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Several protocols have been developed for human induced pluripotent stem cell neuronal differentiation. We compare several methods for forebrain cortical neuronal differentiation by assessing cell morphology, immunostaining and gene expression. We evaluate embryoid aggregate vs. monolayer with dual SMAD inhibition differentiation protocols, manual vs. AggreWell aggregate formation, plating substrates, neural progenitor cell (NPC) isolation methods, NPC maintenance and expansion, and astrocyte co-culture. The embryoid aggregate protocol, using a Matrigel substrate, consistently generates a high yield and purity of neurons. NPC isolation by manual selection, enzymatic rosette selection, or FACS all are efficient, but exhibit some differences in resulting cell populations. Expansion of NPCs as neural aggregates yields higher cell purity than expansion in a monolayer. Finally, co-culture of iPSC-derived neurons with astrocytes increases neuronal maturity by day 40. This study directly compares commonly employed methods for neuronal differentiation of iPSCs, and can be used as a resource for choosing between various differentiation protocols.

Background: The infection due to the COVID-19 virus has shown to cause diverse set of manifestations and effects in various people across the world. Pregnancy being a dynamic, physiological condition of the human body, nudges one to question what happens if a pregnant woman is infected with the COVID-19 virus? Hence, the study aims to probe this very question. Aim and Objectives: The aim of this study was to study the effect of COVID-19 virus infection on pregnancy and its outcomes. Materials and Methods: We did a retrospective review of medical records of 28 pregnant ladies who were admitted in the tertiary care hospital, Mysuru between March 2020 and February 2021. All 28 pregnant ladies were tested positive for severe acute coronavirus disease by use of quantitative RTPCR on samples obtained from nose and throat. The procedure was followed according to ICMR guidelines of collection of samples and reporting of COVID-19 infection. The clinical findings, laboratory findings, and the fetal and maternal outcomes of 28 pregnant ladies were obtained from medical record section of the institution. The reports were entered in excel sheet and subjected to statistical analysis using SPSS version 20. Results: One out of 28 showed maternal death during COVID-19 pandemic. There was one case with abortion which was reported. Twenty out of 28 had normal deliveries and three women had LSCS. Neonatal outcomes seem to be within normal limits with no adverse complications as a consequence of COVID-19 maternal infection. Conclusion: It is seen that COVID-19 infection behaves in a similar fashion to general population and that morbid states may trigger adversity in mother rather than the fetus. It is also noted that due to the effect of the chaos caused by the pandemic, documentation of the cases was incomplete. Transfer of cases depending on status of infection to other hospitals hindered the tracking of these cases. Discharge against medical advice was also observed due to panic of being in a hospital during an ongoing pandemic.

The development of three-dimensional culture methods has allowed for the study of developing cortical morphology in human cells. This provides a new tool to study the neurodevelopmental consequences of disease-associated mutations. Here, we study the effects of isogenic DISC1 mutation in cerebral organoids. DISC1 has been implicated in psychiatric disease based on genetic studies, including its interruption by a balanced translocation that increases the risk of major mental illness. Isogenic wild-type and DISC1-disrupted human-induced pluripotent stem cells were used to generate cerebral organoids, which were then examined for morphology and gene expression. We show that DISC1-mutant cerebral organoids display disorganized structural morphology and impaired proliferation, which is phenocopied by WNT agonism and rescued by WNT antagonism. Furthermore, there are many shared changes in gene expression with DISC1 disruption and WNT agonism, including in neural progenitor and cell fate markers, regulators of neuronal migration, and interneuron markers. These shared gene expression changes suggest mechanisms for the observed morphologic dysregulation with DISC1 disruption and points to new avenues for future studies. The shared changes in three-dimensional cerebral organoid morphology and gene expression with DISC1 interruption and WNT agonism further strengthens the link between DISC1 mutation, abnormalities in WNT signaling, and neuropsychiatric disease.

Insulin/IGF-1 signaling plays a pivotal role in the regulation of cellular homeostasis through its control of glucose metabolism as well as due to its effects on cell proliferation. Aberrant regulation of insulin signaling has been repeatedly implicated in uncontrolled cell growth and malignant transformations. TBC1D3 is a hominoid specific gene previously identified as an oncogene in breast and prostate cancers. Our efforts to identify the molecular mechanisms of TBC1D3-induced oncogenesis revealed the role of TBC1D3 in insulin/IGF-1 signaling pathway. We document here that TBC1D3 intensifies insulin/IGF-1-induced signal transduction through intricate, yet elegant fine-tuning of signaling mechanisms. We show that TBC1D3 expression substantially delayed ubiquitination and degradation of insulin receptor substrate-1 (IRS-1). This effect is achieved through suppression of serine phosphorylation at S636/639, S307 and S312 of IRS-1, which are key phosphorylation sites required for IRS-1 degradation. Furthermore, we report that the effect of TBC1D3 on IRS-1:S636/639 phosphorylation is mediated through TBC1D3-induced activation of protein phosphatase 2A (PP2A), followed by suppression of T389 phosphorylation on p70 S6 kinase (S6K). TBC1D3 specifically interacts with PP2A regulatory subunit B56γ, indicating that TBC1D3 and PP2A B56γ operate jointly to promote S6K:T389 dephosphorylation. These findings suggest that TBC1D3 plays an unanticipated and potentially unique role in the fine-tuning of insulin/IGF-1 signaling, while providing novel insights into the regulation of tumorigenesis by a hominoid-specific protein.

Objectives To describe a 10‐year single center experience with parotid gland malignancies and to determine factors affecting outcomes. Study Design Retrospective review. Methods The institutional cancer registry was used to identify patients treated surgically for malignancies of the parotid gland between January 2005 and December 2014. Clinical and pathologic data were collected retrospectively from patient charts and analyzed for their association with overall survival (OS) and disease‐free survival (DFS). Results Two hundred patients were identified. Mean age at surgery was 57.8 years, and mean follow‐up time was 52 months. One hundred two patients underwent total parotidectomy, while 77 underwent superficial parotidectomy, and 21 underwent deep lobe resection. Seventy patients (35%) required facial nerve (FN) sacrifice. Acinic cell carcinoma was the most common histologic type (22%), followed by mucoepidermoid carcinoma (21.5%) and adenoid cystic carcinoma (12.5%). Twenty‐nine patients (14.5%) experienced recurrences, with mean time to recurrence of 23.6 months (range: 1‐82 months). Five‐ and 10‐year OS were 81% and 73%, respectively. Five‐ and 10‐year DFS were 80% and 73%, respectively. In univariate analyses, age > 60, histologic type, positive margins, high grade, T‐stage, node positivity, perineural invasion, and FN involvement were predictors of OS and DFS. In the multivariate analysis, histology, positive margins, node positivity, and FN involvement were independent predictors of OS and DFS. Conclusions Our single‐center experience of 200 patients suggests that histology, positive margins, node positivity, and FN involvement are independently associated with outcomes in parotid malignancies. Level of Evidence 4

Expression of the hominoid-specific TBC1D3 oncoprotein enhances growth factor receptor signaling and subsequently promotes cellular proliferation and survival. Here we report that TBC1D3 is degraded in response to growth factor signaling, suggesting that TBC1D3 expression is regulated by a growth factor-driven negative feedback loop. To gain a better understanding of how TBC1D3 is regulated, we studied the effects of growth factor receptor signaling on TBC1D3 post-translational processing and turnover. Using a yeast two-hybrid screen, we identified CUL7, the scaffolding subunit of the CUL7 E3 ligase complex, as a TBC1D3-interacting protein. We show that CUL7 E3 ligase ubiquitinates TBC1D3 in response to serum stimulation. Moreover, TBC1D3 recruits F-box 8 (Fbw8), the substrate recognition domain of CUL7 E3 ligase, in pull-down experiments and in an in vitro assay. Importantly, alkaline phosphatase treatment of TBC1D3 suppresses its ability to recruit Fbw8, indicating that TBC1D3 phosphorylation is critical for its ubiquitination and degradation. We conclude that serum- and growth factor-stimulated TBC1D3 ubiquitination and degradation are regulated by its interaction with CUL7-Fbw8.

Essential minerals are cofactors for synthesis of neurotransmitters supporting cognition and mood. An 8-week fully-blind randomised controlled trial of multinutrients for attention-deficit/hyperactivity disorder (ADHD) demonstrated three times as many children (age 6–12) had significantly improved behaviour (‘treatment responders’) on multinutrients (54 %) compared with placebo (18 %). The aim of this secondary study was to evaluate changes in fasted plasma and urinary mineral concentrations following the intervention and their role as mediators and moderators of treatment response. Fourteen essential or trace minerals were measured in plasma and/or urine at baseline and week eight from eighty-six participants (forty-nine multinutrients, thirty-seven placebos). Two-sample t tests/Mann–Whitney U tests compared 8-week change between treatment and placebo groups, which were also evaluated as potential mediators. Baseline levels were evaluated as potential moderators, using logistic regression models with clinical treatment response as the outcome. After 8 weeks, plasma boron, Cr (in females only), Li, Mo, Se and vanadium and urinary iodine, Li and Se increased more with multinutrients than placebo, while plasma phosphorus decreased. These changes did not mediate treatment response. However, baseline urinary Li trended towards moderation: participants with lower baseline urinary Li were more likely to respond to multinutrients (P = 0·058). Additionally, participants with higher baseline Fe were more likely to be treatment responders regardless of the treatment group (P = 0·036.) These results show that multinutrient treatment response among children with ADHD is independent of their baseline plasma mineral levels, while baseline urinary Li levels show potential as a non-invasive biomarker of treatment response requiring further study.

BackgroundThe Micronutrients for Attention-Deficit/Hyperactivity Disorder in Youth (MADDY) study evaluated the efficacy and safety of a multinutrient formula for children with ADHD and emotional dysregulation. The post-RCT open-label extension (OLE) compared the effect of treatment duration (8 weeks vs 16 weeks) on ADHD symptoms, height velocity, and adverse events (AEs).MethodsChildren aged 6–12 years randomized to multinutrients vs. placebo for 8 weeks (RCT), received an 8–week OLE for a total of 16 weeks. Assessments included the Clinical Global Impression-Improvement (CGI-I), Child and Adolescent Symptom Inventory-5 (CASI-5), Pediatric Adverse Events Rating Scale (PAERS), and anthropometric measures (height and weight).ResultsOf the 126 in the RCT, 103 (81%) continued in the OLE. For those initially assigned to placebo, CGI-I responders increased from 23% in the RCT to 64% in the OLE; those who took multinutrients for 16 weeks increased from 53% (RCT) to 66% responders (OLE). Both groups improved on the CASI-5 composite score and subscales from week 8 to week 16 (all p–values < 0.01). The group taking 16 weeks of multinutrients had marginally greater height growth (2.3 cm) than those with 8 weeks (1.8 cm) (p = 0.07). No difference in AEs between groups was found.ConclusionThe response rate to multinutrients by blinded clinician ratings at 8 weeks was maintained to 16 weeks; the response rate in the group initially assigned to placebo improved significantly with 8 weeks of multinutrients and almost caught up with 16 weeks. Longer time on multinutrients did not result in greater AEs, confirming an acceptable safety profile.

ObjectiveWith dual focus on structured, objective quantification of parent observations of child’s behavior and identifying behaviors most amenable to change, this report examines Parent Target Problems (PTP) as a secondary outcome in a randomized clinical trial (RCT) of children with attention-deficit/ hyperactivity disorder (ADHD) in which one primary outcome, Clinical Global Impression-Improvement, showed a significant advantage of multinutrients over placebo and the other, Likert-type parent ratings, showed significant improvement in both groups, without significant difference between them.MethodIn a multisite 8-week RCT of broad-spectrum micronutrients (“multinutrients”), parents of children ages 6–12 (N = 126, 73% male, 88% white) with ADHD and emotional dysregulation nominated their child’s most concerning problem(s) at baseline and quantified them by frequency, duration, impairment, and consequences. At subsequent visits, parents re-quantified the problem(s). Blinded child psychiatrists independently reviewed the PTPs and rated change at two timepoints compared to baseline. PTPs were grouped into 9 categories. Mean ratings were compared between active and placebo groups and explored by category.ResultsBy week 8, a significant separation favored multinutrients: 38% of the multinutrient group were “definitely improved” or better, compared to 25% of the placebo group, and ratings of “no change” or “worse” occurred in 35% with placebo versus 23% with multinutrients (p = 0.04). Inattention (72.2%) and emotional dysregulation (69.1%) were the most frequently reported PTP categories. Inattention and internalizing symptoms improved more with multinutrients than placebo (p = 0.01, d = 0.55; p = 0.03, d = 0.80, respectively). The multinutrient advantage was not significant for 7 other symptoms, including hyperactivity/impulsivity, aggression, autistic symptoms, or emotional dysregulation/irritable oppositionality.ConclusionsThis secondary analysis found that the multinutrients, compared to placebo, were associated with improvements in parental concerns overall, and in two domains specifically: inattention and internalizing symptoms (anxiety/depression), but not in seven domains: hyperactivity/impulsivity, aggression, autistic symptoms or physiological symptoms, peer relationships or emotional dysregulation/irritable oppositionality.