BACKGROUND:Botulinum toxin type A is widely used to block acetylcholine release in the treatment of chronic sialorrhea, muscle spasticity, and dystonia. We aim to develop a user-friendly method for detecting cases of medical botulinum toxin poisoning. METHODS:The mice poisoning model was established by injecting or gavage with Botulax®, and the poisoning dose and symptoms were observed. The residual levels of toxin in poisoned mice were detected by high-resolution mass spectrometry and sandwich ELISA, respectively. RESULTS:Two hours after poisoning, no residual botulinum toxin was found by mass spectrometry (MS) under our specific untargeted workflow and sample preparation conditions, but ELISA detected residual toxin in various tissues of mice. Among them, the muscle tissue had the highest level. There is no noticeable difference in the levels of toxin residues in the same organs of mice, regardless of the route of poisoning. The sandwich ELISA method is user-friendly for detecting medical botulinum toxin poisoning. The presence of toxin residues can be detected in various tissues two hours after exposure, with muscle being the optimal sampling tissue. CONCLUSIONS:Our research indicates that under specific sample preparation, chromatographic separation, and untargeted detection conditions, mass spectrometry may not be effective for detecting Botulinum toxin at concentrations below the ng/ml level. The study demonstrates that ELISA is a sensitive and practical alternative for early detection. Positive results can be detected within 2 h of poisoning, especially when taken from muscle tissue.

Globally, bladder cancer is the tenth most common cancer. Mitophagy, a critical process regulating mitochondrial quantity and quality, has attracted increasing attention for its pivotal function in cancer. Nonetheless, its roles and underlying mechanisms in bladder cancer are yet to be elucidated. Therefore, in this study, 16 mitophagy-related genes were screened to construct a robust prognostic model with exceptional predictive accuracy for the outcomes of patients with bladder cancer. Of these genes, DARS2 was identified as a key regulator that significantly affected cancer progression. The findings established that DARS2 promoted the G1-to-S phase transition by upregulating CDK4 expression, thereby suppressing cellular senescence and driving cell proliferation. In addition, DARS2 augmented PINK1 expression, leading to increased PINK1-mediated mitophagy. Both in vitro and in vivo experiments confirmed that DARS2 inhibited cellular senescence and facilitated tumor progression by enhancing PINK1-mediated mitophagy. The observations from this study have provided novel insights into the multifaceted roles of DARS2-mediated mitophagy in bladder cancer. Targeting DARS2 and its regulation of mitophagy is a promising therapeutic strategy to improve the outcomes for patients with bladder cancer.

Pericytes are multipotent mesenchymal precursor cells that demonstrate tissue-specific properties. In this study, by comparing human adipose tissue- and periosteum-derived pericyte microarrays, we identified T cell lymphoma invasion and metastasis 1 (TIAM1) as a key regulator of cell morphology and differentiation decisions. TIAM1 represented a tissue-specific determinant between predispositions for adipocytic versus osteoblastic differentiation in human adipose tissue-derived pericytes. TIAM1 overexpression promoted an adipogenic phenotype, whereas its downregulation amplified osteogenic differentiation. These results were replicated in vivo, in which TIAM1 misexpression altered bone or adipose tissue generation in an intramuscular xenograft animal model. Changes in pericyte differentiation potential induced by TIAM1 misexpression correlated with actin organization and altered cytoskeletal morphology. Small molecule inhibitors of either small GTPase Rac1 or RhoA/ROCK signaling reversed TIAM1-induced morphology and differentiation in pericytes. In summary, our results demonstrate that TIAM1 regulates the cellular morphology and differentiation potential of human pericytes, representing a molecular switch between osteogenic and adipogenic cell fates.

Akt is commonly activated and serves as a valuable target in human cancer. In this study, OTUD1 is identified as an Akt-associated protein and is downregulated upon Akt activation. Ectopic OTUD1 inhibits Akt phosphorylation; however, its deubiquitinase activity contributes only slightly to this effect. A short peptide (OUN-36) located in the OTUD1 N-terminal intrinsically disordered region strongly binds to the Akt PH domain. The residues in the PH domain, which are required for PtdIns(3,4,5)P3 recognition, are also essential for OUN-36 binding. OUN-36 preferentially inhibits Akt-hyperactive tumor cells' proliferation and interferes with Akt cell membrane localization, presumably by disrupting PH domain-PIP3 interaction. Importantly, OUN-36-based therapy efficiently abrogates Akt feedback reactivation in response to MK-2206 treatment and sensitizes cancer cells to chemotherapy and immunotherapy. We therefore show a mechanism by which OTUD1 modulates Akt activity and suggest a potential peptide-based cancer therapeutic strategy implemented by targeting the Akt PH domain.

Extensive data research is helpful to find sensitive biomarkers for prognostic prediction of metastatic breast cancer. Through analyzing multiple GEO datasets, literature retrieval, and verified in GEPIA datasets, we identify BIRC5 (Baculoviral IAP repeat containing 5) and CDO1 (Cysteine dioxygenase type 1) as DEGs (differentially expressed genes) between breast tumor and normal tissue and DEGs between metastatic breast cancer and breast cancer in situ. Then, we performed a series of in silico studies on BIRC5 and CDO1 using online tools including the UALCAN, TIMER, TCGA-BRCA, LinkedOmics Kaplan-Meier Plotter, and an R script for analysis. To verify the association of 2 genes expression and patients’ clinical data, we detected BIRC5 and CDO1 mRNA in the tissue of 48 breast cancer patients. The results showed the tumor with BIRC5 high CDO1 low expression generally indicated patients’ shorter overall (OS) and relapse-free survival (RFS). Specifically, BIRC5 and CDO1 levels significantly affect OS or RFS in patients with Lymph node metastasis and molecular subtypes of TNBC (triple-negative breast cancer) and Luminal A. A BIRC5 high tumor displayed a purer tumor purity and expressed more KIR receptors on NK cells while activating more FOXP3 + CD25 + Treg cells. The CDO1 low tumors infiltrated with more immunocytes leading to less tumor purity. In our verified experiment, BIRC5 mRNA level in patients with stage III and over was significantly higher than in patients with stage 0 to II, but there were no significant differences among molecular subtyping groups; TNBC tissue expressed lower CDO1 mRNA level than HER2 + and Luminal type cancer tissue. In conclusion, a BIRC5 high CDO1 low expression type in breast cancer tissue indicates a poorer prognosis of patients. The potential mechanism might be increased BIRC5 expression in cancer tissue is likely to accompany NK cells inhibition, activating more Treg cells, and lacking effective CD8 + T cells proliferation. Meanwhile, CDO1 level is positively related to more immunocytes infiltration. Keywords Breast cancer , BIRC5 , CDO1 , prognostic prediction

Extensive data research is helpful to find sensitive biomarkers for prognostic prediction of metastatic breast cancer. Through analyzing multiple GEO datasets, literature retrieval, and verified in GEPIA datasets, we identify BIRC5 (Baculoviral IAP repeat containing 5) and CDO1 (Cysteine dioxygenase type 1) as DEGs (differentially expressed genes) between breast tumor and normal tissue and DEGs between metastatic breast cancer and breast cancer in situ. Then, we performed a series of in silico studies on BIRC5 and CDO1 using online tools including the UALCAN, TIMER, TCGA-BRCA, LinkedOmics Kaplan-Meier Plotter, and an R script for analysis. To verify the association of 2 genes expression and patients' clinical data. we detected BIRC5 and CDO1 mRNA in the tissue of 48 breast cancer patients. The results showed the tumor with BIRC5(high) CDO1(low) expression generally indicated patients' shorter overall (OS) and relapse-free survival (RFS). Specifically. BIRC5 and CDO1 levels significantly affect OS or RFS in patients with Lymph node metastasis and molecular subtypes of TNBC (triple-negative breast cancer) and Luminal A. A BIRC5(high) tumor displayed a purer tumor purity and expressed more KIR receptors on NK cells while activating more FOXP3(+)CD25(+) Treg cells. The CDO1(low) tumors infiltrated with more immunocytes leading to less tumor purity. In our verified experiment. BIRC5 mRNA level in patients with stage III and over was significantly higher than in patients with stage 0 to II, but there were no significant differences among molecular subtyping groups: TNBC tissue expressed lower CDO1 mRNA level than HER2(+) and Luminal type cancer tissue. In conclusion, a BIRC5(high) CDO1(low) expression type in breast cancer tissue indicates a poorer prognosis of patients. The potential mechanism might be increased BIRC5 expression in cancer tissue is likely to accompany NK cells inhibition, activating more Treg cells, and lacking effective CD8(+) T cells proliferation. Meanwhile, CDO1 level is positively related to more immunocytes infiltration.

Abstract Background Stool DNA (sDNA) tests and fecal immunochemical test (FIT) are used for the detection of colorectal cancer (CRC). Here we performed a novel evaluation using sDNA and FIT to assess their performance in CRC screening and monitoring in Hubei, China. Methods Stool samples were collected from a high-risk population in Hubei, China (n = 359). sDNA tests and FIT were performed to test for KRAS mutations, NDRG4 and BMP3 methylation, and check hemoglobin levels. The methylation in BMP3 and NDRG4 genes was detected by TaqMan PCR method from human fecal samples. KRAS gene mutation in human fecal DNA was tested using TaqMan probe and amplification-refractory mutation system method. The colloid gold method was used for detection of hemoglobin in fecal samples. Finally, a novel evaluation by software was used to calculate the comprehensive value of the combined results for CRC detection and monitoring. Results The sensitivity and specificity of the novel evaluation for early CRC (stage I and II), advanced adenoma (AA), and non-colon cancer neoplasm (NA) detection were 95.45% and 81.6%, 29.63% and 75.9%, and 23.08% and 75.17%, respectively. The receiver operating characteristic (ROC) curves of the combined value for the above diseases were 0.945 ± 0.015, 0.543 ± 0.055, and 0.547 ± 0.038, respectively. The levels of the novel evaluation were not significantly associated with the pathology and stage (P > 0.05). In 20 out of 22 CRC patients, the novel evaluation of sDNA and FIT had decreased below threshold (< 165) at after surgery. Discussion The novel evaluation with sDNA test and FIT has increased sensitivity for screening of CRC and AA. The novel evaluation may have potential importance as an indicator of early CRC. Additionally, the dynamic changes of the comprehensive value after surgery were correlated with CRC treatment.

BACKGROUND:Circulating tumor cells (CTCs) and survivin are indicators for tumor stage and metastasis, as well as epitheliomesenchymal transition, in various cancers, including hepatocellular cancer (HCC).AIM:To explore the potential of survivin-positive CTCs, specifically, as a marker for tumor progression in HCC patients.METHODS:We examined the survivin expression pattern in CTCs obtained from 179 HCC patients, and investigated the in vitro effects of survivin silencing and overexpression on the proliferation and invasion of HCC cells. CTC count and survivin expression in patient samples were examined using RNA in situ hybridization.RESULTS:All 179 patients were positive for CTC markers, and 94.41% of the CTCs were positive for survivin. The CTC and survivin-positive CTC counts were significantly higher in the HCC patients than in the normal controls, and were significantly associated with tumor stage and degree of differentiation. Further, survivin overexpression was found to induce HepG2 cell proliferation, reduce apoptosis, and improve invasive ability.CONCLUSION:Survivin shows upregulated expression (indicative of anti-apoptotic effects) in HCC. Thus, survivin-positive CTCs are promising as a predictor of HCC prognosis and metastasis, and their accurate measurement may be useful for the management of this cancer.

Complete autotrophic nitrogen removal over nitrite (CANON) is used in wastewater treatment. However, the performance of the CANON system significantly decreases at low temperatures; thus, a new strategy to improve the resistance of the CANON system is required. To investigate the impact of sludge ratio control (high-granule, equivalent, and high-floc systems) on the resistance of CANON to low temperatures, and their recovery after restoring to normal temperature, the nitrogen removal performance of hybrid systems with different ratios was evaluated. The equivalent system had the lowest nitrite accumulation rate and highest nitrogen removal rate. Anaerobic ammonia oxidation was the rate-limiting step of each system, and hzs was the rate-limiting gene. The higher anaerobic ammonium oxidizing bacteria (AAOB) abundance and hzs expression levels resulted in an equivalent system with better resistance and recovery to short-term low temperatures at the gene level.

Myocardial infarction (MI) will inevitably result in cardiac fibrosis. In this study, we investigated the effect of microRNA-145 (miR-145) and transcription factor sex-determining region Y box 9 (SOX9) in the production of cardiac fibrosis induced by MI. MI rat models were established by left anterior descending coronary artery (LAD) occlusion. Four weeks after LAD, the cardiac fibrosis level was assessed by Masson's trichrome staining. Cardiac fibroblasts (CFs) exposed to hypoxia were used to simulate MI-induced fibrosis. Flow cytometry, cell counting kit-8, and transwell assays were used to examine changes in CF apoptosis, proliferation, and migration, respectively. miR-145 expression was measured by quantitative real-time polymerase chain reaction. Immunofluorescence and Western blot analysis were performed to determine the relative expression of proteins. In comparison to the sham-operated group, the expression of miR-145 was significantly downregulated in the infarction peripheral area, whereas, SOX9 was upregulated. In the infarcted heart, the overexpression of miR-145 significantly ameliorated cardiac fibrosis and cardiac function, and there was a negative correlation between miR-145 and SOX9 expressions in hypoxic CFs in vitro. In addition, SOX9 was verified to be a functional target of miR-145. Overexpression of miR-145 or inhibition of SOX9 decreased CF proliferation, migration, and fibrosis, but augmented their apoptotic rate. Moreover, the upregulation of miR-145 or suppression of SOX9 inhibited AKT and β-catenin signaling in hypoxic CFs. Taken together, this study highlights a potential treatment for cardiac fibrosis through the targeted regulation of SOX9 by miR-145, and our findings indicate that miR-145 exerts anti-fibrotic effects in MI via the negative regulation of SOX9 and its downstream AKT/GSK-3β/β-catenin pathways.

Human osteogenic progenitors are not precisely defined, being primarily studied as heterogeneous multipotent cell populations and termed mesenchymal stem cells (MSCs). Notably, select human pericytes can develop into bone-forming osteoblasts. Here, we sought to define the differentiation potential of CD146+ human pericytes from skeletal and soft tissue sources, with the underlying goal of defining cell surface markers that typify an osteoblastogenic pericyte. CD146+CD31-CD45- pericytes were derived by fluorescence-activated cell sorting from human periosteum, adipose, or dermal tissue. Periosteal CD146+CD31-CD45- cells retained canonical features of pericytes/MSC. Periosteal pericytes demonstrated a striking tendency to undergo osteoblastogenesis in vitro and skeletogenesis in vivo, while soft tissue pericytes did not readily. Transcriptome analysis revealed higher CXCR4 signaling among periosteal pericytes in comparison to their soft tissue counterparts, and CXCR4 chemical inhibition abrogated ectopic ossification by periosteal pericytes. Conversely, enrichment of CXCR4+ pericytes or stromal cells identified an osteoblastic/non-adipocytic precursor cell. In sum, human skeletal and soft tissue pericytes differ in their basal abilities to form bone. Diversity exists in soft tissue pericytes, however, and CXCR4+ pericytes represent an osteoblastogenic, non-adipocytic cell precursor. Indeed, enrichment for CXCR4-expressing stromal cells is a potential new tactic for skeletal tissue engineering.

OBJECTIVE:To evaluate the therapeutic effectiveness and safety of mesotherapy by comparing it with the classic systematic therapy in patients with osteoarthritis (OA).METHODS:Sixty patients were included and classified into two groups based on the existence of contraindications for nonsteroidal anti-inflammatory drugs (NSAIDs). These patients were treated with oral NSAIDs (Group A) or mesotherapy (Group B). After completing the treatment, the patients were followed up for 6 months. Their clinical features, laboratory results, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores were evaluated.RESULTS:A total of 50 patients completed treatment and follow-up. The patients in Group B had significantly fewer gastric acid-related complaints and requested less supplementary treatment for recurrent pain (p < 0.05). The patients in both groups exhibited decreased blood viscosity after treatment (p < 0.05). WOMAC scores, specifically those for pain and stiffness, were found to be significantly improved after either type of treatment (p < 0.05). Mesotherapy also ameliorated physical function (p < 0.05). Furthermore, Group B presented with better outcomes than Group A (p < 0.05 or p < 0.01).CONCLUSION:Our results suggest that mesotherapy is an effective and safe treatment for patients with OA. Clinicians should consider mesotherapy as an alternative therapy for patients with contraindications for NSAID use.

Ficolin-2 is an important serum complement lectin. Here, we describe novel findings indicating that serum ficolin-2 concentrations in multiple tumor patients are significantly lower than those in healthy donors. Administration of exogenous ficolin-2 or ficolin-A (a ficolin-2-like molecule in mouse), with only once, could remarkably inhibit the tumor cells growth in murine tumor models via early macrophages, dendritic cells (DCs) and CD8+ T cells, but not CD4+ T cells. Ficolin-A (FCN-A) knockout (KO) mice exhibits significantly increased tumor cell growth. Ficolin-2 induces macrophage activation, promotes M1 polarization and facilitates proliferation and antigen-specific cytotoxicity of CD8+ T cells. Ficolin-2 binds to Toll-like receptor 4 (TLR4) on macrophages and DCs and promotes their antigen-presenting abilities to CD8+ T cells. Our findings provide a new therapeutic strategy for tumors based on the triggering of immune-mediated antitumor effect by ficolin-2.

The expression of IL-17A and programmed death ligand 1 (PDL1) is increased in estrogen receptor-negative breast cancer. IL-17A promotes tumor cell survival and invasiveness and inhibits the antitumor immune response. The PDL1-PD1 (programmed death protein 1) signaling pathway promotes escape from immune surveillance in tumor cells. The pro-tumor properties of IL-17A and PDL1 in various cancers have been previously examined; however, the relationship and roles of IL-17A and PDL1 in ER-negative breast cancer have not been evaluated. Therefore, we assessed whether IL-17A promotes PDL1 expression in tumor cells and whether targeting of IL-17A could inhibit ER-negative breast cancer progression in a murine model. Our study revealed that IL-17A promoted PDL1 expression in human and mouse cells. In the murine cancer model, targeting of IL-17A inhibited PDL1 expression in the tumor microenvironment, decreased the percentage of Treg cells in tumor-infiltrating lymphocytes, and promoted CD4(+) and CD8(+) T cells to secrete interferon gamma. More importantly, treatment with combined anti-IL-17A and anti-PDL1 antibodies enhanced antitumor effects in favor of tumor eradication. Thus, our study established a pro-tumor role of IL-17A in promoting tumor immune escape and supports the development of a novel cytokine immunotherapy against breast cancer.

To investigate the effects and potential mechanism of L161982 (a kind of EP4 antagonist) on the collagen-induced arthritis (CIA) mice model.

Context: The audiological features and cochlear morphology of individuals with noise-induced hearing loss (NIHL) are well characterized. However, the molecular processes in the cochlea are not well understood. Aims: To explore the role of the endoplasmic reticulum stress (ERS) response in the guinea pig model of cochlear damage induced by exposure to intense noise. Settings and Design: A pilot case–control study. Subjects and Methods: Forty-eight guinea pigs were divided into four equal groups. At 1, 4, or 14 days (d) post-exposure, the auditory brainstem responses (ABRs) were tested before sacrificing the subjects. The expression levels of the binding immunoglobulin protein/glucose-regulated protein 78 (BiP/GRP78) and C/EBP-homologous protein/growth arrest and DNA damage-inducible gene 153 (CHOP/Gadd153) proteins were evaluated using immunohistochemistry and Western blotting. The number of cochlear hair cells with altered nuclei was counted using confocal fluorescence microscopy. Statistical analysis used: One-way analysis of variance (ANOVA) and the least squares difference (LSD) test. Results: The outer hair cells (OHCs) showed changes of apoptosis, necrosis, and loss after noise exposure. In the 1- and 4-d groups, more apoptotic cells were found than necrotic cells (P < 0.01). The level of BiP/GRP78 was significantly higher in all three experimental groups compared to the control group (P < 0.01). The level of CHOP/Gadd153 was increased at 1 d post-exposure, achieving a peak that was maintained until 4 d, after which it returned to baseline levels by 14 d post-exposure. Conclusions: ERS response was activated by inducing the expression of BiP/GRP78 to lessen the extent of the resulting cellular damage and activating the CHOP/Gadd153 pathway to eliminate the most severely damaged cells.

D-dimer level in cancer patients is associated with risk of venous thromboembolism and deep venous thrombosis. Most cancer patients have “abnormal” D-dimer levels based on the current normal reference range. To investigate tumor-specific D-dimer reference range, we compared D-dimer levels for nine different tumour types with healthy controls by using simultaneous quantile regression and constructing a median, 5th percentile, and 95th percentile model of normal tumour D-dimer concentration. Associations with tumour primary site, stage, pathological type, and treatment were also explored. Additionally, 190 patients were tracked to reveal the relevance of initial D-dimer levels to cancer prognosis. D-dimer ranges (median, 5th, 95th) in various cancers (mg/L) were: liver 1.12, 0.27, 5.25; pancreatic 0.96, 0.23, 4.81; breast 0.44, 0.2, 2.17; gastric 0.65, 0.22, 5.03; colorectal 0.73, 0.22, 4.45; lung 0.7, 0.25, 4.0; gynaecological 0.61, 0.22, 3.98; oesophageal 0.23, 0.7, 3.45; and head and neck 0.22, 0.44, 2.19. All were significantly higher than that of healthy controls (0.18, 0.07, 0.57). D-dimer peaked 1–2 days postoperatively but had decreased to the normal range by 1 week. Additionally, cancer patients with high initial D-dimer were shown a tendency of poor prognosis in survival rate. In conclusion, D-dimer levels in cancer depend on patient age, tumour primary site, and tumour stage. Thrombosis prevention is necessary if D-dimer has not decreased to the tumor-specific baseline a week after surgery.

Objective To investigate the effectsandmechanism of EP4 antagonist on the mice model of rheumatoid arthritis ( CIA) .Methods The DBA/1 female mice were randomly divided into the model group, the L161982 ( EP4 antagonist ) group and the control group.Thirty-four days after the injection, the arthritis score ( AS) , the histopathologic examination and the synovitis score were evaluated on the mice.The protein expression of interleukin-17 ( IL-17 ) and monocyte chemoattractant protein-1 ( MCP-1) was detected by ELISA.The quantity of CD4 +CD25 +Foxp3 +Treg cells were determined by flow cytometry.Results On day 29, 31, 33, 35 after the first immunization, the AS of the L161982 group was remarkably lower than that of the model group.The synovitis score of the L161982 group ( 1.80 ±1.70 ) was lower than that of the model group ( 3.50 ±0.50 ) .The expression levels of IL-17 [ ( 13.52 ±3.90 ) pg/ml] and MCP-1[(15.80 ±2.10) pg/ml] in the L161982 group were significantly lower than those in the model group[IL-17:(27.51 ±8.10) pg/ml, F=24.28, P<0.01;MCP-1:(30.20 ±3.40) pg/ml, F=127.78, P <0.01].The percentages of CD4 +CD25 +Foxp3+Treg cells in lymph nodes and spleens in the L161982 group were significantly higher than those in the model group [ lymph nodes ( 4.21 ± 0.52)%vs (3.31 ±0.36)%, F=27.46,P <0.01; spleens (2.63 ±0.41)% vs (1.67 ±0.14)%, F=29.43,P <0.01].Conclusion The L161982 (EP4 antagonist) might mitigate the disease o CIA by inhibiting the serum expression of IL-17 and MCP-1, as well as increasing the differentiation of Treg cells.

Objective To detect the therapeutic effects of IL-17RA specific DNA aptamers on the mouse model of experimental osteoarthritis (OA).Methods A meniscectomy was performed on Balb/C mice to generate an animal model of OA .All the mice received weekly intra-articular ( i.a.) injections of aptamers or the control treatment for six weeks .The synovial membrane was evaluated by histomorphology and the mRNAs of critical inflammatory cytokines were measured by quantitative reverse transcriptase -polymerase chain reaction ( RT-PCR ) .Results The histological examination and quantitative RT-PCR results showed that the OA mice that injected RA 10-6, especially in combination with celecoxib , demonstrated an inhibition of synovial thickening and reduction of IL-6 level in the synovial tissue . Conclusion The results suggest that RA10-6 can inhibit synovial inflammation by blocking IL-17/IL-17RA-mediated IL-6 expression.