Hierarchical DNA branch assembly-encoded fluorescent nanoladders for single-cell transcripts imaging.

Spatial visualization of single-cell transcripts is limited by signal specificity and multiplexing. Here, we report hierarchical DNA branch assembly-encoded fluorescent nanoladders, which achieve denoised and highly multiplexed signal amplification for single-molecule transcript imaging. This method first offers independent RNA-primed rolling circle amplification without nonspecific amplification based on circular DNAzyme. It then executes programmable DNA branch assembly on these amplicons to encode virtual signals for visualizing numbers of targets by FISH. In theory, more virtual signals can be encoded via the increase of detection spectral channels and repeats of the same sequences on barcode. Our method almost eliminates nonspecific amplification in fixed cells (reducing nonspecific spots of single cells from 16 to nearly zero), and achieves simultaneous quantitation of nine transcripts by using only two detection spectral channels. We demonstrate accurate RNA profiling in different cancer cells, and reveal diverse localization patterns for spatial regulation of transcripts.

Chikusetsu Saponin IVa liposomes modified with a retro-enantio peptide penetrating the blood-brain barrier to suppress pyroptosis in acute ischemic stroke rats.

BACKGROUND: The therapeutic strategies for acute ischemic stroke were faced with substantial constraints, emphasizing the necessity to safeguard neuronal cells during cerebral ischemia to reduce neurological impairments and enhance recovery outcomes. Despite its potential as a neuroprotective agent in stroke treatment, Chikusetsu saponin IVa encounters numerous challenges in clinical application. RESULT: Brain-targeted liposomes modified with THRre peptides showed substantial uptake by bEnd. 3 and PC-12 cells and demonstrated the ability to cross an in vitro blood-brain barrier model, subsequently accumulating in PC-12 cells. In vivo, they could significantly accumulate in rat brain. Treatment with C-IVa-LPs-THRre notably reduced the expression of proteins in the P2RX7/NLRP3/Caspase-1 pathway and inflammatory factors. This was evidenced by decreased cerebral infarct size and improved neurological function in MCAO rats. CONCLUSION: The findings indicate that C-IVa-LPs-THRre could serve as a promising strategy for targeting cerebral ischemia. This approach enhances drug concentration in the brain, mitigates pyroptosis, and improves the neuroinflammatory response associated with stroke.

Quality of evidence supporting the role of hyperbaric oxygen therapy for diabetic foot ulcers.

The goal of this overview of systematic reviews (SRs) and meta-analyses (MAs) was to methodically gather, evaluate and summarize the data supporting the use of hyperbaric oxygen therapy (HBOT) to treat diabetic foot ulcers (DFUs). The Cochrane Library, Embase, PubMed, Web of Science and Embase were all searched thoroughly to identify SRs/MAs that qualified. AMSTAR-2 tool, PRISMA checklists and GRADE system were applied by two reviewers independently to assess the methodological quality, reporting and evidence quality of the included SRs/MAs, respectively. Eleven SRs/MAs were enrolled in this overview. According to AMSTAR-2, a very low methodological quality assessment was given to the included SRs/MAs due to the limitations of items 2, 4 and 7. For the PRISMA, the overall quality of reporting is not satisfactory due to missing reporting on protocol, search, as well as additional analysis. The majority of outcomes had low- to moderate-quality evidence, and no high-quality evidence was found to support the role of HBOT for DFUs, according to GRADE. To conclude, the potential of HBOT in treating DFUs is supported by evidence of low to moderate quality. More rigorously designed, high-level studies are needed in the future to determine the evidence for HBOT for DFU, including the timing, frequency and duration of HBOT interventions.

Single-Cell RNA Sequencing Reveals Unique Alterations in the Immune Panorama and Treg Subpopulations in Mice during the Late Stages of Echinococcus granulosus Infection.

Cystic echinococcosis (CE) is a common zoonotic parasitic disease that seriously impacts public health. However, the full spectrum of immune cell changes in Echinococcus granulosus infection, especially the negative immune regulation of subpopulations of regulatory T (Treg) cells, are not yet well understood. In this study, we used single-cell RNA sequencing and immunome repertoire (IR) sequencing to analyze 53,298 cells from the spleens and peripheral blood mononuclear cells (PBMCs) of healthy and E. granulosus-infected mice. We used immunofluorescence combined with RNA fluorescence in situ hybridization and quantitative real-time PCR to verify the sequencing results. Our results showed tissue-specific immune system alterations in mice infected with E. granulosus. E. granulosus-infected mice induced a subpopulation of CD4+ cells with type I interferon production potential. Furthermore, there were six different Treg cell subpopulations in vivo at three stages of differentiation, and Treg subpopulations of different classes and different stages of differentiation showed tissue specificity. After infection, the Lag3hi Treg and Gpr83+Igfbp4+ naive Treg subpopulations were specifically induced in PBMCs and the spleen, respectively. Furthermore, T follicular helper 2 (Tfh2) cells with high expression of Cxxc5 and Spock2 were found in E. granulosus-infected mice. Our data uncovered changes in the full spectrum of immune cells in mice following the late stages of E. granulosus infection, including subpopulations of cells that have not been emphasized in previous studies. These results further enrich the study of the bidirectional immunomodulatory mechanism and offer a different perspective for subsequent studies of infection in E. granulosus.

Lighting Up Nucleic Acid Modifications in Single Cells with DNA-Encoded Amplification.

Nucleic acids are naturally decorated with various chemical modifications at nucleobases. Most nucleic acid modifications (NAMs) do not alter Watson-Crick base pairing but can regulate gene expression known as "epigenetics". Their abundances present a very wide range, approximately 10-2 to 10-6 of total bases. Different NAMs may coexist in spatial proximity (e.g., <20 nm) in the crowded intracellular environment. Considering the highly dynamic chromatin accessibility (physical access to DNA), the NAMs in inaccessible DNA probably plays different roles. These multilayered features of NAMs vary from cell to cell. Our understanding of the function and mechanism of NAMs in biological processes and disease states has largely been driven by the expanding array of sequencing-based methodologies. However, an underexplored aspect is the measurement of the subcellular distribution, spatial proximity, and inaccessibility of NAMs in single cells. In recent years, we have developed new approaches that light up single-cell NAMs with single-site sensitivity. These methods are mainly based on the integration of chemical or chemoenzymatic tools, DNA amplification and nanotechnology, and/or microfluidics. An overview of these methods together with conventional methods such as immunofluorescence (IF) and fluorescence in situ hybridization (FISH) is provided in this Account.Our laboratory has proposed DNA-encoded amplification (DEA) as the main strategy for developing a set of single-cell NAM imaging methods. In brief, DEA transforms the different features of NAMs into unique DNA primers for rolling circle amplification (RCA) followed by FISH imaging. The first method is base-encoded amplifying FISH (BEA-FISH), in which we convert individual NAMs into RCA primers via chemoselective labeling and click bioconjugation. It enables the in situ visualization of low-abundance NAMs (e.g., 5hmU), which is impracticable by conventional methods. We subsequently developed pairwise proximity-differentiated amplifying FISH (PPDA-FISH), which integrates BEA-FISH with DNA nanotechnology. PPDA-FISH utilizes proximity ligation and toehold strand displacement to label the adjacent site of two different NAMs (one-to-one proximity) and their respective residual sites with three unique RCA probes. It achieves simultaneous counting of the above-mentioned three types of modified sites in the same cells. The third method is cellular macromolecule-tethered DNA walking indexing (Cell-TALKING) to probe more than two NAMs within the same nanoenvironments. Cell-TALKING uses dynamic DNA proximity cleavage to continuously activate different preblocked RCA primers (for each NAM) near one walking probe (for one target molecule). We have explored three NAMs around one histone (one-to-many proximity) in different cancer cell lines and clinical specimens. Then, we describe a single-cell hydrogel encoding amplification (scHEA) method by integrating droplet microfluidics with BEA-FISH. This method generates hydrogel beads that encapsulate single cells and their genomic DNA after cell lysis. It realizes the analysis of global (accessible and inaccessible) DNA from the same cells. We find that the global levels of both 5hmC and 5hmU in single cells can distinguish different breast cancer cells. Finally, the current limitations of these strategies and the future development directions are also discussed. We hope that this Account can spark new ideas and invite new efforts from different disciplines for single-cell NAM analysis.

Concentration Effect, Structural Properties, and Driving Force on Aß28 Dimerization with and without Zn2+ Cooperation: Learning from Replica Exchange Sampling.

Zn2+ is a very important factor in promoting the formation of amyloid beta (Aß) aggregates and amyloid plaques. The Zn2+ -bound Aß species generate amorphous or low molecular-weight oligomers. However, it is a lack of studies to approach the starting structural features (dimerization) in Aß nucleation processes with and without Zn2+ , which is the key point in understanding Zn2+ -induced nucleation mechanisms. To better understand the effect of concentration, structural properties, and the driving force, 14 independent replica exchange molecular dynamics simulations were performed in Aß28 dimerization with and without Zn2+ (zAß28 ) cooperation. Our scanning results show that the aggregation propensity is easier in Aß28 -Aß28 and Aß28 -zAß28 systems than zAß28 -zAß28 system. In binding property, the Aß28 -Aß28 model (-61.5 kcal mol-1 ) is stronger than zAß28 -zAß28 (-26.6 kcal mol-1 ) and Aß28 -zAß28 (-7.24 kcal mol-1 ) models. Further analysis confirmed that H13 and H14 residues play specific roles in the three systems. The key point is the orientation of N atom of the imidazole ring in histidine residues. Furthermore, we discovered different driving forces for each system. Our current study contributes to the understanding of how the Aß28 dimer interacts with Zn2+ , which could lead to new insights into Zn2+ -induced nucleation mechanisms.

Novel synergistic mechanism of 11-keto-ß-boswellic acid and Z-Guggulsterone on ischemic stroke revealed by single-cell transcriptomics.

Although strides have been made, the challenge of preventing and treating ischemic stroke continues to persist globally. For thousands of years, the natural substances Frankincense and Myrrh have been employed in Chinese and Indian medicine to address cerebrovascular diseases, with the key components of 11-keto-ß-boswellic acid (KBA) and Z-Guggulsterone (Z-GS) being the active agents. In this study, the synergistic effect and underlying mechanism of KBA and Z-GS on ischemic stroke were examined using single-cell transcriptomics. Fourteen cell types were identified in KBA-Z-GS-treated ischemic penumbra, and microglia and astrocytes account for the largest proportion. They were further re-clustered into six and seven subtypes, respectively. GSVA analysis reflected the distinct roles of each subtype. Pseudo-time trajectory indicated that Slc1a2 and Timp1 were core fate transition genes regulated by KBA-Z-GS. In addition, KBA-Z-GS synergistically regulated inflammatory reactions in microglia and cellular metabolism and ferroptosis in astrocytes. Most notably, we established an innovative drug-gene synergistic regulation pattern, and genes regulated by KBA-Z-GS were divided into four categories based on this pattern. Finally, Spp1 was demonstrated as the hub target of KBA-Z-GS. Taken together, this study reveals the synergistic mechanism of KBA and Z-GS on cerebral ischemia, and Spp1 may be the synergistic target for that. Precise drug development targeting Spp1 may offer a potential therapeutic approach for treating ischemic stroke.

A Knowledge Map of the Relationship between Diabetes and Stroke: A Bibliometric Analysis Study.

INTRODUCTION: The correlation between diabetes and stroke has been studied extensively in epidemiological research. Here we used bibliometric software to visualize and analyze the literature related to diabetic stroke to provide an overview of the current state of research, hot spots, and future trends in the field. METHODS: Based on the Web of Science Core Collection(WoSCC) database, we collected studies related to diabetic stroke from 2007 to May 2022. We used CiteSpace (version 6.1.R5), VOSviewer, and Sci-mago Graphica to create knowledge maps and conduct visual analyses on authors, countries, in-stitutions, cited references, and keywords, and Origin for statistical analysis. RESULTS: We included a total of 5171 papers on diabetic stroke from the WoSCC database. Overall, there was a steady increase in the number of publications, with a high number of emerging scientists. The United States was the most productive and influential country, which dominated national col-laborations. The most common subject category was "neurology". In total, 12 major clusters were generated from the cited references. Keywords analysis showed that keywords related to post-stroke injury and treatment are those with the highest burst intensity and latest burst time. CONCLUSIONS: Individual disease treatment remains a hot topic and how to balance acute stroke treatment and glycemic control is currently a difficult clinical problem. At the same time, the mechanism of their interaction and the prevention and treatment of related causative factors remain a hot topic of current and future research.

Prognostic nomograms for gastric carcinoma after D2 + total gastrectomy to assist decision-making for postoperative treatment: based on Lasso regression.

OBJECTIVE: This study aimed to establish novel nomograms that could be used to predict the prognosis of gastric carcinoma patients who underwent D2 + total gastrectomy on overall survival (OS) and progression-free survival (PFS). METHODS: Lasso regression was employed to construct the nomograms. The internal validation process included bootstrapping, which was used to test the accuracy of the predictions. The calibration curve was then used to demonstrate the accuracy and consistency of the predictions. In addition, the Harrell's Concordance index (C-index) and time-dependent receiver operating characteristic (t-ROC) curves were used to evaluate the discriminative abilities of the new nomograms and to compare its performance with the 8th edition of AJCC-TNM staging. Furthermore, decision curve analysis (DCA) was performed to assess the clinical application of our model. Finally, the prognostic risk stratification of gastric cancer was conducted with X-tile software, and the nomograms were converted into a risk-stratifying prognosis model. RESULTS: LASSO regression analysis identified pT stage, the number of positive lymph nodes, vascular invasion, neural invasion, the maximum diameter of tumor, the Clavien-Dindo classification for complication, and Ki67 as independent risk factors for OS and pT stage, the number of positive lymph nodes, neural invasion, and the maximum diameter of tumor for PFS. The C-index of OS nomogram was 0.719 (95% CI: 0.690-0.748), which was superior to the 8th edition of AJCC-TNM staging (0.704, 95%CI: 0.623-0.783). The C-index of PFS nomogram was 0.694 (95% CI: 0.654-0.713), which was also better than that of the 8th edition of AJCC-TNM staging (0.685, 95% CI: 0.635-0.751). The calibration curves, t-ROC curves, and DCA of the two nomogram models showed that the prediction ability of the two nomogram models was outstanding. The statistical difference in the prognosis between the low- and high-risk groups further suggested that our model had an excellent risk stratification performance. CONCLUSION: We reported the first risk stratification and nomogram for gastric carcinoma patients with total gastrectomy in Chinese population. Our model could potentially be used to guide treatment selections for the low- and high-risk patients to avoid delayed treatment or unnecessary overtreatment.

[Mechanism of Zhenwu Decoction in improving renal inflammatory injury in mice with DN of spleen-kidney Yang deficiency syndrome by regulating ROCK/IKK/NF-κB pathway].

To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1ß(IL-1ß), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P<0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P<0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P<0.05). The levels of inflammatory factors IL-1ß, IL-6, IL-8, and TNF-α increased(P<0.05), while the level of IL-10 decreased(P<0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P<0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P<0.05), increased protein expression of IκB(P<0.05), decreased levels of serum inflammatory factors IL-1ß, IL-6, IL-8, and TNF-α(P<0.05), and increased level of IL-10(P<0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.

Expression profiling of exosomal miRNAs derived from different stages of infection in mice infected with Echinococcus granulosus protoscoleces using high-throughput sequencing.

Echinococcosis is a worldwide zoonosis. The mechanism of the establishment, growth, and persistence of parasites in the host has not been fully understood. Exosomes are found to be a way of information exchange between parasites and hosts. They exist in various body fluids widely. There are few studies on host-derived exosomes and their miRNA expression profiles at different infection time points. In this study, BALB/c mice were intraperitoneally infected with protricercariae. Exosomes were extracted from plasma (0, 3, 9, and 20 weeks post infection), and the expression profiles of exosome miRNA in the peripheral blood of mice were determined using RNA-sequencing. Compared to the 0 week groups, 24, 35, and 22 differentially expressed miRNAs were detected in infected mouse at the three infection stages, respectively. The results showed that there were significant differences in the miRNAs of exosomes at different infection time points. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to annotate the different miRNAs. The results showed that the biological pathways of parasites changed significantly at different stages of infection, with many significant and abundant pathways involved in cell differentiation, inflammation, and immune response, such as MAPK signaling pathway, Th17 cell differentiation, Wnt signaling pathway, FoxO signaling pathway, Notch signaling pathway, etc. These results suggest that miRNA may be an important regulator of interactions between Echinococcus granulosus and host. The data provided here provide valuable information to increase understanding of the regulatory function of microRNAs in the host microenvironment and the mechanism of host-parasite interaction. This may help us to find targets for Echinococcus granulosus to escape host immune attack and control Echinococcus granulosus infection in the future.

Regulation of circular RNAs act as ceRNA in a hypoxic pulmonary hypertension rat model.

To explore potential critical genes and identify circular RNAs (circRNAs) that act as the competitive endogenous RNA (ceRNA) in a hypoxic pulmonary hypertension (HPH) rat model. Constructed rat model, and a bioinformatics method was used to analyse differentially expressed (DE) genes and construct a circRNA-miRNA-mRNA ceRNA regulatory network. Then, qRT-PCR was used to verify. The significant DEcircRNAs/DEmiRNAs/DEmRNAs was showed, and a ceRNA network with 8 DEcircRNAs, 9 DEmiRNAs and 46 DEmRNAs were constructed. The functional enrichment suggested the inflammatory response, NF-κB signalling, MAPK cascade and Toll-like receptor were associated with HPH. Further assessment confirmed that circ_002723, circ_008021, circ_016925 and circ_020581 could have a potential ceRNA mechanism by sponging miR-23a or miR-21 to control downstream target gene and be involved in the pathophysiology of HPH. The qRT-PCR validation results were consistent with the RNA-Seq results. This study revealed potentially important genes, pathways and ceRNA regulatory networks in HPH.

Corporate social performance and firm debt levels: Impacts of the covid-19 pandemic and institutional environments.

This paper examines the relation between corporate social performance (CSP) and firm debt levels and explores the channels between them by focusing on the ongoing health crisis, the COVID-19 pandemic. We use a large sample of public firms from 31 countries between 2002 and 2020. Employing pooled ordinary least squared and firm fixed effects models, after controlling for endogeneity and sample selection bias, we find that during the pre-COVID economic condition, CSP has a significantly positive impact on firm debt levels by reducing financial constraints and enhancing stakeholder engagement. However, during the outbreak, CSP becomes costlier and reveals more managerial agency problems for firms that make such associations attenuated. Furthermore, our evidence suggests that in countries with better institutional environments, the CSP-firm debt levels relation is less pronounced. These results have several implications in terms of investment and capital structure decisions.

Bioorthogonal Chemical Signature Enabling Amplified Visualization of Cellular Oxidative Thymines.

Cellular oxidative thymines, 5-hydroxymethyluracil (5hmU) and 5-formyluracil (5fU), are found in the genomes of a diverse range of organisms, the distribution of which profoundly influence biological processes and living systems. However, the distribution of cellular oxidative thymines has not been explored because of lacking both specific bioorthogonal labeling and sensitivity methods for single-cell analysis. Herein, we report a bioorthogonal chemical signature enabling amplified visualization of cellular oxidative thymines in single cells. The synthesized ATP-γ-alkyne, an ATP analogue with bioorthogonal tag modified on γ-phosphate can be specifically linked to cellular 5hmU by chemoenzymatic labeling. DNA with 5-alkynephosphomethyluracil were then clicked with azide (N3)-modified 5hmU-primer. Identification of 5fU is based on selective reduction from 5fU to 5hmU, subsequent chemoenzymatic labeling of the newly generated 5hmU, and cross-linking with N3-modified 5fU-primer via click chemistry. Then, all of the 5hmU and 5fU sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. The above two kinds of barcodes can be simultaneously amplified for differentiated visualization of 5hmU and 5fU in single cells. We find these two kinds of cellular oxidative thymines are spatially organized in a cell-type-dependent style with cell-to-cell heterogeneity. We also investigate their multilevel subcellular information and explore their dynamic changes during cell cycles. Further, using DNA sequencing instead of fluorescence imaging, our proposed bioorthogonal chemical signature holds great potential to offer the sequence information of these oxidative thymines in cells and may provide a reliable chemical biology approach for studying the whole-genome oxidative thymines profiles and insights into their functional role and dynamics in biology.

The targetable nanoparticle BAF312@cRGD-CaP-NP represses tumor growth and angiogenesis by downregulating the S1PR1/P-STAT3/VEGFA axis in triple-negative breast cancer.

BACKGROUND: Overexpressed vascular endothelial growth factor A (VEGFA) and phosphorylated signal transducer and activator of transcription 3 (P-STAT3) cause unrestricted tumor growth and angiogenesis of breast cancer (BRCA), especially triple-negative breast cancer (TNBC). Hence, novel treatment strategy is urgently needed. RESULTS: We found sphingosine 1 phosphate receptor 1 (S1PR1) can regulate P-STAT3/VEGFA. Database showed S1PR1 is highly expressed in BRCA and causes the poor prognosis of patients. Interrupting the expression of S1PR1 could inhibit the growth of human breast cancer cells (MCF-7 and MDA-MB-231) and suppress the angiogenesis of human umbilical vein endothelial cells (HUVECs) via affecting S1PR1/P-STAT3/VEGFA axis. Siponimod (BAF312) is a selective antagonist of S1PR1, which inhibits tumor growth and angiogenesis in vitro by downregulating the S1PR1/P-STAT3/VEGFA axis. We prepared pH-sensitive and tumor-targeted shell-core structure nanoparticles, in which hydrophilic PEG2000 modified with the cyclic Arg-Gly-Asp (cRGD) formed the shell, hydrophobic DSPE formed the core, and CaP (calcium and phosphate ions) was adsorbed onto the shell; the nanoparticles were used to deliver BAF312 (BAF312@cRGD-CaP-NPs). The size and potential of the nanoparticles were 109.9 ± 1.002 nm and - 10.6 ± 0.056 mV. The incorporation efficacy for BAF312 was 81.4%. Results confirmed BAF312@cRGD-CaP-NP could dramatically inhibit tumor growth and angiogenesis in vitro and in MDA-MB-231 tumor-bearing mice via downregulating the S1PR1/P-STAT3/VEGFA axis. CONCLUSIONS: Our data suggest a potent role for BAF312@cRGD-CaP-NPs in treating BRCA, especially TNBC by downregulating the S1PR1/P-STAT3/VEGFA axis.

Click-encoded rolling FISH for visualizing single-cell RNA polyadenylation and structures.

Spatially resolved visualization of RNA processing and structures is important for better studying single-cell RNA function and landscape. However, currently available RNA imaging methods are limited to sequence analysis, and not capable of identifying RNA processing events and structures. Here, we developed click-encoded rolling FISH (ClickerFISH) for visualizing RNA polyadenylation and structures in single cells. In ClickerFISH, RNA 3' polyadenylation tails, single-stranded and duplex regions are chemically labeled with different clickable DNA barcodes. These barcodes then initiate DNA rolling amplification, generating repetitive templates for FISH to image their subcellular distributions. Combined with single-molecule FISH, the proposed strategy can also obtain quantitative information of RNA of interest. Finally, we found that RNA poly(A) tailing and higher-order structures are spatially organized in a cell type-specific style with cell-to-cell heterogeneity. We also explored their spatiotemporal patterns during cell cycle stages, and revealed the highly dynamic organization especially in S phase. This method will help clarify the spatiotemporal architecture of RNA polyadenylation and structures.

Pairwise Proximity-Differentiated Visualization of Single-Cell DNA Epigenetic Marks.

Spatial positioning and proximity of relevant biomolecules such as DNA epigenetic marks are fundamental to a deeper understanding of life. However, it remains poorly explored and technically challenging. Here we report the pairwise proximity-differentiated visualization of single-cell 5-formylcytosine (5fC) and 5-hydroxymethylcytosine (5hmC). These two marks on chromatin in fixed cells are successively labeled and crosslinked with their DNA primer probes via click chemistry. Based on a pairwise proximity-differentiated mechanism, proximal 5fC/5hmC sites and residual 5fC or 5hmC sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. We thus demonstrate the differentiated visualization of 5fC or 5hmC spatial positioning and their pairwise proximity in single cells. Such multi-level subcellular information may provide insights into regulation functions and mechanisms of chromatin modifications, and the spatial proximity can expose the potential crosstalk or interaction between their reader proteins.

Differentiated Visualization of Single-Cell 5-Hydroxymethylpyrimidines with Microfluidic Hydrogel Encoding.

5-Hydroxymethyluracil ( 5hmU ) is found in the genomes of a diverse range of organisms as another kind of 5-hydroxymethylpyrimidine, with the exception of 5-hydroxymethylcytosine ( 5hmC ). The biological function of 5hmU has not been well explored due to lacking both specific 5hmU recognition and single-cell analysis methods. Here we report differentiated visualization of single-cell 5hmU and 5hmC with microfluidic hydrogel encoding (sc 5hmU / 5hmC -microgel). Single cells and their genomic DNA after cell lysis can be encapsulated in individual agarose microgels. The 5hmU sites are then specifically labeled with thiophosphate for the first time, followed by labeling 5hmC with azide glucose. These labeled bases are each encoded into respective DNA barcode primers by chemical cross-linking. In situ amplification is triggered for single-molecule fluorescence visualization of single-cell 5hmU and 5hmC . On the basis of the sc 5hmU / 5hmC -microgel, we reveal cell type-specific molecular signatures of these two bases with remarkable single-cell heterogeneity. Utilizing machine learning algorithms to decode four-dimensional signatures of 5hmU / 5hmC , we visualize the discrimination of nontumorigenic, carcinoma and highly invasive breast cell lines. This strategy provides a new route to analyze and decode single-cell DNA epigenetic modifications.

RNA-Primed Amplification for Noise-Suppressed Visualization of Single-Cell Splice Variants.

Splice variants visualization is pivotal for a deeper understanding of cell growth and development. However, it remains technically challenging due to short lengths, similar sequences, and low abundance. The existing single-cell imaging strategies suffer from nonspecific amplification that causes considerable noise during visualization of the splice variants. Herein we develop a new RNA-primed amplification strategy for noise-suppressed visualization of single-cell splice variants. Block probes were designed to specifically identify the conjugated region of exons in mRNA, which was then digested by endonuclease and provided a hydroxyl group at the 3' terminal. The RNA target can act as primer to trigger rolling circle amplification, achieving visualization of splice variants with noise suppressed to nearly zero. We further explored the expression and distribution of BRCA1 splice variants in three breast cell lines, revealing cell-type specific mapping of this cancer suppressor gene.

Visualizing Newly Synthesized RNA by Bioorthogonal Labeling-Primed DNA Amplification.

Monitoring RNA synthesis and spatial distribution can help to understand its role in physiology and diseases. However, visualizing newly synthesized RNA in single cells remains a great challenge. Here, we developed a bioorthogonal labeling-primed DNA amplification strategy to visualize newly synthesized RNA in single cells. The new bioorthogonal N6-allyladenosine nucleoside was prepared to metabolically label cellular newly synthesized RNAs. These allyl-functionalized RNAs then reacted with tetrazine-modified primers. These primers could initiate rolling circle amplification, producing tandem periodic long single DNA strands to capture hundreds of fluorescence probes for signal amplification. Using this method, we explored the subcellular distributions of newly synthesized RNAs. And we found that newly synthesized RNAs are spatially organized in a cell type-specific style with cell-to-cell heterogeneity.