S100A6 participates in initiation of autoimmune encephalitis and is under epigenetic control.

INTRODUCTION: Autoimmune encephalitis (AE) is caused by autoantibodies attacking neuronal cell surface antigens and/or synaptic antigens. We previously demonstrated that S100A6 was hypomethylated in patients with AE and that it promoted B lymphocyte infiltration through the simulated blood-brain barrier (BBB). In this study, we focused on the epigenetic regulation of S100A6, the process by which S100A6 affects B lymphocyte infiltration, and the therapeutic potential of S100A6 antibodies. METHODS: We enrolled and collected serum from 10 patients with AE and 10 healthy control (HC) subjects. Promoter methylation and 5-azacytidine treatment assays were conducted to observe the methylation process of S100A6. The effect of S100A6 on B lymphocytes was analyzed using an adhesion assay and leukocyte transendothelial migration (LTEM) assay. A LTEM assay was also used to compare the effects of the serum of HCs, serum of AE patients, S100A6 recombinant protein, and S100A6 antibodies on B lymphocytes. RESULT: The promoter methylation and 5-azacytidine treatment assays confirmed that S100A6 was regulated by DNA methylation. The adhesion study demonstrated that the addition of S100A6 enhanced adhesion between B lymphocytes and a BBB endothelial cell line in a concentration-dependent manner. The LTEM assay showed that the serum of AE patients, as well as S100A6, promoted B lymphocyte infiltration and that this effect could be attenuated by S100A6 antibodies. CONCLUSION: We clarified that S100A6 was under epigenetic regulation in patients with AE and that it helped B lymphocytes to adhere to and infiltrate the BBB endothelial layer, which could be counteracted by S100A6 antibodies. Therefore, the methylation profile of S100A6 could be a marker of the activity of AE, and countering the effect of S100A6 may be a potential treatment target for AE.

Calcyclin-binding protein-promoted degradation of MdFRUCTOKINASE2 regulates sugar homeostasis in apple.

Fructokinase (FRK) activates fructose through phosphorylation, which sends the activated fructose into primary metabolism and regulates fructose signaling capabilities in plants. The apple (Malus × domestica) FRK gene MdFRK2 shows especially high affinity to fructose, and its overexpression decreases fructose levels in the leaves of young plants. However, in the current study of mature plants, fruits of transgenic apple trees overexpressing MdFRK2 accumulated a higher level of fructose than wild-type (WT) fruits (at both young and mature stages). Transgenic apple trees with high mRNA MdFRK2 expression showed no significant differences in MdFRK2 protein abundance or FRK enzyme activity compared to WT in mature leaves, young fruits, and mature fruits. Immunoprecipitation-mass spectrometry analysis identified an skp1, cullin, F-box (SCF) E3 ubiquitin ligase, calcyclin-binding protein (CacyBP), that interacted with MdFRK2. RNA-sequencing analysis provided evidence for ubiquitin-mediated post-transcriptional regulation of MdFRK2 protein for the maintenance of fructose homeostasis in mature leaves and fruits. Further analyses suggested an MdCacyBP-MdFRK2 regulatory module, in which MdCacyBP interacts with and ubiquitinates MdFRK2 to facilitate its degradation by the 26S proteasome, thus decreasing the FRK enzyme activity to elevate fructose concentration in transgenic apple trees. This result uncovered an important mechanism underlying plant fructose homeostasis in different organs through regulating the MdFRK2 protein level via ubiquitination and degradation. Our study provides usable data for the future improvement of apple flavor and expands our understanding of the molecular mechanisms underlying plant fructose content and signaling regulation.

Differential gene expression analysis of RAGE-S100A6 complex for target selection and the design of novel inhibitors for anticancer drug discovery.

Receptor for advanced glycation end products (RAGE), a member of the immunoglobulin family, interactions with its ligands trigger downstream signaling and induce an inflammatory response linked to diabetes, inflammation, carcinogenesis, cardiovascular disease, and a variety of other human disorders. The interaction of RAGE and S100A6 has been associated with a variety of malignancies. For the control of RAGE-related illnesses, there is a great demand for more specialized drug options. To identify the most effective target for combating human malignancies associated with RAGE-S100A6 complex, we conducted single and differential gene expression analyses of S100A6 and RAGE, comparing normal and malignant tissues. Further, a structure-based virtual screening was conducted using the ZINC15 database. The chosen compounds were then subjected to a molecular docking investigation on the RAGE active site region, recognized by the various cancer-related RAGE ligands. An optimized RAGE structure was screened against a library of drug-like molecules. The screening results suggested that three promising compounds were presented as the top acceptable drug-like molecules with a high binding affinity at the RAGE V-domain catalytic region. We depicted that these compounds may be potential RAGE inhibitors and could be used to produce a successful medication against human cancer and other RAGE-related diseases based on their various assorted parameters, binding energy, hydrogen bonding, ADMET characteristics, etc. MD simulation on a time scale of 50 ns was used to test the stability of the RAGE-inhibitor complexes. Therefore, targeting RAGE and its ligands using these drug-like molecules may be an effective therapeutic approach.

SNHG1 functions as an oncogenic lncRNA and promotes osteosarcoma progression by up-regulating S100A6 via miR-493-5p.

The mechanism behind the aberrant expression of S100A6 in osteosarcoma is seldom reported so far. This study sought to explore the regulatory axis targeting S100A6 involved in osteosarcoma progression. Clinical samples collected from osteosarcoma patients were used to detect the expressions of SNHG1, miR-493-5p, and S100A6 by western bolt analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The effects of S100A6 on proliferation and osteogenic differentiation were investigated by the CCK-8 assay, colony formation assay, Ethynyl deoxyuridine staining, matrix mineralization assay, and alkaline phosphatase assay. The potential of lncRNAs/miRNAs targeting S100A6 was identified by the bioinformatics approach, and the results were verified by the dual luciferase assay and RNA immunoprecipitation assay. Both and rescue experiments were performed to investigate the regulatory relationship between the identified lncRNAs and S100A6. The results showed that S100A6 is highly expressed in osteosarcoma. S100A6 overexpression not only increases the proliferation but also reduces the osteogenic differentiation of osteosarcoma cells, while S1006A silence exerts the opposite effects. Then, SNHG1 is identified to directly interact with miR-493-5p to attenuate miR-493-5p binding to the 3'-untranslated region of S100A6. Notably, S100A6 silence partially rescues the effect of SNHG1 overexpression on proliferation and osteogenic differentiation of osteosarcoma cells. Furthermore, the suppressive role of SNHG1 silence in the growth of osteosarcoma xenograft tumors is countered by S100A6 overexpression. Collectively, this study reveals that S100A6 plays an important role in osteosarcoma progression, and SNHG1 promotes S100A6 expression by competitively sponging miR-493-5p.

Overexpression of the dystrophins Dp40 and Dp40L170P modifies neurite outgrowth and the protein expression profile of PC12 cells.

Dp40 is ubiquitously expressed including the central nervous system. In addition to being present in the nucleus, membrane, and cytoplasm, Dp40 is detected in neurites and postsynaptic spines in hippocampal neurons. Although Dp40 is expressed from the same promoter as Dp71, its role in the cognitive impairment present in Duchenne muscular dystrophy patients is still unknown. Here, we studied the effects of overexpression of Dp40 and Dp40L170P during the neuronal differentiation of PC12 Tet-On cells. We found that Dp40 overexpression increased the percentage of PC12 cells with neurites and neurite length, while Dp40L170P overexpression decreased them compared to Dp40 overexpression. Two-dimensional gel electrophoresis analysis showed that the protein expression profile was modified in nerve growth factor-differentiated PC12-Dp40L170P cells compared to that of the control cells (PC12 Tet-On). The proteins α-internexin and S100a6, involved in cytoskeletal structure, were upregulated. The expression of vesicle-associated membrane proteins increased in differentiated PC12-Dp40 cells, in contrast to PC12-Dp40L170P cells, while neurofilament light-chain was decreased in both differentiated cells. These results suggest that Dp40 has an important role in the neuronal differentiation of PC12 cells through the regulation of proteins involved in neurofilaments and exocytosis of synaptic vesicles, functions that might be affected in PC12-Dp40L170P.

c-Myc Upregulated by High Glucose Inhibits HaCaT Differentiation by S100A6 Transcriptional Activation.

Keratinocyte differentiation dysfunction in diabetic skin is closely related to impaired skin barrier functions. We investigated the effects of c-Myc and S100A6 on Human immortal keratinocyte line (HaCaT) or keratinocyte differentiation and potential mechanisms. The expression levels of differentiation makers such as transglutaminase 1 (TGM1), loricrin (LOR), and keratin 1 (K1) were significantly reduced, while the expression of c-Myc was significantly increased in HaCaT cells cultured in high glucose and wound margin keratinocytes from diabetic rats and human patients. Overexpression of c-Myc caused differentiation dysfunction of HaCaT, while knocking down c-Myc promoted differentiation. High glucose increased the expression of c-Myc and inhibited differentiation in HaCaT cells by activating the WNT/ß-catenin pathway. Moreover, inhibition of c-Myc transcriptional activity alleviated the differentiation dysfunction caused by high glucose or overexpression of c-Myc. c-Myc binds to the S100A6 promoter to directly regulate S100A6 expression and high glucose promoted S100A6 transcription. The expression of S100A6 was increased in HaCaT cultured with high glucose and wound margin keratinocytes from diabetic rats and human patients. However, the expression of S100A6 was decreased during normal HaCaT differentiation. HaCaT cells treated with S100A6 recombinant protein showed differentiation dysfunction. The expressions of TGM1, LOR and K1 in knockdown S100A6 HaCaT cells were higher than those in the control group. Overexpression of c-Myc or high glucose caused differentiation dysfunction of HaCaT cells, and was rescued by knocking down S100A6. These findings illustrate a new mechanism by which c-Myc upregulated by high glucose inhibits HaCaT differentiation by directly activating S100A6 transcription. Thus, c-Myc and S100A6 may be potential targets for the treatment of chronic diabetic wounds.

Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target.

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311-342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311-347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

Were Ancestral Proteins Less Specific?

Some have hypothesized that ancestral proteins were, on average, less specific than their descendants. If true, this would provide a universal axis along which to organize protein evolution and suggests that reconstructed ancestral proteins may be uniquely powerful tools for protein engineering. Ancestral sequence reconstruction studies are one line of evidence used to support this hypothesis. Previously, we performed such a study, investigating the evolution of peptide-binding specificity for the paralogs S100A5 and S100A6. The modern proteins appeared more specific than their last common ancestor (ancA5/A6), as each paralog bound a subset of the peptides bound by ancA5/A6. In this study, we revisit this transition, using quantitative phage display to measure the interactions of 30,533 random peptides with human S100A5, S100A6, and ancA5/A6. This unbiased screen reveals a different picture. While S100A5 and S100A6 do indeed bind to a subset of the peptides recognized by ancA5/A6, they also acquired new peptide partners outside of the set recognized by ancA5/A6. Our previous work showed that ancA5/A6 had lower specificity than its descendants when measured against biological targets; our new work shows that ancA5/A6 has similar specificity to the modern proteins when measured against a random set of peptide targets. This demonstrates that altered biological specificity does not necessarily indicate altered intrinsic specificity, and sounds a cautionary note for using ancestral reconstruction studies with biological targets as a means to infer global evolutionary trends in specificity.

Tissue mRNA for S100A4, S100A6, S100A8, S100A9, S100A11 and S100P Proteins in Colorectal Neoplasia: A Pilot Study.

S100 proteins are involved in the pathogenesis of sporadic colorectal carcinoma through different mechanisms. The aim of our study was to assess tissue mRNA encoding S100 proteins in patients with non-advanced and advanced colorectal adenoma. Mucosal biopsies were taken from the caecum, transverse colon and rectum during diagnostic and/or therapeutic colonoscopy. Another biopsy was obtained from adenomatous tissue in the advanced adenoma group. The tissue mRNA for each S100 protein (S100A4, S100A6, S100A8, S100A9, S100A11 and S100P) was investigated. Eighteen biopsies were obtained from the healthy mucosa in controls and the non-advanced adenoma group (six individuals in each group) and thirty biopsies in the advanced adenoma group (ten patients). Nine biopsies were obtained from advanced adenoma tissue (9/10 patients). Significant differences in mRNA investigated in the healthy mucosa were identified between (1) controls and the advanced adenoma group for S100A6 (p = 0.012), (2) controls and the non-advanced adenoma group for S100A8 (p = 0.033) and (3) controls and the advanced adenoma group for S100A11 (p = 0.005). In the advanced adenoma group, differences between the healthy mucosa and adenomatous tissue were found in S100A6 (p = 0.002), S100A8 (p = 0.002), S100A9 (p = 0.021) and S100A11 (p = 0.029). Abnormal mRNA expression for different S100 proteins was identified in the pathological adenomatous tissue as well as in the morphologically normal large intestinal mucosa.

Blocking the interface region amongst S100A6 and RAGE V domain via S100B protein.

The Ca2+-mediated S100 family protein S100A6 has a crucial task in various intracellular and extracellular activities thereby demonstrating a possible involvement in the advancement and development of malignant tumors. S100A6 has been found to associate with receptor for advanced glycation end products, RAGE, through its extracellular extension. This extension is famously identified as a prominent receptor for many S100 family associates. Additionally, S100A6 binds to S100B protein and forms a heterodimer. Thus, we consider the S100B protein to be a prospective drug molecule to obstruct the interacting regions amongst S100A6 and RAGE V domain. We applied the NMR spectroscopy method to locate the binding area amid the S100A6m (mutant S100A6, cysteine at 3rd position of S100A6 is replaced with serine, C3S) and S100B proteins. The 1H-15N HSQC NMR titrations revealed the probable requisite dynamics of S100A6m and S100B interfaces. Utilizing data from the NMR titrations as input parameters, we ran the HADDOCK program and created a S100A6m-S100B heterodimer complex. The obtained complex was then superimposed with the reported complex of S100A6m-RAGE V domain. This superimposition displayed the possibility of S100B to be a potential antagonist that can block the interface area of the S100A6m and the RAGE V domain. Moreover, an in vitro cancer model using SW480 cells in water-soluble tetrazolium-1 assay (WST-1) showed a noticeable change in the cell proliferation as an effect of these proteins. Our study indicates the possibility to develop a S100B-like competitor that could play a key role in the treatment of S100- and RAGE-mediated human diseases.

Dysregulated expression of androgen metabolism genes and genetic analysis in hypospadias.

BACKGROUND: The aberrant expression of genes involved in androgen metabolism and genetic contribution are unclear in hypospadias. METHODS: We compared gene expression profiles by RNA sequencing from five non-hypospadiac foreskins, five mild hypospadiac foreskins, and five severe hypospadiac foreskins. In addition, to identify rare coding variants with large effects on hypospadias risk, we carried out whole exome sequencing in three patients in a hypospadias family. RESULTS: The average expression of androgen receptor (AR) and CYP19A1 were significantly decreased in severe hypospadias (p < .01) and mild hypospadias (p < .05), whereas expression of several other androgen metabolism enzymes, including CYP3A4, HSD17B14, HSD3B7, HSD17B7, CYP11A1 were exclusively significantly expressed in severe hypospadias (p < .05). Compound rare damaging mutants of AR gene with HSD3B1 and SLC25A5 genes were identified in the different severe hypospadias. CONCLUSIONS: In conclusion, our findings demonstrated that dysregulation of AR and CYP19A1 could play a crucial role in the development of hypospadias. Inconsistent AR expression may be caused by the feedback loop of ESR1 signaling or combined genetic effects with other risk genes. This findings complement the possible role of AR triggered mechanism in the development of hypospadias.

Proteomic Characterization of Colorectal Cancer Cells versus Normal-Derived Colon Mucosa Cells: Approaching Identification of Novel Diagnostic Protein Biomarkers in Colorectal Cancer.

In the western world, colorectal cancer (CRC) is the third most common cause of cancer-related deaths. Survival is closely related to the stage of cancer at diagnosis striking the clinical need for biomarkers capable of early detection. To search for possible biological parameters for early diagnosis of CRC we evaluated protein expression for three CREC (acronym: Cab45, reticulocalbin, ERC-55, calumenin) proteins: reticulocalbin, calumenin, and ERC-55 in a cellular model consisting of a normal derived colon mucosa cell line, NCM460, and a primary adenocarcinoma cell line of the colon, SW480. Furthermore, this cellular model was analyzed by a top-down proteomic approach, 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for novel putative diagnostic markers by identification of differentially expressed proteins between the two cell lines. A different colorectal carcinoma cell line, HCT 116, was used in a bottom-up proteomic approach with label-free quantification (LFQ) LC-MS/MS. The two cellular models gave sets of putative diagnostic CRC biomarkers. Various of these novel putative markers were verified with increased expression in CRC patient neoplastic tissue compared to the expression in a non-involved part of the colon, including reticulocalbin, calumenin, S100A6 and protein SET. Characterization of these novel identified biological features for CRC patients may have diagnostic potential and therapeutic relevance in this malignancy characterized by a still unmet clinical need.

Serum S100A6, S100A8, S100A9 and S100A11 proteins in colorectal neoplasia: results of a single centre prospective study.

S100 proteins are involved in biological events related to colorectal carcinogenesis. Aim of this prospective study was to assess serum concentration of S100A6, A8, A9 and A11 proteins in patients with colorectal neoplasia. Eighty-four subjects were enrolled: 20 controls (average risk population with normal findings on colonoscopy; 7 men, 13 women, age 23-74, mean 55 ± 14), 20 patients with non-advanced colorectal adenoma (non-AA, 10 men, 10 women, age 41-82, mean 62 ± 11), 22 with advanced colorectal adenoma (AA, 15 men, 7 women, age 49-80, mean 64 ± 8) and 22 with colorectal cancer (CRC, 12 men, 10 women, age 49-86, mean 69 ± 10). Peripheral venous blood was obtained. Serum S100 proteins were investigated by enzyme immunoassay technique. Serum S100A6 was significantly lower in CRC (mean 8530 ± 4743 ng/L), p = .035 compared to controls (mean 11308 ± 2968 ng/L). Serum S100A8 was significantly higher in AA (median 11955 ng/L, IQR 2681-34756 ng/L), p = .009 and in CRC (median 27532 ng/L, IQR 6794-35092 ng/L), p < .001 compared to controls (median 2513 ng/L, IQR 2111-4881 ng/L). Serum S100A9 concentrations did not differ between any tested group and controls, p > .05. Serum concentration of S100A11 was significantly lower in non-AA (mean 3.5 ± 2.4 µg/L), p = .004 and in CRC (mean 3.4 ± 2.4 µg/L), p = .002 compared to controls (mean 5.9 ± 2.5 µg/L). Sensitivity and specificity for S100A8 protein in patients with CRC were 94% and 73%; positive predictive value 68% and negative predictive value 95%. Patients with colorectal neoplasia have significantly lower serum S100A6 and S100A11 levels, significantly higher S100A8 and unaltered serum S100A9 levels.

Single Cell RNA Sequencing Identifies Subsets of Hepatic Stellate Cells and Myofibroblasts in Liver Fibrosis.

Activation of hepatic stellate cells (HSCs) and their trans-differentiation towards collagen-secreting myofibroblasts (MFB) promote liver fibrosis progression. During chronic liver disease, resting HSCs become activated by inflammatory and injury signals. However, HSCs/MFB not only produce collagen, but also secrete cytokines, participate in metabolism, and have biomechanical properties. We herein aimed to characterize the heterogeneity of these liver mesenchymal cells by single cell RNA sequencing. In vivo resting HSCs or activated MFB were isolated from C57BL6/J mice challenged by carbon tetrachloride (CCl4) intraperitoneally for 3 weeks to induce liver fibrosis and compared to in vitro cultivated MFB. While resting HSCs formed a homogenous population characterized by high platelet derived growth factor receptor ß (PDGFRß) expression, in vivo and in vitro activated MFB split into heterogeneous populations, characterized by α-smooth muscle actin (α-SMA), collagens, or immunological markers. S100 calcium binding protein A6 (S100A6) was a universal marker of activated MFB on both the gene and protein expression level. Compared to the heterogeneity of in vivo MFB, MFB in vitro sequentially and only transiently expressed marker genes, such as chemokines, during culture activation. Taken together, our data demonstrate the heterogeneity of HSCs and MFB, indicating the existence of functionally relevant subsets in hepatic fibrosis.

S100A6 activates EGFR and its downstream signaling in HaCaT keratinocytes.

Epidermal growth factor receptor (EGFR) is a central transmitter of mitogenic signals in epithelial cells; enhanced EGFR activity is observed in many tumors of epithelial origin. S100A6 is a small calcium-binding protein, characteristic mainly of epithelial cells and fibroblasts, strongly implicated in cell proliferation and upregulated in tumors. In this study, using biochemical assays along with immunohistochemical and immunocytochemical analysis of organotypic and standard cultures of HaCaT keratinocytes with S100A6 overexpression or knock-down, we have examined the effect of S100A6 on EGFR activity and downstream signaling. We found that HaCaT cells overexpressing S100A6 had enhanced EGFR, phospho EGFR, and phospho extracellular signal-regulated kinase 1/2 (pERK1/2) staining intensity and level coupled to higher signal transducer and activator of transcription 3 (STAT3) activity. Conversely, S100A6 knockdown cells had impaired EGFR signaling that could be enhanced by addition of recombinant S100A6 to the culture media. Altogether the results show that S100A6 may exert its proproliferative effects through activating EGFR.

Changes in tear biomarker levels in keratoconus after corneal collagen crosslinking.

Purpose: The purpose of this work was to analyze the expressions of matrix metalloproteinase 9 (MMP-9), calcyclin (S100A6), and cystatin S (CST4) in the tears of keratoconus (KC) patients. The correlations between the expressions of these proteins and the values of various ocular surface parameters were examined after accelerated corneal crosslinking (A-CXL) with pulsed ultraviolet light. Methods: This prospective, observational study enrolled patients with different grades of KC, scheduled to undergo the A-CXL procedure, as well as healthy subjects. Tear samples were analyzed by employing customized antibody microarray assays for MMP-9, S100A6, and CST4 proteins. The keratometry readings at the maximum keratometry (Kmax) and the simulated keratometry (SimK) values were obtained for examining the postoperative evolution of corneal topography. The state of the ocular surface was evaluated using the results of the Ocular Surface Disease Index (OSDI) questionnaire, tear osmolarity (OSM) test, Schirmer test (SCH), Tear Break Up Time (TBUT), tear clearance (CLR), and fluorescein (FLUO) and lissamine green (LG) corneal staining. Results: A total of 18 patients (22 eyes) and 10 healthy subjects were studied. The concentrations of MMP-9 and S100A6 decreased in tears, from 104.5 ± 78.98 ng/ml and 350.20 ± 478.08 ng/ml before the surgery to 48.7 ± 24.20 ng/ml and 55.70 ± 103.62 ng/ml, respectively, after 12 months of follow up. There were no changes in the CST4 concentration after 12 months of follow up (2202.75 ± 2863.70 versus 2139.68 ±2719.89 ng/ml). When the patients were divided into three groups according to the evolutive stage of KC, the trends for the three biomarkers in each group were the same as in the general group. Basal concentrations of MMP-9 and S100A6 from healthy subjects and KC patients were compared. The levels of MMP-9 and S100A6 in tears were (9.8 ± 5.11 and 104.55 ± 78.98 ng/ml, p<0.01; and 11.35 ± 3.18 and 350.26 ± 478.06 ng/ml, respectively, p<0.01). This was not the case for CST4, which did not exhibit statistically significant differences between the two groups (2261.94 ± 510.65 and 2176.73 ± 2916.27 ng/ml respectively, p=0.07). Conclusions: A-CXL promoted a decrease in the concentrations of MMP-9 and S100A6 in the tear film. This effect may be related to the restoration of corneal homeostasis and the consequent repair of the tissue damage caused by KC. Moreover, the A-CXL treatment did not produce lasting alterations in the ocular surface, and the values of the evaluated clinical parameters did not change significantly.

Discovering proteins for chemoprevention and chemotherapy by curcumin in liver fluke infection-induced bile duct cancer.

Modulation or prevention of protein changes during the cholangiocarcinoma (CCA) process induced by Opisthorchis viverrini (Ov) infection may become a key strategy for prevention and treatment of CCA. Monitoring of such changes could lead to discovery of protein targets for CCA treatment. Curcumin exerts anti-inflammatory and anti-CCA activities partly through its protein-modulatory ability. To support the potential use of curcumin and to discover novel target molecules for CCA treatment, we used a quantitative proteomic approach to investigate the effects of curcumin on protein changes in an Ov-induced CCA-harboring hamster model. Isobaric labelling and tandem mass spectrometry were used to compare the protein expression profiles of liver tissues from CCA hamsters with or without curcumin dietary supplementation. Among the dysregulated proteins, five were upregulated in liver tissues of CCA hamsters but markedly downregulated in the CCA hamsters supplemented with curcumin: S100A6, lumican, plastin-2, 14-3-3 zeta/delta and vimentin. Western blot and immunohistochemical analyses also showed similar expression patterns of these proteins in liver tissues of hamsters in the CCA and CCA + curcumin groups. Proteins such as clusterin and S100A10, involved in the NF-κB signaling pathway, an important signaling cascade involved in CCA genesis, were also upregulated in CCA hamsters and were then suppressed by curcumin treatment. Taken together, our results demonstrate the important changes in the proteome during the genesis of O. viverrini-induced CCA and provide an insight into the possible protein targets for prevention and treatment of this cancer.

The Apoptotic, Angiogenic and Cell Proliferation Genes CD63, S100A6 e GNB2L1 are Altered in Patients with Endometriosis.

OBJECTIVE: The aim of the present study was to analyze the expression of the CD63, S100A6, and GNB2L1genes, which participate in mechanisms related to the complex pathophysiology of endometriosis. METHODS: A case-control study was conducted with 40 women who were diagnosed with endometriosis, and 15 fertile and healthy women. Paired samples of eutopic endometrium and endometriotic lesions (peritoneal and ovarian endometriotic implants) were obtained from the women with endometriosis in the proliferative (n = 20) or secretory phases (n = 20) of the menstrual cycle. As controls, paired endometrial biopsy samples were collected from the healthy women in the proliferative (n = 15) and secretory (n = 15) phases of the same menstrual cycle. We analyzed the expression levels of the CD63, S100A6, and GNB2L1 genes by real-time polymerase chain reaction. RESULTS: An increase in CD63, S100A6, and GNB2L1 gene transcript levels was observed in the ectopic implants compared with the eutopic endometrium of the women with and without endometriosis, regardless of the phase of the menstrual cycle. CONCLUSION: These findings suggest that the CD63, S100A6, and GNB2L1 genes may be involved in the pathogenesis of endometriosis, since they participate in mechanisms such as inhibition of apoptosis, angiogenesis and cell proliferation, which lead to the loss of cell homeostasis in the ectopic endometrium, thus contributing to the implantation and survival of the tissue in the extrauterine environment.

OBJETIVO: O objetivo do presente estudo foi analisar a expressão dos genes CD63, S100A6 e GNB2L1, que participam em mecanismos relacionados à complexa fisiopatologia da endometriose. MéTODOS: Um estudo caso-controle foi realizado com 40 mulheres diagnosticadas com endometriose e 15 mulheres férteis e saudáveis. Amostras pareadas de endométrio eutópico e de lesões endometrióticas (implantes endometrióticos peritoneais e ovarianos) foram obtidas de mulheres com endometriose nas fases proliferativa (n = 20) ou secretora (n = 20) do ciclo menstrual. Como controle, amostras pareadas de biópsia endometrial foram coletadas de mulheres saudáveis nas fases proliferativa (n = 15) e secretora (n = 15) no mesmo ciclo menstrual. Foram analisados os níveis de expressão dos genes CD63, S100A6 e GNB2L1 por reação em cadeia da polimerase em tempo real. RESULTADOS: Foi observado um aumento nos níveis de transcritos dos genes CD63, S100A6 e GNB2L1 em implantes ectópicos quando comparado ao endométrio eutópico de mulheres com e sem endometriose, independente da fase do ciclo menstrual. CONCLUSãO: Estes achados sugerem que os genes CD63, S100A6 e GNB2L1 podem estar envolvidos na patogênese da endometriose, pois participam de mecanismos como inibição de apoptose, angiogênese e proliferação celular, os quais levam à perda da homeostase celular no endométrio ectópico e, portanto, contribuem para o implante e a sobrevivência do tecido no ambiente extrauterino.

The apoptotic, angiogenic and cell proliferation genes CD63, S100A6 e GNB2L1 are altered in patients with endometriosis / Os genes apoptóticos, angiogênicos e de proliferação celular CD63, S100A6 e GNB2L1 estão alterados em pacientes com endometriose

Abstract Objective The aim of the present study was to analyze the expression of the CD63, S100A6, and GNB2L1genes, which participate in mechanisms related to the complex pathophysiology of endometriosis. Methods A case-control study was conducted with 40 women who were diagnosed with endometriosis, and 15 fertile and healthy women. Paired samples of eutopic endometrium and endometriotic lesions (peritoneal and ovarian endometriotic implants) were obtained from the women with endometriosis in the proliferative (n = 20) or secretory phases (n = 20) of the menstrual cycle. As controls, paired endometrial biopsy samples were collected from the healthy women in the proliferative (n = 15) and secretory (n = 15) phases of the samemenstrual cycle.We analyzed the expression levels of the CD63, S100A6, and GNB2L1 genes by real-time polymerase chain reaction. Results An increase in CD63, S100A6, and GNB2L1 gene transcript levels was observed in the ectopic implants compared with the eutopic endometrium of the women with and without endometriosis, regardless of the phase of the menstrual cycle. Conclusion These findings suggest that the CD63, S100A6, and GNB2L1 genesmay be involved in the pathogenesis of endometriosis, since they participate in mechanisms such as inhibition of apoptosis, angiogenesis and cell proliferation, which lead to the loss of cell homeostasis in the ectopic endometrium, thus contributing to the implantation and survival of the tissue in the extrauterine environment.

Resumo Objetivo O objetivo do presente estudo foi analisar a expressão dos genes CD63, S100A6 e GNB2L1, que participam em mecanismos relacionados à complexa fisiopatologia da endometriose. Métodos Um estudo caso-controle foi realizado com 40 mulheres diagnosticadas com endometriose e 15 mulheres férteis e saudáveis. Amostras pareadas de endométrio eutópico e de lesões endometrióticas (implantes endometrióticos peritoneais e ovarianos) foram obtidas de mulheres com endometriose nas fases proliferativa (n = 20) ou secretora (n = 20) do ciclo menstrual. Como controle, amostras pareadas de biópsia endometrial foram coletadas de mulheres saudáveis nas fases proliferativa (n = 15) e secretora (n = 15) nomesmo ciclomenstrual. Foram analisados os níveis de expressão dos genes CD63, S100A6 e GNB2L1 por reação em cadeia da polimerase em tempo real. Resultados Foi observado um aumento nos níveis de transcritos dos genes CD63, S100A6 e GNB2L1 em implantes ectópicos quando comparado ao endométrio eutópico de mulheres com e sem endometriose, independente da fase do ciclo menstrual. Conclusão Estes achados sugerem que os genes CD63, S100A6 e GNB2L1 podem estar envolvidos na patogênese da endometriose, pois participam de mecanismos como inibição de apoptose, angiogênese e proliferação celular, os quais levam à perda da homeostase celular no endométrio ectópico e, portanto, contribuem para o implante e a sobrevivência do tecido no ambiente extrauterino.

The Neurogenesis Actuator and NR2B/NMDA Receptor Antagonist Ro25-6981 Consistently Improves Spatial Memory Retraining Via Brain Region-Specific Gene Expression.

NR2B-containing NMDA (NR2B/NMDA) receptors are important in controlling neurogenesis and are involved in generating spatial memory. Ro25-6981 is a selective antagonist at these receptors and actuates neurogenesis and spatial memory. Inter-structural neuroanatomical profiles of gene expression regulating adult neurogenesis and neuroapoptosis require examination in the context of memory retrieval and reversal learning. The aim was to investigate spatial memory retrieval and reversal learning in relation to gene expression-linked neurogenetic processes following blockade of NR2B/NMDA receptors by Ro25-6981. Rats were trained in Morris water maze (MWM) platform location for 5 days. Ro25-6981 was administered (protocol days 6-7) followed by retraining (days 15-18 or 29-32). Platform location was tested (on days 19 or 33) then post-mortem brain tissue sampling (on days 20 or 34). The expression of three genes known to regulate cell proliferation (S100a6), differentiation (Ascl1), and apoptosis (Casp-3) were concomitantly evaluated in the hippocampus, prefrontal cortex, and cerebellum in relation to the MWM performance protocol. Following initial training, Ro25-6981 enhanced visuospatial memory retrieval performance during further retraining (protocol days 29-32) but did not influence visuospatial reversal learning (day 33). Hippocampal Ascl1 and Casp-3 expressions were correspondingly increased and decreased while cerebellar S100a6 and Casp-3 activities were decreased and increased respectively 27 days after Ro25-6981 treatment. Chronological analysis indicated a possible involvement of new mature neurons in the reconfiguration of memory processes. This was attended by behavioral/gene correlations which revealed direct links between spatial memory retrieval enhancement and modified gene activity induced by NR2B/NMDA receptor blockade and upregulation.