Apigenin suppresses epithelial-mesenchymal transition in high glucose-induced retinal pigment epithelial cell by inhibiting CBP/p300-mediated histone acetylation.

Epithelial mesenchymal transition (EMT) is a critical process implicated in the pathogenesis of retinal fibrosis and the exacerbation of diabetic retinopathy (DR) within retinal pigment epithelium (RPE) cells. Apigenin (AP), a potential dietary supplement for managing diabetes and its associated complications, has demonstrated inhibitory effects on EMT in various diseases. However, the specific impact and underlying mechanisms of AP on EMT in RPE cells remain poorly understood. In this study, we have successfully validated the inhibitory effects of AP on high glucose-induced EMT in ARPE-19 cells and diabetic db/db mice. Notably, our findings have identified CBP/p300 as a potential therapeutic target for EMT in RPE cells and have further substantiated that AP effectively downregulates the expression of EMT-related genes by attenuating the activity of CBP/p300, consequently reducing histone acetylation alterations within the promoter region of these genes. Taken together, our results provide novel evidence supporting the inhibitory effect of AP on EMT in RPE cells, and highlight the potential of specifically targeting CBP/p300 as a strategy for inhibiting retinal fibrosis in the context of DR.

Establishment of a chip digital PCR detection method for canine circovirus.

Canine circovirus (CanineCV), which is a new mammalian circovirus first reported in the United States in 2012, mainly causes diarrhea and vomiting in dogs. As CanineCV evolves and new subtypes emerge, there is an urgent need for new detection technologies to improve the sensitivity and detection rates of viruses in complex scenarios. A chip digital PCR(cdPCR) assay was established for the detection of CanineCV in this study. The results showed good reproducibility, specificity and a linear relationship; the minimum detection limit of CanineCV by cdPCR was 6.62 copies/µL, which is 10 times more sensitive than quantitative real-time PCR (qPCR). The qPCR-positive detection rate was 1 %, while CanineCV cdPCR (2.1 %) exhibited a greater positive detection rate. Fifteen complete genomes were sequenced and subdivided into CanineCV-1 and CanineCV-3. In conclusion, we developed a rapid, reliable, and specific cdPCR method for screening and monitoring canine CV.

Nitro-oleic acid (NO2-OA) ameliorates erectile dysfunction in a rat model of diabetes mellitus via modulation of fibrosis, inflammation and autophagy.

Background: Inflammation, fibrosis and autophagy represent closely related factors associated with the pathogenesis of diabetes mellitus erectile dysfunction (DMED). In this study, the therapeutic effect of nitro-oleic acid (NO2-OA) in a streptozotocin-induced rat model of DMED was evaluated. Methods: Sixty rats were randomly divided into four groups: control, DMED, DMED + Vehicle and DMED + NO2-OA. DMED was induced by intraperitoneal injection of streptozotocin in male rats. Blood glucose and body weight were measured every 2 weeks. After 4 weeks of NO2-OA treatment, erectile function was measured by electrical stimulation of cavernous nerve (CN). Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), immunofluorescence and Masson's trichrome staining were used to verify the related factors and protein expression levels. Results: We found that NO2-OA could significantly increase erectile pressure in the corpus cavernosum of DMED rats. Results of western blot, confocal immunofluorescence and qRT-PCR assays revealed that NO2-OA significantly reduced inflammatory factors and the expression of nuclear factor kappa B (NF-κB). In addition, Masson staining results indicated that NO2-OA significantly reduced the display of fibrotic tissue in the corpus cavernosum. These beneficial effects may be related to reductions in the expression of transforming growth factor-ß1 (TGF-ß1) and connective tissue growth factor (CTGF) and the increase in the expression of α-smooth muscle actin (α-SMA). Finally, NO2-OA treatment increased the expression of the autophagy marker, LC3, while P62 was decreased, effects suggesting that one of the underlying mechanisms of NO2-OA may involve an activation of the PI3K/AKT/mTOR pathway to enhance the capacity for autophagy within this tissue. Conclusions: NO2-OA enhances erectile function within a rat model of DMED by inhibiting inflammation and fibrosis along with activating autophagy.

Angiocrine signaling in sinusoidal homeostasis and liver diseases.

The hepatic sinusoids are composed of liver sinusoidal endothelial cells (LSEC), which are surrounded by hepatic stellate cells (HSC) and contain liver-resident macrophages called Kupffer cells, and other patrolling immune cells. All these cells communicate with each other and with hepatocytes to maintain sinusoidal homeostasis and a spectrum of hepatic functions under healthy conditions. Sinusoidal homeostasis is disrupted by metabolites, toxins, viruses, and other pathological factors, leading to liver injury, chronic liver diseases, and liver cirrhosis. Alterations in hepatic sinusoids are linked to fibrosis progression and portal hypertension. LSECs are crucial regulators of cellular crosstalk within their microenvironment via angiocrine signaling. This review discusses the mechanisms by which angiocrine signaling orchestrates sinusoidal homeostasis, as well as the development of liver diseases. Here, we summarize the crosstalk between LSECs, HSCs, hepatocytes, cholangiocytes, and immune cells in health and disease and comment on potential novel therapeutic methods for treating liver diseases.

Predicting prognosis in intrahepatic cholangiocarcinoma by the histopathological features.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a highly heterogeneous liver tumor. The associations between histopathological feature and prognosis of ICC are limited. The present study aimed to investigate the prognostic significance of glandular structure and tumor budding in ICC. METHODS: Patients received radical hepatectomy for ICC were included. Glandular structure and tumor budding were detected by Hematoxylin-eosin staining. The Kaplan-Meier method and the Cox proportional hazards regression model were used to calculate the survival and hazard ratio. Based on the results of multivariate analysis, nomograms of OS and DFS were constructed. C-index and Akaike information criterion (AIC) were used to assess accuracy of models. RESULTS: A total of 323 ICC patients who underwent surgery were included in our study. Glandular structure was associated with worse overall survival (OS) [hazard ratio (HR): 2.033, 95% confidence interval (CI): 1.047 to 3.945] and disease-free survival (DFS) [HR: 1.854, 95% CI: 1.082 to 3.176]. High tumor budding was associated with worse DFS [HR: 1.636, 95%CI: 1.060 to 2.525]. Multivariate analysis suggested that glandular structure, tumor number, lymph node metastasis, and CA19-9 were independent risk factors for OS. Independent predictor factors for DFS were tumor budding, glandular structure, tumor number, and lymph node metastasis. The c-index (0.641 and 0.642) and AIC (957.69 and 1188.52) showed that nomograms of OS and DFS have good accuracy. CONCLUSION: High tumor budding and glandular structure are two important histopathological features that serve as prognostic factors for ICC patients undergoing hepatectomy.

Fabrication of soy protein nanoparticles based on metal-phenolic networks for stabilization of nano-emulsions delivery system.

The use of soy protein isolate (SPI) nanoparticles as a stabilizer in nano-emulsion systems has garnered significant interest. While metal-phenolic networks (MPNs) have been explored for their multifunctional surface modification capabilities, their integration with food protein-based delivery systems remains less explored. In this study, we attempt to develop a novel strategy to encapsulate cinnamaldehyde using MPNs (EGCG-Fe3+) with self-assembling soy protein nanoparticles (SE-Fe NPs) as a stabilizer for nano-emulsions. UV, Raman, and X-ray photoelectron spectroscopy analyses demonstrated that SE-Fe NPs were generated through metal-phenolic coordination and covalent interactions. SE-Fe NPs had a narrower particle size distribution and enhanced radical scavenging (up to 3.35-fold), as well as thermal stability. Furthermore, the smaller droplet size, higher modulus, higher cinnamaldehyde encapsulation efficiency (from 63.5% to 84.2%), and improved bio-accessibility of SE-Fe NPs stabilized nano-emulsions delivery system demonstrated in this study shows promising future applications in the food industry.

Analytical approaches for assessing protein structure in protein-rich food: A comprehensive review.

This review focuses on changes in nutrition and functional properties of protein-rich foods, primarily attributed to alterations in protein structures. We provide a comprehensive overview and comparison of commonly used laboratory methods for protein structure identification, aiming to offer readers a convenient understanding of these techniques. The review covers a range of detection technologies employed in food protein analysis and conducts an extensive comparison to identify the most suitable method for various proteins. While these techniques offer distinct advantages for protein structure determination, the inherent complexity of food matrices presents ongoing challenges. Further research is necessary to develop and enhance more robust detection methods to improve accuracy in protein conformation and structure analysis.

"Qi Nan" agarwood restores podocyte autophagy in diabetic kidney disease by targeting EGFR signaling pathway.

BACKGROUND: Diabetic kidney disease (DKD) is a microvascular complication of diabetes mellitus, contributing to end-stage renal disease with limited treatment options. The development of DKD is attributed to podocyte injury resulting from abnormal podocyte autophagy. Consequently, the restoration of podocyte autophagy is deemed a practicable approach in the treatment of DKD. METHODS: Diabetic mice were induced by streptozotocin and high-fat diet feeding. Following 8 weeks of "QN" agarwood treatment, metrics such as albuminuria, serum creatinine (Scr), and blood urea nitrogen (BUN) were evaluated. Renal histological lesions were evaluated by H&E, PAS, Masson, and Sirius red staining. Evaluation of the effects of "QN" agarwood on renal inflammation and fibrosis in DKD mice through WB, q-PCR, and IHC staining analysis. Cytoscape 3.7.1 was used to construct a PPI network. With the DAVID server, the gene ontology (GO) functional annotation and the Kyoto encyclopedia of genes and genomes (KEGG) signaling pathways of the target enrichment were performed. Molecular docking and binding affinity calculations were conducted using AutoDock, while PyMOL software was employed for visualizing the docking results of active compounds and protein targets. RESULTS: The results of this study show that "QN" agarwood reduced albuminuria, Scr, and BUN in DKD mice, and improved the renal pathological process. Additionally, "QN" agarwood was observed to downregulate the mRNA and protein expression levels of pro-inflammatory and pro-fibrotic factors in the kidneys of DKD mice. Network pharmacology predicts that "QN" agarwood modulates the epidermal growth factor receptor (EGFR) signaling pathway. "QN" agarwood can increase the expression of LC3B and Nphs1 in DKD mice while reducing the expression of EGFR. CONCLUSION: The present study demonstrated that "QN" agarwood ameliorated renal injury in DKD by targeting EGFR and restoring podocyte autophagy.

Research progress on the regulatory mechanisms of Irisin on cognitive dysfunction in patients with Alzheimer's disease and the interventional role of Irisin in associated diseases.

Irisin, a peptide produced during exercise, is believed to play a role in regulating energy levels within the body. Moreover, Irisin has the ability to traverse the blood-brain barrier and engage in various pathophysiological processes within the central nervous system. An increasing body of research identifies Irisin as a significant therapeutic target for neurodegenerative diseases, indicating a strong link between Irisin and the development of cognitive impairments. In this paper, we present a concise review of effects of different types of exercise on Irisin production, and the mechanisms underlying the Irisin's intervention in various diseases including metabolic diseases, kidney injury and depression. Following this, we delve into an in-depth exploration of its role in modulating cognitive dysfunction among patients with Alzheimer's disease (AD), focusing on recent advancements in three critical areas: neuroinflammation, mitochondrial dysfunction, and protein misfolding. Finally, we put forth 3 hypotheses: (1) exercise-induced fibronectin type III domain containing protein 5 (FNDC5) stimulation and subsequent Irisin cleavage may be associated with the stress response in energy metabolism; (2) Irisin, as a myokine, likely plays a role in mitochondrial repair mechanisms to ameliorate cognitive impairment in AD patients; (3) Irisin is a homeostatic factor that maintains energy homeostasis and is closely related to the dynamic stability of the body's internal environment.

A Systematic Review of Statins for the Treatment of Nonalcoholic Steatohepatitis: Safety, Efficacy, and Mechanism of Action.

Nonalcoholic fatty liver disease (NAFLD) is the liver component of a cluster of conditions, while its subtype, nonalcoholic steatohepatitis (NASH), emerges as a potentially progressive liver disorder that harbors the risk of evolving into cirrhosis and culminating in hepatocellular carcinoma (HCC). NASH and cardiovascular disease (CVD) have common risk factors, but compared to liver-related causes, the most common cause of death in NASH patients is CVD. Within the pharmacological armamentarium, statins, celebrated for their lipid-modulating prowess, have now garnered attention for their expansive therapeutic potential in NASH. Evidence from a plethora of studies suggests that statins not only manifest anti-inflammatory and antifibrotic properties but also impart a multifaceted beneficial impact on hepatic health. In this review, we used "statin", "NAFLD", "NASH", and "CVD" as the major keywords and conducted a literature search using the PubMed and Web of Science databases to determine the safety and efficacy of statins in patients and animals with NASH and NAFLD, and the mechanism of statin therapy for NASH. Simultaneously, we reviewed the important role of the intestinal microbiota in statin therapy for NASH, as it is hoped that statins will provide new insights into modulating the harmful inflammatory microbiota in the gut and reducing systemic inflammation in NASH patients.

Porous structures inspired by porcupine quill: multiscale design optimization approach.

This paper presents a novel approach for designing a freeform bending-resistant structure from the combination of explicit discrete component-based topology optimization (TO) and the porcupine quill-inspired features. To embed the porcupine quill's features into the TO formulations, the method involves constructing discrete components at various scales to imitate features including solid shell, stochastically distributed pores, and graded stiffeners. The components are iteratively updated, and the optimization process allows for the grading of quill-inspired features while achieving optimal structural compliance under bending loads. The proposed approach is demonstrated to be effective through the resolution of Messershmitt-Bolkow-Blohm (MBB) beam designs, parameterized studies of geometric parameters, and numerical validation of long-span and short-span quill-inspired beam designs. By examining the von Mises stress distribution, the study highlights the mitigation of material yielding at the shell region brought by the geometric features of porcupine quills, leading to the potential theory support for the bending resistance. The optimized MBB beams are manufactured using the material extrusion technique, and three-point bending tests are conducted to explore the failure mitigation capability of the quill-inspired beam under large deformation. Consequently, the study concludes that the proposed quill-inspired component-based TO approach can design a structure with excellent bending resistance according to the improved energy absorption as well as increased deformation after reaching 75% peak load.

Enhancing simultaneous nitrogen and phosphorus availability through biochar addition during Chinese medicinal herbal residues composting: Synergism of microbes and humus.

The disposal of Chinese medicinal herbal residues (CMHRs) derived from Chinese medicine extraction poses a significant environmental challenge. Aerobic composting presents a sustainable treatment method, yet optimizing nutrient conversion remains a critical concern. This study investigated the effect and mechanism of biochar addition on nitrogen and phosphorus transformation to enhance the efficacy and quality of compost products. The findings reveal that incorporating biochar considerably enhanced the process of nutrient conversion. Specifically, biochar addition promoted the retention of bioavailable organic nitrogen and reduced nitrogen loss by 28.1 %. Meanwhile, adding biochar inhibited the conversion of available phosphorus to non-available phosphorus while enhancing its conversion to moderately available phosphorus, thereby preserving phosphorus availability post-composting. Furthermore, the inclusion of biochar altered microbial community structure and fostered organic matter retention and humus formation, ultimately affecting the modification of nitrogen and phosphorus forms. Structural equation modeling revealed that microbial community had a more pronounced impact on bioavailable organic nitrogen, while humic acid exerted a more significant effect on phosphorus availability. This research provides a viable approach and foundation for regulating the levels of nitrogen and phosphorus nutrients during composting, serving as a valuable reference for the development of sustainable utilization technologies pertaining to CMHRs.

Machine learning and experimental validation identified autophagy signature in hepatic fibrosis.

Background: The molecular mechanisms of hepatic fibrosis (HF), closely related to autophagy, remain unclear. This study aimed to investigate autophagy characteristics in HF. Methods: Gene expression profiles (GSE6764, GSE49541 and GSE84044) were downloaded, normalized, and merged. Autophagy-related differentially expressed genes (ARDEGs) were determined using the limma R package and the Wilcoxon rank sum test and then analyzed by GO, KEGG, GSEA and GSVA. The infiltration of immune cells, molecular subtypes and immune types of healthy control (HC) and HF were analyzed. Machine learning was carried out with two methods, by which, core genes were obtained. Models of liver fibrosis in vivo and in vitro were constructed to verify the expression of core genes and corresponding immune cells. Results: A total of 69 ARDEGs were identified. Series functional cluster analysis showed that ARDEGs were significantly enriched in autophagy and immunity. Activated CD4 T cells, CD56bright natural killer cells, CD56dim natural killer cells, eosinophils, macrophages, mast cells, neutrophils, and type 17 T helper (Th17) cells showed significant differences in infiltration between HC and HF groups. Among ARDEGs, three core genes were identified, that were ATG5, RB1CC1, and PARK2. Considerable changes in the infiltration of immune cells were observed at different expression levels of the three core genes, among which the expression of RB1CC1 was significantly associated with the infiltration of macrophage, Th17 cell, natural killer cell and CD56dim natural killer cell. In the mouse liver fibrosis experiment, ATG5, RB1CC1, and PARK2 were at higher levels in HF group than those in HC group. Compared with HC group, HF group showed low positive area in F4/80, IL-17 and CD56, indicating decreased expression of macrophage, Th17 cell, natural killer cell and CD56dim natural killer cell. Meanwhile, knocking down RB1CC1 was found to inhibit the activation of hepatic stellate cells and alleviate liver fibrosis. Conclusion: ATG5, RB1CC1, and PARK2 are promising autophagy-related therapeutic biomarkers for HF. This is the first study to identify RB1CC1 in HF, which may promote the progression of liver fibrosis by regulating macrophage, Th17 cell, natural killer cell and CD56dim natural killer cell.

Paeoniflorin promotes PPARγ expression to suppress HSCs activation by inhibiting EZH2-mediated histone H3K27 trimethylation.

BACKGROUND: The alleviating effect of paeoniflorin (Pae) on liver fibrosis has been established; however, the molecular mechanism and specific target(s) underlying this effect remain elusive. PURPOSE: This study was to investigate the molecular mechanism underlying the regulatory effect of Pae on hepatic stellate cells (HSCs) activation in liver fibrosis, with a specific focus on the role of Pae in modulating histone methylation modifications. METHODS: The therapeutic effect of Pae was evaluated by establishing in vivo and in vitro models of carbon tetrachloride (CCl4)-induced mice and transforming growth factor ß1 (TGF-ß1)-induced LX-2 cells, respectively. Molecular docking, surface plasmon resonance (SPR), chromatin immunoprecipitation-quantitative real time PCR (ChIP-qPCR) and other molecular biological methods were used to clarify the molecular mechanism of Pae regulating HSCs activation. RESULTS: Our study found that Pae inhibited HSCs activation and histone trimethylation modification in liver of CCl4-induced mice and LX-2 cells. We demonstrated that the inhibitory effect of Pae on the activation of HSCs was dependent on peroxisome proliferator-activated receptor γ (PPARγ) expression and enhancer of zeste homolog 2 (EZH2). Mechanistically, Pae directly binded to EZH2 to effectively suppress its enzymatic activity. This attenuation leaded to the suppression of histone H3K27 trimethylation in the PPARγ promoter region, which induced upregulation of PPARγ expression. CONCLUSION: This investigative not only sheds new light on the precise targets that underlie the remission of hepatic fibrogenesis induced by Pae but also emphasizes the critical significance of EZH2-mediated H3K27 trimethylation in driving the pathogenesis of liver fibrosis.

Synthesis, characterization and in vitro antiproliferative effects of isosteviol derivatives.

Nineteen isosteviol derivatives were designed and synthesized by C-16, C-19 and D-ring modifications of isosteviol. These compounds were screened for their cytotoxic activities against Hela and A549 cells in vitro. Among them, the inhibitory effect of compounds 3b and 16 on Hela cells was comparable to that of the positive control gefitinib, and the compounds 3b (IC50=7.84 ± 0.84 µM) and 7a (IC50=6.89 ± 0.33 µM) exhibited significant cytotoxicity superior to gefitinib (IC50=11.02 ± 3.27 µM) against A549 cells.

Sphingolipid metabolism controls mammalian heart regeneration.

Utilization of lipids as energy substrates after birth causes cardiomyocyte (CM) cell-cycle arrest and loss of regenerative capacity in mammalian hearts. Beyond energy provision, proper management of lipid composition is crucial for cellular and organismal health, but its role in heart regeneration remains unclear. Here, we demonstrate widespread sphingolipid metabolism remodeling in neonatal hearts after injury and find that SphK1 and SphK2, isoenzymes producing the same sphingolipid metabolite sphingosine-1-phosphate (S1P), differently regulate cardiac regeneration. SphK2 is downregulated during heart development and determines CM proliferation via nuclear S1P-dependent modulation of histone acetylation. Reactivation of SphK2 induces adult CM cell-cycle re-entry and cytokinesis, thereby enhancing regeneration. Conversely, SphK1 is upregulated during development and promotes fibrosis through an S1P autocrine mechanism in cardiac fibroblasts. By fine-tuning the activity of each SphK isoform, we develop a therapy that simultaneously promotes myocardial repair and restricts fibrotic scarring to regenerate the infarcted adult hearts.

A simple and effective method to enrich endogenous DNA from mammalian faeces.

Utilization of faeces has long been a popular approach for genetic and ecological studies of wildlife. However, the success of molecular marker genotyping and genome resequencing is often unpredictable due to insufficient enrichment of endogenous DNA in the total faecal DNA that is dominated by bacterial DNA. Here, we report a simple and cheap method named PEERS to predominantly lyse animal cells over bacteria by using sodium dodecyl sulphate so as to discharge endogenous DNA into liquid phase before bacterial DNA. By brief centrifugation, total DNA with enriched endogenous fraction can be extracted from the supernatant using routine methods. Our assessments showed that the endogenous DNA extracted by PEERS was significantly enriched for various types of faeces from different species, preservation time and conditions. It significantly improves the genotyping correctness and efficiency of genome resequencing with the total additional cost of $ 0.1 and a short incubation step to treat a faecal sample. We also provide methods to assess the enrichment efficiency of mitochondrial and nuclear DNA and models to predict the usability of faecal DNA for genotyping of short tandem repeat, single-nucleotide polymorphism and whole-genome resequencing.

Recent developments in the management of ascites in cirrhosis.

In recent years, advances have been made for treating ascites in patients with cirrhosis. Recent studies have indicated that several treatments that have been used for a long time in the management of portal hypertension may have beneficial effects that were not previously identified. Long-term albumin infusion may improve survival in patients with cirrhosis and ascites while beta-blockers may reduce ascites occurrence. Transjugular intrahepatic porto-systemic shunt (TIPS) placement may also improve survival in selected patients in addition to the control with ascites. Low-flow ascites pump insertion can be another option for some patients with intractable ascites. In this review, we summarize the latest data related to the management of ascites occurring in cirrhosis. There are still unanswered questions, such as the optimal use of albumin as a long-term therapy, the place of beta-blockers, and the best timing for TIPS placement to improve the natural history of ascites, as well as the optimal stent diameter to reduce the risk of shunt-related side-effects. These issued should be addressed in future studies.

ILT2 and ILT4 Drive Myeloid Suppression via Both Overlapping and Distinct Mechanisms.

Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.