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In the Mexican-American population, the prevalence of Type 2 diabetes mellitus (T2DM) is as high as 50% of the population. This randomized controlled clinical trial was designed to elucidate how treatment of periodontal disease affects HbA1c values in this population.
Materials and Methods
154 T2DM patients with periodontal disease were enrolled in the study. The test group was treated with scaling and root planing (SRP); the control group received oral hygiene instructions. At baseline and 4–6 weeks after therapy, a complete periodontal examination was performed. Blood was collected at baseline and 4 months later for HbA1c levels.
126 individuals completed the study. Baseline mean ± SD HbA1c for the test and control groups were 9.0 ± 2.3% and 8.4 ± 2.0%, respectively. Non-significant difference in HbA1c reductions (0.6±2.1% and 0.3±1.7%) was found between test and control groups at 4 months. Comparisons of the periodontal clinical parameters between the test and control groups found significant differences with improved results in the test subjects.
No statistically significant differences were found in the changes of HbA1c levels between test and control groups. Non-surgical periodontal therapy improved the magnitude of change in periodontal parameters as compared to the control subjects. Identifier: NCT01128374
PMCID: PMC4080623  PMID: 24797222
type 2 diabetes mellitus; periodontitis; scaling and root planning; HbA1c; Hispanic population
2.  Vorapaxar in Patients With Diabetes Mellitus and Previous Myocardial Infarction 
Circulation  2015;131(12):1047-1053.
Supplemental Digital Content is available in the text.
Vorapaxar reduces cardiovascular death, myocardial infarction (MI), or stroke in patients with previous MI while increasing bleeding. Patients with diabetes mellitus (DM) are at high risk of recurrent thrombotic events despite standard therapy and may derive particular benefit from antithrombotic therapies. The Thrombin Receptor Antagonist in Secondary Prevention of Atherothrombotic Ischemic Events-TIMI 50 trial was a randomized, double-blind, placebo-controlled trial of vorapaxar in patients with stable atherosclerosis.
Methods and Results—
We examined the efficacy of vorapaxar in patients with and without DM who qualified for the trial with a previous MI. Because vorapaxar is contraindicated in patients with a history of stroke or transient ischemic attack, the analysis (n=16 896) excluded such patients. The primary end point of cardiovascular death, MI, or stroke occurred more frequently in patients with DM than in patients without DM (rates in placebo group: 14.3% versus 7.6%; adjusted hazard ratio, 1.47; P<0.001). In patients with DM (n=3623), vorapaxar significantly reduced the primary end point (11.4% versus 14.3%; hazard ratio, 0.73 [95% confidence interval, 0.60–0.89]; P=0.002) with a number needed to treat to avoid 1 major cardiovascular event of 29. The incidence of moderate/severe bleeding was increased with vorapaxar in patients with DM (4.4% versus 2.6%; hazard ratio, 1.60 [95% confidence interval, 1.07–2.40]). However, net clinical outcome integrating these 2 end points (efficacy and safety) was improved with vorapaxar (hazard ratio, 0.79 [95% confidence interval, 0.67–0.93]).
In patients with previous MI and DM, the addition of vorapaxar to standard therapy significantly reduced the risk of major vascular events with greater potential for absolute benefit in this group at high risk of recurrent ischemic events.
Clinical Trial Registration—
URL: Unique identifier: NCT00526474.
PMCID: PMC4365950  PMID: 25681464
coronary disease; diabetes mellitus; myocardial infarction; secondary prevention
3.  p38 MAP Kinase Inhibition Reduces Propionibacterium acnes-Induced Inflammation in Vitro 
Dermatology and Therapy  2015;5(1):53-66.
Propionibacterium acnes, a ubiquitous skin bacterium, stimulates keratinocytes to produce a number of proinflammatory cytokines and may contribute to inflammatory acne. The aim of the study was to investigate whether P. acnes-induced proinflammatory cytokine release is mediated by P. acnes-induced activation of p38 mitogen-activated protein kinase (p38 MAPK or p38) in human keratinocytes.
Immunohistochemistry was used to evaluate p38 phosphorylation in human skin samples with or without acne. Primary human keratinocytes and epidermal skin equivalents were exposed to viable P. acnes. Phosphorylation of MAPKs without or with p38 inhibitors was examined by Western blot and cytokine secretion was detected by Enzyme-Linked Immunosorbent Assay (ELISA).
Increased levels of phospho-p38 were observed in human acne lesions, predominantly in follicular and perifollicular keratinocytes. Exposure of cultured human keratinocytes to viable P. acnes resulted in phosphorylation of multiple members of the MAPK family, including rapid and transient activation of p38 and extracellular signal-related kinase (ERK1/2) and relatively slow but sustained activation of c-Jun N-terminal kinases (JNK1/2). Viable P. acnes induced the secretion of interleukin-1α (IL-1α), tumor necrosis factor-α (TNF-α), and IL-8 from human keratinocytes. The phosphorylation of p38 (phospho-p38) and the secretion of cytokines induced by P. acnes in cultured keratinocytes were inhibited by SB203580, a p38α/β inhibitor. Furthermore, SCIO-469, a selective inhibitor of p38α, showed similar effects in cultured keratinocytes. Topical treatment of SCIO-469 inhibited the P. acnes-induced phospho-p38 and cytokine secretion in human epidermal equivalents.
The data demonstrate that P. acnes induces p38-dependent inflammatory responses in keratinocytes, and suggest that p38 may play an important role in the pathogenesis of inflammatory acne.
Johnson & Johnson.
Electronic supplementary material
The online version of this article (doi:10.1007/s13555-015-0072-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4374066  PMID: 25749612
Acne vulgaris; Inflammation; Keratinocyte; p38 mitogen-activated protein kinase; p38 inhibitor; Propionibacterium acnes
4.  Method for the determination of trajectory angles of directional secretory vesicles in cultured astrocytes 
Inquiro  2013;7:48-52.
Astrocytes provide a principal pathway for glutamate uptake in the mammalian brain, a task accomplished by the powerful action of excitatory amino acid transporters (EAAT) 1 and 2. These transporters are synthesized within the endoplasmic reticulum and are then trafficked to the plasma membrane. The characteristics of their intracellular traffic within astrocytes have not been investigated. We monitored the trafficking of secretory vesicles laden with the recombinant fluorescent protein chimera of EAAT2 in cultured astrocytes. Such vesicles appeared as fluorescent puncta, and their trafficking parameters were obtained using original algorithms, which we describe here in detail. We determined the maximal displacement, average instantaneous speed, and trajectory angle of individual puncta/vesicles, with angles near 0° indicating radial movement directly away from or toward the nucleus and angles near 90° indicating tangential movement. Analysis of these trafficking parameters demonstrated that trafficking of EAAT2-laden vesicles has typical characteristics expected of the trafficking of secretory vesicles in cultured astrocytes. The distribution of trajectory angles for directional vesicles, i.e. those with a maximal displacement greater than 1 μm within the 40-s time-lapse imaging, was found to be unimodal, with angles near 0° being the most prominent (mode 7°). These measurements are in good agreement with previous measurements of trajectory angles of similar trafficking vesicles carrying cannabinoid receptor 1, evidencing the validity and robustness of our analytical approach and algorithms.
PMCID: PMC4276363  PMID: 25544968
5.  Patient and Hospital Characteristics Associated with Inappropriate Percutaneous Coronary Interventions 
Journal of the American College of Cardiology  2013;62(24):10.1016/j.jacc.2013.07.086.
To examine whether rates of inappropriate PCI differ by demographic characteristics and insurance status.
Prior studies have found that blacks, women and those with public or no health insurance are less likely to undergo percutaneous coronary intervention (PCI). Whether this reflects potential overuse in whites, men, and privately insured patients, in addition to underuse in disadvantaged populations, is unknown.
Within the NCDR® CathPCI Registry®, we identified 221,254 non-acute PCIs performed between July 2009 and March 2011. PCI appropriateness was determined using Appropriate Use Criteria (AUC) for coronary revascularization. Multivariable hierarchical regression models evaluated the association between patient demographics and insurance status with AUC-defined inappropriate PCI.
Of 211,254 non-acute PCIs, 25,749 (12.2%) were classified as inappropriate. After multivariable adjustment, men (adjusted OR, 1.08 [95% CI: 1.05–1.11]; P<0.001) and whites (adjusted OR, 1.09 [1.05–1.14]; P<0.001) were more likely to undergo an inappropriate PCI, compared with women and non-whites. Compared with privately insured patients, those with Medicare (adjusted OR, 0.85 [0.83–0.88]), other public insurance (adjusted OR, 0.78 [0.73–0.83]) and no insurance (adjusted OR, 0.56 [0.50–0.61]) were less likely to undergo an inappropriate PCI (P<0.001). Additionally, compared with urban hospitals, those admitted at rural hospitals were less likely to undergo inappropriate PCI, whereas those at suburban hospitals were more likely.
For non-acute indications, PCIs categorized as inappropriate were more commonly performed in men, patients of white race, and those with private insurance. Higher rates of PCI in these patient populations may be, in part, due to procedural overuse.
PMCID: PMC3864986  PMID: 24055743
6.  Molecular Studies on the Roles of Runx2 and Twist1 in Regulating FGF signaling 
Supernumerary teeth are often observed in patients suffering from cleidocranial dysplasia due to a mutation in Runx2 that results in haploinsufficiencty. However, the underlying molecular mechanisms are poorly defined. In this study, we assessed the roles of Runx2 and its functional antagonist Twist1 in regulating fibroblast growth factor (FGF) signaling using in vitro biochemical approaches.
We showed that Twist1 stimulated Fgfr2 and Fgf10 expression in a mesenchymal cell line and that it formed heterodimers with ubiquitously expressed E12 (together with E47 encoded by E2A gene) and upregulated Fgfr2 and Fgf10 promoter activities in a dental mesenchyme-derived cell line. We further demonstrated that the bHLH domain of Twist1 was essential for its synergistic activation of Fgfr2 promoter with E12 and that the binding of E12 stabilized Twist1 by preventing it from undergoing lysosomal degradation. Although Runx2 had no apparent effects on Fgfr2 and Fgf10 promoter activities, it inhibited the stimulatory activity of Twist1 on Fgfr2 promoter.
These findings suggest that Runx2 haploinsufficiency might result in excessive unbound Twist1 that can freely bind to E12 and enhance FGF signaling, thereby promoting the formation of extra teeth.
PMCID: PMC4153435  PMID: 22972545
Runx2; Twist1; E12; FGF10; Fgfr2; tooth morphogenesis
7.  West Nile Virus Transmission in Winter: The 2013 Great Salt Lake Bald Eagle and Eared Grebes Mortality Event 
PLoS Currents  2014;6:ecurrents.outbreaks.b0f031fc8db2a827d9da0f30f0766871.
West Nile Virus (WNV) infection has been reported in over 300 species of birds and mammals. Raptors such as eagles, hawks and falcons are remarkably susceptible, but reports of WNV infection in Bald Eagles (Haliaeetus leucocephalus) are rare and reports of WNV infection in grebes (Podicipediformes) even rarer. We report an unusually large wild bird mortality event involving between 15,000-20,000 Eared Grebes (Podiceps nigricollis) and over 40 Bald Eagles around the Great Salt Lake, Utah, in November-December 2013. Mortality in grebes was first reported in early November during a period when the area was unseasonably warm and the grebes were beginning to gather and stage prior to migration. Ten out of ten Eared Grebes collected during this period were WNV RT-PCR and/or isolation positive. This is the first report of WNV infection in Eared Grebes and the associated mortality event is matched in scale only by the combined outbreaks in American White Pelican (Pelecanus erythrorhynchos) colonies in the north central states in 2002-2003. We cannot be sure that all of the grebes were infected by mosquito transmission; some may have become infected through contact with WNV shed orally or cloacally from other infected grebes. Beginning in early December, Bald Eagles in the Great Salt Lake area were observed to display neurological signs such as body tremors, limb paralysis and lethargy. At least 43 Bald Eagles had died by the end of the month. Nine of nine Bald Eagles examined were infected with WNV. To the best of our knowledge, this is the largest single raptor mortality event since WNV became endemic in the USA. Because the majority of the eagles affected were found after onset of below-freezing temperatures, we suggest at least some of the Bald Eagles were infected with WNV via consumption of infected Eared Grebes or horizontal transmission at roost sites.
PMCID: PMC3994192  PMID: 24761310
bald eagle; disease outbreak; eared grebe; Utah; West Nile virus
8.  A Framework Phylogeny of the American Oak Clade Based on Sequenced RAD Data 
PLoS ONE  2014;9(4):e93975.
Previous phylogenetic studies in oaks (Quercus, Fagaceae) have failed to resolve the backbone topology of the genus with strong support. Here, we utilize next-generation sequencing of restriction-site associated DNA (RAD-Seq) to resolve a framework phylogeny of a predominantly American clade of oaks whose crown age is estimated at 23–33 million years old. Using a recently developed analytical pipeline for RAD-Seq phylogenetics, we created a concatenated matrix of 1.40 E06 aligned nucleotides, constituting 27,727 sequence clusters. RAD-Seq data were readily combined across runs, with no difference in phylogenetic placement between technical replicates, which overlapped by only 43–64% in locus coverage. 17% (4,715) of the loci we analyzed could be mapped with high confidence to one or more expressed sequence tags in NCBI Genbank. A concatenated matrix of the loci that BLAST to at least one EST sequence provides approximately half as many variable or parsimony-informative characters as equal-sized datasets from the non-EST loci. The EST-associated matrix is more complete (fewer missing loci) and has slightly lower homoplasy than non-EST subsampled matrices of the same size, but there is no difference in phylogenetic support or relative attribution of base substitutions to internal versus terminal branches of the phylogeny. We introduce a partitioned RAD visualization method (implemented in the R package RADami; to investigate the possibility that suboptimal topologies supported by large numbers of loci—due, for example, to reticulate evolution or lineage sorting—are masked by the globally optimal tree. We find no evidence for strongly-supported alternative topologies in our study, suggesting that the phylogeny we recover is a robust estimate of large-scale phylogenetic patterns in the American oak clade. Our study is one of the first to demonstrate the utility of RAD-Seq data for inferring phylogeny in a 23–33 million year-old clade.
PMCID: PMC3976371  PMID: 24705617
9.  Analysis of phenotypic evolution in Dictyostelia highlights developmental plasticity as a likely consequence of colonial multicellularity 
Colony formation was the first step towards evolution of multicellularity in many macroscopic organisms. Dictyostelid social amoebas have used this strategy for over 600 Myr to form fruiting structures of increasing complexity. To understand in which order multicellular complexity evolved, we measured 24 phenotypic characters over 99 dictyostelid species. Using phylogenetic comparative methods, we show that the last common ancestor (LCA) of Dictyostelia probably erected small fruiting structures directly from aggregates. It secreted cAMP to coordinate fruiting body morphogenesis, and another compound to mediate aggregation. This phenotype persisted up to the LCAs of three of the four major groups of Dictyostelia. The group 4 LCA co-opted cAMP for aggregation and evolved much larger fruiting structures. However, it lost encystation, the survival strategy of solitary amoebas that is retained by many species in groups 1–3. Large structures, phototropism and a migrating intermediate ‘slug’ stage coevolved as evolutionary novelties within most groups. Overall, dictyostelids show considerable plasticity in the size and shape of multicellular structures, both within and between species. This probably reflects constraints placed by colonial life on developmental control mechanisms, which, depending on local cell density, need to direct from 10 to a million cells into forming a functional fructification.
PMCID: PMC3712420  PMID: 23782883
evolution of multicellularity; morphogenetic signalling; phylogenomics; phototropism; encystation; sporulation
10.  A Customized Self-Assembling Peptide Hydrogel for Dental Pulp Tissue Engineering 
Tissue Engineering. Part A  2011;18(1-2):176-184.
Root canal therapy is common practice in dentistry. During this procedure, the inflamed or necrotic dental pulp is removed and replaced with a synthetic material. However, recent research provides evidence that engineering of dental pulp and dentin is possible by using biologically driven approaches. As tissue engineering strategies hold the promise to soon supersede conventional root canal treatment, there is a need for customized scaffolds for stem cell delivery or recruitment. We hypothesize that the incorporation of dental pulp-derived stem cells with bioactive factors into such a scaffold can promote cell proliferation, differentiation, and angiogenesis. In this study, we used a cell adhesive, enzyme-cleavable hydrogel made from self-assembling peptide nanofibers to encapsulate dental pulp stem cells. The growth factors (GFs) fibroblast growth factor basic, transforming growth factor β1, and vascular endothelial growth factor were incorporated into the hydrogel via heparin binding. Release profiles were established, and the influence of GFs on cell morphology and proliferation was assessed to confirm their bioactivity after binding and subsequent release. Cell morphology and spreading in three-dimensional cultures were visualized by using cell tracker and histologic stains. Subcutaneous transplantation of the hydrogel within dentin cylinders into immunocompromised mice led to the formation of a vascularized soft connective tissue similar to dental pulp. These data support the use of this novel biomaterial as a highly promising candidate for future treatment concepts in regenerative endodontics.
PMCID: PMC3525904  PMID: 21827280
11.  TWIST1 Promotes the Odontoblast-like Differentiation of Dental Stem Cells 
Advances in Dental Research  2011;23(3):280-284.
Stem cells derived from the dental pulp of extracted human third molars (DPSCs) have the potential to differentiate into odontoblasts, osteoblasts, adipocytes, and neural cells when provided with the appropriate conditions. To advance the use of DPSCs for dentin regeneration, it is important to replicate the permissive signals that drive terminal events in odontoblast differentiation during tooth development. Such a strategy is likely to restore a dentin matrix that more resembles the tubular nature of primary dentin. Due to the limitations of culture conditions, the use of ex vivo gene therapy to drive the terminal differentiation of mineralizing cells holds considerable promise. In these studies, we asked whether the forced expression of TWIST1 in DPSCs could alter the potential of these cells to differentiate into odontoblast-like cells. Since the partnership between Runx2 and Twist1 proteins is known to control the onset of osteoblast terminal differentiation, we hypothesized that these genes act to control lineage determination of DPSCs. For the first time, our results showed that Twist1 overexpression in DPSCs enhanced the expression of DSPP, a gene that marks odontoblast terminal differentiation. Furthermore, co-transfection assays showed that Twist1 stimulates Dspp promoter activity by antagonizing Runx2 function in 293FT cells. Analysis of our in vitro data, taken together, suggests that lineage specification of DPSCs can be modulated through ex vivo gene modifications.
PMCID: PMC3144037  PMID: 21677079
TWIST1; RUNX2; dental stem cells; gene transfer; odontoblast; tooth development
12.  An expanded phylogeny of social amoebas (Dictyostelia) shows increasing diversity and new morphological patterns 
Social Amoebae or Dictyostelia are eukaryotic microbes with a unique life cycle consisting of both uni- and multicellular stages. They have long fascinated molecular, developmental and evolutionary biologists, and Dictyostelium discoideum is now one of the most widely studied eukaryotic microbial models. The first molecular phylogeny of Dictyostelia included most of the species known at the time and suggested an extremely deep taxon with a molecular depth roughly equivalent to Metazoa. The group was also shown to consist of four major clades, none of which correspond to traditional genera. Potential morphological justification was identified for three of the four major groups, on the basis of which tentative names were assigned.
Over the past four years, the Mycetozoan Global Biodiversity Survey has identified many new isolates that appear to be new species of Dictyostelia, along with numerous isolates of previously described species. We have determined 18S ribosomal RNA gene sequences for all of these new isolates. Phylogenetic analyses of these data show at least 50 new species, and these arise from throughout the dictyostelid tree breaking up many previously isolated long branches. The resulting tree now shows eight well-supported major groups instead of the original four. The new species also expand the known morphological diversity of the previously established four major groups, violating nearly all previously suggested deep morphological patterns.
A greatly expanded phylogeny of Dictyostelia now shows even greater morphological plasticity at deep taxonomic levels. In fact, there now seem to be no obvious deep evolutionary trends across the group. However at a finer level, patterns in morphological character evolution are beginning to emerge. These results also suggest that there is a far greater diversity of Dictyostelia yet to be discovered, including novel morphologies.
PMCID: PMC3073913  PMID: 21453486
13.  Novel expression and transcriptional regulation of FoxJ1 during oro-facial morphogenesis 
Human Molecular Genetics  2008;17(23):3643-3654.
Axenfeld–Rieger syndrome (ARS) patients with PITX2 point mutations exhibit a wide range of clinical features including mild craniofacial dysmorphism and dental anomalies. Identifying new PITX2 targets and transcriptional mechanisms are important to understand the molecular basis of these anomalies. Chromatin immunoprecipitation assays demonstrate PITX2 binding to the FoxJ1 promoter and PITX2C transgenic mouse fibroblasts and PITX2-transfected cells have increased endogenous FoxJ1 expression. FoxJ1 is expressed at embryonic day 14.5 (E14.5) in early tooth germs, then down-regulated from E15.5–E17.5 and re-expressed in the inner enamel epithelium, oral epithelium, tongue epithelium, sub-mandibular salivary gland and hair follicles during E18.5 and neonate day 1. FoxJ1 and Pitx2 exhibit overlapping expression patterns in the dental and oral epithelium. PITX2 activates the FoxJ1 promoter and, Lef-1 and β-catenin interact with PITX2 to synergistically regulate the FoxJ1 promoter. FoxJ1 physically interacts with the PITX2 homeodomain to synergistically regulate FoxJ1, providing a positive feedback mechanism for FoxJ1 expression. Furthermore, FoxJ1, PITX2, Lef-1 and β-catenin act in concert to activate the FoxJ1 promoter. The PITX2 T68P ARS mutant protein physically interacts with FoxJ1; however, it cannot activate the FoxJ1 promoter. These data indicate a mechanism for the activity of the ARS mutant proteins in specific cell types and provides a basis for craniofacial/ tooth anomalies observed in these patients. These data reveal novel transcriptional mechanisms of FoxJ1 and demonstrate a new role of FoxJ1 in oro-facial morphogenesis.
PMCID: PMC2733810  PMID: 18723525
14.  Development of emergency response planning guidelines (ERPGs) 
Journal of Medical Toxicology  2008;4(2):127-131.
Emergency Response Planning Guidelines (ERPGs) are airborne concentrations of chemicals that have been evaluated for three levels of emergency response. These are a nuisance level, and level that would affect egress from an exposure or a level that is near, but below a life threatening concentration. The ERPG is a volunteer committee of the American Industrial Hygiene Association that is comprised of industrial hygienists and toxicologists. This paper describes the history of emergency response guidelines and process of the evaluation.
PMCID: PMC3550139  PMID: 18570174
ERPG; AEGL; emergency response
15.  Using Phylogenetic, Functional and Trait Diversity to Understand Patterns of Plant Community Productivity 
PLoS ONE  2009;4(5):e5695.
Two decades of research showing that increasing plant diversity results in greater community productivity has been predicated on greater functional diversity allowing access to more of the total available resources. Thus, understanding phenotypic attributes that allow species to partition resources is fundamentally important to explaining diversity-productivity relationships.
Methodology/Principal Findings
Here we use data from a long-term experiment (Cedar Creek, MN) and compare the extent to which productivity is explained by seven types of community metrics of functional variation: 1) species richness, 2) variation in 10 individual traits, 3) functional group richness, 4) a distance-based measure of functional diversity, 5) a hierarchical multivariate clustering method, 6) a nonmetric multidimensional scaling approach, and 7) a phylogenetic diversity measure, summing phylogenetic branch lengths connecting community members together and may be a surrogate for ecological differences. Although most of these diversity measures provided significant explanations of variation in productivity, the presence of a nitrogen fixer and phylogenetic diversity were the two best explanatory variables. Further, a statistical model that included the presence of a nitrogen fixer, seed weight and phylogenetic diversity was a better explanation of community productivity than other models.
Evolutionary relationships among species appear to explain patterns of grassland productivity. Further, these results reveal that functional differences among species involve a complex suite of traits and that perhaps phylogenetic relationships provide a better measure of the diversity among species that contributes to productivity than individual or small groups of traits.
PMCID: PMC2682649  PMID: 19479086
16.  A kinase-dead knock-in mutation in mTOR leads to early embryonic lethality and is dispensable for the immune system in heterozygous mice 
BMC Immunology  2009;10:28.
The mammalian target of rapamycin protein (mTOR) is an evolutionarily conserved kinase that regulates protein synthesis, cell cycle progression and proliferation in response to various environmental cues. As a critical downstream mediator of PI3K signaling, mTOR is important for lymphocyte development and function of mature T and B-cells. Most studies of mTOR in immune responses have relied on the use of pharmacological inhibitors, such as rapamycin. Rapamycin-FKBP12 complex exerts its immunosuppressive and anti-proliferative effect by binding outside the kinase domain of mTOR, and subsequently inhibiting downstream mTOR signaling.
To determine the requirement for mTOR kinase activity in the immune system function, we generated knock-in mice carrying a mutation (D2338) in the catalytic domain of mTOR. While homozygous mTOR kd/kd embryos died before embryonic day 6.5, heterozygous mTOR+/kd mice appeared entirely normal and are fertile. mTOR +/kd mice exhibited normal T and B cell development and unaltered proliferative responses of splenocytes to IL-2 and TCR/CD28. In addition, heterozygousity for the mTOR kinase-dead allele did not sensitize T cells to rapamycin in a CD3-mediated proliferation assay. Unexpectedly, mTOR kinase activity towards its substrate 4E-BP1 was not decreased in hearts and livers from heterozygous animals.
Altogether, our findings indicate that mTOR kinase activity is indispensable for the early development of mouse embryos. Moreover, a single wild type mTOR allele is sufficient to maintain normal postnatal growth and lymphocyte development and proliferation.
PMCID: PMC2698930  PMID: 19457267
17.  The use of magical plants by curanderos in the Ecuador highlands 
Although the use of plants for treating supernaturally caused illnesses (e.g., soul loss, evil wind, witchcraft) has been documented in the Ecuador highlands, so-called magical plants have received much less focused attention than plants used for treating naturalistic disorders. Drawing on interviews done in 2002 and 2003 with 116 curanderos residing in the Ecuador highlands, this paper examines the characteristics of plants identified as magical, how they are used, and how the study of magical plants provides insights into the mindscape of residents of the highlands.
PMCID: PMC2642781  PMID: 19161618
Changes in the fibrinolytic system that occur after cardiac transplantation (CTx) and the factors which influence such changes are poorly described, yet may be ultimately important in determining the varying morphologic features of transplant related coronary artery disease (Tx CAD). Baselines demographic, as well as serial clinical information and plasma fibrinolytic levels were prospectively recorded preCTx and multiple times postCTx in 110 de novo cardiac transplant recipients. We noted a biphasic change in fibrinolytic activity over the first year of CTx with an early immediate decline in PAI-1 activity (p > 0.001) matched with stable PAP (plasmin) activity corresponding to an “enhanced” fibrinolytic state early post CTx followed by a significant increase at 6 months (p = 0.004) and 1 year (p < 0.001) in PAI-1 activity concomitant with a significant decline in PAP after 3 months (p = 0.005 at 3 months, p < 0.001 at 6 months, and p < 0.001 at 1 year) corresponding to an “impaired” fibrinolytic state late post CTx. This biphasic nature of the fibrinolytic system could account for the varying morphologic features of Tx CAD.
PMCID: PMC1950728  PMID: 17449419
Cardiac Transplantation; Chronic Rejection; Fibrinolysis; Clinical Transplantation; Allograft Coronary Disease
19.  Molecular phylogeny and evolution of morphology in the social amoebas 
Science (New York, N.Y.)  2006;314(5799):661-663.
The social amoebas (Dictyostelia) display conditional multicellularity in a wide variety of forms. Despite widespread interest in Dictyostelium discoideum as a model system, almost no molecular data exist from the rest of the group. We have constructed the first molecular phylogeny of the Dictyostelia with parallel small subunit ribosomal RNA and α-tubulin datasets, and we found that dictyostelid taxonomy requires complete revision. A mapping of characters onto the phylogeny shows that the dominant trend in dictyostelid evolution is increased size and cell-type specialization of fruiting structures, with some complex morphologies evolving several times independently. Thus, the latter may be controlled by only a few genes, making their underlying mechanisms relatively easy to unravel.
PMCID: PMC2173941  PMID: 17068267
21.  Cross-scale ecological dynamics and microbial size spectra in marine ecosystems. 
Evaluating the component features of 'scaling' planktonic size spectra, commonly observed in marine ecosystems, is crucial for understanding the ecological and evolutionary processes from which they emerge. Here, we develop a theoretical framework that describes such spectra in terms of the size distributions of individual species, and test it against actual datasets of microbial size spectra from the Atlantic Ocean. We describe characteristics of size probability distributions of component species that are sufficient to support the observational evidence and infer that, when a power law describes the community size spectrum (thus suggesting critical self-organization of microbial ecosystem structure and function), a related power law links the total number of individuals of a given species to its mean size.
PMCID: PMC1691128  PMID: 12396505
22.  Regions and Activities of Simian Virus 40 T Antigen That Cooperate with an Activated ras Oncogene in Transforming Primary Rat Embryo Fibroblasts 
Journal of Virology  2002;76(7):3145-3157.
Prolonged expression of a ras oncogene in primary cells accelerates the natural process of senescence. This ras-induced permanent growth arrest is bypassed in cells expressing the simian virus 40 large T antigen. Previously we showed that two regions of T antigen, a region consisting of the N-terminal 147 amino acids and a region consisting of amino acids 251 to 708 (T251-708), independently overcome ras-induced senescence. Coexpression of either T-antigen fragment and Ras results in the appearance of dense foci of transformed cells. Using a series of mutants that produce shorter T-antigen fragments, we show that the C-terminal limit of the N-terminal T-antigen fragment that cooperates with Ras lies between amino acids 83 and 121. The N-terminal limit of the C-terminal T-antigen fragment lies between amino acids 252 and 271. In addition, we present evidence that cooperation between the N-terminal fragment and Ras depends upon an intact T-antigen J domain and the ability of the T antigen to bind and inactivate the growth-suppressive effect of the tumor suppressor Rb. Introduction of specific amino acid substitutions surrounding residue 400 into T251-708 prevented the fragment from cooperating with Ras. T251-708 proteins with these same substitutions inhibited the transcriptional transactivating potential of p53 as effectively as did the wild-type protein. Thus, at least one activity contained within T251-708, other than inactivating p53 as a transcriptional transactivator, is likely to be required to bypass Ras-induced senescence.
PMCID: PMC136032  PMID: 11884539
23.  Transactivation of a Ribosomal Gene by Simian Virus 40 Large-T Antigen Requires at Least Three Activities of the Protein 
Journal of Virology  1999;73(1):214-224.
Simian virus 40 large-T antigen transactivates the ribosomal genes which are transcribed by RNA polymerase (pol I), as well as genes that are dependent on either pol II or pol III. This report identifies regions and activities of T antigen that are required to transactivate a pol I-dependent rat ribosomal gene promoter. By using the rat ribosomal gene (rDNA) promoter linked to a chloramphenicol acetyltransferase gene, we show that at least three separable T-antigen regions are necessary to achieve wild-type levels of transactivation of rDNA in transiently transfected monkey cells. One activity depends on the region of T antigen shared with small-t antigen (T/t common region). A second activity maps to amino acids 109 to 626 and is highly sensitive to mutational inactivation. Complementation analyses suggest that at least one activity in this region is independent of and must be in cis with the activity within the T/t common region. In addition, a functional nuclear localization signal is required for maximal T-antigen-mediated transactivation of rat rDNA. The three activities work in concert to override cellular species-specific controls and transactivate the rat ribosomal gene promoter. Finally, we provide evidence that although the tumor suppressor protein Rb has been shown to repress a pol I-dependent promoter, transactivation of the rat rDNA promoter does not depend on T antigen’s ability to bind the tumor suppressor product Rb.
PMCID: PMC103825  PMID: 9847324
24.  Interleukin (IL)-1 Receptor–associated Kinase (IRAK) Requirement for Optimal Induction of Multiple IL-1 Signaling Pathways and IL-6 Production  
The Journal of Experimental Medicine  1998;187(12):2073-2079.
Interleukin (IL)-1 is a proinflammatory cytokine with pleiotropic effects in inflammation. IL-1 binding to its receptor triggers a cascade of signaling events, including activation of the stress-activated mitogen-activated protein (MAP) kinases, c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase, as well as transcription factor nuclear factor κB (NF-κB). IL-1 signaling results in cellular responses through induction of inflammatory gene products such as IL-6. One of the earliest events in IL-1 signaling is the rapid interaction of IL-1 receptor–associated kinases, IRAK and IRAK-2, with the receptor complex. The relative roles of IRAK and IRAK-2 in IL-1 signaling pathways and subsequent cellular responses have not been previously determined. To evaluate the importance of IRAK in IL-1 signaling, IRAK-deficient mouse fibroblast cells were prepared and studied. Here we report that IL-1–mediated activation of JNK, p38, and NF-κB were all reduced in embryonic fibroblasts deficient in IRAK expression. In addition, IL-6 production in response to IL-1 was also dramatically reduced in IRAK-deficient embryonic fibroblasts and in skin fibroblasts prepared from IRAK-deficient mice. Our results demonstrate that IRAK plays an essential proximal role in coordinating multiple IL-1 signaling pathways for optimal induction of cellular responses.
PMCID: PMC2212370  PMID: 9625767
IRAK; IL-1; JNK; p38; NF-κB
25.  Simian virus 40 large T antigen contains two independent activities that cooperate with a ras oncogene to transform rat embryo fibroblasts. 
Journal of Virology  1995;69(2):923-934.
The simian virus 40 large T antigen immortalizes growing primary cells in culture. In addition, this viral oncoprotein cooperates with an activated ras protein to produce dense foci on monolayers of rat embryo fibroblasts (REF). The relationship between independent immortalization and cooperative transformation with ras has not been defined. Previously, two regions of T antigen were shown to contain immortalization activities. An N-terminal fragment consisting of amino acids 1 to 147 immortalizes rodent cells (L. Sompayrac and K. J. Danna, Virology 181:412-415, 1991). Loss-of-function analysis indicated that immortalization depended on integrity of the T-antigen segments containing amino acids 351 to 450 and 533 to 626 (T. D. Kierstead and M. J. Tevethia, J. Virol. 67:1817-1829, 1993). The experiments described here were directed toward determining whether these same T-antigen regions were sufficient for cooperation with ras. Initially, constructs that produce T antigens containing amino acids 176 to 708 (T176-708) or 1 to 147 were tested in a ras cooperation assay. Both polypeptides cooperated with ras to produce dense foci on monolayers of primary REF. These results showed that T antigen contains two separate ras cooperation activities. In order to determine the N-terminal limit of the ras cooperation activity contained within the T176-708 polypeptide, a series of constructs designed to produce fusion proteins containing T-antigen segments beginning at residues 251, 301, 337, 351, 371, 401, 451, 501, 551, 601, and 651 was generated. Each of these constructs was tested for the capacity to cooperate with ras to produce dense foci on REF monolayers. The results indicated that a polypeptide containing T-antigen amino acids 251 to 708 (T251-708) was sufficient to cooperate with ras, whereas the more extensively truncated products were not. The abilities of the N-terminally truncated T antigens to bind p53 were examined in p53-deficient cells infected with a recombinant vaccinia virus expressing a phenotypically wild-type mouse p53. The results showed that polypeptides containing T-antigen amino acids 251 to 708, 301 to 708, 337 to 708, or 351 to 708 retained p53-binding capacity. The introduction into the T251-708 polypeptide of deletions that either prevented p53 binding (dl434-444) or did not prevent p53 binding (dl400) abrogated ras cooperation. These results indicated that although p53 binding may be necessary for ras cooperation, an additional, as-yet-undefined activity contained within the T251-708 polypeptide is needed.
PMCID: PMC188661  PMID: 7815561

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