Citations

Abstract Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases.

Abstract Replication stress and DNA damage activate the ATR-Chk1 checkpoint signaling pathway that licenses repair and cell survival processes. In this study, we examined the respective roles of the ATR and Chk1 kinases in ovarian cancer cells using genetic and pharmacologic inhibitors in combination with cisplatin, topotecan, gemcitabine, and the PARP inhibitor veliparib (ABT-888), four agents with clinical activity in ovarian cancer. RNA interference (RNAi)–mediated depletion or inhibition of ATR sensitized ovarian cancer cells to all four agents. In contrast, while cisplatin, topotecan, and gemcitabine each activated Chk1, RNAi-mediated depletion or inhibition of this kinase in cells sensitized them only to gemcitabine. Unexpectedly, we found that neither the ATR kinase inhibitor VE-821 nor the Chk1 inhibitor MK-8776 blocked ATR-mediated Chk1 phosphorylation or autophosphorylation, two commonly used readouts for inhibition of the ATR-Chk1 pathway. Instead, their ability to sensitize cells correlated with enhanced CDC25A levels. In addition, we also found that VE-821 could further sensitize BRCA1-depleted cells to cisplatin, topotecan, and veliparib beyond the potent sensitization already caused by their deficiency in homologous recombination. Taken together, our results established that ATR and Chk1 inhibitors differentially sensitize ovarian cancer cells to commonly used chemotherapy agents and that Chk1 phosphorylation status may not offer a reliable marker for inhibition of the ATR-Chk1 pathway. A key implication of our work is the clinical rationale it provides to evaluate ATR inhibitors in combination with PARP inhibitors in BRCA1/2-deficient cells. Cancer Res; 73(12); 3683–91. ©2013 AACR.

Background: Early recognition and treatment of social and emotional disorders in children is significant for school preparation. These disorders are frequently underdetected without the use of standardized screening instruments. The purpose of our study is to describe the implementation of the Ages and Stages Questionnaire: Social–Emotional (ASQ:SE) in primary care practice by mail when children are 30 months old. Methods: In this 4-month study, parents of all 30-month-old children who receive primary care at our study site were mailed the ASQ:SE. In children who did not pass screening or received a call from a registered nurse for parental concerns documented on the questionnaire, short-term clinical outcomes were obtained from the electronic medical record. During the last month of the study, the demographics variables of race and insurance type were analyzed for an association with questionnaire completion by mail. Results: Of the 870 families mailed 30-month ASQ:SE screens, 507 (58.3%) were returned by mail. Out of the children with returned screens, 38 (7.5%) of parents were contacted for either elevated scores or concerning comments and 6 (1.2%) were referred to Early Intervention. Parents of children with government insurance returned the ASQ:SE questionnaire 34.2% (13/38) of the time compared with 65.5% (76/116) of those with private insurance ( P < .001). Conclusion: Our results indicate that mental health screening can be effectively managed in primary care practice by a registered nurse using a follow-up protocol. Mailing the ASQ:SE is likely not an effective way to comprehensively screen most primary care populations.

Objective To evaluate carbonic anhydrase IX ( CAIX ) expression as an independent prognostic marker for clear cell renal cell carcinoma ( ccRCC ). With recent smaller studies showing conflicting results, we aimed to update our initial analysis in 2007 with an additional 5‐year follow‐up. Patients and Methods We provided long‐term follow‐up of the same cohort used in our 2007 study (730 patients with unilateral, sporadic ccRCC treated surgically between 1990 and 1999). Associations of CAIX expression with RCC death and distant metastases were evaluated using C ox proportional hazards regression models. Results CAIX was expressed in 708 (97.0%) of the specimens; 163 tumours (22.3%) had low (≤85%) expression and 567 (77.7%) high (>85%) expression. There were 483 deaths and 265 RCC ‐specific deaths. The median follow‐up for the 247 patients still under observation was 13.8 years. Univariately, low CAIX expression was associated with an increased risk of RCC death vs high expression (hazard ratio 1.62; P < 0.001). Low CAIX expression was not statistically significantly associated with RCC death or distant metastases after adjusting for nuclear grade or coagulative tumour necrosis. Conclusion After additional long‐term follow‐up of our large cohort, our results continue to suggest that CAIX is not an independent prognostic marker for ccRCC .