Elevated expression of human bHLH factor ATOH7 accelerates cell cycle progression of progenitors and enhances production of avian retinal ganglion cells.

The production of vertebrate retinal projection neurons, retinal ganglion cells (RGCs), is regulated by cell-intrinsic determinants and cell-to-cell signaling events. The basic-helix-loop-helix (bHLH) protein Atoh7 is a key neurogenic transcription factor required for RGC development. Here, we investigate whether manipulating human ATOH7 expression among uncommitted progenitors can promote RGC fate specification and thus be used as a strategy to enhance RGC genesis. Using the chicken retina as a model, we show that cell autonomous expression of ATOH7 is sufficient to induce precocious RGC formation and expansion of the neurogenic territory. ATOH7 overexpression among neurogenic progenitors significantly enhances RGC production at the expense of reducing the progenitor pool. Furthermore, forced expression of ATOH7 leads to a minor increase of cone photoreceptors. We provide evidence that elevating ATOH7 levels accelerates cell cycle progression from S to M phase and promotes cell cycle exit. We also show that ATOH7-induced ectopic RGCs often exhibit aberrant axonal projection patterns and are correlated with increased cell death during the period of retinotectal connections. These results demonstrate the high potency of human ATOH7 in promoting early retinogenesis and specifying the RGC differentiation program, thus providing insight for manipulating RGC production from stem cell-derived retinal organoids

Single-Cell RNA Sequencing of hESC-Derived 3D Retinal Organoids Reveals Novel Genes Regulating RPC Commitment in Early Human Retinogenesis.

The development of the mammalian retina is a complicated process involving the generation of distinct types of neurons from retinal progenitor cells (RPCs) in a spatiotemporal-specific manner. The progression of RPCs during retinogenesis includes RPC proliferation, cell-fate commitment, and specific neuronal differentiation. In this study, by performing single-cell RNA sequencing of cells isolated from human embryonic stem cell (hESC)-derived 3D retinal organoids, we successfully deconstructed the temporal progression of RPCs during early human retinogenesis. We identified two distinctive subtypes of RPCs with unique molecular profiles, namely multipotent RPCs and neurogenic RPCs. We found that genes related to the Notch and Wnt signaling pathways, as well as chromatin remodeling, were dynamically regulated during RPC commitment. Interestingly, our analysis identified that CCND1, a G1-phase cell-cycle regulator, was coexpressed with ASCL1 in a cell-cycle-independent manner. Temporally controlled overexpression of CCND1 in retinal organoids demonstrated a role for CCND1 in promoting early retinal neurogenesis. Together, our results revealed critical pathways and novel genes in early retinogenesis of humans

Modulating Linker Composition of Haptens Resulted in Improved Immunoassay for Histamine.

Histamine (HA) is an important food contaminant generated during food fermentation or spoilage. However, an immunoassay for direct (derivatization free) determination of HA has rarely been reported due to its small size to induce the desired antibodies by its current hapten-protein conjugates. In this work, despite violating the classical hapten design criteria which recommend introducing a linear aliphatic (phenyl free) linker into the immunizing hapten, a novel haptens, HA-245 designed and synthesized with a phenyl-contained linker, exhibited significantly enhanced immunological properties. Thus, a quality-improved monoclonal antibody (Mab) against HA was elicited by its hapten-carrier conjugates. Then, as the linear aliphatic linker contained haptens, Hapten B was used as linker-heterologous coating haptens to eliminate the recognition of linker antibodies. Indirect competitive ELISA (ic-ELISA) was developed with a 50% inhibition concentration (IC50) of 0.21 mg/L and a limit of detection (LOD) of 0.06 mg/L in buffer solution. The average recoveries of HA from spiked food samples for this ic-ELISA ranged from 84.1% and 108.5%, and the analysis results agreed well with those of referenced LC-MS/MS. This investigation not only realized derivatization-free immunoassay for HA, but also provided a valuable guidance for hapten design and development of immunoassay for small molecules

Dynamic Pax6 expression during the neurogenic cell cycle influences proliferation and cell fate choices of retinal progenitors

BACKGROUND: The paired homeobox protein Pax6 is essential for proliferation and pluripotency of retinal progenitors. However, temporal changes in Pax6 protein expression associated with the generation of various retinal neurons have not been characterized with regard to the cell cycle. Here, we examine the dynamic changes of Pax6 expression among chicken retinal progenitors as they progress through the neurogenic cell cycle, and determine the effects of altered Pax6 levels on retinogenesis. RESULTS: We provide evidence that during the preneurogenic to neurogenic transition, Pax6 protein levels in proliferating progenitor cells are down-regulated. Neurogenic retinal progenitors retain a relatively low level of Pax6 protein, whereas postmitotic neurons either elevate or extinguish Pax6 expression in a cell type-specific manner. Cell imaging and cell cycle analyses show that neurogenic progenitors in the S phase of the cell cycle contain low levels of Pax6 protein, whereas a subset of progenitors exhibits divergent levels of Pax6 protein upon entering the G2 phase of the cell cycle. We also show that M phase cells contain varied levels of Pax6, and some correlate with the onset of early neuronal marker expression, forecasting cell cycle exit and cell fate commitment. Furthermore, either elevating or knocking down Pax6 attenuates cell proliferation and results in increased cell death. Reducing Pax6 decreases retinal ganglion cell genesis and enhances cone photoreceptor and amacrine interneuron production, whereas elevating Pax6 suppresses cone photoreceptor and amacrine cell fates. CONCLUSION: These studies demonstrate for the first time quantitative changes in Pax6 protein expression during the preneurogenic to neurogenic transition and during the neurogenic cell cycle. The results indicate that Pax6 protein levels are stringently controlled in proliferating progenitors. Maintaining a relatively low Pax6 protein level is necessary for S phase re-entry, whereas rapid accumulation or reduction of Pax6 protein during the G2/M phase of the cell cycle may be required for specific neuronal fates. These findings thus provide novel insights on the dynamic regulation of Pax6 protein among neurogenic progenitors and the temporal frame of neuronal fate determination

Guiding Clinical Reasoning with Large Language Models via Knowledge Seeds

Clinical reasoning refers to the cognitive process that physicians employ in evaluating and managing patients. This process typically involves suggesting necessary examinations, diagnosing patients' diseases, and deciding on appropriate therapies, etc. Accurate clinical reasoning requires extensive medical knowledge and rich clinical experience, setting a high bar for physicians. This is particularly challenging in developing countries due to the overwhelming number of patients and limited physician resources, contributing significantly to global health inequity and necessitating automated clinical reasoning approaches. Recently, the emergence of large language models (LLMs) such as ChatGPT and GPT-4 have demonstrated their potential in clinical reasoning. However, these LLMs are prone to hallucination problems, and the reasoning process of LLMs may not align with the clinical decision path of physicians. In this study, we introduce a novel framework, In-Context Padding (ICP), designed to enhance LLMs with medical knowledge. Specifically, we infer critical clinical reasoning elements (referred to as knowledge seeds) and use these as anchors to guide the generation process of LLMs. Experiments on two clinical question datasets demonstrate that ICP significantly improves the clinical reasoning ability of LLMs

Associations between impulsivity, aggression, and suicide in Chinese college students

BACKGROUND: Although there are accumulating data regarding the epidemiology of suicide in China, there are meager data on suicidal ideation and attempts among college students. Interestingly, elevated impulsivity is thought to facilitate the transition from suicidal thoughts to suicidal behavior. Therefore, the objective of this research was to identify the associations between suicide and the personality factors of impulsivity and aggression. METHODS: This study’s sampling method employed stratified random cluster sampling. A multi-stage stratified sampling procedure was used to select participants (n = 5,245). We conducted structured interviews regarding a range of socio-demographic characteristics and suicidal morbidity. The Patient Health Questionnaire depression module (PHQ-9) was used to acquire the information about thoughts of being better off dead or hurting themselves in some ways during the past two weeks. The impulsivity symptoms in this study were assessed with the BIS-11-CH (i.e., the Chinese version of the BIS-11), and the Aggressive symptoms were assessed with the BAQ. The statistical package for social science (SPSS) v.13.0 program (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Socio-demographic variables such as ethnic and gender were compared between groups, through the use of χ(2) tests. The nonparametric test (k Independent Sample test, Kruskal-Wallis H) was performed to determine differences between the personality factors of impulsivity and aggression and suicide. RESULTS: In total, 9.1% (n = 479) of the 5,245 students reported they have ever thought about committing suicide; and 1% (n = 51) reported a history of attempted suicide (attempters). The analyses detected significant differences in scores on cognitive impulsivity (p < 0.01), when comparing individuals who only had suicidal ideation and individuals who had attempted suicide. Moreover, significant differences were found between ideators only and attempters on scores of self-oriented attack (p < .001). CONCLUSIONS: Suicidal ideation is prevalent among Chinese university students. Students with high aggression scores were more susceptible to committing suicide. Scores on self-oriented attack and cognitive impulsivity may be important factors for differentially predicting suicide ideation and suicide attempts

FSD-C10: A more promising novel ROCK inhibitor than Fasudil for treatment of CNS autoimmunity.

Rho-Rho kinase (Rho-ROCK) triggers an intracellular signalling cascade that regulates cell survival, death, adhesion, migration, neurite outgrowth and retraction and influences the generation and development of several neurological disorders. Although Fasudil, a ROCK inhibitor, effectively suppressed encephalomyelitis (EAE), certain side effects may limit its clinical use. A novel and efficient ROCK inhibitor, FSD-C10, has been explored. In the present study, we present chemical synthesis and structure of FSD-C10, as well as the relationship between compound concentration and ROCK inhibition. We compared the inhibitory efficiency of ROCKI and ROCK II, the cell cytotoxicity, neurite outgrowth and dendritic formation, neurotrophic factors and vasodilation between Fasudil and FSD-C10. The results demonstrated that FSD-C10, like Fasudil, induced neurite outgrowth of neurons and dendritic formation of BV-2 microglia and enhanced the production of neurotrophic factor brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3). However, the cell cytotoxicity and vasodilation of FSD-C10 were relatively small compared with Fasudil. Although Fasudil inhibited both ROCK I and ROCK II, FSD-C10 more selectively suppressed ROCK II, but not ROCK I, which may be related to vasodilation insensitivity and animal mortality. Thus, FSD-C10 may be a safer and more promising novel ROCK inhibitor than Fasudil for the treatment of several neurological disorders