The mitochondrial NADH shuttle system is a targetable vulnerability for Group 3 medulloblastoma in a hypoxic microenvironment.

Medulloblastoma is a cancerous brain tumor that affects mostly children. Among the four groups defined by molecular characteristics, Group 3, the least well characterized, is also the least favorable, with a survival rate of 50%. Current treatments, based on surgery, radiotherapy, and chemotherapy, are not adequate and the lack of understanding of the different molecular features of Group 3 tumor cells makes the development of effective therapies challenging. In this study, the problem of medulloblastoma is approached from a metabolic standpoint in a low oxygen microenvironment. We establish that Group 3 cells use both the mitochondrial glycerol-3 phosphate (G3PS) and malate-aspartate shuttles (MAS) to produce NADH. Small molecules that target G3PS and MAS show a greater ability to decrease cell proliferation and induce apoptosis specifically of Group 3 cells. In addition, as Group 3 cells show improved respiration in hypoxia, the use of Phenformin, a mitochondrial complex 1 inhibitor, alone or in combination, induced significant cell death. Furthermore, inhibition of the cytosolic NAD+ recycling enzyme lactate dehydrogenase A (LDHA), enhanced the effects of the NADH shuttle inhibitors. In a 3D model using Group 3 human cerebellar organoids, tumor cells also underwent apoptosis upon treatment with NADH shuttle inhibitors. Our study demonstrates metabolic heterogeneity depending on oxygen concentrations and provides potential therapeutic solutions for patients in Group 3 whose tumors are the most aggressive.

HIF-1 inactivation empowers HIF-2 to drive hypoxia adaptation in aggressive forms of medulloblastoma.

Medulloblastoma (MB) is the most prevalent brain cancer in children. Four subgroups of MB have been identified; of these, Group 3 is the most metastatic. Its genetics and biology remain less clear than the other groups, and it has a poor prognosis and few effective treatments available. Tumor hypoxia and the resulting metabolism are known to be important in the growth and survival of tumors but, to date, have been only minimally explored in MB. Here we show that Group 3 MB tumors do not depend on the canonical transcription factor hypoxia-inducible factor-1α (HIF-1α) to mount an adaptive response to hypoxia. We discovered that HIF-1α is rendered inactive either through post-translational methylation, preventing its nuclear localization specifically in Group 3 MB, or by a low expression that prevents modulation of HIF-target genes. Strikingly, we found that HIF-2 takes over the role of HIF-1 in the nucleus and promotes the activation of hypoxia-dependent anabolic pathways. The exclusion of HIF-1 from the nucleus in Group 3 MB cells enhances the reliance on HIF-2's transcriptional role, making it a viable target for potential anticancer strategies. By combining pharmacological inhibition of HIF-2α with the use of metformin, a mitochondrial complex I inhibitor to block respiration, we effectively induced Group 3 MB cell death, surpassing the effectiveness observed in Non-Group 3 MB cells. Overall, the unique dependence of MB cells, but not normal cells, on HIF-2-mediated anabolic metabolism presents an appealing therapeutic opportunity for treating Group 3 MB patients with minimal toxicity.

Quantitative profiling of the UGT transcriptome in human drug-metabolizing tissues.

Alternative splicing as a mean to control gene expression and diversify function is suspected to considerably influence drug response and clearance. We report the quantitative expression profiles of the human UGT genes including alternatively spliced variants not previously annotated established by deep RNA-sequencing in tissues of pharmacological importance. We reveal a comprehensive quantification of the alternative UGT transcriptome that differ across tissues and among individuals. Alternative transcripts that comprise novel in-frame sequences associated or not with truncations of the 5'- and/or 3'- termini, significantly contribute to the total expression levels of each UGT1 and UGT2 gene averaging 21% in normal tissues, with expression of UGT2 variants surpassing those of UGT1. Quantitative data expose preferential tissue expression patterns and remodeling in favor of alternative variants upon tumorigenesis. These complex alternative splicing programs have the strong potential to contribute to interindividual variability in drug metabolism in addition to diversify the UGT proteome.

Unravelling the transcriptomic landscape of the major phase II UDP-glucuronosyltransferase drug metabolizing pathway using targeted RNA sequencing.

A comprehensive view of the human UDP-glucuronosyltransferase (UGT) transcriptome is a prerequisite to the establishment of an individual's UGT metabolic glucuronidation signature. Here, we uncover the transcriptome landscape of the 10 human UGT gene loci in normal and tumoral metabolic tissues by targeted RNA next-generation sequencing. Alignment on the human hg19 reference genome identifies 234 novel exon-exon junctions. We recover all previously known UGT1 and UGT2 enzyme-coding transcripts and identify over 130 structurally and functionally diverse novel UGT variants. We further expose a revised genomic structure of UGT loci and provide a comprehensive repertoire of transcripts for each UGT gene. Data also uncover a remodelling of the UGT transcriptome occurring in a tissue- and tumor-specific manner. The complex alternative splicing program regulating UGT expression and protein functions is likely critical in determining detoxification capacity of an organ and stress-related responses, with significant impact on drug responses and diseases.

Pharmacogenomics of human uridine diphospho-glucuronosyltransferases and clinical implications.

Glucuronidation by uridine diphospho-glucuronosyltransferase enzymes (UGTs) is a major phase II biotransformation pathway and, complementary to phase I metabolism and membrane transport, one of the most important cellular defense mechanisms responsible for the inactivation of therapeutic drugs, other xenobiotics, and endogenous molecules. Interindividual variability in UGT pathways is significant and may have profound pharmacological and toxicological implications. Several genetic and genomic processes underlie this variability and are discussed in relation to drug metabolism and diseases such as cancer.

Regulation of skin aging and heart development by TAp63.

Since the discovery of the TP63 gene in 1998, many studies have demonstrated that ΔNp63, a p63 isoform of the p53 gene family, is involved in multiple functions during skin development and in adult stem/progenitor cell regulation. In contrast, TAp63 studies have been mostly restricted to its apoptotic function and more recently as the guardian of oocyte integrity. TAp63 endogenous expression is barely detectable in embryos and adult (except in oocytes), presumably because of its rapid degradation and the lack of antibodies able to detect weak expression. Nevertheless, two recent independent studies have demonstrated novel functions for TAp63 that could have potential implications to human pathologies. The first discovery is related to the protective role of TAp63 on premature aging. TAp63 controls skin homeostasis by maintaining dermal and epidermal progenitor/stem cell pool and protecting them from senescence, DNA damage and genomic instability. The second study is related to the role of TAp63, expressed by the primitive endoderm, on heart development. This unexpected role for TAp63 has been discovered by manipulation of embryonic stem cells in vitro and confirmed by the severe cardiomyopathy observed in brdm2 p63-null embryonic hearts. Interestingly, in both cases, TAp63 acts in a cell-nonautonomous manner on adjacent cells. Here, we discuss these findings and their potential connection during development.

Alternative-splicing forms of the major phase II conjugating UGT1A gene negatively regulate glucuronidation in human carcinoma cell lines.

The UDP-glucuronosyltransferase UGT1A gene is a major biotransformation gene involved in the metabolism of a vast array of molecules. Recently, we uncovered a new series of alternative spliced isoforms referred to as isoforms 2 or UGT1As_i2 that use an alternative exon 5 (5b). The function of such mRNAs and the corresponding 45 kDa proteins still remains unclear. Although devoid of glucuronosyltransferase activity, UGT1As_i2 are widely co-expressed with the enzymatically active and classical UGT1A isoforms (UGT1As_i1). In this study, we observed abundant signal in human colon tissue samples, predominantly along intestinal crypts. In human cells, UGT1A_i2 proteins are expressed in similar subcellular compartments as UGT1As_i1. Cellular properties of i2-spliced forms were then studied using synthetic small-interfering RNA (siRNA) in two human colon cancer cell lines that show a significant amount of exon 5a- and exon 5b-containing mRNAs and that display enzymatic activities for UGT1As substrates. We observed that siRNA-mediated knockdown of endogenous i2 upregulates cellular glucuronidation activities by 120-170% (P<0.01) for all substrates tested. Functional data support a dominant-negative function for endogenous exon 5b-spliced forms of UGT1A, hence potentially affecting in vivo glucuronidation capacity. This new regulatory strategy may ensure an additional mean to modulate cellular response to endo/xeno stimulus.

Practice patterns in the management of chronic obstructive pulmonary disease in primary practice: the CAGE study.

BACKGROUND: The information on usual care for patients with chronic obstructive pulmonary disease (COPD) in primary care is limited in Canada. OBJECTIVE: To evaluate primary care practice in patients with COPD in Quebec and Ontario compared with recommended care. METHODS: The COPD Care Gap Evaluation (CAGE) was a prospective, cross-sectional study. Physicians' self-reported data of enrolled COPD patients were compared with the recommended care for the level of disease severity (using the Canadian Thoracic Society classification by symptoms) and stability, derived from Canadian Thoracic Society COPD guidelines. Pharmacological treatment, spirometric confirmation of diagnosis and nonpharmacological management, including smoking cessation counselling, influenza immunization and referral for pulmonary rehabilitation, were assessed. RESULTS: Participating physicians (n=161; 44 in Quebec, 117 in Ontario) recruited 1090 patients (320 in Quebec, 770 in Ontario). The mean (+/- SD) age of the patients was 69.9+/-10.4 years; 60% were male and 40% were currently smoking. Pharmacological treatment that matched guideline recommendations was identified in 34% of patients. Discrepancies between reported and recommended treatment stemmed from nonprescription of long-acting bronchodilators (LABDs) for patients with moderate (27%) and severe (21%) COPD, nonprescription of two long-acting beta agonists (a beta(2)-agonist and an anticholinergic) for patients with severe COPD (51%), and prescription of inhaled corticosteroids (63%) and LABDs (47%) for patients with mild COPD for which the treatment is not recommended. Spirometric confirmation of diagnosis, as recommended by the guidelines, was reported in 56% of patients. For nonpharmacological management, smoking cessation counselling (95%) and influenza immunization (80%) were near optimal. Referral for pulmonary rehabilitation (9%) was not common. Differences between provinces were seen mainly in the prescription of short-acting bronchodilators (89% in Quebec, 76% in Ontario) and LABDs (60% in Quebec, 80% in Ontario). CONCLUSIONS: Substantial gaps between recommended and current care exist in the management of COPD patients in primary care practice. Undertreatment of patients with severe COPD has potential clinical implications, including loss of autonomy and hospitalization.

Assessing the impact of pulmonary rehabilitation on functional status in COPD.

BACKGROUND: The optimal way of assessing the impact of pulmonary rehabilitation on functional status in chronic obstructive pulmonary disease (COPD) is currently unknown. The minimal clinically important difference for the constant work rate cycling exercise test also needs to be investigated to facilitate its interpretation. A study was undertaken to evaluate the changes in the 6-min walking test and in the constant work rate cycle endurance test immediately following and 1 year after pulmonary rehabilitation, together with the importance of these changes in terms of health status in patients with COPD. METHODS: Patients with COPD of mean (SD) age 65 (8) years and mean (SD) forced expiratory volume in 1 s (FEV1) 45 (15)% predicted were recruited from a multicentre prospective cohort study and evaluated at baseline, immediately after a pulmonary rehabilitation programme (n = 157) and at 1 year (n = 106). The 6-min walking test and the cycle endurance test were performed at each evaluation. Health status was evaluated with the St George Respiratory Questionnaire. RESULTS: Following pulmonary rehabilitation, cycle endurance time increased (198 (352) s, p<0.001) and stayed over baseline values at 1 year (p<0.001). The 6-min walking distance also showed improvements following rehabilitation (25 (52) m, p<0.001) but returned to baseline values at the 1-year follow-up. Changes in cycle endurance time were more closely associated with changes in health status than with the 6-min walking test. An improvement of 100-200 s in the cycle endurance time was associated with clinically meaningful changes in the St George Respiratory Questionnaire scores. CONCLUSIONS: The cycle endurance test was more responsive than the 6-min walking test in detecting improvement in exercise tolerance following pulmonary rehabilitation, and was also better correlated with improvements in health status. An improvement in the cycle endurance time of 100-200 s appeared to be clinically meaningful.

PARP-3 associates with polycomb group bodies and with components of the DNA damage repair machinery.

Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synthesize poly(ADP-ribose) on themselves and other acceptor proteins. Very little is known about this PARP, which is closely related to PARP-1 and PARP-2. By sequence analysis, we find that PARP-3 may be expressed in two isoforms which we studied in more detail to gain insight into their possible functions. We find that both PARP-3 isoforms, transiently expressed as GFP or FLAG fusions, are nuclear. Detection of endogenous PARP-3 with a specific antibody also shows a widespread nuclear distribution, appearing in numerous small foci and a small number of larger foci. Through co-localization experiments and immunoprecipitations, the larger nuclear foci were identified as Polycomb group bodies (PcG bodies) and we found that PARP-3 is part of Polycomb group protein complexes. Furthermore, using a proteomics approach, we determined that both PARP-3 isoforms are part of complexes comprising DNA-PKcs, PARP-1, DNA ligase III, DNA ligase IV, Ku70, and Ku80. Our findings suggest that PARP-3 is a nuclear protein involved in transcriptional silencing and in the cellular response to DNA damage.

BMP-4 induces a Smad-dependent apoptotic cell death of mouse embryonic stem cell-derived neural precursors.

Embryonic ectoderm is fated to become either neural or epidermal, depending on patterning processes that occur before and during gastrulation. It has been stated that epidermal commitment proceeds from a bone morphogenetic protein-4 (BMP-4)-dependent inhibition of dorsal ectoderm neuralization. We recently demonstrated that murine embryonic stem (ES) cells treated with BMP-4 undergo effective keratinocyte commitment and epidermogenesis. Focusing on the precise role of BMP-4 in the early choice between neural and epidermal commitment, we show here that BMP-4 treatment of ES cells leads to a dramatic apoptotic death of Sox-1+ neural precursors with concomitant epidermal engagement. In addition, neutralization of the Smad pathway prevents both the BMP-4 apoptotic process and the inhibition of neural differentiation. Our results suggest that, in mammals, BMP-4, as an active inducer of epidermal commitment, interferes with the survival of neural precursors through induction of their apoptotic cell death.

Self-management reduces both short- and long-term hospitalisation in COPD.

The aim of the present study was to assess the long-term impact on hospitalisation of a self-management programme for chronic obstructive pulmonary disease (COPD) patients. A multicentre, randomised clinical trial was carried out involving 191 COPD patients from seven hospitals. Patients who had one or more hospitalisations in the year preceding study enrolment were assigned to a self-management programme "Living Well with COPD(TM)" or to standard care. Hospitalisations from all causes were the primary outcome and were documented from the provincial hospitalisation database; emergency visits were recorded from the provincial health insurance database. Most patients were elderly, not highly educated, had advanced COPD (reflected by a mean forced expiratory volume in one second of 1 L), and almost half reported a dyspnoea score of 5/5 (modified Medical Research Council). At 2 years, there was a statistically significant and clinically relevant reduction in all-cause hospitalisations of 26.9% and in all-cause emergency visits of 21.1% in the intervention group as compared to the standard-care group. After adjustment for the self-management intervention effect, the predictive factors for reduced hospitalisations included younger age, sex (female), higher education, increased health status and exercise capacity. In conclusion, in this study, patients with chronic obstructive pulmonary disease who received educational intervention with supervision and support based on disease-specific self-management maintained a significant reduction in hospitalisations after a 2-year period.

The Lac repressor provides a reversible gene expression system in undifferentiated and differentiated embryonic stem cell.

Control of mammalian gene promoters by the bacterial LacI repressor provides reversible regulation and dose-response levels of derepressed expression by the lactose analog isopropyl thiogalactose (IPTG). Here, we show that insertion of LacI-binding sites in the ubiquitous beta-actin promoter confers a strong and dose-dependent IPTG-regulatable expression of transiently transfected reporter genes in mouse embryonic stem (ES) cells expressing LacI. We established ES cell lines stably expressing reporter genes under inducible control and found a five- to tenfold IPTG induction of transgene expression. The kinetics of induction is rapid and stable, and can be rapidly reversed after IPTG removal. Importantly, this regulatable expression was maintained throughout the differentiation process of ES cells, and observed in individual differentiated cardiomyocyte-like cells and neuronal-like cells. This reversible system is the first to function from undifferentiated to individual well-differentiated ES cells, providing a very useful tool to understand molecular mechanisms underlying ES cell self-renewal, commitment and differentiation.

Cognitive factors related to suicidal ideation and resolution in psychiatrically hospitalized children and adolescents.

OBJECTIVE: This study was conducted to determine whether there are changes in the cognitive factors of attributional style, hopelessness, and self-esteem when suicidal ideation fades in psychiatrically hospitalized children and adolescents. METHOD: The cognitive factors of attributional style, hopelessness, and self-esteem were assessed in subjects aged 7-17 years (50 with and 50 without suicidal ideation) at admission and discharge from a psychiatric hospital. RESULTS: For subjects with suicidal ideation, attributional style became significantly more positive and hopelessness was decreased from admission to discharge, by which time suicidal ideation had faded. There was no association between self-esteem and suicidal ideation after control for depression. These changes in cognitive factors were not seen in the group without suicidal ideation. There were no significant differences between children and adolescents in the pattern of results. CONCLUSIONS: Change in attributional style was shown to be a factor significantly related to the resolution of suicidal ideation in children and adolescents. This cognitive style could be specifically addressed in psychotherapy with depressed children and adolescents as a means of reducing suicidal ideation. These results may have an implication for reducing the length of psychiatric inpatient stays.

Influence of asthma education on asthma severity, quality of life and environmental control.

BACKGROUND: Several studies have examined the influence of asthma education, focusing mainly on the use of health services. OBJECTIVES: To assess the influence of an asthma education program (AEP) on airway responsiveness, asthma symptoms, patient quality of life (QOL) and environmental control. DESIGN: A prospective, randomized, controlled study with parallel groups. SETTING: Three tertiary care hospitals in Quebec. POPULATION: One hundred and eighty-eight patients with moderate to severe asthma. INTERVENTION: After optimization of asthma treatment with inhaled corticosteroids, patients were randomly assigned to receive either an education program based on self-management (group E) or usual care (control group C). RESULTS: One year after an AEP, there was a significant decrease in the number of days per month without daytime asthma symptoms in group E only (P=0.03). Asthma daily symptom scores decreased significantly in group E in comparison with group C (P=0. 006). QOL scores improved markedly in both groups after treatment optimization during the run-in period (P<0.01). After an AEP, the QOL score increased further in group E patients in comparison with group C patients (P=0.04). The concentration of methacholine that induces a 20% fall in forced expiratory volume in 1 s (PC20) improved significantly in both groups (group E 1.2+/-1.1 to 2.4+/-0. 2, group C 1.5+/-1.2 to 2.4+/-1.3, P<0.01). After one year, 26 of 37 patients from group E sensitized to house dust mites (HDM) adopted the specific measures recommended to reduce their exposure to HDM, while none of the 21 subjects from group C did (P<0.001). Among the patients sensitized to cats or dogs, 15% of patients from group E and 23% of patients in group C no longer had a pet at home at the final visit (P>0.5). CONCLUSIONS: One year after the educational intervention, it was observed that the program had added value over and above that of optimization of medication and regular clinical follow-ups. The education program was highly effective in promoting HDM avoidance measures but minimally effective for removing domestic animals, suggesting that more efficient strategies need to be developed for the latter.

Parameters of suicidal crises vary as a function of previous suicide attempts in youth inpatients.

OBJECTIVES: Although suicidal crises demand an enormous amount of clinical attention, surprisingly little empirical research has been conducted on the parameters of suicidal crises in general, and in children and adolescents in particular. On the basis of past conceptual work on the unique characteristics of multiple suicide attempters, as well as work on the effect of previous suicidal and depressive experience on later functioning, the authors developed predictions regarding the intensity and duration of suicidal crises in youths presenting to inpatient psychiatry units. Specifically, it was hypothesized that multiple attempt status would relate significantly to intensity of suicidal crises and would relate more strongly to intensity than to duration of crises. METHOD: Data on past suicide history and self-rated symptoms were collected for 50 suicidal patients, all of whom were available at follow-up. RESULTS: Findings conformed to prediction: Multiple attempters experienced more intense but not more long-lasting crises; the relation between multiple attempt status and crisis intensity exceeded that between multiple attempt status and crisis duration. CONCLUSIONS: Previous suicidal experience may alter the parameters of current suicidal crises. Implications of these findings for suicide risk and clinical assessment and management are discussed.

Caspase-independent commitment phase to apoptosis in activated blood T lymphocytes: reversibility at low apoptotic insult.

Little is known about the mechanisms of programmed death triggered in T lymphocytes by stimuli that can bypass caspase activation. Anti-CD2 monoclonal antibody and staurosporine are such apoptosis inducers because they operate in the presence of broad-spectrum caspase inhibitors BOC-D.fmk and Z-VAD.fmk. A system was devised, based on the isolation according to density of activated blood T cells progressively engaged in the apoptotic process. This allowed definition of a sequence of caspase-dependent and caspase-independent apoptogenic events that are triggered by anti-CD2 and staurosporine. Thus, a commitment phase to apoptosis was defined that is entirely caspase independent and that is characterized by cell volume loss, partial chromatin condensation, and release into the cytosol and the nucleus of mitochondrial "apoptosis-inducing factor " (AIF). Committed cells were viable, displayed a high mitochondrial inner transmembrane potential (triangle upPsim), and lacked large-scale and oligonucleosomal DNA fragmentation. Mitochondrial release of AIF was selective because cytochrome c was retained in mitochondria of the very same cells. Mitochondrial release of cytochrome c occurred later, at the onset of the execution phase of apoptosis, concurrently with triangle upPsim collapse, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation. The apoptogenic events of this commitment phase are reversible if the strength of the stimulus is low and of short duration.

Antibody-mediated endocytosis of G250 tumor-associated antigen allows targeted gene transfer to human renal cell carcinoma in vitro.

Specific gene transfer into targeted tumor cells remains a critical issue for the development of systemic gene therapy protocols. With this end in view, we have tested the possibility of selectively directing genes to tumor cells through the recognition of tumor-associated antigens (TAA). This was approached in vitro on four human renal cell carcinoma (RCC) lines by means of the highly specific mouse G250 monoclonal antibody (mAb) chemically conjugated to a plasmid DNA conveying a reporter activity. This mAb directed to a TAA that is present on 95% of primary RCCs and on 60% of metastatic human RCCs was extensively characterized, including during clinical trials. Epifluorescence microscopy analysis indicated that upon specific binding to G250 TAA, G250 mAb alone or conjugated to plasmid DNA was internalized by an active endocytic process and colocalized with the transferrin concentrated in the late recycling perinuclear compartment. We also observed that both unconjugated G250 mAb or G250 mAb conjugated to plasmid DNA remained in the perinuclear region of the cells for > or = 20 hours and were not rapidly translocated to lysosomes or recycled to the plasma membrane. In contrast, unconjugated plasmid DNA was not internalized. After transfection of G250 TAA-positive RCC lines with G250 mAb conjugated to a plasmid cDNA encoding mouse interleukin-2, a significant and sustained production of mouse interleukin-2 protein was detected from days 5-15 and was abrogated by inhibiting the internalization process. Altogether, our data showed that endocytosis of G250 TAA should be the basis of gene transfer to RCC, suggesting that targeting of TAA capable of internalization may be the basis of new approaches for designing alternative cancer gene therapy procedures.