Emended description of porcine [Pasteurella] aerogenes, [Pasteurella] mairii and [Actinobacillus] rossii

The aim of this study was to improve the definition and identification of a group of veterinarily important bacteria referred to as the [Pasteurella] aerogenes-[Pasteurella] mairii-[Actinobacillus] rossii complex. These organisms have mainly been isolated from the reproductive and intestinal tracts of pigs and in most cases have been considered as opportunistic pathogens. A collection of 87 strains were characterized by phenotypic analysis from which 41 strains were selected for 16S rRNA gene sequence comparison, out of which 23 have been sequenced in the present study. One group of 21 strains phenotyped as biovars 1, 3-5, 9-11, 19 and 25-27, including the type strain of [P.] aerogenes, showed 16S rRNA gene sequence similarities of 99.6 % or higher; another group of 18 strains including biovars 2, 6-8, 12-15, 21, 23, 24 and 26A and the type strain of [A.] rossii showed 97.8 % or higher 16S rRNA gene sequence similarity. Between the two groups, 93.8-95.7 % 16S rRNA gene sequence similarity was observed. Strains of [P.] mairii showed 99.5 % similarity, with 95.5-97.2 and 93.8-95.5 % similarity to strains of [P.] aerogenes and [A.] rossii, respectively. Four strains could not be classified with any of these groups and belonged to other members of Pasteurellaceae. Comparisons were also made to DNA-DNA hybridization data. Biovars 1, 9, 10, 11 and 19, including the type strain of [P.] aerogenes, linked at 70 % DNA reassociation, whereas strains identified as biovars 2, 6, 7, 8, 12, 15 and 21 of [P.] aerogenes linked at 81 %. The latter group most likely represents [A.] rossii based on the 16S rRNA gene sequence comparisons. DNA reassociation between the [P.] aerogenes and [A.] rossii groups was at most 37 %, whereas 47 % was the highest DNA reassociation found between [P.] aerogenes and [P.] mairii. The study showed that [P.] aerogenes, [P.] mairii and [A.] rossii can not be easily separated and may consequently be misidentified based on current knowledge of their phenotypic characteristics. In addition, these taxa are difficult to separate from other taxa of the Pasteurellaceae. A revised scheme for separation based upon phenotypic characters is suggested for the three species [P.] aerogenes emend., [P.] mairii emend. and [A.] rossii emend., with the respective type strains ATCC 27883T, NCTC 10699T and ATCC 27072T.

MQN-Mapplet: Visualization of Chemical Space with Interactive Maps of DrugBank, ChEMBL, PubChem, GDB-11, and GDB-13

The MQN-mapplet is a Java application giving access to the structure of small molecules in large databases via color-coded maps of their chemical space. These maps are projections from a 42-dimensional property space defined by 42 integer value descriptors called molecular quantum numbers (MQN), which count different categories of atoms, bonds, polar groups, and topological features and categorize molecules by size, rigidity, and polarity. Despite its simplicity, MQN-space is relevant to biological activities. The MQN-mapplet allows localization of any molecule on the color-coded images, visualization of the molecules, and identification of analogs as neighbors on the MQN-map or in the original 42-dimensional MQN-space. No query molecule is necessary to start the exploration, which may be particularly attractive for nonchemists. To our knowledge, this type of interactive exploration tool is unprecedented for very large databases such as PubChem and GDB-13 (almost one billion molecules). The application is freely available for download at www.gdb.unibe.ch.

Molecular epidemiology and antibiotic susceptibility of livestock Brucella melitensis isolates from Naryn Oblast, Kyrgyzstan.

The incidence of human brucellosis in Kyrgyzstan has been increasing in the last years and was identified as a priority disease needing most urgent control measures in the livestock population. The latest species identification of Brucella isolates in Kyrgyzstan was carried out in the 1960s and investigated the circulation of Brucella abortus, B. melitensis, B. ovis, and B. suis. However, supporting data and documentation of that experience are lacking. Therefore, typing of Brucella spp. and identification of the most important host species are necessary for the understanding of the main transmission routes and to adopt an effective brucellosis control policy in Kyrgyzstan. Overall, 17 B. melitensis strains from aborted fetuses of sheep and cattle isolated in the province of Naryn were studied. All strains were susceptible to trimethoprim-sulfamethoxazole, gentamicin, rifampin, ofloxacin, streptomycin, doxycycline, and ciprofloxacin. Multilocus variable number tandem repeat analysis showed low genetic diversity. Kyrgyz strains seem to be genetically associated with the Eastern Mediterranean group of the Brucella global phylogeny. We identified and confirmed transmission of B. melitensis to cattle and a close genetic relationship between B. melitensis strains isolated from sheep sharing the same pasture.

Using state variables to model the response of tumour cells to radiation and heat: a novel multi-hit-repair approach

In order to overcome the limitations of the linear-quadratic model and include synergistic effects of heat and radiation, a novel radiobiological model is proposed. The model is based on a chain of cell populations which are characterized by the number of radiation induced damages (hits). Cells can shift downward along the chain by collecting hits and upward by a repair process. The repair process is governed by a repair probability which depends upon state variables used for a simplistic description of the impact of heat and radiation upon repair proteins. Based on the parameters used, populations up to 4-5 hits are relevant for the calculation of the survival. The model describes intuitively the mathematical behaviour of apoptotic and nonapoptotic cell death. Linear-quadratic-linear behaviour of the logarithmic cell survival, fractionation, and (with one exception) the dose rate dependencies are described correctly. The model covers the time gap dependence of the synergistic cell killing due to combined application of heat and radiation, but further validation of the proposed approach based on experimental data is needed. However, the model offers a work bench for testing different biological concepts of damage induction, repair, and statistical approaches for calculating the variables of state.

Patterns and controls of sediment production, transfer and yield in the Illgraben

Quantification of the volumes of sediment removed by rock–slope failure and debris flows and identification of their coupling and controls are pertinent to understanding mountain basin sediment yield and landscape evolution. This study captures a multi-decadal period of hillslope erosion and channel change following an extreme rock avalanche in 1961 in the Illgraben, a catchment prone to debris flows in the Swiss Alps. We analyzed photogrammetrically-derived datasets of hillslope and channel erosion and deposition along with climatic and seismic variables for a 43 year period from 1963 to 2005. Based on these analyses we identify and discuss (1) patterns of hillslope production, channel transfer and catchment sediment yield, (2) their dominant interactions with climatic and seismic variables, and (3) the nature of hillslope–channel coupling and implications for sediment yield and landscape evolution in this mountain basin. Our results show an increase in the mean hillslope erosion rate in the 1980s from 0.24 ± 0.01 m yr− 1 to 0.42 ± 0.03 m yr− 1 that coincided with a significant increase in air temperature and decrease in snow cover depth and duration, which we presume led to an increase in the exposure of the slopes to thermal weathering processes. The combination of highly fractured slopes close to the threshold angle for failure, and multiple potential triggering mechanisms, means that it is difficult to identify an individual control on slope failure. On the other hand, the rate of channel change was strongly related to variables influencing runoff. A period of particularly high channel erosion rate of 0.74 ± 0.02 m yr− 1 (1992–1998) coincided with an increase in the frequency and magnitude of intense rainfall events. Hillslope erosion exceeded channel erosion on average, indicative of a downslope-directed coupling relationship between hillslope and channel, and demonstrating the first order control of rock–slope failure on catchment sediment yield and landscape evolution.

DEVELOPMENTAL AND CELLULAR FUNCTIONS OF DELTA-CATENIN

Catenins have diverse and powerful roles in embryogenesis, homeostasis or disease progression, as best exemplified by the well-known beta-catenin. The less studied delta-catenin likewise contains a central Armadillo-domain. In common with other p120 sub-class members, it acts in a variety of intracellular compartments and modulates cadherin stability, small GTPase activities and gene transcription. In mammals, delta-catenin exhibits neural specific expression, with its knock-out in mice correspondingly producing cognitive defects and synaptic dysfunctions. My work instead employed the amphibian, Xenopus laevis, to explore delta-catenin’s physiological functions in a distinct vertebrate system. Initial isolation and characterization indicated delta-catenin’s expression in Xenopus. Unlike the pattern observed for mammals, delta-catenin was detected in most adult Xenopus tissues, although enriched in embryonic structures of neural fate as visualized using RNA in-situ hybridization. To determine delta-catenin’s requirement in amphibian development, I employed anti-sense morpholinos to knock-down gene products, finding that delta-catenin depletion results in developmental defects in gastrulation, neural crest migration and kidney tubulogenesis, phenotypes that were specific based upon rescue experiments. In biochemical and cellular assays, delta-catenin knock-down reduced cadherin levels and cell adhesion, and impaired activation of RhoA and Rac1, small GTPases that regulate actin dynamics and morphogenetic movements. Indeed, exogenous C-cadherin, or dominant-negative RhoA or dominant-active Rac1, significantly rescued delta-catenin depletion. Thus, my results indicate delta-catenin’s essential roles in Xenopus development, with contributing functional links to cadherins and Rho family small G proteins. In examining delta-catenin’s nuclear roles, I identified delta-catenin as an interacting partner and substrate of the caspase-3 protease, which plays critical roles in apoptotic as well as non-apoptotic processes. Delta-catenin’s interaction with and sensitivity to caspase-3 was confirmed using assays involving its cleavage in vitro, as well as within Xenopus apoptotic extracts or mammalian cell lines. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo-repeat 6 of delta-catenin, was identified through peptide sequencing. Cleavage thus generates an amino- (1-816) and carboxyl-terminal (817-1314) fragment each containing about half of the central Armadillo-domain. I found that cleavage of delta-catenin both abolishes its association with cadherins, and impairs its ability to modulate small GTPases. Interestingly, the carboxyl-terminal fragment (817-1314) possesses a conserved putative nuclear localization signal that I found is needed to facilitate delta-catenin’s nuclear targeting. To probe for novel nuclear roles of delta-catenin, I performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating its interaction with an uncharacterized KRAB family zinc finger protein I named ZIFCAT. My results indicate that ZIFCAT is nuclear, and suggest that it may associate with DNA as a transcriptional repressor. I further determined that other p120 sub-class catenins are similarly cleaved by caspase-3, and likewise bind ZIFCAT. These findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120 sub-family members, facilitating the coordinate modulation of cadherins, small GTPases and nuclear functions. Together, my work suggested delta-catenin’s essential roles in Xenopus development, and has revealed its novel contributions to cell junctions (via cadherins), cytoskeleton (via small G proteins), and nucleus (via ZIFCAT). Future questions include the larger role and gene targets of delta-catenin in nucleus, and identification of upstream signaling events controlling delta-catenin’s activities in development or disease progression.

Mitochondrial outer membrane proteome of Trypanosoma brucei reveals macromolecular transport machineries and novel factors required to maintain mitochondrial morphology

The mitochondrial outer membrane (MOM) separates the mitochondria from the cytoplasm, serving both as a barrier and as a gateway. Protein complexes — believed to be universally conserved in all eukaryotes — reside in the MOM to orchestrate and control metabolite exchange, lipid metabolism and uptake of biopolymers such as protein and RNA. African trypanosomes are the causative agent of the sleeping sickness in humans. The parasites are among the earliest diverging eukaryotes that have bona fide mitochondria capable of oxidative phosphorylation. Trypanosomes have unique mitochondrial biology that concerns their mitochondrial metabolism and their unusual mitochondrial morphology that differs to great extent between life stages. Another striking feature is the organization of the mitochondrial genome that does not encode any tRNA genes, thus all tRNAs needed for mitochondrial translation have to be imported. However, the MOM of T. brucei is essentially unchartered territory. It lacks a canonical protein import machinery and facilitation of tRNA translocation remains completely elusive. Using biochemical fractionation and label-free quantitative mass spectrometry for correlated protein abundance-profiling we were able to identify a cluster of 82 candidate proteins that can be localized to the trypanosomal MOM with high confidence. This enabled us to identify a highly unusual, potentially archaic protein import machinery that might also transport tRNAs. Moreover, two-thirds of the identified polypeptides present on the MOM have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the MOM of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of insect-stage parasites and therefore directly or indirectly are involved in the regulation of mitochondrial morphology.

Geochemical variability in distal and proximal glass from the Youngest Toba Tuff eruption

The Youngest Toba Tuff (YTT, erupted ca. 74 ka ago) is a distinctive and widespread tephra marker across south and southeast Asia. The climatic, human and environmental consequences of the YTT eruption are widely debated. Although a considerable body of geochemical data is available for this unit, there has not been a systematic study of the variability of the ash geochemistry. Intrinsic (magmatic) and extrinsic (post-depositional) chemical variations bring fundamental information regarding the petrogenesis of the magma, the distribution of the tephra and the interaction between the ash and the receiving environment. Considering the importance of the geochemistry of the YTT for stratigraphic correlations and eruptive models, it is central to the YTT debate to quantify and interpret such variations. Here we collate all published geochemical data on the YTT glass, including analyses from 68 sites described in the literature and three new samples. Two principal sources of chemical variation are investigated: (i) compositional zonation of the magma reservoir, and (ii) post-depositional alteration. Post-depositional leaching is responsible for up to ca. 11% differences in Na2O/K2O and ca. 1% differences in SiO2/Al2O3 ratios in YTT glass from marine sites. Continental tephra are 2% higher in Na2O/K2O and 3% higher in SiO2/Al2O3 respect to the marine tephra. We interpret such post-depositional glass alteration as related to seawater induced alkali migration in marine environments, or to site-specific water pH. Crystal fractionation and consequential magmatic differentiation, which produced order-of-magnitude variations in trace element concentrations reported in the literature, also produced major element differences in the YTT glass. FeO/Al2O3 ratios vary by about 50 %, which is analytically significant. These variations represent magmatic fractionation involving Fe-bearing phases. We also compared major element concentrations in YTT and Oldest Toba Tuff (OTT) ash samples, to identify potential compositional differences that could constrain the stratigraphic identity of the Morgaon ash (Western India); no differences between the OTT and YTT samples were observed.

Feasibility of cardiopulmonary exercise testing and training using a robotics-assisted tilt table in dependent-ambulatory stroke patients

BACKGROUND: We evaluated the feasibility of an augmented robotics-assisted tilt table (RATT) for incremental cardiopulmonary exercise testing (CPET) and exercise training in dependent-ambulatory stroke patients. METHODS: Stroke patients (Functional Ambulation Category ≤ 3) underwent familiarization, an incremental exercise test (IET) and a constant load test (CLT) on separate days. A RATT equipped with force sensors in the thigh cuffs, a work rate estimation algorithm and real-time visual feedback to guide the exercise work rate was used. Feasibility assessment considered technical feasibility, patient tolerability, and cardiopulmonary responsiveness. RESULTS: Eight patients (4 female) aged 58.3 ± 9.2 years (mean ± SD) were recruited and all completed the study. For IETs, peak oxygen uptake (V'O2peak), peak heart rate (HRpeak) and peak work rate (WRpeak) were 11.9 ± 4.0 ml/kg/min (45 % of predicted V'O2max), 117 ± 32 beats/min (72 % of predicted HRmax) and 22.5 ± 13.0 W, respectively. Peak ratings of perceived exertion (RPE) were on the range "hard" to "very hard". All 8 patients reached their limit of functional capacity in terms of either their cardiopulmonary or neuromuscular performance. A ventilatory threshold (VT) was identified in 7 patients and a respiratory compensation point (RCP) in 6 patients: mean V'O2 at VT and RCP was 8.9 and 10.7 ml/kg/min, respectively, which represent 75 % (VT) and 85 % (RCP) of mean V'O2peak. Incremental CPET provided sufficient information to satisfy the responsiveness criteria and identification of key outcomes in all 8 patients. For CLTs, mean steady-state V'O2 was 6.9 ml/kg/min (49 % of V'O2 reserve), mean HR was 90 beats/min (56 % of HRmax), RPEs were > 2, and all patients maintained the active work rate for 10 min: these values meet recommended intensity levels for bouts of training. CONCLUSIONS: The augmented RATT is deemed feasible for incremental cardiopulmonary exercise testing and exercise training in dependent-ambulatory stroke patients: the approach was found to be technically implementable, acceptable to the patients, and it showed substantial cardiopulmonary responsiveness. This work has clinical implications for patients with severe disability who otherwise are not able to be tested.

Phosphatidylethanolamine and phosphatidylcholine biosynthesis by the Kennedy pathway occurs at different sites in Trypanosoma brucei

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are among the most abundant phospholipids in biological membranes. In many eukaryotes, the CDP-ethanolamine and CDP-choline branches of the Kennedy pathway represent major and often essential routes for the production of PE and PC, with ethanolamine and choline/ethanolamine phosphotransferases (EPT and CEPT, respectively) catalysing the last reactions in the respective pathways. Although the site of PE and PC synthesis is commonly known to be the endoplasmic reticulum (ER), detailed information on the localization of the different phosphotransferases is lacking. In the unicellular parasite, Trypanosoma brucei, both branches of the Kennedy pathway are essential for cell growth in culture. We have previously reported that T. brucei EPT (TbEPT) catalyses the production of ether-type PE molecular species while T. brucei CEPT (TbCEPT) synthesizes diacyl-type PE and PC molecular species. We now show that the two enzymes localize to different sub-compartments of the ER. By expressing a series of tagged forms of the two enzymes in T. brucei parasites, in combination with sub-cellular fractionation and enzyme activity measurements, TbEPT was found exclusively in the perinuclear ER, a distinct area located close to but distinct from the nuclear membrane. In contrast, TbCEPT was detected in the bulk ER.

Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

Firearm legislation and firearm mortality in the USA: a cross-sectional, state-level study.

BACKGROUND In an effort to reduce firearm mortality rates in the USA, US states have enacted a range of firearm laws to either strengthen or deregulate the existing main federal gun control law, the Brady Law. We set out to determine the independent association of different firearm laws with overall firearm mortality, homicide firearm mortality, and suicide firearm mortality across all US states. We also projected the potential reduction of firearm mortality if the three most strongly associated firearm laws were enacted at the federal level. METHODS We constructed a cross-sectional, state-level dataset from Nov 1, 2014, to May 15, 2015, using counts of firearm-related deaths in each US state for the years 2008-10 (stratified by intent [homicide and suicide]) from the US Centers for Disease Control and Prevention's Web-based Injury Statistics Query and Reporting System, data about 25 firearm state laws implemented in 2009, and state-specific characteristics such as firearm ownership for 2013, firearm export rates, and non-firearm homicide rates for 2009, and unemployment rates for 2010. Our primary outcome measure was overall firearm-related mortality per 100 000 people in the USA in 2010. We used Poisson regression with robust variances to derive incidence rate ratios (IRRs) and 95% CIs. FINDINGS 31 672 firearm-related deaths occurred in 2010 in the USA (10·1 per 100 000 people; mean state-specific count 631·5 [SD 629·1]). Of 25 firearm laws, nine were associated with reduced firearm mortality, nine were associated with increased firearm mortality, and seven had an inconclusive association. After adjustment for relevant covariates, the three state laws most strongly associated with reduced overall firearm mortality were universal background checks for firearm purchase (multivariable IRR 0·39 [95% CI 0·23-0·67]; p=0·001), ammunition background checks (0·18 [0·09-0·36]; p<0·0001), and identification requirement for firearms (0·16 [0·09-0·29]; p<0·0001). Projected federal-level implementation of universal background checks for firearm purchase could reduce national firearm mortality from 10·35 to 4·46 deaths per 100 000 people, background checks for ammunition purchase could reduce it to 1·99 per 100 000, and firearm identification to 1·81 per 100 000. INTERPRETATION Very few of the existing state-specific firearm laws are associated with reduced firearm mortality, and this evidence underscores the importance of focusing on relevant and effective firearms legislation. Implementation of universal background checks for the purchase of firearms or ammunition, and firearm identification nationally could substantially reduce firearm mortality in the USA. FUNDING None.

Cerebellar Pathology Does Not Impair Performance on Identification or Categorization Tasks

In comparison to the basal ganglia, prefrontal cortex, and medial temporal lobes, the cerebellum has been absent from recent research on the neural substrates of categorization and identification, two prominent tasks in the learning and memory literature. To investigate the contribution of the cerebellum to these tasks, we tested patients with cerebellar pathology (seven with bilateral degeneration, six with unilateral lesions, and two with midline damage) on rule-based and information-integration categorization tasks and an identification task. In rule-based tasks, it is assumed that participants learn the categories through an explicit reasoning process. In information-integration tasks, optimal performance requires the integration of information from multiple stimulus dimensions, and participants are typically unaware of the decision strategy. The identification task, in contrast, required participants to learn arbitrary, color-word associations. The cerebellar patients performed similar to matched controls on all three tasks and performance did not vary with the extent of cerebellar pathology. Although the interpretation of these null results requires caution, these data contribute to the current debate on cerebellar contributions to cognition by providing boundary conditions on understanding the neural substrates of categorization and identification, and help define the functional domain of the cerebellum in learning and memory.

Health care policy analysis of Medicaid's early and periodic screening, diagnosis, and treatment (EPSDT) benefit in Colorado

The purpose of this thesis is to identify "best practice" recommendations for successful implementation of the EPSDT outreach program at Memorial Health System's Hospital for Children in Colorado Springs through a policy analysis of Medicaid EPSDT services in Colorado. A successful program at Memorial will increase education and awareness of EPSDT services, enrollment, and access to and utilization of health care services for eligible children. Methodology utilized in this study included questionnaires designed for the EPSDT contract administrator and outreach coordinators/workers; analysis of current federal and state policies; and studies conducted at the federal and state level, and by various advocacy groups. The need for this analysis of EPSDT came about in part through an awareness of increasingly high numbers of children in poverty and who are uninsured. Though the percentage of children living in poverty in Colorado is slightly below the national average (see Table 2), according to data analyzed by The Annie E. Casey Foundation, the percentage of children (0-18) living in poverty in Colorado increased from 10% in 2000 to 16% in 2006, a dramatic increase of 60% surpassed by only one other state in the nation (The Annie E. Casey Foundation, 2008). By comparison, the U.S. percentage of children in poverty during the same time frame rose from 17% to 18% (The Annie E. Casey Foundation, 2008). What kind of health care services are available to this vulnerable and growing group of Coloradans, and what are the barriers that affect their enrollment in, access to and utilization of these health care services? Barriers identified included difficulty with the application process; system and process issues; a lack of providers; and a lack of awareness and knowledge of EPSDT. Fiscal restraints and legislation at the federal and state level are also barriers to increasing enrollment and access to services. Outreach services are a critical component of providing EPSDT services, and there were several recommendations regarding outreach and case management that will benefit the program in the future. Through this analysis and identification of a broad range of barriers, a clearer picture emerged of current challenges within the EPSDT program as well as a broad range of strategies and recommendations to address these challenges. Through increased education and advocacy for EPSDT and the services it encompasses; stronger collaboration and cooperation between all groups involved, including providing a Medical Home for all eligible children; and new legislation putting more money and focus on comprehensive health care for low-income uninsured children; enrollment, access to and utilization of developmentally appropriate and quality health care services can be achieved. ^

PRKCa: Identification of a Novel Downstream Target of WT1

Wilms tumor is a childhood tumor of the kidney arising from the undifferentiated metanephric mesenchyme. Tumorigenesis is attributed to a number of genetic and epigenetic alterations. In 20% of Wilms tumors, Wilms tumor gene 1 (WT1) undergoes inactivating homozygous mutations causing loss of function of the zinc finger transcription factor it encodes. It is hypothesized that mutations in WT1 result in dysregulation of downstream target genes, leading to aberrant kidney development and/or Wilms tumor. These downstream target genes are largely unknown, and identification is important for further understanding Wilms tumor development. Heatmap data of human Wilms tumor protein expression, generated by reverse phase protein assay analysis (RPPA), show significant correlation between WT1 mutation status and low PRKCα expression (p= 0.00013); additionally, p-PRKCα (S657) also shows decreased expression in these samples (p= 0.00373). These data suggest that the WT1 transcription factor regulates PRKCα expression, and that PRKCα plays a potential role in Wilms tumor tumorigenesis. We hypothesize that the WT1 transcription factor directly/indirectly regulates PRKCα and mutations occurring in WT1 lead to decreased expression of PRKCα. Prkcα and Wt1 have been shown to co-localize in E14.5 mesenchymal cells of the developing kidney. siRNA knockdown, in-vivo ablation, and tet-inducible expression of Wt1 each independently confirm regulation of Prkcα expression by Wt1 at both RNA and protein levels, and investigation into possible WT1 binding sites in PRKCα regulatory regions has identified multiple sites to be confirmed by luciferase reporter constructs. With the goal of identifying WT1 and PRKCα downstream targets, RPPA analysis of protein expression in mesenchymal cell culture, following lentiviral delivered shRNA knockdown of Wt1 and shRNA knockdown of Prkcα, will be carried out. Apart from Wilms tumor, WT1 also plays an important role in Acute Myeloid Leukemia (AML). WT1 mutation status has been implicated, controversially, as an independent poor-prognosis factor in leukemia, leading to decreased probability of overall survival, complete remission, and disease free survival. RPPA analysis of AML patient samples showed significant decreases in PRKCα/p-PRKCα protein expression in a subset of patients (Kornblau, personal communication); therefore, the possible role of WT1 and PRKCα in leukemia disease progression is an additional focus of this study. WT1 mutation analysis of diploid leukemia patient samples revealed two patients with mutations predicted to affect WT1 activity; of these two samples, only one corresponded to the low PRKCα expression cohort. Further characterization of the role of WT1 in AML, and further understanding of WT1 regulated PRKCα expression, will be gained following RPPA analysis of protein expression in HL60 leukemia cell lines with lentiviral delivered shRNA knockdown of WT1 and shRNA knockdown of PRKCα.