Fecal Protein Analysis of Dusp6 Knockout C57BL/6J Mice by Metaproteomics.

The research of obesity and gut microbiota has been carried out for years, yet the study process was in a slow pace for several challenges to conquer. As a complex status of disorder, the contributing factors refer to gut microbiota about obesity were controversial in a wide range. In terms of proteomics, 2D-DIGE technology is a powerful method for this study to identify fecal proteins from lean microbiota in Dusp6 knockout C57BL/6J mice, exploring the protein markers of the ability resisting to diet-induced obesity (DIO) transferred to the host mice after fecal microbiota transplantation. The results showed that the fecal microbiota expressed 289 proteins differentially with 23 proteins identified, which were considered to be the reasons to assist the microbiota exhibiting distinct behavior. By means of proteomics technology, we had found that differentially expressed proteins of lean microbiota determined the lean microbial behavior might be able to resist leaky gut. To sum up our study, the proteomics strategies offered as a tool to demonstrate and analyze the features of lean microbiota, providing new speculations in the behavior about the gut microbiota reacting to DIO.

Characterization of TGF-ß by Induced Oxidative Stress in Human Trabecular Meshwork Cells.

Oxidative stress generated by reactive oxygen species (ROS) plays a critical role in the pathomechanism of glaucoma, which is a multifactorial blinding disease that may cause irreversible damage within human trabecular meshwork cells (HTMCs). It is known that the transforming growth factor-ß (TGF-ß) signaling pathway is an important component of oxidative stress-induced damage related to extracellular matrix (ECM) fibrosis and activates cell antioxidative mechanisms. To elucidate the dual potential roles and regulatory mechanisms of TGF-ß in effects on HTMCs, we established an in vitro oxidative model using hydrogen peroxide (H2O2) and further focused on TGF-ß-related oxidative stress pathways and the related signal transduction. Via a series of cell functional qualitative analyses to detect related protein level alterations and cell fibrosis status, we illustrated the role of TGF-ß1 and TGF-ß2 in oxidative stress-induced injury by shTGF-ß1 and shTGF-ß2 knockdown or added recombinant human TGF-ß1 protein (rhTGF-ß1). The results of protein level showed that p38 MAPK, TGF-ß, and its related SMAD family were activated after H2O2 stimulation. Cell functional assays showed that HTMCs with H2O2 exposure duration had a more irregular actin architecture compared to normal TM cells. Data with rhTGF-ß1 (1 ng/mL) pretreatment reduced the cell apoptosis rate and amount of reactive oxygen species (ROS), while it also enhanced survival. Furthermore, TGF-ß1 and TGF-ß2 in terms of antioxidant signaling were related to the activation of collagen I and laminin, which are fibrosis-response proteins. Succinctly, our study demonstrated that low concentrations of TGF-ß1 (1 ng/mL) preserves HTMCs from free radical-mediated injury by p-p38 MAPK level and p-AKT signaling balance, presenting a signaling transduction mechanism of TGF-ß1 in HTMC oxidative stress-related therapies.

The Role of Transforming Growth Factor-Beta in Retinal Ganglion Cells with Hyperglycemia and Oxidative Stress.

A characteristic of diabetes mellitus is hyperglycemia, which is considered with an emphasis on the diabetic retinopathy of progressive neurodegenerative disease. Retinal ganglion cells (RGCs) are believed to be important cells affected in the pathogenesis of diabetic retinopathy. Transforming growth factor-beta (TGF-ß) is a neuroprotective protein that helps to withstand various neuronal injuries. To investigate the potential roles and regulatory mechanisms of TGF-ß in hyperglycemia-triggered damage of RGCs in vitro, we established RGCs in 5.5, 25, 50, and 100 mM D-glucose supplemented media and focused on the TGF-ß-related oxidative stress pathway in combination with hydrogen peroxide (H2O2). Functional experiments showed that TGF-ß1/2 protein expression was upregulated in RGCs with hyperglycemia. The knockdown of TGF-ß enhanced the accumulation of reactive oxygen species (ROS), inhibited the cell proliferation rate, and reduced glutathione content in hyperglycemia. Furthermore, the results showed that the TGF-ß-mediated enhancement of antioxidant signaling was correlated with the activation of stress response proteins and the antioxidant pathway, such as aldehyde dehydrogenase 3A1 (ALDH3A1), heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor (Nrf2), and hypoxia-inducible factor (HIF-1α). Summarizing, our results demonstrated that TGF-ß keeps RGCs from hyperglycemia-triggered harm by promoting the activation of the antioxidant pathway, suggesting a potential anti-diabetic therapy for the treatment of diabetic retinopathy.

TGF-ß1 signaling protects retinal ganglion cells from oxidative stress via modulation of the HO-1/Nrf2 pathway.

Oxidative stress provides a major contribution to the pathogenesis of glaucoma and may induce retinal ganglion cell (RGC) damage. Transforming growth factor ß (TGF-ß) has appeared as a neuroprotective protein in various indignities. However, the TGF-ß mechanism of protective effects against oxidative stress damage in RGCs still undetermined. In our research, we investigated the regulatory mechanisms and potential effects of TGF-ß1 & TGF-ß2 in hydrogen peroxide (H2O2)-stimulated oxidative stress of RGCs in vitro. By a series of cell functional qualitative analysis, such as MTT cell viability assay, wound healing ability assay, apoptosis assay, intracellular ROS detection, immunoblot analysis, intracellular GSH content, and high-resolution respirometry, we illustrated the cell state in oxidative stress-induced injury. Results of protein expression showed that TGF-ß1 & TGF-ß2 was upregulated in RGCs after H2O2 stimulation. Cell functional assays resulted that knockdown of TGF-ß1 & TGF-ß2 reduced survival rate whereas enhanced apoptosis and accumulation of reactive oxygen species (ROS). Especially TGF-ß1 upregulation promoted the protein expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and increased the activity of antioxidant and neuroprotection pathways. Additionally, TGF-ß1 & TGF-ß2 on antioxidant signaling was related to activation of heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor (Nrf2), which are stress-response proteins. ROS accumulation followed by the accumulation of hypoxia-inducible factor (HIF-1α) caused mitochondrial damage and led to neurodegeneration. In summary, our results demonstrated that TGF-ß1 preserves RGCs from free radicals-mediated injury by upregulating the activation of Nrf2 expression and HO-1 signaling balance HIF-1α upregulation, implying a prospective role of TGF-ß1 in retinal neuroprotection-related therapies.

Targeting UDP-glucose dehydrogenase inhibits ovarian cancer growth and metastasis.

More than 70% of patients with ovarian cancer are diagnosed in advanced stages. Therefore, it is urgent to identify a promising prognostic marker and understand the mechanism of ovarian cancer metastasis development. By using proteomics approaches, we found that UDP-glucose dehydrogenase (UGDH) was up-regulated in highly metastatic ovarian cancer TOV21G cells, characterized by high invasiveness (TOV21GHI ), in comparison to its parental control. Previous reports demonstrated that UGDH is involved in cell migration, but its specific role in cancer metastasis remains unclear. By performing immunohistochemical staining with tissue microarray, we found overexpression of UGDH in ovarian cancer tissue, but not in normal adjacent tissue. Silencing using RNA interference (RNAi) was utilized to knockdown UGDH, which resulted in a significant decrease in metastatic ability in transwell migration, transwell invasion and wound healing assays. The knockdown of UGDH caused cell cycle arrest in the G0 /G1 phase and induced a massive decrease of tumour formation rate in vivo. Our data showed that UGDH-depletion led to the down-regulation of epithelial-mesenchymal transition (EMT)-related markers as well as MMP2, and inactivation of the ERK/MAPK pathway. In conclusion, we found that the up-regulation of UGDH is related to ovarian cancer metastasis and the deficiency of UGDH leads to the decrease of cell migration, cell invasion, wound healing and cell proliferation ability. Our findings reveal that UGDH can serve as a prognostic marker and that the inhibition of UGDH is a promising strategy for ovarian cancer treatment.

Proteomic analysis of Antrodia Cinnamomea-induced ER stress in liver cancer cells.

Antrodia Cinnamomea is a fungus species widely used as a herb medicine for hypertension, cancer and handover. Nevertheless, the biological roles of Antrodia Cinnamomea on the molecular mechanism of liver cancer are not entirely understood. To determine whether Antrodia Cinnamomea is able to be used for the treatment of liver cancer and its molecular mechanism, we examined the effect of Antrodia Cinnamomea on the differential proteomic patterns in liver cancer cell lines HepG2 and C3A as well as in Chang's liver cell, a normal liver cell, using quantitative proteomic approach. The proteomic analysis demonstrated that abundance of 82, 125 and 125 proteins was significantly altered in Chang's liver cells, C3A and HepG2, respectively. The experimental outcomes also demonstrated that Antrodia Cinnamomea-induced cytotoxicity in liver cancer cells mostly involved dysregulation of protein folding, cytoskeleton regulation, redox-regulation, glycolysis pathway as well as transcription regulation. Further analysis also revealed that Antrodia Cinnamomea promoted misfolding of intracellular proteins and dysregulate of cellular redox-balance resulting in ER-stress. To sum up our studies demonstrated that the proteomic strategy used in this study offered a tool to investigate the molecular mechanisms of Antrodia Cinnamomea-induced liver cancer cytotoxicity. The proteomic results might be further evaluated as prospective targets in liver cancer treatment.

Identification of hyperglycemia-associated microbiota alterations in saliva and gingival sulcus.

Oral microbes are a contributing factor to hyperglycemia by inducing an increase in insulin resistance resulting in uncontrolled blood glucose levels. However, the relationship between the distribution of oral flora and hyperglycemia is still controversial. Combining the power of MALDI-Biotyper with anaerobic bacterial culture, this study explores the correlation between anaerobic bacteria in the oral cavity and blood glucose levels. The results demonstrated that altered blood glucose levels contributed to a varied bacterial distribution in the oral cavity. Specifically, Veillonella spp. and Prevotella spp. were identified in a higher proportion in people with elevated blood glucose levels. Six bacterial species identified in this study (Prevotella melaninogenica, Campylobacter rectus, Streptococcus gordonii, Streptococcus mitis, Streptococcus salivarius, and Veillonella parvula) not only demonstrated a positive association with higher blood glucose levels, but also likely contribute to the development of the condition. The data demonstrated MALDI-TOF MS to be a simpler, faster, and more economical clinical identification tool that provides clarity and depth to the research on blood glucose and oral microbiota.

Proteomic investigating the cooperative lethal effect of EGFR and MDM2 inhibitors on ovarian carcinoma.

With the concept of precision medicine, combining multiple molecular-targeting therapies has brought new approaches to current cancer treatments. Malfunction of the tumor suppressor protein, p53 is a universal hallmark in human cancers. Under normal conditions, p53 is degraded through an ubiquitin-proteosome pathway regulated by its negative regulator, MDM2. In contrast, cellular stress such as DNA damage will activate p53 to carry out DNA repair, cell cycle arrest, and apoptosis. In this study, we focused on ovarian carcinoma with high EGFR and MDM2 overexpression rate. We assessed the effects of combined inhibition by MDM2 (JNJ-26854165) and EGFR (gefitinib) inhibitors on various ovarian cell lines to determine the importance of these two molecular targets on cell proliferation. We then used a proteomic strategy to investigate the relationship between MDM2 and EGFR inhibition to explore the underlying mechanisms of how their combined signaling blockades work together to exert cooperative inhibition. Our results demonstrated that all four cell lines were sensitive to both individual and combined, MDM2 and EGFR inhibition. The proteomic analysis also showed that gefitinib/JNJ-treated CAOV3 cells exhibited downregulation of proteins involved in nucleotide biosynthesis such as nucleoside diphosphate kinase B (NME2). In conclusion, our study showed that the combined treatment with JNJ and gefitinib exerted synergistic inhibition on cell proliferation, thereby suggesting the potential application of combining MDM2 inhibitors with EGFR inhibitors for enhancing efficacy in ovarian cancer treatment.

Proteomic Analysis of Various Rat Ocular Tissues after Ischemia-Reperfusion Injury and Possible Relevance to Acute Glaucoma.

Glaucoma is a group of eye diseases that can cause vision loss and optical nerve damage. To investigate the protein expression alterations in various intraocular tissues (i.e., the cornea, conjunctiva, uvea, retina, and sclera) during ischemia-reperfusion (IR) injury, this study performed a proteomic analysis to qualitatively investigate such alterations resulting from acute glaucoma. The IR injury model combined with the proteomic analysis approach of two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to monitor the protein expression alterations in two groups of specimens (an IR injury group and a control group). The analysis results revealed 221 unique differentially expressed proteins of a total of 1481 proteins in the cornea between the two groups. In addition, 97 of 1206 conjunctival proteins, 90 of 1354 uveal proteins, 61 of 1180 scleral proteins, and 37 of 1204 retinal proteins were differentially expressed. These findings imply that different ocular tissues have different tolerances against IR injury. To sum up, this study utilized the acute glaucoma model combined with 2D-DIGE and MALDI-TOF MS to investigate the IR injury affected protein expression on various ocular tissues, and based on the ratio of protein expression alterations, the alterations in the ocular tissues were in the following order: the cornea, conjunctiva, uvea, sclera, and retina.