Chk1 suppresses a caspase-2 apoptotic response to DNA damage that bypasses p53, Bcl-2, and caspase-3.

Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. We report that depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore gamma-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after gamma-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved "Chk1-suppressed" pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy.

Noninvasive prenatal detection of chromosomal aneuploidies using different next generation sequencing strategies and algorithms.

OBJECTIVE: Here we describe the successful application of massively parallel sequencing for noninvasive prenatal detection of trisomy 21. In addition, for the detection of a broader spectrum of fetal aneuploidies, a target enrichment approach was successfully tested. METHODS: The circulating cell-free DNA was prepared from 53 maternal blood samples and analysed using Illumina's sequencing systems Genome Analyzer(IIx) and HiSeq2000, respectively. In a first experiment the SureSelect Target Enrichment System was tested. RESULTS: In our initial study analysing 42 samples on the Genome Analyzer(IIx) , all eight samples from women carrying a trisomy 21 fetus were correctly identified. On the basis of our HiSeq2000 sequence data, we discussed new algorithms for detection of fetal trisomy 21. In addition, we successfully used the combination of a target enrichment system followed by sequencing and were able to identify fetal trisomy 13 and fetal trisomy 21. CONCLUSIONS: Our results confirm previous reports that massively parallel sequencing of cell-free fetal DNA allows the reliably noninvasive detection of trisomy 21 from maternal blood with the potential to enhance test selectivity and specificity by bioinformatic means. According to our preliminary results, targeted sequencing might be an alternative strategy to detect chromosomal aneuploidies besides trisomy 21.

Interplay of pu.1 and gata1 determines myelo-erythroid progenitor cell fate in zebrafish.

The zebrafish is a powerful model system for investigating embryonic vertebrate hematopoiesis, allowing for the critical in vivo analysis of cell lineage determination. In this study, we identify zebrafish myeloerythroid progenitor cells (MPCs) that are likely to represent the functional equivalent of mammalian common myeloid progenitors. Utilizing transgenic pu.1-GFP fish, real-time MPC differentiation was correlated with dynamic changes in cell motility, morphology, and gene expression. Unlike mammalian hematopoiesis, embryonic zebrafish myelopoiesis and erythropoiesis occur in anatomically separate locations. Gene knockdown experiments and transplantation assays demonstrated the reciprocal negative regulation of pu.1 and gata1 and their non-cell-autonomous regulation that determines myeloid versus erythroid MPC fate in the distinct blood-forming regions. Furthermore, forced expression of pu.1 in the bloodless mutant cloche resulted in myelopoietic rescue, providing intriguing evidence that this gene can function in the absence of some stem cell genes, such as scl, in governing myelopoiesis.

Reference Curve for the Mean Uterine Artery Pulsatility Index in Singleton Pregnancies.

INTRODUCTION: Doppler sonography of the uterine artery (UA) is done to monitor pregnancies, because the detected flow patterns are useful to draw inferences about possible disorders of trophoblast invasion. Increased resistance in the UA is associated with an increased risk of preeclampsia and/or intrauterine growth restriction (IUGR) and perinatal mortality. In the absence of standardized figures, the normal ranges of the various available reference curves sometimes differ quite substantially from one another. The causes for this are differences in the flow patterns of the UA depending on the position of the pulsed Doppler gates as well as branching of the UA. Because of the discrepancies between the different reference curves and the practical problems this poses for guideline recommendations, we thought it would be useful to create our own reference curves for Doppler measurements of the UA obtained from a singleton cohort under standardized conditions. MATERIAL AND METHODS: This retrospective cohort study was carried out in the Department of Obstetrics of the Charité - Universitätsmedizin Berlin, the Department for Obstetrics and Prenatal Medicine of the University Hospital Halle (Saale) and the Center for Prenatal Diagnostics and Human Genetics Kurfürstendamm 199. Available datasets from the three study locations were identified and reference curves were generated using the LMS method. Measured values were correlated with age of gestation, and a cubic model and Box-Cox power transformation (L), the median (M) and the coefficient of variation (S) were used to smooth the curves. RESULTS: 103 720 Doppler examinations of the UA carried out in singleton pregnancies from the 11th week of gestation (10 + 1 GW) were analyzed. The mean pulsatility index (Mean PI) showed a continuous decline over the course of pregnancy, dropping to a plateau of around 0.84 between the 23rd and 27th GW, after which it decreased again. CONCLUSION: Age of gestation, placental position, position of pulsed Doppler gates and branching of the UA can all change the flow pattern. The mean pulsatility index (Mean PI) showed a continuous decrease over time. There were significant differences between our data and alternative reference curves. A system of classifying Doppler studies and a reference curve adapted to the current technology are urgently required to differentiate better between physiological and pathological findings.

The pu.1 promoter drives myeloid gene expression in zebrafish.

PU.1 is a member of the Ets family of transcription factors and plays an essential role in the development of both myeloid and lymphoid cells. To examine zebrafish pu.1 (zpu.1) expression in subpopulations of blood cells during zebrafish development, we linked a 9-kb zebrafish genomic fragment upstream of the zpu.1 initiator codon to green fluorescent protein (GFP) and microinjected this construct to generate stable transgenic lines. GFP-positive fluorescent myeloid precursors were observed migrating from the anterolateral mesoderm in living embryos from 16 to 28 hours after fertilization (hpf) in a pattern that overlaps the expression pattern of endogenous zpu.1 mRNA. Analysis of larval histologic sections revealed GFP-expressing hematopoietic cells in the developing zebrafish kidney. Flow cytometric analysis of cells from adult whole kidney marrow revealed 2 discrete subpopulations of GFP-positive cells, which after cell sorting exhibited either myeloid or early lymphoid morphology. Thus, the zebrafish zpu.1 promoter fragment used here is capable of driving reporter gene expression in subsets of embryonic and adult hematopoietic cells. These transgenic lines will be useful to dissect the cellular and molecular control of myeloid cell differentiation, and this promoter fragment may prove useful in the development of zebrafish models of acute myeloid leukemia.

Long-term tolerability of benazepril in dogs with congestive heart failure.

OBJECTIVES: To test the tolerability of long-term administration of benazepril in dogs with congestive heart failure (CHF). METHODS: The study was a prospective, randomized, double-blinded, placebo-controlled clinical trial. A total of 162 dogs with New York Heart Association (NYHA) class II-IV heart failure caused by chronic valvular disease (CVD) or dilated cardiomyopathy (DCM) were enrolled. Benazepril (minimum dosage, 0.25 mg/kg) or placebo were administered orally once daily for up to 34 months. In this paper, we report results of plasma alanine aminotransferase (ALT), creatinine, potassium and urea. RESULTS: The two groups were matched at baseline (p>/=0.18). Plasma creatinine concentrations were lower during treatment with benazepril versus placebo for all dogs (p=0.14) and every sub-group tested (NYHA II, III or IV; CVD; DCM; initial creatinine >124 mumol/L), although statistical significance was not reached (p=0.14-0.6). However, significantly (p=0.035) more cases of creatinine >124 mumol/L during treatment occurred with placebo (47%) as compared to benazepril (30%). Plasma ALT and urea values did not differ between groups for all dogs (p>0.5) or any sub-group (p=0.23-1.0). Plasma potassium values did not differ between groups for all dogs (p>0.5). Although differences approached statistical significance for potassium in some sub-groups (p=0.07-0.1), there were no consistent differences between groups. CONCLUSIONS: Benazepril was well tolerated during long-term therapy in dogs with CHF and no specific precautions appear to be necessary regarding plasma ALT, creatinine, potassium or urea. The possible action of benazepril in improving renal function (evidenced via lower plasma creatinine) merits further investigation.