract.
doi:10.2147/JIR.S75484
PMCID: PMC4298291  PMID: 25609991
in vitro; TNF-α; COX-2; PGE2; artemisinin; Arthrem
2.  A Novel Method for Simulating Insulin Mediated GLUT4 Translocation 
Biotechnology and bioengineering  2014;111(12):2454-2465.
Glucose transport in humans is a vital process which is tightly regulated by the endocrine system. Specifically, the insulin hormone triggers a cascade of intracellular signals in target cells mediating the uptake of glucose. Insulin signaling triggers cellular relocalization of the glucose transporter protein GLUT4 to the cell surface, which is primarily responsible for regulated glucose import. Pathology associated with the disruption of this pathway can lead to metabolic disorders, such as type II diabetes mellitus, characterized by the failure of cells to appropriately uptake glucose from the blood. We describe a novel simulation tool of the insulin intracellular response, incorporating the latest findings regarding As160 and GEF interactions. The simulation tool differs from previous computational approaches which employ algebraic or differential equations; instead, the tool incorporates statistical variations of kinetic constants and initial molecular concentrations which more accurately mimic the intracellular environment. Using this approach, we successfully recapitulate observed in vitro insulin responses, plus the effects of Wortmannin-like inhibition of the pathway. The developed tool provides insight into transient changes in molecule concentrations throughout the insulin signaling pathway, and may be employed to identify or evaluate potentially critical components of this pathway, including those associated with insulin resistance. In the future, this highly tractable platform may be useful for simulating other complex cell signaling pathways.
doi:10.1002/bit.25310
PMCID: PMC4213344  PMID: 24917169
insulin; metabolism; computational modeling; GLUT4; queuing theory
3.  TRAMP Prostate Tumor Growth Is Slowed by Walnut Diets Through Altered IGF-1 Levels, Energy Pathways, and Cholesterol Metabolism 
Journal of Medicinal Food  2014;17(12):1281-1286.
Abstract
Dietary changes could potentially reduce prostate cancer morbidity and mortality. Transgenic adenocarcinoma of the mouse prostate (TRAMP) prostate tumor responses to a 100 g of fat/kg diet (whole walnuts, walnut oil, and other oils; balanced for macronutrients, tocopherols [α-and γ]) for 18 weeks ad libitum were assessed. TRAMP mice (n=17 per group) were fed diets with 100 g fat from either whole walnuts (diet group WW), walnut-like fat (diet group WLF, oils blended to match walnut's fatty acid profile), or as walnut oil (diet group WO, pressed from the same walnuts as WW). Fasted plasma glucose was from tail vein blood, blood was obtained by cardiac puncture, and plasma stored frozen until analysis. Prostate (genitourinary intact [GUI]) was weighed and stored frozen at −80°C. Plasma triglyceride, lipoprotein cholesterol, plasma multianalyte levels (Myriad RBM Rat Metabolic MAP), prostate (GUI), tissue metabolites (Metabolon, Inc., Durham, NC, USA), and mRNA (by Illumina NGS) were determined. The prostate tumor size, plasma insulin-like growth factor-1 (IGF-1), high density lipoprotein, and total cholesterol all decreased significantly (P<.05) in both WW and WO compared to WLF. Both WW and WO versus WLF showed increased insulin sensitivity (Homeostasis Model Assessment [HOMA]), and tissue metabolomics found reduced glucose-6-phosphate, succinylcarnitine, and 4-hydroxybutyrate in these groups suggesting effects on cellular energy status. Tissue mRNA levels also showed changes suggestive of altered glucose metabolism with WW and WO diet groups having increased PCK1 and CIDEC mRNA expression, known for their roles in gluconeogenesis and increased insulin sensitivity, respectively. WW and WO group tissues also had increased MSMB mRNa a tumor suppressor and decreased COX-2 mRNA, both reported to inhibit prostate tumor growth. Walnuts reduced prostate tumor growth by affecting energy metabolism along with decreased plasma IGF-1 and cholesterol. These effects are not due to the walnut's N-3 fatty acids, but due to component(s) found in the walnut's fat component.
doi:10.1089/jmf.2014.0061
PMCID: PMC4259176  PMID: 25354213
chemoprevention; fat; insulin-like growth factor 1; prostate cancer; transgenic adenocarcinoma of the mouse prostate model; walnut; whole foods
4.  Site Specific Discrete PEGylation of 124I-Labeled mCC49 Fab′ Fragments Improves Tumor MicroPET/CT Imaging in Mice 
Bioconjugate chemistry  2013;24(11):1945-1954.
The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2–4 kDa discrete, branched PEGylation reagents on mCC49 Fab′ (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab′ (Fab′-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 (124I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab′ with Mal-dPEG-A (Fab′-A) reduced the binding affinity of the non PEGylated Fab′ by 30%; however, in vivo, Fab′-A significantly lengthened the blood retention vs Fab′-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab′-NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A.
doi:10.1021/bc400375f
PMCID: PMC4240220  PMID: 24175669
5.  ESTIMATING BACTERIAL DIVERSITY IN SCIRTOTHRIPS DORSALIS (THYSANOPTERA: THRIPIDAE) VIA NEXT GENERATION SEQUENCING 
The Florida entomologist  2014;97(2):362-366.
The last 2 decades have produced a better understanding of insect-microbial associations and yielded some important opportunities for insect control. However, most of our knowledge comes from model systems. Thrips (Thysanoptera: Thripidae) have been understudied despite their global importance as invasive species, plant pests and disease vectors. Using a culture and primer independent next-generation sequencing and metagenomics pipeline, we surveyed the bacteria of the globally important pest, Scirtothrips dorsalis Hood. The most abundant bacterial phyla identified were Actinobacteria and Proteobacteria and the most abundant genera were Propionibacterium, Stenotrophomonas, and Pseudomonas. A total of 189 genera of bacteria were identified. The absence of any vertically transferred symbiont taxa commonly found in insects is consistent with other studies suggesting that thrips primarilly acquire resident microbes from their environment. This does not preclude a possible beneficial/intimate association between S. dorsalis and the dominant taxa identified and future work should determine the nature of these associations.
PMCID: PMC4222051  PMID: 25382863
Next Generation Sequencing; Metagenomics; chilli thrips
6.  Recurrence of Differentiated Thyroid Carcinoma During Full TSH Suppression: Is the Tumor Now Thyroid Hormone Dependent? 
Hormones & Cancer  2014;6:7-12.
Well-standardized primary treatment and long-term management of differentiated thyroid carcinoma (DTC) include lowering or suppression of host thyrotropin (TSH) with exogenous L-thyroxine (T4). This treatment recognizes the trophic action of TSH on DTC cells. Suppression of endogenous TSH with T4 is continued in recurrent disease. However, T4 can induce proliferation of follicular and papillary thyroid carcinoma cell lines and of other human carcinoma cells. The proliferative mechanism is initiated at a cell surface receptor for T4 on integrin αvβ3, a receptor by which the hormone also inhibits p53-dependent apoptosis in tumor cells. In recurrent DTC with satisfactory suppression of endogenous TSH, we discuss here the possibility that the tumor is no longer TSH dependent and that T4 has become a critical growth factor for the cancer.
doi:10.1007/s12672-014-0204-z
PMCID: PMC4309911  PMID: 25292307
7.  Tetraiodothyroacetic acid-conjugated PLGA nanoparticles: a nanomedicine approach to treat drug-resistant breast cancer 
Nanomedicine (London, England)  2013;8(12):10.2217/nnm.12.200.
Aim
The aim was to evaluate tetraiodothyroacetic acid (tetrac), a thyroid hormone analog of l-thyroxin, conjugated to poly(lactic-co-glycolic acid) nanoparticles (T-PLGA-NPs) both in vitro and in vivo for the treatment of drug-resistant breast cancer.
Materials & methods
The uptake of tetrac and T-PLGA-NPs in doxorubicin-resistant MCF7 (MCF7-Dx) cells was evaluated using confocal microscopy. Cell proliferation assays and a chick chorioallantoic membrane model of FGF2-induced angiogenesis were used to evaluate the anticancer effects of T-PLGA-NPs. In vivo efficacy was examined in a MCF7-Dx orthotopic tumor BALBc nude mouse model.
Results
T-PLGA-NPs were restricted from entering into the cell nucleus, and T-PLGA-NPs inhibited angiogenesis by 100% compared with 60% by free tetrac. T-PLGA-NPs enhanced inhibition of tumor-cell proliferation at a low-dose equivalent of free tetrac. In vivo treatment with either tetrac or T-PLGA-NPs resulted in a three- to five-fold inhibition of tumor weight.
Conclusion
T-PLGA-NPs have high potential as anticancer agents, with possible applications in the treatment of drug-resistant cancer.
doi:10.2217/nnm.12.200
PMCID: PMC3825799  PMID: 23448245
angiogenesis; breast cancer; chick chorioallantoic membrane; MCF7 breast cancer cell; nanoparticle; tetrac; thyroid hormone
8.  De novo Assembly and Analysis of the Northern Leopard Frog Rana pipiens Transcriptome 
Journal of Genomics  2014;2:141-149.
The northern leopard frog Rana (Lithobates) pipiens is an important animal model, being used extensively in cancer, neurology, physiology, and biomechanical studies. R. pipiens is a native North American frog whose range extends from northern Canada to southwest United States, but over the past few decades its populations have declined significantly and is now considered uncommon in large portions of the United States and Canada. To aid in the study and conservation of R. pipiens, this paper describes the first R. pipiens transcriptome. The R. pipiens transcriptome was annotated using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Eukaryotic Orthologous Groups (KOG). Differential expression analysis revealed universal and tissue specific genes, and endocrine-related genes were identified. Transcriptome assemblies and other sequence data are available for download.
doi:10.7150/jgen.9760
PMCID: PMC4218947  PMID: 25371763
Northern Leopard Frog; Rana pipiens; Transcriptome.
9.  Biology of Infantile Hemangioma 
Frontiers in Surgery  2014;1:38.
Infantile hemangioma (IH), the most common tumor of infancy, is characterized by an initial proliferation during infancy followed by spontaneous involution over the next 5–10 years, often leaving a fibro-fatty residuum. IH is traditionally considered a tumor of the microvasculature. However, recent data show the critical role of stem cells in the biology of IH with emerging evidence suggesting an embryonic developmental anomaly due to aberrant proliferation and differentiation of a hemogenic endothelium with a neural crest phenotype that possesses the capacity for endothelial, hematopoietic, mesenchymal, and neuronal differentiation. Current evidence suggests a putative placental chorionic mesenchymal core cell embolic origin of IH during the first trimester. This review outlines the emerging role of stem cells and their interplay with the cytokine niche that promotes a post-natal environment conducive for vasculogenesis involving VEGFR-2 and its ligand VEGF-A and the IGF-2 ligand in promoting cellular proliferation, and the TRAIL-OPG anti-apoptotic pathway in preventing cellular apoptosis in IH. The discovery of the role of the renin–angiotensin system in the biology of IH provides a plausible explanation for the programed biologic behavior and the β-blocker-induced accelerated involution of this enigmatic condition. This crucially involves the vasoactive peptide, angiotensin II, that promotes cellular proliferation in IH predominantly via its action on the ATIIR2 isoform. The role of the RAS in the biology of IH is further supported by the effect of captopril, an ACE inhibitor, in inducing accelerated involution of IH. The discovery of the critical role of RAS in IH represents a novel and fascinating paradigm shift in the understanding of human development, IH, and other tumors in general.
doi:10.3389/fsurg.2014.00038
PMCID: PMC4286974  PMID: 25593962
infantile hemangioma; renin–angiotensin system; beta-blocker; angiotensin-converting enzyme inhibitor; propranolol; captopril; hemogenic endothelium; placenta
10.  Nanotetrac targets integrin αvβ3 on tumor cells to disorder cell defense pathways and block angiogenesis 
OncoTargets and therapy  2014;7:1619-1624.
The extracellular domain of integrin αvβ3 contains a receptor for thyroid hormone and hormone analogs. The integrin is amply expressed by tumor cells and dividing blood vessel cells. The proangiogenic properties of thyroid hormone and the capacity of the hormone to promote cancer cell proliferation are functions regulated nongenomically by the hormone receptor on αvβ3. An L-thyroxine (T4) analog, tetraiodothyroacetic acid (tetrac), blocks binding of T4 and 3,5,3′-triiodo-L-thyronine (T3) by αvβ3 and inhibits angiogenic activity of thyroid hormone. Covalently bound to a 200 nm nanoparticle that limits its activity to the cell exterior, tetrac reformulated as Nanotetrac has additional effects mediated by αvβ3 beyond the inhibition of binding of T4 and T3 to the integrin. These actions of Nanotetrac include disruption of transcription of cell survival pathway genes, promotion of apoptosis by multiple mechanisms, and interruption of repair of double-strand deoxyribonucleic acid breaks caused by irradiation of cells. Among the genes whose expression is suppressed by Nanotetrac are EGFR, VEGFA, multiple cyclins, catenins, and multiple cytokines. Nanotetrac has been effective as a chemotherapeutic agent in preclinical studies of human cancer xenografts. The low concentrations of αvβ3 on the surface of quiescent nonmalignant cells have minimized toxicity of the agent in animal studies.
doi:10.2147/OTT.S67393
PMCID: PMC4172128  PMID: 25258542
integrin; thyroid hormone; thyroxine; antiangiogenesis; proapoptosis
11.  Thyroid hormone regulates adhesion, migration and matrix metalloproteinase 9 activity via αvβ3 integrin in myeloma cells 
Oncotarget  2014;5(15):6312-6322.
Thyroid hormone (3,5,3′-triiodothyronine, T3; L-thyroxine, T4) enhances cancer cell proliferation, invasion and angiogenesis via a discrete receptor located near the RGD recognition site on αvβ3 integrin. Tetraiodothyroacetic acid (tetrac) and its nanoparticulate formulation interfere with binding of T3/T4 to the integrin. This integrin is overexpressed in multiple myeloma (MM) and other cancers. MM cells interact with αvβ3 integrin to support growth and invasion. Matrix metalloproteinases (MMPs) are a family of enzymes active in tissue remodeling and cancer. The association between integrins and MMPs secretion and action is well established. In the current study, we examined the effects of thyroid hormone on myeloma cell adhesion, migration and MMP activity.
We show that T3 and T4 increased myeloma adhesion to fibronectin and induced αvβ3 clustering. In addition, the hormones induced MMP-9 expression and activation via αvβ3 and MAPK induction. Bortezomib, a standard myeloma treatment, caused a decrease in activity/quantity of MMPs and thyroid hormone opposed this effect. RGD peptide and tetrac impaired the production of MMP-9 in cell lines and in primary BM cells from myeloma patients.
In conclusion, thyroid hormone-dependent regulation via αvβ3 of myeloma cell adhesion and MMP-9 production may play a role in myeloma migration and progression.
PMCID: PMC4171632  PMID: 25071016
Integrin; myeloma; thyroid hormone; MMP-9; adhesion
12.  THE SPRING RUNOFF IN NEBRASKA’S ELKHORN RIVER WATERSHED AND ITS IMPACT ON TWO SENTINEL ORGANISMS 
The objectives of the present study were to determine 1) if temporal variability influenced the toxicity of Elkhorn River water and 2) if the toxic effect was consistent between two sentinel organisms, the fathead minnow (Pimephales promelas) and the northern leopard frog (Rana pipiens). During spring 2012, atrazine indicator strips were used to document the occurrence of agrichemical pulses in the Elkhorn River. Polar organic chemical integrative samplers (POCIS) were deployed for 14 d during both a pulse and post-pulse period as indicated by the atrazine strips. Pesticide concentrations detected in the POCIS extracts ranged from 1.6 to 281 fold higher during the pulse period compared to the post-pulse period. Fish and frog bioassays were conducted for 7 d, and hepatic mRNA expression of vitellogenin (Vtg) and estrogen receptor-α (ERα) was determined by quantitative real-time PCR (RT-qPCR). Compared to lab water controls, fish exposed to water collected during an agrichemical pulse experienced significant reductions in Vtg and ERα, whereas exposed female frogs did not. Male leopard frogs, in contrast experienced significant increases in the expression of ERα, whereas pulse exposed male minnows did not. The significant effects observed following agrichemical pulse exposure demonstrate 1) that episodic agrichemical runoff adversely impacts sentinel organisms, and 2) that the adverse impacts observed depends upon the sex and species of the sentinel organism.
doi:10.1002/etc.2220
PMCID: PMC3683351  PMID: 23504772
Agricultural runoff; POCIS; Northern leopard frog; Fathead minnow; Gene expression
13.  Diversity and Expression of MicroRNAs in the Filarial Parasite, Brugia malayi 
PLoS ONE  2014;9(5):e96498.
Human filarial parasites infect an estimated 120 million people in 80 countries worldwide causing blindness and the gross disfigurement of limbs and genitals. An understanding of RNA-mediated regulatory pathways in these parasites may open new avenues for treatment. Toward this goal, small RNAs from Brugia malayi adult females, males and microfilariae were cloned for deep-sequencing. From ∼30 million sequencing reads, 145 miRNAs were identified in the B. malayi genome. Some microRNAs were validated using the p19 RNA binding protein and qPCR. B. malayi miRNAs segregate into 99 families each defined by a unique seed sequence. Sixty-one of the miRNA families are highly conserved with homologues in arthropods, vertebrates and helminths. Of those miRNAs not highly conserved, homologues of 20 B. malayi miRNA families were found in vertebrates. Nine B. malayi miRNA families appear to be filarial-specific as orthologues were not found in other organisms. The miR-2 family is the largest in B. malayi with 11 members. Analysis of the sequences shows that six members result from a recent expansion of the family. Library comparisons found that 1/3 of the B. malayi miRNAs are differentially expressed. For example, miR-71 is 5–7X more highly expressed in microfilariae than adults. Studies suggest that in C.elegans, miR-71 may enhance longevity by targeting the DAF-2 pathway. Characterization of B. malayi miRNAs and their targets will enhance our understanding of their regulatory pathways in filariads and aid in the search for novel therapeutics.
doi:10.1371/journal.pone.0096498
PMCID: PMC4019659  PMID: 24824352
14.  Single molecular weight discrete PEG compounds: emerging roles in molecular diagnostics, imaging and therapeutics 
“The use of PEGylation has subsequently become commonplace in the development and modification of numerous biopharmaceuticals and has led to many advancements in molecular diagnostics, imaging and therapeutics.”
doi:10.1586/erm.13.19
PMCID: PMC3748965  PMID: 23638813
branched; linear; PEG; PEG conjugation; PEG length; PEGylated antibodies; PEGylated peptides; PEGylated protein; PEGylation; polyethylene glycol
15.  Characterization of the Asian Citrus Psyllid Transcriptome 
Journal of Genomics  2014;2:54-58.
The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is a vector for the causative agents of Huanglongbing, which threatens citrus production worldwide. This study reports and discusses the first D. citri transcriptomes, encompassing the three main life stages of D. citri, egg, nymph and adult. The transcriptomes were annotated using Gene Ontology (GO) and insecticide-related genes within each life stage were identified to aid the development of future D. citri insecticides. Transcriptome assemblies and other sequence data are available for download at the International Asian Citrus Psyllid Genome Consortium website [http://psyllid.org/download] and at NCBI [http://www.ncbi.nlm.nih.gov/bioproject/29447].
doi:10.7150/jgen.7692
PMCID: PMC3914308  PMID: 24511328
Asian Citrus Psyllid; Diaphorina citri Kuwayama
16.  Characterization of the Asian Citrus Psyllid Transcriptome 
Journal of genomics  2014;2:54-58.
The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is a vector for the causative agents of Huanglongbing, which threatens citrus production worldwide. This study reports and discusses the first D. citri transcriptomes, encompassing the three main life stages of D. citri, egg, nymph and adult. The transcriptomes were annotated using Gene Ontology (GO) and insecticide-related genes within each life stage were identified to aid the development of future D. citri insecticides. Transcriptome assemblies and other sequence data are available for download at the International Asian Citrus Psyllid Genome Consortium website [http://psyllid.org/download] and at NCBI [http://www.ncbi.nlm.nih.gov/bioproject/29447].
doi:10.7150/jgen.7692
PMCID: PMC3914308  PMID: 24511328
Asian Citrus Psyllid; Diaphorina citri Kuwayama
17.  Design, Assessment, and in vivo Evaluation of a Computational Model Illustrating the Role of CAV1 in CD4+ T-lymphocytes 
Caveolin-1 (CAV1) is a vital scaffold protein heterogeneously expressed in both healthy and malignant tissue. We focus on the role of CAV1 when overexpressed in T-cell leukemia. Previously, we have shown that CAV1 is involved in cell-to-cell communication, cellular proliferation, and immune synapse formation; however, the molecular mechanisms have not been elucidated. We hypothesize that the role of CAV1 in immune synapse formation contributes to immune regulation during leukemic progression, thereby warranting studies of the role of CAV1 in CD4+ T-cells in relation to antigen-presenting cells. To address this need, we developed a computational model of a CD4+ immune effector T-cell to mimic cellular dynamics and molecular signaling under healthy and immunocompromised conditions (i.e., leukemic conditions). Using the Cell Collective computational modeling software, the CD4+ T-cell model was constructed and simulated under CAV1+/+, CAV1+/−, and CAV1−/− conditions to produce a hypothetical immune response. This model allowed us to predict and examine the heterogeneous effects and mechanisms of CAV1 in silico. Experimental results indicate a signature of molecules involved in cellular proliferation, cell survival, and cytoskeletal rearrangement that were highly affected by CAV1 knock out. With this comprehensive model of a CD4+ T-cell, we then validated in vivo protein expression levels. Based on this study, we modeled a CD4+ T-cell, manipulated gene expression in immunocompromised versus competent settings, validated these manipulations in an in vivo murine model, and corroborated acute T-cell leukemia gene expression profiles in human beings. Moreover, we can model an immunocompetent versus an immunocompromised microenvironment to better understand how signaling is regulated in patients with leukemia.
doi:10.3389/fimmu.2014.00599
PMCID: PMC4257089  PMID: 25538703
caveolin-1; CD4+ T-lymphocyte; the cell collective; adult T-cell leukemia; immunosuppression; immunotherapy; computational biology; logical models
18.  Cancer Cell Gene Expression Modulated from Plasma Membrane Integrin αvβ3 by Thyroid Hormone and Nanoparticulate Tetrac 
Integrin αvβ3 is generously expressed by cancer cells and rapidly dividing endothelial cells. The principal ligands of the integrin are extracellular matrix proteins, but we have described a cell surface small molecule receptor on αvβ3 that specifically binds thyroid hormone and thyroid hormone analogs. From this receptor, thyroid hormone (l-thyroxine, T4; 3,5,3′-triiodo-l-thyronine, T3) and tetraiodothyroacetic acid (tetrac) regulate expression of specific genes by a mechanism that is initiated non-genomically. At the integrin, T4 and T3 at physiological concentrations are pro-angiogenic by multiple mechanisms that include gene expression, and T4 supports tumor cell proliferation. Tetrac blocks the transcriptional activities directed by T4 and T3 at αvβ3, but, independently of T4 and T3, tetrac modulates transcription of cancer cell genes that are important to cell survival pathways, control of the cell cycle, angiogenesis, apoptosis, cell export of chemotherapeutic agents, and repair of double-strand DNA breaks. We have covalently bound tetrac to a 200 nm biodegradable nanoparticle that prohibits cell entry of tetrac and limits its action to the hormone receptor on the extracellular domain of plasma membrane αvβ3. This reformulation has greater potency than unmodified tetrac at the integrin and affects a broader range of cancer-relevant genes. In addition to these actions on intra-cellular kinase-mediated regulation of gene expression, hormone analogs at αvβ3 have additional effects on intra-cellular protein-trafficking (cytosol compartment to nucleus), nucleoprotein phosphorylation, and generation of nuclear coactivator complexes that are relevant to traditional genomic actions of T3. Thus, previously unrecognized cell surface-initiated actions of thyroid hormone and tetrac formulations at αvβ3 offer opportunities to regulate angiogenesis and multiple aspects of cancer cell behavior.
doi:10.3389/fendo.2014.00240
PMCID: PMC4290672  PMID: 25628605
integrin; thyroid hormone; tetraiodothyroacetic acid; nanoparticle; gene transcription
19.  WormBase 2014: new views of curated biology 
Nucleic Acids Research  2013;42(Database issue):D789-D793.
WormBase (http://www.wormbase.org/) is a highly curated resource dedicated to supporting research using the model organism Caenorhabditis elegans. With an electronic history predating the World Wide Web, WormBase contains information ranging from the sequence and phenotype of individual alleles to genome-wide studies generated using next-generation sequencing technologies. In recent years, we have expanded the contents to include data on additional nematodes of agricultural and medical significance, bringing the knowledge of C. elegans to bear on these systems and providing support for underserved research communities. Manual curation of the primary literature remains a central focus of the WormBase project, providing users with reliable, up-to-date and highly cross-linked information. In this update, we describe efforts to organize the original atomized and highly contextualized curated data into integrated syntheses of discrete biological topics. Next, we discuss our experiences coping with the vast increase in available genome sequences made possible through next-generation sequencing platforms. Finally, we describe some of the features and tools of the new WormBase Web site that help users better find and explore data of interest.
doi:10.1093/nar/gkt1063
PMCID: PMC3965043  PMID: 24194605
20.  A Hybrid Online Intervention for Reducing Sedentary Behavior in Obese Women 
Sedentary behavior (SB) has emerged as an independent risk factor for cardiovascular disease and type 2 diabetes. While exercise is known to reduce these risks, reducing SB through increases in non-structured PA and breaks from sitting may appeal to obese women who have lower self-efficacy for PA. This study examined effects of a combined face-to-face and online intervention to reduce SB in overweight and obese women. A two-group quasi-experimental study was used with measures taken pre and post. Female volunteers (M age = 58.5, SD = 12.5 years) were enrolled in the intervention (n = 40) or waitlisted (n = 24). The intervention, based on the Social Cognitive Theory, combined group sessions with email messages over 6 weeks. Individualized feedback to support mastery and peer models of active behaviors were included in the emails. Participants self-monitored PA with a pedometer. Baseline and post measures of PA and SB were assessed by accelerometer and self-report. Standard measures of height, weight, and waist circumference were conducted. Repeated measures ANOVA was used for analyses. Self-reported SB and light PA in the intervention group (I) changed significantly over time [SB, F(1, 2) = 3.81, p = 0.03, light PA, F(1, 2) = 3.39, p = 0.04]. Significant Group × Time interactions were found for light PA, F(1, 63) = 5.22, p = 0.03, moderate PA, F(1, 63) = 3.90, p = 0.05, and for waist circumference, F(1, 63) = 16.0, p = 0.001. The intervention group decreased significantly while the comparison group was unchanged. Hybrid computer interventions to reduce SB may provide a non-exercise alternative for increasing daily PA and potentially reduce waist circumference, a risk factor for type 2 diabetes. Consumer-grade accelerometers may aide improvements to PA and SB and should be tested as part of future interventions.
doi:10.3389/fpubh.2013.00045
PMCID: PMC3859962  PMID: 24350214
computer; accelerometer; inactivity; physical activity; waist circumference
21.  Ensembl Genomes 2013: scaling up access to genome-wide data 
Nucleic Acids Research  2013;42(Database issue):D546-D552.
Ensembl Genomes (http://www.ensemblgenomes.org) is an integrating resource for genome-scale data from non-vertebrate species. The project exploits and extends technologies for genome annotation, analysis and dissemination, developed in the context of the vertebrate-focused Ensembl project, and provides a complementary set of resources for non-vertebrate species through a consistent set of programmatic and interactive interfaces. These provide access to data including reference sequence, gene models, transcriptional data, polymorphisms and comparative analysis. This article provides an update to the previous publications about the resource, with a focus on recent developments. These include the addition of important new genomes (and related data sets) including crop plants, vectors of human disease and eukaryotic pathogens. In addition, the resource has scaled up its representation of bacterial genomes, and now includes the genomes of over 9000 bacteria. Specific extensions to the web and programmatic interfaces have been developed to support users in navigating these large data sets. Looking forward, analytic tools to allow targeted selection of data for visualization and download are likely to become increasingly important in future as the number of available genomes increases within all domains of life, and some of the challenges faced in representing bacterial data are likely to become commonplace for eukaryotes in future.
doi:10.1093/nar/gkt979
PMCID: PMC3965094  PMID: 24163254
22.  Antimitochondrial Antibodies in Acute Liver Failure: Implications for Primary Biliary Cirrhosis 
Hepatology (Baltimore, Md.)  2007;46(5):1436-1442.
In our previous work, including analysis of more than 10,000 sera from control patients and patients with a variety of liver diseases, we have demonstrated that with the use of recombinant autoantigens, antimitochondrial autoantibodies (AMA) are only found in PBC and that a positive AMA is virtually pathognomonic of either PBC or future development of PBC. Although the mechanisms leading to the generation of AMA are enigmatic, we have postulated that xenobiotic-induced and/or oxidative modification of mitochondrial autoantigens is a critical step leading to loss of tolerance. This thesis suggests that a severe liver oxidant injury would lead to AMA production. We analyzed 217 serum samples from 69 patients with acute liver failure (ALF) collected up to 24 months post-ALF, compared with controls, for titer and reactivity with the E2 subunits of pyruvate dehydrogenase (PDC-E2), branched chain 2-oxo-acid dehydrogenase (BCOADC-E2) and 2-oxo-glutarate dehydrogenase (OGDC-E2). AMA were detected in 28/69 (40.6%) ALF patients with reactivity found against all of the major mitochondrial autoantigens. In addition, and as further controls, sera were also analyzed for autoantibodies to gp210, Sp100, centromere, chromatin, soluble liver antigen (SLA), tissue transglutaminase (tTG) and deaminated gliadin peptides (DGP) where the most frequently detected non-mitochondrial autoantibody was against tTG (57.1% of ALF patients). In conclusion, the strikingly high frequency of AMA in ALF supports the thesis that oxidative stress-induced liver damage may lead to AMA induction. The rapid disappearance of AMA in these patients provides further support for the contention that PBC pathogenesis requires additional factors including genetic susceptibility.
doi:10.1002/hep.21828
PMCID: PMC3731127  PMID: 17657817
Tolerance; Oxidative injury; Self-reactivity; Epitopes
23.  A simplified counter-selection recombineering protocol for creating fluorescent protein reporter constructs directly from C. elegans fosmid genomic clones 
BMC Biotechnology  2013;13:1.
Background
Recombineering is a genetic engineering tool that enables facile modification of large episomal clones, e.g. BACs, fosmids. We have previously adapted this technology to generate, directly from fosmid-based genomic clones, fusion gene reporter constructs designed to investigate gene expression patterns in C. elegans. In our adaptation a rpsL-tet(A) positive/negative-selection cassette (RT-cassette) is first inserted and then, under negative selection, seamlessly replaced with the desired sequence. We report here on the generation and application of a resource comprising two sets of constructs designed to facilitate this particular recombineering approach.
Results
Two complementary sets of constructs were generated. The first contains different fluorescent protein reporter coding sequences and derivatives while the second set of constructs, based in the copy-number inducible vector pCC1Fos, provide a resource designed to simplify RT-cassette-based recombineering. These latter constructs are used in pairs the first member of which provides a template for PCR-amplification of an RT-cassette while the second provides, as an excised restriction fragment, the desired fluorescent protein reporter sequence. As the RT-cassette is flanked by approximately 200 bp from the ends of the reporter sequence the subsequent negative selection replacement step is highly efficient. Furthermore, use of a restriction fragment minimizes artefacts negating the need for final clone sequencing. Utilizing this resource we generated single-, double- and triple-tagged fosmid-based reporters to investigate expression patterns of three C. elegans genes located on a single genomic clone.
Conclusions
We describe the generation and application of a resource designed to facilitate counter-selection recombineering of fosmid-based C. elegans genomic clones. By choosing the appropriate pair of ‘insertion’ and ‘replacement’ constructs recombineered products, devoid of artefacts, are generated at high efficiency. Gene expression patterns for three genes located on the same genomic clone were investigated via a set of fosmid-based reporter constructs generated with the modified protocol.
doi:10.1186/1472-6750-13-1
PMCID: PMC3561212  PMID: 23281894
C. elegans; Recombineering; Fosmid; Fluorescent protein; Deoxyribose-phosphate aldolase; Peroxiredoxin; Metallocarboxypeptidase
24.  Targeted Disruption of Toxoplasma gondii Serine Protease Inhibitor 1 Increases Bradyzoite Cyst Formation In Vitro and Parasite Tissue Burden in Mice 
Infection and Immunity  2012;80(3):1156-1165.
As an intracellular protozoan parasite, Toxoplasma gondii is likely to exploit proteases for host cell invasion, acquisition of nutrients, avoidance of host protective responses, escape from the parasitophorous vacuole, differentiation, and other activities. T. gondii serine protease inhibitor 1 (TgPI1) is the most abundantly expressed protease inhibitor in parasite tachyzoites. We show here that alternative splicing produces two TgPI1 isoforms, both of which are secreted via dense granules into the parasitophorous vacuole shortly after invasion, become progressively more abundant over the course of the infectious cycle, and can be detected in the infected host cell cytoplasm. To investigate TgPI1 function, the endogenous genomic locus was disrupted in the RH strain background. ΔTgPI1 parasites replicate normally as tachyzoites but exhibit increased bradyzoite gene transcription and labeling of vacuoles with Dolichos biflorus lectin under conditions promoting in vitro differentiation. The differentiation phenotype can be partially complemented by either TgPI1 isoform. Mice infected with the ΔTgPI1 mutant display ∼3-fold-increased parasite burden in the spleen and liver, and this in vivo phenotype is also complemented by either TgPI1 isoform. These results demonstrate that TgPI1 influences both parasite virulence and bradyzoite differentiation, presumably by inhibiting parasite and/or host serine proteases.
doi:10.1128/IAI.06167-11
PMCID: PMC3294639  PMID: 22202120
25.  Advancing clinical decision support using lessons from outside of healthcare: an interdisciplinary systematic review 
Background
Greater use of computerized decision support (DS) systems could address continuing safety and quality problems in healthcare, but the healthcare field has struggled to implement DS technology. This study surveys DS experience across multiple non-healthcare disciplines for new insights that are generalizable to healthcare provider decisions. In particular, it sought design principles and lessons learned from the other disciplines that could inform efforts to accelerate the adoption of clinical decision support (CDS).
Methods
Our systematic review drew broadly from non-healthcare databases in the basic sciences, social sciences, humanities, engineering, business, and defense: PsychINFO, BusinessSource Premier, Social Sciences Abstracts, Web of Science, and Defense Technical Information Center. Because our interest was in DS that could apply to clinical decisions, we selected articles that (1) provided a review, overview, discussion of lessons learned, or an evaluation of design or implementation aspects of DS within a non-healthcare discipline and (2) involved an element of human judgment at the individual level, as opposed to decisions that can be fully automated or that are made at the organizational level.
Results
Clinical decisions share some similarities with decisions made by military commanders, business managers, and other leaders: they involve assessing new situations and choosing courses of action with major consequences, under time pressure, and with incomplete information. We identified seven high-level DS system design features from the non-healthcare literature that could be applied to CDS: providing broad, system-level perspectives; customizing interfaces to specific users and roles; making the DS reasoning transparent; presenting data effectively; generating multiple scenarios covering disparate outcomes (e.g., effective; effective with side effects; ineffective); allowing for contingent adaptations; and facilitating collaboration. The article provides examples of each feature. The DS literature also emphasizes the importance of organizational culture and training in implementation success. The literature contrasts “rational-analytic” vs. “naturalistic-intuitive” decision-making styles, but the best approach is often a balanced approach that combines both styles. It is also important for DS systems to enable exploration of multiple assumptions, and incorporation of new information in response to changing circumstances.
Conclusions
Complex, high-level decision-making has common features across disciplines as seemingly disparate as defense, business, and healthcare. National efforts to advance the health information technology agenda through broader CDS adoption could benefit by applying the DS principles identified in this review.
doi:10.1186/1472-6947-12-90
PMCID: PMC3524755  PMID: 22900537

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