•Human PSCs readily aggregate to form spheroids using microscale technologies.•Homogeneous populations of aggregates are maintained using rotary orbital culture.•Aggregation enables selective ...enrichment of cells to generate homotypic constructs.•Aggregation allows for co-assembly of cells to yield heterotypic microtissues.•Human PSC-derived progeny readily form 3D multicellular aggregates.
Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types.
In this review, we define hypercalcemia levels, common causes for hypercalcemia in children, and treatment in order to aid the practicing pediatrician.
One rare cause of hypercalcemia in the child is ...familial hypocalciuric hypercalcemia (also termed familial benign hypercalcemia). Mutations that inactivate the Ca-sensing receptor gene FHH have been described as an autosomal dominant disorder, but recently milder mutations in the CASR have been shown to cause hypercalcemia when homozygous.
Normal serum levels of calcium are maintained through the interplay of parathyroid, renal, and skeletal factors. In this review, we have distinguished the neonate and infant from the older child and adolescent because the causes and clinical features of hypercalcemia can differ in these two age groups. However, the initial approach to the medical treatment of severe or symptomatic hypercalcemia is to increase the urinary excretion of calcium in both groups. In most cases, hypercalcemia is due to osteoclastic bone resorption, and agents that inhibit or destroy osteoclasts are, therefore, effective treatments. Parathyroid surgery, the conventional treatment for adults with symptomatic primary hyperparathyroidism, is recommended for all children with primary hyperparathyroidism.
Osteogenesis imperfecta（OI） comprises a group of heritable connective tissue disorders generally defined by recurrent fractures, low bone mass, short stature and skeletal fragility. Beyond the ...skeletal complications of OI,many patients also report intolerance to physical activity, fatigue and muscle weakness. Indeed, recent studies have demonstrated that skeletal muscle is also negatively affected by OI, both directly and indirectly. Given the well-established interdependence of bone and skeletal muscle in both physiology and pathophysiology and the observations of skeletal muscle pathology in patients with OI, we investigated the therapeutic potential of simultaneous anabolic targeting of both bone and skeletal muscle using a soluble activin receptor 2B（ACVR2B） in a mouse model of type Ⅲ OI（oim）. Treatment of 12-week-old oim mice with ACVR2 B for 4 weeks resulted in significant increases in both bone and muscle that were similar to those observed in healthy,wild-type littermates. This proof of concept study provides encouraging evidence for a holistic approach to treating the deleterious consequences of OI in the musculoskeletal system.
Myeloproliferative neoplasms (MPNs) are unique hematopoietic stem cell disorders sharing mutations that constitutively activate the signal-transduction pathways involved in haematopoiesis. They are ...characterized by stem cell-derived clonal myeloproliferation. The key MPNs comprise chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). CML is defined by the presence of the Philadelphia (Ph) chromosome and
fusion gene. Despite effective cytoreductive agents and targeted therapy, complete CML/MPN stem cell eradication is rarely achieved. In this review article, we discuss the novel agents and combination therapy that can potentially abnormal hematopoietic stem cells in CML and MPNs and the CML/MPN stem cell-sustaining bone marrow microenvironment.
This study examined the extent to which social standing based on reputation (i.e., popularity), affective regard (i.e., peer acceptance and peer rejection), and affiliation‐network centrality (i.e., ...degree, betweenness, and closeness centrality), in addition to gender and grade in school, predicted children's accuracy in detecting affiliation patterns within peer networks. Third through fifth‐grade students (n = 400) were from three semi‐rural elementary schools and clustered in eight grade‐level units. The relationship between a child's perception of the affiliation (i.e., “hanging‐out”) network and the collective perception of the affiliation network served as the indicator of network perception accuracy. In general, girls, older children, and more popular and well‐connected children had more accurate perceptions of the social environment. In particular, girls, popular children, children with high degrees centrality, and fifth graders, were more likely to report ties that existed in the collectively perceived network (made fewer false‐negative reports). Fifth graders’ specific patterns of accuracy/inaccuracy suggested a more developed cognitive model of social network structures. Children experiencing peer rejection made more false negative errors (failed to report existing ties). Interestingly, net of the other network variables in the model, closeness centrality was related to a decrease in falsely reporting ties (improving accuracy) but an increase in failing to report ties that existed (worsening accuracy). These relationships between children's social standing and grade level on their accuracy of network perception has conceptual and methodological implications for studies of children's social functioning within their school‐based peer networks.
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Zika virus has emerged as a potential threat to human health globally. A previous drug repurposing screen identified the approved anthelminthic drug niclosamide as a small molecule ...inhibitor of Zika virus infection. However, as antihelminthic drugs are generally designed to have low absorption when dosed orally, the very limited bioavailability of niclosamide will likely hinder its potential direct repurposing as an antiviral medication. Here, we conducted SAR studies focusing on the anilide and salicylic acid regions of niclosamide to improve physicochemical properties such as microsomal metabolic stability, permeability and solubility. We found that the 5-bromo substitution in the salicylic acid region retains potency while providing better drug-like properties. Other modifications in the anilide region with 2′-OMe and 2′-H substitutions were also advantageous. We found that the 4′-NO2 substituent can be replaced with a 4′-CN or 4′-CF3 substituents. Together, these modifications provide a basis for optimizing the structure of niclosamide to improve systemic exposure for application of niclosamide analogs as drug lead candidates for treating Zika and other viral infections. Indeed, key analogs were also able to rescue cells from the cytopathic effect of SARS-CoV-2 infection, indicating relevance for therapeutic strategies targeting the COVID-19 pandemic.
The molecular mechanism of the hepatic tropism of hepatitis C virus (HCV) remains incompletely defined. In vitro hepatic differentiation of pluripotent stem cells produces hepatocyte-like cells ...(HLCs) permissive for HCV infection, providing an opportunity for studying liver development and host determinants of HCV susceptibility. We previously identified the transition stage of HCV permissiveness and now investigate whether a host protein whose expression is induced during this transition stage is important for HCV infection. We suppressed the expression of a liver-specific protein, cell death-inducing DFFA-like effector b (CIDEB), and performed hepatocyte function and HCV infection assays. We also used a variety of cell-based assays to dissect the specific step of the HCV life cycle that potentially requires CIDEB function. We found CIDEB to be an essential cofactor for HCV entry into hepatocytes. Genetic interference with CIDEB in stem cells followed by hepatic differentiation leads to HLCs that are refractory to HCV infection, and infection time course experiments revealed that CIDEB functions in a late step of HCV entry, possibly to facilitate membrane fusion. The role of CIDEB in mediating HCV entry is distinct from those of the well-established receptors, as it is not required for HCV pseudoparticle entry. Finally, HCV infection effectively downregulates CIDEB protein through a posttranscriptional mechanism.
This study identifies a hepatitis C virus (HCV) entry cofactor that is required for HCV infection of hepatocytes and potentially facilitates membrane fusion between viral and host membranes. CIDEB and its interaction with HCV may open up new avenues of investigation of lipid droplets and viral entry.
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•Extended exploration of 1H-pyrrolo1,2-cimidazol-1-one scaffold were conducted.•Two tetrahydroquinoline-fused imidazolone derivatives are active against ZIKV.•It seems that N10′s ...basicity is not the key for antiviral activity in this work.•Tuning of some pharmacophores results in promising candidates for anti-Zika agents.
Zika virus (ZIKV) infection represents a significant threat to the global health system, and the search for efficient antivirals to ZIKV remains necessary and urgent. In this study, we extended the exploration of our previously discovered scaffold of 1H-pyrrolo1,2-cimidazol-1-one and revealed that two trans isomers of compounds 2 and 7 and one mixture with major trans isomer of compound 3 as novel tetrahydroquinoline-fused imidazolone derivatives are active against ZIKV infection but they are not virucidal. Western Blot and ELISA analyses of ZIKV NS5 and NS1 further demonstrate that compounds of (±)-2, (±)-3 and (±)-7 act as effective agents against ZIKV infection. We show that the N10′s basicity is not the basic requirement for these compounds’ antiviral activity in the current work. Importantly, tuning of some pharmacophores including substituents at arene can generate promising candidates for anti-ZIKV agents.
Policy makers are increasingly using performance feedback that compares physicians to their peers as part of payment policy reforms. However, it is not known whether peer comparisons can improve ...broad outcomes, beyond changing specific individual behaviors such as reducing inappropriate prescribing of antibiotics. We conducted a cluster-randomized controlled trial with Blue Cross Blue Shield of Hawaii to examine the impact of providing peer comparisons feedback on the quality of care to primary care providers in the setting of a shift from fee-for-service to population-based payment. Over 74,000 patients and eighty-eight primary care providers across sixty-three sites were included over a period of nine months in 2016. Patients in the peer comparisons intervention group experienced a 3.1-percentage-point increase in quality scores compared to the control group-whose members received individual feedback only. This result underscores the effectiveness of peer comparisons as a way to improve health care quality, and it supports Medicare's decisions to provide comparative feedback as part of recently implemented primary care and specialty payment reform programs.
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