The extraction of keratin from natural feathers has been studied for its use in various cosmetics and drug delivery applications. There are various reducing agents to dissolve the hard keratin such as sodium dodecyl sulfate and 2-mercaptoethanol, in the present work, a novel extraction methodhas been developed using sodium sulphite, sodium bisulphite, and sodium dodecyl sulfate in the presence of urea, 2-mercaptoethanol, Ethylenediaminetetraacetic acid (EDTA), and thiourea. To increase extraction yield, the weight of feathers, time of incubation, pH, and temperature were investigated using a Central Composite Design and Mixture plan for Optimization. With the present process, we evaluated the apport of keratin treatment and extraction techniques utilizing sodium sulphite, sodium bisulphite, and sodium dodecyl sulfate in the presence of urea, 2-mercaptoethanol, Ethylenediaminetetraacetic acid (EDTA), and thiourea. The percentage yield and keratin concentration were measured using UV-Vis absorbance, Bradford, and Biuret assays. Then, the protein profile and their functional groups were characterized using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Fourier Transform Infrared Spectroscopy (FTIR). The purpose was to compare the different procedures in terms of keratin protein quality and quantity, as well as their cost-effectiveness, and to determine the optimum conditions for the keratin extraction process. The results proved that the yield of white chicken feathers keratin (81.2 %) increased using sodium sulphite (1M), sodium bisulphite (0.1 M), and Sodium Dodecyl Sulfate (0.1 M). The highest protein production was measured at 80 degrees C in 10 h with 5 g of feathers at pH 10. This process of keratin extraction can be used from the laboratory to industrial production with high recoverability and stable properties.

The aim of this study was to investigate the phytochemical composition of dried Roselle calyx (Hibiscus sabdariffa L.) using both ethanolic and aqueous extracts. We report the antimicrobial activities against a wide range of bacteria, yeast, and fungi. The antioxidant activities were tested using 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, and 2–2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging assays. We report also for the first time the effect of the swarming motility in Pseudomonas aeruginosa PAO1. Our results showed that the tested two extracts were a rich source of phenols, flavonoids, and tannins with different degrees. Additionally, eleven phytoconstituents were identified by LC/MS technique (Hibiscus acid: 3-caffeoylquinic acid, 5-caffeoylquinic acid, 5-feruloylquinic acid, cyanidin 3-o-glucoside, myricetin, quercetin 7-o-rutinoside, quercetin 3-o-glucoside, delphinidin 3-o-sambubioside, and kaempferol 3-o-p-coumaroyl-glucoside). Also, it was shown that the calyx extract can scavenge 86% of the DPPH radical, while the rate of 53% and 23% of inhibition of the DPPH was obtained only at the concentration of 125 and 50 µg/mL, and a small inhibition was made at a concentration of 5 μg/mL. Roselle extracts inhibited the growth of the selected microorganisms at low concentrations, while higher concentrations are needed to completely kill them. However, no activity against CVB-3 was recorded for both extracts. In addition, the obtained extracts reduced the swarming motility of P. aeruginosa at 2.5 mg/ml. The docking simulation showed acceptable binding affinities (up to −9.6 kcal/mol) and interaction with key residues of 1JIJ, 2QZW, and 2UVO. The obtained results highlighted the potential use of Roselle extract as a source of phytoconstituents with promising antimicrobial, antioxidant, and anti-quorum sensing activities.

Background and Objectives: In this work, our aims were to investigate the antimicrobial resistance, and anti-quorum sensing activities of Punica granatum L. methanolic extract. Materials and Methods: Antibacterial and antifungal activities were performed against thirteen bacteria and five fungal pathogens. Ultra-high performance liquid chromatography was used to identify the polyphenolic extract. The inhibition of pyocyanin production, proteolytic and elastolytic activity and swarming motility in Pseudomonas aeruginosa PAO1 test strain were estimated. Results: The methanolic extract from P. granatum L. was dominated by chlorogenic acid (34.028 mg/g), rutin (26.05 mg/g), epicatechin (12.207 mg/g), gallic acid (11.157 mg/g), and caffeic acid 9.768 mg/g). Results showed antibacterial activities against almost all tested microorganisms with mean diameter of growth inhibition zone ranging from 6 ± 0 to 30 ± 0 mm for Candida species and from 6 ± 0 to 22.66 ± 0.57 for bacterial strains. The lowest minimal inhibitory concentrations were recorded for Listeria monocytogenes ATCC 19115 and Salmonella enterica CECT 529 (0.14 mg/ml, respectively). The anti-quorum sensing activity of methanolic extract against P. aeruginosa showed a significant inhibition of swarming motility and an attenuation in virulence factors like pyocyanin production at low concentrations. Conclusion: The obtained results indicates that P. granatum L. extracts is a rich source of phenolic compounds and highlighted the possibilities uses of pomegranate to attenuate the expression of quorum sensing controlled factors in P. aeruginosa PAO1 strain.

Aims: Bladder toxicity is one of the major toxicity effects associated with cancer chemotherapeutic treatments with Cyclophosphamide (CP) and Buthionine-SR-Sulfoximine (BSO). In the current study, we utilized a Swiss albino mouse model to investigate the effects of Nigella sativa seeds (NSE) pre-treatment on the urotoxicity produced by acute concentrations of CP and Buthionine-SRSulfoximine (BSO). Methods: To evaluate the toxicity modulation, we carried out several measurements such as Lipid Peroxidation (LPO), Peroxide Hydrogen production (H2O2), and antioxidants in the urinary bladder of the mice. Results: The findings revealed that NSE pre-treatment induced a protective effect in terms of LPO, H2O2 and enzymatic antioxidant activities. The levels of Glutathione S-Transferase (GST), Glutathione Reductase (GR), Glutathione Peroxidase (GP), and Catalase (CAT) in mice exposed to CP were significantly lower than in controls. The data obtained also show a decrease in Glutathione (GSH) levels and an increase in LPO activity in the CP-treated mice group. BSO treatment has shown an additive toxic impact in CP-treated mice. Our findings emphasize NSE's ability to restore enzymatic activity and hence provide protection against CP and BSO. Conclusion: From the outcome of our investigation, it is possible to conclude that NSE has a powerful preventive effect against CP and BSO urotoxcicity. As a pre-treatment, NSE restores GSH which might contribute to the CP-induced apoptosis reversal and the free radical-mediated LPO.

Coxsackievirus B4 (CV-B4) is suspected to be an environmental factor that has the intrinsic capacity to damage the pancreatic beta cells and therefore causes insulitis and type 1 diabetes (T1D). Although vaccination against CV-B4 could reduce the incidence of this chronic auto-immune disease, there is currently no therapeutic reagent or vaccine in clinical use. By the employment of the Bac-to-Bac® vector system to express the major viral capsid protein, we contributed towards the development of a CV-B4 vaccine by producing CV-B4 virus-like particles (VLPs) from recombinant baculovirus in infected insect cells. In fact Western blot and Immunofluorescence analysis detected the viral protein 1 (VP1) in the cells resulting from the construction of a recombinant bacmid DNA carrying the key immunogenic protein then transfected in the insect cells. Sucrose gradient ultracentrifugation fractions of the infected cell lysates contained the recombinant protein and the electron microscopy demonstrated the presence of VLPs in these sucrose fractions. This study clearly shows for the first time the expression of CVB4 VP1 structure protein alone can form VLPs in the baculovirus-infected insect cell keeping conserved both characteristics and morphology.

The aim of this study is to evaluate tropomyosin-4 (TM4) expression in infiltrating ductal breast carcinomas (IDCAs), as well as its prognostic significance. Using a 2-DE/MALDI-TOF mass spectrometry investigation coupled with an immunohistochemical approach, we have assessed the expression of TM4 in IDCAs, as well as in other types of breast tumors. Proteomic analyses revealed an increased expression of tropomyosin-4 in IDCA tumors. Using immunohistochemistry, overexpression of tropomyosin-4 was confirmed in 51 additional tumor specimens. Statistical analyses revealed, however, no significant correlations between tropomyosin-4 expression and clinicopathological parameters of the disease including tumor stage, patient age, estrogen and progesterone receptor status, and lymph node metastasis occurrence. A significant association was found, however, with a high Scarf–Bloom–Richardson (SBR) grade, a known marker of tumor severity. Additionally, the SBR component showing a correlation with TM4 expression was the tubular differentiation status. This study demonstrates the upregulation of tropomyosin-4 in IDCA tissues, which may highlight its involvement in breast cancer development. Our findings also support a link between tropomyosin-4 expression and aggressiveness of IDCA tumors.

The aim of this study was to evaluate calreticulin expression in infiltrating ductal breast carcinomas (IDCAs), as well as its relationships with clinicopathological parameters of the disease. Using a two-dimensional gel electrophoresis/matrix-assisted laser desorption ionization time of flight mass spectrometry investigation coupled to an immunohistochemical approach, we have assessed the expression of calreticulin in IDCAs, as well as in other types of breast tumors. The humoral immune response against calreticulin was estimated using a serological proteomics-based strategy. Proteomic analyses revealed an increased expression of calreticulin in IDCA tumors. Using immunohistochemistry, overexpression of calreticulin was confirmed in 51 additional tumor specimens. Statistical analyses revealed, however, no significant correlations between calreticulin expression and clinicopathological parameters of the disease including tumor stage, patient age, SBR grade, and lymph node metastasis occurrence. A significant association was found, however, with estrogen receptor status. This study demonstrates the upregulation of calreticulin in IDCA tissues which may highlight its involvement in breast cancer development. Our findings also support a link between calreticulin expression and estrogen transduction pathways. Our results do not, however, support the involvement of calreticulin in the development of a humoral immune response in IDCAs.

This study aims to evaluate αB-crystallin expression in infiltrating ductal breast carcinomas (IDCAs), as well as, its prognostic significance. Using a two-dimensional electrophoresis matrix-assisted laser desorption/ionisation-time of flight mass spectrometry investigation coupled to an immunohistochemical approach, we have assessed the expression of αB-crystallin in IDCAs, as well as, in other types of breast tumors (invasive lobular carcinomas, medullary carcinomas, and in situ ductal carcinomas). Correlation between αB-crystallin expression and clinicopathological parameters of breast cancer has also been investigated. Proteomic analyses revealed an increased expression of αB-crystallin in IDCA tumors compared to adjacent nontumor tissues. Overexpression of this molecular chaperone was further confirmed in 51 tumor specimens. Statistical analyses revealed, however, no significant correlations between αB-crystallin expression and clinicopathological parameters of the disease (tumor stage, patient age, hormone receptors, SBR grade, and lymph node metastases). This study demonstrates the upregulation of αB-crystallin in IDCA tissues which may highlight its possible involvement in breast cancer development. Our findings do not, however, support the involvement of this molecular chaperone in the progression of this disease.

Urotoxicity is a troublesome complication associated with cylophosphamide (CP) andL-buthionine-SR-sulfoximine (BSO) treatment in chemotherapy. With this concern in mind, this present study investigated the potential effects of diosgenin for the first time on urotoxicity induced by acute CP and BSO doses using a Swiss albino mouse model. Toxicity modulation was evaluated through measuring lipid peroxidation (LPO) and anti-oxidants in urinary bladder. The findings reveal that the diosgenin exerted a protective effect not only on LPO but also on enzymatic anti-oxidants. When compared to the controls, the CP-treated animals underwent significant decrease in the glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GP) and catalase (CAT) activities. The level of reduced glutathione (GSH) was also decreased with an increase in LPO in the CP-treated animals. BSO treatment exerted an additive toxic effect in the CP-treated animals. Interestingly, pre-treatment with the diosgenin restored the activities of all enzymes back to normal levels and to exhibit an overall protective effect on the CP and BSO induced toxicities in urinary bladder. The restoration of GSH through the treatment with the diosgenincan play an important role in reversing CP-induced apoptosis and free radical mediated LPO. Key words: Diosgenin, cyclophosphamide, L-buthionine-SR-sulfoximine, urinary bladder anti-oxidants

Breast cancer represents a major public health problem. Approximately one woman in ten is likely to develop a malignant tumor of the breast in their lifetime. The frequency of breast cancer is rising steadily for 20 years and the practical benefits in the diagnosis, prognosis and treatment of this disease are still too limited. Actually, there is no tumor marker with a specificity and sensitivity sufficient to have an utility in clinical and early diagnosis of breast cancer, although, carcinoembryonic antigen (CEA), MUC-1 and CA 15-3 were reported to be useful as markers for monitoring this disease. Thus, proteomics approaches are needed for the discovery and the identification of new protein biomarkers that may allow a better understanding of biological mechanisms of breast tumor development and serve as potential therapeutic targets. This article reviews advances in this field, as well as, the major contribution of these markers in breast pathology, with a focus on their biological characteristics and their clinical and therapeutic involvement.

Breast cancer is the most common malignancy in women, comprising about 18% of all female cancers. The most important tools in screening and early detection are imaging techniques: mammography, ultrasonography and magnetic resonance imaging. However, up to 20% of new breast cancer incidents cannot be detected by these methods. Up to now, poor diagnosis of breast cancer is due to a lack of specific biomarkers. Therefore, there is much interest in identifying useful pathological markers that can help in detection and treatment of this disease. Up to now, detecting minor proteins in human plasma is a difficult strategy due to the presence of several highly abundant proteins such as albumin and haptoglobins. The objective of this study was to develop a reproducible method that depletes high-abundant proteins and improves detection of minor ones. For this purpose, we evaluated the impact of using Affi-gel blue affinity columns and organic solvent (acetonitrile) precipitation on the removal of high abundant proteins. Fractionated plasma protein extracts were subsequently analyzed by SDS-PAGE and two dimensional gel electrophoresis. Our findings confirm that Affi-gel blue can enhance detection of minor plasma proteins and could thus be a useful strategy in the discovery of new breast cancer markers. Key words: Affigel blue, albumin, acetonitrile, plasma, proteomic separation, breast cancer.

Objectives: To determine the clinical significance of alpha 1-proteinase inhibitor (alpha 1-Pi) in infiltrating ductal breast carcinoma patients.Design and methods: Serum levels of alpha 1-Pi, tryptic specific inhibitory capacity and alpha 1-Pi circulating immune complexes were determined using radial immunodiffusion, BAPNA assays and ELISA, respectively. 2-DE-MS and immunohistochemistry were performed to examine alpha 1-Pi protein expression.Results: A decreased serum level of alpha 1-Pi was found among breast cancer patients in comparison to controls. In addition, we found a significantly decreased mean level of alpha 1-Pi in the node metastatic group when compared to node negative patients. However, the functional activity of the inhibitor did not decrease proportionately. Through 2-DE analyses, a differential expression of alpha 1-Pi isoforms according to tumor stage and node metastatic development was found.Conclusions: Both alpha 1-Pi levels and specific activity could be a source of complementary clinical information and may provide useful information for a better understanding of the mechanisms of metastasis. (C) 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Apolipoprotein A1 (ApoA1) is the major apoprotein constituent of high-density lipoprotein that can play important roles in tumor invasion and metastasis. In the current report, we evaluated the role of the functional ApoA1 polymorphisms (-75 G/A and +83 C/T) as genetic markers for breast cancer susceptibility and prognosis. We used the polymerase chain reaction and restriction enzyme digestion (RFLP-PCR) to characterize the variations of the ApoA1 gene in 295 unrelated Tunisian patients with breast carcinoma and 197 healthy control subjects. No association was found between the +83 C/T genetic variation in ApoA1 gene and the risk of breast cancer occurrence. The presence of the (+83) T allele appeared however to be associated with an increased risk of lymph node metastasis occurrence (OR = 2.94; P = 0.01). Furthermore, a positive association was found between ApoA1 -75 A allele carriers and breast cancer risk (OR = 1.57; P = 0.02). Regarding prognostic indicators, a significant association was found between ApoA1 (-75) A allele carriers and the premenopausal status of breast cancer patients (OR = 1.73; P = 0.03). Additionally, the presence of the -75 A allele was correlated with the oestrogen receptor status among premenopausal women (OR = 2.45; P = 0.02). This is the first report on the studies of ApoA1 single nucleotide polymorphisms (SNPs) in breast carcinomas. Our data suggest that these genetic variations of ApoA1 may represent a marker for the increased risk of breast cancer.

Up to now, there are no protein tumor markers with a specificity and sensitivity sufficient to have a utility in prognosis and early diagnosis of cancer. Recent advances in proteomics approaches have led to the identification of novel tumor markers of cancer that may have a utility in screening strategies and treatment. The purpose of the current review is to describe the major advances in cancer proteomics, especially those related to the study of serum biomarkers, immune-related responses (autoantibodies) and alterations in cellular proteins. (C) 2011 Elsevier Masson SAS. All rights reserved.

Cancer epidemiology has undergone marked development since the nineteen-fifties. One of the most spectacular and specific contributions was the demonstration of the massive effect of smoking and genetic polymorphisms on the occurrence of bladder cancer. The tobacco carcinogens are metabolized by various xenobiotic metabolizing enzymes, such as the super-families of N-acetyltransferases (NAT) and glutathione S-transferases (GST). DNA repair is essential to an individual's ability to respond to damage caused by tobacco carcinogens. Alterations in DNA repair genes may affect cancer risk by influencing individual susceptibility to this environmental exposure. Polymorphisms in NAT2, GST and DNA repair genes alter the ability of these enzymes to metabolize carcinogens or to repair alterations caused by this process. We have conducted a case-control study to assess the role of smoking, slow NAT2 variants, GSTM1 and GSTT1 null, and XPC, XPD, XPG nucleotide excision-repair (NER) genotypes in bladder cancer development in North Tunisia. Taken alone, each gene unless NAT2 did not appear to be a factor affecting bladder cancer susceptibility. For the NAT2 slow acetylator genotypes, the NAT2*5/*7 diplotype was found to have a 7-fold increased risk to develop bladder cancer (OR = 7.14; 95% CI: 1.30-51.41). However, in tobacco consumers, we have shown that Null GSTM1, Wild GSTT1, Slow NAT2, XPC (CC) and XPG (CC) are genetic risk factors for the disease. When combined together in susceptible individuals compared to protected individuals these risk factors give an elevated OR (OR = 61). So, we have shown a strong cumulative effect of tobacco and different combinations of studied genetic risk factors which lead to a great susceptibility to bladder cancer.

Urotoxicity is a troublesome complication associated with cyclophosphamide (CP) and L-buthionine-SR-sulfoximine (BSO) treatment in chemotherapy. With this concern in mind, the present study investigated the potential effects of a hydroxytyrosol extract from olive mill waste (OMW) on urotoxicity induced by acute CP and BSO doses using a Swiss albino mouse model. Toxicity modulation was evaluated by measuring lipid peroxidation (LPO) and antioxidants in urinary bladder. The findings revealed that the hydroxytyrosol extract exerted a protective effect not only on LPO but also on enzymatic antioxidants. When compared to the controls, the CP-treated animals underwent significant decreases in the glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GP), and catalase (CAT) activities. The level of glutathione (GSH) was also reduced with increased doses of LPO in the CP-treated animals. L-Buthionine-SR-sulfoximine treatment exerted an additive toxic effect on the CP-treated animals. Interestingly, pretreatment with the hydroxytyrosol extract restored the activities of all enzymes back to normal levels and exhibited an overall protective effect on the CP- and BSO-induced toxicities in urinary bladder. The restoration of GSH through the treatment with the hydroxytyrosol extract can play an important role in reversing CP-induced apoptosis and free radical-mediated LPO.

OBJECTIVES:In the current study, we have used an immunoproteomics approach to identify proteins that commonly elicit a humoral response in patients with infiltrating ductal carcinomas of the breast. DESIGN AND METHODS:Sera obtained at the time of diagnosis from 40 patients with invasive breast cancer and 42 healthy controls were screened for the presence of IgG antibodies to MCF-7 cell line proteins using a serological proteomics-based approach. RESULTS:An immunoreactive protein detected in sera from 21 of 40 patients was isolated and subsequently identified as elongation factor-Tu. CONCLUSIONS:The immunoproteomic approach implemented here offers a powerful tool for determining novel tumor antigens that induce a humoral immune response in cancer patients. From our findings, the immunoreactive EF-Tu protein and/or the related circulating antibodies may display clinical usefulness as potential diagnostic markers and provide a means for a better understanding of the molecular mechanisms underlying breast cancer development.

There is an important need to find relevant biomarkers that show high sensitivity and specificity for early diagnosis and prognosis of many cancers. To day, immunoproteomics is a relatively new concept in the field of proteomics and an increasingly powerful technology in the hunt for new and novel diagnostic and prognostic biomarkers. For cancer diagnostic, it is important that circulating autoantibodies reflect a molecular imprint of those antigens (lowabundance) that are specifically related to tumor. Identification of tumor-associated antigens and their cognate autoantibodies is a promising strategy for the discovery of relevant biomarkers. Importantly, sera fromtumor patients contain tumor specific antibodies directly against antigenic proteins. Also, autoantibodies are highly stable compared to many other serum proteins, they seem ideal to be implemented in clinical diagnostic assays for the detection of antigen-associated diseases. To facilitate autoantibody discovery, approaches that allow the simultaneous identification ofmultiple autoantibodies are preferred. During the past few years, proteomic approaches, including SEREX and SERPA, have been the dominant strategies used to identify tumor-associated antigens and their cognate autoantibodies. In this review, we will outline the advances applications of immunoproteomics for the discovery and analysis of clinically relevant protein targets and discuss their merits in clinical applications of the most common cancers.

UNLABELLED Breast cancer is the most diagnosed cancer in women, accounting for approximately 40,000 deaths annually in the USA. In Tunisia, the incidence of breast cancer is approximately 19 new cases per 100,000 women per year. Significant advances have been made in the areas of detection and treatment, but a significant number of breast cancers are detected late. The advent of proteomics provides the hope of discovering novel biological markers that can be used for early detection, prognosis, diagnosis, and therapy. Several proteomics technologies have been used to uncover molecular mechanisms associated with breast. INTRODUCTION Breast cancer is a major health problem and one of the leading causes of death among women worldwide. Its incidence is steadily rising in developing countries. In Tunisia, the incidence of breast cancer is approximately 19 new cases per 100,000 women per year(1). Invasive carcinomas represent 70-80% of all breast cancer and among these, infiltrating ductal carcinomas (IDCA) are the most aggressive forms and have a poor prognosis(2). Histopathologically identical breast cancers show a different biological behavior in terms of aggressiveness, progression, and response to therapy. Thus, there is a great need for new breast cancer biomarkers that might help detect this cancer at an earlier stage, to uncover prognostically distinct subclasses, and to provide best individual treatment(2). Currently, the search for specific cancer-related alterations are largely carcinoma at the global level to discover protein patterns that distinguish disease and disease-free states with high sensitivity and specificity. Two dimensional gel electrophoresis coupled with mass spectrometry constitute a new proteomics based paradigm for detecting disease in pathology specimens and monitoring disease response to therapy. This review describes these proteomics technologies and their application in the analysis of breast carcinoma.