Reversal of mitochondrial malate dehydrogenase 2 enables anaplerosis via redox rescue in respiration-deficient cells.

Inhibition of the electron transport chain (ETC) prevents the regeneration of mitochondrial NAD+, resulting in cessation of the oxidative tricarboxylic acid (TCA) cycle and a consequent dependence upon reductive carboxylation for aspartate synthesis. NAD+ regeneration alone in the cytosol can rescue the viability of ETC-deficient cells. Yet, how this occurs and whether transfer of oxidative equivalents to the mitochondrion is required remain unknown. Here, we show that inhibition of the ETC drives reversal of the mitochondrial aspartate transaminase (GOT2) as well as malate and succinate dehydrogenases (MDH2 and SDH) to transfer oxidative NAD+ equivalents into the mitochondrion. This supports the NAD+-dependent activity of the mitochondrial glutamate dehydrogenase (GDH) and thereby enables anaplerosis-the entry of glutamine-derived carbon into the TCA cycle and connected biosynthetic pathways. Thus, under impaired ETC function, the cytosolic redox state is communicated into the mitochondrion and acts as a rheostat to support GDH activity and cell viability.

ITPase deficiency causes a Martsolf-like syndrome with a lethal infantile dilated cardiomyopathy.

Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated "mutation negative" probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA-and by implication rI production-correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.

Recovery High School Attendance Effects on Student Delinquency and Substance Use: the Moderating Role of Social Problem Solving Styles.

Recovery high schools (RHSs) provide educational programming and therapeutic support services for young people in recovery from substance use disorders (SUDs). The objectives of this study were to examine whether students with SUDs who attended RHSs report less delinquency and substance use than students with SUDs who attended non-RHSs, and how students' social problem solving styles might moderate those associations. Participants were students from a longitudinal quasi-experimental study of adolescents who enrolled in high schools after receiving treatment for SUDs. The propensity-score balanced sample included 260 adolescents (143 in RHSs, 117 in non-RHSs) enrolled in schools in Minnesota, Wisconsin, or Texas (M age = 16; 83% White; 44% female). Negative binomial regression models were used to compare delinquency and substance use outcomes for RHS and non-RHS students at 6-month and 12-month follow-ups. The results indicated that students attending RHSs after discharge from SUD treatment reported less frequent delinquent behavior while intoxicated, and fewer days of substance use relative to students attending non-RHSs. Negative problem solving styles moderated the effect of RHS attendance on substance use outcomes, with RHSs providing minimal beneficial effects for those students endorsing maladaptive problem solving styles. We conclude that RHSs offer a promising continuing care approach for adolescents in recovery from SUD problems, but may vary in their effectiveness for students with impulsive, careless, or avoidant problem solving styles.

Net Benefits of Recovery High Schools: Higher Cost but Increased Sobriety and Educational Attainment.

BACKGROUND: Recovery high schools (RHS) provide a supportive educational and therapeutic environment for students subsequent to treatment for substance use disorders (SUDs). Most students served by RHSs have concurrent mental health disorders and are at risk for school failure or dropout and substance use relapse. AIMS OF THE STUDY: The central question addressed is whether RHSs are economically efficient alternatives to other high school settings for students in recovery. The aim is to estimate the incremental cost-benefit of RHSs. METHODS: A quasi-experimental non-equivalent pretest-posttest comparison group design was used. We compared substance use and educational outcomes for adolescents who had received specialty SUD treatment; 143 who enrolled in an RHS were compared to 117 who enrolled in a non-RHS school. Groups were balanced by use of a propensity score to drop students who were not similar to those in the other group. The propensity score was also used as a covariate in multiple regression to estimate cost and outcome parameters and standard errors. To take account of uncertainties in impacts and shadow prices, we used Monte Carlo simulations to estimate the distribution of incremental benefits of RHS relative to non-RHS schooling. RESULTS: Two beneficial impacts of statistical and substantive importance were identified: increased probability of high school graduation and increased sobriety. RHS students had significantly (p<.05) less substance use during the study period -- at 12-month follow-up, 55% of RHS and 26% of comparison students reported 3 month abstinence from alcohol and drugs. Urinalysis confirmed abstinence from THC (cannabis) for 68% of RHS versus 37% of comparison students. RHS students' high school graduation rates were 21 to 25 percentage points higher than comparison students. Adopting a societal perspective, incremental benefits of RHSs were estimated by monetizing the increased probability of high school graduation and comparing it to incremental costs. Mean net benefits ranged from USD16.1 thousand to USD51.9 thousand per participant; benefit-to-cost ratios ranged from 3.0 to 7.2. DISCUSSION: Monetizing the benefits and the incremental costs of RHS relative to conventional schooling show substantial positive net benefits from RHS participation. Two factors lend credibility to the results. First, the RHS improvement in substance use indicates a mechanism through which the increased probability of high school graduation can plausibly occur. Second, the estimated increases in the probability of high school graduation were large and statistically significant. As the productivity gains from high school graduation are also large, the dominant benefit category is very plausible. Limitations include the non-randomized design; selection bias into the study conditions not fully controlled by the propensity scores; generalizability only to young people with treated behavioral health disorders; lack of estimates for direct monetization of reduced substance use among adolescents; possible attenuation of the value of education among individuals with behavioral health issues; and uncertainty in calculation of school costs. IMPLICATIONS FOR BEHAVIORAL HEALTH POLICIES: This research provides evidence that the recovery high school model provides cost beneficial support for high school students after primary SUD treatment. The students who enroll in RHSs typically have co-occurring mental health and substance use disorders, adding complexity to their continuing care. Funding policies recognizing the multiple systems of care (behavioral health, education, child and family services, juvenile justice) responsible for these young people are called for.

Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome.

Removal of the assembly factor eukaryotic initiation factor 6 (eIF6) is critical for late cytoplasmic maturation of 60S ribosomal subunits. In mammalian cells, the current model posits that eIF6 release is triggered following phosphorylation of Ser 235 by activated protein kinase C. In contrast, genetic studies in yeast indicate a requirement for the ortholog of the SBDS (Shwachman-Bodian-Diamond syndrome) gene that is mutated in the inherited leukemia predisposition disorder Shwachman-Diamond syndrome (SDS). Here, by isolating late cytoplasmic 60S ribosomal subunits from Sbds-deleted mice, we show that SBDS and the GTPase elongation factor-like 1 (EFL1) directly catalyze eIF6 removal in mammalian cells by a mechanism that requires GTP binding and hydrolysis by EFL1 but not phosphorylation of eIF6 Ser 235. Functional analysis of disease-associated missense variants reveals that the essential role of SBDS is to tightly couple GTP hydrolysis by EFL1 on the ribosome to eIF6 release. Furthermore, complementary NMR spectroscopic studies suggest unanticipated mechanistic parallels between this late step in 60S maturation and aspects of bacterial ribosome disassembly. Our findings establish a direct role for SBDS and EFL1 in catalyzing the translational activation of ribosomes in all eukaryotes, and define SDS as a ribosomopathy caused by uncoupling GTP hydrolysis from eIF6 release.

Recovery high schools: Effect of schools supporting recovery from substance use disorders.

BACKGROUND: Recovery high schools (RHSs) provide post-treatment education and recovery support for young people with substance use disorders (SUDs). This is the first quasi-experimental outcome study to determine RHS effectiveness relative to students in non-RHSs. OBJECTIVES: To examine effects of RHS attendance on academic and substance use outcomes among adolescents treated for SUDs 6 months after recruitment to the study. METHODS: A quasi-experimental design comparing outcomes for adolescents with treated SUDs who attended RHSs for at least 28 days versus a propensity-score balanced sample of students with treated SUDs who did not attend RHSs. The sample included 194 adolescents (134 in RHSs, 60 in non-RHSs) enrolled in Minnesota, Wisconsin, or Texas schools (M age = 16; 86% White; 49% female). Multilevel linear regression models were used to examine the effect of RHS attendance on students' outcomes, after adjusting for a range of potential confounders. RESULTS: Adolescents attending RHSs were significantly more likely than non-RHS students to report complete abstinence from alcohol, marijuana, and other drugs at the 6-month follow-up (OR = 4.36, p = .026), significantly lower levels of marijuana use (d = -0.51, p = .034) and less absenteeism from school (d = -0.56, p = .028). CONCLUSION: These results indicate that RHSs have significantly beneficial effects on substance use and school absenteeism after 6 months for adolescents treated for SUDs.

AssayR: A Simple Mass Spectrometry Software Tool for Targeted Metabolic and Stable Isotope Tracer Analyses.

Metabolic analyses generally fall into two classes: unbiased metabolomic analyses and analyses that are targeted toward specific metabolites. Both techniques have been revolutionized by the advent of mass spectrometers with detectors that afford high mass accuracy and resolution, such as time-of-flights (TOFs) and Orbitraps. One particular area where this technology is key is in the field of metabolic flux analysis because the resolution of these spectrometers allows for discrimination between 13C-containing isotopologues and those containing 15N or other isotopes. While XCMS-based software is freely available for untargeted analysis of mass spectrometric data sets, it does not always identify metabolites of interest in a targeted assay. Furthermore, there is a paucity of vendor-independent software that deals with targeted analyses of metabolites and of isotopologues in particular. Here, we present AssayR, an R package that takes high resolution wide-scan liquid chromatography-mass spectrometry (LC-MS) data sets and tailors peak detection for each metabolite through a simple, iterative user interface. It automatically integrates peak areas for all isotopologues and outputs extracted ion chromatograms (EICs), absolute and relative stacked bar charts for all isotopologues, and a .csv data file. We demonstrate several examples where AssayR provides more accurate and robust quantitation than XCMS, and we propose that tailored peak detection should be the preferred approach for targeted assays. In summary, AssayR provides easy and robust targeted metabolite and stable isotope analyses on wide-scan data sets from high resolution mass spectrometers.

'Pickle or a cucumber?' administrator and practitioner views of successful adolescent recovery.

INTRODUCTION: Adolescent substance use disorders often involve a recurring cycle of treatment and relapse. The academic and practical definition of addition recovery for adults has been debated; yet, elements determining a successful adolescent recovery aside from abstinence have not been delineated. Thus, we sought to explore how practitioners and administrators define "success" in recovery and how they foster youth progress towards success. METHODS: Using a qualitative design, we purposively selected and visited treatment and recovery services sites and interviewed practitioners and administrators (N = 13). Sites included recovery high schools (N = 2), alternative peer groups (N = 4), and one treatment center. Two authors analyzed the data using the constant comparative method. RESULTS: Success emerged from the interviews in three primary themes (1) factors demonstrating success, (2) progress that highlights success, and (3) factors enabling success and two sub-themes (1) use of metaphors and (2) use of specific examples. A variety of factors and processes were discussed as indicators of success. Multiple practitioners stated that sobriety and length of abstinence were not the best success measures; yet, sobriety and education were mentioned most often. CONCLUSIONS: A key finding of this study, which has not been addressed in existing qualitative studies of youth recovery, is that the understanding of recovery was so diverse and multi-dimensional and provided a view of success beyond sobriety, highlighting the various facets from which practitioners must operate and address recovery. This demonstrates the need for researchers to carefully conceptualize how they operationalize adolescent recovery.

Aberrant 3' oligoadenylation of spliceosomal U6 small nuclear RNA in poikiloderma with neutropenia.

The recessive disorder poikiloderma with neutropenia (PN) is caused by mutations in the C16orf57 gene that encodes the highly conserved USB1 protein. Here, we present the 1.1 Å resolution crystal structure of human USB1, defining it as a member of the LigT-like superfamily of 2H phosphoesterases. We show that human USB1 is a distributive 3'-5' exoribonuclease that posttranscriptionally removes uridine and adenosine nucleosides from the 3' end of spliceosomal U6 small nuclear RNA (snRNA), directly catalyzing terminal 2', 3' cyclic phosphate formation. USB1 measures the appropriate length of the U6 oligo(U) tail by reading the position of a key adenine nucleotide (A102) and pausing 5 uridine residues downstream.We show that the 3' ends of U6 snRNA in PN patient lymphoblasts are elongated and unexpectedly carry nontemplated 3' oligo(A) tails that are characteristic of nuclear RNA surveillance targets. Thus, our study reveals a novel quality control pathway in which posttranscriptional 3'-end processing by USB1 protects U6 snRNA from targeting and destruction by the nuclear exosome. Our data implicate aberrant oligoadenylation of U6 snRNA in the pathogenesis of the leukemia predisposition disorder PN.

Mdm2 is critically and continuously required to suppress lethal p53 activity in vivo.

There is currently much interest in the idea of restoring p53 activity in tumor cells by inhibiting Hdm2/Mdm2. However, it has remained unclear whether this would also activate p53 in normal cells. Using a switchable endogenous p53 mouse model, which allows rapid and reversible toggling of p53 status between wild-type and null states, we show that p53 is spontaneously active in all tested tissues of mdm2-deficient mice, triggering fatal pathologies that include ablation of classically radiosensitive tissues. In apoptosis-resistant tissues, spontaneous unbuffered p53 activity triggers profound inhibition of cell proliferation. Such acute spontaneous p53 activity occurs in the absence of any detectable p53 posttranslational modification, DNA damage, or p19ARF signaling and triggers rapid p53 degradation.

Modelling Myc inhibition as a cancer therapy.

Myc is a pleiotropic basic helix-loop-helix leucine zipper transcription factor that coordinates expression of the diverse intracellular and extracellular programs that together are necessary for growth and expansion of somatic cells. In principle, this makes inhibition of Myc an attractive pharmacological approach for treating diverse types of cancer. However, enthusiasm has been muted by lack of direct evidence that Myc inhibition would be therapeutically efficacious, concerns that it would induce serious side effects by inhibiting proliferation of normal tissues, and practical difficulties in designing Myc inhibitory drugs. We have modelled genetically both the therapeutic impact and the side effects of systemic Myc inhibition in a preclinical mouse model of Ras-induced lung adenocarcinoma by reversible, systemic expression of a dominant-interfering Myc mutant. We show that Myc inhibition triggers rapid regression of incipient and established lung tumours, defining an unexpected role for endogenous Myc function in the maintenance of Ras-dependent tumours in vivo. Systemic Myc inhibition also exerts profound effects on normal regenerating tissues. However, these effects are well tolerated over extended periods and rapidly and completely reversible. Our data demonstrate the feasibility of targeting Myc, a common downstream conduit for many oncogenic signals, as an effective, efficient and tumour-specific cancer therapy.

Continuing Care in High Schools: A Descriptive Study of Recovery High School Programs.

Data from 17 recovery high schools suggest programs are dynamic and vary in enrollment, fiscal stability, governance, staffing, and organizational structure. Schools struggle with enrollment, funding, lack of primary treatment accessibility, academic rigor, and institutional support. Still, for adolescents having received treatment for substance abuse, recovery schools appear to successfully function as continuing care providers reinforcing and sustaining therapeutic benefits gained from treatment. Small size and therapeutic programming allow for a potentially broader continuum of services than currently exists in most of the schools. Recovery schools thus provide a useful design for continuing care warranting further study and policy support.

Awareness of peers in recovery and of a campus collegiate recovery community at a university in the southeastern United States.

Objective: Little is known about the extent of student awareness about collegiate recovery communities (CRCs) and of peers in recovery. Participants: A convenience sample of 237 undergraduate students from a diverse major at a private university participated in an anonymous online survey in Fall 2019. Methods: Participants reported whether they knew about the local CRC, whether they knew a peer in recovery, sociodemographic characteristics, and other information. Multivariable modified Poisson regression models were fitted to estimate correlates of awareness of the CRC and of peers in recovery. Results: Overall, 34% were aware of the CRC and 39% knew a peer in recovery. The latter was associated with being a member of Greek life, a junior or senior, using substances regularly, and personally being in recovery. Conclusions: Future research should explore ways to increase awareness of CRCs and assess the role of connections between students in recovery and peers across campus.

Spotlight on New Therapeutic Opportunities for MYC-Driven Cancers.

MYC can be considered to be one of the most pressing and important targets for the development of novel anti-cancer therapies. This is due to its frequent dysregulation in tumors and due to the wide-ranging impact this dysregulation has on gene expression and cellular behavior. As a result, there have been numerous attempts to target MYC over the last few decades, both directly and indirectly, with mixed results. This article reviews the biology of MYC in the context of cancers and drug development. It discusses strategies aimed at targeting MYC directly, including those aimed at reducing its expression and blocking its function. In addition, the impact of MYC dysregulation on cellular biology is outlined, and how understanding this can underpin the development of approaches aimed at molecules and pathways regulated by MYC. In particular, the review focuses on the role that MYC plays in the regulation of metabolism, and the therapeutic avenues offered by inhibiting the metabolic pathways that are essential for the survival of MYC-transformed cells.

Vitamin B5 and succinyl-CoA improve ineffective erythropoiesis in SF3B1-mutated myelodysplasia.

Patients with myelodysplastic syndrome and ring sideroblasts (MDS-RS) present with symptomatic anemia due to ineffective erythropoiesis that impedes their quality of life and increases morbidity. More than 80% of patients with MDS-RS harbor splicing factor 3B subunit 1 (SF3B1) mutations, the founder aberration driving MDS-RS disease. Here, we report how mis-splicing of coenzyme A synthase (COASY), induced by mutations in SF3B1, affects heme biosynthesis and erythropoiesis. Our data revealed that COASY was up-regulated during normal erythroid differentiation, and its silencing prevented the formation of erythroid colonies, impeded erythroid differentiation, and precluded heme accumulation. In patients with MDS-RS, loss of protein due to COASY mis-splicing led to depletion of both CoA and succinyl-CoA. Supplementation with COASY substrate (vitamin B5) rescued CoA and succinyl-CoA concentrations in SF3B1mut cells and mended erythropoiesis differentiation defects in MDS-RS primary patient cells. Our findings reveal a key role of the COASY pathway in erythroid maturation and identify upstream and downstream metabolites of COASY as a potential treatment for anemia in patients with MDS-RS.

MYC sensitises cells to apoptosis by driving energetic demand.

The MYC oncogene is a potent driver of growth and proliferation but also sensitises cells to apoptosis, which limits its oncogenic potential. MYC induces several biosynthetic programmes and primary cells overexpressing MYC are highly sensitive to glutamine withdrawal suggesting that MYC-induced sensitisation to apoptosis may be due to imbalance of metabolic/energetic supply and demand. Here we show that MYC elevates global transcription and translation, even in the absence of glutamine, revealing metabolic demand without corresponding supply. Glutamine withdrawal from MRC-5 fibroblasts depletes key tricarboxylic acid (TCA) cycle metabolites and, in combination with MYC activation, leads to AMP accumulation and nucleotide catabolism indicative of energetic stress. Further analyses reveal that glutamine supports viability through TCA cycle energetics rather than asparagine biosynthesis and that TCA cycle inhibition confers tumour suppression on MYC-driven lymphoma in vivo. In summary, glutamine supports the viability of MYC-overexpressing cells through an energetic rather than a biosynthetic mechanism.

Spatiofunctional Dynamics of NKX3.1 to Safeguard the Prostate from Cancer.

A novel role of NKX3.1 in the mitochondria regulating the transcription of the electron transport chain components is reported. Mechanistically, HSPA9 chaperones NKX3.1 into the mitochondria in response to oxidative stress to regulate reactive oxygen species and suppress tumor initiation.See related article by Papachristodoulou et al., p. 2316.

Defining the fetal origin of MLL-AF4 infant leukemia highlights specific fatty acid requirements.

Infant MLL-AF4-driven acute lymphoblastic leukemia (ALL) is a devastating disease with dismal prognosis. A lack of understanding of the unique biology of this disease, particularly its prenatal origin, has hindered improvement of survival. We perform multiple RNA sequencing experiments on fetal, neonatal, and adult hematopoietic stem and progenitor cells from human and mouse. This allows definition of a conserved fetal transcriptional signature characterized by a prominent proliferative and oncogenic nature that persists in infant ALL blasts. From this signature, we identify a number of genes in functional validation studies that are critical for survival of MLL-AF4+ ALL cells. Of particular interest are PLK1 because of the readily available inhibitor and ELOVL1, which highlights altered fatty acid metabolism as a feature of infant ALL. We identify which aspects of the disease are residues of its fetal origin and potential disease vulnerabilities.

De novo DNA methyltransferase activity in colorectal cancer is directed towards H3K36me3 marked CpG islands.

The aberrant gain of DNA methylation at CpG islands is frequently observed in colorectal tumours and may silence the expression of tumour suppressors such as MLH1. Current models propose that these CpG islands are targeted by de novo DNA methyltransferases in a sequence-specific manner, but this has not been tested. Using ectopically integrated CpG islands, here we find that aberrantly methylated CpG islands are subject to low levels of de novo DNA methylation activity in colorectal cancer cells. By delineating DNA methyltransferase targets, we find that instead de novo DNA methylation activity is targeted primarily to CpG islands marked by the histone modification H3K36me3, a mark associated with transcriptional elongation. These H3K36me3 marked CpG islands are heavily methylated in colorectal tumours and the normal colon suggesting that de novo DNA methyltransferase activity at CpG islands in colorectal cancer is focused on similar targets to normal tissues and not greatly remodelled by tumourigenesis.

The hepatic compensatory response to elevated systemic sulfide promotes diabetes.

Impaired hepatic glucose and lipid metabolism are hallmarks of type 2 diabetes. Increased sulfide production or sulfide donor compounds may beneficially regulate hepatic metabolism. Disposal of sulfide through the sulfide oxidation pathway (SOP) is critical for maintaining sulfide within a safe physiological range. We show that mice lacking the liver- enriched mitochondrial SOP enzyme thiosulfate sulfurtransferase (Tst-/- mice) exhibit high circulating sulfide, increased gluconeogenesis, hypertriglyceridemia, and fatty liver. Unexpectedly, hepatic sulfide levels are normal in Tst-/- mice because of exaggerated induction of sulfide disposal, with associated suppression of global protein persulfidation and nuclear respiratory factor 2 target protein levels. Hepatic proteomic and persulfidomic profiles converge on gluconeogenesis and lipid metabolism, revealing a selective deficit in medium-chain fatty acid oxidation in Tst-/- mice. We reveal a critical role of TST in hepatic metabolism that has implications for sulfide donor strategies in the context of metabolic disease.