A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions.

The endoplasmic reticulum (ER) is an important regulator of Ca2+ in cells and dysregulation of ER calcium homeostasis can lead to numerous pathologies. Understanding how various pharmacological and genetic perturbations of ER Ca2+ homeostasis impacts cellular physiology would likely be facilitated by more quantitative measurements of ER Ca2+ levels that allow easier comparisons across conditions. Here, we developed a ratiometric version of our original ER-GCaMP probe that allows for more quantitative comparisons of the concentration of Ca2+ in the ER across cell types and sub-cellular compartments. Using this approach we show that the resting concentration of ER Ca2+ in primary dissociated neurons is substantially lower than that in measured in embryonic fibroblasts.

Astrocyte growth is driven by the Tre1/S1pr1 phospholipid-binding G protein-coupled receptor.

Astrocytes play crucial roles in regulating neural circuit function by forming a dense network of synapse-associated membrane specializations, but signaling pathways regulating astrocyte morphogenesis remain poorly defined. Here, we show the Drosophila lipid-binding G protein-coupled receptor (GPCR) Tre1 is required for astrocytes to establish their intricate morphology in vivo. The lipid phosphate phosphatases Wunen/Wunen2 also regulate astrocyte morphology and, via Tre1, mediate astrocyte-astrocyte competition for growth-promoting lipids. Loss of s1pr1, the functional analog of Tre1 in zebrafish, disrupts astrocyte process elaboration, and live imaging and pharmacology demonstrate that S1pr1 balances proper astrocyte process extension/retraction dynamics during growth. Loss of Tre1 in flies or S1pr1 in zebrafish results in defects in simple assays of motor behavior. Tre1 and S1pr1 are thus potent evolutionarily conserved regulators of the elaboration of astrocyte morphological complexity and, ultimately, astrocyte control of behavior.

Detection in seized samples, analytical characterization, and in vitro metabolism of the newly emerged 5-bromo-indazole-3-carboxamide synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS) and new structural scaffolds have emerged on the recreational drug market since the enactment of Chinese SCRA analog controls in 2021. This study reports the first SCRAs to be detected with a bromide at the 5 position (5'Br) on the phenyl ring of the indazole core and without a tail moiety. ADB-5'Br-INACA (ADMB-5'Br-INACA) and MDMB-5'Br-INACA were detected in seized samples from Scottish prisons, Belgian customs, and US forensic casework. The brominated analog with a tail moiety, ADB-5'Br-BUTINACA (ADMB-5'Br-BUTINACA), was also detected in Scottish prisons and US forensic casework. The metabolites of these compounds and the predicted compound MDMB-5'Br-BUTINACA were identified through incubation with primary human hepatocytes to aid in their toxicological identification. The bromide on the indazole remains intact on metabolites, allowing these compounds to be easily distinguished in toxicological samples from their non-brominated analogs. Glucuronidation was more common for tail-less analogs than their butyl tail-containing counterparts. Forensic toxicologists are advised to update their analytical methods with the characteristic ions for these compounds, as well as their anticipated urinary markers: amide hydrolysis and monoOH at tert-butyl metabolites (after ß-glucuronidase treatment) for ADB-5'Br-INACA; monoOH at tert-butyl and amide hydrolysis metabolites for ADB-5'Br-BUTINACA; and ester hydrolysis metabolites with additional metabolites for MDMB-5'Br-INACA and MDMB-5'Br-BUTINACA. Toxicologists should remain vigilant to the emergence of new SCRAs with halogenation of the indazole core and tail-less analogs, which have already started to emerge.

Maternal Diabetes in Youth-Onset Type 2 Diabetes Is Associated With Progressive Dysglycemia and Risk of Complications.

CONTEXT: Prenatal exposures, including undernutrition, overnutrition, and parental diabetes, are recognized risk factors for future cardiometabolic disease. There are currently no data on effects of parental diabetes on disease progression or complications in youth-onset type 2 diabetes (T2D). OBJECTIVE: We analyzed effects of parental diabetes history on glycemic outcomes, ß-cell function, and complications in a US cohort of youth-onset T2D. METHODS: Participants (N = 699) aged 10 to 17 years with T2D were enrolled at 15 US centers and followed for up to 12 years as part of the TODAY (Treatment Options for type 2 Diabetes in Adolescents and Youth) and TODAY2 follow-up studies. Information about diabetes diagnosis in biological mothers was available for 621 participants (never = 301; before or during pregnancy = 218; after pregnancy = 102) and in biological fathers for 519 (no diabetes = 352; paternal diabetes = 167). RESULTS: Maternal, but not paternal, diabetes was associated with loss of glycemic control over time, defined as glycated hemoglobin A1c greater than or equal to 8% for more than 6 months (P = .001). Similarly, maternal, but not paternal, diabetes was associated with increased risk of glomerular hyperfiltration (P = .01) and low heart rate variability (P = .006) after 12 years of follow-up. Effects were largely independent of age, sex, race/ethnicity, and household income. Maternal diabetes during vs after pregnancy had similar effects on outcomes. CONCLUSION: Maternal diabetes, regardless of whether diagnosed during vs after pregnancy, is associated with worse glycemic control, glomerular hyperfiltration, and reduced heart rate variability in youth with T2D in TODAY. The strong associations of diabetes outcomes with maternal diabetes suggest a possible role for in utero programming.

The majority of ruptured aneurysms are small with low rupture risk scores.

BACKGROUND: Understanding the rupture risk of unruptured intracranial aneurysms has important clinical implications given the morbidity and mortality associated with subarachnoid hemorrhage (SAH). The ISUIA, UCAS, and PHASES studies provide rupture risk calculations. OBJECTIVE: We apply the risk calculations to a series ruptured intracranial aneurysms to assess the rupture risk for each aneurysm (had they been discovered in the unruptured state). METHODS: This is a retrospective study of 246 patients with SAH from a ruptured saccular aneurysm. The ISUIA, UCAS, and PHASES calculators were applied to each patient/aneurysm to demonstrate a theoretical annual risk of rupture dichotomized by aneurysm location. RESULTS: The average diameter of the aneurysms was 5.5 ± 3.1 mm. Three quarters (75%) of the aneurysms measured <7 mm and 48.8% were <5 mm. The anterior communicating artery (Acomm) was the most common location of rupture (24.7%). Posterior communicating artery aneurysms (Pcomm) were the third most common at 16.2%. The average ISUIA 1-year rupture risk was 0.46 ± 0.008%. The average UCAS 1-year rupture risk was 0.93% ± 0.01. The annualPHASESrupture risk was0.32 ± 0.004%. The highest risk locations were the vertebral artery (up to 10.3% per year) and superior cerebellar artery (up to 2.7% per year). On average, Acomm aneurysms had 1 year risk no higher than 1.1% and Pcomm aneurysms no higher than 1.2% per year. CONCLUSION: We observed that in a small retrospective series of ruptured aneurysms, the majority were <7 mm and that the theoretical rupture risk of these aneurysms, had they been discovered in the unruptured state, is low (<1% per year). Our study has a number of limitations and these results should be validated in a larger multicenter study.

Diabetes Distress in Young Adults With Youth-Onset Type 2 Diabetes: TODAY2 Study Results.

OBJECTIVE: To assess the prevalence of high diabetes distress and associated factors in the Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY2) study cohort of young adults with youth-onset type 2 diabetes. RESEARCH DESIGN AND METHODS: Participants completed the Diabetes Distress Scale (DDS) at end-of-study visits. Factors examined for association with high distress were demographic (sex, race/ethnicity, age, education, income), medical (HbA1c, BMI, complications), psychological (depressive and anxiety symptoms), and social (number in household, offspring, health care coverage, established with diabetes care provider). Univariate logistic regression identified factors associated with high distress that were controlled for in multivariate logistic regressions. RESULTS: Of 438 participants, 66% were female (mean age 26.8 years, 18% non-Hispanic White, 37% non-Hispanic Black, 38% Hispanic). High distress (DDS ≥2) was reported by 105 (24%) participants. Subscales identified 40% with high regimen distress and 29.7% with high emotional burden. A greater percentage of those with high distress were female (P = 0.002), diagnosed with hypertension (P = 0.037) and retinopathy (P = 0.005), treated with insulin, had higher HbA1c, and had moderate to severe depressive and anxiety symptoms (all P < 0.001). In multivariate analyses, female sex (P < 0.001), HbA1c (P < 0.001), anxiety symptoms (P = 0.036), and lack of health care coverage (P = 0.019) were associated with high distress, after controlling for potential confounders. Moderate to severe depressive symptoms were associated with high regimen distress (P = 0.018) and emotional burden (P < 0.001); insulin treatment was associated with high emotional burden (P = 0.027). CONCLUSIONS: Future research should identify modifiable factors associated with high diabetes distress in young adults with youth-onset type 2 diabetes that may inform distress interventions with this medically vulnerable group.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity is required for V(D)J recombination.

A whole-genome CRISPR/Cas9 screen identified ATP2A2, the gene encoding the Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2 protein, as being important for V(D)J recombination. SERCAs are ER transmembrane proteins that pump Ca2+ from the cytosol into the ER lumen to maintain the ER Ca2+ reservoir and regulate cytosolic Ca2+-dependent processes. In preB cells, loss of SERCA2 leads to reduced V(D)J recombination kinetics due to diminished RAG-mediated DNA cleavage. SERCA2 deficiency in B cells leads to increased expression of SERCA3, and combined loss of SERCA2 and SERCA3 results in decreased ER Ca2+ levels, increased cytosolic Ca2+ levels, reduction in RAG1 and RAG2 gene expression, and a profound block in V(D)J recombination. Mice with B cells deficient in SERCA2 and humans with Darier disease, caused by heterozygous ATP2A2 mutations, have reduced numbers of mature B cells. We conclude that SERCA proteins modulate intracellular Ca2+ levels to regulate RAG1 and RAG2 gene expression and V(D)J recombination and that defects in SERCA functions cause lymphopenia.

Local Sourcing of Secretory Proteins in Faraway Places.

A recent paper by Carter et al. identifies a novel organelle, the ribosome-associated vesicle (RAV), that might serve as a portable, local factory for producing proteins destined for the secretory pathway. The appearance of RAVs in dendrites suggests they may serve to generate membrane and secreted proteins in distal processes.

Molecular Tuning of the Axonal Mitochondrial Ca2+ Uniporter Ensures Metabolic Flexibility of Neurotransmission.

The brain is a vulnerable metabolic organ and must adapt to different fuel conditions to sustain function. Nerve terminals are a locus of this vulnerability, but how they regulate ATP synthesis as fuel conditions vary is unknown. We show that synapses can switch from glycolytic to oxidative metabolism, but to do so, they rely on activity-driven presynaptic mitochondrial Ca2+ uptake to accelerate ATP production. We demonstrate that, whereas mitochondrial Ca2+ uptake requires elevated extramitochondrial Ca2+ in non-neuronal cells, axonal mitochondria readily take up Ca2+ in response to small changes in external Ca2+. We identified the brain-specific protein MICU3 as a critical driver of this tuning of Ca2+ sensitivity. Ablation of MICU3 renders axonal mitochondria similar to non-neuronal mitochondria, prevents acceleration of local ATP synthesis, and impairs presynaptic function under oxidative conditions. Thus, presynaptic mitochondria rely on MICU3 to facilitate mitochondrial Ca2+ uptake during activity and achieve metabolic flexibility.

Heart Rate Variability and Cardiac Autonomic Dysfunction: Prevalence, Risk Factors, and Relationship to Arterial Stiffness in the Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) Study.

OBJECTIVE: To determine whether prior type 2 diabetes (T2D) treatment or glycemic control over time are independently associated with heart rate variability (HRV) and whether the presence of cardiac autonomic dysfunction is associated with arterial stiffness in young adults with youth-onset T2D enrolled in the Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) study. RESEARCH DESIGN AND METHODS: Heartbeats over 10 min were measured to derive the normal R-Rs (NN intervals). Outcomes included the standard deviation of the NN intervals (SDNN), the root mean square differences of successive NN intervals (RMSSD), percent of NN beats that differ by more than 50 ms (PNN50), and the low-frequency (LF) power domain, high-frequency (HF) power domain, and their ratio (LF:HF). Autonomic dysfunction was defined as ≥3 of 5 abnormal HRV indices compared with obese controls from a separate study. RESULTS: A total of 397 TODAY participants were evaluated 7 years after randomization. TODAY participants had reduced HRV (SDNN 58.1 ± 29.6 ms vs. controls 67.1 ± 25.4 ms; P < 0.0001) with parasympathetic loss (RMSSD 53.2 ± 36.7 ms vs. controls 67.9 ± 35.2 ms; P < 0.0001) with sympathetic overdrive (LF:HF ratio 1.4 ± 1.7 vs. controls 1.0 ± 1.1; P < 0.0001). Cardiac autonomic dysfunction was present in 8% of TODAY participants, and these participants had greater pulse wave velocity compared with those without dysfunction (P = 0.0001). HRV did not differ by randomized treatment, but higher hemoglobin A1c (HbA1c) over time was independently associated with lower SDNN and RMSSD and higher LF:HF ratio after adjustment for age, race-ethnicity, sex, and BMI. CONCLUSIONS: Young adults with youth-onset T2D show evidence of cardiac autonomic dysfunction with both parasympathetic and sympathetic impairments that are associated with higher HbA1c.

SynGO: An Evidence-Based, Expert-Curated Knowledge Base for the Synapse.

Synapses are fundamental information-processing units of the brain, and synaptic dysregulation is central to many brain disorders ("synaptopathies"). However, systematic annotation of synaptic genes and ontology of synaptic processes are currently lacking. We established SynGO, an interactive knowledge base that accumulates available research about synapse biology using Gene Ontology (GO) annotations to novel ontology terms: 87 synaptic locations and 179 synaptic processes. SynGO annotations are exclusively based on published, expert-curated evidence. Using 2,922 annotations for 1,112 genes, we show that synaptic genes are exceptionally well conserved and less tolerant to mutations than other genes. Many SynGO terms are significantly overrepresented among gene variations associated with intelligence, educational attainment, ADHD, autism, and bipolar disorder and among de novo variants associated with neurodevelopmental disorders, including schizophrenia. SynGO is a public, universal reference for synapse research and an online analysis platform for interpretation of large-scale -omics data (https://syngoportal.org and http://geneontology.org).

Acute liver failure in neonates with undiagnosed hereditary fructose intolerance due to exposure from widely available infant formulas.

Hereditary fructose intolerance (HFI) is an autosomal recessive disorder caused by aldolase B (ALDOB) deficiency resulting in an inability to metabolize fructose. The toxic accumulation of intermediate fructose-1-phosphate causes multiple metabolic disturbances, including postprandial hypoglycemia, lactic acidosis, electrolyte disturbance, and liver/kidney dysfunction. The clinical presentation varies depending on the age of exposure and the load of fructose. Some common infant formulas contain fructose in various forms, such as sucrose, a disaccharide of fructose and glucose. Exposure to formula containing fructogenic compounds is an important, but often overlooked trigger for severe metabolic disturbances in HFI. Here we report four neonates with undiagnosed HFI, all caused by the common, homozygous mutation c.448G>C (p.A150P) in ALDOB, who developed life-threatening acute liver failure due to fructose-containing formulas. These cases underscore the importance of dietary history and consideration of HFI in cases of neonatal or infantile acute liver failure for prompt diagnosis and treatment of HFI.

GLUT4 Mobilization Supports Energetic Demands of Active Synapses.

The brain is highly sensitive to proper fuel availability as evidenced by the rapid decline in neuronal function during ischemic attacks and acute severe hypoglycemia. We previously showed that sustained presynaptic function requires activity-driven glycolysis. Here, we provide strong evidence that during action potential (AP) firing, nerve terminals rely on the glucose transporter GLUT4 as a glycolytic regulatory system to meet the activity-driven increase in energy demands. Activity at synapses triggers insertion of GLUT4 into the axonal plasma membrane driven by activation of the metabolic sensor AMP kinase. Furthermore, we show that genetic ablation of GLUT4 leads to an arrest of synaptic vesicle recycling during sustained AP firing, similar to what is observed during acute glucose deprivation. The reliance on this biochemical regulatory system for "exercising" synapses is reminiscent of that occurring in exercising muscle to sustain cellular function and identifies nerve terminals as critical sites of proper metabolic control.

Barriers to participation in industry-sponsored clinical trials in pediatric type 2 diabetes.

BACKGROUND: The rapid emergence of type 2 diabetes (T2D) in the pediatric population has left pediatric endocrinologists with limited artillery in terms of management. While multiple medications are available for adults, Food and Drug Administration (FDA)-approved medications in children are limited to only metformin and insulin. Additional treatment options require randomized controlled trials, yet heretofore several barriers at the participant and institutional level have impeded these studies from proceeding in children and adolescents. Identification of the most challenging obstacles that pediatric endocrinologists experience in participating in industry-sponsored T2D trials may facilitate development of feasible platforms for future studies. MATERIALS AND METHODS: We conducted an anonymous online survey consisting of 31 questions that assessed potential barriers to industry-sponsored clinical trials in pediatric patients with T2D. The survey was sent to members of the Pediatric Endocrine Society (PES), and members conducted the survey between October and November of 2014. As part of the survey, respondents rated the significance of several possible barriers to participation in industry-sponsored T2D studies. RESULTS: We received a total of 207 responses from members of PES. Baseline demographics showed that 50% of represented institutions care for 50 or fewer T2D patients age 18 years and younger; 70% of institutions diagnose 20 or fewer new T2D cases per year; and 3 racial groups predominated: African American, Hispanic, and Caucasian. A total of 70% of responders have a research infrastructure to participate in clinical trials, but only half have dedicated research nurses. Protocol restrictions on participant recruitment due to current glycemic control or medication use as well as frequent visit schedules were reported to be major obstacles. In addition, the financial support provided to centers to carry out the studies is insufficient. CONCLUSIONS: Efforts must be made to ease the burden of research participation on both pediatric T2D patients as well as pediatric endocrinologists.

Hashimoto's encephalopathy: a rare pediatric brain disease.

We report a 9-year-old female who presented with new onset intractable seizure activity followed by a prolonged encephalopathic state. After ruling out common etiologies, Hashimoto's encephalopathy (HE) was considered, and antibody levels to thyroid peroxidase and thyroglobulin were both markedly elevated in her serum. She was euthyroid at the time of presentation. Upon treatment with high dose methylprednisolone, the patient demonstrated a significant improvement in her encephalopathy. The diagnosis of HE requires strong clinical suspicion with evidence of antithyroid antibodies, as well as an encephalopathy not explained by another etiology. While well documented in the adult literature, only a handful of pediatric cases have been described to date. Patients with HE have a nearly universal response to high dose glucocorticoids. HE should be considered in the differential diagnosis of any patient, adult or pediatric, who displays prolonged, unexplainable encephalopathy.

Stereoselective [4 + 1] annulation reactions with silyl vinylketenes derived from Fischer carbene complexes.

Stable silyl vinylketenes were prepared via the thermal reaction of Fischer carbene complexes with triisopropylsilyl- or tert-butyldimethylsilyl-substituted alkynes. The ability of these silyl vinylketenes to participate with carbenoid reagents in [4 + 1] annulation reactions was investigated. The best results were obtained with diazomethane and substituted diazomethane reagents, which provided cyclopentenone products in excellent yields and essentially complete stereoselectivity.