Novel correlative analysis identifies multiple genomic variations impacting ASD with macrocephaly.

Autism spectrum disorders (ASD) display both phenotypic and genetic heterogeneity, impeding the understanding of ASD and development of effective means of diagnosis and potential treatments. Genes affected by genomic variations for ASD converge in dozens of gene ontologies (GOs), but the relationship between the variations at the GO level have not been well elucidated. In the current study, multiple types of genomic variations were mapped to GOs and correlations among GOs were measured in ASD and control samples. Several ASD-unique GO correlations were found, suggesting the importance of co-occurrence of genomic variations in genes from different functional categories in ASD etiology. Combined with experimental data, several variations related to WNT signaling, neuron development, synapse morphology/function and organ morphogenesis were found to be important for ASD with macrocephaly, and novel co-occurrence patterns of them in ASD patients were found. Furthermore, we applied this gene ontology correlation analysis method to find genomic variations that contribute to ASD etiology in combination with changes in gene expression and transcription factor binding, providing novel insights into ASD with macrocephaly and a new methodology for the analysis of genomic variation.

VISTA is an acidic pH-selective ligand for PSGL-1.

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.

Deletion of the Dishevelled family of genes disrupts anterior-posterior axis specification and selectively prevents mesoderm differentiation.

The Dishevelled proteins transduce both canonical Wnt/ß-catenin and non-canonical Wnt/planar cell polarity (PCP) signaling pathways to regulate many key developmental processes during embryogenesis. Here, we disrupt both canonical and non-canonical Wnt pathways by targeting the entire Dishevelled family of genes (Dvl1, Dvl2, and Dvl3) to investigate their functional roles in the early embryo. We identified several defects in anterior-posterior axis specification and mesoderm patterning in Dvl1+/-; Dvl2-/-; Dvl3-/- embryos. Homozygous deletions in all three Dvl genes (Dvl TKO) resulted in defects in distal visceral endoderm migration and a complete failure to induce mesoderm formation. To identify potential mechanisms that lead to the defects in the developmental processes preceding gastrulation, we generated Dvl TKO mouse embryonic stem cells (mESCs) and compared the transcriptional profile of these cells with wild-type (WT) mESCs during germ lineage differentiation into 3D embryoid bodies (EBs). While the Dvl TKO mESCs displayed similar morphology, self-renewal properties, and minor transcriptional variation from WT mESCs, we identified major transcriptional dysregulation in the Dvl TKO EBs during differentiation in a number of genes involved in anterior-posterior pattern specification, gastrulation induction, mesenchyme morphogenesis, and mesoderm-derived tissue development. The absence of the Dvls leads to specific down-regulation of BMP signaling genes. Furthermore, exogenous activation of canonical Wnt, BMP, and Nodal signaling all fail to rescue the mesodermal defects in the Dvl TKO EBs. Moreover, endoderm differentiation was promoted in the absence of mesoderm in the Dvl TKO EBs, while the suppression of ectoderm differentiation was delayed. Overall, we demonstrate that the Dvls are dispensable for maintaining self-renewal in mESCs but are critical during differentiation to regulate key developmental signaling pathways to promote proper axis specification and mesoderm formation.

Factors that influence minority use of high-volume hospitals for colorectal cancer care.

BACKGROUND: Previous studies suggest that minorities cluster in low-quality hospitals despite living close to better performing hospitals. This may contribute to persistent disparities in cancer outcomes. OBJECTIVE: The purpose of this work was to examine how travel distance, insurance status, and neighborhood socioeconomic factors influenced minority underuse of high-volume hospitals for colorectal cancer. DESIGN: The study was a retrospective, cross-sectional, population-based study. SETTINGS: All hospitals in California from 1996 to 2006 were included. PATIENTS: Patients with colorectal cancer diagnosed and treated in California between 1996 and 2006 were identified using California Cancer Registry data. MAIN OUTCOME MEASURES: Multivariable logistic regression models predicting high-volume hospital use were adjusted for age, sex, race, stage, comorbidities, insurance status, and neighborhood socioeconomic factors. RESULTS: A total of 79,231 patients treated in 417 hospitals were included in the study. High-volume hospitals were independently associated with an 8% decrease in the hazard of death compared with other settings. A lower proportion of minorities used high-volume hospitals despite a higher proportion living nearby. Although insurance status and socioeconomic factors were independently associated with high-volume hospital use, only socioeconomic factors attenuated differences in high-volume hospital use of black and Hispanic patients compared with white patients. LIMITATIONS: The use of cross-sectional data and racial and ethnic misclassifications were limitations in this study. CONCLUSIONS: Minority patients do not use high-volume hospitals despite improved outcomes and geographic access. Low socioeconomic status predicts low use of high-volume settings in select minority groups. Our results provide a roadmap for developing interventions to increase the use of and access to higher quality care and outcomes. Increasing minority use of high-volume hospitals may require community outreach programs and changes in physician referral practices.

What factors influence minority use of National Cancer Institute-designated cancer centers?

BACKGROUND: National Cancer Institute (NCI) cancer centers provide high-quality care and are associated with better outcomes. However, racial and ethnic minority populations tend not to use these settings. The current study sought to understand what factors influence minority use of NCI cancer centers. METHODS: A data set containing California Cancer Registry (CCR) data linked to patient discharge abstracts identified all patients with colorectal cancer (CRC) who were treated from 1996 through 2006. Multivariable models were generated to predict the use of NCI settings by race. Geographic proximity to an NCI center and patient sociodemographic and clinical characteristics were assessed. RESULTS: Approximately 5% of all identified patients with CRC (n = 79,231) were treated in NCI settings. The median travel distance for treatment for all patients in all hospitals was ≤ 5 miles. A higher percentage of minorities lived near an NCI cancer center compared with whites. A baseline multivariable model predicting use showed a negative association between Hispanic ethnicity and NCI center use (odds ratio, 0.71; 95% confidence interval, 0.64-0.79). Asian/Pacific Islander patients were more likely to use NCI centers (odds ratio, 1.41; 95% confidence interval, 1.28-1.54). There was no difference in use noted among black patients. Increasing living distance from an NCI cancer center was found to be predictive of lower odds of use for all populations. Medicare and Medicaid insurance statuses were positively associated with NCI center use. Neighborhood-level education was found to be a more powerful predictor of NCI use than poverty or unemployment. CONCLUSIONS: Select minority groups underuse NCI cancer centers for CRC treatment. Sociodemographic factors and proximity to NCI centers are important predictors of use. Interventions to address these factors may improve minority attendance to NCI cancer centers for care.

Adequacy of lymph node examination in colorectal surgery: contribution of the hospital versus the surgeon.

BACKGROUND: Examination of at least 12 lymph nodes (LNs) in the staging of colon cancer (CC) was recommended by the National Comprehensive Cancer Network in 2000; however, rates of an adequate examination remain low. This study compares the impact of the hospital contextual variance against that of the operating surgeon on delivery of an adequate LN examination. STUDY DESIGN: Retrospective analysis of California Cancer Registry data for all CC operations (2001-2006). Hierarchical models predicted the adequacy of LN examination as a function of patient, surgeon, and hospital characteristics. Models were created using penalized quasi-likelihood approximation with second order Taylor linearization as implemented in MLwiN 2.15. RESULTS: A total of 25,606 resections involving 3376 surgeons operating in 346 hospitals were analyzed. Half of cases had an adequate examination. Hierarchical models showed the median odds of an adequate examination associated with the hospital context [(MORhosp 2.05; 95% confidence interval, 1.9-2.2) was much higher than that associated with the surgeon (MORsurg 1.34; 95% confidence interval, 1.2-1.4)]. Hospital characteristics teaching and high volume predicted higher odds of an adequate examination. There was no association with hospital revenue. CONCLUSIONS: Approximately half of patients undergoing surgery for CC received an adequate LN examination. Hospital contextual factors had a stronger association with receipt of an adequate examination than surgeon factors. Our results suggest that quality improvement initiatives and incentives should be targeted at the hospital level to achieve the highest impact. Furthermore, we have identified nonteaching and low volume settings as rational targets for these efforts.

Racial and ethnic differences in lymph node examination after colon cancer resection do not completely explain disparities in mortality.

BACKGROUND: In 1999, a multidisciplinary panel of experts in colorectal cancer reviewed the relevant medical literature and issued a consensus recommendation for a 12-lymph node (LN) minimum examination after resection for colon cancer. Some authors have shown racial/ethnic differences in receipt of this evidence-based care. To date, however, none has investigated the correlation between disparities in LN examination and disparities in outcomes after colon cancer treatment. METHODS: This retrospective analysis used California Cancer Registry linked to California Office of Statewide Health Planning and Development discharge data (1996-2006). Chi-square analysis, logistic regression, and Cox proportional hazard models predicted disparities in receipt of an adequate examination and the effect of an inadequate exam on mortality and disparities. Patients with stage I and II colon cancers undergoing surgery in California were included; patients with stage III and IV disease were excluded. RESULTS: A total of 37,911 records were analyzed. Adequate staging occurred in fewer than half of cases. An inadequate examination (<12 LNs) was associated with higher mortality rates. Hispanics had the lowest odds of receiving an adequate exam; however, blacks, not Hispanics, had the highest risk of mortality compared with whites. This disparity was not completely explained by inadequate LN examination. CONCLUSIONS: Inadequate LN exam occurs often and is associated with increased mortality. There are disparities in receipt of the minimum exam, but this only explains a small part of the observed disparity in mortality. Improving the quality of LN examination alone is unlikely to correct colon cancer disparities.

Adipocyte-specific deletion of zinc finger protein 407 results in lipodystrophy and insulin resistance in mice.

PPARγ deficiency in humans and model organisms impairs the transcriptional control of adipogenesis and mature adipocyte function resulting in lipodystrophy and insulin resistance. Zinc finger protein 407 (ZFP407) positively regulates PPARγ target gene expression and insulin-stimulated glucose uptake in cultured adipocytes. The in vivo physiological role of ZFP407 in mature adipocytes, however, remains to be elucidated. Here we generated adipocyte-specific ZFP407 knockout (AZKO) mice and discovered a partial lipodystrophic phenotype with reduced fat mass, hypertrophic adipocytes in inguinal and brown adipose tissue, and reduced adipogenic gene expression. The lipodystrophy was further exacerbated in AZKO mice fed a high-fat diet. Glucose and insulin tolerance tests revealed decreased insulin sensitivity in AZKO mice compared to control littermates. Cell-based assays demonstrated that ZFP407 is also required for adipogenesis, which may also contribute to the lipodystrophic phenotype. These results demonstrate an essential in vivo role of ZFP407 in brown and white adipose tissue formation and organismal insulin sensitivity.

Highly efficient methods to obtain homogeneous dorsal neural progenitor cells from human and mouse embryonic stem cells and induced pluripotent stem cells.

BACKGROUND: Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been widely used to generate cellular models harboring specific disease-related genotypes. Of particular importance are ESC and iPSC applications capable of producing dorsal telencephalic neural progenitor cells (NPCs) that are representative of the cerebral cortex and overcome the challenges of maintaining a homogeneous population of cortical progenitors over several passages in vitro. While previous studies were able to derive NPCs from pluripotent cell types, the fraction of dorsal NPCs in this population is small and decreases over several passages. Here, we present three protocols that are highly efficient in differentiating mouse and human ESCs, as well as human iPSCs, into a homogeneous and stable population of dorsal NPCs. These protocols will be useful for modeling cerebral cortical neurological and neurodegenerative disorders in both mouse and human as well as for high-throughput drug screening for therapeutic development. METHODS: We optimized three different strategies for generating dorsal telencephalic NPCs from mouse and human pluripotent cell types through single or double inhibition of bone morphogenetic protein (BMP) and/or SMAD pathways. Mouse and human pluripotent cells were aggregated to form embryoid bodies in suspension and were treated with dorsomorphin alone (BMP inhibition) or combined with SB431542 (double BMP/SMAD inhibition) during neural induction. Neural rosettes were then selected from plated embryoid bodies to purify the population of dorsal NPCs. We tested the expression of key dorsal NPC markers as well as nonectodermal markers to confirm the efficiency of our three methods in comparison to published and commercial protocols. RESULTS: Single and double inhibition of BMP and/or SMAD during neural induction led to the efficient differentiation of dorsal NPCs, based on the high percentage of PAX6-positive cells and the NPC gene expression profile. There were no statistically significant differences in the variation of PAX6 and SOX1-positive NPCs between the two human pluripotent cell-derived methods; therefore, both methods are suitable for producing stable dorsal NPCs. When further differentiated into mature neurons, NPCs gave rise to a population of almost exclusively forebrain cortical neurons, confirming the dorsal fate commitment of the progenitors. CONCLUSIONS: The methods described in this study show improvements over previously published studies and are highly efficient at differentiating human and mouse pluripotent cell types into dorsal PAX6-positive NPCs and eventually into forebrain cortical neurons.

Bax-induced apoptosis shortens the life span of DNA repair defect Ku70-knockout mice by inducing emphysema.

Cells with DNA damage undergo apoptosis or cellular senescence if the damage cannot be repaired. Recent studies highlight that cellular senescence plays a major role in aging. However, age-associated diseases, including emphysema and neurodegenerative disorders, are caused by apoptosis of lung alveolar epithelial cells and neurons, respectively. Therefore, enhanced apoptosis also promotes aging and shortens the life span depending on the cell type. Recently, we reported that ku70(-) (/) (-)bax(-) (/) (-) and ku70(-) (/) (-)bax(+/) (-) mice showed significantly extended life span in comparison with ku70(-) (/) (-)bax(+/+) mice. Ku70 is essential for non-homologous end joining pathway for DNA double strand break repair, and Bax plays an important role in apoptosis. Our study suggests that Bax-induced apoptosis has a significant impact on shortening the life span of ku70(-) (/) (-) mice, which are defective in one of DNA repair pathways. The lung alveolar space gradually enlarges during aging, both in mouse and human, and this age-dependent change results in the decrease of respiration capacity during aging that can lead to emphysema in more severe cases. We found that emphysema occurred in ku70(-) (/) (-) mice at the age of three-months old, and that Bax deficiency was able to suppress it. These results suggest that Bax-mediated apoptosis induces emphysema in ku70(-) (/) (-) mice. We also found that the number of cells, including bronchiolar epithelial cells and type 2 alveolar epithelial cells, shows a higher DNA double strand break damage response in ku70 KO mouse lung than in wild type. Recent studies suggest that non-homologous end joining activity decreases with increased age in mouse and rat model. Together, we hypothesize that the decline of Ku70-dependent DNA repair activity in lung alveolar epithelial cells is one of the causes of age-dependent decline of lung function resulting from excess Bax-mediated apoptosis of lung alveolar epithelial cells (and their progenitor cells).

Do hospitals that serve a high percentage of Medicaid patients perform well on evidence-based guidelines for colon cancer care?

BACKGROUND: Previous work suggests hospitals serving high percentages of patients with Medicaid are associated with worse colon cancer survival. It is unclear if practice patterns in these settings explain differential outcomes. HYPOTHESIS: High Medicaid hospitals (HMH) have lower compliance with evidence-based care processes (examining 12 or more lymph nodes (LN) during surgical staging and providing appropriate chemo-therapy). METHODS: Retrospective analysis of stage I-III colon cancers from California Cancer Registry (1996-2006) linked to discharge abstracts and hospital profiles predicted hospital compliance with guidelines and trends in compliance over time. RESULTS: Cases (N=60,000) in 439 hospitals analyzed. High Medicaid hospital settings had lower odds of compliance with the 12 LN exam (OR(HMH)0.91, CI(HMH)[0.85, 0.98]) and with the delivery of appropriate chemotherapy (OR(HMH)0.76, CI(HMH)[0.67, 0.86]). CONCLUSIONS: High Medicaid hospital status is associated with poor performance on evidence-based colon cancer care. Policies to improve the quality of colon cancer care should target these settings.

Cell-Penetrating Penta-Peptides (CPP5s): Measurement of Cell Entry and Protein-Transduction Activity.

Previously, we developed cell-penetrating penta-peptides (CPP5s). In the present study, VPTLK and KLPVM, two representative CPP5s, were used to characterize the cell-penetration and protein-transduction activities of these small molecules. Various inhibitors of endocytosis and pinocytosis (chlorpromazine, cytochalasin D, Filipin III, amiloride, methyl-ß-cyclodextrin, and nocodazole) were tested. Only cytochalasin D showed suppression of CPP5 entry, though the effect was partial. In addition, CPP5s were able to enter a proteoglycan-deficient CHO cell line. These results suggest that pinocytosis and endocytosis may play only a minor role in the cell entry of CPP5s. By mass spectrometry, we determined that the intracellular concentration of VPTLK ranged from 20 nM to 6.0 µM when the cells were cultured in medium containing 1 µM - 1.6 mM VPTLK. To determine the protein-transduction activity of CPP5s, the Tex-LoxP EG cell line, which has a Cre-inducible green fluorescent protein (GFP) gene, was used. VPTLK and KLPVM were added to the N-terminus of Cre, and these fusion proteins were added to the culture medium of Tex-LoxP EG cells. Both VPTLK-Cre and KLPVM-Cre were able to turn on GFP expression in these cells, suggesting that CPP5s have protein-transduction activity. Since CPP5s have very low cytotoxic activity, even at a concentration of 1.6 mM in the medium, CPP5s could be utilized as a new tool for drug delivery into cells.