The Critical Role of the Shroom Family Proteins in Morphogenesis, Organogenesis and Disease.

The Shroom (Shrm) family of actin-binding proteins has a unique and highly conserved Apx/Shrm Domain 2 (ASD2) motif. Shroom protein directs the subcellular localization of Rho-associated kinase (ROCK), which remodels the actomyosin cytoskeleton and changes cellular morphology via its ability to phosphorylate and activate non-muscle myosin II. Therefore, the Shrm-ROCK complex is critical for the cellular shape and the development of many tissues, including the neural tube, eye, intestines, heart, and vasculature system. Importantly, the structure and expression of Shrm proteins are also associated with neural tube defects, chronic kidney disease, metastasis of carcinoma, and X-link mental retardation. Therefore, a better understanding of Shrm-mediated signaling transduction pathways is essential for the development of new therapeutic strategies to minimize damage resulting in abnormal Shrm proteins. This paper provides a comprehensive overview of the various Shrm proteins and their roles in morphogenesis and disease.

Gradient inflammation in the pancreatic stump after pancreaticoduodenectomy: Two case reports and review of literature.

BACKGROUND: Postoperative pancreatic fistula (POPF) contributes significantly to morbidity and mortality after pancreaticoduodenectomy (PD). However, the underlying mechanisms remain unclear. This study explored this pathology in the pancreatic stumps and elucidated the mechanisms of POPF following PD. CASE SUMMARY: Pathological analysis and 16S rRNA gene sequencing were performed on specimens obtained from two patients who underwent complete pancreatectomy for grade C POPF after PD. Gradient inflammation is present in the pancreatic stump. The apoptosis was lower than that in the normal pancreas. Moreover, neutrophil-dominated inflammatory cells are concentrated in the ductal system. Notably, neutrophils migrated through the ductal wall in acinar duct metaplasia-formed ducts. Additionally, evidence indicates that gut microbes migrate from the digestive tract. Gradient inflammation occurs in pancreatic stumps after PD. CONCLUSION: The mechanisms underlying POPF include high biochemical activity in the pancreas, mechanical injury, and digestive reflux. To prevent POPF and address pancreatic inflammation and reflux, breaking the link with anastomotic dehiscence is practical.

The m6 RNA methylation regulator KIAA1429 is associated with autophagy-mediated drug resistance in lung cancer.

N6-methyladenosine (m6A) modification plays a crucial role in cancer progression. However, the role of m6A modification-mediated autophagy underlying non-small cell lung cancer (NSCLC) gefitinib resistance remains unknown. Here, we discovered that m6A methyltransferase KIAA1429 was highly expressed in NSCLC gefitinib-resistant cells (PC9-GR) as well as tissues, and KIAA1429 high expression was associated with poor survival. In addition, silent KIAA1429 repressed gefitinib resistance in NSCLC and reduced tumor growth in vivo. Mechanistically, KIAA1429 stabilized WTAP, a significant player in autophagy, by binding to the 3' untranslated regions (3'-UTR) of WTAP. In a word, our findings indicated that KIAA1429 could elevate NSCLC gefitinib resistance, which may provide a promising targeted therapy for NSCLC patients.

Antioncogenic roles of USP9Y and DDX3Y in lung cancer: USP9Y stabilizes DDX3Y by preventing its degradation through deubiquitination.

In previous studies, downregulation of USP9Y and DDX3Y in lung cancer (LC) tissues was identified, while their function in LC progression remains elusive. In our current work, we intended to elucidate the effect and mechanisms of USP9Y and DDX3Y in LC. Gene downregulation has been confirmed in our LC tissues and cells. The effect of USP9Y or DDX3Y on LC cell malignancies was analyzed by functional assay. Both USP9Y and DDX3Y overexpression showed suppressive impact on LC cell malignancies. USP9Y overexpression has also been demonstrated to inhibit tumorigenesis in vivo. Based on GEPIA database, it was found that there was a positive correlation between the levels of USP9Y and DDX3Y in LC tissues. The mRNA expression of DDX3Y was not affected by USP9Y overexpression, while its protein levels were significantly up-regulated in USP9Y overexpressed LC cells. Moreover, USP9Y interacted with DDX3Y and has been demonstrated to stabilize DDX3Y expression by preventing its degradation via deubiquitination. In conclusion, USP9Y and DDX3Y exerted antioncogenic effects on the cell proliferation potential, cell cycle process, apoptosis, and tumorigenesis of LC. USP9Y binds to DDX3Y to prevent DDX3Y degradation through deubiquitination.

Effect of laparoscopic pancreaticoduodenectomy on the incidence of surgical-site wound infection: A meta-analysis.

A meta-analysis was conducted to assess the impact of robotic and laparoscopic pancreaticoduodenectomies on postoperative surgical site wound infections. A comprehensive computerised search of databases, such as PubMed, EMBASE, Cochrane Library, Web of Science, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, and Wanfang Data, was performed to identify studies comparing robotic pancreaticoduodenectomy (PD) with laparoscopicPD. Relevant studies were searched from the inception of the database construction until April 2023. The meta-analysis outcomes were analysed using odds ratios (OR) with corresponding 95% confidence intervals (CI). The RevMan 5.4 software was used for the meta-analysis. The findings of the meta-analysis showed that patients who underwent laparoscopic PD had a significantly lower incidence of surgical-site wound (16.52% vs. 18.92%, OR: 0.78, 95% CI: 0.68-0.90, P = .0005), superficial wound (3.65% vs. 7.57%, OR: 0.51, 95% CI: 0.39-0.68, P < .001), and deep wound infections (1.09% vs. 2.23%, OR: 0.53, 95% CI: 0.34-0.85, P = .008) than those who received robotic PD. However, because of variations in sample size between studies, some studies suffered from methodological quality deficiencies. Therefore, further validation of this result is needed in future studies with higher quality and larger sample sizes.

The molecular mechanisms of alpha-lipoic acid on ameliorating aflatoxin B1-induced liver toxicity and physiological dysfunction in northern snakehead (Channa argus).

This research aimed to evaluate the protective mechanism of alpha-lipoic acid (α-LA) on the food-borne aflatoxin B1 (AFB1) exposure-induced liver toxicity and physiological dysfunction in the northern snakehead (Channa argus). 480 fish (9.24±0.01 g) were randomly assigned to four treatment groups and fed with four experimental diets for 56 d including the control group (CON), AFB1 group (200 ppb AFB1), 600 α-LA group (600 ppm α-LA+200 ppb AFB1), and 900 α-LA group (900 ppm α-LA+200 ppb AFB1). The results revealed that 600 and 900 ppm α-LA attenuated AFB1-induced growth inhibition and immunosuppression in northern snakehead. 600 ppm α-LA significantly decreased the serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase levels, and AFB1 bioaccumulation, and attenuated the changes of hepatic histopathological and ultrastructure induced by AFB1. Moreover, 600 and 900 ppm α-LA significantly up-regulated phase I metabolism genes (cytochrome P450-1a, 1b, and 3a) mRNA expression, inhibited the levels of malondialdehyde, 8­hydroxy-2 deoxyguanosine and reactive oxygen species in the liver. Notably, 600 ppm α-LA significantly up-regulated the expression levels of nuclear factor E2 related factor 2 and its related downstream antioxidant molecules (heme oxygenase 1 and NAD(P)H: quinone oxidoreductase 1, etc.), increased the phase II detoxification enzyme-related molecules (glutathione-S-transferase and glutathione), antioxidant parameters (catalase and superoxide dismutase, etc.), and the expressions of Nrf2 and Ho-1 protein in the presence of AFB1 exposure. Furthermore, 600 and 900 ppm α-LA significantly reduced the characteristic indices of AFB1-induced endoplasmic reticulum stress (glucose-regulated protein 78 and inositol requiring enzyme 1, etc.), apoptosis (caspase-3 and cytochrome c, etc.) and inflammation (nuclear factor kappa B and tumor necrosis factor α, etc.), while increased the B-cell lymphoma-2 and inhibitor of κBα in the liver after being exposed to AFB1. To summarize, the above results indicate that dietary α-LA could modulate the Nrf2 signaling pathway to ameliorate AFB1-induced growth inhibition, liver toxicity, and physiological dysfunction in northern snakehead. Although the concentration of α-LA increased to 900 ppm from 600 ppm, the protective effects of the 900 ppm α-LA do not show an advantage over the 600 ppm α-LA, and even show inferiority in some respects. So that the recommended concentration of α-LA is 600 ppm. The present study provides the theoretical foundation for developing α-LA as the prevention and treatment of AFB1-induced liver toxicity in aquatic animals.

Novel exopolysaccharide derived from probiotic Lactobacillus pantheris TCP102 strain with immune-enhancing and anticancer activities.

Probiotics are gaining attention due to their functions of regulating the intestinal barrier and promoting human health. The production of exopolysaccharide (EPS) is one of the important factors for probiotics to exert beneficial properties. This study aimed to screen exopolysaccharides-producing lactic acid bacteria (LAB) and evaluate the probiotic potential. we obtained three exopolysaccharide fractions (EPS1, EPS2, and EPS3) from Lactobacillus pantheris TCP102 and purified by a combination of ion-exchange chromatography and gel permeation chromatography. The structures of the fractions were characterized by FT-IR, UV, HPLC, and scanning electron microscopy (SEM) analysis. The Mw of EPS1, EPS2, and EPS3 were approximately 20.3, 23.0, and 19.3 kDa, and were mainly composed of galactose, glucose, and mannose, with approximate molar ratios of 2.86:1:1.48, 1.26:1:1, 1.58:1.80:1, respectively. Furthermore, SEM analysis demonstrated that the three polysaccharide fractions differ in microstructure and surface morphology. Additionally, preliminary results for immune-enhancing and anticancer activities reveal that these EPSs significantly induced the production of nitric oxide (NO), TNF-α, and IL-6 in Ana-1 cells and peritoneal macrophage cells. Meanwhile, the EPSs also significantly suppressed the proliferation of HCT-116, BCG-803, and particularly A-2780 cells. The results suggest that the three novel EPSs isolated from Lactobacillus pantheris TCP102 can be regarded as potential application value in functional food and natural antitumor drugs.

Exopolysaccharides from Lactobacillus kiferi as adjuvant enhanced the immuno-protective against Staphylococcus aureus infection.

Exopolysaccharides from lactic acid bacteria (LAB) have gained more attention due to their health benefits. Most research on LAB EPS focuses on antitumor and antioxidant activities. To our knowledge, the immunoadjuvant activity of LAB EPS has not been thoroughly studied. In this study, the EPS produced by Lactobacillus kiferi WXD029 were purified by ethanol precipitation and column chromatography fractionation. The molecular weight of the EPS was 3.423 × 105 Da and was mainly composed of Glu, GlcN, and GalN in a molar ratio of 3.1:1:1. In vitro, EPS could significantly enhance the proliferation and phagocytic activity as well as induce the production of NO, TNF-α, IL-1ß, and IL-6 in RAW264.7 cells. In vivo, the EPS adjuvant could increase the titers of S.aureus antigen-specific antibodies and markedly enhanced T cell proliferation. Notably, EPS adjuvant also induced a strong potential Th1, Th2 and Th17-cell mixture responses. Furthermore, immunization with S.aureus antigen plus EPS adjuvant induced a protective effect when compared with S.aureus antigen alone in murine bacteremia, pneumonia and mastitis model. Collectively, these results suggest that EPS derived from probiotic Lactobacillus kiferi strain is promising as an efficient adjuvant candidate for the prevention of S. aureus infections.

Protective effect of triterpenes of Ganoderma lucidum on lipopolysaccharide-induced inflammatory responses and acute liver injury.

Ganoderma lucidum is a popular medicinal mushroom, which has been used as therapeutic for centuries in traditional Chinese medicine. Although G. lucidum showed strong protective effects in prevention or treatment of a variety of inflammatory diseases, the mechanisms underlying the anti-inflammatory properties of triterpenes of G. lucidum remain undefined. In the current study, we demonstrated that ethanol extract and triterpenes of G. lucidum specifically suppressed LPS-mediated inflammatory responses. Notably, ganodermanontriol inhibited the expressions and interactions of TLR4 and MyD88, NF-κB translocation to nucleus and its DNA binding activity, phosphorylation of p38, ErK1/2 and JNK. In vivo, we showed that ganodermanontriol effectively prevented LPS/D-Galactosamine-induced liver injury by reducing TNF-α and IL-6 production, and decrease of ALT/AST release. Collectively, our results revealed a novel role in inhibition of inflammatory diseases for triterpenes that may act through potential inhibition of TLR4-MyD88-mediated NF-κB and MAPK signaling pathways.

Anti-cancer effect of marchantin C via inducing lung cancer cellular senescence associated with less secretory phenotype.

BACKGROUND: Lung cancer is the leading cause of global cancer deaths. Current chemotherapeutic agents for lung cancer treatment are generally accompanied with severe side effects. Here, we report that marchantin C (Mar-C), a potential natural compound with little chemotherapeutic toxicity, exerts a well anti-tumor effect against lung cancer via inducing cellular senescence. METHODS: The antitumor activity of Mar-C was evaluated by MTT and colony formation in vitro cytotoxicity assays, and xenograft and homograft in vivo model. Antitumor mechanisms of Mar-C were investigated through SA-ß-gal staining, Q-PCR, immunoblotting, immunofluorescence, protein array and siRNA knocking-down analysis. RESULTS: Mar-C selectively induces senescence of lung cancer cells with limited cytotoxicity on normal or non-neoplastic cells. Mar-C-induced senescence was associated with the elevation of ROS and activation of DNA-damage, and largely dependent of prolonged p21CIP1 accumulation. The senescence-associated secretory phenotype (SASP) induced by Mar-C was distinct from doxorubicin-induced. Furthermore, Mar-C exhibited an inhibitory activity on tumor growth with little toxicity in animal studies, and significantly prolonged the survival time of tumor-bearing mice than that of doxorubicin or vehicle treatments. CONCLUSION: Mar-C selectively inhibited tumor growth via the induction of cancer cell senescence and had little chemotherapeutic toxicity, suggesting the potential of Mar-C as a promising anticancer agent. GENERAL SIGNIFICANCE: This study provided evidence to identify a novelty of Mar-C that exerted antitumor activity on lung cancer through induction of senescence with limited toxicity.

α-Pyrone Derivatives from a Streptomyces Strain Resensitize Tamoxifen Resistance in Breast Cancer Cells.

Tamoxifen resistance (TamR) is the underlying cause of treatment failure in many breast cancer patients receiving tamoxifen. In order to look for noncytotoxic natural products with the ability to reverse TamR, an extract from strain Streptomyces sp. KIB-H0495 was detected to be active. Subsequent large scale fermentation and isolation led to the isolation of four α-pyrone derivatives including two new compounds, violapyrones J (2) and K (3), and two known analogues, violapyrones B (1) and I (4). Further bioactivity assays indicated that only 1 and 3 exerted potent resensitization effects on MCF-7/TamR cells at a concentration of 1 µM. Owing to the simple structures of 1 and 3, these two compounds might have potential for further investigation as novel tamoxifen resensitization agent in breast cancer chemotherapy.

HuR mediates motility of human bone marrow-derived mesenchymal stem cells triggered by sphingosine 1-phosphate in liver fibrosis.

Sphingosine 1-phosphate (S1P) participates in migration of bone marrow (BM)-derived mesenchymal stem cells (BMSCs) toward damaged liver via upregulation of S1P receptor 3 (S1PR3) during mouse liver fibrogenesis. But, the molecular mechanism is still unclear. HuR, as an RNA-binding protein, regulates tumor cell motility. Here, we examined the role of HuR in migration of human BMSCs (hBMSCs) in liver fibrosis. Results showed that HuR messenger RNA (mRNA) level was increased in human or mouse fibrotic livers, and correlated with S1PR3 mRNA expression. Using immunofluorescence, we found that HuR mainly localized in the nuclei of hepatocytes and non-parenchymal cells in normal livers. However, in fibrotic livers, we detected an increased HuR cytoplasmic localization in non-parenchymal cells. In chimeric mice of BM cell-labeled by EGFP, significant numbers of EGFP-positive cells (BM origin) were positive for HuR in fibrotic areas. Meanwhile, HuR-positive cells were also positive for α-SMA (myofibroblasts). In vitro, S1P induced hBMSCs migration via S1PR3 upregulation. HuR involved in S1P-induced hBMSCs migration and increased stabilization of S1PR3 mRNA via competing with miR-30e. RNA immunoprecipitation showed that HuR interacted with S1PR3 mRNA 3'UTR. Moreover, S1P resulted in phosphorylation and cytoplasmic translocation of HuR via S1PR3 and p38MAPK. Furthermore, we transplanted EGFP+ BMSCs with or without HuR small interfering RNA (siRNA) into carbon tetrachloride-treated mice and found that knockdown of HuR inhibited the migration of BMSCs toward injured livers by flow cytometric analysis in vivo. We identified a positive feedback regulation mechanism between HuR and S1PR3 in S1P-induced BMSCs migration. HuR participates in upregulation of S1PR3 induced by S1P. S1P results in phosphorylation and translocation of HuR via S1PR3. Our results provide a new regulatory manner to the mechanism of liver fibrogenesis. KEY MESSAGE: HuR expression and cytoplasmic localization were increased in fibrotic livers. S1P induced migration of human bone marrow Mesenchymal Stem Cells via S1PR3 and HuR. HuR regulated S1PR3 mRNA expression by binding with S1PR3 mRNA 3'UTR. S1P induced HuR phosphorylation and cytoplasmic translocation via S1PR3. HuR regulated S1PR3 expression by competing with miR-30e.

Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform.

Large-scale genomics studies have generated vast resources for in-depth understanding of vital biological and pathological processes. A rising challenge is to leverage such enormous information to rapidly decipher the intricate protein-protein interactions (PPIs) for functional characterization and therapeutic interventions. While a number of powerful technologies have been employed to detect PPIs, a singular PPI biosensor platform with both high sensitivity and robustness in a mammalian cell environment remains to be established. Here we describe the development and integration of a highly sensitive NanoLuc luciferase-based bioluminescence resonance energy transfer technology, termed BRET(n), which enables ultra-high-throughput (uHTS) PPI detection in live cells with streamlined co-expression of biosensors in a miniaturized format. We further demonstrate the application of BRET(n) in uHTS format in chemical biology research, including the discovery of chemical probes that disrupt PRAS40 dimerization and pathway connectivity profiling among core members of the Hippo signaling pathway. Such hippo pathway profiling not only confirmed previously reported PPIs, but also revealed two novel interactions, suggesting new mechanisms for regulation of Hippo signaling. Our BRET(n) biosensor platform with uHTS capability is expected to accelerate systematic PPI network mapping and PPI modulator-based drug discovery.

Levamisole promotes murine bone marrow derived dendritic cell activation and drives Th1 immune response in vitro and in vivo.

Our lab previously found that levamisole (LMS) as an adjuvant enhanced the efficacy of vaccine against infectious pathogens. However, the cellular and molecular mechanisms remain to be defined. In this study, we showed that BALB/c bone marrow-derived DC stimulated with LMS resulted in enhanced cell-surface expression of CD80, CD86, CD40 and MHC class II, as well as enhanced production of IL-12p70, TNF-α and IL-1ß. Interestingly, the LMS activated DCs were able to stimulate CD4(+) T cell proliferation and facilitated Th1 differentiation by increasing the secretion of IFN-γ in an allogeneic mixed leukocyte reaction. Furthermore, to confirm the in vitro data, we investigated the effect of LMS on antigen-specific antibody and cytokine production in BALB/c mice. Immunization with LMS plus OVA showed that anti-OVA IgG2a and IFN-γ were increased significantly compared with OVA alone in BALB/c mice. In conclusion, our results suggested that murine bone marrow-derived DC, played a crucial role in the effect of LMS on the induction of Th1 responses, which probably was due to its ability to promote DC maturation and secrete proinflammatory cytokines.

Hepcidin and iron metabolism in non-diabetic obese and type 2 diabetic rats.

The aim of this study was to investigate the changes of iron levels and hepatic regulatory molecules expression involved in iron metabolism in non-diabetic obese/type 2 diabetic rat models. Male Wistar rats were divided into 3 groups: control group, non-diabetic obese group and type 2 diabetic group (n=20 each). The rats were evaluated physiologically and biochemically. The hepatic histopathological changes were observed using haematoxylin and eosin (HE) staining. The mRNA expression patterns of hepcidin, interleukin-6 (IL-6), hypoxia-inducible factor (HIF) and ferroportin (Fpn) in the rat liver in control group, non-diabetic obese group and type 2 diabetic group were analyzed by real-time RT-PCR. The protein expression patterns of hepcidin in liver of each group were further analyzed by immunohistochemistry and Western blotting. As compared with control group, the ferritin in non-diabetic obese group and type 2 diabetic group was increased significantly (P<0.001). However, there was no significant difference in soluble transferring receptor (sTfR):ferritin ratio among the three groups (P>0.05). The real-time RT-PCR, immunohistochemistry and Western blotting results all revealed that the expression levels of hepcidin in non-diabetic obese group and type 2 diabetic group were elevated significantly as compared with those in control group (P<0.001). The expression levels of hepcidin mRNA between non-diabetic obese group and type 2 diabetic group showed no significant difference (P>0.05). However, the protein expression levels of hepcidin in type 2 diabetic group were significantly higher than those in non-diabetic obese group (P<0.05). Compared to control group, the expression levels of IL-6 mRNA in non-diabetic obese group and type 2 diabetic group were increased significantly and the expression levels of Fpn mRNA decreased (P<0.05). However, the expression levels of HIF mRNA had no significant difference among three groups. It is suggested that iron metabolism is substantially disturbed in non-diabetic obese and type 2 diabetic rats probably by the abnormal expression of hepcidin in chronic inflammatory status. The increased hepcidin may restrain the iron release from the cells by affecting the expression of Fpn, which probably associates with the development of diabetic complication.

[Effect of cervical disc arthroplasty and anterior cervical decompression and fusion on adjacent segment degeneration].

OBJECTIVE: To compare the effect on adjacent segment degeneration after cervical disc arthroplasty (CDA) and anterior cervical decompression and fusion (ACDF) for treatment of cervical spondylosis. METHODS: Between August 2009 and February 2012, 60 cases of single segmental cervical spondylosis accorded with the inclusion criteria were included. Of 60 patients, 28 patients underwent CDA (CDA group) and 32 patients underwent ACDF (ACDF group). There was no significant difference in gender, age, disease duration, pathological type, pathological segment, the time of conservation treatment, preoperative neck disability index (NDI), preoperative Japanese Orthopaedic Association (JOA) score, and degeneration of the adjacent segment and disc between 2 groups (P > 0.05). The NDI and JOA score were used to evaluate effectiveness. The range of motion (ROM) of adjacent segment was measured, and degeneration of the adjacent segment and disc was evaluated according to Kellgren grading system based on X-ray and Miyazaki grading system based on T2-weighted MRI, respectively. RESULTS: The follow-up time was 24-50 months (mean, 34 months) in 2 groups. All patients had no complication of prosthesis loosening, dislocation, or fracture of plate. The NDI and JOA scores from 12 months after operation were significantly improved compared with preoperative scores in 2 groups (P < 0.05), but no significant difference was found at each time point between 2 groups (P > 0.05). The improvement rate of JOA was 80.68% ± 4.01% in ACDF group and was 79.44% ± 3.76% in CDA group at last follow-up, showing no significant difference (t = -1.237, P = 0.221). And the improvement rate of JOA in 2 groups were excellent. There was no significant difference in ROM and degeneration grading of adjacent segments between at last follow-up and at pre-operation in 2 groups (P > 0.05), and between 2 groups at pre-operation and at last follow-up (P > 0.05). The degeneration grading of disc at last follow-up showed significant difference in 2 groups compared with preoperative ones (P < 0.05), but no significant difference was found between 2 groups (Z = 0.132, P = 0.895). CONCLUSION: Both CDA and ACDF can achieve good effectiveness in treating cervical spondylosis, but CDA can not significantly slow down the degeneration of adjacent segment disc.

Multiplex real-time PCR for RRM1, XRCC1, TUBB3 and TS mRNA for prediction of response of non-small cell lung cancer to chemoradiotherapy.

BACKGROUND: This study was aimed to establish a novel method to simultaneously detect expression of four genes, ribonucleotide reductase subunit M1(RRM1), X-ray repair cross-complementing gene 1 (XRCC1), thymidylate synthase (TS) and class III ß-tubulin (TUBB3), and to assess their application in the clinic for prediction of response of non-small cell lung cancer (NSCLC) to chemoradiotherapy. MATERIALS AND METHODS: We have designed four gene molecular beacon (MB) probes for multiplex quantitative real-time polymerase chain reactions to examine RRM1, XRCC1, TUBB3 and TS mRNA expression in paraffin-embedded specimens from 50 patients with advanced or metastatic carcinomas. Twenty one NSCLC patients receiving cisplatin- based first-line treatment were analyzed. RESULTS: These molecular beacon probes could specially bind to their target genes in homogeneous solutions. Patients with low RRM1 and XRCC1 mRNA levels were found to have apparently higher response rates to chemoradiotherapy compared with those with high levels of RRM1 and XRCC1 expression (p<0.05). The TS gene expression level was not significantly associated with chemotherapy response (p>0.05). CONCLUSIONS: A method of simultaneously detecting four molecular markers was successfully established and applied for evaluation of chemoradiotherapy response. It may be a useful tool in personalized cancer therapy.

Staphylococcus aureus induces apoptosis in primary bovine mammary epithelial cells through Fas-FADD death receptor-linked caspase-8 signaling.

Infections with Staphylococcus aureus, a common inducer of mastitis, often result in mammary gland damage and death of various cell types. Although S. aureus was suggested to induce apoptosis in a bovine mammary epithelial cell (BMEC) line, MAC-T, it is unknown whether primary BMECs (pBMECs) apoptosis is triggered by S. aureus and the associated underlying molecular mechanisms have not been determined. Here, we demonstrated that S. aureus induced apoptosis in pBMECs in a time- and dose-dependent manner. Further, S. aureus-induced apoptosis in pBMECs was associated with activation of caspase-3 and caspase-8, but caspase-9 was not. In addition, pBMECs apoptosis was mitigated by caspase-3 and caspase-8 inhibitors, suggesting that apoptosis is initiated via caspase-8 activation. Moreover, S. aureus infection significantly increased expressions of Fas and Fas-associated death domain (FADD) of pBMECs. Taken together, our results demonstrated that S. aureus induced apoptosis in pBMECs via the Fas-FADD death receptor and subsequently triggered the caspase-8-dependent signaling.

Sphingosine 1-phosphate receptors negatively regulate collagen type I/III expression in human bone marrow-derived mesenchymal stem cell.

Collagen is the most abundant structural protein in mammals and is expressed in various tissues. In recent years, sphingosine 1-phosphate receptors (S1PRs) have been proven to play an important role in the regulation of collagen expression. Our previous studies reported that S1PRs are involved in TGF-ß1-induced collagen expression via up-regulating S1PR1/3 in mouse bone marrow-derived mesenchymal stem cells (BMSCs), and result in experimental mouse liver fibrogenesis. But it remains unclear whether this process happens in human bone marrow-derived mesenchymal stem cells (hMSCs). In this study, we provide evidences that S1PR1/3, but not S1PR2, negatively regulate the expression of collagen in hMSCs using cellular and molecular approaches in vitro. We find that treatment of hMSCs with TGF-ß1 up-regulated collagen expression in a dose- and time-dependent manner. Meanwhile, TGF-ß1 inhibited the expression of S1PR1/3, but not S1PR2, in hMSCs in a time-dependent manner. Furthermore, either selective knock-down of S1PR1 or silencing S1PR3 induced collagen α1(I) and collagen α1(III) expression in hMSCs. In contrast, inhibition of S1PR2 by siRNA had no effects on the expression of collagen. Altogether, all these findings demonstrated that collagen expression was negatively regulated by S1PR1 and S1PR3 in hMSCs. This study highlights the differences between hMSCs and mouse BMSCs, provides a new regulation mechanism for collagen expression, and points out the risk of utilizing hMSCs in clinical applications.

[EXPRESSIONS OF TRANSFORMING GROWTH FACTOR ß1 AND CONNECTIVE TISSUE GROWTH FACTOR IN HUMAN LUMBAR INTERVERTEBRAL DISCS IN DIFFERENT DEGREES OF DEGENERATION].

OBJECTIVE: To investigate the role of transforming growth factor ß1 (TGF-ß1) and connective tissue growth factor (CTGF) in pathogenesis and progression of human intervertebral disc degeneration by detecting the expressions of these two factors in different degrees of degenerative discs. METHODS: The lumbar intervertebral discs were collected from 33 patients with lumbar disc herniation and 12 patients with lumbar vertebral fracture between November 2012 and April 2013. All samples were observed under the microscope after HE staining, and then were divided into different subgroups according to the degenerative degree. The expressions of TGF-ß1 and CTGF were detected by Western blot. RESULTS: According to the pathological features, 10 discs were defined as normal discs, 10 as mild degenerative discs, 9 as moderate degenerative discs, and 16 as severe degenerative discs. The histological observation showed that rounded nucleus pulposus cells with similar size evenly distributed in the cartilage-like matrix, and no hyperplastic collagenous fiber was seen in normal discs; mild degenerative discs characterized by slightly larger nucleus pulposus cells in the matrix, but cells did not decrease, a small quantity of inflammatory cells infiltrated in the matrix, hyperplasia of collagenous fiber was not seen; most of the nucleus pulposus cells became bigger, some showed a bulb form, the number of nucleus pulposus cells was significantly reduced, low grade hyperplasia of collagenous fiber emerged in the matrix, new vessels and inflammatory cells were both found in some specific areas of discs in moderate degenerative discs; there was no nucleus pulposus cells in the matrix of severe degenerative discs, the hyperplasia of collagenous fiber was obvious. The relative expression of TGF-ß1 in 3 degeneration discs was significantly higher than that in normal discs (P < 0.05), and the expression of TGF-ß1 was significantly higher in severe degenerative discs than in moderate and mild degenerative discs (P < 0.05), but no significant difference between moderate and mild degenerative discs (P > 0.05). The relative expression of CTGF in moderate and severe degeneration discs was significantly higher than that in normal discs (P < 0.05); and the expression of CTGF in mild degenerative discs was higher than that in normal discs, but there was no significant difference (P > 0.05); and significant difference in CTGF expression was found among 3 degeneration discs (P < 0.05). CONCLUSION: The expressions of TGF-ß1 and CTGF are closely related to the degree of human lumbar disc degeneration, these two factors may play an important role in promoting lumbar intervertebral disc degeneration.