Prevention of mercaptopurine-induced hypoglycemia using allopurinol to reduce methylated thiopurine metabolites.

Skewing of mercaptopurine (6-MP) metabolism preferentially toward the 6-methylmercaptopurine (6-MMP) metabolite over the antileukemic metabolite 6-thioguanine (6-TGN) is associated with 6-MP-related hepatotoxocity. Allopurinol when coadministered with 6-MP can reduce this skewing and ameliorate the associated adverse effects. The cases we report here demonstrate that aberrant overproduction of 6-MMP is also associated with profound 6-MP-associated hypoglycemia, which can be reversed by administration of allopurinol. This case series contributes to the scant literature on 6-MP-induced hypoglycemia and provides evidence that addition of allopurinol to reduced dose 6-MP can successfully manage this severe toxicity.

24-Methylenecyclopropane steroidal inhibitors: A Trojan horse in ergosterol biosynthesis that prevents growth of Trypanosoma brucei.

A new class of steroidal therapeutics based on phylogenetic-guided design of covalent inhibitors that target parasite-specific enzymes of ergosterol biosynthesis is shown to prevent growth of the protozoan-Trypanosoma brucei, responsible for sleeping sickness. In the presence of approximately 15±5µM 26,27-dehydrolanosterol, T. brucei procyclic or blood stream form growth is inhibited by 50%. This compound is actively converted by the parasite to an acceptable substrate of sterol C24-methyl transferase (SMT) that upon position-specific side chain methylation at C26 inactivates the enzyme. Treated cells show dose-dependent depletion of ergosterol and other 24ß-methyl sterols with no accumulation of intermediates in contradistinction to profiles typical of tight binding inhibitor treatments to azoles showing loss of ergosterol accompanied by accumulation of toxic 14-methyl sterols. HEK cells accumulate 26,27-dehydrolanosterol without effect on cholesterol biosynthesis. During exposure of cloned TbSMT to 26,27-dehydrozymosterol, the enzyme is gradually inactivated (kcat/kinact=0.13min-1/0.08min-1; partition ratio of 1.6) while 26,27-dehydrolanosterol binds nonproductively. GC-MS analysis of the turnover product and bound intermediate released as a C26-methylated diol (C3-OH and C24-OH) confirmed substrate recognition and covalent binding to TbSMT. This study has potential implications for design of a novel class of chemotherapeutic leads functioning as mechanism-based inhibitors of ergosterol biosynthesis to treat neglected tropical diseases.

Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth.

Ergosterol biosynthesis and homeostasis in the parasitic protozoan Trypanosoma brucei was analyzed by RNAi silencing and inhibition of sterol C24ß-methyltransferase (TbSMT) and sterol 14α-demethylase [TbSDM (TbCYP51)] to explore the functions of sterols in T. brucei growth. Inhibition of the amount or activity of these enzymes depletes ergosterol from cells at <6 fg/cell for procyclic form (PCF) cells or <0.01 fg/cell for bloodstream form (BSF) cells and reduces infectivity in a mouse model of infection. Silencing of TbSMT expression by RNAi in PCF or BSF in combination with 25-azalanosterol (AZA) inhibited parasite growth and this inhibition was restored completely by adding synergistic cholesterol (7.8 µM from lipid-depleted media) with small amounts of ergosterol (1.2 µM) to the medium. These observations are consistent with the proposed requirement for ergosterol as a signaling factor to spark cell proliferation while imported cholesterol or the endogenously formed cholesta-5,7,24-trienol act as bulk membrane components. To test the potential chemotherapeutic importance of disrupting ergosterol biosynthesis using pairs of mechanism-based inhibitors that block two enzymes in the post-squalene segment, parasites were treated with AZA and itraconazole at 1 µM each (ED50 values) resulting in parasite death. Taken together, our results demonstrate that the ergosterol pathway is a prime drug target for intervention in T. brucei infection.

Dose-dependent effects of Dnmt3a in an inducible murine model of KrasG12D-driven leukemia.

DNMT3A mutations are frequently found in clonal hematopoiesis and a variety of hematologic malignancies, including acute myeloid leukemia. An assortment of mouse models have been engineered to explore the tumorigenic potential and malignant lineage bias due to loss of function of DNMT3A in consort with commonly comutated genes in myeloid malignancies, such as Flt3, Nras, Kras, and c-Kit. We employed several tamoxifen-inducible Cre-ERT2 murine model systems to study the effects of constitutively active KrasG12D-driven myeloid leukemia (Kras) development together with heterozygous (3aHet) or homozygous Dnmt3a deletion (3aKO). Due to the rapid generation of diverse nonhematologic tumors appearing after tamoxifen induction, we employed a transplantation model. With pretransplant tamoxifen induction, most Kras mice died quickly of T-cell malignancies regardless of Dnmt3a status. Using posttransplant induction, we observed a dose-dependent effect of DNMT3A depletion that skewed the leukemic phenotype toward a myeloid lineage. Specifically, 64% of 3aKO/Kras mice had exclusively myeloid disease compared with 36% of 3aHet/Kras and only 13% of Kras mice. Here, 3aKO combined with Kras led to increased disease burden, multiorgan infiltration, and faster disease progression. DOT1L inhibition exerted profound antileukemic effects in malignant 3aKO/Kras cells, but not malignant cells with Kras mutation alone, consistent with the known sensitivity of DNMT3A-mutant leukemia to DOT1L inhibition. RNAseq from malignant myeloid cells revealed that biallelic Dnmt3a deletion was associated with loss of cell-cycle regulation, MYC activation, and TNF⍺ signaling. Overall, we developed a robust model system for mechanistic and preclinical investigations of acute myeloid leukemia with DNMT3A and Ras-pathway lesions.

Effects of Multicomponent Injury Prevention Programs on Children and Adolescents' Fundamental Movement Skills: A Systematic Review With Meta-Analyses.

OBJECTIVE: Fundamental movement skills (FMS) are essential to participate in physical activity. Understanding the effects of multicomponent injury prevention programs (MIPP) on FMS may help promote safe physical activity. Our objective was to synthesize the evidence on the effects of MIPP on biomechanical outcomes and neuromuscular performance measured on children and adolescents while performing FMS. DATA SOURCE: We searched PubMed, SPORTDiscus, Web of Science, and SCOPUS. STUDY INCLUSION AND EXCLUSION CRITERIA: We included peer-reviewed randomized controlled trials, published in English, that analyzed the effects of MIPP on biomechanics and neuromuscular performance of FMS in participants under 18 years of age. DATA EXTRACTION: Two reviewers screened the articles, assessed the quality of the evidence using the Physiotherapy Evidence Database (PEDro) scale, and synthesized the data. DATA SYNTHESIS: We conducted meta-analyses and reported the characteristics, outcomes, and risk of bias of studies. RESULTS: We included 27 articles that reported data from 1,427 participants. Positive effects on FMS were reported in 23 of the 27 included articles. Vertical Jump, running speed, acceleration, and dynamic balance presented positive-significant pooled effect sizes. Dribbling and horizontal jump presented non-significant pooled effect sizes. CONCLUSION: MIPP can positively affect FMS in children and adolescents in sports-related settings. Lack of participant compliance and implementation fidelity may affect MIPP effectiveness. Including MIPP in physical literacy interventions, physical education classes, and organized physical activity may lead to functional adaptations that help promote safe physical activity.

Evolutionarily conserved Delta(25(27))-olefin ergosterol biosynthesis pathway in the alga Chlamydomonas reinhardtii.

Ergosterol is the predominant sterol of fungi and green algae. Although the biosynthetic pathway for sterol synthesis in fungi is well established and is known to use C24-methylation-C24 (28)-reduction (Δ(24(28))-olefin pathway) steps, little is known about the sterol pathway in green algae. Previous work has raised the possibility that these algae might use a novel pathway because the green alga Chlamydomonas reinhardtii was shown to possess a mevalonate-independent methylerythritol 4-phosphate not present in fungi. Here, we report that C. reinhardtii synthesizes the protosterol cycloartenol and converts it to ergosterol (C24ß-methyl) and 7-dehydroporiferasterol (C24ß-ethyl) through a highly conserved sterol C24- methylation-C25-reduction (Δ(25(27))-olefin) pathway that is distinct from the well-described acetate-mevalonate pathway to fungal lanosterol and its conversion to ergosterol by the Δ(24(28))-olefin pathway. We isolated and characterized 23 sterols by a combination of GC-MS and proton nuclear magnetic resonance spectroscopy analysis from a set of mutant, wild-type, and 25-thialanosterol-treated cells. The structure and stereochemistry of the final C24-alkyl sterol side chains possessed different combinations of 24ß-methyl/ethyl groups and Δ(22(23))E and Δ(25(27))-double bond constructions. When incubated with [methyl-(2)H(3)]methionine, cells incorporated three (into ergosterol) or five (into 7-dehydroporiferasterol) deuterium atoms into the newly biosynthesized 24ß-alkyl sterols, consistent only with a Δ(25(27))-olefin pathway. Thus, our findings demonstrate that two separate isoprenoid-24-alkyl sterol pathways evolved in fungi and green algae, both of which converge to yield a common membrane insert ergosterol.

The Impact of an After-School Physical Activity Program on Children's Physical Activity and Well-Being during the COVID-19 Pandemic: A Mixed-Methods Evaluation Study.

INTRODUCTION: This study evaluated the impact of the Build Our Kids' Success (BOKS) after-school program on children's physical activity (PA) and well-being during the COVID-19 pandemic. METHODS: Program leaders, children, and their parents were recruited from after-school programs in Nova Scotia, Canada, that delivered BOKS programming in Fall 2020. After participating, Grade 4-6 children (n = 14) completed the Physical Literacy Assessment for Youth Self (PLAYself), Physical Activity Questionnaire for Older Children (PAQ-C), the Physical Activity Enjoyment Scale (PACES), and 5 National Institutes of Health (NIH) Patient-Reported Outcomes Measures Information System (PROMIS) scales. Children (n = 7), parents (n = 5), and program leaders (n = 3) completed interviews, which were analyzed for themes inductively. RESULTS: The average PAQ-C score was 2.70 ± 0.48, PLAYself was 68.23 ± 13.12, and PACES was 4.22 ± 0.59 (mean ± SD). NIH PROMIS scores were below standard means (cognitive function, family relationships) or within normal limits (peer relationships, positive affect, and life satisfaction). A thematic analysis of interviews revealed that children's PA levels were impacted by the pandemic and that BOKS positively impacted children's physical well-being and integrated well with school-based activities. CONCLUSIONS: Participation in BOKS provided an overall positive experience and may have mitigated COVID-19-related declines in PA in well-being. The results of this evaluation can inform future physically-active after-school programming.

The Effect of an After-School Physical Activity Program on Children's Cognitive, Social, and Emotional Health during the COVID-19 Pandemic in Nova Scotia.

Children's physical activity participation declined during the COVID-19 pandemic, and these negative changes could lead to longer-term impacts on children's cognitive, social, and emotional health. PURPOSE: To determine parent/caregivers' perceptions of their children's cognitive function, peer and family relationships, life satisfaction, physical activity, sleep, positive affect, and global health, before and after participating in the Build Our Kids' Success (BOKS) programming at after-school programs in Fall 2020. METHODS: Parents of children participating in the BOKS programming at after-school programs in Nova Scotia, Canada, were recruited. At baseline, 159 parents completed the National Institutes of Health (NIH) Patient-Reported Outcomes Measures Information System (PROMIS) parent-proxy questionnaire, and 75 parents completed the measures at follow-up. Independent t-tests were used to determine if there were differences between baseline and follow-up Parent Proxy Questionnaire data. RESULTS: All NIH PROMIS outcome variables at baseline and follow-up were within normal limits (Adjusted T-Scores: 46.67 ± 7.15 to 50.04 ± 7.13). There were no significant differences in life satisfaction (t(188) = -1.05, p = 0.30), family relationships (t(189) = 0.31, p = 0.76), cognitive function (t(199) = -1.16, p = 0.25), peer relationships (t(192) = -1.86, p = 0.06), positive affect (t(195) = 0.25, p = 0.81), global health (t(216) = -0.43, p = 0.67), physical activity (t(202) = 0.787, p = 0.732), sleep disturbance (t(193) = 1.72, p = 0.087), or psychological stress (t(196) = 1.896, p = 0.059), from baseline to follow-up. CONCLUSIONS: Parent-proxy questionnaires suggested that the BOKS programming had a protective effect on children's health behaviours and cognitive, social, and emotional health as values remained within normal limits and were not impacted by the public health restrictions during the second wave of the COVID-19 pandemic in Nova Scotia.

Chronic Pain Patients' Kinesiophobia and Catastrophizing are Associated with Activity Intensity at Different Times of the Day.

PURPOSE: To examine the relationship between baseline kinesiophobia and baseline pain catastrophizing with the 4-day average activity intensity at different times of the day while accounting for different wake and sleep-onset times in chronic pain patients. METHODS: Twenty-one participants suffering from idiopathic chronic pain completed baseline questionnaires about kinesiophobia, catastrophizing, disability, depression, and pain. We measured the participants' activity using accelerometers and calculated activity intensity in the morning, afternoon, and evening. We performed a 2-way repeated measures ANOVA to compare activity levels at different times of the day, and multiple linear regressions. RESULTS: Baseline kinesiophobia was significantly associated with 4-day average evening light activity and sedentary activity at all time periods while baseline catastrophizing was significantly associated with increased 4-day average light activity in the evening and more moderate to vigorous activity in the morning. Our participants engaged in more light activity on average than sedentary activity, and very little moderate-vigorous activity. Participants were most active in the afternoon. CONCLUSION: Baseline kinesiophobia and baseline catastrophizing were not associated with the 4-day average total daily activity; however, they were associated with 4-day average activity intensities at different times throughout the day. Segmenting daily activity into morning, afternoon, evening may influence the relationship between daily activity, and kinesiophobia and pain catastrophizing. Individuals with chronic pain are less sedentary than previously thought which may affect future interventions.

Overall safety of relamorelin in adults with diabetic gastroparesis: Analysis of phase 2a and 2b trial data.

BACKGROUND: Relamorelin, a pentapeptide ghrelin receptor agonist, accelerated gastric emptying significantly and improved symptoms in adults with diabetic gastroparesis in phase 2 trials. AIM: To assess the safety and tolerability of relamorelin across phase 2 trials. METHODS: Safety assessments in patients aged 18-75 years (weight, adverse events [AEs] and laboratory tests) from two randomised, double-blind phase 2 trials (NCT01571297, NCT02357420; results published previously) were reviewed descriptively. Analysis of covariance assessed treatment effect on glycated haemoglobin (HbA1c) and blood glucose post hoc. Phase 2a and 2b trial durations were, respectively, 4 weeks (relamorelin 10 µg once or twice daily [b.d.] or placebo b.d.) and 12 weeks (relamorelin 10, 30 or 100 µg or placebo b.d.) with 1- and 2-week, single-blind placebo run-ins. RESULTS: Among 204 phase 2a and 393 phase 2b patients, respectively, 67% and 62% were female, and 88% and 89% had type 2 diabetes. Proportions of patients reporting serious AEs were similar across treatment groups, as were those with ≥1 treatment-emergent AE (TEAE). TEAE-related discontinuations were proportionally higher in relamorelin groups than placebo. Of 12 serious TEAEs in phase 2a, none occurred in >1 patient. In phase 2b, five serious TEAEs were reported in >1 patient, and one (100 µg) died (urosepsis), all unrelated to relamorelin. In phase 2b, increased HbA1c and fasting blood glucose levels were dose-related (P < 0.0001 and P = 0.0043, respectively). CONCLUSIONS: Relamorelin showed acceptable safety and tolerability in phase 2 trials. Relamorelin may elevate blood glucose: this should be managed proactively in relamorelin-treated patients.

Non-pharmacological rehabilitation interventions for concussion in children: a scoping review.

PURPOSE: To summarise the extent, nature, and quality of current scholarly literature related to non-pharmacological, rehabilitation interventions following concussion, or mild traumatic brain injury in children. METHODS: An electronic search was conducted from 1987 to 24 October 2017. Studies were included if they met the following criteria: (1) full text, peer reviewed, and written in English, (2) original research, (3) diagnosed concussion or mild traumatic brain injury, (4) described the evaluation of an intervention, (5) the outcome was a concussion impairment, and (6) the mean/median age was under 19. Quality assessment using the Down's and Black criteria was conducted. RESULTS: Twenty-six studies published between 2001 and 2017 were identified. Interventions included rest, active rehabilitation, exercise, vestibular, oculomotor, cervicospinal therapy, education, early intervention, telephone counselling, mobile health application, Web-based Self-Management program, multimodal physical therapy, cognitive behavioural therapy, transcranial direct current stimulation, and acupuncture. The quality assessments ranged from poor to good. CONCLUSIONS: The literature describing interventions following concussion in children is scarce. While both positive and negative results were obtained, there were methodological concerns in most studies limiting the ability to draw conclusions. Interventions incorporating aerobic exercise show promise as a concussion management strategy. Implications for rehabilitation Few studies have examined rehabilitation interventions for youth following concussion. Research ranging from rest to exercise highlights the uncertainty of the field. Low quality research limits the generalizability of results. The use of physical activity appears to be an emerging area of interest. Individualised, aerobic exercise should be used as part of clinical management.

Fluorinated Sterols Are Suicide Inhibitors of Ergosterol Biosynthesis and Growth in Trypanosoma brucei.

Trypanosoma brucei, the causal agent for sleeping sickness, depends on ergosterol for growth. Here, we describe the effects of a mechanism-based inhibitor, 26-fluorolanosterol (26FL), which converts in vivo to a fluorinated substrate of the sterol C24-methyltransferase essential for sterol methylation and function of ergosterol, and missing from the human host. 26FL showed potent inhibition of ergosterol biosynthesis and growth of procyclic and bloodstream forms while having no effect on cholesterol biosynthesis or growth of human epithelial kidney cells. During exposure of cloned TbSMT to 26-fluorocholesta-5,7,24-trienol, the enzyme is gradually killed as a consequence of the covalent binding of the intermediate C25 cation to the active site (kcat/kinact = 0.26 min(-1)/0.24 min(-1); partition ratio of 1.08), whereas 26FL is non-productively bound. These results demonstrate that poisoning of ergosterol biosynthesis by a 26-fluorinated Δ(24)-sterol is a promising strategy for developing a new treatment for trypanosomiasis.

Characterization, mutagenesis and mechanistic analysis of an ancient algal sterol C24-methyltransferase: Implications for understanding sterol evolution in the green lineage.

Sterol C24-methyltransferases (SMTs) constitute a group of sequence-related proteins that catalyze the pattern of sterol diversity across eukaryotic kingdoms. The only gene for sterol alkylation in green algae was identified and the corresponding catalyst from Chlamydomonas reinhardtii (Cr) was characterized kinetically and for product distributions. The properties of CrSMT were similar to those predicted for an ancient SMT expected to possess broad C3-anchoring requirements for substrate binding and formation of 24ß-methyl/ethyl Δ(25(27))-olefin products typical of primitive organisms. Unnatural Δ(24(25))-sterol substrates, missing a C4ß-angular methyl group involved with binding orientation, convert to product ratios in favor of Δ(24(28))-products. Remodeling the active site to alter the electronics of Try110 (to Leu) results in delayed timing of the hydride migration from methyl attack of the Δ(24)-bond, that thereby produces metabolic switching of product ratios in favor of Δ(25(27))-olefins or impairs the second C1-transfer activity. Incubation of [27-(13)C]lanosterol or [methyl-(2)H3]SAM as co-substrates established the CrSMT catalyzes a sterol methylation pathway by the "algal" Δ(25(27))-olefin route, where methylation proceeds by a conserved SN2 reaction and de-protonation proceeds from the pro-Z methyl group on lanosterol corresponding to C27. This previously unrecognized catalytic competence for an enzyme of sterol biosynthesis, together with phylogenomic analyses, suggest that mutational divergence of a promiscuous SMT produced substrate- and phyla-specific SMT1 (catalyzes first biomethylation) and SMT2 (catalyzes second biomethylation) isoforms in red and green algae, respectively, and in the case of SMT2 selection afforded modification in reaction channeling necessary for the switch in ergosterol (24ß-methyl) biosynthesis to stigmasterol (24α-ethyl) biosynthesis during the course of land plant evolution.

The effect of a short-term high-intensity circuit training program on work capacity, body composition, and blood profiles in sedentary obese men: a pilot study.

The objective of this study was to determine how a high-intensity circuit-training (HICT) program affects key physiological health markers in sedentary obese men. Eight obese (body fat percentage >26%) males completed a four-week HICT program, consisting of three 30-minute exercise sessions per week, for a total of 6 hours of exercise. Participants' heart rate (HR), blood pressure (BP), rating of perceived exertion, total work (TW), and time to completion were measured each exercise session, body composition was measured before and after HICT, and fasting blood samples were measured before throughout, and after HICT program. Blood sample measurements included total cholesterol, triacylglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, glucose, and insulin. Data were analyzed by paired t-tests and one-way ANOVA with repeated measures. Statistical significance was set to P < 0.05. Data analyses revealed significant (P < 0.05) improvements in resting HR (16% decrease), systolic BP (5.5% decrease), TW (50.7%), fat tissue percentage (3.6%), lean muscle tissue percentage (2%), cholesterol (13%), triacylglycerol (37%), and insulin (18%) levels from before to after HICT program. Overall, sedentary obese males experienced a significant improvement in biochemical, physical, and body composition characteristics from a HICT program that was only 6 hours of the total exercise.

Biosynthesis of phytosterols. Kinetic mechanism for the enzymatic C-methylation of sterols.

Cloned soybean sterol methyltransferase was purified from Escherichia coli to gel electrophoretic homogeneity. From initial velocity experiments, catalytic constants for substrates best suited for the first and second C1 transfer activities, cycloartenol and 24(28)-methylenelophenol, were 0.01 and 0.001 s-1, respectively. Two-substrate kinetic analysis using cycloartenol and S-adenosyl-l-methionine (AdoMet) generated an intersecting line pattern characteristic of a ternary complex kinetic mechanism. The high energy intermediate analog 25-azacycloartanol was a noncompetitive inhibitor versus cycloartenol and an uncompetitive inhibitor versus AdoMet. The dead end inhibitor analog cyclolaudenol was competitive versus cycloartenol and uncompetitive versus AdoMet. 24(28)-Methylenecycloartanol and AdoHcy generated competitive and noncompetitive kinetic patterns, respectively, with respect to AdoMet. Therefore, 24(28)-methylenecycloartanol combines with the same enzyme form as does cycloartenol and must be released from the enzyme before AdoHcy. 25-Azacycloartanol inhibited the first and second C1 transfer activities with about equal efficacy (Ki = 45 nm), suggesting that the successive C-methylation of the Delta 24 bond occurs at the same active center. Comparison of the initial velocity data using AdoMet versus [2H3-methyl]AdoMet as substrates tested against saturating amounts of cycloartenol indicated an isotope effect on VCH3/VCD3 close to unity. [25-2H]24(28)-Methylenecycloartanol, [28E-2H]24 (28)-methylenelanosterol, and [28Z-2H]24(28)-methylene lanosterol were prepared and paired with AdoMet or [methyl-3H3]AdoMet to examine the kinetic isotope effects attending the C-28 deprotonation in the enzymatic synthesis of 24-ethyl(idene) sterols. The stereochemical features as well as the observation of isotopically sensitive branching during the second C-methylation suggests that the two methylation steps can proceed by a change in chemical mechanism resulting from differences in sterol structure, concerted versus carbocation; the kinetic mechanism remains the same during the consecutive methylation of the Delta 24 bond.