AGS3 and Gαi3 Are Concomitantly Upregulated as Part of the Spindle Orientation Complex during Differentiation of Human Neural Progenitor Cells.

Adult neurogenesis is modulated by many Gi-coupled receptors but the precise mechanism remains elusive. A key step for maintaining the population of neural stem cells in the adult is asymmetric cell division (ACD), a process which entails the formation of two evolutionarily conserved protein complexes that establish the cell polarity and spindle orientation. Since ACD is extremely difficult to monitor in stratified tissues such as the vertebrate brain, we employed human neural progenitor cell lines to examine the regulation of the polarity and spindle orientation complexes during neuronal differentiation. Several components of the spindle orientation complex, but not those of the polarity complex, were upregulated upon differentiation of ENStem-A and ReNcell VM neural progenitor cells. Increased expression of nuclear mitotic apparatus (NuMA), Gαi subunit, and activators of G protein signaling (AGS3 and LGN) coincided with the appearance of a neuronal marker (ß-III tubulin) and the concomitant loss of neural progenitor cell markers (nestin and Sox-2). Co-immunoprecipitation assays demonstrated that both Gαi3 and NuMA were associated with AGS3 in differentiated ENStem-A cells. Interestingly, AGS3 appeared to preferentially interact with Gαi3 in ENStem-A cells, and this specificity for Gαi3 was recapitulated in co-immunoprecipitation experiments using HEK293 cells transiently overexpressing GST-tagged AGS3 and different Gαi subunits. Moreover, the binding of Gαi3 to AGS3 was suppressed by GTPγS and pertussis toxin. Disruption of AGS3/Gαi3 interaction by pertussis toxin indicates that AGS3 may recognize the same site on the Gα subunit as G protein-coupled receptors. Regulatory mechanisms controlling the formation of spindle orientation complex may provide novel means to manipulate ACD which in turn may have an impact on neurogenesis.

Role of G Protein-Coupled Receptors in the Regulation of Structural Plasticity and Cognitive Function.

Cognition and other higher brain functions are known to be intricately associated with the capacity of neural circuits to undergo structural reorganization. Structural remodelling of neural circuits, or structural plasticity, in the hippocampus plays a major role in learning and memory. Dynamic modifications of neuronal connectivity in the form of dendritic spine morphology alteration, as well as synapse formation and elimination, often result in the strengthening or weakening of specific neural circuits that determine synaptic plasticity. Changes in dendritic complexity and synapse number are mediated by cellular processes that are regulated by extracellular signals such as neurotransmitters and neurotrophic factors. As many neurotransmitters act on G protein-coupled receptors (GPCRs), it has become increasingly apparent that GPCRs can regulate structural plasticity through a myriad of G protein-dependent pathways and non-canonical signals. A thorough understanding of how GPCRs exert their regulatory influence on dendritic spine morphogenesis may provide new insights for treating cognitive impairment and decline in various age-related diseases. In this article, we review the evidence of GPCR-mediated regulation of structural plasticity, with a special emphasis on the involvement of common as well as distinct signalling pathways that are regulated by major neurotransmitters.

PRAgmatic Clinical Trial Design of Integrative MediCinE (PRACTICE): A Focus Group Series and Systematic Review on Trials of Diabetes and Kidney Disease.

Background: Pragmatic trials inform clinical decision with better generalizability and can bridge different streams of medicine. This study collated the expectations regarding pragmatic trial design of integrative medicine (IM) for diabetes and kidney diseases among patients and physicians. Dissonance between users' perspective and existing pragmatic trial design was identified. The association between risk of bias and pragmatism of study design was assessed. Method: A 10-group semi-structured focus group interview series [21 patients, 14 conventional medicine (ConM) and 15 Chinese medicine (CM) physicians] were purposively sampled from private and public clinics in Hong Kong. Perspectives were qualitatively analyzed by constant comparative method. A systematic search of four databases was performed to identify existing IM pragmatic clinical trials in diabetes or kidney disease. Primary outcomes were the pragmatism, risk of bias, and rationale of the study design. Risk of bias and pragmatism were assessed based on Cochrane risk-of-bias tool and PRECIS-2, respectively. The correlation between risk of bias and pragmatism was assessed by regression models with sensitivity analyses. Results: The subtheme on the motivation to seek IM service was analyzed, covering the perceived limitation of ConM effect, perceived benefits of IM service, and assessment of IM effectiveness. Patients expected IM service to retard disease progression, stabilize concomitant drug dosage, and reduce potential side effects associated with ConM. In the systematic review, 25 studies from six countries were included covering CM, Korean medicine, Ayurvedic medicine, and western herbal medicine. Existing study designs did not include a detailed assessment of concomitant drug change and adverse events. Majority of studies either recruited a non-representative proportion of patients as traditional, complementary, and integrative medicine (TCIM) diagnosis was used as inclusion criteria, or not reflecting the real-world practice of TCIM by completely dropping TCIM diagnosis in the trial design. Consultation follow-up frequency is the least pragmatic domain. Increase in pragmatism did not associate with a higher risk of bias. Conclusion: Existing IM pragmatic trial design does not match the patients' expectation in the analysis of incident concomitant drug change and adverse events. A two-layer design incorporating TCIM diagnosis as a stratification factor maximizes the generalizability of evidence and real-world translation of both ConM and TCIM.

Patients' and clinicians' expectations on integrative medicine Services for Diabetes: a focus group study.

BACKGROUND: Difference of perspective between patients and physicians over integrative medicine (IM) research and service provision remains unclear despite significant use worldwide. We observed an exceptionally low utilisation of IM and potential underreporting in diabetes. We aimed to explore the barriers and recommendations regarding service delivery and research of IM service among diabetes patients and physicians. METHODS: A 10-group, 50-participant semi-structured focus group interview series was conducted. Twenty-one patients with diverse severity of disease, comorbidities and education levels; and 29 physicians (14 conventional medicine (ConM) and 15 Chinese medicine (CM)) with diverse clinical experience, academic background and affiliation were purposively sampled from private and public clinics. Their perspectives were qualitatively analysed by constant comparative method. RESULTS: Seven subthemes regarding barriers towards IM service were identified including finance, service access, advice from medical professionals, uncertainty of service quality, uncertainty of CM effect, difficulty in understanding CM epistemology and access to medical records. Patients underreported the use of CM due to the concern over neutrality of medical advice among physicians. Inconvenience of service access, frequent follow-up, use of decoction and long-term financial burden were identified as key obstacles among patients. Regarding research design, ConM physicians emphasised standardisation and reproducibility while CM physicians emphasised personalisation. Some CM-related outcome measurements were suggested as non-communicable. Both physicians acknowledged the discordance in epistemology should be addressed by pragmatic approach. CONCLUSION: Key obstacles of CAM clinical utilisation are different between patients. Further assessment on IM should be pragmatic to balance between standardisation, reproducibility and real-world practice. Evidence-based IM programs and research should merge with existing infrastructure.

A Hierarchical Machine Learning Model to Discover Gleason Grade-Specific Biomarkers in Prostate Cancer.

(1) Background:One of the most common cancers that affect North American men and men worldwide is prostate cancer. The Gleason score is a pathological grading system to examine the potential aggressiveness of the disease in the prostate tissue. Advancements in computing and next-generation sequencing technology now allow us to study the genomic profiles of patients in association with their different Gleason scores more accurately and effectively. (2) Methods: In this study, we used a novel machine learning method to analyse gene expression of prostate tumours with different Gleason scores, and identify potential genetic biomarkers for each Gleason group. We obtained a publicly-available RNA-Seq dataset of a cohort of 104 prostate cancer patients from the National Center for Biotechnology Information's (NCBI) Gene Expression Omnibus (GEO) repository, and categorised patients based on their Gleason scores to create a hierarchy of disease progression. A hierarchical model with standard classifiers in different Gleason groups, also known as nodes, was developed to identify and predict nodes based on their mRNA or gene expression. In each node, patient samples were analysed via class imbalance and hybrid feature selection techniques to build the prediction model. The outcome from analysis of each node was a set of genes that could differentiate each Gleason group from the remaining groups. To validate the proposed method, the set of identified genes were used to classify a second dataset of 499 prostate cancer patients collected from cBioportal. (3) Results: The overall accuracy of applying this novel method to the first dataset was 93.3%; the method was further validated to have 87% accuracy using the second dataset. This method also identified genes that were not previously reported as potential biomarkers for specific Gleason groups. In particular, PIAS3 was identified as a potential biomarker for Gleason score 4 + 3 = 7, and UBE2V2 for Gleason score 6. (4) Insight: Previous reports show that the genes predicted by this newly proposed method strongly correlate with prostate cancer development and progression. Furthermore, pathway analysis shows that both PIAS3 and UBE2V2 share similar protein interaction pathways, the JAK/STAT signaling process.

Synthesis and Evaluation of 2-Alkylthio-4-(N-substituted sulfonamide)pyrimidine Hydroxamic Acids as Anti-myeloma Agents.

A series of pyrimidine hydroxamic acids with a sulfide substituent at the second position and a sulfonamide substituent at the fourth position have been synthesized and evaluated for their activity against human myeloma cell line RPMI 8226. Several compounds exhibited significant anti-cancer potency. It was found that representative compound 6a selectively killed cancerous but not normal cells. Moreover, compound 6a was effective in causing apoptosis in RPMI 8226 cells and exhibited promising HDAC-inhibitory activities.

Sox9 is critical for suppression of neurogenesis but not initiation of gliogenesis in the cerebellum.

BACKGROUND: The high mobility group (HMG) family transcription factor Sox9 is critical for induction and maintenance of neural stem cell pool in the central nervous system (CNS). In the spinal cord and retina, Sox9 is also the master regulator that defines glial fate choice by mediating the neurogenic-to-gliogenic fate switch. On the other hand, the genetic repertoire governing the maintenance and fate decision of neural progenitor pool in the cerebellum has remained elusive. RESULTS: By employing the Cre/loxP strategy, we specifically inactivated Sox9 in the mouse cerebellum. Unexpectedly, the self-renewal capacity and multipotency of neural progenitors at the cerebellar ventricular zone (VZ) were not perturbed upon Sox9 ablation. Instead, the mutants exhibited an increased number of VZ-derived neurons including Purkinje cells and GABAergic interneurons. Simultaneously, we observed continuous neurogenesis from Sox9-null VZ at late gestation, when normally neurogenesis ceases to occur and gives way for gliogenesis. Surprisingly, glial cell specification was not affected upon Sox9 ablation. CONCLUSION: Our findings suggest Sox9 may mediate the neurogenic-to-gliogenic fate switch in mouse cerebellum by modulating the termination of neurogenesis, and therefore indicate a functional discrepancy of Sox9 between the development of cerebellum and other major neural tissues.

CD44v6 regulates growth of brain tumor stem cells partially through the AKT-mediated pathway.

Identification of stem cell-like brain tumor cells (brain tumor stem-like cells; BTSC) has gained substantial attention by scientists and physicians. However, the mechanism of tumor initiation and proliferation is still poorly understood. CD44 is a cell surface protein linked to tumorigenesis in various cancers. In particular, one of its variant isoforms, CD44v6, is associated with several cancer types. To date its expression and function in BTSC is yet to be identified. Here, we demonstrate the presence and function of the variant form 6 of CD44 (CD44v6) in BTSC of a subset of glioblastoma multiforme (GBM). Patients with CD44(high) GBM exhibited significantly poorer prognoses. Among various variant forms, CD44v6 was the only isoform that was detected in BTSC and its knockdown inhibited in vitro growth of BTSC from CD44(high) GBM but not from CD44(low) GBM. In contrast, this siRNA-mediated growth inhibition was not apparent in the matched GBM sample that does not possess stem-like properties. Stimulation with a CD44v6 ligand, osteopontin (OPN), increased expression of phosphorylated AKT in CD44(high) GBM, but not in CD44(low) GBM. Lastly, in a mouse spontaneous intracranial tumor model, CD44v6 was abundantly expressed by tumor precursors, in contrast to no detectable CD44v6 expression in normal neural precursors. Furthermore, overexpression of mouse CD44v6 or OPN, but not its dominant negative form, resulted in enhanced growth of the mouse tumor stem-like cells in vitro. Collectively, these data indicate that a subset of GBM expresses high CD44 in BTSC, and its growth may depend on CD44v6/AKT pathway.