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Soil salinity harmfully effect bell pepper production being salt sensitive crop. The aim of this study was to compare capsicum (bell-pepper) cultivars for their ability to tolerate salt stress. Electrical conductivity of different levels i.e., control (no sodium chloride), 1.5, 3.0, 4.5, and 6.0 dS m-1, was developed by using NaCl, after 30 days of seedling emergence of six cultivars (Yolo Wonder, California Wonder, Cop-amber Hybrid, F1 Pangs No. 206, Sweet Pepper F1, Kaka F1). These cultivars were evaluated based on morphological (shoot and root length, plant fresh and dry biomass), physiological (chlorophyll content) and biochemical attributes (nitrogen concentration, and protein contents). Minimum chlorophyll contents (SPAD units) in Kaka F1 (16.77 SPAD units) at 1.5 dSm-1. However, maximum value of chlorophyll contents was observed in Sweet Pepper F1 (49.23 SPAD units) at control followed by “Yolo Wonder” (41.53 SPAD units) at control. Maximum dry biomass was found in California Wonder (0.468 g) at 1.5 dSm-1, while minimum value of dry biomass (g) was observed in Kaka F1 (0.095 g) at 4.5 dSm-1. Six cultivars of capsicum were categorized into three groups. Salt tolerant cultivars included “California Wonder” followed by “Yolo Wonder”. “Kaka F1” was proved most salt sensitive and all others were moderately salt tolerant.

Objectives Ready-to-eat (RTE) food sold in Quetta, Pakistan was assessed for microbial contamination. Methods Equal numbers of samples were collected from four categories of RTE food – burgers, shawarma, pizza and sandwiches – from January 2018 to December 2018. Microbial contamination of individual food samples was assessed by quantifying the total aerobic count obtained from plating samples on bacterial growth medium. Salmonella spp. serovars were identified using polymerase chain reaction. Results Approximately 38% (121/320) of RTE food samples were not fit for human consumption. The most contaminated type of RTE food was shawarma (49%). Microbial contamination of food samples was higher in summer compared with the other seasons. Approximately 40% (49/121) of food samples that were not fit for human consumption were contamined with Salmonella spp. Salmonella enteritidis (69%) and Salmonella typhimurium (31%) were the only serovars among the samples testing positive for Salmonella spp. Of the 49 samples with high microbial counts, S. enteritidis was present in 34 samples and S. typhimurium was present in 15 samples. The antibiotic sensitivity results demonstrated that both S. enteritidis and S. typhimurium were resistant to amoxicillin. In addition, S. enteritidis was resistant to chloramphenicol and erythromycin, and S. typhimurium presented high resistance to erythromycin. Both S. typhimurium and S. enteritidis were highly sensitive to kanamycin. Conclusion RTE food sold by street vendors in Quetta was found to be contaminated with Salmonella spp. and poses a great health risk to consumers. As such, consumption should be avoided, and the health authorities should take stringent action to ensure the quality of street food in order to reduce the healthcare burden.

Background/objectives: Orobanche is one of the important parasites of solanaceae crops and causes heavy yield losses. This study aimed to use DNA barcoding technique for identification of Orobanche species. Methods: Surveillance study was conducted in the year 2018 on tomato crops grown in four different districts in Balochistan. From these tomato fields, 15 Orobanche plant samples were collected and put in polyethylene bags containing silica gel for drying. The dried samples were processed for DNA extraction using CTAB method. The extracted DNA was confirmed through gel electrophoresis which were further amplified on PCR using rbcL, matK primers. Then, the PCR products were processed for DNA sequencing using Sanger method. Further, the DNA sequences were edited and aligned with BioEdit and MegaX software and the aligned sequences were matched on NCBI for BLAST. The FASTA results of both partial and/or complete genome were run on MegaX for phylogenetic analysis. Findings: The success rates for PCR amplification was 100% for rbcl primer, while it was 68% for matK primer. However, the resolution power of matK was higher than that obtained from rbcL as manifested in the rate of DNA acquisition sequence. The DNA barcode results of 15 samples revealed 99.16% rate of DNA acquisition sequence for identification of P. ramosa and 96.41 for P. purpurea. While, Rbcl rate of DNA sequencing show no results. Application/Improvement: from this study it was inferred that matK gene performed better than rbcL in Orobanche species identification and proved as a potential gene for other pseudogene plants species identification. Keywords: Orobanche; species; DNA barcode; rbcL; matK; tomato

The Rhodococcus erythropolis gene DYC18_RS18060 (1437 bp) putatively codes for a secondary transporter of the Nucleobase Cation Symporter-1 (NCS-1) protein family (478 amino acids). The DYC18_RS18060 gene was successfully cloned from R. erythropolis genomic DNA with addition of EcoRI and PstI restriction sites at the 5′ and 3′ ends, respectively, using PCR technology. The amplified gene was introduced into IPTG-inducible plasmid pTTQ18 immediately upstream of the sequence coding for a His6-tag. The construct was transformed into Escherichia coli BL21(DE3), then amplified expression of the DYC18_RS18060-His6 protein was achieved with detection by SDS-PAGE and western blotting. Computational methods predicted that DYC18_RS18060 has a molecular weight of 51.1 kDa and isoelectric point of 6.58. The protein was predicted to be hydrophobic in nature (aliphatic index 113.24, grand average of hydropathicity 0.728) and to form twelve transmembrane spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. Whilst database sequence similarity searches and phylogenetic analysis suggested that the substrate of DYC18_RS18060 could be cytosine, this was not certain based on comparisons of residues involved in substrate binding in experimentally characterised NCS-1 proteins. This study has laid foundations for further structural and functional studies of DYC18_RS18060 and other NCS-1 proteins. Copyright(c) The Authors

One of the main goals of bioinformatics is to understand and analyze the 3D structure of proteins and the relationship between amino acid sequences. With the help of amino acid sequences, the protein structure can easily be predicted as proteins are essential in natural science research and they are linked with evolution, drug development, mutation and the occurrence of different diseases directly or indirectly. Biologists used bioinformatics tools to discover different diseases by knowing protein’s structure and functions rather than using different technologies/experimental tools which can’t completely explains proteins, its structure and role in several diseases. Prostate Cancer is the leading cause of cancer deaths in males worldwide, it’s least common in Asia and more common in western countries. The study was conducted for the bioinformatics analysis of Prostate cancer proteins.

Critical for diverse biological processes, proteases represent one of the largest families of pharmaceutical targets. To inhibit pathogenic proteases with desired selectivity, monoclonal antibodies (mAbs) hold great promise as research tools and therapeutic agents. However, identification of mAbs with inhibitory functions is challenging because current antibody discovery methods rely on binding rather than inhibition. This study developed a highly efficient selection method for protease inhibitory mAbs by coexpressing 3 recombinant proteins in the periplasmic space of Escherichia coli—an antibody clone, a protease of interest, and a β-lactamase modified by insertion of a protease cleavable peptide sequence. During functional selection, inhibitory antibodies prevent the protease from cleaving the modified β-lactamase, thereby allowing the cell to survive in the presence of ampicillin. Using this method to select from synthetic human antibody libraries, we isolated panels of mAbs inhibiting 5 targets of 4 main protease classes: matrix metalloproteinases (MMP-14, a predominant target in metastasis; MMP-9, in neuropathic pain), β-secretase 1 (BACE-1, an aspartic protease in Alzheimer’s disease), cathepsin B (a cysteine protease in cancer), and Alp2 (a serine protease in aspergillosis). Notably, 37 of 41 identified binders were inhibitory. Isolated mAb inhibitors exhibited nanomolar potency, exclusive selectivity, excellent proteolytic stability, and desired biological functions. Particularly, anti-Alp2 Fab A4A1 had a binding affinity of 11 nM and inhibition potency of 14 nM, anti-BACE1 IgG B2B2 reduced amyloid beta (Aβ(40)) production by 80% in cellular assays, and IgG L13 inhibited MMP-9 but not MMP-2/-12/-14 and significantly relieved neuropathic pain development in mice.

— Using wastewater for agricultural irrigation presents potential risks to public health and the environment. The aim of this study was to compare the microbial burden and nutritional components of cabbage grown in fresh and wastewater in Quetta, Pakistan. Cabbage samples randomly collected from fields irrigated with fresh and wastewater sources were analysed for viable bacterial and fungal counts, pH and vitamin contents. There was a significant difference in viable bacterial (3.0 × 106, 1.9 × 107 CFU g-1) and fungal (2.4 × 103, 1.0 × 105 CFU g-1) counts for cabbage grown in fresh and wastewater, respectively. There were no significant differences in pH (7.88/7.86) and contents of the vitamins thiamin (0.31/0.30 mg/100 g), riboflavin (0.05/0.04 mg/100 g), niacin (0.56/.54 mg/100 g), pyridoxine (0.13/0.12 mg/100 g) and ascorbic acid (57.00/56.60 mg/100 g). Results demonstrated that cabbage grown in wastewater had a significantly higher microbial burden than cabbage grown in fresh water, although there were no significant differences in nutritional components of cabbage from the different water sources. These observations emphasise the potential dangers to public health in using wastewater for agricultural irrigation.

Matrix metalloproteinase-12 (MMP-12), also known as macrophage elastase, is a potent inflammatory mediator and therefore an important pharmacological target. Clinical trial failures of broad-spectrum compound MMP inhibitors suggested that specificity is the key for a successful therapy. To provide the required selectivity, monoclonal antibody (mAb)-based inhibitors are on the rise. However, poor production of active recombinant human MMP-12 catalytic domain (cdMMP-12) presented a technical hurdle for its inhibitory mAb development. We hypothesized that this problem could be solved by designing an expression-optimized cdMMP-12 mutant without structural disruptions at its reaction cleft and surrounding area, and thus isolated active-site inhibitory mAbs could maintain their binding and inhibition functions toward wild-type MMP-12. We combined three advances in the field—PROSS algorithm for cdMMP-12 mutant design, convex paratope antibody library construction, and functional selection for inhibitory mAbs. As a result, isolated Fab inhibitors showed nanomolar affinity and potency toward cdMMP-12 with high selectivity and high proteolytic stability. Particularly, Fab LH11 targeted the reaction cleft of wild-type cdMMP-12 with 75 nM binding K(D) and 23 nM inhibition IC(50). We expect that our methods can promote the development of mAbs inhibiting important proteases, many of which are recalcitrant to functional production.

Escherichia coli (E. coli) is a widespread bacterial species that comprise a broad variety of strains and can be highly pathogenic often. Diarrhea caused by E. coli pathotypes is a leading public health problem in underdeveloped countries such as Pakistan. Five different diarrheagenic E. coli pathotypes (Enterohaemorrhagic, Enteropathogenic, Enteroaggregative, Enteroinvasive and Enterotoxigenic E. coli) were isolated from 200 samples of diarrheal children in Quetta, Pakistan. Total-cell protein profiles of all the strains isolated were resolved via SDS-Polyacrylamide Gel Electrophoresis (PAGE). More than 15 protein bands were found ranging from 15 kDa to 153 kDa in size. A dendrogram plot explained the relatedness of the pathotypes amongst them. The protein bands of 70 and 15 kDa, analyzed through GelAnalyzer 2010a© were found common among all the isolated pathotypes. Purified bands of these two proteins (70 and 15 kDa) from the gel were injected in experimental mice groups for immunization purposes. During immunogenicity tests, the mice that were immunized with 70 and 15 kDa proteins stayed alive for seven days despite subsequently being challenged with the E. coli pathotypes, while higher mortality 80% were observed in the non-immunized control group. Upon the postmortem it was revealed that all the vital organs were covered by the E. coli strains injected for challenge studies. The immunized mice and control mice sera subjected to the Agar Gel Immunodiffusion assay (AGID) test for antibody/antigen reaction were found positive for antibodies presence. The exploration of immunogenic proteins among all the prevalent pathotypes can be a breakthrough in the development of a combined, effective, and safe vaccine. Further investigation of these immunogenic proteins would be of great importance for future immunization endeavors.

Reem Abdullah Alamro,1 Mohammad Mustafa,2 Abdulrahman K Al-Asmari2 1Deparment of Gastroenterology, Prince Sultan Military Medical City, Riyadh, Saudi Arabia; 2Research Centre, Prince Sultan Military Medical City, Riyadh, Saudi Arabia Objective: The present study planned to investigate the changes in the mRNA expression of inflammatory genes and their association with colorectal cancer (CRC). Our findings could be useful for noninvasive early screening of CRC patients. Patients and methods: Venous blood of 20 CRC cases and 15 healthy controls was collected. The mRNA expression of COX-2, TNF-α, NF-κB and IL-6 genes was carried out by using real-time polymerase chain reaction. Relative quantification was done to find out the fold change of these genes. Results: The mean age of cases and controls was 55 and 50 years, respectively. The ΔCt of COX-2, TNF-α, NF-κB and IL-6 genes was significantly (p < 0.05) lower in cases as compared to controls. Subsequently, the mRNA expression of these genes was, respectively, 3.56-, 3.4-, 1.71- and 3.86-fold higher in CRC cases as compared to controls. Positive correlation of ΔCt of COX-2 was found with ΔCt of TNF-α (r = 0.461, p = 0.041) and NF-κB (r = 0.536, p=0.015) in CRC cases. The mRNA expression of COX-2 was significantly lower in T2 stage, while mRNA expression of NF-κB was significantly lower in both T2 and T3 stages of CRC as compared to T4 stage. Conclusion: The increased mRNA expression of COX-2 along with the high mRNA expression of TNF-α, NF-κB and IL-6 genes may be associative risk factors for CRC. COX-2 and NF-κB genes were more expressed in advanced stages of CRC indicating their role in tumor progression. Our findings support the possible role of blood biomarker in the screening of CRC patients in the early stages. Keywords: colorectal cancer, mRNA expression, COX-2, TNF-α, NF-κB, IL-6, blood biomarker

A thorough characterisation of the genetics, physiology and metabolism of Escherichia coli has led to the availability of a large number of strains and vectors suitable for recombinant protein expression. Despite the relative ease in using E. coli for achieving amplified expression of many recombinant proteins, for some proteins this can be a frustrating and time-consuming process leading to very low expression or no expression at all. This is especially true for membrane proteins, which introduce additional challenges. A number of factors can be considered and optimised for achieving required levels of amplified expression of recombinant proteins in E. coli that are broadly classified as host strain, expression vector and growth conditions. In this paper we summarise these factors and consolidate the common challenges encountered and approaches to overcome them, focusing in particular on cases where there is low amplified expression or no expression at all of the desired recombinant protein, due to various reasons.

Balochistan is the largest province of Pakistan with almost 44% of the country land. Most of the area is rangeland and only 5-7 % area is hardly cultivable and more than 70% population depend upon livestock for their livelihood. Animal rearing is the centuries old occupation of farmers. The province produces 45% and 22% sheep and goat population of the country. Different diseases such as, Enterotoxaemia, sheep and goat Pox, Contagious Caprine Pleuropneumonia, Anthrax and Hemorrhagic Septicemia, Peste des Petits ruminants and Foot and Mouth disease are the most common infections of small and large ruminants. Vaccination covers hardly 10% of the population. The role of livestock in poverty reduction inevitable. Although animal health care facilities are available throughout the province but still it needs to be expanded and improved. Establishment of new Vaccine production centers in the province may be helpful to cover the vaccination of animals.

GeneXpert MTB/RIF has revolutionized the tuberculosis diagnosis by simultaneous detection of Mycobacterium tuberculosis and resistance to RIF (rifampicin), a surrogate marker for multidrug-resistant TB in less than two hours. The RIF-resistance pattern in Balochistan, Pakistan, is not documented. This study was aimed to detect RIF-resistant TB and mutations in RNA polymerase beta (rpoB) gene of M. tuberculosis within 81-bp RRDR in Quetta, Pakistan using GeneXpert® MTB/RIF assay. In total, 2300 clinical specimens were collected from suspected TB patients at Fatima Jinnah General and Chest Hospital Quetta, Pakistan between January and August 2017. These specimens were analyzed by GeneXpert® MTB/RIF assay. The data was statistically analyzed using SPSS software. Out of 2300 clinical specimens, M. tuberculosis was positive in in 899 (39.1%) cases by GeneXpert® MTB/RIF assay [positive respiratory cases 42.9% (871/2032) and non-respiratory 10.4% (28/268) with statistically significant difference (χ2= 104.5, p

International audience; Infectious diseases caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) are a worldwide burden to health, especially in developing countries. Pakistan has one of the highest levels of HBV and HCV infection, causing a severe health problem with significant challenges and priorities. To prevent vertical transmission of infection, regular screening of pregnant women for HBV and HCV is vital. Objectives: The objective of this study was to evaluate the prevalence of HBV and HCV virus in pregnant women having prenatal care. Study Design: Cross-Sectional study. Setting: Bolan Medical Complex Hospital and Sandeman Provincial Civil Hospital Quetta. Period: August 2017 to July 2018. Material & Methods: Blood serum samples were screened for HBV surface antigen (HBsAg) and for anti-HCV using immunochromatography methods. Results: A total of 12,209 pregnant women were tested over a period of one year (August 2017 to July 2018). The overall HBV infections frequency was 1.3% (95% CI 1.1-1.4%) and for HCV infections it was 0.6% (95% CI 0.6-0.7%). Whilst there was only small month-wise variation in the occurrence of HBV and HCV infections, HBV prevalence was highest in May (1.7%) and HCV prevalence was highest in August and December (0.8%). Conclusions: Screening of all pregnant women for HBV and HCV is essential for reducing and eliminating vertical transmission of infection. Risk factors for infection need to be avoided and managed properly.

International audience

Matrix metalloproteinase (MMP)-14 is an important target for cancer treatment due to its critical roles in tumor invasion and metastasis. Previous failures of all compound-based broad-spectrum MMP inhibitors in clinical trials suggest that selectivity is the key for a successful therapy. With inherent high specificity, monoclonal antibodies (mAbs) therefore arise as attractive inhibitors able to target the particular MMP of interest. As a routine screening method, enzyme-linked immunosorbent assays (ELISA) have been applied to panned phage libraries for the isolation of mAbs inhibiting MMP-14. However, because of suboptimal growth conditions and insufficient antibody expression associated with monoclonal ELISA, a considerable number of potentially inhibitory clones might not be identified. Taking advantage of next-generation sequencing (NGS), we monitored enrichment profiles of millions of antibody clones along three rounds of phage panning, and identified 20 Fab inhibitors of MMP-14 with inhibition IC50 values of 10-4000 nM. Among these inhibitory Fabs, 15 were not found by monoclonal phage ELISA. Particularly, Fab R2C7 exhibited an inhibition potency of 100 nM with an excellent selectivity to MMP-14 over MMP-9. Inhibition kinetics and epitope mapping suggested that as a competitive inhibitor, R2C7 directly bound to the vicinity of the MMP-14 catalytic site. This study demonstrates that deep sequencing is a powerful tool to facilitate the systematic discovery of mAbs with protease inhibition functions. This article is protected by copyright. All rights reserved

Background: Proteases are one of the largest pharmaceutical targets for drug developments. Their dysregulations result in a wide variety of diseases. Because proteolytic networks usually consist of protease family members that share high structural and catalytic homology, distinguishing them using small molecule inhibitors is often challenging. To achieve specific inhibition, this study described a novel approach for the generation of protease inhibitory antibodies. As a proof of concept, we aimed to convert a matrix metalloproteinase (MMP)-14 specific inhibitor to MMP-9 specific inhibitory antibodies with high selectivity. Methods: An error-prone single-chain Fv (scFv) library of an MMP-14 inhibitor 3A2 was generated for yeast surface display. A dual-color competitive FACS was developed for selection on MMP-9 catalytic domain (cdMMP-9) and counter-selection on cdMMP-14 simultaneously, which were fused/conjugated with different fluorophores. Isolated MMP-9 inhibitory scFvs were biochemically characterized by inhibition assays on MMP-2/-9/-12/-14, proteolytic stability tests, inhibition mode determination, competitive ELISA with TIMP-2 (a native inhibitor of MMPs), and paratope mutagenesis assays. Results: We converted an MMP-14 specific inhibitor 3A2 into a panel of MMP-9 specific inhibitory antibodies with dramatic selectivity shifts of 690–4,500 folds. Isolated scFvs inhibited cdMMP-9 at nM potency with high selectivity over MMP-2/-12/-14 and exhibited decent proteolytic stability. Biochemical characterizations revealed that these scFvs were competitive inhibitors binding to cdMMP-9 near its reaction cleft via their CDR-H3s. Conclusions: This study developed a novel approach able to convert the selectivity of inhibitory antibodies among closely related protease family members. This methodology can be directly applied for mAbs inhibiting many proteases of biomedical importance. Statement of Significance To achieve high selectivity required for therapies, we developed a novel approach for the generation of protease inhibitory antibodies with nM potency and decent proteolytic stability. The methodology demonstrated here can be readily applied to many proteases of biomedical importance.

Introduction: Tuberculosis is a chronic debilitating infectious disease causing a severe challenge to public health, especially in developing countries. The aim of this study was to examine genetic diversity in Mycobacterium tuberculosis strains circulating in the Balochistan region of Pakistan. Methodology: One hundred isolates collected from patients visiting the Fatima Jinnah TB Hospital in Quetta were subjected to genotype analysis by spoligotyping. Results: Three main genotypes were identified: Central Asian Strain 1 (CAS1) (n = 89), East African Indian (EAI) strain (n = 7) and Latin American Mediterranean (LAM) strain (n = 3). The CAS1 clade (ST 26) had high genetic diversity represented by seven different spoligopatterns, of which one had major predominace (n = 75). Conclusions: This is the first insight into the genotype of M. tuberculosis strains in the Balochistan region that might serve as a base line study for control of tuberculosis in the community.

Background: Viral hepatitis is the major cause of morbidity and mortility. Pakistan ranked in intermediate prevalence zone for Hepatitis B and C. Random screening of Viral Hepatitis B and C can be a suitable approach to monitor the prevalence. The present study is conducted to determine the Seroprevalence of HBV and HCV in Females of Quetta. Methodology: A total of 100 samples of blood were tested for the presence of anti-HBV and anti-HCV antibodies using rapid ICT (SD Korean) kits. Results: Seroprevalence of HBV was 5% and HCV was 6%. Age wise prevalence of HBV indicates that, one patient was HBV positive in age group of 41 to 50 years and 3% were positive in age group of 51 to 60 years, while 1% patient was found HBV positive in age group of 61 to 70 years. Whereas in case of HCV one patient was found in 51 to 60 years, three (3) subjects were in 61 to 70 years and 1% was in 71 to 80 years of age group. Out of 67 married patients 6% were HBV positive and only 9% were HCV positive. Out of 33 single patient 3% were HBV positive and none of the single patient were HCV positive. Out of 84 literate patients 2% were HBV positive and 1% were HCV positive. On the other hand, out 16 illiterate 1% were HBV positive and 30% were HCV positive. Keywords: Serosurvillance, Viral Hepatitis, females, Quetta.

International audience; Camel shows a seasonal breeding pattern with enhanced reproductive activity during the period of low climatic temperature, high rainfall and better food conditions. Therefore, the study was conducted to explore the underlying seasonal effects on histological dimensions of reproductive tract in adult female one-humped camel (Camelus dromedarius) kept in the natural environment of Pakistan. A total 25 reproductive tracts were collected during spring, summer, autumn and winter seasons and were analysed for histo-morphometric parameters during different environmental conditions. A significant increase in number (p

Hepatocellular carcinoma (HCC) is considered as one of the major liver cancers prevailing in the world. The survival rate for the patients diagnosed with HCC is very low as average survival time is only one year. Due to the lack of early diagnosis and poor knowledge of pathophysiological mechanisms, screening of HCC remains possible only at advanced stages. Furthermore, due to non-effective treatment and poor diagnostic techniques led to high fatality rate. Previously, number of HCC markers has been identified but have very less specificity. This study was designed to look into and find out certain HCC specific serological proteins which may be used as biomarker for the early diagnosis of the said disease. In this study all collected samples were analyzed by SDS-PAGE. The results revealed that four different proteins were up-regulated in HCC diagnosed patients compared to the control sample. These proteins may be used as potential biomarkers for the early diagnosis of HCC. The further investigations of the relevant proteins may reveal important clues for the early diagnosis of HCC. Keywords: Biomarker proteins; HCC; SDS-PAGE; Early diagnosis; Liver Cancer http://dx.doi.org/10.19045/bspab.2017.60006

BACKGROUND: Cholangiocarcinoma (CCA) is the second most common primary hepatic neoplasia. Antibodies are normally produced in response to a foreign protein or substance within the body. Autoantibodies can be used clinically for cancer detection which can be identified by proteomic analysis of tumor associated antigens (TAAs) that are potentially involved in malignant transformation. The purpose of the present study was to detect the autoantibodies in sera of patients with cholangiocarcinoma by serological proteomic approach for early cancer diagnosis. METHODS: Serum samples were obtained from 9 patients with CCA and sera from 5 healthy volunteers were used as controls. The European Cell Culture Line provided the two human cholangiocarcinoma cell lines (CCSW1 and CCLP1). Histologically, both originate from extra-hepatic bile duct adenocarcinoma. The protein concentration of the samples was determined using the 2D Quant kit. Proteins were separated using 2-DE (2-dimensional gel electrophoresis). Then proteins were transferred on to nitrocellulose membrane and proteins were detected using enzyme conjugated secondary antibody and ECL substrate (enhanced chemiluminescent) generated signals were visualized by X-films. Siler staining was used to see the migration pattern on gels. RESULTS: 2-D electrophoresis maps of CCA cell lines shown different protein distributions. After silver staining, an average of 160–180 spots were detected from CCSW1, mainly between pH 4 and 8, with molecular weight ranging from 40 to 90 kDa. With CCLP1, between 80 and 100 spots were distributed in acidic parts of the map with MW from 20 to 90 kDa. By comparative blotting analysis, four common immunoreactive spots were found in CCSW1 blots with and two out of nine CCA patient sera but not with control sera, whereas two common immunoreactive spots were found only with control sera (n=4). With CCLP1, no common spot found between the immunoblots with CCA sera (n=5). CONCLUSIONS: The detected immunoreactive protein spots might correspond to the autoantibodies produced to the homologous TAAs. These immunoreactive protein spots might be identified by mass spectrometry and used clinically for early CCA diagnosis.

Background: Hepatocellular carcinoma (HCC) and Cholangiocarcinoma (CC) are primary liver cancers. HCC develops in hepatocytes while CC originates from bile duct epithelium. Due to the lack of early diagnosis, the incidence of HCC and CC are ever increasing and providing effective therapy to the patients suffering from these cancers have increasingly become difficult. The significance of clinical diagnosis of HCC by the detection of tumor markers has not been well documented. So far, none of the studies that indicate the presence of specific autoantibodies in CC, on the contrary to HCC, has been reported. The aim of our current study is to identify new immunological tumor markers for the diagnosis of HCC and CC.

Matrix metalloproteinase (MMP)-14 is an important target for cancer treatment due to its critical roles in tumor invasion and metastasis. Previous failures of all compound-based broad-spectrum MMP inhibitors in clinical trials suggest that selectivity is the key for a successful therapy. With inherent high specificity, monoclonal antibodies (mAbs) therefore arise as attractive inhibitors able to target the particular MMP of interest. As a routine screening method, enzyme-linked immunosorbent assays (ELISA) have been applied to panned phage libraries for the isolation of mAbs inhibiting MMP-14. However, because of suboptimal growth conditions and insufficient antibody expression associated with monoclonal ELISA, a considerable number of potentially inhibitory clones might not be identified. Taking advantage of next-generation sequencing (NGS), we monitored enrichment profiles of millions of antibody clones along three rounds of phage panning, and identified 20 Fab inhibitors of MMP-14 with inhibition IC50 values of 10–4000 nM. Among these inhibitory Fabs, 15 were not found by monoclonal phage ELISA. Particularly, Fab R2C7 exhibited an inhibition potency of 100 nM with an excellent selectivity to MMP-14 over MMP-9. Inhibition kinetics and epitope mapping suggested that as a competitive inhibitor, R2C7 directly bound to the vicinity of the MMP-14 catalytic site. This study demonstrates that deep sequencing is a powerful tool to facilitate the systematic discovery of mAbs with protease inhibition functions.