Brain organoid protocols and limitations.

Stem cell-derived organoid technology is a powerful tool that revolutionizes the field of biomedical research and extends the scope of our understanding of human biology and diseases. Brain organoids especially open an opportunity for human brain research and modeling many human neurological diseases, which have lagged due to the inaccessibility of human brain samples and lack of similarity with other animal models. Brain organoids can be generated through various protocols and mimic whole brain or region-specific. To provide an overview of brain organoid technology, we summarize currently available protocols and list several factors to consider before choosing protocols. We also outline the limitations of current protocols and challenges that need to be solved in future investigation of brain development and pathobiology.

Buprenorphine and methadone differentially alter early brain development in human cortical organoids.

Buprenorphine (BUP) and methadone (MTD) are used for medication-assisted treatment (MAT) in opioid use disorder. Although both medications show improved maternal and neonatal outcomes compared with illicit opioid use during pregnancy, BUP has exhibited more favorable outcomes to newborns than MTD. The underlying cellular and molecular mechanisms for the difference between BUP and MTD are largely unknown. Here, we examined the growth and neuronal activity in human cortical organoids (hCOs) exposed to BUP or MTD. We found that the growth of hCOs was significantly restricted in the MTD-treated but not in the BUP-treated hCOs and BUP attenuated the growth-restriction effect of MTD in hCOs. Furthermore, a κ-receptor agonist restricted while an antagonist alleviated the growth-restriction effect of MTD in hCOs. Since BUP is not only a µ-agonist but a κ-antagonist, the prevention of this growth-restriction by BUP is likely due to its κ-receptor-antagonism. In addition, using multielectrode array (MEA) technique, we discovered that both BUP and MTD inhibited neuronal activity in hCOs but BUP showed suppressive effects only at higher concentrations. Furthermore, κ-receptor antagonist nBNI did not prevent the MTD-induced suppression of neuronal activity in hCOs but the NMDA-antagonism of MTD (that BUP lacks) plays a role in the inhibition of neuronal activity. We conclude that, although both MTD and BUP are µ-opioid agonists, a) the additional κ-receptor antagonism of BUP mitigates the MTD-induced growth restriction during neurodevelopment and b) the lack of NMDA antagonism of BUP (in contrast to MTD) induces much less suppressive effect on neural network communications.

Making a 'sex-difference fact': Ambien dosing at the interface of policy, regulation, women's health, and biology.

The U.S. Food and Drug Administration's (FDA) 2013 decision to lower recommended Ambien dosing for women has been widely cited as a hallmark example of the importance of sex differences in biomedicine. Using regulatory documents, scientific publications, and media coverage, this article analyzes the making of this highly influential and mobile 'sex-difference fact'. As we show, the FDA's decision was a contingent outcome of the drug approval process. Attending to how a contested sex-difference fact came to anchor elite women's health advocacy, this article excavates the role of regulatory processes, advocacy groups, and the media in producing perceptions of scientific agreement while foreclosing ongoing debate, ultimately enabling the stabilization of a binary, biological sex-difference fact and the distancing of this fact from its conditions of construction.

Dopamine transporter and synaptic vesicle sorting defects underlie auxilin-associated Parkinson's disease.

Auxilin participates in the uncoating of clathrin-coated vesicles (CCVs), thereby facilitating synaptic vesicle (SV) regeneration at presynaptic sites. Auxilin (DNAJC6/PARK19) loss-of-function mutations cause early-onset Parkinson's disease (PD). Here, we utilized auxilin knockout (KO) mice to elucidate the mechanisms through which auxilin deficiency and clathrin-uncoating deficits lead to PD. Auxilin KO mice display cardinal features of PD, including progressive motor deficits, α-synuclein pathology, nigral dopaminergic loss, and neuroinflammation. Significantly, treatment with L-DOPA ameliorated motor deficits. Unbiased proteomic and neurochemical analyses of auxilin KO brains indicated dopamine dyshomeostasis. We validated these findings by demonstrating slower dopamine reuptake kinetics in vivo, an effect associated with dopamine transporter misrouting into axonal membrane deformities in the dorsal striatum. Defective SV protein sorting and elevated synaptic autophagy also contribute to ineffective dopamine sequestration and compartmentalization, ultimately leading to neurodegeneration. This study provides insights into how presynaptic endocytosis deficits lead to dopaminergic vulnerability and pathogenesis of PD.

Alzheimer's disease like neuropathology in Down syndrome cortical organoids.

Introduction: Down syndrome (DS) is a genetic disorder with an extra copy of chromosome 21 and DS remains one of the most common causes of intellectual disabilities in humans. All DS patients have Alzheimer's disease (AD)-like neuropathological changes including accumulation of plaques and tangles by their 40s, much earlier than the onset of such neuropathological changes in AD patients. Due to the lack of human samples and appropriate techniques, our understanding of DS neuropathology during brain development or before the clinical onset of the disease remains largely unexplored at the cellular and molecular levels. Methods: We used induced pluripotent stem cell (iPSC) and iPSC-derived 3D cortical organoids to model Alzheimer's disease in Down syndrome and explore the earliest cellular and molecular changes during DS fetal brain development. Results: We report that DS iPSCs have a decreased growth rate than control iPSCs due to a decreased cell proliferation. DS iPSC-derived cortical organoids have a much higher immunoreactivity of amyloid beta (Aß) antibodies and a significantly higher amount of amyloid plaques than control organoids. Although Elisa results did not detect a difference of Aß40 and Aß42 level between the two groups, the ratio of Aß42/Aß40 in the detergent-insoluble fraction of DS organoids was significantly higher than control organoids. Furthermore, an increased Tau phosphorylation (pTau S396) in DS organoids was confirmed by immunostaining and Western blot. Elisa data demonstrated that the ratio of insoluble Tau/total Tau in DS organoids was significantly higher than control organoids. Conclusion: DS iPSC-derived cortical organoids mimic AD-like pathophysiologyical phenotype in vitro, including abnormal Aß and insoluble Tau accumulation. The molecular neuropathologic signature of AD is present in DS much earlier than predicted, even in early fetal brain development, illustrating the notion that brain organoids maybe a good model to study early neurodegenerative conditions.

ARID1B, a molecular suppressor of erythropoiesis, is essential for the prevention of Monge's disease.

At high altitude Andean region, hypoxia-induced excessive erythrocytosis (EE) is the defining feature of Monge's disease or chronic mountain sickness (CMS). At the same altitude, resides a population that has developed adaptive mechanism(s) to constrain this hypoxic response (non-CMS). In this study, we utilized an in vitro induced pluripotent stem cell model system to study both populations using genomic and molecular approaches. Our whole genome analysis of the two groups identified differential SNPs between the CMS and non-CMS subjects in the ARID1B region. Under hypoxia, the expression levels of ARID1B significantly increased in the non-CMS cells but decreased in the CMS cells. At the molecular level, ARID1B knockdown (KD) in non-CMS cells increased the levels of the transcriptional regulator GATA1 by 3-fold and RBC levels by 100-fold under hypoxia. ARID1B KD in non-CMS cells led to increased proliferation and EPO sensitivity by lowering p53 levels and decreasing apoptosis through GATA1 mediation. Interestingly, under hypoxia ARID1B showed an epigenetic role, altering the chromatin states of erythroid genes. Indeed, combined Real-time PCR and ATAC-Seq results showed that ARID1B modulates the expression of GATA1 and p53 and chromatin accessibility at GATA1/p53 target genes. We conclude that ARID1B is a novel erythroid regulator under hypoxia that controls various aspects of erythropoiesis in high-altitude dwellers.

Why "sex as a biological variable" conflicts with precision medicine initiatives.

Policies that require male-female sex comparisons in all areas of biomedical research conflict with the goal of improving health outcomes through context-sensitive individualization of medical care. Sex, like race, requires a rigorous, contextual approach in precision medicine. A "sex contextualist" approach to gender-inclusive medicine better aligns with this aim.

Law, policy, biology, and sex: Critical issues for researchers.

Researchers should be aware of how sex-difference science is (mis)applied in legal and policy contexts.

Species-Resolved Metagenomics of Kindergarten Microbiomes Reveal Microbial Admixture Within Sites and Potential Microbial Hazards.

Microbiomes on surfaces in kindergartens, the intermediate transfer medium for microbial exchange, can exert significant impact on the hygiene and wellbeing of young children, both individually and as a community. Here employing 2bRAD-M, a novel species-resolved metagenomics approach for low-biomass microbiomes, we surveyed over 100 samples from seven frequently contacted surfaces by children, plus individual children's palms, in two kindergartens. Microbiome compositions, although kindergarten-specific, were grouped closely based on the type of surface within each kindergarten. Extensive microbial admixture were found among the various sampled sites, likely facilitated by contact with children's hands. Notably, bacterial species with potential human health concerns and potentially antibiotic-resistant, although found across all sampled locations, were predominantly enriched on children's hands instead of on the environmental sites. This first species-resolved kindergarten microbiome survey underscores the importance of good hand hygiene practices in kindergartens and provides insights into better managing hygiene levels and minimizing spread of harmful microbes in susceptible indoor environments.

Identification of substrates of palmitoyl protein thioesterase 1 highlights roles of depalmitoylation in disulfide bond formation and synaptic function.

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause neuronal ceroid lipofuscinosis (NCL), a devastating neurodegenerative disease. The substrates of PPT1 are largely undescribed, posing a limitation on molecular dissection of disease mechanisms and therapeutic development. Here, we provide a resource identifying >100 novel PPT1 substrates. We utilized Acyl Resin-Assisted Capture (Acyl RAC) and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout (KO) mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated diverse PPT1 substrates at the synapse, including channels and transporters, G-protein-associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation plays a role in disulfide bond formation in a tertiary screen analyzing posttranslational modifications (PTMs). Collectively, these data highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of NCL and other neurodegenerative diseases, and advance our basic understanding of the purpose of depalmitoylation.

Global intercompany assessment of ICIEF platform comparability for the characterization of therapeutic proteins.

An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data.

Species-resolved sequencing of low-biomass or degraded microbiomes using 2bRAD-M.

Microbiome samples with low microbial biomass or severe DNA degradation remain challenging for amplicon-based or whole-metagenome sequencing approaches. Here, we introduce 2bRAD-M, a highly reduced and cost-effective strategy which only sequences ~ 1% of metagenome and can simultaneously produce species-level bacterial, archaeal, and fungal profiles. 2bRAD-M can accurately generate species-level taxonomic profiles for otherwise hard-to-sequence samples with merely 1 pg of total DNA, high host DNA contamination, or severely fragmented DNA from degraded samples. Tests of 2bRAD-M on various stool, skin, environmental, and clinical FFPE samples suggest a successful reconstruction of comprehensive, high-resolution microbial profiles.

Impact of dermoscopy-aided pediatric teledermatology program on the accessibility and efficiency of dermatology care at community health centers.

BACKGROUND: There are few studies focusing on pediatric teledermatology, and the impact of a large-scale pediatric teledermatology program on the accessibility and efficiency of dermatology care remains unclear. This study evaluated the impact of a state-wide implementation of a store-and-forward teledermatology program augmented by the incorporation of dermoscopy in pediatric patients visiting community health centers. METHODS: It was a descriptive, retrospective cohort study of 876 pediatric dermatology referrals. RESULTS: In the traditional referral system, only 60 patients (17.6%) were seen by dermatologists with average waiting times of 75 days due to limited access. In comparison, with an implementation of dermoscopy-aided teledermatology, all 536 teledermatology referrals received dermatological care within 24 h, of which only 64 (12%) patients requires face-to-face (F2F) consultation. Patients referred for F2F consultation via eConsults had a much lower no-show rate as compared to the traditional referral system (39% vs. 71%). Side by side comparison between general population and pediatric population has demonstrated shared features in efficiency and access improvement but revealed specific characteristics of pediatric teledermatology in terms of diagnosis and treatment. CONCLUSION: Coordinated store-and-forward teledermatology platform with incorporation of dermoscopy between large community care network and dermatology provider can greatly improve access to pediatric dermatology care especially in underserved population. The efficiency of teledermatology in access improvement for pediatric population is comparable with adult population in eConsults. There are also unique features and challenges in pediatric teledermatology that require further research.

Modeling Input Factors in Second Language Acquisition of the English Article Construction.

Based on the Competition Model, the current study investigated how cue availability and cue reliability as two important input factors influenced second language (L2) learners' cue learning of the English article construction. Written corpus data of university-level Chinese-L1 learners of English were sampled for a comparison of English majors and non-English majors who demonstrated two levels of L2 competence in English article usage. The path model analysis in structural equation modeling was utilized to investigate the relationship between the input factors and L2 usage (frequency and accuracy of article cue production). The findings contribute novel and scarce empirical evidence that confirms a central claim of the Competition Model, i.e., the changing importance of cue availability and cue reliability in the frequency and accuracy of production. Cue availability was found to determine L2 production frequency regardless of level of L2 competence. Cue reliability was the input factor that differentiated competence levels. When learners stayed at a relatively lower L2 proficiency, cue reliability played an important role in influencing L2 frequency of usage rather than accuracy of usage. When learners developed increased exposure to and stronger competence in the target language, cue reliability played a significant role in determining learners' success of cue learning. The study is methodologically innovative and expands the empirical applicability of the Competition Model to the domain of second language production and construction learning.

Sonographic evaluation of leprosy of ulnar nerve.

Leprosy is a chronic granulomatous infection caused by Mycobacterium Leprae that predominantly affects the skin and peripheral nerves. The disease is prevalent in developing countries of Asia and Africa, with occasional cases reported from United States. In this case report we highlight a 33-year-old man from Qatar, who presented with symptoms of progressive weakness in his left wrist and hand for six months and was later found to have a granuloma of the ulnar nerve secondary to leprosy. We will discuss the etiopathogenesis, clinical features, role of imaging, and management of neuropathy due to leprosy. Our case provides an excellent learning example of the utility of ultrasonography to characterize leprosy granulomas of the ulnar nerve.

Informational advantages in social networks: The core-periphery divide in peer performance ratings.

Organizations frequently rely on peer performance ratings to capture employees' unique and difficult to observe contributions at work. Though useful, peers exhibit meaningful variance in the accuracy and informational utility they offer about ratees. In this research, we develop and test theory which suggests that raters' social network positions explains this variance in systematic ways. Drawing from information processing theory, we posit that members who occupy core (peripheral) positions in the network have greater (less) access to firsthand and secondhand performance information about ratees, which is in turn associated with more (less) accurate performance ratings. To overcome difficulties in obtaining a "true" performance score in interdependent field settings, we employ an external criterion comparison method to benchmark our arguments, such that larger validity coefficients between established predictors of performance (i.e., a ratee's general mental ability [GMA] and conscientiousness) and peer performance ratings should reflect more (less) accurate ratings for core (peripheral) members. In Study 1, we use an organization-wide network in a technology startup company to examine the validity coefficient of a ratee's GMA on performance as rated by central versus peripheral members. In Study 2, we attempt to replicate and extend Study 1's conclusions in team networks using ratee conscientiousness as a benchmark indicator. Findings from both studies generally support the hypotheses that core network members provide distinct, and presumably more accurate, peer performance ratings than peripheral network members. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Methadone Suppresses Neuronal Function and Maturation in Human Cortical Organoids.

Accumulating evidence has suggested that prenatal exposure to methadone causes multiple adverse effects on human brain development. Methadone not only suppresses fetal neurobehavior and alters neural maturation, but also leads to long-term neurological impairment. Due to logistical and ethical issues of accessing human fetal tissue, the effect of methadone on brain development and its underlying mechanisms have not been investigated adequately and are therefore not fully understood. Here, we use human cortical organoids which resemble fetal brain development to examine the effect of methadone on neuronal function and maturation during early development. During development, cortical organoids that are exposed to clinically relevant concentrations of methadone exhibited suppressed maturation of neuronal function. For example, organoids developed from 12th week till 24th week have an about 7-fold increase in AP firing frequency, but only half and a third of this increase was found in organoids exposed to 1 and 10 µM methadone, respectively. We further demonstrated substantial increases in I Na (4.5-fold) and I KD (10.8-fold), and continued shifts of Na+ channel activation and inactivation during normal organoid development. Methadone-induced suppression of neuronal function was attributed to the attenuated increase in the densities of I Na and I KD and the reduced shift of Na+ channel gating properties. Since normal neuronal electrophysiology and ion channel function are critical for regulating brain development, we believe that the effect of prolonged methadone exposure contributes to the delayed maturation, development fetal brain and potentially for longer term neurologic deficits.

Methadone interrupts neural growth and function in human cortical organoids.

Prenatal opioids exposure can lead to both neonatal abstinence syndrome in newborns and neurological deficits later in life. Although opioids have been well studied in general, the cellular and molecular mechanisms by which opioids affect human fetal brain development has not been well understood. In this work, we have taken advantage of a human 3D-brain cortical organoid (hCO) that facilitated enormously the investigation of early human brain development. Using imaging, immunofluorescence, multi-electrode array (MEA) and patch clamp recording techniques, we have investigated the effect of methadone, a frequently used opioid during pregnancy, on early neural development, including neuronal growth, neural network activity and synaptic transmission in hCOs. Our results demonstrated that methadone dose-dependently halted the growth of hCOs and induced organoid disintegration after a prolonged exposure. In addition, methadone dose-dependently suppressed the firing of spontaneous action potentials in hCOs and this suppression could be reversed upon methadone withdrawal in hCOs treated with lower dosages. Further investigation using patch clamp whole cell configuration revealed that, at clinically relevant concentrations, methadone decreased the frequency and amplitude of excitatory postsynaptic currents in neurons, indicating a critical role of methadone in weakening synaptic transmission in neural networks in hCOs. In addition, methadone significantly attenuated the voltage-dependent Na+ current in hCOs. We conclude that methadone interrupts neural growth and function in early brain development.

Interleukin-10 control of pre-miR155 maturation involves CELF2.

The anti-inflammatory cytokine interleukin-10 (IL10) is essential for attenuating inflammatory responses, which includes reducing the expression of pro-inflammatory microRNA-155 (miR155) in lipopolysaccharide (LPS) activated macrophages. miR155 enhances the expression of pro-inflammatory cytokines such as TNFα and suppresses expression of anti-inflammatory molecules such as SHIP1 and SOCS1. We previously found that IL10 interfered with the maturation of pre-miR155 to miR155. To understand the mechanism by which IL10 interferes with pre-miR155 maturation we isolated proteins that associate with pre-miR155 in response to IL10 in macrophages. We identified CELF2, a member of the CUGBP, ELAV-Like Family (CELF) family of RNA binding proteins, as protein whose association with pre-miR155 increased in IL10 treated cells. CRISPR-Cas9 mediated knockdown of CELF2 impaired IL10's ability to inhibit both miR155 expression and TNFα expression.