SRT1720 inhibits bladder cancer cell progression by impairing autophagic flux.

Bladder cancer (BC) is the most common cancer of the urinary tract, with poor survival, high recurrence rates, and lacking of targeted drugs. In this study, we constructed a library to screen compounds inhibiting bladder cancer cells growth. Among them, SRT1720 was identified to inhibit bladder cancer cell proliferation in vitro and in vivo. SRT1720 treatment also suppressed bladder cancer cells migration, invasion and induced apoptosis. Mechanism studies shown that SRT1720 promoted autophagosomes accumulation by inducing early-stage autophagy but disturbed the late-stage of autophagy by blocking fusion of autophagosomes and lysosomes. SRT1720 appears to induce autophagy related proteins expression and alter autophagy-related proteins acetylation to impede the autophagy flux. LAMP2, an important lysosomal associated membrane protein, may mediate SRT1720-inhibited autophagy flux as SRT1720 treatment significantly deacetylated LAMP2 which may influence its activity. Taken together, our results demonstrated that SRT1720 mediated apoptosis and autophagy flux inhibition may be a novel therapeutic strategy for bladder cancer treatment.

Raltitrexed induces apoptosis through activating ROS-mediated ER stress by impeding HSPA8 expression in prostate cancer cells.

Prostate cancer is the most common malignant tumor in males, which frequently develops into castration-resistant prostate cancer (CRPC). CRPC metastasis is the main reason for its high mortality rate. At present, it lacks effective treatment for patients with CRPC. Raltitrexed (RTX) has been shown to be effective in the treatment of colorectal cancer. However, the effect of RTX on prostate cancer and the underlying mechanism remain unknown. In the current study, we found that RTX could dose-dependently inhibit proliferation, migration, colony formation and induce apoptosis in DU145 and PC-3 cells. RTX also increased ROS generation in prostate cancer cells. Pretreatment with N-acetyl-L-cysteine (NAC) significantly prevented RTX-induced cell apoptosis and endoplasmic reticulum (ER) stress signaling activation in prostate cancer cells. Additionally, we found RTX-induced ROS generation and ER stress activation depended on the expression of heat shock protein family A member 8 (HSPA8). Over-expression of HSPA8 could alleviate RTX-induced cell apoptosis, ROS generation and ER stress signaling activation. Finally, our study also showed that RTX attenuated the tumor growth of prostate cancer in the DU145 xenograft model and significantly downregulated HSPA8 expression and activated ER stress signaling pathway in tumor tissues. Our study is the first to reveal that RTX induces prostate cancer cells apoptosis through inhibiting the expression of HSPA8 and further inducing ROS-mediated ER stress pathway action. This study suggests that RTX may be a novel promising candidate drug for prostate cancer therapy.

Ultrasensitive fluorescence microRNA biosensor by coupling hybridization-initiated exonuclease I protection and tyramine signal amplification.

Tyramine signal amplification (TSA) has made its mark in immunoassay due to its excellent signal amplification ability and short reaction time, but its application in nucleic acid detection is still very limited. Herein, an ultrasensitive microRNA (miRNA) biosensor by coupling hybridization-initiated exonuclease I (Exo I) protection and TSA strategy was established. Target miRNA is complementarily hybridized to the biotin-modified DNA probe to form a double strand, which protects the DNA probe from Exo I hydrolysis. Subsequently, horseradish peroxidase (HRP) is attached to the duplex via the biotin-streptavidin reaction and catalyzes the deposition of large amounts of biotin-tyramine in the presence of hydrogen peroxide (H2O2), followed by the conjugation of signal molecule streptavidin-phycoerythrin (SA-PE), which generates an intense fluorescence signal upon laser excitation. This method gave broad linearity in the range of 0.1 fM - 10 pM, yielding a detection limit as low as 74 aM. An increase in sensitivity of 4 orders of magnitude was observed compared to the miRNA detection without TSA amplification. This biosensor was successfully applied to the determination of miR-21 in breast cancer cells and human serum. By further design of specific DNA probes and coupling with the Luminex xMAP technology, it could be easily extended to multiplex miRNA assay, which possesses great application potential in clinical diagnosis.

Microvascular rarefaction caused by the NOTCH signaling pathway is a key cause of TKI-apatinib-induced hypertension and cardiac damage.

With the advancement of tumour-targeted therapy technology, the survival of cancer patients has continued to increase, and cardiovascular events have gradually become an important cause of death in cancer patients. This phenomenon occurs due to adverse cardiovascular reactions caused by the cardiovascular toxicity of antitumour therapy. Moreover, the increase in the proportion of elderly patients with cancer and cardiovascular diseases is due to the extension of life expectancy. Hypertension is the most common cardiovascular side effect of small molecule tyrosine kinase inhibitors (TKIs). The increase in blood pressure induced by TKIs and subsequent cardiovascular complications and events affect the survival and quality of life of patients and partly offset the benefits of antitumour therapy. Many studies have confirmed that in the pathogenesis of hypertension, arterioles and capillary thinness are involved in its occurrence and development. Our previous findings showing that apatinib causes microcirculation rarefaction of the superior mesenteric artery and impaired microvascular growth may inspire new therapeutic strategies for treating hypertension. Thus, by restoring microvascular development and branching patterns, total peripheral resistance and blood pressure are reduced. Therefore, exploring the key molecular targets of TKIs that inhibit the expression of angiogenic factors and elucidating the specific molecular mechanism involved are key scientific avenues for effectively promoting endothelial cell angiogenesis and achieving accurate repair of microcirculation injury in hypertension patients.

Downregulation of S100A11 promotes T cell infiltration by regulating cancer-associated fibroblasts in prostate cancer.

OBJECTIVE: This study aims at revealing the relationship between S100A11 and cancer-associated fibroblasts (CAFs) in prostate cancer and improving T cell infiltration into solid tumors. METHODS: H&E, IHC and Sirius red staining were used to detect the stroma content in prostate cancer tissues. Stable S100A11 knockdown cell lines DU 145, 22Rv1, RM-1 and NOR-10 were established by lentivirus transfection. Co-culture system of RM-1 and CAFs was established. CCK-8, wound healing and transwell were proceeded to determine proliferation, migration and invasion of prostate cancer cells. Stably knocked-down RM-1 and CAFs were co-injected into C57BL/6 mice to detect the role of S100A11 in vivo. CAFs, CD4+ T cell and CD8+ T cell in these tumors were assessed by IF. T cell profile was analyzed by flow cytometry. RESULTS: A significant amount of stroma exists in prostate cancer tissues. Downregulation of S100A11 inhibits proliferation, migration and invasion of human prostate cancer cells in vitro, and suppresses the expression of cancer-associated fibroblasts (CAFs) in vivo. Knockdown of S100A11 enhances the inhibitory effect of Erdafitinib on CAFs in both the co-culture system and in vivo. The combined knockdown of S100A11 in tumor cells and CAFs shows a superior therapeutic effect compared to the individual knockdown in tumor cells alone. Knockdown of S100A11, both in RM-1 and CAFs, combined with Erdafitinib treatment reduces tumorigenicity by suppressing the content of CAFs and increasing the infiltration of CD4+ T cell and effective CD8+ T cell in tumor. CONCLUSION: Downregulation of S100A11 plays a crucial role in enhancing the therapeutic response to Erdafitinib and reversing immunosuppressive tumor microenvironment.

Sanguinarine chloride induces ferroptosis by regulating ROS/BACH1/HMOX1 signaling pathway in prostate cancer.

BACKGROUND: Sanguinarine chloride (S.C) is a benzophenanthrine alkaloid derived from the root of sanguinaria canadensis and other poppy-fumaria species. Studies have reported that S.C exhibits antioxidant, anti-inflammatory, proapoptotic, and growth inhibitory effects, which contribute to its anti-cancer properties. Recent studies suggested that the antitumor effect of S.C through inducing ferroptosis in some cancers. Nevertheless, the precise mechanism underlying the regulation of ferroptosis by S.C remains poorly understood. METHODS: A small molecule library was constructed based on FDA and CFDA approved small molecular drugs. CCK-8 assay was applied to evaluate the effects of the small molecule compound on tumor cell viability. Prostate cancer cells were treated with S.C and then the cell viability and migration ability were assessed using CCK8, colony formation and wound healing assay. Reactive oxygen species (ROS) and iron accumulation were quantified through flow cytometry analysis. The levels of malondialdehyde (MDA) and total glutathione (GSH) were measured using commercially available kits. RNA-seq analysis was performed to identify differentially expressed genes (DEGs) among the treatment groups. Western blotting and qPCR were utilized to investigate the expression of relevant proteins and genes. In vivo experiments employed a xenograft mice model to evaluate the anti-cancer efficacy of S.C. RESULTS: Our study demonstrated that S.C effectively inhibited the viability of various prostate cancer cells. Notably, S.C exhibited the ability to enhance the cytotoxicity of docetaxel in DU145 cells. We found that S.C-induced cell death partially relied on the induction of ferroptosis, which was mediated through up-regulation of HMOX1 protein. Additionally, our investigation revealed that S.C treatment decreased the stability of BACH1 protein, which contributed to HMOX1expression. We further identified that S.C-induced ROS caused BACH1 instability by suppressing USP47expression. Moreover, In DU145 xenograft model, we found S.C significantly inhibited prostate cancer growth, highlighting its potential as a therapeutic strategy. Collectively, these findings provide evidence that S.C could induce regulated cell death (RCD) in prostate cancer cells and effectively inhibit tumor growth via triggering ferroptosis. This study provides evidence that S.C effectively suppresses tumor progression and induces ferroptosis in prostate cancer cells by targeting ROS/USP47/BACH1/HMOX1 axis. CONCLUSION: This study provides evidence that S.C effectively suppresses tumor progression and induces ferroptosis in prostate cancer cells by targeting the ROS/USP47/BACH1/HMOX1 axis. These findings offer novel insights into the underlying mechanism by which S.C inhibits the progression of prostate cancer. Furthermore, leveraging the potential of S.C in targeting ferroptosis may present a new therapeutic opportunity for prostate cancer. This study found that S.C induces ferroptosis by targeting the ROS/USP47/BACH1/HMOX1 axis in prostate cancer cells.

Polymer selection for amorphous solid dispersion of a new drug candidate by investigation of drug polymer molecular interactions.

The antitumor drug candidate X-05 is being developed as an innovative anti-lung cancer drug candidate due to its excellent antitumour activity. A Caco-2 cell permeability study and solubility study confirmed that X-05 belonged to BCS class or compounds. Therefore, the main challenge is to develop appropriate preparations for preclinical studies and further clinical phase research. By evaluating the preliminary results of kinetic solubility in biorelevant media and the structural analysis of X-05 and polymers, three polymers PVP K30, PVP VA 64 and HPMCAS, which may have intermolecular interactions with X-05, were chosen to select the optimal carrier for X-05 to prepare amorphous solid dispersions (ASDs). ASD X-05-PVP VA 64 was selected as the optimal polymer by evaluating its kinetic solubility in biorelevant media and solid stability. The physical and chemical properties of ASD X-05-PVP VA 64 remain stable when the drug loading is as high as 50%. The drug-polymer interactions of ASD X-05-PVP VA 64 were studied by ultraviolet spectrophotometry, nuclear magnetic resonance spectrometry, infrared and Raman spectrophotometry, and the results indicated that the intermolecular hydrogen bond interaction between the drug and polymer was the foundation of the solubilization and stabilization of X-05 in PVP VA 64.

CAB39 promotes cisplatin resistance in bladder cancer via the LKB1-AMPK-LC3 pathway.

Systemic therapy for muscle-invasive bladder cancer (BC) remains dominated by cisplatin-based chemotherapy. However, resistance to cisplatin therapy greatly limits long-term survival. Resistance to cisplatin-based chemotherapy still needs to be addressed. In this study, we established three cisplatin-resistant BC cell lines by multiple cisplatin pulse treatments. Interestingly, after exposure to cisplatin, all cisplatin-resistant cell lines showed lower reactive oxygen species (ROS) levels than the corresponding parental cell lines. Using proteomic analysis, we identified 35 proteins that were upregulated in cisplatin-resistant BC cells. By knocking down eleven of these genes, we found that after CAB39 knockdown, BC cisplatin-resistant cells were more sensitive to cisplatin. Overexpression of CAB39 had the opposite effect. Then, the knockdown of six genes downstream of CAB39 revealed that CAB39 promoted cisplatin resistance in BC through LKB1. Moreover, a key cause of cisplatin-induced cell death is damage to mitochondria and increased ROS levels. In our study, cisplatin-resistant cells exhibited higher autophagic flux and healthier mitochondrial status after cisplatin exposure. We demonstrated that the CAB39-LKB1-AMPK-LC3 pathway plays a critical role in enhancing autophagy to maintain the health of mitochondria and reduce ROS levels. In addition, the autophagy inhibitor chloroquine (CQ) can significantly enhance the killing effect of cisplatin on BC cells. Compared with gemcitabine plus cisplatin (GC), GC plus CQ significantly reduced tumor burden in vivo. In conclusion, our study shows that CAB39 counteracts the killing of cisplatin by enhancing the autophagy of BC cells to damaged mitochondria and other organelles to alleviate the damage of cells caused by harmful substances such as ROS.

Formin-related protein 1 facilitates proliferation and aggressive phenotype of clear cell renal cell carcinoma through MAPK/MMP2 pathway.

BACKGROUND: Formin-related protein-1(FRL1) has reportedly been overexpressed in a variety of malignancies, such as clear cell renal cell carcinoma (ccRCC). However, the clinical value and molecular mechanisms underlying ccRCC tumorigenesis and progression in association with FRL1 remain poorly understood. METHODS: Immunohistochemical analysis was performed on 119 paraffin-embedded RCC tissue samples to detect FRL1 expression and analyze its prognostic value. Colony formation, the CCK-8 assay, flow cytometry, and in vivo nude mice subcutaneous experiments were used to identify the effects of FRL1 on growth and proliferation. In vitro tests for wound healing, migration, and invasion were used to assess the involvement of FRL1 in invasion and metastatic potential. The process of epithelial-mesenchymal transition process (EMT) and the MMP2 expression were detected in stably transfected RCC cells via western blotting, as well as in tumor tissue paraffin sections from xenograft model. RESULTS: Both FRL1 mRNA and protein levels were noticeably elevated in ccRCC cell lines and samples. Aberrant overexpression of FRL1 was associated with unfavorable clinicopathological features of ccRCC and indicated poor prognosis. Ectopic overexpression of FRL1 increased the growth-promoting traits of ccRCC cells as well as the migratory and invasive capacity of RCC cells, whereas FRL1-silencing caused the opposite results. In addition, FRL1 promoted epithelial-mesenchymal transition (EMT) and upregulated the expression of matrix metalloproteinase 2 (MMP2). Finally, overexpression of FRL1 upregulated phosphorylation level of ERK1/2 with no effect on total level of ERK1/2 in the RCC cells. MAPK/ERK inhibitor reversed the promotional effects of FRL1. CONCLUSION: FRL1 was overexpressed in ccRCC tissues and predicted poor prognosis. FRL1 contributes to invasion and aggressive phenotype of ccRCC by facilitating EMT through MAPK/MMP2 axis.

A chemiluminescent sensor for imaging endogenous hydrogen polysulfides in a living system.

By constructing 2-(benzoylthio)benzoate and a 2-fluoro-4-nitrobenzoate structure in an adamantylidene-dioxetane system, we designed and synthesized two novel chemiluminescent probes for the detection of H2Sn from other RSS. Under the same conditions, the maximum luminescence emission intensity of the probe CL-HP2 could reach 150 times that of the probe CL-HP1, and the chemiluminescence signal still existed at low concentrations. Therefore, CL-HP2 was more suitable for H2Sn detection as a chemiluminescent probe. The probe CL-HP2 exhibited a good linear relationship with Na2S4 in a wide range (0.025-10 mM). Interestingly, a good linear relationship (R2 = 0.997) was also observed at low concentrations (0-100 µM) with a LOD as low as 0.23 µM. CL-HP2 has been effectively employed to visualize endogenous H2Sn within living cells. Moreover, it has been applied for live imaging of bacterially infected murine models and the ferroptosis process in tumor-bearing mouse models.

A prognostic model based on tumor microenvironment-related lncRNAs predicts therapy response in pancreatic cancer.

Pancreatic cancer is an aggressive malignant tumor with high mortality and a low survival rate. The immune and stromal cells that infiltrate in the tumor microenvironment (TME) significantly impact immunotherapy and drug responses. Therefore, we identify the TME-related lncRNAs to develop a prognostic model for predicting the therapy efficacy in pancreatic cancer patients. Firstly, we identified differentially expressed genes (DEGs) for weighted gene co-expression network analysis (WGCNA) to identify the TME-related module eigengenes. According to the module eigengenes, the TME-related prognostic lncRNAs were screened through the univariate Cox, least absolute shrinkage and selection operator (LASSO), and multivariate Cox analyses to construct a prognostic risk score (RS) model. Next, the predictive power of this model was evaluated by the time-dependent receiver operating characteristic (ROC) curve and Kaplan-Meier analyses. In addition, functional enrichment, immune cell infiltration, and somatic mutation analyses were performed. Finally, tumor immune dysfunction and exclusion (TIDE) score and drug sensitivity analyses were applied to predict therapy response. In this study, 11 TME-related prognostic lncRNAs were identified to develop the prognostic RS model. According to the RS, the low-risk patients had a better prognosis, lower rates of somatic mutation, lower TIDE scores, and higher sensitivity to gemcitabine and paclitaxel compared to high-risk patients. The findings above suggested that low-risk patients may benefit more from immunotherapy, and high-risk patients may benefit more from chemotherapy. Within this study, we established a prognostic RS model based on 11 TME-related lncRNAs, which may help improve clinical decision-making.

ADAM12 promotes gemcitabine resistance by activating EGFR signaling pathway and induces EMT in bladder cancer.

BACKGROUND: Gemcitabine (GEM)-based chemotherapy regimens is widely used in bladder cancer (BC) patients. However, GEM resistance may occur and result in treatment failure and disease progression. A disintegrin and metalloprotease 12 (ADAM12) plays a critical role in many cancers. However, the role of ADAM12 in GEM resistance of BC remains unclear. METHODS: We analyzed the relationship between ADAM12 expression and tumor characteristics using the data downloaded from The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database. Then, we established GEM resistant BC cell lines and used quantitative real-time PCR, western blot, cell counting kit-8, immunohistochemistry, and xenograft mouse model to investigate the role of ADAM12 in GEM resistance. RESULTS: In general, ADAM12 was found to be upregulated in GEM resistant BC cells. ADAM12 knockdown increased the chemosensitivity of BC cells. We further proved that ADAM12 could promote GEM resistance by activating the epidermal growth factor receptor (EGFR) signaling pathway in BC. Furthermore, the epithelial-mesenchymal transition (EMT) phenotype was observed in GEM resistant BC cells. ADAM12 induced EMT process and promotes tumor progression in BC. CONCLUSION: Our findings suggested that ADAM12 was a key gene for GEM resistance and positively correlated with malignancy of BC. It might serve as a novel and valuable therapeutic target for BC.

Radical nucleophilic substitution/cyclization: A novel strategy for selective and ultrafast fluorescence imaging of cysteine levels in ferroptosis process.

Diphenylisoindolo[2,1-a]quinoline can be used to detect cysteine among homocysteine, glutathione, and other 19 natural amino acids. Unlike other reported probes, the response mechanism involves sulfhydryl radical nucleophilic substitution and cyclization, and thus the differences in ring-formation kinetics enable high selectivity. After treated with Cys, the response process was completed rapidly and the maximum fluorescence intensity (at 496 nm) was reached extremely fast (<1 s) when excited at 380 nm in MeCN-PBS buffer (10.0 mM, pH = 7.4, 3:7 (v/v)). The quantum yield after the reaction was increased almost 7 times to be 0.02 from 0.003. Fluorescence intensity displayed a good quantitative linear relationship in the range 1-10 µM Cys with a detection limit of 270 nM. Furthermore, the probe was demonstrated for real-time monitoring of intracellular cysteine levels within HepG2 cells in ferroptosis process.

A Prognostic Ferroptosis-Related lncRNA Model Associated With Immune Infiltration in Colon Cancer.

Colon cancer (CC) is a common malignant tumor worldwide, and ferroptosis plays a vital role in the pathology and progression of CC. Effective prognostic tools are required to guide clinical decision-making in CC. In our study, gene expression and clinical data of CC were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. We identified the differentially expressed ferroptosis-related lncRNAs using the differential expression and gene co-expression analysis. Then, univariate and multivariate Cox regression analyses were used to identify the effective ferroptosis-related lncRNAs for constructing the prognostic model for CC. Gene set enrichment analysis (GSEA) was conducted to explore the functional enrichment analysis. CIBERSORT and single-sample GSEA were performed to investigate the association between our model and the immune microenvironment. Finally, three ferroptosis-related lncRNAs (XXbac-B476C20.9, TP73-AS1, and SNHG15) were identified to construct the prognostic model. The results of the validation showed that our model was effective in predicting the prognosis of CC patients, which also was an independent prognostic factor for CC. The GSEA analysis showed that several ferroptosis-related pathways were significantly enriched in the low-risk group. Immune infiltration analysis suggested that the level of immune cell infiltration was significantly higher in the high-risk group than that in the low-risk group. In summary, we established a prognostic model based on the ferroptosis-related lncRNAs, which could provide clinical guidance for future laboratory and clinical research on CC.

A facile turn-on chemiluminescence probe for sensitive imaging on aminopeptidase N activity.

Aminopeptidase N, as a target for drug discovery, shows marked relationships with many diseases, especially liver injury and cancer. Here, we explored a chemiluminescence (CL) probe for sensing APN by tethering the APN-specific substrate group to the ortho-acrylated phenoxy-dioxetane scaffold. In this way, two CL probes (APN-CL and BAPN-CL) were designed with noncapped leucine and butoxy-carbonyl capped leucine as the protecting group to preserve the chemiexcitation energy. The uncovered leucine was demonstrated to be essential for detection of APN activity by comparing the CL intensity of two CL probes. Probe APN-CL was turned on upon APN cleavage, resulting in a high chemiluminescent emission, whereas the chemiexcitation energy of probe BAPN-CL was still restrained even with the high-level APN. The result was further elucidated by molecular docking simulations. Probe APN-CL exhibited a fast response and high sensitivity with a detection limit of 0.068 U/L, and an excellent specificity for the discrimination of APN from biological ions, small molecules, and other proteases commonly found in living system. By virtue of good stability and cell viability, probe APN-CL imaged abnormal levels of APN in tumour cells and tumour-bearing mice. Moreover, this probe APN-CL could be easily used to evaluate APN inhibitors and APN levels in plasma samples from 20 patients. Overall, as a facile and cost-effective probe, APN-CL will be a promising alternative in the early diagnosis of pathologies and for cost-effective screening of inhibitors.

FOXO3a-ROS pathway is involved in androgen-induced proliferation of prostate cancer cell.

BACKGROUND: Although FOXO3a can inhibit the cell proliferation of prostate cancer, its relationship with reactive oxygen species (ROS) in prostate cancer (PCa) has not been reported. METHODS: We analyzed the correlation between the expression of FOXO3a and the antioxidant enzyme catalase in prostate cancer with the TCGA and GEPIA databases. We also constructed a PPI network of FOXO3a via the STRING database. The mRNA and protein expression of FOXO3a and catalase were detected by qRT-PCR or western blotting in LNCaP and 22RV1 cells treated with DHT, R1881, or Enzalutamide. The effects of FOXO3a on catalase expression were tested by over-expressing or knocking down FOXO3a in LNCaP cells. Furthermore, the catalase activity and ROS level were detected in LNCaP cells treated with DHT. Cell proliferation and ROS were also analyzed in LNCaP which was treated with antioxidant. RESULTS: Results showed that the catalase expression was down-regulated in prostate cancer. A positive correlation between FOXO3a and catalase existed. DHT treatment could significantly reduce FOXO3a and catalase expression at mRNA and protein level in LNCaP cells. Catalase expression partly depended on FOXO3a as over-expression and knockdown of FOXO3a could result in the expresssion change of catalase. DHT treatment was found to inhibit catalase activity and increase ROS level in prostate cancer cell. Our study also demonstrated that antioxidant treatment reduced DHT-induced proliferation and ROS production in prostate cancer cell. CONCLUSIONS: We discovered a novel mechanism by which DHT promotes prostate cancer cell proliferation via suppressing catalase activity and activating ROS signaling via a FOXO3a dependent manner.

Base excision-initiated terminal deoxynucleotide transferase-assisted amplification for simultaneous detection of multiple DNA glycosylases.

Various DNA glycosylases involved in base excision repair may be associated with a wide disease spectrum that includes cancer, myocardial infarction, neurodegenerative disorders, etc. In this paper, we developed a sensitive method for simultaneous detection of multiple DNA glycosylases based on the target-initiated removal of damaged base and terminal deoxynucleotidyl transferase (TdT)-assisted labeling and signal amplification. We designed three specific stem-loop probes which contained specific targeting damaged bases in the stem for uracil DNA glycosylase (UDG), human alkyladenine DNA glycosylase (hAAG), and human 8-oxoguanine DNA glycosylase 1 (hOGG1), respectively. Target DNA glycosylase can initiate the recognition and clearance of damaged base on immobilized 3' blocked stem-loop probe, releasing apurine/apyrimidine (AP) site which can be hydrolyzed by AP endonuclease to produce 3'OH probe fragment for TdT extension. Numerous biotin-modified dUTPs were successively labeled on the 3' terminus of the probe fragments, and then reacted with streptavidin-phycoerythrin (SA-PE) for analysis by using the Luminex xMAP array platform. The amplification strategy based on TdT has been utilized to simultaneously and sensitively detect three different DNA glycosylases with detection limits of 10-3 U/ml. Moreover, it could be applied for analyzing DNA glycosylase activity in complex HeLa cell lysate samples. Therefore, this strategy possesses the advantages of high sensitivity, specificity, and multiplex, holding great potential for DNA glycosylase-related biomedical research.

Immune Cell Landscape in Gastric Cancer.

BACKGROUND: The tumor-infiltrating immune cells are closely associated with the prognosis of gastric cancer (GC). This article is aimed at determining the composition change of immune cells and immune regulatory factors in GC and normal tissues, depicting their prognosis value in GC, and revealing the relationship between them and GC clinical parameters. METHODS: We used CIBERSORT to calculate the proportion of 22 immune cells in the GC or normal tissues; a t-test was applied to assess the expression difference of immune cells and immune regulatory factors in normal and GC tissues. The relationship of the immune cells, immune regulatory factors, and GC patients' clinical characteristics was assessed by univariate analysis. RESULTS: In this study, we found that the proportion of macrophages increased, while plasma cells and monocytes decreased in GC tissues. In these immune fractions, Tregs and naïve B cells were found to be correlated with GC patients' prognosis. Interestingly, the expression of immune regulatory factors was ambiguous with their classical function in GC tissues. For example, TIM-3, FOXP3, and CMTM6 were overexpressed, while CD27 and PD-1 were underexpressed in GC tissues. We also found that IDO1, PD-1, TIGIT, and TIM-3 were highly expressed in high-grade GC tissues, the HERC2 expression level was related to patients' gender, and the TIGIT expression level was sensitive to targeted therapy. Furthermore, our results suggested that the infiltration of Tregs and naive B cells was strongly correlated with the T stage, radiation therapy, targeted molecular therapy, and the expression levels of TIM-3 and FOXP3 in GC. CONCLUSION: The expression pattern of tumor-infiltrating immune cells and immune regulatory factors was systematically depicted in the GC tumor microenvironment, indicating that individualized treatment based on the tumor-infiltrating immune cells and immune regulatory factors may be beneficial to GC patients.

Electrophysiological assessment and pharmacological treatment of blast-induced tinnitus.

Tinnitus, the phantom perception of sound, often occurs as a clinical sequela of auditory traumas. In an effort to develop an objective test and therapeutic approach for tinnitus, the present study was performed in blast-exposed rats and focused on measurements of auditory brainstem responses (ABRs), prepulse inhibition of the acoustic startle response, and presynaptic ribbon densities on cochlear inner hair cells (IHCs). Although the exact mechanism is unknown, the "central gain theory" posits that tinnitus is a perceptual indicator of abnormal increases in the gain (or neural amplification) of the central auditory system to compensate for peripheral loss of sensory input from the cochlea. Our data from vehicle-treated rats supports this rationale; namely, blast-induced cochlear synaptopathy correlated with imbalanced elevations in the ratio of centrally-derived ABR wave V amplitudes to peripherally-derived wave I amplitudes, resulting in behavioral evidence of tinnitus. Logistic regression modeling demonstrated that the ABR wave V/I amplitude ratio served as a reliable metric for objectively identifying tinnitus. Furthermore, histopathological examinations in blast-exposed rats revealed tinnitus-related changes in the expression patterns of key plasticity factors in the central auditory pathway, including chronic loss of Arc/Arg3.1 mobilization. Using a formulation of N-acetylcysteine (NAC) and disodium 2,4-disulfophenyl-N-tert-butylnitrone (HPN-07) as a therapeutic for addressing blast-induced neurodegeneration, we measured a significant treatment effect on preservation or restoration of IHC ribbon synapses, normalization of ABR wave V/I amplitude ratios, and reduced behavioral evidence of tinnitus in blast-exposed rats, all of which accorded with mitigated histopathological evidence of tinnitus-related neuropathy and maladaptive neuroplasticity.

A label-free mass spectrometry detection of microRNA by signal switching from high-molecular-weight polynucleotides to highly sensitive small molecules.

MicroRNAs (miRNAs) are associated with various cellular processes and have been recognized as potential biomarkers for many human diseases. The sensitive and accurate determination of miRNA expression levels in biological specimens is highly significant for understanding their biological functions and clinical diagnosis. Mass spectrometry (MS) has shown its potential to study bioactive molecules, however, direct MS analysis of miRNAs is often hampered by limited sensitivity. For sensitive detection of miRNAs, indirect methods are generally employed through the use of DNA probes labeled with peptides or metal elements. In this work, we proposed a novel MS-based label-free strategy for miRNA quantification. A dual-amplification system was developed by using a padlock probe containing the poly(thymine) sequence in combination with rolling circle amplification (RCA). The specific recognition of target miRNA by the padlock probes produced long single-stranded DNAs containing poly (adenine) segments, which guaranteed the specificity of detection and realized primary amplification. Then the RCA products were extracted and treated with acid to release a large number of free adenines as reporter molecules for secondary signal amplification. Overall, the quantification of target miRNA was carried out by signal switching from high-molecular-weight RCA products to highly sensitive small molecule of adenine. The developed method achieved a linear detection range from 200 amol to 100 fmol for miRNA-21 with a limit of detection of 50 amol, and successfully applied to detect endogenous miRNA-21 levels from lung cancer cells. Overall, the present study provides a sensitive, specific MS-based method for miRNA detection and holds great potential for further application of MS technology to detect other biomarkers in biomedical research and early clinical diagnosis.