Whole-genome sequence of Pseudomonas benzopyrenica MLY92: isolation from diseased leaves of tobacco in China.

Here we present a sketch of the whole-genome sequence of Pseudomonas benzopyrenica. The strain comes from the leaf veins of a diseased tobacco plant. This study has significant research implications for gaining insights into the characteristics of microorganisms belonging to the genus Pseudomonas.

Adeno-to-squamous transition drives resistance to KRAS inhibition in LKB1 mutant lung cancer.

KRASG12C inhibitors (adagrasib and sotorasib) have shown clinical promise in targeting KRASG12C-mutated lung cancers; however, most patients eventually develop resistance. In lung patients with adenocarcinoma with KRASG12C and STK11/LKB1 co-mutations, we find an enrichment of the squamous cell carcinoma gene signature in pre-treatment biopsies correlates with a poor response to adagrasib. Studies of Lkb1-deficient KRASG12C and KrasG12D lung cancer mouse models and organoids treated with KRAS inhibitors reveal tumors invoke a lineage plasticity program, adeno-to-squamous transition (AST), that enables resistance to KRAS inhibition. Transcriptomic and epigenomic analyses reveal ΔNp63 drives AST and modulates response to KRAS inhibition. We identify an intermediate high-plastic cell state marked by expression of an AST plasticity signature and Krt6a. Notably, expression of the AST plasticity signature and KRT6A at baseline correlates with poor adagrasib responses. These data indicate the role of AST in KRAS inhibitor resistance and provide predictive biomarkers for KRAS-targeted therapies in lung cancer.

(Tn5-)FISH-based imaging in the era of 3D/spatial genomics.

3D genomics mainly focuses on the 3D position of single genes at the cell level, while spatial genomics focuses more on the tissue level. In this exciting new era of 3D/spatial genomics, half-century old FISH and its derivative methods, including Tn5-FISH, play important roles. In this review, we introduce the Tn5-FISH we developed recently, and present six different applications published by our collaborators and us, based on (Tn5-)FISH, which can be either general BAC clone-based FISH or Tn5-FISH. In these interesting cases, (Tn5-)FISH demonstrated its vigorous ability of targeting sub-chromosomal structures across different diseases and cell lines (leukemia, mESCs (mouse embryonic stem cells), and differentiation cell lines). Serving as an effective tool to image genomic structures at the kilobase level, Tn5-FISH holds great potential to detect chromosomal structures in a high-throughput manner, thus bringing the dawn for new discoveries in the great era of 3D/spatial genomics.

Development and Experimental Validation of a Novel Prognostic Signature for Gastric Cancer.

BACKGROUND: Gastric cancer is a malignant tumor with high morbidity and mortality. Therefore, the accurate recognition of prognostic molecular markers is the key to improving treatment efficacy and prognosis. METHODS: In this study, we developed a stable and robust signature through a series of processes using machine-learning approaches. This PRGS was further experimentally validated in clinical samples and a gastric cancer cell line. RESULTS: The PRGS is an independent risk factor for overall survival that performs reliably and has a robust utility. Notably, PRGS proteins promote cancer cell proliferation by regulating the cell cycle. Besides, the high-risk group displayed a lower tumor purity, higher immune cell infiltration, and lower oncogenic mutation than the low-PRGS group. CONCLUSIONS: This PRGS could be a powerful and robust tool to improve clinical outcomes for individual gastric cancer patients.

Single-cell and spatial transcriptome analyses revealed cell heterogeneity and immune environment alternations in metastatic axillary lymph nodes in breast cancer.

BACKGROUND: Tumor heterogeneity plays essential roles in developing cancer therapies, including therapies for breast cancer (BC). In addition, it is also very important to understand the relationships between tumor microenvironments and the systematic immune environment. METHODS: Here, we performed single-cell, VDJ sequencing and spatial transcriptome analyses on tumor and adjacent normal tissue as well as axillar lymph nodes (LNs) and peripheral blood mononuclear cells (PBMCs) from 8 BC patients. RESULTS: We found that myeloid cells exhibited environment-dependent plasticity, where a group of macrophages with both M1 and M2 signatures possessed high tumor specificity spatially and was associated with worse patient survival. Cytotoxic T cells in tumor sites evolved in a separate path from those in the circulatory system. T cell receptor (TCR) repertoires in metastatic LNs showed significant higher consistency with TCRs in tumor than those in nonmetastatic LNs and PBMCs, suggesting the existence of common neo-antigens across metastatic LNs and primary tumor cites. In addition, the immune environment in metastatic LNs had transformed into a tumor-like status, where pro-inflammatory macrophages and exhausted T cells were upregulated, accompanied by a decrease in B cells and neutrophils. Finally, cell interactions showed that cancer-associated fibroblasts (CAFs) contributed most to shaping the immune-suppressive microenvironment, while CD8+ cells were the most signal-responsive cells. CONCLUSIONS: This study revealed the cell structures of both micro- and macroenvironments, revealed how different cells diverged in related contexts as well as their prognostic capacities, and displayed a landscape of cell interactions with spatial information.

1-Tetradecanol, Diethyl Phthalate and Tween 80 Assist in the Formation of Thermo-Responsive Azoxystrobin Nanoparticles.

The occurrence of crop fungal diseases is closely related to warm environmental conditions. In order to control the release of fungicides in response to warm conditions, and enhance the efficacy, a series of thermo-responsive fungicide-loaded nanoparticles were developed. The fungicide azoxystrobin, solvent DEP, emulsifier Tween 80 and thermo-responsive component TDA were combined to create thermal-response oil phases, conditions for emulsification were then optimized. LDLS, zeta potential, FTIR, DSC, TGA, XRD, SEM and antifungal efficacy assays were carried out to investigate the characteristics and forming mechanism. The results indicated that the formula with 5 g azoxystrobin, 10 mL DEP, 6 mL Tween 80 and 2.5 g TDA constructed the proposed oil phase with the ability to transform from solid at 20 °C to softerned at 31.5 °C. Both DEP and TDA played key roles in interfering with the crystallization of azoxystrobin. The optimal T3t-c12 nanoparticles had a mean particle size of 162.1 nm, thermo-responsive morphological transformation between 20 °C and 30 °C, AZO crystal reforming after drying, the ability to attach to fungal spores and satisfied antifungal efficacy against P. nicotiana PNgz07 and A. niger A1513 at 30 °C. This report provides referable technical support for the construction of smart-release nanoparticles of other agrochemicals.

SOTIP is a versatile method for microenvironment modeling with spatial omics data.

The rapidly developing spatial omics generated datasets with diverse scales and modalities. However, most existing methods focus on modeling dynamics of single cells while ignore microenvironments (MEs). Here we present SOTIP (Spatial Omics mulTIPle-task analysis), a versatile method incorporating MEs and their interrelationships into a unified graph. Based on this graph, spatial heterogeneity quantification, spatial domain identification, differential microenvironment analysis, and other downstream tasks can be performed. We validate each module's accuracy, robustness, scalability and interpretability on various spatial omics datasets. In two independent mouse cerebral cortex spatial transcriptomics datasets, we reveal a gradient spatial heterogeneity pattern strongly correlated with the cortical depth. In human triple-negative breast cancer spatial proteomics datasets, we identify molecular polarizations and MEs associated with different patient survivals. Overall, by modeling biologically explainable MEs, SOTIP outperforms state-of-art methods and provides some perspectives for spatial omics data exploration and interpretation.

The transcriptional coactivator RUVBL2 regulates Pol II clustering with diverse transcription factors.

RNA polymerase II (Pol II) apparatuses are compartmentalized into transcriptional clusters. Whether protein factors control these clusters remains unknown. In this study, we find that the ATPase-associated with diverse cellular activities (AAA + ) ATPase RUVBL2 co-occupies promoters with Pol II and various transcription factors. RUVBL2 interacts with unphosphorylated Pol II in chromatin to promote RPB1 carboxy-terminal domain (CTD) clustering and transcription initiation. Rapid depletion of RUVBL2 leads to a decrease in the number of Pol II clusters and inhibits nascent RNA synthesis, and tethering RUVBL2 to an active promoter enhances Pol II clustering at the promoter. We also identify target genes that are directly linked to the RUVBL2-Pol II axis. Many of these genes are hallmarks of cancers and encode proteins with diverse cellular functions. Our results demonstrate an emerging activity for RUVBL2 in regulating Pol II cluster formation in the nucleus.

[Establishment of a CFTR-based detection method for the second messenger cAMP in the cytoplasm].

Objective: To establish a detection method based on Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that can sensitively detect the second messenger cyclic AMP (cAMP) in the cytoplasm. Methods: The eukaryotic expression vectors of CFTR and YFP-H148Q / I152L were constructed respectively. FRT cells co-expressing CFTR and YFP-H148Q / I152L were obtained by liposome transfection. The expression of CFTR and YFP-H148Q / I152L in FRT cells was observed by an inverted fluorescence microscopy, and flow cytometry was used to detect the purity of cells; The cell model was identified by the fluorescence quenching kinetics test. The validation of the cell model which could screen CFTR modulators was verified by the fluorescence quenching kinetics experiments. The radioimmunoassay was used to detect the cAMP concentration in cytoplasm after adding CFTR activator. Results: The results of the inverted fluorescence microscope showed that CFTR was expressed in the cell membrane and YFP-H148Q / I152L was expressed in the cytoplasm of FRT cells. The FRT cell model stably co-expressing ANO1 and YFP-H148Q / I152L was successfully constructed. The model could screen CFTR modulators, and the slope of fluorescence change and the concentration of CFTR modulators were in a dose-dependent manner. The slope of the fluorescence could reflect the cAMP concentration in the cytoplasm. The cell model could sensitively detect the intracellular cAMP concentration. Conclusion: The cell model could efficiently and sensitively detect the second messenger cAMP concentration in the cytoplasm, and it provided a simple and efficient method for the study of other targets associated cAMP signal.

Polarization modulation with optical lock-in detection reveals universal fluorescence anisotropy of subcellular structures in live cells.

The orientation of fluorophores can reveal crucial information about the structure and dynamics of their associated subcellular organelles. Despite significant progress in super-resolution, fluorescence polarization microscopy remains limited to unique samples with relatively strong polarization modulation and not applicable to the weak polarization signals in samples due to the excessive background noise. Here we apply optical lock-in detection to amplify the weak polarization modulation with super-resolution. This novel technique, termed optical lock-in detection super-resolution dipole orientation mapping (OLID-SDOM), could achieve a maximum of 100 frames per second and rapid extraction of 2D orientation, and distinguish distance up to 50 nm, making it suitable for monitoring structural dynamics concerning orientation changes in vivo. OLID-SDOM was employed to explore the universal anisotropy of a large variety of GFP-tagged subcellular organelles, including mitochondria, lysosome, Golgi, endosome, etc. We found that OUF (Orientation Uniformity Factor) of OLID-SDOM can be specific for different subcellular organelles, indicating that the anisotropy was related to the function of the organelles, and OUF can potentially be an indicator to distinguish normal and abnormal cells (even cancer cells). Furthermore, dual-color super-resolution OLID-SDOM imaging of lysosomes and actins demonstrates its potential in studying dynamic molecular interactions. The subtle anisotropy changes of expanding and shrinking dendritic spines in live neurons were observed with real-time OLID-SDOM. Revealing previously unobservable fluorescence anisotropy in various samples and indicating their underlying dynamic molecular structural changes, OLID-SDOM expands the toolkit for live cell research.

ScaffComb: A Phenotype-Based Framework for Drug Combination Virtual Screening in Large-Scale Chemical Datasets.

Combinational therapy is used for a long time in cancer treatment to overcome drug resistance related to monotherapy. Increased pharmacological data and the rapid development of deep learning methods have enabled the construction of models to predict and screen drug pairs. However, the size of drug libraries is restricted to hundreds to thousands of compounds. The ScaffComb framework, which aims to bridge the gaps in the virtual screening of drug combinations in large-scale databases, is proposed here. Inspired by phenotype-based drug design, ScaffComb integrates phenotypic information into molecular scaffolds, which can be used to screen the drug library and identify potent drug combinations. First, ScaffComb is validated using the US food and drug administration dataset and known drug combinations are successfully reidentified. Then, ScaffComb is applied to screen the ZINC and ChEMBL databases, which yield novel drug combinations and reveal an ability to discover new synergistic mechanisms. To our knowledge, ScaffComb is the first method to use phenotype-based virtual screening of drug combinations in large-scale chemical datasets.

A novel cytogenetic method to image chromatin interactions at subkilobase resolution: Tn5 transposase-based fluorescence in situ hybridization.

There is an increasing interest in understanding how three-dimensional organization of the genome is regulated. Different strategies have been used to identify genome-wide chromatin interactions. However, owing to current limitations in resolving genomic contacts, visualization and validation of these genomic loci at subkilobase resolution remain unsolved to date. Here, we describe Tn5 transposase-based fluorescence in situ hybridization (Tn5-FISH), a polymerase chain reaction-based, cost-effective imaging method, which can colocalize the genomic loci at subkilobase resolution, dissect genome architecture, and verify chromatin interactions detected by chromatin configuration capture-derived methods. To validate this method, short-range interactions in the keratin-encoding gene (KRT) locus in the topologically associated domain were imaged by triple-color Tn5-FISH, indicating that Tn5-FISH is very useful to verify short-range chromatin interactions inside the contact domain and TAD. Therefore, Tn5-FISH can be a powerful molecular tool for clinical detection of cytogenetic changes in numerous genetic diseases such as cancers.

Super-resolution imaging of fluorescent dipoles via polarized structured illumination microscopy.

Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality.

Deletion of the type 2 metabotropic glutamate receptor increases heroin abuse vulnerability in transgenic rats.

Opioid abuse is a rapidly growing public health crisis in the USA. Despite extensive research in the past decades, little is known about the etiology of opioid addiction or the neurobiological risk factors that increase vulnerability to opioid use and abuse. Recent studies suggest that the type 2 metabotropic glutamate receptor (mGluR2) is critically involved in substance abuse and addiction. In the present study, we evaluated whether low-mGluR2 expression may represent a risk factor for the development of opioid abuse and addiction using transgenic mGluR2-knockout (mGluR2-KO) rats. Compared to wild-type controls, mGluR2-KO rats exhibited higher nucleus accumbens (NAc) dopamine (DA) and locomotor responses to heroin, higher heroin self-administration and heroin intake, more potent morphine-induced analgesia and more severe naloxone-precipitated withdrawal symptoms. In contrast, mGluR2-KO rats displayed lower motivation for heroin self-administration under high price progressive-ratio (PR) reinforcement conditions. Taken together, these findings suggest that mGluR2 may play an inhibitory role in opioid action, such that deletion of this receptor results in an increase in brain DA responses to heroin and in acute opioid reward and analgesia. Low-mGluR2 expression in the brain may therefore be a risk factor for the initial development of opioid abuse and addiction.

Polarization-based super-resolution imaging of surface-enhanced Raman scattering nanoparticles with orientational information.

Raman scattering provides key information of the biological environment through light-molecule interaction; yet, it is generally very weak to detect. Surface-enhanced Raman scattering (SERS) can boost the Raman signal by several orders-of-magnitude, and thus is highly attractive for biochemical sensing. However, conventional super-resolution imaging of SERS is challenging as the Raman signal is generated from the virtual state which cannot be easily modulated as fluorescence. Here, we demonstrate super-resolution microscopy with a surface-enhanced Raman scattering (SERS) signal, with a resolution of approximately 50 nm. By modulating the polarization angle of the excitation laser, the SERS nanorods display a dramatic anisotropy effect, allowing nanoscale orientation determination of multiple dipoles with dense concentration. Furthermore, a well-established defocused analysis was performed to reconfirm the orientation accuracy of super-resolved SERS nanorods. Sub-diffraction resolution was achieved in the imaging of SERS nanorod labeled vesicles in fixed macrophages. Finally, we demonstrate dynamic SERS nanorod tracking in living macrophages, which provides not only the particle trajectory with high spatial resolution but also the rotational changes at the nanometer scale. This pioneering study paves a new way for subcellular super-resolution imaging with the SERS effect, shedding light on wider biological applications.

Alterations of specific chromatin conformation affect ATRA-induced leukemia cell differentiation.

Chromatin conformation plays a key role in regulating gene expression and controlling cell differentiation. However, the whole-genome chromatin conformation changes that occur during leukemia cell differentiation are poorly understood. Here, we characterized the changes in chromatin conformation, histone states, chromatin accessibility, and gene expression using an all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation model. The results showed that the boundaries of topological associated domains (TADs) were stable during differentiation; however, the chromatin conformations within several specific TADs were obviously changed. By combining H3K4me3, H3K27ac, and Hi-C signals, we annotated the differential gene-regulatory chromatin interactions upon ATRA induction. The gains and losses of the gene-regulatory chromatin interactions are significantly correlated with gene expression and chromatin accessibility. Finally, we found that the loss of GATA2 expression and DNA binding are crucial for the differentiation process, and changes in the chromatin structure around the GATA2 regulate its expression upon ATRA induction. This study provided both statistical insights and experimental details regarding the relationship between chromatin conformation changes and transcription regulation during leukemia cell differentiation, and the results suggested that the chromatin conformation is a new type of potential drug target for cancer therapy.

Spatiotemporally Controllable Peptide-Based Nanoassembly in Single Living Cells for a Biological Self-Portrait.

Simultaneous precise localization and activity evaluation of a biomolecule in a single living cell is through an enzyme-specific signal-amplification process, which involves the localized, site-specific self-assembly, and activation of a presignaling molecule. The inactive presignaling tetraphenylethylene (TPE)-peptide derivative, TPE-YpYY, is nondetectable and highly biocompatible and these small molecules rapidly diffuse into living cells. Upon safely arriving at an active site, and accessing the catalytic pocket of an enzyme, TPE-YpYY immediately and quantitatively accumulates in situ in response to enzymatic activity, forms an enzyme anchor TPE-YYY nanoassembly, displays aggregation-induced emission behavior, and finally lights up the active enzyme, indicating its activity, and allowing its status in living cells to be tracked. This simple and direct self-portrait method can be used to monitor dynamic self-assembly processes in individual living cells and may provide new insights that reveal undiscovered biological processes and that aid in developing biomedical hybrid devices. In the future, this strategy of molecular design can be further expanded to the noninvasive investigation of other bioactive molecules, thus facilitating quantitative imaging.

Quantitative combination of natural anti-oxidants prevents metabolic syndrome by reducing oxidative stress.

Insulin resistance and abdominal obesity are present in the majority of people with the metabolic syndrome. Antioxidant therapy might be a useful strategy for type 2 diabetes and other insulin-resistant states. The combination of vitamin C (Vc) and vitamin E has synthetic scavenging effect on free radicals and inhibition effect on lipid peroxidation. However, there are few studies about how to define the best combination of more than three anti-oxidants as it is difficult or impossible to test the anti-oxidant effect of the combination of every concentration of each ingredient experimentally. Here we present a math model, which is based on the classical Hill equation to determine the best combination, called Fixed Dose Combination (FDC), of several natural anti-oxidants, including Vc, green tea polyphenols (GTP) and grape seed extract proanthocyanidin (GSEP). Then we investigated the effects of FDC on oxidative stress, blood glucose and serum lipid levels in cultured 3T3-L1 adipocytes, high fat diet (HFD)-fed rats which serve as obesity model, and KK-ay mice as diabetic model. The level of serum malondialdehyde (MDA) in the treated rats was studied and Hematoxylin-Eosin (HE) staining or Oil red slices of liver and adipose tissue in the rats were examined as well. FDC shows excellent antioxidant and anti-glycation activity by attenuating lipid peroxidation. FDC determined in this investigation can become a potential solution to reduce obesity, to improve insulin sensitivity and be beneficial for the treatment of fat and diabetic patients. It is the first time to use the math model to determine the best ratio of three anti-oxidants, which can save much more time and chemical materials than traditional experimental method. This quantitative method represents a potentially new and useful strategy to screen all possible combinations of many natural anti-oxidants, therefore may help develop novel therapeutics with the potential to ameliorate the worldwide metabolic abnormalities.

R-modafinil attenuates nicotine-taking and nicotine-seeking behavior in alcohol-preferring rats.

(±)-Modafinil (MOD) is used clinically for the treatment of sleep disorders and has been investigated as a potential medication for the treatment of psychostimulant addiction. However, the therapeutic efficacy of (±)-MOD for addiction is inconclusive. Herein we used animal models of self-administration and in vivo microdialysis to study the pharmacological actions of R-modafinil (R-MOD) and S-modafinil (S-MOD) on nicotine-taking and nicotine-seeking behavior, and mechanisms underlying such actions. We found that R-MOD is more potent and effective than S-MOD in attenuating nicotine self-administration in Long-Evans rats. As Long-Evans rats did not show a robust reinstatement response to nicotine, we used alcohol-preferring rats (P-rats) that display much higher reinstatement responses to nicotine than Long-Evans rats. We found that R-MOD significantly inhibited intravenous nicotine self-administration, nicotine-induced reinstatement, and nicotine-associated cue-induced drug-seeking behavior in P-rats. R-MOD alone neither sustained self-administration in P-rats previously self-administering nicotine nor reinstated extinguished nicotine-seeking behavior. The in vivo brain microdialysis assays demonstrated that R-MOD alone produced a slow-onset moderate increase in extracellular DA. Pretreatment with R-MOD dose-dependently blocked nicotine-induced dopamine (DA) release in the nucleus accumbens (NAc) in both naive and nicotine self-administrating rats, suggesting a DA-dependent mechanism underlying mitigation of nicotine's effects. In conclusion, the present findings support further investigation of R-MOD for treatment of nicotine dependence in humans.

ModuleRole: a tool for modulization, role determination and visualization in protein-protein interaction networks.

UNLABELLED: Rapidly increasing amounts of (physical and genetic) protein-protein interaction (PPI) data are produced by various high-throughput techniques, and interpretation of these data remains a major challenge. In order to gain insight into the organization and structure of the resultant large complex networks formed by interacting molecules, using simulated annealing, a method based on the node connectivity, we developed ModuleRole, a user-friendly web server tool which finds modules in PPI network and defines the roles for every node, and produces files for visualization in Cytoscape and Pajek. For given proteins, it analyzes the PPI network from BioGRID database, finds and visualizes the modules these proteins form, and then defines the role every node plays in this network, based on two topological parameters Participation Coefficient and Z-score. This is the first program which provides interactive and very friendly interface for biologists to find and visualize modules and roles of proteins in PPI network. It can be tested online at the website http://www.bioinfo.org/modulerole/index.php, which is free and open to all users and there is no login requirement, with demo data provided by "User Guide" in the menu Help. Non-server application of this program is considered for high-throughput data with more than 200 nodes or user's own interaction datasets. Users are able to bookmark the web link to the result page and access at a later time. As an interactive and highly customizable application, ModuleRole requires no expert knowledge in graph theory on the user side and can be used in both Linux and Windows system, thus a very useful tool for biologist to analyze and visualize PPI networks from databases such as BioGRID. AVAILABILITY: ModuleRole is implemented in Java and C, and is freely available at http://www.bioinfo.org/modulerole/index.php. Supplementary information (user guide, demo data) is also available at this website. API for ModuleRole used for this program can be obtained upon request.