Human XPR1 structures reveal phosphate export mechanism.

Inorganic phosphate (Pi) is a fundamental macronutrient for all living organisms, the homeostasis of which is critical for numerous biological activities1-3. As the only known human Pi exporter to date, XPR1 has an indispensable role in cellular Pi homeostasis4,5. Dysfunction of XPR1 is associated with neurodegenerative disease6-8. However, the mechanisms underpinning XPR1-mediated Pi efflux and regulation by the intracellular inositol polyphosphate (InsPP) sensor SPX domain remain poorly understood. Here we present cryo-electron microscopy structures of human XPR1 in Pi-bound closed, open and InsP6-bound forms, revealing the structural basis for XPR1 gating and regulation by InsPPs. XPR1 consists of an N-terminal SPX domain, a dimer-formation core domain and a Pi transport domain. Within the transport domain, three basic clusters are responsible for Pi binding and transport, and a conserved W573 acts as a molecular switch for gating. In addition, the SPX domain binds to InsP6 and facilitates Pi efflux by liberating the C-terminal loop that limits Pi entry. This study provides a conceptual framework for the mechanistic understanding of Pi homeostasis by XPR1 homologues in fungi, plants and animals.

Single-cell multi-modal chromatin profiles revealing epigenetic regulations of cells in hepatocellular carcinoma.

BACKGROUND: Various epigenetic regulations systematically govern gene expression in cells involving various biological processes. Dysregulation of the epigenome leads to aberrant transcriptional programs and subsequently results in diseases, such as cancer. Therefore, comprehensive profiling epigenomics is essential for exploring the mechanisms underlying gene expression regulation during development and disease. METHODS: In this study, we developed single-cell chromatin proteins and accessibility tagmentation (scCPA-Tag), a multi-modal single-cell epigenetic profile capturing technique based on barcoded Tn5 transposases and a droplet microfluidics platform. scCPA-Tag enables the simultaneous capture of DNA profiles of histone modification and chromatin accessibility in the same cell. RESULTS: By applying scCPA-Tag to K562 cells and a hepatocellular carcinoma (HCC) sample, we found that the silence of several chromatin-accessible genes can be attributed to lysine-27-trimethylation of the histone H3 tail (H3K27me3) modification. We characterized the epigenetic features of the tumour cells and different immune cell types in the HCC tumour tissue by scCPA-Tag. Besides, a tumour cell subtype (C2) with more aggressive features was identified and characterized by high chromatin accessibility and a lower abundance of H3K27me3 on tumour-promoting genes. CONCLUSIONS: Our multi-modal scCPA-Tag provides a comprehensive approach for exploring the epigenetic landscapes of heterogeneous cell types and revealing the mechanisms of gene expression regulation during developmental and pathological processes at the single-cell level. HIGHLIGHTS: scCPA-Tag offers a highly efficient and high throughput technique to simultaneously profile histone modification and chromatin accessibility within a single cell. scCPA-Tag enables to uncover multiple epigenetic modification features of cellular compositions within tumor tissues. scCPA-Tag facilitates the exploration of the epigenetic landscapes of heterogeneous cell types and provides the mechanisms governing gene expression regulation.

A single-cell chromatin accessibility dataset of human primed and naïve pluripotent stem cell-derived teratoma.

Teratoma, due to its remarkable ability to differentiate into multiple cell lineages, is a valuable model for studying human embryonic development. The similarity of the gene expression and chromatin accessibility patterns in these cells to those observed in vivo further underscores its potential as a research tool. Notably, teratomas derived from human naïve (pre-implantation epiblast-like) pluripotent stem cells (PSCs) have larger embryonic cell diversity and contain extraembryonic lineages, making them more suitable to study developmental processes. However, the cell type-specific epigenetic profiles of naïve PSC teratomas have not been yet characterized. Using single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), we analyzed 66,384 cell profiles from five teratomas derived from human naïve PSCs and their post-implantation epiblast-like (primed) counterparts. We observed 17 distinct cell types from both embryonic and extraembryonic lineages, resembling the corresponding cell types in human fetal tissues. Additionally, we identified key transcription factors specific to different cell types. Our dataset provides a resource for investigating gene regulatory programs in a relevant model of human embryonic development.

A violet light-emitting diode-based gas-phase molecular absorption device for measurement of nitrate and nitrite in environmental water.

A simple and sensitive device for the detection of nitrite and nitrate in environmental waters was developed based on visible light gas-phase molecular absorption spectrometry. By integrating a detection cell (DC), semiconductor refrigeration temperature-controlling system (SRTCY), and nitrite reactor into a sequential injection analysis system, trace levels of nitrite and nitrate in complex matrices were successfully measured. A low energy-consuming light-emitting diode (violet, 400-405 nm) was coupled with a visible light-to-voltage converter (TSL257) to measure the gas-phase molecular absorption. To reduce the interference of water vapor, an SRTCY was used to condense the water vapor on-line before the gas-phase analyte entered the DC. The DC was radiatively heated by the SRTCY to avoid water vapor condensation in the light path. As a result, the obtained baseline noise reduced 3.75 times than that of without SRTCY. Under the optimized conditions, the device achieved limits of detection (3σ/k) of 0.055 and 0.36 mmol/L (0.77 and 5.04 mg N/L) for nitrite and nitrate, respectively, and the linear calibration ranges were 0.1-15 mmol/L (R2 = 0.9946) and 1-10 mmol/L (R2 = 0.9995), respectively. Precisions of 5.2 % and 9.0 % were achieved for ten successive determinations of 0.3 mmol/L nitrite and 1.0 mmol/L nitrate, and the analytical times for nitrite and nitrate determination were 5 and 13 min, respectively. This method was validated against standard methods and recovery tests, and it was applied to the measurement of nitrite and nitrate in environmental waters. Moreover, a device was designed to enable the field measurement of nitrite and nitrate in complex matrices.

Single cell multi-omics reveal intra-cell-line heterogeneity across human cancer cell lines.

Human cancer cell lines have long served as tools for cancer research and drug discovery, but the presence and the source of intra-cell-line heterogeneity remain elusive. Here, we perform single-cell RNA-sequencing and ATAC-sequencing on 42 and 39 human cell lines, respectively, to illustrate both transcriptomic and epigenetic heterogeneity within individual cell lines. Our data reveal that transcriptomic heterogeneity is frequently observed in cancer cell lines of different tissue origins, often driven by multiple common transcriptional programs. Copy number variation, as well as epigenetic variation and extrachromosomal DNA distribution all contribute to the detected intra-cell-line heterogeneity. Using hypoxia treatment as an example, we demonstrate that transcriptomic heterogeneity could be reshaped by environmental stress. Overall, our study performs single-cell multi-omics of commonly used human cancer cell lines and offers mechanistic insights into the intra-cell-line heterogeneity and its dynamics, which would serve as an important resource for future cancer cell line-based studies.

Aloe-emodin targets multiple signaling pathways by blocking ubiquitin-mediated degradation of DUSP1 in nasopharyngeal carcinoma cells.

Aloe-emodin (AE) has been shown to inhibit the proliferation of several cancer cell lines, including human nasopharyngeal carcinoma (NPC) cell lines. In this study, we confirmed that AE inhibited malignant biological behaviors, including cell viability, abnormal proliferation, apoptosis, and migration of NPC cells. Western blotting analysis revealed that AE upregulated the expression of DUSP1, an endogenous inhibitor of multiple cancer-associated signaling pathways, resulting in blockage of the extracellular signal-regulated kinase (ERK)-1/2, protein kinase B (AKT), and p38-mitogen activated protein kinase（p38-MAPK） signaling pathways in NPC cell lines. Moreover, the selective inhibitor of DUSP1, BCI-hydrochloride, partially reversed the AE-induced cytotoxicity and blocked the aforementioned signaling pathways in NPC cells. In addition, the binding between AE and DUSP1 was predicted via molecular docking analysis using AutoDock-Vina software and further verified via a microscale thermophoresis assay. The binding amino acid residues were adjacent to the predicted ubiquitination site (Lys192) of DUSP1. Immunoprecipitation with the ubiquitin antibody, ubiquitinated DUSP1 was shown to be upregulated by AE. Our findings revealed that AE can stabilize DUSP1 by blocking its ubiquitin-proteasome-mediated degradation and proposed an underlying mechanism by which AE-upregulated DUSP1 may potentially target multiple pathways in NPC cells.

Local Axial Scale-Attention for Universal Lesion Detection.

Universal lesion detection (ULD) in computed tomography (CT) images is an important and challenging prerequisite for computer-aided diagnosis (CAD) to find abnormal tissue, such as tumors of lymph nodes, liver tumors, and lymphadenopathy. The key challenge is that lesions have a tiny size and high similarity with non-lesions, which can easily lead to high false positives. Specifically , non-lesions are nearby normal anatomy that include the bowel, vasculature, and mesentery, which decrease the conspicuity of small lesions since they are often hard to differentiate. In this study, we present a novel scale-attention module that enhances feature discrimination between lesion and non-lesion regions by utilizing the domain knowledge of radiologists to reduce false positives effectively. Inspired by the domain knowledge that radiologists tend to divide each CT image into multiple areas, then detect lesions in these smaller areas separately, a local axial scale-attention (LASA) module is proposed to re-weight each pixel in a feature map by aggregating local features from multiple scales adaptively. In addition, to keep the same weight, a combination of axial pixels in the height- and width-axes is designed, attached with position embedding. The model can be used in CNNs easily and flexibly. We test our method on the DeepLesion dataset. The sensitivities at 0.5, 1, 2, 4, 8, and 16 false positives (FPs) per image and average sensitivity at [0.5, 1, 2, 4] are calculated to evaluate the accuracy. The sensitivities are 78.30%, 84.96%, 89.86%, 93.14%, 95.36%, and 95.54% at 0.5, 1, 2, 4, 8, and 16 FPs per image; the average sensitivity is 86.56%, outperforming the former methods. The proposed method enhances feature discrimination between lesion and non-lesion regions by adding LASA modules. These encouraging results illustrate the potential advantage of exploiting the domain knowledge for lesion detection.

CCR5 as a prognostic biomarker correlated with immune infiltrates in head and neck squamous cell carcinoma by bioinformatic study.

BACKGROUND: C-C chemokine receptor 5 (CCR5) has recently been recognized as an underlying therapeutic target for various malignancies. However, the association of CCR5 with prognosis in the head and neck squamous cell carcinoma (HNSC) patients and tumor-infiltrating lymphocytes (TILs) is unclear. METHODS: In the current experiment, methods such as the Tumor Immune Estimation Resource Analysis (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN, and Kaplan-Meier plotter Analysis were used to comprehensively evaluate the expression of CCR5 in human various malignancies and the clinical prognosis in HNSC patients. Subsequently, we used the TIMER database and the TISIDB platform to investigate the correlation between CCR5 expression levels and immune cell infiltration in the HNSC tumor microenvironment. Furthermore, immunomodulatory and chemokine profiling were performed using the TISIDB platform to analyse the correlation between CCR5 expression levels and immunomodulation in HNSC patients. RESULTS: We found that CCR5 expression in HNSC tumor tissues was significantly upregulated than in normal tissues. In HNSC, patients with high CCR5 expression levels had worse overall survival (OS, HR = 0.59, p = 0.00015) and worse recurrence-free survival (RFS, HR = 3.27, p = 0.00098). Upregulation of CCR5 expression is closely associated with immunomodulators, chemokines, and infiltrating levels of CD4+ T cells, neutrophils, macrophages, and myeloid dendritic cells. Furthermore, upregulated CCR5 was significantly associated with different immune markers in the immune cell subsets of HNSC. CONCLUSIONS: High expression of CCR5 plays an important prognostic role in HNSC patients and may serve as a prognostic biomarker correlated with immune infiltration, and further studies are still needed to investigate therapeutic targeting HNSC patients in the future.

Rolling back human pluripotent stem cells to an eight-cell embryo-like stage.

After fertilization, the quiescent zygote experiences a burst of genome activation that initiates a short-lived totipotent state. Understanding the process of totipotency in human cells would have broad applications. However, in contrast to in mice1,2, demonstration of the time of zygotic genome activation or the eight-cell (8C) stage in in vitro cultured human cells has not yet been reported, and the study of embryos is limited by ethical and practical considerations. Here we describe a transgene-free, rapid and controllable method for producing 8C-like cells (8CLCs) from human pluripotent stem cells. Single-cell analysis identified key molecular events and gene networks associated with this conversion. Loss-of-function experiments identified fundamental roles for DPPA3, a master regulator of DNA methylation in oocytes3, and TPRX1, a eutherian totipotent cell homeobox (ETCHbox) family transcription factor that is absent in mice4. DPPA3 induces DNA demethylation throughout the 8CLC conversion process, whereas TPRX1 is a key executor of 8CLC gene networks. We further demonstrate that 8CLCs can produce embryonic and extraembryonic lineages in vitro or in vivo in the form of blastoids5 and complex teratomas. Our approach provides a resource to uncover the molecular process of early human embryogenesis.

Recombinant chimpanzee adenovirus AdC7 expressing dimeric tandem-repeat spike protein RBD protects mice against COVID-19.

A safe and effective vaccine is urgently needed to control the unprecedented COVID-19 pandemic. Four adenovirus-vectored vaccines expressing spike (S) protein have been approved for use. Here, we generated several recombinant chimpanzee adenovirus (AdC7) vaccines expressing S, receptor-binding domain (RBD), or tandem-repeat dimeric RBD (RBD-tr2). We found vaccination via either intramuscular or intranasal route was highly immunogenic in mice to elicit both humoral and cellular immune responses. AdC7-RBD-tr2 showed higher antibody responses compared to either AdC7-S or AdC7-RBD. Intranasal administration of AdC7-RBD-tr2 additionally induced mucosal immunity with neutralizing activity in bronchoalveolar lavage fluid. Either single-dose or two-dose mucosal administration of AdC7-RBD-tr2 protected mice against SARS-CoV-2 challenge, with undetectable subgenomic RNA in lung and relieved lung injury. AdC7-RBD-tr2-elicted sera preserved the neutralizing activity against the circulating variants, especially the Delta variant. These results support AdC7-RBD-tr2 as a promising COVID-19 vaccine candidate.

Single-Nucleus Chromatin Accessibility Landscape Reveals Diversity in Regulatory Regions Across Distinct Adult Rat Cortex.

Rats have been widely used as an experimental organism in psychological, pharmacological, and behavioral studies by modeling human diseases such as neurological disorders. It is critical to identify and characterize cell fate determinants and their regulatory mechanisms in single-cell resolutions across rat brain regions. Here, we applied droplet-based single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) to systematically profile the single-cell chromatin accessibility across four dissected brain areas in adult Sprague-Dawley (SD) rats with a total of 59,023 single nuclei and identified 16 distinct cell types. Interestingly, we found that different cortex regions exhibit diversity in both cellular compositions and gene regulatory regions. Several cell-type-specific transcription factors (TFs), including SPI1, KLF4, KLF6, and NEUROD2, have been shown to play important roles during the pathogenesis of various neurological diseases, such as Alzheimer's disease (AD), astrocytic gliomas, autism spectrum disorder (ASD), and intellectual disabilities. Therefore, our single-nucleus atlas of rat cortex could serve as an invaluable resource for dissecting the regulatory mechanisms underlying diverse cortex cell fates and further revealing the regulatory networks of neuropathogenesis.

Single-Cell Sequencing of Peripheral Mononuclear Cells Reveals Distinct Immune Response Landscapes of COVID-19 and Influenza Patients.

The coronavirus disease 2019 (COVID-19) pandemic poses a current world-wide public health threat. However, little is known about its hallmarks compared to other infectious diseases. Here, we report the single-cell transcriptional landscape of longitudinally collected peripheral blood mononuclear cells (PBMCs) in both COVID-19- and influenza A virus (IAV)-infected patients. We observed increase of plasma cells in both COVID-19 and IAV patients and XIAP associated factor 1 (XAF1)-, tumor necrosis factor (TNF)-, and FAS-induced T cell apoptosis in COVID-19 patients. Further analyses revealed distinct signaling pathways activated in COVID-19 (STAT1 and IRF3) versus IAV (STAT3 and NFκB) patients and substantial differences in the expression of key factors. These factors include relatively increase of interleukin (IL)6R and IL6ST expression in COVID-19 patients but similarly increased IL-6 concentrations compared to IAV patients, supporting the clinical observations of increased proinflammatory cytokines in COVID-19 patients. Thus, we provide the landscape of PBMCs and unveil distinct immune response pathways in COVID-19 and IAV patients.

Phenomenon of dual- and single-retention behaviors of solutes and its validation by computational simulation in linear programmed temperature gas chromatography.

The current theory of programmed temperature gas chromatography considers that solutes are focused by the stationary phase at the column head completely and does not explicitly recognize the different effects of initial temperature (To ) and heating rate (rT ) on the retention time or temperature of a homologue series. In the present study, n-alkanes, 1-alkenes, 1-alkyl alcohols, alkyl benzenes, and fatty acid methyl esters standards were used as model chemicals and were separated on two nonpolar columns, one moderately polar column and one polar column. Effects of To and rT on the retention of nonstationary phase focusing solutes can be explicitly described with isothermal and cubic equation models, respectively. When the solutes were in the stationary phase focusing status, the single-retention behavior of solutes was observed. It is simple, dependent upon rT only and can be well described by the cubic equation model that was visualized through four sequential slope analyses. These observed dual- and single-retention behaviors of solutes were validated by various experimental data, physical properties, and computational simulation.

NR2B antagonist CP-101,606 inhibits NR2B phosphorylation at tyrosine-1472 and its interactions with Fyn in levodopa-induced dyskinesia rat model.

The augmented tyrosine phosphorylation of NR2B subunit of N-methyl-d-aspartate receptors (NMDAR) dependent on Fyn kinase has been associated with levodopa (l-dopa)-induced dyskinesia (LID). CP-101,606, one selective NR2B subunit antagonist, can improve dyskinesia. Yet, the accurate action mechanism is less well understood. In the present study, the evidences were investigated. Valid 6-hydroxydopamine-lesioned parkinsonian rats were treated with l-dopa intraperitoneally for 22 days to induce LID rat model. On day 23, rats received either CP-101,606 (0.5mg/kg) or vehicle with each l-dopa dose. On the day of 1, 8, 15, 22, and 23 during l-dopa treatment, we determined abnormal involuntary movements (AIMs) in rats. The levels of NR2B phosphorylation at tyrosine-1472 (pNR2B-Tyr1472) and interactions of NR2B with Fyn in LID rat model were detected by immunoblotting and immunoprecipitation. Results showed that CP-101,606 attenuated l-dopa-induced AIMs. In agreement with behavioral analysis, CP-101,606 reduced the augmented pNR2B-Tyr1472 and its interactions with Fyn triggered during the l-dopa administration in the lesioned striatum of parkinsonian rats. Moreover, CP-101,606 also decreased the level of Ca(2+)/calmodulin-dependent protein kinase II at threonine-286 hyperphosphorylation (pCaMKII-Thr286), which was the downstream signaling amplification molecule of NMDAR overactivation and closely associated with LID. However, the protein level of NR2B and Fyn had no difference under the above conditions. These data indicate that the inhibition of the interactions of NR2B with Fyn and NR2B tyrosine phosphorylation may contribute to the CP-101,606-induced downregulation of NMDAR function and provide benefit for the therapy of LID.

Transfected early growth response gene-1 DNA enzyme prevents stenosis and occlusion of autogenous vein graft in vivo.

The aim of this study was to detect the inhibitory action of the early growth response gene-1 DNA enzyme (EDRz) as a carrying agent by liposomes on vascular smooth muscle cell proliferation and intimal hyperplasia. An autogenous vein graft model was established. EDRz was transfected to the graft vein. The vein graft samples were obtained on each time point after surgery. The expression of the EDRz transfected in the vein graft was detected using a fluorescent microscope. Early growth response gene-1 (Egr-1) mRNA was measured using reverse transcription-PCR and in situ hybridization. And the protein expression of Egr-1 was detected by using western blot and immunohistochemistry analyses. EDRz was located at the media of the vein graft from 2 to 24 h, 7 h after grafting. The Egr-1 protein was mainly located in the medial VSMCs, monocytes, and endothelium cells during the early phase of the vein graft. The degree of VSMC proliferation and thickness of intima were obviously relieved compared with the no-gene therapy group. EDRz can reduce Egr-1 expression in autogenous vein grafts, effectively restrain VSMC proliferation and intimal hyperplasia, and prevent vascular stenosis and occlusion after vein graft.

Effect of transcranial magnetic stimulation on the expression of c-Fos and brain-derived neurotrophic factor of the cerebral cortex in rats with cerebral infarct.

The effect of transcranial magnetic stimulation (TMS) on the neurological functional recovery and expression of c-Fos and brain-derived neurotrophic factor (BDNF) of the cerebral cortex in rats with cerebral infarction was investigated. Cerebral infarction models were established by using left middle cerebral artery occlusion (MCAO) and were randomly divided into a model group (n=40) and a TMS group (n=40). TMS treatment (2 times per day, 30 pulses per time) with a frequency of 0.5 Hz and magnetic field intensity of 1.33 Tesla was carried out in TMS group after MCAO. Modified neurological severity score (NSS) were recorded before and 1, 7, 14, 21, and 28 day(s) after MCAO. The expression of c-Fos and BDNF was immunohistochemically detected 1, 7, 14, 21, and 28 day(s) after infarction respectively. Our results showed that a significant recovery of NSS (P<0.05) was found in animals treated by TMS on day 7, 14, 21, and 28 as compared with the animals in the model group. The positive expression of c-Fos and BDNF was detected in the cortex surrounding the infarction areas, while the expression of c-Fos and BDNF increased significantly in TMS treatment group in comparison with those in model group 7, 14, 21, and 28 days (P<0.05) and 7, 14, 21 days (P<0.01) after infarction, respectively. It is concluded that TMS has therapeutic effect on cerebral infarction and this may have something to do with TMS's ability to promote the expression of c-Fos and BDNF of the cerebral cortex in rats with cerebral infarction.