Spatial barcoding technologies have the potential to reveal histological details of transcriptomic profiles; however, they are currently limited by their low resolution. Here, we report Seq-Scope, a ...spatial barcoding technology with a resolution comparable to an optical microscope. Seq-Scope is based on a solid-phase amplification of randomly barcoded single-molecule oligonucleotides using an Illumina sequencing platform. The resulting clusters annotated with spatial coordinates are processed to expose RNA-capture moiety. These RNA-capturing barcoded clusters define the pixels of Seq-Scope that are ∼0.5–0.8 μm apart from each other. From tissue sections, Seq-Scope visualizes spatial transcriptome heterogeneity at multiple histological scales, including tissue zonation according to the portal-central (liver), crypt-surface (colon) and inflammation-fibrosis (injured liver) axes, cellular components including single-cell types and subtypes, and subcellular architectures of nucleus and cytoplasm. Seq-Scope is quick, straightforward, precise, and easy-to-implement and makes spatial single-cell analysis accessible to a wide group of biomedical researchers.
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•Seq-Scope repurposes Illumina sequencing platform for spatial transcriptomics•Seq-Scope achieves submicrometer resolution and efficient transcriptome capture•Seq-Scope enabled spatial single cell and subcellular analysis of liver and colon
Seq-Scope uses spatial barcoding and the Illumina sequencing platform to achieve sub-micron resolution spatial transcriptomics, enabling the visualization of transcriptomic heterogeneity at the cellular and subcellular level in various tissues.
Inverted colloidal crystal (ICC) hydrogel scaffolds represent unique opportunities in modeling lymphoid tissues and expanding hematopoietic‐lymphoid cells. Fully interconnected spherical pore arrays ...direct the formation of stromal networks and facilitate interactions between stroma and hematopoietic‐lymphoid cells. However, due to the intricate architecture of these materials, release of expanded cells is restricted and requires mechanical disruption or chemical dissolution of the hydrogel scaffold. One potent biomaterials strategy to release pore‐entrapped hematopoietic‐lymphoid cells without breaking the scaffolds apart is to transiently increase the dimensions of these materials using stimuli‐responsive polymers. Having this mindset, thermoresponsive ICC scaffolds that undergo rapid (<1 min) and substantial (>300%) diameter change over a physiological temperature range (4–37 °C) by using poly(N‐isopropylacrylamide) (PNIPAM) with nanogel crosslinkers is developed. For a proof‐of‐concept study, the stromal niche by creating osteospheroids, aggregates of osteoblasts, and bone chips is first replicated, and subsequently Nalm‐6 model hematopoietic‐lymphoid cells are introduced. A sixfold increase in cell count is harvested when ICC hydrogel scaffolds are expanded without termination of the established 3D stromal cell culture. It is envisioned that thermoresponsive ICC hydrogel scaffolds will enable for scalable and sustainable ex vivo expansion of hematopoietic‐lymphoid cells.
Thermoresponsive inverted colloidal crystal hydrogel scaffolds with rapid response rate and large volumetric change enable selective retrieval of expanded model hematopoietic‐lymphoid cells from coculture with osteospheroids via a temperature switch in a repeatable fashion.
Severe acute respiratory syndrome (SARS) is an infectious disease with a strong potential for transmission upon close personal contact and is caused by the SARS-coronavirus (CoV). However, there are ...no natural or synthetic compounds currently available that can inhibit SARS-CoV. We examined the inhibitory effects of 64 purified natural compounds against the activity of SARS helicase, nsP13, and the hepatitis C virus (HCV) helicase, NS3h, by conducting fluorescence resonance energy transfer (FRET)-based double-strand (ds) DNA unwinding assay or by using a colorimetry-based ATP hydrolysis assay. While none of the compounds, examined in our study inhibited the DNA unwinding activity or ATPase activity of human HCV helicase protein, we found that myricetin and scutellarein potently inhibit the SARS-CoV helicase protein in vitro by affecting the ATPase activity, but not the unwinding activity, nsP13. In addition, we observed that myricetin and scutellarein did not exhibit cytotoxicity against normal breast epithelial MCF10A cells. Our study demonstrates for the first time that selected naturally-occurring flavonoids, including myricetin and scultellarein might serve as SARS-CoV chemical inhibitors.
As a first step toward understanding how rare variants contribute to risk for complex diseases, we sequenced 15,585 human protein-coding genes to an average median depth of 111 x in 2440 individuals ...of European (n = 1351) and African (n = 1088) ancestry. We identified over 500,000 single-nucleotide variants (SNVs), the majority of which were rare (86% with a minor allele frequency less than 0.5%), previously unknown (82%), and population-specific (82%). On average, 2.3% of the 13,595 SNVs each person carried were predicted to affect protein function of -313 genes per genome, and -95.7% of SNVs predicted to be functionally important were rare. This excess of rare functional variants is due to the combined effects of explosive, recent accelerated population growth and weak purifying selection. Furthermore, we show that large sample sizes will be required to associate rare variants with complex traits.
Cleft lip with or without cleft palate (CL/P) is one of the most common congenital birth defects. This study aims to identify novel pathogenic microRNAs associated with cleft palate (CP). Through ...data analyses of miRNA-sequencing for developing palatal shelves of C57BL/6J mice, we found that miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p were significantly upregulated, and that miR-19a-3p, miR-130a-3p, miR-301a-3p, and miR-486b-5p were significantly downregulated, at embryonic day E14.5 compared to E13.5. Among them, overexpression of the miR-449 family (miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p) and miR-486b-5p resulted in reduced cell proliferation in primary mouse embryonic palatal mesenchymal (MEPM) cells and mouse cranial neural crest cell line O9-1. On the other hand, inhibitors of miR-130a-3p and miR-301a-3p significantly reduced cell proliferation in MEPM and O9-1 cells. Notably, we found that treatment with dexamethasone, a glucocorticoid known to induce CP in mice, suppressed miR-130a-3p expression in both MEPM and O9-1 cells. Moreover, a miR-130a-3p mimic could ameliorate the cell proliferation defect induced by dexamethasone through normalization of
expression. Taken together, our results suggest that miR-130-3p plays a crucial role in dexamethasone-induced CP in mice.
This study aimed to compare the accuracy of novel lipid indices, including the visceral adiposity index (VAI), lipid accumulation product (LAP), triglycerides and glucose (TyG) index, TyG-body mass ...index (TyG-BMI), and TyG-waist circumference (TyG-WC), in identifying insulin resistance and establish valid cutoff values. This cross-sectional study used the data of 11,378 adults, derived from the United States National Health and Nutrition Examination Survey (1999-2016). Insulin resistance was defined as a homeostasis model assessment-insulin resistance value above the 75th percentile for each sex and race/ethnicities. The area under the curves (AUCs) were as follows: VAI, 0.735; LAP, 0.796; TyG index, 0.723; TyG-BMI, 0.823, and; TyG-WC, 0.822. The AUCs for TyG-BMI and TyG-WC were significantly higher than those for VAI, LAP, and TyG index (vs. TyG-BMI, p < 0.001; vs. TyG-WC, p < 0.001). The cutoff values were as follows: VAI: men 1.65, women 1.65; LAP: men 42.5, women 42.5; TyG index: men 4.665, women 4.575; TyG-BMI: men 135.5, women 135.5; and TyG-WC: men 461.5, women 440.5. Given that lipid indices can be easily calculated with routine laboratory tests, these values may be useful markers for insulin resistance risk assessments in clinical settings.
The analysis of next-generation sequencing data is computationally and statistically challenging because of the massive volume of data and imperfect data quality. We present GotCloud, a pipeline for ...efficiently detecting and genotyping high-quality variants from large-scale sequencing data. GotCloud automates sequence alignment, sample-level quality control, variant calling, filtering of likely artifacts using machine-learning techniques, and genotype refinement using haplotype information. The pipeline can process thousands of samples in parallel and requires less computational resources than current alternatives. Experiments with whole-genome and exome-targeted sequence data generated by the 1000 Genomes Project show that the pipeline provides effective filtering against false positive variants and high power to detect true variants. Our pipeline has already contributed to variant detection and genotyping in several large-scale sequencing projects, including the 1000 Genomes Project and the NHLBI Exome Sequencing Project. We hope it will now prove useful to many medical sequencing studies.
Chemoresistance is a significant problem in the effective treatment of bone metastasis. Adipocytes are a major stromal cell type in the bone marrow and may play a crucial role in developing ...microenvironment-driven chemoresistance. However, detailed investigation remains challenging due to the anatomical inaccessibility and intrinsic tissue complexity of the bone marrow microenvironment. In this study, we developed 2D and 3D in vitro models of bone marrow adipocytes to examine the mechanisms underlying adipocyte-induced chemoresistance. We first established a protocol for the rapid and robust differentiation of human bone marrow stromal cells (hBMSCs) into mature adipocytes in 2D tissue culture plastic using rosiglitazone (10 μM), a PPARγ agonist. Next, we created a 3D adipocyte culture model by inducing aggregation of hBMSCs and adipogenesis to create adipocyte spheroids in porous hydrogel scaffolds that mimic bone marrow sinusoids. Simulated chemotherapy treatment with doxorubicin (2.5 μM) demonstrated that mature adipocytes sequester doxorubicin in lipid droplets, resulting in reduced cytotoxicity. Lastly, we performed direct coculture of human multiple myeloma cells (MM1.S) with the established 3D adipocyte model in the presence of doxorubicin. This resulted in significantly accelerated multiple myeloma proliferation following doxorubicin treatment. Our findings suggest that the sequestration of hydrophobic chemotherapeutics by mature adipocytes represents a potent mechanism of bone marrow microenvironment-driven chemoresistance.
Background and Purpose
In this study, we examined the possibility that 4‐hydroxynonenal (4‐HNE) acting as a ligand for the HCA2 receptor (GPR109A) elicits both anti‐inflammatory and cell death ...responses.
Experimental Approach
Agonistic activity of 4‐HNE was determined by observing the inhibition of cAMP generation in CHO‐K1‐GPR109A‐Gi cell line, using surface plasmon resonance (SPR) binding and competition binding assays with 3H‐niacin. 4‐HNE‐mediated signalling pathways and cellular responses were investigated in cells expressing GPR109A and those not expressing these receptors.
Key Results
Agonistic activity of 4‐HNE was stronger than that of niacin or 3‐OHBA at inhibiting forskolin‐induced cAMP production and SPR binding affinity. In ARPE‐19 and CCD‐841 cells, activation of GPR109A by high concentrations of the agonists 4‐HNE (≥10 μM), niacin (≥1000 μM) and 3‐OHBA (≥1000 μM) induced apoptosis accompanied by elevated Ca2+ and superoxide levels. This 4‐HNE‐induced cell death was blocked by knockdown of GPR109A or NOX4 genes, or treatment with chemical inhibitors of Gβγ (gallein), intracellular Ca2+ (BAPTA‐AM), NOX4 (VAS2870) and JNK (SP600125), but not by the cAMP analogue 8‐CPT‐cAMP. By contrast, low concentrations of 4‐HNE, niacin and 3‐OHBA down‐regulated the expression of pro‐inflammatory cytokines IL‐6 and IL‐8. These 4‐HNE‐induced inhibitory effects were blocked by a cAMP analogue but not by inhibitors of Gβγ‐downstream signalling molecules.
Conclusions and Implications
These results revealed that 4‐HNE is a strong agonist for GPR109A that induces Gαi‐dependent anti‐inflammatory and Gβγ‐dependent cell death responses. Moreover, the findings indicate that specific intracellular signalling molecules, but not GPR109A, can serve as therapeutic targets to block 4‐HNE‐induced cell death.
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