Gene features of tumor-specific T cells relevant to immunotherapy, targeted therapy and chemotherapy in lung cancer.

1 Background: In lung cancer, the use of small-molecule inhibitors, chemotherapy and immunotherapy has led to unprecedented survival benefits in selected patients. Considering most patients will experience a relapse within a short period of time due to single drug resistance, combination therapy is also particularly important to improve patient prognosis. Therefore, more robust biomarkers to predict responses to immunotherapy, targeted therapy, chemotherapy and rationally drug combination therapies may be helpful in clinical treatment choices. 2 Methods: We defined tumor-specific T cells (TSTs) and their features (TSTGs) by single-cell RNA sequencing. We applied LASSO regression to filter out the most survival-relevant TSTGs to form the Tumor-specific T cell score (TSTS). Immunological characteristics, enriched pathways, and mutation were evaluated in high- and low TSTS groups. 3 Results: We identified six clusters of T cells as TSTs in lung cancer, and four most robust genes from 9 feature genes expressed only on tumor-specific T cells were screened to construct a tumor-specific T cells score (TSTS). TSTS was positively correlated with immune infiltration and angiogenesis and negatively correlated with malignant cell proliferation. Moreover, potential vascular-immune crosstalk in lung cancer provides the theoretical basis for combined anti-angiogenic and immunotherapy. Noticeable, patients in high TSTS had better response to ICB and targeted therapy and patients in the low TSTS group often benefit from chemotherapy. 4 Conclusion: The proposed TSTS is a promising indicator to predict immunotherapy, targeted therapy and chemotherapy responses in lung cancer patients for helping clinical treatment choices.

In vitro determination of osteo-adipogenic lineage choice of bone marrow stromal/stem cells (BMSCs).

Bone marrow stromal/stem cells (BMSCs) are primitive and heterogeneous cells that can be differentiated into osteoblasts, adipocytes and other subsets. Their bone-fat lineage commitment is responsible for the homeostasis of bone marrow microenvironment. However, there are little effective methods and evidence to simultaneously visualise the lineage commitment of BMSCs. Here we provide a bivalent differentiation medium that can enable BMSCs differentiation into osteoblasts and adipocytes in vitro, and establish a method to simultaneously distinguish osteoblasts or adipocytes from the heterogeneous BMSCs based on Alizarin red S and Oil red O staining, which have been used for detection of specific mineralized nodules and lipid droplets, respectively. This assay provides a specifically simple but effective and low-cost method to evaluate the efficiency of osteo-adipogenic (OA) allocation of BMSCs.►Researchers can utilize the bivalent differentiation medium to evaluate the efficiency of osteogenic and adipogenic differentiation of BMSCs in vitro.

Rapid Access to Chiral Spiro[2.3] Lactams: Stereoselective Hydroborylation and Hydrosilylation and Remote Control of Axial Chirality by Copper-Catalyzed Desymmetrization of Spirocyclopropenes.

Chiral spirocyclopropyl ß-lactams are common motifs in bioactive compounds and pharmaceuticals. Here we disclose a diastereoselective and enantioselective hydroborylation and hydrosilylation of spirocyclopropenes, via a Cu-catalyzed desymmetrization strategy, for the rapid preparation of enantio-enriched spirocyclopropyl ß-lactams. The efficient desymmetrization strategy allows the remote control of axial chirality, offering the borylated and silylated products bearing central, spiro, and axial chirality. The combination of multichiral elements would provide a novel motif for biological evaluation in potential drug discovery.

Mitigating life-cycle environmental impacts and increasing net ecosystem economic benefits via optimized fertilization combined with lime in pomelo production in Southeast China.

Excessive fertilization is acknowledged as a significant driver of heightened environmental pollution and soil acidification in agricultural production. Combining fertilizer optimization with soil acidity amendment can effectively achieve sustainable crop production in China, especially in Southeast China. However, there is a lack of long-term studies assessing the environmental and economic sustainability of combining fertilizer optimization with soil acidity amendment strategies, especially in fruit production. A four-year field experiment was conducted to explore pomelo yield, fruit quality, and environmental and economic performance in three treatments, e.g., local farmer practices (FP), optimized NPK fertilizer application (OPT), and OPT with lime (OPT+L). The results showed that the OPT+L treatment exhibited the highest pomelo yield and fruit quality among the three treatments. The OPT treatment had the lowest net greenhouse gas (GHG) emissions among the three treatments, which were 90.1 % and 42.6 % lower than those in FP and OPT+L, respectively. It is essential to note that GHG emissions associated with lime production constitute 40.7 % of the total emissions from fertilizer production. The OPT+L treatment reduced reactive nitrogen (Nr) emissions and phosphorus (P) losses, compared to FP and OPT. Moreover, the OPT+L treatment increased the net ecosystem economic benefit by 220.3 % and 20.3 % compared with the FP and OPT treatments, respectively. Overall, the OPT and OPT+L treatments underscore the potential to achieve environmentally friendly and economically sustainable pomelo production. Our study provides science-based evidence to achieve better environmental and economic performance in pomelo production through optimized NPK fertilization and alleviating soil acidification by lime.

Novel-miR-81 Promotes the Chondrocytes Differentiation of Bone Marrow Mesenchymal Stem Cells Through Inhibiting Rac2 Expression.

OBJECTIVE: MicroRNAs (miRNAs) play a key role in the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into chondrocytes. Our previous study found that novel-miR-81 can relieve osteoarthritis, but its role in chondrogenic differentiation of BMSCs remains unclear. The purpose of this study was to explore the role of novel-miR-81 in chondrogenic differentiation of BMSCs. METHODS: We used a model in which transforming growth factor (TGF)-ß3-induced BMSCs differentiation into chondrocytes. We detected the expression Sox9, Collagen Ⅱ, Aggrecan, novel-miR-81, and Rac2 by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Western blot was performed to detect the expression of Sox9, Collagen Ⅱ, and Rac2. Dual-luciferase reporter gene assay confirmed that the association between novel-miR-81 and Rac2. In addition, the ectopic chondrocyte differentiation of BMSCs was performed subcutaneously in nude mice. The effect of novel-miR-81 and Rac2 on ectopic chondrogenic differentiation of BMSCs was determined by immunohistochemical staining. RESULTS: Novel-miR-81 upregulated in chondrogenic differentiation of BMSCs. Rac2 was a key target of novel-miR-81. Mimic novel-miR-81 and siRac2 upregulated the expression of Sox9, Collagen Ⅱ, and Aggrecan. CONCLUSION: Novel-miR-81 promotes the chondrocytes differentiation of BMSCs by inhibiting the expression of target gene Rac2, which provides potential targets for BMSCs transplantation to repair cartilage defects.

Three-Component Domino Acylation/Trifluoromethylation of Bicyclic Amidines.

We herein describe a three-component reaction involving bicyclic amidines such as DBN/DBU, acyl fluorides, and TMSCF3 for access to a novel class of N-acyl trifluoromethylated bicyclic aminals. Under mild and operationally simple conditions, bicyclic amidines can undergo difunctionalization (acylation/trifluoromethylation) using readily available reagents. Further Lewis acid-promoted nucleophilic ring-opening can lead to diverse products with quaternary carbon centers containing CF3. The corresponding pentafluoroethylation is also achieved by using TMSC2F5.

Palladium-Catalyzed Stereoselective Defluoroborylation of gem-Difluoroalkenes with Unsymmetrical Diboron: Access to Tetrasubstituted Monofluorinated Vinyl-B(dan) Derivatives.

The stereoselective C-F bond borylation of tetrasubstituted ß,ß-difluoroacrylates has been achieved. In contrast to the previously used B2pin2 reagent, which only led to dimerization products, this work employs the unsymmetrical diboron reagent (pin)B-B(dan) under the palladium(0)-catalyzed conditions to access novel boronamides containing the monofluorinated vinyl-B(dan) functionality. These compounds can cross-couple directly with gem-difluoroalkenes without reactivation in Suzuki-Miyaura reactions to afford vincinal difluoro 1,3-dienes with modular control of the substituents.

Isotope-coded derivatization with designed Girard-type reagent as charged isobaric mass tags for non-targeted profiling and discovery of natural aldehydes by liquid chromatography-tandem mass spectrometry.

Aldehyde-containing metabolites are reactive electrophiles that have attracted extensive attention due to their widespread occurrence in organisms and natural foods. Herein we described a newly-designed Girard's reagent, 1-(4-hydrazinyl-4-oxobutyl)pyridin-1-ium bromide (HBP), as charged tandem mass (MS/MS) tags to facilitate selective capture, sensitive detection and semi-targeted discovery of aldehyde metabolites via hydrazone formation. After HBP labeling, the detection signals of the test aldehydes were increased by 21-2856 times, with the limits of detection were 2.5-7 nM. Upon isotope-coded derivatization with a pair of labeling reagents, HBP-d0 and its deuterium-labeled counterpart HBP-d5, the aldehyde analytes were converted to hydrazone derivatives, which generated characteristic neutral fragments of 79 Da and 84 Da, respectively. The isobaric HBP-d0/HBP-d5 labeling based LC-MS/MS method was validated by relative quantification of human urinary aldehydes (slope=0.999, R2 > 0.99, RSDs ≤ 8.5%) and discrimination analysis between diabetic and control samples. The unique isotopic doubles (Δm/z = 5 Da) by dual neutral loss scanning (dNLS) provided a generic reactivity-based screening strategy that allowed non-targeted profiling and identification of endogenous aldehydes even amidst noisy data. The LC-dNLS-MS/MS screening of cinnamon extracts led to finding 61 possible natural aldehydes and guided discovery of 10 previously undetected congeners in this medicinal plant.

IL16 Regulates Osteoarthritis Progression as a Target Gene of Novel-miR-81.

OBJECTIVE: Functional polymorphisms of interleukin 16 (IL16) have been reported to be closely related to the risk of osteoarthritis (OA). However, how IL16 affects OA remains unclear. In this study, the role of IL16 in OA and the possible mechanisms were examined. METHODS: We established a meniscal/ligament injury (MLI) post-traumatic OA model in Sprague Dawley rats and an IL1ß-induced ADTC5 cells OA model. We detected the expression of IL16, novel-miR-81, MMP3, and MMP13 by quantitative real-time polymerase chain reaction. Western blot was performed to detect the expression of IL16, MMP3, and MMP13. The association between IL16 and novel-miR-81 was confirmed by luciferase reporter assay. Hematoxylin and eosin staining, Safranin O and Fast Green staining, and immunohistochemical staining were performed to clarify the effect of intra-articular injection of novel-miR-81 agomir in rats OA model. RESULTS: IL16 was upregulated in OA model. Knockdown of IL16 and overexpression of novel-miR-81 downregulated the expression of MMP3 and MMP13. Importantly, IL16 was a key target of novel-miR-81. Intra-articular injection of novel-miR-81 agomir could attenuate OA progression in rats OA model. CONCLUSION: Novel-miR-81 targeted IL16 to relieve OA, suggesting that novel-miR-81and IL16 may be new therapeutic targets for OA.

Palladium-Catalyzed Stereoselective Defluorosilylation of gem-Difluoroalkenes for the Synthesis of Tetrasubstituted Monofluorinated Vinylsilanes.

Stereoselective synthesis of tetrasubstituted vinylsilanes is a challenging task. We herein report a novel palladium(0)-catalyzed defluorosilylation of ß,ß-difluoroacrylates to access tetrasubstituted vinylsilanes containing the monofluoroalkene motif in excellent diastereoselectivities (>99:1). This is our first example of C-heteroatom bond formation from the C-F bond under such a Pd catalytic manifold.

Copper-Catalyzed Visible-Light-Induced Allylic Difluoromethylation of Unactivated Alkenes Using Difluoroacetic Acid.

We herein describe a straightforward allylic difluoromethylation reaction of unactivated alkenes. Compared to cross-couplings of prefunctionalized allylic substrates for the construction of allylic CF2H bonds, this reaction employs readily available alkenes as substrates under mild conditions. Difluoroacetic acid is used as an inexpensive and easy-to-handle source of CF2H radical under visible light irradiation with PIDA. The copper catalyst plays an important role of diverting the reaction pathway toward allylic difluoromethylation as opposed to previously found hydrodifluoromethylation.

Prediction of microvascular invasion in hepatocellular carcinoma based on preoperative Gd-EOB-DTPA-enhanced MRI: Comparison of predictive performance among 2D, 2D-expansion and 3D deep learning models.

Purpose: To evaluate and compare the predictive performance of different deep learning models using gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced MRI in predicting microvascular invasion (MVI) in hepatocellular carcinoma. Methods: The data of 233 patients with pathologically confirmed hepatocellular carcinoma (HCC) treated at our hospital from June 2016 to June 2021 were retrospectively analyzed. Three deep learning models were constructed based on three different delineate methods of the region of interest (ROI) using the Darwin Scientific Research Platform (Beijing Yizhun Intelligent Technology Co., Ltd., China). Manual segmentation of ROI was performed on the T1-weighted axial Hepatobiliary phase images. According to the ratio of 7:3, the samples were divided into a training set (N=163) and a validation set (N=70). The receiver operating characteristic (ROC) curve was used to evaluate the predictive performance of three models, and their sensitivity, specificity and accuracy were assessed. Results: Among 233 HCC patients, 109 were pathologically MVI positive, including 91 men and 18 women, with an average age of 58.20 ± 10.17 years; 124 patients were MVI negative, including 93 men and 31 women, with an average age of 58.26 ± 10.20 years. Among three deep learning models, 2D-expansion-DL model and 3D-DL model showed relatively good performance, the AUC value were 0.70 (P=0.003) (95% CI 0.57-0.82) and 0.72 (P<0.001) (95% CI 0.60-0.84), respectively. In the 2D-expansion-DL model, the accuracy, sensitivity and specificity were 0.7143, 0.739 and 0.688. In the 3D-DL model, the accuracy, sensitivity and specificity were 0.6714, 0.800 and 0.575, respectively. Compared with the 3D-DL model (based on 3D-ResNet), the 2D-DL model is smaller in scale and runs faster. The frames per second (FPS) for the 2D-DL model is 244.7566, which is much larger than that of the 3D-DL model (73.3374). Conclusion: The deep learning model based on Gd-EOB-DTPA-enhanced MRI could preoperatively evaluate MVI in HCC. Considering that the predictive performance of 2D-expansion-DL model was almost the same as the 3D-DL model and the former was relatively easy to implement, we prefer the 2D-expansion-DL model in practical research.

Atractylodes lancea volatile oils target ADAR2-miR-181a-5p signaling to mesenchymal stem cell chondrogenic differentiation.

Atractylodeslancea Rhizoma (Rhizoma atractylodis [RA]) has long been recommended for the treatment of arthritis in traditional Chinese medicine, but its mechanism of action is still unclear. RA contains a large amount of Atractylodes lancea volatile oils (Atr). In this study, we investigated whether Atr can promote mesenchymal stem cells (MSCs) chondrogenic differentiation. The Atr were extracted from RA by steam distillation method, and the effect of Atr on MSCs was detected by the CCK8 assay. The optimal concentration of Atr for MSCs cultivation was 3 µg/ml. The differentially expressed miR-181a-5p was screened by miRNA microarray assay, and its mimics and inhibitors were transfected into MSCs. It was found that the inhibitor of miR-181a-5p could upregulate cartilage-specific genes such as SOX9, COL2A1, and ACAN. Meanwhile, we also found that the expression of gene editing enzyme ADAR2 was significantly increased in the chondrogenic differentiation of MSCs induced by Atr, and the bases of precursor sequence of miR-181a-5p were changed from A to G. After ADAR2 deletion, the expression of cartilage-specific genes was significantly down-regulated and the precursor sequence bases of miR-181a-5p were not changed. Bioinformatics analysis revealed that the predicted target gene of miR-181a-5p was yingyang1 (YY1), and the targeting relationship was verified by dual-luciferase reporter assay. After deleting YY1, the expression of cartilage-specific genes was significantly down-regulated. In conclusion, our study demonstrated that Atr can promote chondrogenic differentiation of MSC through regulation of the ADAR2-miR-181a-5p signaling pathway. This may provide a new insight into the possible mechanism of traditional Chinese medicine (Atr) in treating inflammatory joint diseases.

TIGER: A Web Portal of Tumor Immunotherapy Gene Expression Resource.

Immunotherapy is a promising cancer treatment method; however, only a few patients benefit from it. The development of new immunotherapy strategies and effective biomarkers of response and resistance is urgently needed. Recently, high-throughput bulk and single-cell gene expression profiling technologies have generated valuable resources. However, these resources are not well organized and systematic analysis is difficult. Here, we present TIGER, a tumor immunotherapy gene expression resource, which contains bulk transcriptome data of 1508 tumor samples with clinical immunotherapy outcomes and 11,057 tumor/normal samples without clinical immunotherapy outcomes, as well as single-cell transcriptome data of 2,116,945 immune cells from 655 samples. TIGER provides many useful modules for analyzing collected and user-provided data. Using the resource in TIGER, we identified a tumor-enriched subset of CD4+ T cells. Patients with melanoma with a higher signature score of this subset have a significantly better response and survival under immunotherapy. We believe that TIGER will be helpful in understanding anti-tumor immunity mechanisms and discovering effective biomarkers. TIGER is freely accessible at http://tiger.canceromics.org/.

Electroacupuncture of the Baihui and Shenting acupoints for vascular dementia in rats through the miR-81/IL-16/PSD-95 pathway.

Background: There is currently no effective treatment for vascular dementia (VaD). Scalp electroacupuncture (EA) has served clinically as an alternative treatment for VaD, but its mechanism is still unclear. In this study, we investigated the effect of EA at the Baihui (GV 20) and Shenting (GV 24) acupoints on spatial learning and memory ability, and the expression level of microRNA-81 (miR-81), interleukin-16 (IL-16), and postsynaptic density protein-95 (PSD-95) in the frontal cortex of VaD rats. Methods: Male Sprague-Dawley rats were randomly divided into four groups, sham, VaD, non-acupuncture (non-AP) and EA group. The VaD model was established by permanent bilateral occlusion of the common carotid arteries. Morris Water Maze was used to assess the rats' spatial learning and memory. Immunochemistry (IHC), quantitative reverse transcription polymerase chain reaction (qRT-PCR), and western blot analysis were performed to detect the expression level of miR-81, IL-16, and PSD-95. Finally, luciferase assay was used to determine the effect of miR-81 on IL-16 expression in PC12 cells. Results: The space exploration experiment of MWM showed the time and distance of the rat's activities around the platform were decreased in the EA group. Compared to the VaD and non-AP group, the number of terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL)-positive frontal cortical neurons was significantly decreased in EA group. The number of the PSD-95-positive cells and the miR-81 expression level in the frontal cortical in the EA group was dramatically increased in comparison with the other groups. In the PC12 cell validation experiment, IL-16 expression level was reduced under the condition of the miR-81 mimic treatment, while increased in the miR-81 inhibitor group. The PSD-95 protein level was up-regulated in the small interfering (si)RNA-IL16 group compared to the NC-IL16 groups with or without oxygen/glucose deprivation/reperfusion (OGD/R) conditions (P<0.05). However, this was abolished by miR-81 mimic. Conclusions: In VaD rats, EA may improve spatial learning and memory through miR-81/IL-16/PSD-95 pathway.

G-Protein Subunit Gamma 4 as a Potential Biomarker for Predicting the Response of Chemotherapy and Immunotherapy in Bladder Cancer.

BACKGROUND: GNG4, a member of the G-protein γ family, is a marker of poor overall survival (OS) rates in some malignancies. However, the potential role of GNG4 in bladder cancer (BLCA) is unknown. It is also unclear whether GNG4 may be utilized as a marker to guide chemotherapy or immunotherapy. METHODS: Single-cell RNA sequencing data were used to explore the expression of GNG4 in tumor microenvironment of BLCA. Bulk RNA sequencing data from TCGA were used to evaluate the relationship between GNG4 expression and biological features, such as immune cell infiltrations and gene mutations. The associations between GNG4 expression and survival in BLCA patients under or not under immunotherapy were evaluated using seven BLCA cohorts. RESULTS: GNG4 was specifically expressed in exhausted CD4+ T cells. And the high expression of the GNG4 was associated with high level of immune cell infiltration. The high-GNG4-expression group displayed a better response to immunotherapy, whereas patients in the low-GNG4-expression group often benefited from chemotherapy. Moreover, the high-GNG4 group was more similar to the basal group, whereas the low-GNG4 group was similar to the luminal group. CONCLUSIONS: GNG4 may be a potential biomarker for the prediction of the response to therapy in BLCA. Higher GNG4 expression can be used as a predictor of response to immunotherapy, and lower GNG4 expression can be used as a predictor of response to chemotherapy.

Dual-acting antitumor agents targeting the A2A adenosine receptor and histone deacetylases: Design and synthesis of 4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine derivatives.

Based on its inhibition by antagonists, the A2A adenosine receptor (A2AAR) has attracted attention as an anti-tumor drug target; however, in preclinical models and clinical trials, A2AAR antagonists have so far shown only limited efficacy as standalone therapies. The design of dual-acting compounds, targeting the A2AAR and histone deacetylases (HDACs), is used here as an approach to the discovery of novel and more potent antitumor agents. Based on the core structures of the A2AAR antagonists V-2006 and CPI-444, novel 4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine derivatives were designed as such dual-acting compounds. The binding affinities for A2AAR of all the new compounds were tested, and their HDAC inhibitory activity was evaluated. Compounds with balanced A2AAR antagonism and HDAC inhibition were tested for their in vitro anti-proliferative activity and pharmacokinetic properties. One of the compounds, 14c (4-(2-(6-Amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-N-(2-amino-phenyl)benzamide) showed an overall favorable pharmacokinetic profile; in the mouse MC38 xenograft model, it showed potent anti-tumor effects with inhibition rates of 44% (90 mg/kg, po, bid) and 85% (60 mg/kg, ip, bid), respectively.

MeRIPseqPipe: an integrated analysis pipeline for MeRIP-seq data based on Nextflow.

SUMMARY: MeRIPseqPipe is an integrated and automatic pipeline that can provide users a friendly solution to perform in-depth mining of MeRIP-seq data. It integrates many functional analysis modules, range from basic processing to downstream analysis. All the processes are embedded in Nextflow with Docker support, which ensures high reproducibility and scalability of the analysis. MeRIPseqPipe is particularly suitable for analyzing a large number of samples at once with a simple command. The final output directory is structured based on each step and tool. And visualization reports containing various tables and plots are provided as HTML files. AVAILABILITY AND IMPLEMENTATION: MeRIPseqPipe is freely available at https://github.com/canceromics/MeRIPseqPipe. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Soil chemical quality assessment and spatial distribution of pomelo orchards in acidic red soil hilly regions of China.

BACKGROUND: Soil quality assessment is a critical strategy for determining optimum fertilization in intensive pomelo production. In this study, we evaluated the soil quality status and mapped the spatial distribution of 347 soil samples collected from pomelo orchards in Pinghe County, southern China. We analyzed nine chemical parameters and an altitude indicator. RESULTS: The mean soil quality index (SQI) was 0.355 in the total data set (TDS) and 0.292 in the minimum data set (MDS). Available Ca (Avail-Ca), pH value, organic matter and altitude were selected as indicators of soil quality in the MDS. The SQI in mature orchards (>10 years) was higher than that in young orchards (<10 years), while no differences between soil types and altitude gradients were identified. We detected a significant positive correlation between the SQI based on TDS (SQITDS ) and the SQI based on MDS (SQIMDS ), and the spatial distribution of soil properties and SQITDS showed a uniform pattern, except for Avail-N, Avail-B and SQIMDS . Overall, unfavorable soil quality indicators, including rich in Avail-P, deficient in Avail-Ca, -Mg and -B, soil acidification and high altitude, were considered to be limiting factors for pomelo production. CONCLUSION: The soil chemical quality in pomelo orchards is generally low, indicating that integrated management by controlling acidification, reducing planting altitude, regulating fertilization and monitoring soil properties is required for sustainable pomelo production. © 2021 Society of Chemical Industry.