Peripheral nerve fibroblasts secrete neurotrophic factors to promote axon growth of motoneurons.

Peripheral nerve fibroblasts play a critical role in nerve development and regeneration. Our previous study found that peripheral nerve fibroblasts have different sensory and motor phenotypes. Fibroblasts of different phenotypes can guide the migration of Schwann cells to the same sensory or motor phenotype. In this study, we analyzed the different effects of peripheral nerve-derived fibroblasts and cardiac fibroblasts on motoneurons. Compared with cardiac fibroblasts, peripheral nerve fibroblasts greatly promoted motoneuron neurite outgrowth. Transcriptome analysis results identified 491 genes that were differentially expressed in peripheral nerve fibroblasts and cardiac fibroblasts. Among these, 130 were significantly upregulated in peripheral nerve fibroblasts compared with cardiac fibroblasts. These genes may be involved in axon guidance and neuron projection. Three days after sciatic nerve transection in rats, peripheral nerve fibroblasts accumulated in the proximal and distal nerve stumps, and most expressed brain-derived neurotrophic factor. In vitro, brain-derived neurotrophic factor secreted from peripheral nerve fibroblasts increased the expression of ß-actin and F-actin through the extracellular regulated protein kinase and serine/threonine kinase pathways, and enhanced motoneuron neurite outgrowth. These findings suggest that peripheral nerve fibroblasts and cardiac fibroblasts exhibit different patterns of gene expression. Peripheral nerve fibroblasts can promote motoneuron neurite outgrowth.

Liver injury changes the biological characters of serum small extracellular vesicles and reprograms hepatic macrophages in mice.

BACKGROUND: Serum small extracellular vesicles (sEVs) and their small RNA (sRNA) cargoes could be promising biomarkers for the diagnosis of liver injury. However, the dynamic changes in serum sEVs and their sRNA components during liver injury have not been well characterized. Given that hepatic macrophages can quickly clear intravenously injected sEVs, the effect of liver injury-related serum sEVs on hepatic macrophages deserves to be explored. AIM: To identify the characteristics of serum sEVs and the sRNAs during liver injury and explore their effects on hepatic macrophages. METHODS: To identify serum sEV biomarkers for liver injury, we established a CCL4-induced mouse liver injury model in C57BL/6 mice to simulate acute liver injury (ALI), chronic liver injury (CLI) and recovery. Serum sEVs were obtained and characterized by transmission electron microscopy and nanoparticle tracking analysis. Serum sEV sRNAs were profiled by sRNA sequencing. Differentially expressed microRNAs (miRNAs) were compared to mouse liver-enriched miRNAs and previously reported circulating miRNAs related to human liver diseases. The biological significance was evaluated by Ingenuity Pathway Analysis of altered sEV miRNAs and conditioned cultures of ALI serum sEVs with primary hepatic macrophages. RESULTS: We found that both ALI and CLI changed the concentration and morphology of serum sEVs. The proportion of serum sEV miRNAs increased upon liver injury, with the liver as the primary contributor. The altered serum sEV miRNAs based on mouse studies were consistent with human liver disease-related circulating miRNAs. We established serum sEV miRNA signatures for ALI and CLI and a panel of miRNAs (miR-122-5p, miR-192-5p, and miR-22-3p) as a common marker for liver injury. The differential serum sEV miRNAs in ALI contributed mainly to liver steatosis and inflammation, while those in CLI contributed primarily to hepatocellular carcinoma and hyperplasia. ALI serum sEVs decreased both CD86 and CD206 expression in monocyte-derived macrophages but increased CD206 expression in resident macrophages in vitro. CONCLUSION: Serum sEVs acquired different concentrations, sizes, morphologies and sRNA contents upon liver injury and could change the phenotype of liver macrophages. Serum sEVs therefore have good diagnostic and therapeutic potential for liver injury.

[Research on quality difference of standard decoction of raw and fried Paeoniae Radix Alba based on fingerprint and multicomponent determination].

The extract rates, multicomponent content and fingerprint were determined in this study to investigate the quality diffe-rence between standard decoction of raw Paeoniae Radix Alba and fried Paeoniae Radix Alba. UPLC fingerprint was established for 17 batches of standard decoction of raw and fried Paeoniae Radix Alba, and the contents of gallic acid, catechin, albiflorin, paeoniflorin and benzoyl paeoniflorin were determined. The peak areas of standard decoction were analyzed by the independent t-test and orthogonal partial least squares discriminant analysis. There was no significant difference in extract rates between the standard decoction of raw and fried Paeoniae Radix Alba. After fried processing, the content of albiflorin increased by 0.26%, while the contents of gallic acid, catechin, paeoniflorin and benzoyl paeoniflorin decreased by 13.04%, 27.97%, 10.30% and 18.79% respectively. There were 14 common peaks in the fingerprint of standard decoction of raw Paeoniae Radix Alba, and 16 common peaks in the fried Paeoniae Radix Alba. Peak 1 and peak 3 were new ones after processing, among which the peak 3 was 5-hydroxymethylfurfural. The results showed that peak 1, peak 3, peak 11 and peak 15 were the key compounds to distinguish standard decoction of raw and fried Paeoniae Radix Alba. In conclusion, this method is stable and can be used for the study of quantity transfer and quality control in the preparation process of standard decoction, granules and other dosage forms for raw and fried Paeoniae Radix Alba, providing reference for the identification of raw and fried Paeoniae Radix Alba and related preparations.

Evaluation of the diagnostic performance of panfungal polymerase chain reaction assay in invasive fungal diseases.

Timely diagnosis of invasive fungal diseases (IFDs) is important, as delays in treatment initiation are associated with increased mortality rates. However, early diagnosis of IFDs in immunocompromised patients remains difficult. The conventional diagnostic methods currently used for IFDs are not sufficiently effective. Molecular tests, such as polymerase chain reaction (PCR)-based assays, have great potential to improve the early diagnosis of IFDs due to their sensitivity and specificity. In the present study, the diagnostic performance of panfungal PCR assays in IFD patients who received bone marrow transplantation was evaluated. The results suggested that panfungal PCR assay offered a quick and convenient guide for clinical decision-making by identifying higher numbers of fungal species in comparison with the conventional blood culture method. Furthermore, panfungal PCR assay exhibited a sensitivity of 93% and a specificity of 71% in the diagnosis of IFD patients based on the EORTC/MSG criteria. Thus, the present study concluded that the reported PCR-based method was effective and sensitive in early IFD diagnosis and should be integrated into clinical decision-making for the treatment of IFDs in the future.

Valproic acid synergistically enhances the cytotoxicity of gossypol in DU145 prostate cancer cells: an iTRTAQ-based quantitative proteomic analysis.

Gossypol (GOS), a BH3 mimetic, has been investigated as a sensitizing co-therapy to radiation and chemotherapy in treatment of metastatic prostate cancer. In this study, we found that valproic acid (VPA), a histone deacetylase inhibitor (HDACI), counteracted the suppressive effect of GOS on histone H3 acetylation and enhanced the cytotoxicity of GOS to DU145 prostate cancer cells. Significant synergistic effects were observed in combined GOS and VPA treatment, culminating in more DNA damage and cell death. The iTRAQ-based quantitative proteomic analysis revealed differential proteomic profiles in cells treated with VPA, GOS or their combination. In GOS-treated cells, oxidative phosphorylation-related proteins were depressed and endoplasmic reticulum stress markers were upregulated. In the presence of VPA, the GOS-induced mitochondrial stress was further enhanced since glycolysis- and hypoxia-associated proteins were upregulated, suggesting a disruption of energy metabolism in these cells. Furthermore, the DNA damage repair ability of cells co-treated with GOS and VPA was also decreased, as evidenced by the downregulation of DNA damage repair proteins and the enhancement of DNA fragmentation and cell death. These findings suggest that GOS in combination with an HDACI has the potential to increase its clinical efficacy in the treatment of prostate cancer.

Autophagy is involved in anti-viral activity of pentagalloylglucose (PGG) against Herpes simplex virus type 1 infection in vitro.

Pentagalloylglucose (PGG) is a natural polyphenolic compound with broad-spectrum anti-viral activity, however, the mechanisms underlying anti-viral activity remain undefined. In this study, we investigated the effects of PGG on anti-viral activity against Herpes simplex virus type 1 (HSV-1) associated with autophagy. We found that the PGG anti-HSV-1 activity was impaired significantly in MEF-atg7-/- cells (autophagy-defective cells) derived from an atg7-/- knockout mouse. Transmission electron microscopy revealed that PGG-induced autophagosomes engulfed HSV-1 virions. The mTOR signaling pathway, an essential pathway for the regulation of autophagy, was found to be suppressed following PGG treatment. Data presented in this report demonstrated for the first time that autophagy induced following PGG treatment contributed to its anti-HSV activity in vitro.

Quantitative proteomics analysis of chondrogenic differentiation of C3H10T1/2 mesenchymal stem cells by iTRAQ labeling coupled with on-line two-dimensional LC/MS/MS.

The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.

[Comprehensive proteome profile of activated rat hepatic stellate cells].

OBJECTIVE: To profile the protein expression in activated rat hepatic stellate cells (HSCs). METHODS: Primary rat HSCs were isolated and cultured in vitro. After 10 days in vitro culture, the HSCs were activated. Total protein extracted from these activated HSCs were digested, and the obtained peptides were analyzed by using online 2D nanoLC-MS/MS. The identified proteins were classified according to their distributions and functions. RESULTS: 1014 proteins were identified from 50 microg HSCs protein extract, the molecular weights of these proteins ranged from 7832 Da to 588,364 Da. Most of these proteins resided in nucleus, cytoskeleton, mitochondrion and endoplasmic reticulum. And these proteins were mainly involved in nucleic acid metabolism, organelle organization, signal transduction and energy generation. Among these proteins, alpha-smooth muscle actin, vimentin and desmin were specifically expressed in activated HSCs. CONCLUSION: To the best of our knowledge, this is the most comprehensive protein expression profile of activated rat HSCs.

Incidence of cytomegalovirus infection in Shanghai, China.

A city-wide cytomegalovirus serosurvey was conducted in Shanghai, China, and associated parameters were calculated by employing the catalytic model. The lowest seroprevalence was 60.37%, found in the >1- to 3-year age group. The value increased rapidly with age until 25 years, when a value of 97.03% was found, caused by the high force of infection (12.69) and by the reproductive rate (8.89).

[Effect of a soluble Jagged 1/Fc chimera protein on the activation, proliferation and cell cycle of lymphocytes in mice].

AIM: To investigate effect of a soluble Jagged 1/Fc chimera protein (Jagged 1/Fc) on activation, proliferation and cell cycles of lymphocytes in BALB/c mice. METHODS: A model to evaluate the lymphocyte proliferation stimulated with a polyclonal activator, concanavalin A (ConA), was established by a carboxy-fluorescein diacetate-succinimidyl ester (CFDA-SE)-labeling technique. Under an effective dose of 500 mug/L of Jagged 1/Fc, the effect of it on the lymphocyte proliferation was analyzed by flow cytometry. A propidium iodide-labeling technique was applied to estimate the influence of Jagged 1/Fc on the lymphocyte cell-cycle stimulated by phorbol 12,13-dibutyrate (PDB) plus Ionomycin (Ion). Expression levels of CD69 and CD25 molecules on surface of CD3(+) lymphocytes activated with ConA in the presence of Jagged 1/Fc were observed by a fluorescein-conjugated monoclonal antibody-labeling technique. RESULTS: Jagged 1/Fc had no effect on the expression levels of CD69 and CD25 of the CD3(+) lymphocytes stimulated with or without ConA, and no effect on the proliferation index of the lymphocytes stimulated by ConA or PDB plus Ion. It led to the increase of sub-G0 phase cell proportion and the decrease of S phase cell proportion of the lymphocytes, but did not influence the changes of the lymphocyte cell-cycle induced by PDB plus Ion. CONCLUSION: The results suggest that Jagged 1/Fc has no obvious effect on the activation and proliferation of lymphocytes in mice, but may promote the apoptosis of them, cause the G0/G1 phase arrest and block the S phase entry.

Phosphodiesterase type 4 inhibitors cause proinflammatory effects in vivo.

Phosphodiesterase type 4 (PDE(4)) inhibitors are currently being evaluated as potential therapies for inflammatory airway diseases. However, this class of compounds has been shown to cause an arteritis/vasculitis of unknown etiology in rats and cynomolgus monkeys. Studies in rodents have demonstrated the anti-inflammatory effects of PDE(4) inhibitors on lipopolysaccharide (LPS)-induced airway inflammation. The aim of this work was to assess the direct effects of PDE(4) inhibitors on inflammatory cells and cytokine levels in the lung in relation to therapeutic effects. The effects of the PDE(4) inhibitors 3-cyclo-propylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide (roflumilast) and 3-(cyclopentyloxy)-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide (piclamilast) were assessed in vivo, using BALB/c mice, and in vitro, in unstimulated human endothelial and epithelial cell lines. In BALB/c mice, LPS challenge caused an increase in neutrophils in bronchoalveolar lavage (BAL) and lung tissue and BAL tumor necrosis factor-alpha levels, which were inhibited by treatment with either roflumilast or piclamilast (30-100 mg/kg subcutaneously). However, roflumilast and piclamilast alone (100 mg/kg) caused a significant increase in plasma and lung tissue keratinocyte-derived chemokine (KC) levels, and lung tissue neutrophils. In vitro, both piclamilast and roflumilast caused an increase in interleukin (IL)-8 release from human umbilical vein endothelial cells but not BEAS-2B cells, suggesting that one source of the increased KC may be endothelial cells. At doses that antagonized an LPS-induced inflammatory response, the PDE(4) inhibitors possessed proinflammatory activities in the lung that may limit their therapeutic potential. The proinflammatory cytokines KC and IL-8 therefore may provide surrogate biomarkers, both in preclinical animal models and in the clinic, to assess potential proinflammatory effects of this class of compounds.

[The effects of resveratrol on the activation, proliferation and cytokine expression of murine T lymphocytes].

AIM: To study the effects of resveratrol (RSV) on the activation, proliferation and cytokine expression of murine T lymphocytes. METHODS: Murine T lymphocytes were isolated from lymph nodes and cultured in vitro. The lymphocytes were pre-treated with RSV for 1 h prior to activation with PDB plus ionomycin. (3)H-TdR incorporation was used to detect T cell proliferation. IL-2 and IFN-gamma mRNA were detected by RT-PCR, IL-2 and IFN-gamma proteins were detected by intracellular cytokine staining with flow cytometry. RESULTS: RSV can inhibit the proliferation and expression of IL-2 and IFN-gamma of T lymphocytes. CONCLUSION: The immunosuppressive action of RSV may be related with its inhibitory effects on T cell activation, proliferation and cytokine expression.

Design of selective phenylglycine amide tissue factor/factor VIIa inhibitors.

Proof of concept experiments have shown that tissue factor/factor VIIa inhibitors have antithrombotic activity without enhancing bleeding propensity. Starting from lead compounds generated by a biased combinatorial approach, phenylglycine amide tissue factor/factor VIIa inhibitors with low nanomolar affinity and good selectivity against other serine proteases of the coagulation cascade were designed, using the guidance of X-ray structural analysis and molecular modelling.

Synthesis and structure-activity studies of novel orally active non-terpenoic 2,3-oxidosqualene cyclase inhibitors.

New orally active non-terpenoic inhibitors of human 2,3-oxidosqualene cyclase (hOSC) are reported. The starting point for the optimization process was a set of compounds derived from a fungicide project, which in addition to showing high affinity for OSC from Candida albicans showed also high affinity for human OSC. Common structural elements of these inhibitors are an amine residue and an electrophilic carbonyl C atom embedded in a benzophenone system, which are at a distance of about 10.7 A. Considering that the keto moiety is in a potentially labile position, modifications of the substitution pattern at the benzophenone as well as annelated heteroaryl systems were explored. Our approach combined testing of the compounds first for increased binding affinity and for increased stability in vitro. Most promising compounds were then evaluated for their efficacy in lowering plasma total cholesterol (TC) and plasma low-density lipoprotein cholesterol (LDL-C) in hyperlipidemic hamsters. In this respect, the most promising compounds are the benzophenone derivative 1.fumarate and the benzo[d]isothiazol 24.fumarate, which lowered TC by 40% and 33%, respectively.