Maspin Research Articles

Effects of smoking on the tissue regeneration-associated functions of human endometrial stem cells via a novel target gene SERPINB2

BackgroundSmokers directly inhale mainstream cigarette smoke, which contains numerous known and potential toxic substances, and thus, smoking is expected to have broad harmful effects that cause tissue injury and dysfunction. Interestingly, many studies have suggested that the recent decline in female fertility and increased rate of spontaneous abortion could be associated with increased smoking rates. Indeed, women that smoked for 10 years or more were reported to have a ~ 20% higher infertility rate than women that had never smoked. However, the reasons for the underlying harmful aspects of smoking on female fertility remain a matter of debate. Importantly, a previous study revealed that resident endometrial stem cell deficiency significantly limits the cyclic regeneration potential of endometrium, which, in turn, decreases successful pregnancy outcomes. In this context, we postulated that exposure to mainstream cigarette smoke extracts might decrease female fertility by inhibiting the functions of resident endometrial stem cells.MethodsWe investigated whether cigarette mainstream smoke exposure directly inhibits various tissue regeneration-associated functions of endometrial stem cells, such as self-renewal, migration, pluripotency, and differentiation capacity in vitro. Next, we determined whether SERPINB2 mediates cigarette smoke-induced suppressive effects on various tissue regeneration-associated functions by depleting SERPINB2 expression with specific shRNA targeting SERPINB2. Mice were injected intraperitoneally with low (0.5 mg/kg) or high (1 mg/kg) doses of cigarette smoke extract (10 times for two weeks), and endometrial stem cells were then isolated from mice uterine tissues.ResultsWe found that exposure to cigarette smoke extracts remarkably suppressed various tissue regeneration-associated functions of endometrial stem cells, such as self-renewal, migration, multilineage differentiation ability, and pluripotency in vitro and in vivo by activating the SERPINB2 gene. Indeed, cigarette smoke-induced inhibitory effects on various endometrial stem cell functions were significantly abolished by SERPINB2 knockdown.ConclusionsThese findings provide valuable information on the harmful effects of cigarette smoking on resident endometrial stem cells and hopefully will facilitate the developments of promising therapeutic strategies for subfertile or infertile women that smoke cigarettes.

Proteomic analysis of inhibitory protein profiles in the urine of children with nephrolithiasis: implication for disease prevention.

In this study we aimed to screen for the presence of biomarkers that are downregulated in children with nephrolithiasis (RS) compared to healthy controls (HC) using a proteomic approach. We hypothesized that RS and HC would display unique inhibitory protein profiles that could be used for comparative pathway analysis. This is a prospective, controlled, pilot study of pooled urine from RS (N = 30, 24 females, mean age 12.95 ± 4.03years) versus age- and gender-matched HC, using liquid chromatography-mass spectrometry. The criteria for protein selection were: (1) patient/control abundance ratio of < 0.5; and (2) ≤ 0.05 p-value for the Fisher's Exact Test. Results were confirmed by ELISA testing in individual samples. 67 proteins were downregulated in RS group, and 17 of those were significantly different compared to controls. Of those seventeen proteins, five (two actins, annexin A5, keratin 6B, and serpin B4) were completely absent in the urine of stone patients but were found in controls. The remaining twelve proteins were significantly less abundant in the patient's urine compared to healthy controls. Protein-protein interaction modeling of significant proteins identified syndecan-1 as the key node, a protein associated with adhesion pathways. ELISA analysis by subgroups showed statistically significant difference in the urinary excretion of osteopontin (5.1 ± 3.22ng/mg creatinine vs 14.1 ± 9.5ng/mg creatinine, p = 0.046) between stone patients with hypocitraturia and controls. Urinary osteopontin concentration was positively correlated with urinary citrate excretion (r = 0.417, p = 0.03). Children with RS have a different urinary inhibitory polypeptide profile compared to HC. Decreased urinary excretion of these proteins indicates their potential inhibitory role in renal stone formation, especially of the adhesion phase. Lower concentration of urinary osteopontin in children with nephrolithiasis and hypocitraturia suggests its potential involvement in the pathogenesis of this disease. Further characterization of these proteins in a larger sample is imperative.

SerpinB3 as a Pro-Inflammatory Mediator in the Progression of Experimental Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease worldwide. In 20-30% of patients, NAFLD can progress into non-alcoholic steatohepatitis (NASH), eventually leading to fibrosis, cirrhosis and hepatocellular carcinoma development. SerpinB3 (SB3), a hypoxia-inducible factor-2α dependent cysteine protease inhibitor, is up-regulated in hepatocytes during progressive NAFLD and proposed to contribute to disease progression. In this study we investigated the pro-inflammatory role of SB3 by employing phorbol-myristate acetate-differentiated human THP-1 macrophages exposed in vitro to human recombinant SB3 (hrSB3) along with mice overexpressing SB3 in hepatocytes (TG/SB3) or knockout for SB3 (KO/SB3) in which NASH was induced by feeding methionine/choline deficient (MCD) or a choline-deficient, L-amino acid defined (CDAA) diets. In vivo experiments showed that the induction of NASH in TG/SB3 mice was characterized by an impressive increase of liver infiltrating macrophages that formed crown-like aggregates and by an up-regulation of hepatic transcript levels of pro-inflammatory cytokines. All these parameters and the extent of liver damage were significantly blunted in KO/SB3 mice. In vitro experiments confirmed that hrSB3 stimulated macrophage production of M1-cytokines such as TNFα and IL-1β and reactive oxygen species along with that of TGFβ and VEGF through the activation of the NF-kB transcription factor. The opposite changes in liver macrophage activation observed in TG/SB3 or KO/SB3 mice with NASH were associated with a parallel modulation in the expression of triggering receptor expressed on myeloid cells-2 (TREM2), CD9 and galectin-3 markers, recently detected in NASH-associated macrophages. From these results we propose that SB3, produced by activated/injured hepatocytes, may operate as a pro-inflammatory mediator in NASH contributing to the disease progression.

Age-Related Increase of Collagen/Fibrin Deposition and High PAI-1 Production in Human Nasal Polyps.

Objective: Our previous studies showed an age-related increased prevalence of nasal polyps (NP) and reduced production of S100A8/9 in elderly patients with chronic rhinosinusitis with NP (CRSwNP). In this study, we investigated an unbiased age-related gene expression profile in CRSwNP subjects and healthy controls, and further identified the differences in their tissue remodeling. Methods: Microarrays using NP and uncinate tissues from health controls (elderly, age ≥65 vs. non-elderly, age 18–49) were performed, and differentially regulated genes were analyzed. Quantitative real-time PCR (qPCR), Immunostaining, Periodic acid-Schiff (PAS), trichrome staining, Western blot, and ELISA were performed for further investigation. Results: Microarrays identified differentially expressed genes according to disease and age; 278 in NP vs. controls, 75 in non-elderly NP vs. non-elderly controls, and 32 in elderly NP vs. elderly controls. qPCR confirmed that the PLAT gene was downregulated and the SERPINB2 gene upregulated in NP vs. controls. The serous glandular cell-derived antimicrobial protein/peptide-related genes such as BPIFB3, BPIFB2, LPO, and MUC7 were remarkably reduced in NP, regardless of age. SERPINE1 gene (plasminogen activator inhibitor-1, PAI-1) expression was significantly increased in elderly NP versus elderly controls. IHC and western blot confirmed significantly decreased production of MUC7 and LPO in NP versus controls. There was a trend of age-related reduction of submucosal gland cells in normal controls. Trichrome and immunofluorescence staining demonstrated an age-related increase of collagen and fibrin deposition in NP, consistent with increased PAI-1 production. Conclusion: This study demonstrated age-related differential glandular remodeling patterns and fibrosis in NP and normal controls. PAI-1 expression was significantly increased in elderly NP versus elderly controls, suggesting PAI-1 as a potential treatment target in elderly NP.

The Role of Granzyme B Activity and Concentration, and Serpin B9 Concentration with Hepatocellular Carcinoma in Patients with Chronic Hepatitis B Virus

Backgrounds: Granzyme B (GzmB) is the main effective weapon of adaptive immune response that involved in killing of the infective as well as cancerous cells. Serum concentration and/or activity of GzmB may influence the course of chronic hepatitis B (CHB). Aims of the study: To investigate the role of serum level of GzmB and its activity as well as the effect of serpin B9 inhibitor on serum level of GzmB in patients with HCC. Patients and Methods: This is a cross-sectional study which included 85 patients with CHB. Those patients were divided into two groups: Group 1: includes 45 patients diagnosed with HCC, group 2: includes 40 Patients without HCC. Demographic data of the patients were collected from direct interview. Liver function test were measured spectrophotometrically. Granzyme B activity was estimated using spectrophotometry. Enzyme linked immunosorbent assay was used to measure Granzyme B concentration, serum level of and serpin B9. Results: Patients without HCC had significantly higher activity (median: 58 pmol, range: 34.0-130.0 pmol) and concentration (median; 31 pg/ml, range: 0.0-383.0 pg/ml) than those without HCC (median activity: 26, range: 7.0-63.0, median concentration: 18.7 pg/ml, range: 0.0-52.0 pg/ml) with highly significant differences. Conclusions: Granzyme B activity and concentration are significantly reduced in CHB patients with HCC compared with those without HCC. Thus, the reduction in GzmB activity cannot be attributed to increase Serpin B9, rather the GzmB polymorphism may be the main effector. Keywords: Chronic hepatitis B, Hepatocellular carcinoma, Granzyme B activity and concentration, Serpin B9

Proteome and secretome analysis of pancreatic cancer cells.

Pancreatic cancer is a lethal malignancy and no screening biomarker or targeted therapy is currently available. Here, we performed a shotgun proteomic label-free quantification (LFQ) to define protein changes in the cellular proteome and secretome of four pancreatic cancer cell lines (PANC1, Paca44, Paca2, and BXPC3) versus normal human pancreatic ductal epithelial cells (HPDE). In the cellular proteome and secretome, 149 and 43 proteins were dysregulated in the most cancer cell lines, respectively. Using Ingenuity Pathway Analysis (IPA), the most dysregulated signaling pathways in pancreatic cancer cells included the activation of epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), extracellular regulated kinase (ERK), and the deactivation of type-I interferon (IFN) pathways, which could promote cancer cell progression and decrease antitumor immunity. Parallel reaction monitoring (PRM) mass spectrometry was used to confirm the changes of seven regulated proteins quantified by LFQ: EGFR, growth/differentiation factor 15 (GDF15), protein-glutamine gamma-glutamyltransferase 2 (TGM2), leukemia inhibitory factor (LIF), interferon-induced GTP-binding protein Mx1 (MX1), signal transducer and activator of transcription 1 (STAT1), and serpin B5 (SERPINB5). Together, this proteomic analysis highlights protein changes associated with pancreatic cancer cells that should be further investigated as potential biomarkers or therapeutic targets.

Association and functional significance of genetic variants present in regulatory elements of SERPINB5 gene in gallbladder cancer.

SERPINB5 is a mammary serine protease inhibitor, which is involved in various cellular functions. The aberrant expression of SERPINB5 is reported in many cancers along with GBC but limited information is available about its role in genetic predisposition for GBC. We carried out case-control study in 206 cases and 219 controls. Promoter SNPs were genotyped by Sanger's sequencing. In-silico promoter analysis and luciferase reporter assay were done to elucidate the role of promoter variants in regulation of SERPINB5 expression. Out of four SNPs, three SERPINB5 promoter variants showed association with GBC in different models. The 'C' allele of variant rs17071138 was found to be significantly associated with GBC (p=0.017). The 'T' allele of rs3744940 significantly increased the risk for GBC in dominant (p=0.035) and additive models (p=0.005). Also, rs3744941 'T' allele increased the risk for GBC by dominant (p=0.042) as well as additive models (p=0.016). In-silico promoter analysis and luciferase reporter assay revealed the probable regulatory role of the SERPINB5 promoter variant rs17071138 on the expression. Overall, our study reveals the genetic association of SERPINB5 promoter variants with GBC and possible role of rs17071138 in the regulation of expression.

Anti-osteoblastogenic, pro-inflammatory and pro-angiogenic effect of extracellular vesicles isolated from the human osteosarcoma cell line MNNG/HOS.

Extracellular Vesicles (EVs) are becoming increasingly recognized as integral signaling vehicles in several types of cancers, including bone malignancies. However, the specific mechanisms by which EVs influence osteosarcoma progression have not been fully determined. We evaluated the effects of EVs derived from the human osteosarcoma cell line MNNG/HOS (MNNG/HOS-EVs) on bone resident cells. We found that MNNG/HOS-EVs are internalized by osteoblasts and osteoclasts in vitro, with potent inhibitory effects on osteoblast metabolic activity, cell density and alkaline phosphatase activity. Consistently, MNNG/HOS-EVs reduced the expression of cell cycle and pro-osteoblastogenic genes, whilst increasing transcriptional expression and protein release of pro-osteoclastogenic/inflammatory cytokines (RankL, Il1b, Il6 and Lcn2), pro-tumoral cytokines (CCL2,5,6,12 and CXCL1,2,5) and the metalloproteinase MMP3. MNNG/HOS-EVs did not induce osteoclast differentiation, while promoting in vitro and in vivo angiogenesis. Intriguingly, EVs derived from another osteosarcoma cell line (U2OS) reduced ALP activity but had no other effect on osteoblast phenotype. MNNG/HOS-EVs were also found to dramatically increase Serpin b2 expression in osteoblasts. To evaluate the significance of this finding, osteoblasts were forced to overexpress Serpin b2, which however did not affect osteoblast differentiation, while Il6 and Lcn2 mRNAs were up regulated. Overall, we shed light on the interactions of osteosarcoma EVs with the cells of the bone microenvironment, identifying key anti-osteoblastogenic, pro-inflammatory and pro-angiogenic factors that could contribute to osteosarcoma expansion.

Detection of hepatitis through proteases and protease inhibitors genes expression and identification of HCV untypable genotype in Abbottabad, Pakistan.

Liver cancer is the 5th most common cancer caused mainly due to the late detection of hepatitis. Therefore, the early detection of hepatitis through genetic markers boosts effective and remedial management. In addition, to determine the occurrence of hepatitis C virus (HCV), genotyping is indispensable as majority of hepatitis cases remain undiagnosed. The current study was designed to find the gene expression of proteases and proteases inhibitors in different hepatitis patients and to determine HCV genotypes mainly focusing on untypeable genotypes of HCV in Abbottabad, Pakistan. PCR was conducted to find the expression of proteases and protease inhibitors genes in hepatitis patients and healthy individuals. HCV genotyping was done by PCR based method and untypeable genotypes were sequenced and verified using online tools. Controlled individuals showed normal expression of cystatin C and leptin, low expression of cathepsin B while high expression of other studied genes including cathepsin D and G, TPP1 and serpin B1 could be seen. Hepatitis A patients showed high expression of leptin while other genes showed low expression. Hepatitis B patients revealed considerable variations in the cathepsin and cystatin C gene expression. Therefore, low cystatin C (high cathepsin B) and/or high cystatin C (low cathepsin B) levels can be regarded as a potential marker for hepatitis B. Hepatitis C infected patients showed high gene expression of cystatin C and leptin, so they could be useful markers for the diagnostics and prediction of the severity of HCV infections. While genotyping findings showed that about 45% of total PCR positive samples (110) were found to be of 3a genotype followed by 3b in 18%, 1a in 13.6% and 1b in 10%. About 9% of infections turned out to be mixed infections, whereas only 4.5% were untraceable by our genotyping system. Sequencing of untypeable genotypes and applying online tools revealed that the described untypeable genotypes of HCV were in fact variants of 3a genotype. Furthermore, full length characterization of these variants could help to classify them into types and subtypes. Keywords: hepatitis; genotyping; genes expression; proteases and protease inhibitors; ML; NJ.

Knockdown of SERPINB2 enhances the osteogenic differentiation of human bone marrow mesenchymal stem cells via activation of the Wnt/\u03b2-catenin signalling pathway

BackgroundGlobally, bone fractures are the most common musculoskeletal trauma, and approximately 8–10% of cases that fall into the categories of delayed or non-union healing. To date, there are no efficient pharmacological agents to accelerate the healing of bone fractures. Thus, it is necessary to find new strategies that accelerate bone healing and reduce the incidence of non-union or delayed fracture healing. Previous studies have revealed that the plasminogen activation system has been demonstrated to play an important role in bone metabolism. However, the function of SERPINB2 in the osteogenesis of hBMSCs remains unclear. Therefore, in this study, we investigated the effects and mechanism of SERPINB2 on osteogenic differentiation.MethodsWe investigated the osteogenesis effects of hBMSCs by both exogenous SerpinB2 protein and SERPINB2 gene silencing in vitro. Cell proliferation assay was used to assess the effect of exogenous SerpinB2 or SERPINB2 silencing on proliferation of hBMSCs. qPCR and Western blotting analysis detected the expression of target genes and proteins respectively. ALP staining was used to evaluated ALP activity and Alizarin Red staining (ARS) was used to evaluate mineral deposition. In vivo, a murie tibial fracture model was established, histological evaluation and radiographic analysis was used to confirm the therapeutic effects of SERPINB2 silencing in fracture healing. Statistical significance between two groups was determined by Student’s t test, one-way ANOVA or Bonferroni’s post-hoc test according to the distribution of the tested population.ResultsThe addition of exogenous SerpinB2 protein inhibted osteoblast differentiation of hBMSCs in vitro, while SERPINB2 gene silencing significant promote osteoblast differentiation of hBMSCs in vitro. And silenced SERPINB2 gene also increased mineral deposits. Moreover, β-catenin levels were up-regulated by SERPINB2 gene depletion. And the enhancement of osteogenic differentiation induced by SERPINB2 silencing was almost inhibited by specific Wnt/β-catenin signaling pathway inhibitor. In a murine tibial fracture model, local injection of SERPINB2 siRNA improved bone fracture healing.ConclusionsTaken together, these findings indicate that SERPINB2 silencing promoted osteogenic differentiation of BMSCs via the Wnt/β-catenin signaling pathway, and silenced SERPINB2 in vivo effectively promotes fracture healing, suggesting that SERPINB2 may be a novel target for bone fracture healing.

The protease-inhibitor SerpinB3 as a critical modulator of the stem-like subset in human cholangiocarcinoma.

Background and aimsCholangiocarcinoma (CCA) is a form of primary liver cancer with limited therapeutic options. Recently, cancer stem cells (CSCs) have been proposed as a driving force of tumour initiation and dissemination, thus representing a crucial therapeutic target. The protease inhibitor SerpinB3 (SB3) has been identified in several malignancies including hepatocellular carcinoma. SB3 has been involved in the early events of hepatocarcinogenesis and is highly expressed in hepatic progenitor cells and in a mouse model of liver progenitor cell activation. However, only limited information on the possible role of SB3 in CCA stem‐like compartment is available.MethodsEnrichment of CCA stem‐like subset was performed by sphere culture (SPH) in CCA cell lines (CCLP1, HUCCT1, MTCHC01 and SG231). Quantitative RT‐PCR and Western blotting were used to detect SB3 in both SPH and parental monolayer (MON) cells. Acquired CSC‐like features were analysed using an endogenous and a paracrine in vitro model, with transfection of SB3 gene or addition of recombinant SB3 to cell medium respectively. SB3 tumorigenic role was explored in an in vivo mouse model of CCA by subcutaneous injection of SB3‐transfected MON (MONSB3+) cells in immune‐deficient NOD‐SCID/IL2Rg null (NSG) mice. SB3 expression in human CCA sections was investigated by immunohistochemistry. Overall survival (OS) and time to recurrence (TTR) analyses were carried out from a transcriptome database of 104 CCA patients.ResultsSB3, barely detected in parental MON cells, was overexpressed in the same CCA cells grown as 3D SPH. Notably, MONSB3+ showed significant overexpression of genes associated with stemness (CD24, CD44, CD133), pluripotency (c‐MYC, NOTCH1, STAT3, YAP, NANOG, BMI1, KLF4, OCT4, SOX2), epithelial mesenchymal transition (β‐catenin, SLUG) and extracellular matrix remodelling (MMP1, MMP7, MMP9, ADAM9, ADAM10, ADAM17, ITGB3). SB3‐overexpressing cells showed superior spherogenic capacity and invasion ability compared to control. Importantly, MONSB3+ exhibited activation of MAP kinases (ERK1/2, p38, JNK) as well as phosphorylation of NFκB (p65) in addition to up‐regulation of the proto‐oncogene β‐catenin. All these effects were reversed after transient silencing of SB3. According to the in vitro finding, MONSB3+ cells retained high tumorigenic potential in NSG mice. SB3 overexpression was observed in human CCA tissues and analysis of OS as well as TTR indicated a worse prognosis in SB3+ CCA patients.ConclusionThese findings indicate a SB3 role in mediating malignant phenotype of CCA and identify a new therapeutic target.

Chemical Modifications Induced by Phthalic Anhydride, a Respiratory Sensitizer, in Reconstructed Human Epidermis: A Combined HRMAS NMR and LC-MS/MS Proteomic Approach.

Chemical skin and respiratory allergies are becoming a major health problem. To date our knowledge on the process of protein haptenation is still limited and mainly derived from studies performed in solution using model nucleophiles. In order to better understand chemical interactions between chemical allergens and the skin, we have investigated the reactivity of phthalic anhydride 1 (PA), a chemical respiratory sensitizer, toward reconstructed human epidermis (RHE). This study was performed using a new approach combining HRMAS NMR to investigate the in situ chemical reactivity and LC-MS/MS to identify modified epidermal proteins. In RHE, the reaction of PA appeared to be quite fast and the major product formed was phthalic acid. Two amide type adducts on lysine residues were observed and after 8h of incubation, we also observed the formation of an imide type cyclized adducts with lysine. In parallel, RHE samples topically exposed to phthalic anhydride (13C)-1 were analyzed using the shotgun proteomics method. Thus, 948 different proteins were extracted and identified, 135 of which being modified by PA, i.e., 14.2% of the extracted proteome. A total of 211 amino acids were modified by PA and validated by fragmentation spectra. We thus identified 154 modified lysines, 22 modified histidines, 30 modified tyrosines, and 5 modified arginines. The rate of modified residues, as a proportion of the total number of modifiable nucleophilic residues in RHE, was rather low (1%). At the protein level, modified proteins were mainly type I and type II keratins and other proteins which are abundant in the epidermis such as protein S100A, Caspase 14, annexin A2, serpin B3, fatty-acid binding protein 5, histone H2, H3, H4, etc. However, the most modified protein, mainly on histidine residues, was filaggrin, a protein that is of low abundance (0.0266 mol %) and rich in histidine.

Overview of serpinB9 and its roles in cancer (Review).

Serine proteinase inhibitorB9 (serpinB9) is a member of the serine protease inhibitor superfamily, which is widely found in animals, plants and microorganisms. SerpinB9 has been reported to protect cells from the immune‑killing effect of granzymeB (GrB) released by lymphocytes. In recent years, an increasing number of studies have indicated that serpinB9 is involved in tumour apoptosis, immune evasion, tumorigenesis, progression, metastasis, drug resistance and even in maintaining the stemness of cancer stem cells (CSCs). Moreover, according to clinical studies, serpinB9 has been demonstrated to be significantly associated with the development of precancerous lesions, a poor prognosis and ineffective therapies, suggesting that serpinB9 may be a potential target for cancer treatment and an indicator of cancer diagnosis; thus, it has begun to attract increased attention from scholars. The present review concisely described the structure and biological functions of the serpin superfamily and serpinB9. In addition, related research on serpins in cancer is discussed in order to provide a comprehensive understanding of the role of serpinB9 in cancer, as well as its clinical significance for cancer diagnosis and prognosis.

Landscape condition influences energetics, reproduction, and stress biomarkers in grizzly bears

Environmental change has been shown to influence mammalian distribution, habitat use, and behavior; however, few studies have investigated the impact on physiological function. This study aimed to determine the influence of landscape condition on the expression of target proteins related to energetics, reproduction, and stress in grizzly bears. We hypothesized that changes in landscape condition explains protein expression. Skin biopsies were collected from free-ranging grizzly bears in Alberta, Canada from 2013–2019 (n = 86 individuals). We used an information theoretic approach to develop 11 a priori candidate generalized linear mixed models to explain protein expression. We compared models using Akaike Information Criteria (AICc) weights and averaged models with ΔAICc < 2 for each protein. Food resources, represented by increased distance to coal mines and decreased crown closure, positively influenced energetic proteins (adiponectin and alpha-1-acid glycoprotein). Proteins related to reproduction (ceruloplasmin and serpin B5) were positively associated with increased wetland and upland food resources in addition to movement, but negatively associated with increased distance to roads. One stress related protein, complement C3, was positively influenced by increased percent conifer. Given the need to detect emerging threats to wildlife, we suggest the assessment of physiological function will lead to improved monitoring of species in rapidly changing landscapes.

Urine Proteomic Analysis of Differences Between Patients With Hyperthyroidism Before and After Carbimazole Treatment

Abstract Background: Hyperthyroidism, characterised by increased circulating thyroid hormone (TH) levels, alters the body’s metabolic and systemic haemodynamic balance and directly influences renal function However, the underlying mechanisms and metabolic implications of these changes are not well understood. Objective: In the present study we aimed to study the changes occurring in the urinary proteome of patients with hyperthyroidism before and after treatment. The levels of the excreted proteins in the urine were studied using an untargeted 2D DIGE MALDI TOF proteomic approach with network analysis. Methods: The study included 9 age matched patients with mean age 38.6 ± 12.1 years with newly diagnosed hyperthyroidism. The patients were evaluated at baseline and after receiving treatment with carbimazole. Urine samples were obtained from the same patient at baseline (hyperthyroid state) with serum FT4 levels of 35.4 + 9.9 pmol/L and TSH 0.014 + 0.014 mIU/L (mean + SD), and post treatment with anti-thyroid drugs (euthyroid state) with levels of FT4 17.0 + 2.8 pmol/L and TSH 0.6 + 0.5 mIU/L (mean + SD). Results: Alterations in the abundance of urinary proteins, analyzed by Progenesis software, revealed statistically significant differential abundance in a total of 40 spots corresponding to 33 proteins, 26 up and 7 down (≥1.5-fold change, ANOVA, p ≤ 0.05). The proteins identified in the study are known to regulate processes related to cellular metabolism, transport, acute phase response. The urinary proteins upregulated with hyperthyroidism included serotransferrin, transthyretin, serum albumin, ceruloplasmin, α1B glycoprotein, syntenin-1, nesprin, and glutamilnyl peptide cyclotransferase while the 3 notable down regulated proteins were plasma kallikrein, protein glutamine gamma-glutamyl transferase and serpin B3. Bioinformatic analysis using Ingenuity Pathway Analysis (IPA) identified dysregulation of pathways related to cellular compromise, inflammatory response, cellular assembly and organization and identified the involvement of the APP and AKT signaling pathways via their interactions with interleukins as the central nodes. Conclusion: The urine proteomic profiling between the hyperthyroid and euthyroid states demonstrates alteration in the protein levels involved in acute phase response and in maintaining an individual’s haemodynamic state.

A Comprehensive Urine Proteome Database Generated From Patients With Various Renal Conditions and Prostate Cancer.

Urine proteins can serve as viable biomarkers for diagnosing and monitoring various diseases. A comprehensive urine proteome database, generated from a variety of urine samples with different disease conditions, can serve as a reference resource for facilitating discovery of potential urine protein biomarkers. Herein, we present a urine proteome database generated from multiple datasets using 2D LC-MS/MS proteome profiling of urine samples from healthy individuals (HI), renal transplant patients with acute rejection (AR) and stable graft (STA), patients with non-specific proteinuria (NS), and patients with prostate cancer (PC). A total of ~28,000 unique peptides spanning ~2,200 unique proteins were identified with a false discovery rate of <0.5% at the protein level. Over one third of the annotated proteins were plasma membrane proteins and another one third were extracellular proteins according to gene ontology analysis. Ingenuity Pathway Analysis of these proteins revealed 349 potential biomarkers in the literature-curated database. Forty-three percentage of all known cluster of differentiation (CD) proteins were identified in the various human urine samples. Interestingly, following comparisons with five recently published urine proteome profiling studies, which applied similar approaches, there are still ~400 proteins which are unique to this current study. These may represent potential disease-associated proteins. Among them, several proteins such as serpin B3, renin receptor, and periostin have been reported as pathological markers for renal failure and prostate cancer, respectively. Taken together, our data should provide valuable information for future discovery and validation studies of urine protein biomarkers for various diseases.

Proteomics Profiling of the Urine of Patients with Hyperthyroidism after Anti-Thyroid Treatment.

Hyperthyroidism, which is characterized by increased circulating thyroid hormone levels, alters the body’s metabolic and systemic hemodynamic balance and directly influences renal function. In this study, the urinary proteome of patients with hyperthyroidism was characterized using an untargeted proteomic approach with network analysis. Urine samples were collected from nine age-matched patients before and after carbimazole treatment. Differences in the abundance of urinary proteins between hyperthyroid and euthyroid states were determined using a 2D-DIGE coupled to MALDI-TOF mass spectrometry. Alterations in the abundance of urinary proteins, analyzed via Progenesis software, revealed a statistically significant difference in abundance in a total of 40 spots corresponding to 32 proteins, 25 up and 7 down (≥1.5-fold change, ANOVA, p ≤ 0.05). The proteins identified in the study are known to regulate processes associated with cellular metabolism, transport, and acute phase response. The notable upregulated urinary proteins were serotransferrin, transthyretin, serum albumin, ceruloplasmin, alpha-1B-glycoprotein, syntenin-1, and glutaminyl peptide cyclotransferase, whereas the three notable downregulated proteins were plasma kallikrein, protein glutamine gamma-glutamyl transferase, and serpin B3 (SERPINB3). Bioinformatic analysis using ingenuity pathway analysis (IPA) identified the dysregulation of pathways associated with cellular compromise, inflammatory response, cellular assembly, and organization and identified the involvement of the APP and AKT signaling pathways via their interactions with interleukins as the central nodes.

Identification of CD56dim subpopulation marked with high expression of GZMB/PRF1/PI-9 in CD56+ interferon-α-induced dendritic cells.

As the sentinels of innate and adaptive immune system, dendritic cells (DCs) have been considered to hold a great promise for medical application. Among the diverse types of DCs, monocyte-derived DCs (mo-DCs) generated in vitro have been most commonly employed. We have been improving the culture protocol and devised a protocol to produce mature interferon-α-induced DCs (IFN-DCs), hereinafter called (mat)IFN-DCs. While exploring the relationship between the expression of CD56 and the cytotoxic activity of (mat)IFN-DCs, we unexpectedly found that sorting of (mat)IFN-DCs with CD56 antibody-coated microbeads (MB) resulted in fractionating cells with tumoricidal activity into the flow-through (FT) but not MB-bound fraction. We uncovered that the FT fraction contains cells expressing low but substantial level of CD56. Moreover, those cells express granzyme B (GrB), perforin (PFN), and serpin B9 at high levels. By employing a specific inhibitor of PFN, we confirmed that direct tumoricidal activity relies on the GrB/PFN pathway. We designated subpopulation in FT fraction as CD56dim and that in CD56 positively sorted fraction as CD56bright , respectively. This is the first time, to our knowledge, to identify subpopulations of CD56-positive IFN-DCs with distinct tumoricidal activity which is ascribed to high expression of the components of GrB/PFN pathway.

The protease-inhibitor serpinB3 as a critical modulator of the stem-like subset in human cholangiocarcinoma

Background and Aim: Cancer stem cells (CSCs) have been proposed as a driving force of tumor initiation, dissemination and drug resistance. The protease inhibitor SerpinB3 (SB3) has been involved in the early events of hepatocarcinogenesis, and is highly expressed in hepatic progenitor cells. However, only limited information on the possible role of SB3 in cholangiocarcinoma (CCA) stem-like compartment is available.

Subcellular Expression of Maspin in Colorectal Cancer: Friend or Foe.

Simple SummaryIn this article we have highlighted the possible role of nuclear maspin in identification of tumor cells “on the point of budding” and the epithelial mesenchymal transition phenomenon of these cells, along with a deep exploration of the maspin-molecular mediated mechanisms in colorectal cancer.In this review the authors aimed to emphasize the practical value of nuclear expression of the mammary serine protease inhibitor (maspin), also known as serpin B5 protein, in colorectal carcinoma (CRC), from pre-malignant disorders to carcinogenesis and metastasis. As the role of maspin is controversial and not yet understood, the present update highlights the latest data revealed by literature which were filtrated through the daily experience of the authors, which was gained at microscopic examination of maspin expression in CRCs and other tumors for daily diagnosis. Data regarding the subcellular localization of maspin, in correlation with the microsatellite status, grade of tumor dedifferentiation, and epithelial-mesenchymal transition (EMT) phenomenon of the tumor buds were presented with details. An original observation refers to the maspin capacity to mark the tumor cells which are “at the point of budding” that were previously considered as having “hybrid EMT phenotype”. It refers to the transitional status of tumor cell that is between “epithelial status” and “mesenchymal status”. The second original hypothesis highlights the possible role of maspin in dysregulating the intestinal microbiota, in patients with idiopathic inflammatory bowel diseases (IBD) and inducing IBD-related CRC. The dynamic process of budding and EMT of tumor buds, possible mediated by maspin, needs further investigation and validation in many human CRC samples. The histological and molecular data reveal that synthesis of maspin-based therapeutics might represent a novel individualized therapeutic strategy for patients with CRC.