Research on Chinese medicinal materials cultivation: A bibliometric and visual analysis.

Chinese medicinal materials (CMMs) are important strategic resource in China. The cultivation process of medicinal plants is the key link which directly affect the quality and efficacy. The literatures of CMMs cultivation were acquired from China National Knowledge Infrastructure (CNKI) database and State Intellectual Property Office (SIPO) patent database for the years between 2001 and 2021. All the articles found were subjected to bibliometric analysis. The development trends and key topics were analyzed and visualized by VOSviewer and CiteSpace software. The results indicate that ecological planting, under-forest economy, intercropping patterns and industrialization production are the research hotspots in this field; cultivation technology and nutritional fertilization technology are the main areas addressed in recent years. Therefore, the high-quality and sustainable development of CMMs cultivation should be examined in terms of theoretical approaches, technical innovation, multi-cooperation, and intellectual property protection.

Investigating the Metabolic Heterogeneity of Cancer Cells Using Functional Single-Cell Selection and nLC Combined with Multinozzle Emitter Mass Spectrometry.

Tumor metastasis and cancer recurrence are often a result of cell heterogeneity, where specific subpopulations of tumor cells may be resistant to radio- or chemotherapy. To investigate this physiological and phenotypic diversity, single-cell metabolomics provides a powerful approach at the chemical level, where distinct lipid profiles can be found in different tumor cells. Here, we established a highly sensitive platform using nanoflow liquid chromatography (nLC) combined with multinozzle emitter electrospray ionization mass spectrometry for more in-depth metabolomics profiling. Our platform identified 15 and 17 lipids from individual osteosarcoma (U2OS) and glioblastoma (GBM) cells when analyzing single-cell samples. Additionally, we used the functional single-cell selection (fSCS) pipeline to analyze the subpopulations of cells with a DNA damage response (DDR) in U2OS cells and fast migration in GBM cells. Specifically, we observed a down-regulation of polyunsaturated fatty acids (PUFAs) in U2OS cells undergoing DDR, such as fatty acids FA 20:3; O2 and FA 17:4; O3. Furthermore, ceramides (Cer 38:0; O3) and triglycerides (TG 36:0) were found to be down-regulated in fast-migrating GBM cells compared to the slow-migrating subpopulation. These findings suggest the potential roles of these metabolites and/or lipids in the cellular behavior of the subpopulations.

Genomic Exploration of Distinct Molecular Phenotypes Steering Temozolomide Resistance Development in Patient-Derived Glioblastoma Cells.

Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells' molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as "adaptive" (ADA) or "non-adaptive" (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor's ability to survive. Depending on the tumor's adaptability potential, subpopulations with acquired resistance mechanisms may arise.

Instant processing of large-scale image data with FACT, a real-time cell segmentation and tracking algorithm.

Quantifying cellular characteristics from a large heterogeneous population is essential to identify rare, disease-driving cells. A recent development in the combination of high-throughput screening microscopy with single-cell profiling provides an unprecedented opportunity to decipher disease-driving phenotypes. Accurately and instantly processing large amounts of image data, however, remains a technical challenge when an analysis output is required minutes after data acquisition. Here, we present fast and accurate real-time cell tracking (FACT). FACT can segment ∼20,000 cells in an average of 2.5 s (1.9-93.5 times faster than the state of the art). It can export quantifiable features minutes after data acquisition (independent of the number of acquired image frames) with an average of 90%-96% precision. We apply FACT to identify directionally migrating glioblastoma cells with 96% precision and irregular cell lineages from a 24 h movie with an average F1 score of 0.91.

Deciphering spatio-seasonal patterns, driving forces, and human health risks of nitrate and fluoride enriched water bodies in the Inner Mongolia Reaches of the Yellow River Basin, China.

The ecology and environment of the Yellow River Basin is threatened by fluoride and nitrate contamination induced by anthropogenic activity and geogenic factors. As a result, deciphering the spatio-temporal variability of fluoride and nitrate contamination in this area remains a challenge. Three hundred eighty-six samples of surface water and groundwater from the Inner Mongolia Reaches of the Yellow River Basin were taken for this investigation. According to the results of the multivariate statistical and geostatistical analyses, the fluoride pollution was primarily discovered in the middle and lower reaches of the study area and was determined to be more severe during the dry season. In contrast, nitrate contamination was found to be more severe during the wet season while being widely distributed in groundwater and concentrated in areas with intensive agricultural activities. The primary mechanisms governing the spatial-seasonal patterns of NO3- and F- pollution were shown by the principal component analysis, isotopic, and hydrochemical diagrams. The water-rock interaction or evaporation was crucial in the enrichment of F-. The human inputs (e.g., fertilizer or sewage) dominated fluoride and nitrate contamination. Additionally, the alkaline environment played a role in the generation of NO3- and F-. The health risk assessment concluded that the threat of fluoride contamination was greater than that of nitrate contamination. Children faced the greatest health risks, followed by females and males. These findings would serve as a guide for water management and pollution control in the Yellow River Basin.

[Water Chemical Isotope Characteristics and Water Transformation Relationship in Mongolian Section of the Yellow River Basin].

The Yellow River in Inner Mongolia was selected as the study area in this study. In July (wet season) and October (dry season) of 2021, the acquisition of seasonal rivers, the Yellow River tributaries and precipitation, the Yellow River, Wuliangsuhai, Lake Hasuhai, Lake Daihai, an irrigation canal system, and underground water and sea water samples were collected to test the water chemical composition and hydrogen and oxygen isotopic values of different water types. Using the Piper triplot, Gibbs plot, ion ratio, and MixSIAR model methods, the evolution of water chemistry in the Mongolian section of the Yellow River Basin was analyzed, and the transformation relationship between precipitation, surface water, and groundwater was revealed. The results showed that both groundwater and surface water in the study area were slightly alkaline; the dominant anion in water was Cl-, and the dominant cation was Na+. The main hydrochemical types of surface water were Cl·SO4-Na·Mg and SO4·HCO3-Na·Mg, whereas those of groundwater were Cl·SO4-Na·Mg and SO4·HCO3-Na·Ca. Groundwater Ca2+ and Mg2+ were primarily derived from the dissolution of silicate and evaporite, and surface water Ca2+ and Mg2+ were primarily derived from carbonate karst dissolution and carbonate and sulfuric acid in water participating in the dissolution process of carbonate and sulfide minerals. Na+ and Cl- in different water bodies were all affected by anthropogenic pollution sources. Owing to the seasonal effect, δD and δ18O of surface water and groundwater were higher in the wet season than in the dry season. The results showed that surface water was affected by evaporative fractionation after receiving precipitation recharge, and the groundwater recharge sources were complex. The MixSIAR model revealed that surface water was the main recharge source of groundwater, accounting for 52.4%-62.2% of the total recharge, and atmospheric precipitation was the main recharge source of surface water, accounting for 85.4%-97.1% of the total recharge.

Spatial Transcriptomic Technologies.

Spatial transcriptomic technologies enable measurement of expression levels of genes systematically throughout tissue space, deepening our understanding of cellular organizations and interactions within tissues as well as illuminating biological insights in neuroscience, developmental biology and a range of diseases, including cancer. A variety of spatial technologies have been developed and/or commercialized, differing in spatial resolution, sensitivity, multiplexing capability, throughput and coverage. In this paper, we review key enabling spatial transcriptomic technologies and their applications as well as the perspective of the techniques and new emerging technologies that are developed to address current limitations of spatial methodologies. In addition, we describe how spatial transcriptomics data can be integrated with other omics modalities, complementing other methods in deciphering cellar interactions and phenotypes within tissues as well as providing novel insight into tissue organization.

Protocol for profiling in vitro intratumor heterogeneity using spatially annotated single-cell sequencing.

Here, we present a protocol for spatially annotated single-cell sequencing, a technique for spatially profiling intratumor heterogeneity with deep single-cell RNA sequencing and single-cell resolution. By combining live-cell imaging and photopatterned illumination, we describe steps to identify regions of interest in an in vitro tumor model, label the selected cells with photoactivatable dyes, and isolate and subject them to scRNAseq. This protocol can be applied to a range of cell lines and could be expanded to tissue sections. For complete details on the use and execution of this protocol, please refer to Smit et al. (2022).1.

Correlation between lipid-lowering therapy and cerebral microbleeds.

OBJECTIVE: To investigate if there is a correlation between lipid-lowering treatment with statins and the occurrence, number, and location of cerebral microbleeds (CMBs) among patients with ischemic cerebrovascular disease (ICVD), and also to compare treatment with atorvastatin and rosuvastatin in terms of the occurrence of CMBs and their differences. METHODS: In this retrospective study, we included patients who were diagnosed with ICVD and underwent susceptibility weighted imaging (SWI) in a grade A tertiary hospital from October 1, 2014 to October 1, 2022. We collected information on previous statin use, past medical history, clinical test indicators, and imaging data. RESULTS: We found that out of 522 patients, 310 patients (59.4%) had no CMB and 212 patients (40.6%) had CMBs. There was no statistically significant correlation between prior statin use, the occurrence, and number of CMBs in patients diagnosed with ICVD (P < 0.05). As for the location of CMB, there was a statistically significant correlation between prior statin use and lobar CMBs (P < 0.048). However, there was no statistically significant correlation between the use of atorvastatin and rosuvastatin and the occurrence of CMBs (P > 0.05). CONCLUSION: There was no independent correlation between previous statin use, and the occurrence, and number of CMBs in patients with ICVD. As for CMBs in different locations, there was a correlation between previous use of statin and lobar CMBs. There was no significant difference between atorvastatin and rosuvastatin in the occurrence of CMBs in patients with ICVD.

Local Structure in α-BIMEVOXes (ME = Ge, Sn).

The BIMEVOXes are among the best oxide ion conductors at low and intermediate temperatures. Their high conductivity is associated with local defect structure. In this work, the local structures of two BIMEVOX compositions, Bi2V0.9Ge0.1O5.45 and Bi2V0.95Sn0.05O5.475, are examined using total neutron and X-ray scattering methods, with both compositions exhibiting the ordered α-phase at 25 °C and the disordered γ-phase at 700 °C. While the diffraction data for the α-phase do not allow for the polar (C2) and nonpolar (C2/m) structures to be readily distinguished, measurements of dielectric permittivity suggest the α-phase is weakly ferroelectric in character, consistent with calculations of spontaneous polarization based on a combination of density functional calculations and machine learning methodology. Reverse Monte Carlo (RMC) analysis of total scattering data reveals Ge preferentially adopts tetrahedral geometry at both temperatures, while Sn is found to predominantly adopt octahedral coordination in the α-phase and tetrahedral coordination in the γ-phase. In all cases, V polyhedra are found to consist of tetrahedral, pentacoordinate, and octahedral geometries, as also predicted by the crystallographic analysis and confirmed by 51V solid state NMR spectroscopy. Although similar long-range structures are observed at room temperature, the oxide ion vacancy distributions were found to be quite different between the two studied compositions, with a nonrandom deficiency in vacancy pairs in the second-nearest shell along the ⟨100⟩ tetragonal direction for BIGEVOX10, compared with a long-distance (>8.0 Å) ordering of equatorial vacancies for BISNVOX05. This is attributed to the differences in the preferred coordination geometries of the substituent cations in the two systems. Impedance spectroscopy measurements reveal both compositions show high conductivity in the order of 10-1 S cm-1 at 600 °C.

Exacerbation of allergic rhinitis by the commensal bacterium Streptococcus salivarius.

Allergic rhinitis (AR)-commonly called hay fever-is a widespread condition that affects the quality of life of millions of people. The pathophysiology of AR remains incompletely understood. In particular, it is unclear whether members of the colonizing nasal microbiota contribute to AR. Here, using 16S ribosomal RNA sequencing, we show that the nasal microbiome of patients with AR (n = 55) shows distinct differences compared with that from healthy individuals (n = 105), including decreased heterogeneity and the increased abundance of one species, Streptococcus salivarius. Using ex vivo and in vivo models of AR, we demonstrate that this commensal bacterium contributes to AR development, promoting inflammatory cytokine release and morphological changes in the nasal epithelium that are characteristic of AR. Our data indicate that this is due to the ability of S. salivarius to adhere to the nasal epithelium under AR conditions. Our study indicates the potential of targeted antibacterial approaches for AR therapy.

Hydrochemical and isotopic fingerprints of groundwater origin and evolution in the Urangulan River basin, China's Loess Plateau.

The origin and evolution of groundwater in the Urangulan River basin area under growing concern as its situated in an economically and ecologically crucial area of China. In the present study, a combination of different methods (i.e. self-organizing maps (SOM), piper diagrams, ionic ratios, multiple isotopic analyses and Bayesian isotope mixing model) provided an efficient way for analysing groundwater origin and evolution. The hydrochemical type was found to be Ca-HCO3 in low TDS and Na + K-Cl or Na + K-SO4 in high TDS groundwater. According to the δ2H and δ18Owater values, groundwater in the study area mainly originated from atmospheric precipitation and was influenced by evaporation. In addition, the rock weathering in conjunction with the cation exchange completely dominated the geochemical evolution process. The dual SO42- isotope and Bayesian isotope mixing model showed that gypsum dissolution, fertilizer input and sewage input were the main sources of SO42- in the study area, accounting for an average of 30.2 %, 28.5 %, and 17.3 % of SO42- in the groundwater, respectively. Other than water-rock interactions, human activity (mining and irrigation) distributed throughout the study area in combination with the spatial characteristics was the dominant factor controlling the hydrochemical evolution. The results of this study provided a basis for understanding groundwater origin and evolution while facilitating the effective management and utilization of groundwater.

Changes in Soil Organic C Fractions and C Pool Stability Are Mediated by C-Degrading Enzymes in Litter Decomposition of Robinia pseudoacacia Plantations.

Litter decomposition is the main source of soil organic carbon (SOC) pool, regarding as an important part of terrestrial ecosystem C dynamics. The turnover of SOC is mainly regulated by extracellular enzymes secreted by microorganisms. However, the response mechanism of soil C-degrading enzymes and SOC in litter decomposition remains unclear. To clarify how SOC fraction dynamics respond to C-degrading enzymes in litter decomposition, we used field experiments to collect leaf litter and SOC fractions from the underlying layer in Robinia pseudoacacia plantations on the Loess Plateau. Our results showed that SOC, easily oxidizable organic C, dissolved organic C, and microbial biomass C increased significantly during the decomposition process. Litter decomposition significantly decreased soil hydrolase activity, but slightly increased oxidase activity. Correlation analysis results showed that SOC fractions were significantly positively correlated with the litter mass, lignin, soil moisture, and oxidase activity, but significantly negatively correlated with cellulose content and soil pH. Partial least squares path models revealed that soil C-degrading enzymes can directly or indirectly affect the changes of soil C fractions. The most direct factors affecting the SOC fractions of topsoil during litter decomposition were litter lignin and cellulose degradation, soil pH, and C-degrading enzymes. Furthermore, regression analysis showed that the decrease of SOC stability in litter decomposition was closely related to the decrease of soil hydrolase to oxidase ratio. These results highlighted that litter degradation-induced changes in C-degrading enzyme activity significantly affected SOC fractions. Furthermore, the distribution of soil hydrolases and oxidases affected the stability of SOC during litter decomposition. These findings provided a theoretical framework for a more comprehensive understanding of C turnover and stabilization mechanisms between plant and soil.

Two-dimensional quantum universality in the spin-1/2 triangular-lattice quantum antiferromagnet Na2BaCo(PO4)2.

An interplay of geometrical frustration and strong quantum fluctuations in a spin-1/2 triangular-lattice antiferromagnet (TAF) can lead to exotic quantum states. Here, we report the neutron-scattering, magnetization, specific heat, and magnetocaloric studies of the recently discovered spin-1/2 TAF Na2BaCo(PO4)2, which can be described by a spin-1/2 easy axis XXZ model. The zero-field neutron diffraction experiment reveals an incommensurate antiferromagnetic ground state with a significantly reduced ordered moment of about 0.54(2) µB/Co. Different magnetic phase diagrams with magnetic fields in the ab plane and along the easy c-axis were extracted based on the magnetic susceptibility, specific heat, and elastic neutron-scattering results. In addition, two-dimensional (2D) spin dispersion in the triangular plane was observed in the high-field polarized state, and microscopic exchange parameters of the spin Hamiltonian have been determined through the linear spin wave theory. Consistently, quantum critical behaviors with the universality class of d = 2 and νz = 1 were established in the vicinity of the saturation field, where a Bose-Einstein condensation (BEC) of diluted magnons occurs. The newly discovered quantum criticality and fractional magnetization phase in this ideal spin-1/2 TAF present exciting opportunities for exploring exotic quantum phenomena.

Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics.

Single-cell proteomics has the potential to decipher tumor heterogeneity, and a method like single-cell proteomics by mass spectrometry (SCoPE-MS) allows profiling several tens of single cells for >1,000 proteins per cell. This method, however, cannot link the proteome of individual cells with phenotypes of interest. Here, we developed a microscopy-based functional single-cell proteomic-profiling technology, called FUNpro, to address this. FUNpro enables screening, identification, and isolation of single cells of interest in a real-time fashion, even if the phenotypes are dynamic or the cells of interest are rare. We applied FUNpro to proteomically profile a newly identified small subpopulation of U2OS osteosarcoma cells displaying an abnormal, prolonged DNA damage response (DDR) after ionizing radiation (IR). With this, we identified the PDS5A protein contributing to the abnormal DDR dynamics and helping the cells survive after IR.

Sling Exercise Can Drive Cortical Representation of the Transversus Abdominis and Multifidus Muscles in Patients With Chronic Low Back Pain.

Objective: The transversus abdominis (TrA) and multifidus (MF) muscles are essential in preventing chronic low back pain (CLBP) recurrence by maintaining segmental stabilization and stiffness. Sling exercise is a high-level core stability training to effectively improve the activities of the TrA and MF muscles. However, the neural mechanism for sling exercise-induced neural plasticity change in the primary motor cortex (M1) remains unclear. This study aimed to investigate the role of sling exercise in the reorganization of the motor cortical representation of the TrA and MF muscles. Methods: Twenty patients with CLBP and 10 healthy individuals were recruited. For map volume, area, the center of gravity (CoG) location (medial-lateral location and anterior-posterior location), and latency, two-way ANOVA was performed to compare the effects of groups (the CLBP-pre, CLBP-post, and healthy groups) and the two muscles (the TrA and MF muscles). The Visual Analog Scale (VAS), the Oswestry Disability Index (ODI), and postural balance stability were assessed at baseline and at the end of 2 weeks of sling exercise. Linear correlations between VAS or ODI and CoG locations were assessed by Pearson's correlation test. Results: 2 weeks of sling exercise induced both the anterior-medial (P < 0.001) and anterior-posterior (P = 0.025) shifts of the MF muscle representation at the left motor cortex in patients with CLBP. Anterior-medial (P = 0.009) shift of the TrA muscle representation at the right motor cortex was observed in patients with CLBP. The motor cortical representation of the two muscles in patients with CLBP after sling exercise (TrA: 2.88 ± 0.27 cm lateral and 1.53 ± 0.47 cm anterior of vertex; MF: 3.02 ± 0.48 cm lateral and 1.62 ± 0.40 cm anterior of vertex) closely resembled that observed in healthy individuals (TrA: 2.83 ± 0.48 cm lateral and 2.00 ± 0.43 cm anterior of vertex; MF: 2.94 ± 0.43 cm lateral and 1.77 ± 0.48 cm anterior of vertex). The VAS and the ODI were reduced following the sling exercise (VAS: P < 0.001; ODI: P < 0.001). Conclusion: This study provides evidence that sling training can drive plasticity changes in the motor system, which corresponds with the reduction in pain and disability levels in patients with CLBP. This study was registered in the Chinese Clinical Trial Registry (Clinical Trial Registration Number: ChiCTR2100045904, http://www.chictr.org.cn/showproj.aspx?proj=125819). Clinical Trial Registration: ChiCTR2100045904.

Effects of methyl halide flux characteristics following Spartina alterniflora invasion in a seaward direction in a temperate salt marsh, China.

In this study, we explored the source-sink characteristics of methyl halide (CH3X; X = Cl, Br, I) in coastal wetlands located in temperate regions, and identified key factors affecting the spatio-temporal variation of CH3X during the invasion of Spartina alterniflora. We used static chamber-gas chromatography to monitor CH3X fluxes in the S. alterniflora area and bare flat area of the Jiaozhou Bay salt marsh for a long time from August 2015 to May 2017. Our results indicated that CH3X emissions showed obvious seasonal and diurnal variations. The S. alterniflora area was a source of CH3X, with higher fluxes in the spring and autumn seasons. CH3X fluxes were higher during the daytime than at night, and the diurnal difference in CH3Br was the most significant (4.51 times). The bare flat area was mainly a sink for CH3X, and the maximum absorption flux occurred in summer. At this time, the microbial activity was greater, and the consumption rate during the day was higher than that at night. Extreme linear correlations existed between the fluxes of CH3Cl, CH3Br, and CH3I (P < 0.01), indicating that the production and consumption of the three gases were likely to have similar mechanisms and were affected by the same factors. S. alterniflora invasion increased CH3X emissions and shifted the original bare flat area from a sink to a source of CH3X. The biomass of S. alterniflora, especially the leaf, significantly affects CH3X fluxes. Additionally, S. alterniflora increased the content of total organic carbon, total sulfur, available sulfur, and iron (III) in the soil, which were the main factors promoting the source-sink transformation of CH3X. Based on the current invasive area of S. alterniflora in China, we estimated that the annual emissions of CH3Cl, CH3Br, and CH3I from S. alterniflora into the troposphere were 9.04 × 106, 2.42 × 105 and 2.06 × 105 mol, respectively.

Glycyrrhiza uralensis promote the metabolism of toxic components of Aconitum carmichaeli by CYP3A and alleviate the development of chronic heart failure.

Aconitum, as "the first drug of choice for invigorating Yang and saving lives", has been widely used for the treatment of heart failure. However, toxic components of Aconitum can easily lead to serious arrhythmia, even death (Y. CT., 2009; Zhang XM., 2018). In this study, a High Performance Liquid Chromatography (HPLC) method for the determination of aconitine (AC), mesaconitine (MA) and hypaconitine (HA) was established; The effect of Glycyrrhiza on CYP3A1 / 2 mRNA expression was detected by RT-PCR; SD rats were given Aconitum and compatibility of Glycyrrhizae and Aconitum by gavage respectively, the blood concentration of toxic components were determined by LC-MS / MS; The CHF rat model was established by intraperitoneal injection of adriamycin (2.5 mg / kg), and were randomly divided into model, Aconitum, the compatibility of Glycyrrhizae and Aconitum and Captopril group, 5 mice/group. After 4 weeks of gavage, the corresponding indexes were detected by ELISA and HPLC. The results showed that Ketoconazole significantly inhibited the metabolites of AC, MA and HA; Glycyrrhiza induced CYP3A gene expression; The level of ALD in the compatibility of Glycyrrhizae and Aconitum group was significantly lower than that in Aconitum group. After intervention with the compatibility of Glycyrrhizae and Aconitum, ATP increased, ADP decreased significantly. In conclusion, we found Glycyrrhiza promoted the metabolism of toxic components of Aconitum by up regulating the expression of CYP3A, and reduced the content of BNP, Ang II and ALD, improved the energy metabolism disorder of myocardium, alleviated the development of CHF.

Dynamic Visualization of TGF-ß/SMAD3 Transcriptional Responses in Single Living Cells.

Transforming growth factor-ß (TGF-ß) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-ß/SMAD3 signaling kinetics, we developed a dynamic TGF-ß/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-ß/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-ß challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-ß/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes.

Spatially Annotated Single Cell Sequencing for Unraveling Intratumor Heterogeneity.

Intratumor heterogeneity is a major obstacle to effective cancer treatment. Current methods to study intratumor heterogeneity using single-cell RNA sequencing (scRNA-seq) lack information on the spatial organization of cells. While state-of-the art spatial transcriptomics methods capture the spatial distribution, they either lack single cell resolution or have relatively low transcript counts. Here, we introduce spatially annotated single cell sequencing, based on the previously developed functional single cell sequencing (FUNseq) technique, to spatially profile tumor cells with deep scRNA-seq and single cell resolution. Using our approach, we profiled cells located at different distances from the center of a 2D epithelial cell mass. By profiling the cell patch in concentric bands of varying width, we showed that cells at the outermost edge of the patch responded strongest to their local microenvironment, behaved most invasively, and activated the process of epithelial-to-mesenchymal transition (EMT) to migrate to low-confluence areas. We inferred cell-cell communication networks and demonstrated that cells in the outermost ∼10 cell wide band, which we termed the invasive edge, induced similar phenotypic plasticity in neighboring regions. Applying FUNseq to spatially annotate and profile tumor cells enables deep characterization of tumor subpopulations, thereby unraveling the mechanistic basis for intratumor heterogeneity.