Deubiquitinase UCHL1 stabilizes KDM4B to augment VEGF signaling and confer bevacizumab resistance in clear cell renal cell carcinoma.

BACKGROUND: Bevacizumab resistance poses barriers to targeted therapy in clear cell renal cell carcinoma (ccRCC). Whether there exist epigenetic targets that modulate bevacizumab sensitivity in ccRCC remains indefinite. The focus of this study is to explore the role of UCHL1 in ccRCC. METHODS: Both in vitro and in vivo experiments were utilized to investigate the roles of UCHL1 in ccRCC. In vivo ubiquitination assays were performed to validate the posttranslational modification of KDM4B by UCHL1. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were utilized to explore KDM4B/VEGFA epigenetic regulations. RESULTS: UCHL1 was increased in ccRCC and associated with unfavorable survival outcomes in patients. UCHL1 was required for ccRCC growth and migration. Mechanistically, the wild-type UCHL1, but not C90A mutant, mediated the deubiquitination of KDM4B and thereby stabilized its proteins. KDM4B was up-regulated in ccRCC and potentiated cell growth. UCHL1 depended on KDM4B to augment ccRCC malignancies. Targeting UCHL1 suppressed tumor growth, colony formation, and migration abilities, which could be rescued by KDM4B. Furthermore, KDM4B was directly bound to the promoter region of VEGFA, abolishing repressive H3K9me3 modifications. KDM4B coordinated with HIF2α to activate VEGFA transcriptional levels. UCHL1-KDM4B axis governs VEGFA levels to sustain the angiogenesis phenotypes. Finally, a specific small-molecule inhibitor (6RK73) targeting UCHL1 remarkably inhibited ccRCC progression and further sensitized ccRCC to bevacizumab treatment. CONCLUSION: Overall, this study defined an epigenetic mechanism of UCHL1/KDM4B in activating VEGF signaling. The UCHL1-KDM4B axis represents a novel target for treating ccRCC and improving the efficacy of anti-angiogenesis therapy.

IGF2BP2-Shox2 axis regulates hippocampal-neuronal senescence to alleviate microgravity-induced recognition disturbance.

During space travel, microgravity leads to disturbances in cognitive function, while the underlying mechanism is still unclear. Simulated microgravity mice showed neuronal age-like changes in the hippocampus of our study. In the context of microgravity, we discovered m6A modification reshapes in the hippocampal region. When paired with RNA-seq and MeRIP-seq, Shox2 was found to be a powerful regulator in hippocampal neuron that respondes to microgravity. Decreased expression of senescence-associated secretory phenotype factors and improved genes related to synapses led to the restoration of memory function in the hippocampus upon increased expression of Shox2. Moreover, we discovered that IGF2BP2 was required for the m6A modification of the Shox2, and overexpressed IGF2BP2 in the hippocampus protected against both neuronal senescence and learning and memory decline caused by loss of gravity. Accordingly, our research identified the hippocampal IGF2BP2-Shox2 axis as a possible therapeutic approach to maintaining cognitive function during space travel.

Mitophagy Regulates the Circadian Rhythms by Degrading NR1D1 in Simulated Microgravity and Isolation Environments.

Long-term spaceflight is known to induce disruptions in circadian rhythms, which are driven by a central pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus, but the underlying molecular mechanisms remain unclear. Here, we developed a rat model that simulated microgravity and isolation environments through tail suspension and isolation (TSI). We found that the TSI environment imposed circadian disruptions to the core body temperature, heart rate, and locomotor-activity rhythms of rats, especially in the amplitude of these rhythms. In TSI model rats' SCNs, the core circadian gene NR1D1 showed higher protein but not mRNA levels along with decreased BMAL1 levels, which indicated that NR1D1 could be regulated through post-translational regulation. The autophagosome marker LC3 could directly bind to NR1D1 via the LC3-interacting region (LIR) motifs and induce the degradation of NR1D1 in a mitophagy-dependent manner. Defects in mitophagy led to the reversal of NR1D1 degradation, thereby suppressing the expression of BMAL1. Mitophagy deficiency and subsequent mitochondrial dysfunction were observed in the SCN of TSI models. Urolithin A (UA), a mitophagy activator, demonstrated an ability to enhance the amplitude of core body temperature, heart rate, and locomotor-activity rhythms by prompting mitophagy induction to degrade NR1D1. Cumulatively, our results demonstrate that mitophagy exerts circadian control by regulating NR1D1 degradation, revealing mitophagy as a potential target for long-term spaceflight as well as diseases with SCN circadian disruption.

Assessment of the changes in growth, photosynthetic traits and gene expression in Cynodon dactylon against drought stress.

Drought stress considered a key restrictive factor for a warm-season bermudagrass growth during summers in China. Genotypic variation against drought stress exists among bermudagrass (Cynodon sp.), but the selection of highly drought-tolerant germplasm is important for its growth in limited water regions and for future breeding. Our study aimed to investigate the most tolerant bermudagrass germplasm among thirteen, along latitude and longitudinal gradient under a well-watered and drought stress condition. Current study included high drought-resistant germplasm, "Tianshui" and "Linxiang", and drought-sensitive cultivars; "Zhengzhou" and "Cixian" under drought treatments along longitude and latitudinal gradients, respectively. Under water deficit conditions, the tolerant genotypes showed over-expression of a dehydrin gene cdDHN4, antioxidant genes Cu/ZnSOD and APX which leads to higher antioxidant activities to scavenge the excessive reactive oxygen species and minimizing the membrane damage. It helps in maintenance of cell membrane permeability and osmotic adjustment by producing organic osmolytes. Proline an osmolyte has the ability to keep osmotic water potential and water use efficiency high via stomatal conductance and maintain transpiration rate. It leads to optimum CO2 assimilation rate, high chlorophyll contents for photosynthesis and elongation of leaf mesophyll, palisade and thick spongy cells. Consequently, it results in elongation of leaf length, stolon and internode length; plant height and deep rooting system. The CdDHN4 gene highly expressed in "Tianshui" and "Youxian", Cu/ZnSOD gene in "Tianshui" and "Linxiang" and APX gene in "Shanxian" and "Linxiang". The genotypes "Zhongshan" and "Xiaochang" showed no gene expression under water deficit conditions. Our results indicate that turfgrass show morphological modifications firstly when subjected to drought stress; however the gene expression is directly associated and crucial for drought tolerance in bermudagrass. Hence, current research has provided excellent germplasm of drought tolerant bermudagrass for physiological and molecular study and future breeding.

Inhibition of SUV39H1 reduces tumor angiogenesis via Notch1 in oral squamous cell carcinoma.

Targeting tumor angiogenesis is an important approach in advanced tumor therapy. Here we investigated the effect of the suppressor of variegation 3-9 homolog 1 (SUV39H1) on tumor angiogenesis in oral squamous cell carcinoma (OSCC). The GEPIA database was used to analyze the expression of SUV39H1 in various cancer tissues. The expression of SUV39H1 in OSCC was detected by immunohistochemistry, and the correlation between SUV39H1 and Notch1 and microvascular density (MVD) was analyzed. The effect of SUV39H1 inhibition on OSCC was investigated in vivo by chaetocin treatment. The migration and tube formation of vascular endothelial cells by conditioned culture-medium of different treatments of oral squamous cell cells were measured. The transcriptional level of SUV39H1 is elevated in various cancer tissues. The transcription level of SUV39H1 in head and neck squamous cell carcinoma was significantly higher than that in control. Immunohistochemistry result showed increased SUV39H1 expression in OSCC, which was significantly correlated with T staging. The expression of SUV39H1 was significantly correlated with Notch1 and CD31. In vivo experiment chaetocin treatment significantly inhibit the growth of tumor, and reduce SUV39H1, Notch1, CD31 expression. The decreased expression of SUV39H1 in OSCC cells lead to the decreased expression of Notch1 and VEGF proteins, as well as the decreased migration and tube formation ability of vascular endothelial cells. Inhibition of Notch1 further enhance this effect. Our results suggest inhibition of SUV39H1 may affect angiogenesis by regulating Notch1 expression. This study provides a foundation for SUV39H1 as a potential therapeutic target for OSCC.

Immunotherapy of M2 macrophage derived from exosome-based nanoparticles for spinal cord injury.

Developing biomimetic nanoparticles without off-target side-effects remains a major challenge in spinal cord injury (SCI) immunotherapy. In this paper, we have conducted a drug carrier which is biocompatible macrophages-exocytosed exosome-biomimetic manganese (Mn)-iron prussian blue analogues (MPBs) for SCI immunotherapy. Exosome-sheathed MPBs (E-MPBs) exhibit promoted microglia accumulation, alleviation from H2O2-induced microenvironment and inhibition of apoptosis and inflammation in vitro. In addition, E-MPBs possessed significant tissue repair and neuroprotection in vivo. These properties endowed E-MPBs with great improvement in vivo in function recovery, resulting in anti-neuroinflammation activity and excellent biocompatibility in mice SCI model. As a promising treatment for efficient SCI immunotherapy, these results demonstrate the use of exosome-sheathed biomimetic nanoparticles exocytosed by anti-inflammation cells is feasible.

Methionine secreted by tumor-associated pericytes supports cancer stem cells in clear cell renal carcinoma.

Here, we identify a subset of vascular pericytes, defined by expression of platelet-derived growth factor receptor beta (PDGFR-ß) and G-protein-coupled receptor 91 (GPR91), that promote tumorigenesis and tyrosine kinase inhibitors (TKIs) resistance by functioning as the primary methionine source for cancer stem cells (CSCs) in clear cell renal cell carcinoma (ccRCC). Tumor-cell-derived succinate binds to GPR91 on pericyte to activate autophagy for methionine production. CSCs use methionine to create stabilizing N6-methyladenosine in ATPase-family-AAA-domain-containing 2 (ATAD2) mRNA, and the resulting ATAD2 protein complexes with SRY-box transcription factor 9 to assemble super enhancers and thereby dictate its target genes that feature prominently in CSCs. Targeting PDGFR-ß+GPR91+ pericytes with specific GRP91 antagonists reduce intratumoral methionine level, eliminate CSCs, and enhance TKIs sensitivity. These results unraveled the mechanisms by which PDGFR-ß+GPR91+ pericytes provide supportive niche for CSCs and could be used to develop targets for treating ccRCC.

A Targeted Exosome Therapeutic Confers Both CfDNA Scavenging and Macrophage Polarization for Ameliorating Rheumatoid Arthritis.

Only a minority of rheumatoid arthritis (RA) patients achieve disease remission, so the exploration of additional pathogenic factors and the development of new therapeutics are needed. Here, strong correlations among the cell-free DNA (cfDNA) level and the inflammatory response in clinical synovial fluid samples and RA disease activity are discovered. The important role of cfDNA in disease development in a collagen-induced arthritis (CIA) murine model is also demonstrated. Building on these findings, a novel therapeutic based on anti-inflammatory (M2) macrophage-derived exosomes as chassis, that are modified with both oligolysine and matrix metalloproteinase (MMP)-cleavable polyethylene glycol (PEG) on the membrane, is developed. After intravenous injection, PEG-enabled prolonged circulation and C─C motif chemokine ligand-directed accumulation together result in enrichment at inflamed joints. Following subsequent MMP cleavage, the positively charged oligolysine is exposed for cfDNA scavenging, while exosomes induce M2 polarization. By using a classical CIA murine model and a newly established CIA canine model, it is demonstrated that the rationally designed exosome therapeutic substantially suppresses inflammation in joints and provides strong chondroprotection and osteoprotection, revealing its potential for effective CIA amelioration.

Curcumin-Loaded Macrophage-Derived Exosomes Effectively Improve Wound Healing.

This study aims to investigate the potential therapeutic effect of exosomes derived from macrophages loaded with curcumin (Exos-cur) on the healing of diabetic wounds. As a new type of biomaterial, Exos-cur has better stability, anti-inflammation, and antioxidation biological activity. In in vitro experiments, Exos-cur can promote the proliferation, migration, and angiogenesis of HUVECs (human umbilical vein endothelial cells) while reducing the ROS (reactive oxygen species) produced by HUVECs induced by high glucose, regulating the mitochondrial membrane potential, reducing cell oxidative damage, and inhibiting oxidative stress and inflammation. In the in vivo experiment, the Exos-cur treatment group had an increased percentage of wound closure and contraction compared with the diabetic wound control group. Hematoxylin-eosin staining (HE) and Masson staining showed that the Exos-cur treatment group had more advanced re-epithelialization, and the generated mature granulation tissue was rich in a large number of capillaries and newly deposited collagen fibers. Western blot and immunofluorescence analyses showed that Exos-cur can inhibit inflammation by activating the Nrf2/ARE pathway, upregulate the expression of wound healing-related molecules, promote angiogenesis, and accelerate wound healing in diabetic rats. These results show that Exos-cur has a good therapeutic effect on diabetic skin defects and provide experimental evidence for the potential clinical benefits of Exos-cur.

Impact of genetic polymorphisms on tacrolimus trough blood concentration in Chinese liver transplant recipients.

Purpose: The aim of this study was to analyze the effects of various genetic polymorphisms and clinical factors on tacrolimus (TAC) concentration in the convalescence period (CP) and stabilization period (SP) post-liver transplantation. Patients & methods: A total of 13 SNPs were genotyped in 97 Chinese liver transplant recipients. Associations between SNPs and TAC trough blood concentration/dose ratio (C0/D) were analyzed using different genetic models in both CP and SP. Results: Only five SNPs were significantly associated with TAC log (C0/D) in the CP, and none showed a significant association in the SP. We identified rs15524 (CYP3A5), rs9200 (C6), albumin and creatinine as independent predictors of TAC C0/D in the CP. Furthermore, a final model in the CP explained a total of 30.5% TAC variation. Conclusion: Our study results suggest that in the early stages post-transplantation surgery, recipients' genetic and clinical factors exert a short-term impact on TAC metabolism that gradually decreases with time.

Screening and identification of multiple abiotic stress responsive candidate genes based on hybrid-sequencing in Vicia sativa.

Common vetch is an important leguminous forage for both livestock fodder and green manure and has a tremendous latent capacity in a sustainable agroecosystem. In the present study, a comprehensive transcriptome analysis of the aboveground leaves and underground roots of common vetch under multiple abiotic stress treatments, including NaCl, drought, cold, and cold drought, was performed using hybrid-sequencing technology, i. e. single-molecule real-time sequencing technology (SMRT) and supplemented by next-generation sequencing (NGS) technology. A total of 485,038 reads of insert (ROIs) with a mean length of 2606 bp and 228,261 full-length nonchimeric (FLNC) reads were generated. After deduplication, 39,709 transcripts were generated. Of these transcripts, we identified 1059 alternative splicing (AS) events, 17,227 simple sequence repeats (SSRs), and 1647 putative transcription factors (TFs). Furthermore, 640 candidates long noncoding RNAs (lncRNAs) and 28,256 complete coding sequences (CDSs) were identified. In gene annotation analyses, a total of 38,826 transcripts (97.78%) were annotated in eight public databases. Finally, seven multiple abiotic stress-responsive candidate genes were obtained through gene expression, annotation information, and protein-protein interaction (PPI) networks. Our research not only enriched the structural information of FL transcripts in common vetch, but also provided useful information for exploring the molecular mechanism of multiple abiotic stress tolerance between aboveground and underground tissues in common vetch and related legumes.

LysM protein BdLM1 of Botryosphaeria dothidea plays an important role in full virulence and inhibits plant immunity by binding chitin and protecting hyphae from hydrolysis.

Botryosphaeria dothidea infects hundreds of woody plants and causes a severe economic loss to apple production. In this study, we characterized BdLM1, a protein from B. dothidea that contains one LysM domain. BdLM1 expression was dramatically induced at 6 h post-inoculation in wounded apple fruit, strongly increased at 7 d post-inoculation (dpi), and peaked at 20 dpi in intact shoots. The knockout mutants of BdLM1 had significantly reduced virulence on intact apple shoots (20%), wounded apple shoots (40%), and wounded apple fruit (40%). BdLM1 suppressed programmed cell death caused by the mouse protein BAX through Agrobacterium-mediated transient expression in Nicotiana benthamiana, reduced H2O2 accumulation and callose deposition, downregulated resistance gene expression, and promoted Phytophthora nicotianae infection in N. benthamiana. Moreover, BdLM1 inhibited the active oxygen burst induced by chitin and flg22, bound chitin, and protected fungal hyphae against degradation by hydrolytic enzymes. Taken together, our results indicate that BdLM1 is an essential LysM effector required for the full virulence of B. dothidea and that it inhibits plant immunity. Moreover, BdLM1 could inhibit chitin-triggered plant immunity through a dual role, i.e., binding chitin and protecting fungal hyphae against chitinase hydrolysis.

Exploration of the cancer genome atlas database reveals genes of interest related with cancer cell stemness indices in clear cell renal cell carcinoma.

Heterogeneity of kidney cancer poses great challenges in clinical management. Practicians are still in need of an effective way to identify high-risk patients. Here we browsed big data from The Cancer Genome Atlas database with reference to cancer cell stemness and identified genes of interest in clear cell renal cell carcinoma. We further analyzed these genes to uncover their role in cancer promotion and progression and presented an interaction network. The results highlighted the NOTCH signaling pathway and functions related with epithelial cell migration. Finally, we managed to construct a predictive model consisting of a reasonable number of genes that successfully recognized patients at higher risk, rendering these genes suitable as subjects in future research.

Coxsackievirus A6 was the most common enterovirus serotype causing hand, foot, and mouth disease in Shiyan City, central China.

BACKGROUND: Hand, foot, and mouth disease (HFMD) has become one of the most common infectious diseases in China. Before 2016, the primary causal serotypes were enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16). Following the introduction of EV-A71 vaccines in China since 2016, the situation could change. CV-A6 has recently replaced EV-A71 and CV-A16 in some areas of China. However, the epidemiological characteristics of central China remain unknown. AIM: To investigate the clinical symptoms and pathogen spectrum of HFMD in Shiyan City, central China, in recent years. METHODS: The epidemiological, clinical, and laboratory data from HFMD cases reported to the Shiyan Center for Disease Control and Prevention between January 2016 and December 2020 were analyzed. 196 throat swab specimens were collected from hospitalized HFMD patients between January 2018 and December 2020. To detect and genotype enteroviruses, real-time reverse transcription-polymerase chain reaction and sequencing of the 5'-untranslated region were used. In Shiyan, 168 laboratory-confirmed HFMD cases were studied using a logistic regression model to determine the effect of predominant enterovirus serotypes. Based on the logistic regression model, the least absolute shrinkage and selection operator model was used to analyze the correlation between CV-A6 infection and various clinical characteristics in HFMD patients in Shiyan. RESULTS: From 2016 to 2020, 35840 HFMD cases were reported in Shiyan. The number of cases decreased by 48.4% from 2016 to 2017. Approximately 1.58-fold increases were found in 2018 and 2019 when compared to the previous year, respectively. In 2020, a decrease of about 85.5% was reported when compared to 2019. The most common serotypes shifted from EV-A71 and CV-A16 (about 60%-80% in 2016 and 2018) to others (more than 80.0% in 2017, 2019, and 2020). EV-A71 lost its dominance in 2017 in Shiyan. Among 196 confirmed HFMD cases, 85.7% tested positive for enterovirus, with CV-A6 being the most common serotype (121/168, 72.0%). The positive rates for CV-A16 and CV-A10 were 4.8% and 3.0%, respectively. There was no EV-A71 discovered. Infection with CV-A6 was linked to fever, myocardial damage, increased creatine kinase MB isoenzyme, and lactate dehydrogenase levels. CONCLUSION: CV-A6 was the most common enterovirus serotype in Shiyan City, replacing EV-A71 and CV-A16 as the HFMD pathogen. Developing vaccines against CV-A6 or multiple pathogens, as well as rising CV-A6 surveillance, will help prevent HFMD in central China.

Agronomic evaluation of shade tolerance of 16 spring Camelina sativa (L.) Crantz genotypes under different artificial shade levels using a modified membership function.

Camelina [Camelina sativa (L.) Crantz] is currently gaining considerable attention as a potential oilseed feedstock for biofuel, oil and feed source, and bioproducts. Studies have shown the potential of using camelina in an intercropping system. However, there are no camelina genotypes evaluated or bred for shade tolerance. The objective of this study was to evaluate and determine the shade tolerance of sixteen spring camelina genotypes (growth stage: BBCH 103; the plants with 4-5 leaves) for intercropping systems. In this study, we simulated three different shade levels, including low (LST), medium (MST), and high shade treatments (HST; 15, 25, and 50% reduction of natural light intensity, respectively), and evaluated the photosynthetic and physiological parameters, seed production, and seed quality. The mean chlorophyll pigments, including the total chlorophyll and chlorophyll a and b across the 16 genotypes increased as shade level increased, while the chlorophyll fluorescence parameter Fv/Fm, chlorophyll a/b, leaf area, the number of silicles and branches plant-1 decreased as shade level increased. The first day of anthesis and days of flowering duration of camelina treated with shade were significantly delayed and shortened, respectively, as shade increased. The shortened lifecycle and altered flowering phenology decreased camelina seed yield. Additionally, the shade under MST and HST reduced the seed oil content and unsaturated fatty acids, but not saturated fatty acids. The dendrograms constructed using the comprehensive tolerance membership values revealed that CamK9, CamC4, and 'SO-40' were the relatively shade-tolerant genotypes among the 16 camelina genotypes. These camelina genotypes can grow under the shade level up to a 25% reduction in natural light intensity producing a similar seed yield and seed oil quality, indicating the potential to intercrop with maize or other small grain crops. The present study provided the baseline information on the response of camelina genotypes to different shade levels, which would help in selecting or breeding shade-tolerant genotypes.

Phylogeographical Pattern and Population Evolution History of Indigenous Elymus sibiricus L. on Qinghai-Tibetan Plateau.

Elymus sibiricus L. is a perennial allotetraploid belonging to Triticeae of Poaceae, Elymus L., as the type species of genus Elymus L. The existing geographical distribution pattern and genetic spatial structure of E. sibiricus on Qinghai-Tibetan Plateau (QTP) are not yet clear. In this study, population genetic structure and demography history of 216 individuals from 44 E. sibiricus populations on QTP were studied used specific-locus amplified fragment sequencing (SLAF-seq). The result of genetic diversity showed that there was no single genetic diversity center was observed across all E. sibiricus populations. The results of genetic variation showed that 44 populations were clearly divided into the following three groups: Qinghai Plateau (Group I), South Tibet (Group II), and Hengduan Mountains (Group III). From the three analyses of AMOVA, Mantel test and Treemix, strong genetic differentiation across all populations and low genetic differentiation among populations within three groups. Molecular dating indicated that E. sibiricus diverged at 16.08 Ma (during the early Miocene) can be linked to the Himalayan Motion stage of QTP uplift. It is speculated that the reasons affecting the current phylogeographical pattern are as follows: (1) The environmental changes due to the uplift of the QTP; (2) The geographic distance between the populations (Groups I and III are close in geographic distance, and gene flow are frequent); (3) Geographical barriers (the Tanggula and Bayangela Mountains between Groups I and II). This study provides new evidence and historical perspective to the future exploration of the evolution and geographic distribution pattern of Elymus L.

Comprehensive Genomic Characterization of Tumor Microenvironment and Relevant Signature in Clear Cell Renal Cell Carcinoma.

Purpose: To systematically investigate the characterization of tumor microenvironment (TME) in clear cell renal cell carcinoma (ccRCC), we performed a comprehensive analysis incorporating genomic alterations, cellular interactions, infiltrating immune cells, and risk signature. Patients and Methods: Multi-omics data including RNA-seq, single-nucleotide variant (SNV) data, copy number variation (CNV) data, miRNA, and corresponding prognostic data were obtained from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) database. The CIBERSORT algorithm was utilized to identify prognostic TME subclusters, and TMEscore was further quantified. Moreover, the mutational landscape of TCGA-KIRC was explored. Lastly, TIDE resource was applied to assess the significance of TMEscore in predicting immunotherapeutic benefits. Results: We analyzed the TME infiltration patterns from 621 ccRCC patients and identified 5 specific TME subclusters associated with clinical outcomes. Then, we found that TMEcluster5 was significantly related to favorable prognosis and enriched memory B-cell infiltration. Accordingly, we depicted the clustering landscape of TMEclusters, TMEscore levels, tumor mutation burden (TMB), tumor grades, purity, and ploidy in all patients. Lastly, TIDE was used to assess the efficiency of immune checkpoint blockers (ICBs) and found that the TMEscore has superior predictive significance to TMB, making it an essential independent prognostic biomarker and drug indicator for clinical use. Conclusions: Our study depicted the clustering landscape of TMEclusters, TMEscore levels, TMB, tumor grades, purity, and ploidy in total ccRCC patients. The TMEscore was proved to have promising significance for predicting prognosis and ICB responses, in accordance with the goal of developing rationally individualized therapeutic interventions.

Preparation of Highly Substituted Sulfated Alfalfa Polysaccharides and Evaluation of Their Biological Activity.

Alfalfa polysaccharides (AP) receive wide attention in the field of medicine, because of their anti-inflammatory property. However, AP has high molecular weight and poor water solubility, resulting in low biological activity. We wanted to obtain highly bioactive alfalfa polysaccharides for further research. Herein, we successfully synthesized highly substituted sulfated alfalfa polysaccharides (SAP) via the chlorosulfonic acid (CSA)-pyridine (Pyr) method, which was optimized using response surface methodology (RSM). Under the best reaction conditions, that is, the reaction temperature, time, and ratio of CSA to Pyr being 55 °C, 2.25 h, and 1.5:1, respectively, the maximum degree of substitution of SAP can reach up to 0.724. Fourier transform infrared spectroscopy also confirmed the existence of sulfonic acid groups on SAP. Despite the increased average molecular weight of SAP, its water solubility is improved, which is beneficial for its biological activity. Further in vitro results showed that SAP exhibited better antioxidant activity and antibacterial ability than AP. Besides, the former can efficiently enhance the viability of oxidatively stressed intestinal epithelial cells compared with the latter. Furthermore, SAP has the potential to inhibit obesity. It is concluded that sulfation modification could improve the antioxidant, antibacterial, bovine intestinal epithelial cells' proliferation-promoting, and the obesity inhibition abilities of AP. The improvement of AP biological activity may provide references for the utilization of plant extracts that have weaker biological activity.

Acid-Responsive Dissociation of Ferrocene Compounds: Diels-Alder Diene Equivalents for Selective Preparation of [60]Fullerene-Fused Bicyclo[2.2.1]hept-5-enes.

Herein, an unprecedented transformation of [60]fullerene with ferrocene compounds giving access to various [60]fullerene-fused bicyclo[2.2.1]hept-5-enes is presented. In the presence of an acid, ferrocene compounds undergo an unusual dissociation process and serve as Diels-Alder diene equivalents to react with [60]fullerene for the selective construction of a class of novel fullerene-fused bridged carbocyclic derivatives. The reaction is easy to perform and has a wide substrate scope as well as excellent functional group compatibility, representing a new application of ferrocene compounds in synthetic chemistry.