Multimodal Nature of the Single-cell Primate Brain Atlas: Morphology, Transcriptome, Electrophysiology, and Connectivity.

Primates exhibit complex brain structures that augment cognitive function. The neocortex fulfills high-cognitive functions through billions of connected neurons. These neurons have distinct transcriptomic, morphological, and electrophysiological properties, and their connectivity principles vary. These features endow the primate brain atlas with a multimodal nature. The recent integration of next-generation sequencing with modified patch-clamp techniques is revolutionizing the way to census the primate neocortex, enabling a multimodal neuronal atlas to be established in great detail: (1) single-cell/single-nucleus RNA-seq technology establishes high-throughput transcriptomic references, covering all major transcriptomic cell types; (2) patch-seq links the morphological and electrophysiological features to the transcriptomic reference; (3) multicell patch-clamp delineates the principles of local connectivity. Here, we review the applications of these technologies in the primate neocortex and discuss the current advances and tentative gaps for a comprehensive understanding of the primate neocortex.

A primate nigrostriatal atlas of neuronal vulnerability and resilience in a model of Parkinson's disease.

The degenerative process in Parkinson's disease (PD) causes a progressive loss of dopaminergic neurons (DaNs) in the nigrostriatal system. Resolving the differences in neuronal susceptibility warrants an amenable PD model that, in comparison to post-mortem human specimens, controls for environmental and genetic differences in PD pathogenesis. Here we generated high-quality profiles for 250,173 cells from the substantia nigra (SN) and putamen (PT) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonian macaques and matched controls. Our primate model of parkinsonism recapitulates important pathologic features in nature PD and provides an unbiased view of the axis of neuronal vulnerability and resistance. We identified seven molecularly defined subtypes of nigral DaNs which manifested a gradient of vulnerability and were confirmed by fluorescence-activated nuclei sorting. Neuronal resilience was associated with a FOXP2-centered regulatory pathway shared between PD-resistant DaNs and glutamatergic excitatory neurons, as well as between humans and nonhuman primates. We also discovered activation of immune response common to glial cells of SN and PT, indicating concurrently activated pathways in the nigrostriatal system. Our study provides a unique resource to understand the mechanistic connections between neuronal susceptibility and PD pathophysiology, and to facilitate future biomarker discovery and targeted cell therapy.

Neural cell isolation from adult macaques for high-throughput analyses and neurosphere cultures.

The low number of neural progenitor cells (NPCs) present in the adult and aged primate brains represents a challenge for generating high-yield and viable in vitro cultures of primary brain cells. Here we report a step-by-step approach for the fast and reproducible isolation of high-yield and viable primary brain cells, including mature neurons, immature cells and NPCs, from adult and aged macaques. We describe the anesthesia, transcardial perfusion and brain tissue preparation; the subsequent microdissection of the regions of interest and their enzymatic dissociation, leading to the separation of single cells. The cell isolation steps of our protocol can also be used for routine cell culturing, in particular for NPC expansion and differentiation, suitable for studies of hippocampal neurogenesis in the adult macaque brain. The purified primary brain cells are largely free from myelin debris and erythrocytes, paving the way for multiple downstream applications in vitro and in vivo. When combined with single-cell profiling techniques, this approach allows an unbiased and comprehensive mapping of cell states in the adult and aged macaque brain, which is needed to advance our understanding of human cognitive and neurological diseases. The neural cell isolation protocol requires 4 h and a team of four to six users with expertize in primary brain cell isolation to avoid tissue hypoxia during the time-sensitive steps of the procedure.

Multi-Layer Graph Attention Network for Sleep Stage Classification Based on EEG.

Graph neural networks have been successfully applied to sleep stage classification, but there are still challenges: (1) How to effectively utilize epoch information of EEG-adjacent channels owing to their different interaction effects. (2) How to extract the most representative features according to confused transitional information in confused stages. (3) How to improve classification accuracy of sleep stages compared with existing models. To address these shortcomings, we propose a multi-layer graph attention network (MGANet). Node-level attention prompts the graph attention convolution and GRU to focus on and differentiate the interaction between channels in the time-frequency domain and the spatial domain, respectively. The multi-head spatial-temporal mechanism balances the channel weights and dynamically adjusts channel features, and a multi-layer graph attention network accurately expresses the spatial sleep information. Moreover, stage-level attention is applied to easily confused sleep stages, which effectively improves the limitations of a graph convolutional network in large-scale graph sleep stages. The experimental results demonstrated classification accuracy; MF1 and Kappa reached 0.825, 0.814, and 0.775 and 0.873, 0.801, and 0.827 for the ISRUC and SHHS datasets, respectively, which showed that MGANet outperformed the state-of-the-art baselines.

Identification of visual cortex cell types and species differences using single-cell RNA sequencing.

The primate neocortex exerts high cognitive ability and strong information processing capacity. Here, we establish a single-cell RNA sequencing dataset of 133,454 macaque visual cortical cells. It covers major cortical cell classes including 25 excitatory neuron types, 37 inhibitory neuron types and all glial cell types. We identified layer-specific markers including HPCAL1 and NXPH4, and also identified two cell types, an NPY-expressing excitatory neuron type that expresses the dopamine receptor D3 gene; and a primate specific activity-dependent OSTN + sensory neuron type. Comparisons of our dataset with humans and mice show that the gene expression profiles differ between species in relation to genes that are implicated in the synaptic plasticity and neuromodulation of excitatory neurons. The comparisons also revealed that glutamatergic neurons may be more diverse across species than GABAergic neurons and non-neuronal cells. These findings pave the way for understanding how the primary cortex fulfills the high-cognitive functions.

Vessel state and immune infiltration of the angiogenesis subgroup and construction of a prediction model in osteosarcoma.

Angiogenesis has been recognized as a pivotal contributor to tumorigenesis and progression. However, the role of angiogenesis-related genes (ARGs) in vessel state, immune infiltration, and prognosis remains unknown in osteosarcoma (OS). Bulk RNA sequencing data of osteosarcoma patients were obtained from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database, and patients were divided into two angiogenesis subgroups according to the expression of ARGs. We compared their vessel state and used two independent algorithms to evaluate the tumor microenvironment (TME) in the two subgroups. Furthermore, hub genes of differentially expressed genes (DEGs) in the two subgroups were selected to perform LASSO regression and multivariate Cox stepwise regression, and two prognostic hub genes were found. An ARG_score based on prognostic hub genes was calculated and proved to be reliable in the overall survival prediction in OS patients. Furthermore, the ARG_score was significantly associated with ARGs, immune infiltration, response to immunotherapy, and drug sensitivity. To make our prediction model perform well, clinical features were added and a highly accurate interactive nomogram was constructed. Immunohistochemistry and qRT-PCR were utilized to verify the expression of prognostic hub genes. GSE21257 from the Gene Expression Omnibus (GEO) database was used as a validation dataset to verify its robustness. In conclusion, our comprehensive analysis of angiogenesis subgroups in OS illustrated that angiogenesis may lead to different vessel states and further affect immune infiltration and prognosis of OS patients. Our findings may bring a novel perspective for the immunotherapy strategies for OS patients.

Integrated microfluidic-SERS for exosome biomarker profiling and osteosarcoma diagnosis.

Osteosarcoma is one of the most frequent primary sarcoma of bone among adolescents. Early diagnosis of osteosarcoma is the key factor to achieve high survival rate of patients. Nevertheless, traditional histological biopsy is highly invasive and associated with the risk of arousing tumor spread. Herein, we develop a method integrating microfluidics and surface-enhanced Raman spectroscopy (SERS) to isolate plasma-derived exosomes and profile multiple exosomal biomarkers for the diagnosis of osteosarcoma. The method showed highly efficient isolation of exosomes directly from human plasma and can profile exosomes based on protein biomarkers, with the detection limit down to 2 exosomes per µL. The whole assay can be performed in 5 h and only consumed 50 µL of plasma for one analysis. With the method, we analyzed the level of three protein biomarkers, i.e., CD63, vimentin (VIM) and epithelial cell adhesion molecule (EpCAM), on plasma-derived exosomes from 20 osteosarcoma patients and 20 heathy controls. Significantly higher levels of CD63, VIM and EpCAM were observed on plasma exosomes from the osteosarcoma patients compared to the healthy controls. Based on the level of the exosomal biomarkers, a classification model was built for the rapid diagnosis of osteosarcoma, with the sensitivity, specificity and accuracy of 100%, 90% and 95%, respectively. The proposed method does not require complex operations nor expensive equipment, and has great promise in clinical diagnosis of cancer as a liquid biopsy technique.

Linking transcriptomes with morphological and functional phenotypes in mammalian retinal ganglion cells.

Retinal ganglion cells (RGCs) are the brain's gateway to the visual world. They can be classified into different types on the basis of their electrophysiological, transcriptomic, or morphological characteristics. Here, we characterize the transcriptomic, morphological, and functional features of 472 high-quality RGCs using Patch sequencing (Patch-seq), providing functional and morphological annotation of many transcriptomic-defined cell types of a previously established RGC atlas. We show a convergence of different modalities in defining the RGC identity and reveal the degree of correspondence for well-characterized cell types across multimodal data. Moreover, we complement some RGC types with detailed morphological and functional properties. We also identify differentially expressed genes among ON, OFF, and ON-OFF RGCs such as Vat1l, Slitrk6, and Lmo7, providing candidate marker genes for functional studies. Our research suggests that the molecularly distinct clusters may also differ in their roles of encoding visual information.

Design, synthesis and biological evaluation of novel macrocyclic derivatives bearing aniline pyrimidine scaffolds as EGFR-TKIs.

Based on EGFR-TKI Osimertinib as lead compound, a series of novel macrocyclic derivatives bearing aniline pyrimidine scaffolds were designed and synthesized by macrocyclization. Their structures were identified by 1H NMR, 13C NMR, 19F NMR and HRMS. The pharmacological activities of the target compounds were tested and the preliminary structure-activity relationship was discussed. Among them, 17-membered ring compound H1 displayed the best inhibitory activities against EGFRL858R/T790M and EGFRd746-750/T790M with IC50 value of 2.92 nM and 0.34 nM, respectively. Exhilaratingly, 17-membered ring compound H7 possessed the most potent antiproliferative activity against BaF3-EGFRdel19/T790M cell lines (IC50 = 0.035 µm), which rivaled that of Osimertinib (IC50 = 0.033 µm).

Identification of the Transcription Co-Factor-Related Gene Signature and Risk Score Model for Osteosarcoma.

Osteosarcoma is a malignant tumor with a poor prognosis. Nowadays, there is a lack of good methods to assess the prognosis of osteosarcoma patients. Transcription co-factors (TcoFs) play crucial roles in transcriptional regulation through the interaction with transcription factors (TFs). Many studies have revealed that TcoFs are related to many diseases, especially cancer. However, few studies have been reported about prognostic prediction models of osteosarcoma by using TcoF-related genes. In order to construct a prognostic risk model with TcoF-related genes, the mRNA expression data and matched clinical information of osteosarcoma were downloaded from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database and the Gene Expression Omnibus (GEO) database. TARGET was used as a training set and GSE21257 from GEO was used as a validation set. Univariate Cox regression was performed to select 13 TcoF-related candidate genes, of which five genes (LMO2, MAML3, MTF2, RBPMS, and SIRT1) were finally used to construct the prognostic risk model by LASSO Cox regression analysis. The Kaplan-Meier (K-M) survival curves showed an obvious difference between high- and low-risk groups. The receiver operating characteristic (ROC) curves based on TARGET demonstrated that this risk model was credible (1-year AUC: 0.607; 3-years AUC: 0.713; 5-years AUC: 0.736). Meanwhile, the risk model was associated with immune cells and immune-related functions. By combining the risk score and clinical factors, the nomogram of osteosarcoma was assessed with a C-index of 0.738 to further support the reliability of this 5-gene prognostic risk model. Finally, the expression of TcoF-related genes was validated in different cell lines by quantitative real-time PCR (qRT-PCR) and also in different tissue samples by immunohistochemistry (IHC). In conclusion, the model can predict the prognosis of osteosarcoma patients and may provide novel targets for the treatment of osteosarcoma patients.

Construction and validation of a novel apoptosis-associated prognostic signature related to osteosarcoma metastasis and immune infiltration.

BACKGROUND: Apoptosis played vital roles in the formation and progression of osteosarcoma. However, no studies elucidated the prognostic relationships between apoptosis-associated genes (AAGs) and osteosarcoma. METHODS: The differentially expressed genes associated with osteosarcoma metastasis and apoptosis were identified from GEO and MSigDB databases. The apoptosis-associated prognostic signature was established through univariate and multivariate cox regression analyses. The Kaplan-Meier (KM) survival curve, ROC curve and nomogram were constructed to investigate the predictive value of this signature. CIBERSORT algorithm and ssGSEA were used to explore the relationships between immune infiltration and AAG signature. The above results were validated in another GEO dataset and the expression of AAGs was also validated in osteosarcoma patient samples by immunohistochemistry. RESULTS: HSPB1 and IER3 were involved in AAG signature. In training and validation datasets, apoptosis-associated risk scores were negatively related to patient survival rates and the AAG signature was regarded as the independent prognostic factor. ROC and calibration curves demonstrated the signature and nomogram were reliable. GSEA revealed the signature related to immune-associated pathways. ssGSEA indicated that one immune cell and three immune functions were significantly dysregulated. The immunohistochemistry analyses of patients' samples revealed that AAGs were significantly differently expressed between metastasis and non-metastasis osteosarcomas. CONCLUSIONS: The present study identified and validated a novel apoptosis-associated prognostic signature related to osteosarcoma metastasis. It could serve as the potential biomarker and therapeutic targets for osteosarcoma in the future.

Single-cell transcriptomics of adult macaque hippocampus reveals neural precursor cell populations.

The extent to which neurogenesis occurs in adult primates remains controversial. In this study, using an optimized single-cell RNA sequencing pipeline, we profiled 207,785 cells from the adult macaque hippocampus and identified 34 cell populations comprising all major hippocampal cell types. Analysis of their gene expression, specification trajectories and gene regulatory networks revealed the presence of all key neurogenic precursor cell populations, including a heterogeneous pool of radial glia-like cells (RGLs), intermediate progenitor cells (IPCs) and neuroblasts. We identified HMGB2 as a novel IPC marker. Comparison with mouse single-cell transcriptomic data revealed differences in neurogenic processes between species. We confirmed that neurogenesis is recapitulated in ex vivo neurosphere cultures from adult primates, further supporting the existence of neural precursor cells (NPCs) that are able to proliferate and differentiate. Our large-scale dataset provides a comprehensive adult neurogenesis atlas for primates.

[Nursing Digital Transformation: An Example of an Electronic Whiteboard in a Regional Hospital in Central Taiwan].

The electronic whiteboard system is an important part of smart medical care. This system has been digitized and upgraded gradually over time, and now functions as a dashboard, incorporating sound effects, touch control, image display, face recognition, and other functions that maximize usage efficiency. In hospitals, electronic whiteboards are specialized for dedicated use in one of two areas: nursing stations and wards. Those used in nursing stations may upload data into the medical information system based on departmental and institutional requirements. Systems are built to the specific needs of different clinical departments and thus differ widely in terms of settings and functionality. Therefore, hospitals should promote regular communication among doctors, nurses, and patients.

The Outcome of Discontinuing Tyrosine Kinase Inhibitors in Advanced Sarcoma Following a Favorable Tumor Response to Antiangiogenics Therapy.

(1) Background: The use of antiangiogenic TKIs (AA-TKIs) has recently emerged as a major paradigm shift in the treatment of advanced sarcoma. However, the feasibility of drug holidays for patients demonstrating a very favorable response remains unknown. (2) Methods: We aim to explore the outcomes of patients with advanced sarcoma who discontinued AA-TKIs after a (near-) complete remission or were long-term responders. Patients with advanced disease were included if they had bilateral or multiple lung metastases, extrapulmonary recurrence, a short disease-free interval, etc., at the initiation of AA-TKIs. (3) Results: A total of 22 patients with AA-TKI discontinuation were analyzed, with a median follow-up of 22.3 months post-discontinuation. Prior to discontinuation, there were four drug-induced complete remissions (CRs), twelve surgical CRs, and six long-term responders. Disease progression was observed in 17/22 (77.3%) patients, with a median of 4.2 months. However, since the majority were still sensitive to the original AA-TKIs and amenable to a second surgical remission, 7 out of these 17 patients achieved a second CR after disease progression and were thus considered as relapse-free post-discontinuation (pd-RFS). Therefore, the pd-RFS and post-discontinuation overall survival (pd-OS) in the last follow-up were 12/22 (54.5%) and 16/22 (72.7%), respectively. Remarkably, surgical CR and drug tapering off (versus abrupt stopping) were associated with a greater pd-RFS and pd-OS (p < 0.05). Furthermore, higher necrosis rates (p = 0.040) and lower neutrophil-to-lymphocyte ratios (NLR) (p = 0.060) before discontinuation tend to have a better pd-RFS. (4) Conclusions: Our results suggest that AA-TKI discontinuation with a taper-off strategy might be safe and feasible in highly selected patients with advanced sarcoma. Surgical CR, NLR, and tumor necrosis rates before discontinuation were potential biomarkers for AA-TKI withdrawal.

SERS and MALDI-TOF MS based plasma exosome profiling for rapid detection of osteosarcoma.

Osteosarcoma is the most frequent primary bone cancer, particularly among children and adolescents. The early diagnosis of osteosarcoma is significant for timely clinical treatment to reduce the mortality of patients. Exosomes play a significant role in intercellular communication and serve as promising biomarkers in liquid biopsy for the diagnosis and monitoring of tumors. Herein, we report the utility of surface-enhanced Raman scattering (SERS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of osteosarcoma. We firstly profiled the intrinsic SERS signals and MALDI-TOF mass fingerprints of different subgroups of extracellular vesicles (EVs) and the corresponding cells, demonstrating that the SERS signals and MALDI-TOF mass spectra of exosomes from different types of cells were more discriminative compared to those of large and medium EVs and the cells themselves. Then, we characterized plasma-derived exosomes of 15 osteosarcoma patients and 15 healthy volunteers using SERS and MALDI-TOF MS, revealing distinctive biochemical differences in the spectra. We further utilized a data fusion approach to combine the two types of spectroscopic techniques, differentiating osteosarcoma patients from healthy controls with higher precision than either technique. The results reveal that the non-invasive liquid biopsy method using SERS and MALDI-TOF MS fingerprinting of exosomes has great potential for rapid diagnosis of osteosarcoma.

Simplifying and Testing the Psychometric Psychiatric Patients' Fall Risk Scale: An Analysis of One-Year Admissions.

This study aimed to simplify the number of items evaluated by fall risk assessment scales for psychiatric patients, conduct associated reliability, validity, and receiver operating characteristic analyses, and determine fall predictors for psychiatric patients. This methodological study was conducted in a hospital specializing in psychiatry, using data from 1101 patients who were hospitalized in 2018. This fall risk assessment scale was modified by the hospital for use in psychiatric patients. The mean age of the sample population was 44.88 (SD = 12.05) years, and the mean duration of hospital stay was 44.04 (SD = 48.14) days. Men comprised 66% of the study population, and women were 34%. Item reduction, psychometric testing for validity and reliability, and receiver operating characteristic analyses were conducted. Logistic regressions were used to analyze fall predictors, including "having anti-epileptic drugs", "need for walking aids", and "having experienced fall occurrence within one year". This study successfully reduced the number of items assessed by the previous scale. The optimal cutoff point was reduced, and the sensitivity and accuracy of the newly revised scale were good. Three fall predictors for psychiatric patients were identified. The revised scale can facilitate the rapid and accurate identification of high-risk, fall-prone psychiatric patients by psychiatric nurses. Hospital information screening should include each patient's fall history.

A Novel Microfluidic Chip for Fast, Sensitive Quantification of Plasma Extracellular Vesicles as Biomarkers in Patients With Osteosarcoma.

Plasma circulating extracellular vesicle (EV) has emerged as a promising biomarker for diagnosis and prognosis of various epithelial tumors. However, fast and efficient capture of EVs with microfluidic chip in sarcoma remains to be established. Herein, we reported a ZnO-nanorods integrated (ZNI) microfluidic chip, where EV capture antibody was uniformly grafted to the surface of the ZnO-nanorods of the chip to enhance the plasma turbulence formation and the capture efficiency at the micro-scale. Based on osteosarcoma (OS) cell line, we demonstrated that a combination of CD81 and CD63 antibody on ZNI chip yielded the greatest amount of total EVs, with an extra sensitive limit of detection (LOD) of ~104 particles mL-1. Furthermore, the addition of fluorescent labeling of Vimentin (VIM), a previously reported sarcoma cell surface biomarker, could enabled the dual visualization of total plasma EVs and VIM-positive EVs from OS patients' plasma. Based on our ZNI chip, we found that the amount of plasma total EVs was significantly different between OS and healthy donors (1562 a.u. versus 639 a.u., p< 0.05), but not between metastatic and nonmetastatic OS (p> 0.05). Interestingly, patients with metastatic disease had a significantly greater amount of VIM-positive EVs (1411 a.u. versus 231 a.u.., p< 0.05) and increased VIM-positive/total EVs ratio (0.943 versus 0.211, p< 0.05) in comparison with the nonmetastatic counterpart. Therefore, our ZNI microfluidic chip has great potential for the fast quantification of plasma EVs, and the microfluidic-based quantification of total and VIM-positive EVs might serve as a promising biomarker for the diagnosis and surveillance in OS patients.

Matrix-assisted laser desorption ionization mass spectrometry profiling of plasma exosomes evaluates osteosarcoma metastasis.

Osteosarcoma is the most common primary sarcoma of bone among adolescents, often characterized by early lung metastasis resulting in high mortality. Recently, exosomes have been used in liquid biopsy to monitor tumors. Herein, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to profile human plasma exosomes for the evaluation of osteosarcoma lung metastasis. Forty patients with osteosarcoma with (n = 20) or without (n = 20) lung metastasis as well as 12 heathy controls were recruited. Exosomes were isolated from human plasma for MALDI-TOF MS analysis. Multivariate statistical analyses were performed based on the MALDI-TOF mass spectra. The strategy can efficiently differentiate osteosarcomas from healthy controls and further discriminate osteosarcoma lung metastasis from non-lung metastasis. We identified seven exosomal proteins as potential biomarkers of osteosarcoma lung metastasis. The proposed method holds great promise to clinically diagnose osteosarcoma and monitor osteosarcoma lung metastasis.