Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment

The protein-tyrosine phosphatase Shp2 plays an essential role in growth factor and integrin signaling, and Shp2 mutations cause developmental defects and/or malignancy. Previous work has placed Shp2 upstream of Ras. However, the mechanism of Shp2 action and its substrate(s) are poorly defined. Additional Shp2 functions downstream of, or parallel to, Ras/Erk activation also are proposed. Here, we show that Shp2 promotes Src family kinase (SFK) activation by regulating the phosphorylation of the Csk regulator PAG/Cbp, thereby controlling Csk access to SFKs. In Shp2-deficient cells, SFK inhibitory C-terminal tyrosines are hyperphosphorylated, and the tyrosyl phosphorylation of multiple SFK substrates, including Plcgamma1, is decreased. Decreased Plcgamma1 phosphorylation leads to defective Ras activation on endomembranes, and may help account for impaired Erk activation in Shp2-deficient cells. Decreased phosphorylation/activation of other SFK substrates may explain additional consequences of Shp2 deficiency, including altered cell spreading, stress fibers, focal adhesions, and motility.     

Chromosome 7 and 19 Trisomy in Cultured Human Neural Progenitor Cells

Background

Stem cell expansion and differentiation is the foundation of emerging cell therapy technologies. The potential applications of human neural progenitor cells (hNPCs) are wide ranging, but a normal cytogenetic profile is important to avoid the risk of tumor formation in clinical trials. FDA approved clinical trials are being planned and conducted for hNPC transplantation into the brain or spinal cord for various neurodegenerative disorders. Although human embryonic stem cells (hESCs) are known to show recurrent chromosomal abnormalities involving 12 and 17, no studies have revealed chromosomal abnormalities in cultured hNPCs. Therefore, we investigated frequently occurring chromosomal abnormalities in 21 independent fetal-derived hNPC lines and the possible mechanisms triggering such aberrations.

Methods and Findings

While most hNPC lines were karyotypically normal, G-band karyotyping and fluorescent in situ hybridization (FISH) analyses revealed the emergence of trisomy 7 (hNPC⁺⁷) and trisomy 19 (hNPC⁺¹⁹), in 24% and 5% of the lines, respectively. Once detected, subsequent passaging revealed emerging dominance of trisomy hNPCs. DNA microarray and immunoblotting analyses demonstrate epidermal growth factor receptor (EGFR) overexpression in hNPC⁺⁷ and hNPC⁺¹⁹ cells. We observed greater levels of telomerase (hTERT), increased proliferation (Ki67), survival (TUNEL), and neurogenesis (β_III-tubulin) in hNPC⁺⁷ and hNPC⁺¹⁹, using respective immunocytochemical markers. However, the trisomy lines underwent replicative senescence after 50–60 population doublings and never showed neoplastic changes. Although hNPC⁺⁷ and hNPC⁺¹⁹ survived better after xenotransplantation into the rat striatum, they did not form malignant tumors. Finally, EGF deprivation triggered a selection of trisomy 7 cells in a diploid hNPC line.

Conclusions

We report that hNPCs are susceptible to accumulation of chromosome 7 and 19 trisomy in long-term cell culture. These results suggest that micro-environmental cues are powerful factors in the selection of specific hNPC aneuploidies, with trisomy of chromosome 7 being the most common. Given that a number of stem cell based clinical trials are being conducted or planned in USA and a recent report in PLoS Medicine showing the dangers of grafting an inordinate number of cells, these data substantiate the need for careful cytogenetic evaluation of hNPCs (fetal or hESC-derived) before their use in clinical or basic science applications.     

Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival

Borrelia burgdorferi (Bb) is the causative agent of Lyme disease in the United States, a disease that can result in carditis, and chronic and debilitating arthritis and/or neurologic symptoms if left untreated. Bb survives in the midgut of the Ixodes scapularis tick, or within tissues of immunocompetent hosts. In the early stages of infection, the bacteria are present in the bloodstream where they must resist clearance by the innate immune system of the host. We have found a novel role for outer surface protein C (OspC) from B. burgdorferi and B. garinii in interactions with the complement component C4b and bloodstream survival in vivo. Our data show that OspC inhibits the classical and lectin complement pathways and competes with complement protein C2 for C4b binding. Resistance to complement is important for maintenance of the lifecycle of Bb, enabling survival of the pathogen within the host as well as in the midgut of a feeding tick when ospC expression is induced.     

Reversal of response to artificial selection on body size in a wild passerine

A general assumption in quantitative genetics is the existence of an intermediate phenotype with higher mean individual fitness in the average environment than more extreme phenotypes. Here, we investigate the evolvability and presence of such a phenotype in wild bird populations from an eleven‐year experiment with four years of artificial selection for long and short tarsus length, a proxy for body size. The experiment resulted in strong selection in the imposed directions. However, artificial selection was counteracted by reduced production of recruits in offspring of artificially selected parents. This resulted in weak natural selection against extreme trait values. Significant responses to artificial selection were observed at both the phenotypic and genetic level, followed by a significant return toward preexperimental means. During artificial selection, the annual observed phenotypic response closely followed the predicted response from quantitative genetic theory ( = 0.96, = 0.56). The rapid return to preexperimental means was induced by three interacting mechanisms: selection for an intermediate phenotype, immigration, and recombination between selected and unselected individuals. The results of this study demonstrates the evolvability of phenotypes and that selection may favor an intermediate phenotype in wild populations.     

Snowflake Vitreoretinal Degeneration (SVD) Mutation R162W Provides New Insights into Kir7.1 Ion Channel Structure and Function

Snowflake Vitreoretinal Degeneration (SVD) is associated with the R162W mutation of the Kir7.1 inwardly-rectifying potassium channel. Kir7.1 is found at the apical membrane of Retinal Pigment Epithelial (RPE) cells, adjacent to the photoreceptor neurons. The SVD phenotype ranges from RPE degeneration to an abnormal b-wave to a liquid vitreous. We sought to determine how this mutation alters the structure and function of the human Kir7.1 channel. In this study, we expressed a Kir7.1 construct with the R162W mutation in CHO cells to evaluate function of the ion channel. Compared to the wild-type protein, the mutant protein exhibited a non-functional Kir channel that resulted in depolarization of the resting membrane potential. Upon co-expression with wild-type Kir7.1, R162W mutant showed a reduction of I_Kir7.1 and positive shift in ‘0’ current potential. Homology modeling based on the structure of a bacterial Kir channel protein suggested that the effect of R162W mutation is a result of loss of hydrogen bonding by the regulatory lipid binding domain of the cytoplasmic structure.     

Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation

Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin–fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular adhesion. These data indicate that Wnt/Glypican4/Frizzled signaling regulates ECM assembly through effects on cadherin-mediated cell cohesion. Together, our results demonstrate that zebrafish Vangl2/Prickle1a and non-canonical Wnt/Frizzled signaling have opposing effects on ECM organization underlying PCP and gastrulation cell movements.     

Phosphorylation of glucose by a guanosine-5′-triphosphate (GTP)-dependent glucokinase in Fibrobacter succinogenes subsp. succinogenes S85

Cell extracts of Fibrobacter succinogenes subsp. succinogenes S85 phosphorylated glucose with a GTP-dependent glucokinase. The enzyme showed little activity with ATP (12% of that with GTP). Of other phosphate donors tested, only dGTP and ITP gave high glucokinase activities. Dialyzed extracts required Mg⁺² and K⁺ for maximal activity. In potassium phosphate buffer, glucokinase showed maximum activity at pH 7.5 with glucose-6-phosphate dehydrogenase as the coupling enzyme. In this assay, glucokinase was active with glucose (100%), 2-deoxy-d-glucose (40%), and mannose (20%). Partially purified glucokinase had a molecular weight of 82,000 and a pl of 4.82. Double-reciprocal plots of substrate concentration versus velocity were linear and the enzyme had apparent K_m values of 55 M for glucose and 72 M for GTP. Dialyzed cell extracts of Fibrobacter intestinalis C1A also contained a GTP-dependent glucokinase that showed little activity with ATP. Potassium also stimulated the activity of this enzyme. These results suggest that this unusual glucokinase may be characteristic of the genus Fibrobacter.Abbreviations CHES cyclohexylaminoethanesulfonic acid - GK glucokinase - PEP phosphoenolpyruvate Published with the approval of the Director of the North Dakota Agricultural Experiment Station as journal article no. 2186     

Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington’s Disease

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Severe and rapid population declines in exotic birds

A particularly vexing phenomenon within invasion ecology is the occurrence of spontaneous collapses within seemingly well-established exotic populations. Here, we assess the frequency of collapses among 68 exotic bird populations established in Hawaii, Puerto Rico, Los Angeles and Miami. Following other published definitions, we define a ‘collapse’ as a decline in abundance of ≥90 % within ≤10 years that lasts for at least 3 years. We show that 44 of the 68 exotic bird populations have exhibited declines at some point within their time series. Sixteen of the populations declined sufficiently to be defined as collapsed. It took on average 3.8 ± 1.8 years for populations to decline into a collapsed state, and this state persisted on average for 7.1 ± 6.3 years across (collapsed) populations. We compared the severity and duration of declines across all 44 declining populations according to taxonomic Order and geographic region. Neither variable explained substantial variation in the metrics of collapse. Our results indicate that severe, rapid, and persistent population declines may be common among exotic populations. We suggest that incorporating the probability and persistence of collapses into management decisions can inform efforts to enact control or eradication measures. We also suggest that applying our approach to other taxa and locations is crucial for improving our understanding of when and where collapses are likely to occur.     

Fusing Data Mining,Machine Learning and Traditional Statistics to Detect Biomarkers Associated with Depression

Background

Atheoretical large-scale data mining techniques using machine learning algorithms have promise in the analysis of large epidemiological datasets. This study illustrates the use of a hybrid methodology for variable selection that took account of missing data and complex survey design to identify key biomarkers associated with depression from a large epidemiological study.

Methods

The study used a three-step methodology amalgamating multiple imputation, a machine learning boosted regression algorithm and logistic regression, to identify key biomarkers associated with depression in the National Health and Nutrition Examination Study (2009–2010). Depression was measured using the Patient Health Questionnaire-9 and 67 biomarkers were analysed. Covariates in this study included gender, age, race, smoking, food security, Poverty Income Ratio, Body Mass Index, physical activity, alcohol use, medical conditions and medications. The final imputed weighted multiple logistic regression model included possible confounders and moderators.

Results

After the creation of 20 imputation data sets from multiple chained regression sequences, machine learning boosted regression initially identified 21 biomarkers associated with depression. Using traditional logistic regression methods, including controlling for possible confounders and moderators, a final set of three biomarkers were selected. The final three biomarkers from the novel hybrid variable selection methodology were red cell distribution width (OR 1.15; 95% CI 1.01, 1.30), serum glucose (OR 1.01; 95% CI 1.00, 1.01) and total bilirubin (OR 0.12; 95% CI 0.05, 0.28). Significant interactions were found between total bilirubin with Mexican American/Hispanic group (p = 0.016), and current smokers (p<0.001).

Conclusion

The systematic use of a hybrid methodology for variable selection, fusing data mining techniques using a machine learning algorithm with traditional statistical modelling, accounted for missing data and complex survey sampling methodology and was demonstrated to be a useful tool for detecting three biomarkers associated with depression for future hypothesis generation: red cell distribution width, serum glucose and total bilirubin.