Phage antibody library technology in tumor therapy: a review / 生物工程学报

Tumor is a serious threat to human health. At present, surgical resection, chemoradiotherapy, targeted therapy and immunotherapy are the main therapeutic strategies. Monoclonal antibody has gradually become an indispensable drug type in the clinical treatment of cancer due to its high efficiency and low toxicity. Phage antibody library technology (PALT) is a novel monoclonal antibody preparation technique. The recombinant immunoglobulin variable region of heavy chain (VH)/variable region of light chain (VL) gene is integrated into the phage vector, and the antibody is expressed on the phage surface in the form of fusion protein to obtain a diverse antibody library. Through the process of adsorption-elution-amplification, the antibody library can be screened to obtain the antibody molecule with specific binding antigen as well as its gene sequence. PALT has the advantages of short antibody production cycle, strong plasticity of antibody structure, large antibody yield, high diversity and direct production of humanized antibodies. It has been used in screening tumor markers and preparation of antibody drugs for breast cancer, gastric cancer, lung cancer and liver cancer. This article reviews the recent progress and the application of PALT in tumor therapy.

Single chain antibody fragment display systems: a review / 生物工程学报

Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.

Preliminary Study on Screening and Identification of Lewis a Antigen Mimic Epitope in Alpaca Phage Display Nanobody Library / 中国实验血液学杂志

OBJECTIVE@#To establish a new method for synthesizing Lewis blood group antigens, that is, the mimotopes of Lewis blood group antigens were screened by using an alpaca phage display nanobody library.@*METHODS@#We selected mimotopes of the Lewis a (lea) antigen by affinity panning of an alpaca phage display nanobody library using a monoclonal anti-lea antibody. Enzyme-linked immunosorbent assay (ELISA) was used to test the affinity of the positive clones for the monoclonal anti-lea antibody, and the high-affinity positive clones were selected for sequencing and synthesis. Finally, the sensitivity, specificity and reactivity of the synthesized lea mimotope in clinical samples were verified by ELISA.@*RESULTS@#A total of 96 phage clones were randomly selected, and 24 were positive. Fourteen positive clones with the highest affinity were selected for sequencing. The result showed that there were 5 different sequences, among which 3 sequences with the highest frequency, largest difference and highest affinity were selected for expression and synthesis. The sensitivity and specificity of lea mimic antigen by ELISA showed that, the minimum detection limit of gel microcolumn assay (GMA) and ELISA method were 25 times different, and the lea mimic antigen had no cross reacted with the other five unrelated monoclonal antibodies(P<0.001). Finally, 30 clinical plasma samples were analyzed. The mean absorbance of the 15 positive plasma samples was significantly higher than that of the 15 negative plasma samples (P=0.02). However, the positive signal values of the clinical samples were much lower than those of the monoclonal antibodies.@*CONCLUSION@#A new method of screening lea mimic antigen by using alpaca phage nanoantibody library has been established, which is expected to realize the screening of lea mimotopes, thus realizing the application of high-sensitivity detection methods such as ELISA and chemiluminescence in blood group antibody identification.

Identification of High-affinity Novel Targeted Peptides for the Treatment of Human Ovarian Cancer

Background: To investigate the potential of the phage display-identified tumor cellbinding peptide as a biomarker of epithelial ovarian cancer using phage display technology. Method: The Ph.D.-7 Phage Display Peptide Library was used to identify the specific conjugated phages with SKOV3 epithelial ovarian cancer cells, while Chinese hamster ovary cells formed the basis. After employing the rapid differential screening method invitro, the enzyme-linked immunosorbent assay (ELISA), DNA sequencing, immunohistochemistry, immunofluorescence, and the competitive inhibition test of synthetic peptides were used to determine the affinity and specificity of the phages with SKOV3 cells. Results: Using bio panning, we screened the phages, showing a 3590-fold increase after the third round. A total of 61 titers of the phage were randomly selected for ELISA and 10 kinds of the phages with an optical density >0.5 were used for DNA sequencing. Clones of the phage TRRNIPN were derived from DNA sequencing based on ELISA, exhibiting both the brown granular phenomenon and green fluorescence. The specific targeted peptide TRRNIPN was incorporated in tumor cells through the competitive inhibition test. Conclusion: The results of our study indicate that the phage display identified polypeptide TRRNIPN may be an effective biomarker for the early diagnosis and targeted therapy of ovarian cancer

A Phage Display-Identified Peptide Selectively Binds to Kidney Injury Molecule-1 (KIM-1) and Detects KIM-1–Overexpressing Tumors In Vivo / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: This study was carried out to identify a peptide that selectively binds to kidney injury molecule-1 (KIM-1) by screening a phage-displayed peptide library and to use the peptide for the detection of KIM-1overexpressing tumors in vivo. MATERIALS AND METHODS: Biopanning of a phage-displayed peptide library was performed on KIM-1–coated plates. The binding of phage clones, peptides, and a peptide multimer to the KIM-1 protein and KIM-1–overexpressing and KIM-1–low expressing cells was examined by enzyme-linked immunosorbent assay, fluorometry, and flow cytometry. A biotin-peptide multimer was generated using NeutrAvidin. In vivo homing of the peptide to KIM-1–overexpressing and KIM1–low expressing tumors in mice was examined by whole-body fluorescence imaging. RESULTS: A phage clone displaying the CNWMINKEC peptide showed higher binding affinity to KIM-1 and KIM-1–overexpressing 769-P renal tumor cells compared to other phage clones selected after biopanning. The CNWMINKEC peptide and a NeutrAvidin/biotin-CNWMINKEC multimer selectively bound to KIM-1 over albumin and to KIM-1–overexpressing 769-P cells and A549 lung tumor cells compared to KIM-1–low expressing HEK293 normal cells. Co-localization and competition assays using an anti–KIM-1 antibody demonstrated that the binding of the CNWMINKEC peptide to 769-P cells was specifically mediated by KIM-1. The CNWMINKEC peptide was not cytotoxic to cells and was stable for up to 24 hours in the presence of serum. Whole-body fluorescence imaging demonstrated selective homing of the CNWM-INKEC peptide to KIM-1–overexpressing A498 renal tumor compared to KIM1–low expressing HepG2 liver tumor in mice. CONCLUSION: The CNWMINKEC peptide is a promising probe for in vivo imaging and detection of KIM-1‒overexpressing tumors.

Sheep polyclonal antibody to map Haemonchus contortus mimotopes using phage display library / Anticorpo policlonal de ovinos para mapear mimetopos de Haemonchus contortus usando a biblioteca de Phage display

Abstract The aim of this study was to evaluate phage display technology for mapping Haemonchus contortus mimotopes. We screened the PhD-7 Phage Display Peptide Library Kit with a sheep polyclonal antibody against H. contortus. After four rounds of selection, 50 phage peptide clones were selected by biopanning and sequenced. Two clones displaying peptide mimotopes of H. contortus proteins were chosen for sheep immunization: clone 6 - mimotope of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and clone 17 - mimotope of a disorganized muscle family member (Dim 1). Twelve sheep were allocated into 3 groups of 4 animals as follow: G1: control group; G2/GAPDH: immunized with clone 6; and G3/Dim1: immunized with clone 17. Four immunizations were performed at intervals of seven days (0, 7, 14, and 21 days). On day 28 post initial vaccination, all groups were orally challenged with 2500 H. contortus infective larvae. The mimotope peptides selected by phage display were recognized by IgG from sheep naturaly infected with H. contortus. The immunization protocol showed an increasein IgG anti-M13 phage titers, but no effect was observed in IgG-specific for the anti-mimotope peptides. This is the first report of successful use of a phage display library for the identification of mimotopes of H. contortus proteins.

Resumo O objetivo deste estudo foi avaliar a tecnologia de phage display no mapeamento de mimetopos de Haemonchus contortus. Anticorpo policlonal de ovinos anti-H. contortus foi usado para seleção a partir da biblioteca PhD-7 Phage Display Peptide Library Kit (New England BioLabs). Após quatro rodadas, 50 clones de fagos expressando peptídeos foram selecionados e sequenciados. Dois clones que exibiram mimetopos de H. contortus foram escolhidos para imunização de ovinos: clone 6 - mimetopo de gliceraldeído-3-fosfato desidrogenase (GAPDH) e clone 17 - mimetopo da família do músculo desorganizado (Dim 1). Doze ovinos foram alocados em 3 grupos de 4 animais, da seguinte forma: G1: grupo controle, G2/GAPDH: imunizado com o clone 6 e G3/Dim1: imunizado com o clone 17. Quatro imunizações foram realizadas (0, 7, 14 e 21 dias). No dia 28 após a primeira imunização, todos os grupos foram desafiados oralmente com 2500 larvas infectantes de H. contortus . Os peptídeos mimetopos selecionados foram reconhecidos por IgG de ovinos naturalmente infectados por H. contortus. O ensaio de imunização revelou um aument dos títulos de IgG anti-fago M13, mas não ocorreu aumento de IgG anti-peptídeos mimetopos. Este é o primeiro relato de uso bem sucedido da biblioteca de Phage display para a identificação de mimetopos de H. contortus.

Preparation and identification of anti-human ICAM-1 scFv / 生物工程学报

To screen the specific anti-human intercellular adhesion molecule-1 (ICAM-1) single chain fragment variable (scFv) using phage display library technology and to identify its biological activity. P1 peptide was used as antigen, and the phage antibodies against human ICAM-1 antigen were panned by four binding-eluting-amplifying cycles using Tomlinson I+J phage display library. After four rounds of selective enrichment screening, the positive clones were determined by PCR, enzyme linked immunosorbent assay (ELISA)-based antigenic cross reaction and Dot blotting. Then the binding specificity and biological activity of purified scFv were identified by Western blotting, competitive ELISA and cell adhesion inhibition assay respectively. Furthermore, four positive clones were first panned through P1 peptide coated-ELISA assay, and then J-A1 was obtained and identified by PCR, ELISA-based antigenic cross reaction and Dot blotting, which could show a specific binding between P1 peptide and human ICAM-1 protein antigen. Subsequently, the purified scFv showed a satisfactory specificity and anti-adhesive activity in competitive ELISA and the cell adhesion inhibition assay. The specific anti-human ICAM-1 scFv was prepared successfully from Tomlinson I+J phage display library, which pave the way for further application of anti-human ICAM-1 scFv for inflammation diseases therapeutics.

Reconhecimento molecular na doença de chagas do ponto de vista do parasita e do hospedeiro / Molecular recognition in Chagas disease from the point of view of the parasite and the host

A doença de Chagas, causada pelo parasita protozoário Trypanosoma cruzi, afeta milhões de pessoas, a maioria delas vivendo na América latina. Apesar dos avanços da medicina e da biotecnologia, ainda existem poucas opções de tratamento para indivíduos com a doença. Assim, é importante compreendermos os detalhes moleculares da infecção parasitária, para que novas alternativas terapêuticas e de diagnóstico possam ser desenvolvidas para esses pacientes. Neste trabalho estudamos esta doença em duas frentes, uma do ponto de vista do parasita, e a outra, da resposta do hospedeiro. Utilizando bioinformática, identifcamos um peptídeo conservado (denominado TS9) presente nas proteínas de superfície gp85/transsialidases do parasita. Este peptídeo é capaz de promover adesão celular e, na sua forma sintética, inibe a entrada do T. cruzi na célula hospedeira. Análise da estrutura proteica revelou que o peptídeo TS9 encontra-se num domínio do tipo laminina-G, lado-a-lado com o peptídeo FLY, outro peptídeo conservado desta grande família, previamente descrito pelo nosso grupo. Juntos, eles formam um sítio de adesão a citoqueratinas e proteínas de flamento intermediário. Na segunda parte, investigamos os antígenos e epítopos reconhecidos pelas imunoglobulinas de pacientes portadores da doença nas suas diferentes formas clínicas: assintomática e cardiomiopatias, leve ou grave. Criamos uma biblioteca de phage display contendo, virtualmente, todos os fragmentos proteicos existentes no T. cruzi, que foi varrida contra imunoglobulinas para a construção de um mapa da resposta humoral dos pacientes com a doença de Chagas. Nossos resultados mostram que a resposta dos pacientes é complexa, e mais de dois mil epítopos foram mapeados. Muitos deles, como os antígenos B13, SAPA e FRA já foram previamente descritos, validando nosso método. Porém, um grande número de novos epítopos, inclusive contra proteína descritas como hipotéticas ou sem função conhecida, também foram encontrados. Seus papéis na infecção e resposta imune da doença merecem, portanto, atenção. Em resumo, as abordagens e técnicas utilizadas nesta tese são inovadoras, e permitiram a identificação de peptídeos e moléculas que poderão ser úteis para o desenvolvimento de novos métodos diagnósticos e terapêuticos para a doença de Chagas

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, afects millions of people, most of them living in Latin America. Despite advances in medicine and biotechnology, there are still few treatment options for individuals with the disease. Thus, it is important to understand the molecular details of the parasitic infection, so that new therapeutic and diagnostic alternatives can be developed for these patients. In this work, we study this disease in two fronts, one from the point of view of the parasite, and the other, of the response of the host. Using bioinformatics, we identifed a conserved peptide (called TS9) present in the surface proteins gp85 / trans-sialidases of the parasite. This peptide is capable of promoting cell adhesion and, in its synthetic form, inhibits the entry of T. cruzi into the host cell. Analysis of the protein structure revealed that the TS9 peptide is in a laminin-G-like domain, side-by-side with the peptide FLY, another conserved peptide of this large family, previously described by our group. Together, they form an adhesion site to cytokeratins and intermediate flament proteins. In the second part, we investigated the antigens and epitopes recognized by the immunoglobulins of patients with the disease in their diferent clinical forms: asymptomatic and cardiomyopathies, mild or severe. We created a phage display library containing virtually all existing protein fragments in T. cruzi. This library was screened against immunoglobulins for the construction of a humoral response map of patients with Chagas disease. Our results show that the response of the patients is complex, and more than 2,000 epitopes have been mapped. Many of them, such as the B13, SAPA and FRA antigens have been previously described, validating our method. However, a large number of new epitopes, including many against proteins described as hypothetical or with no known function, were also found. Their roles in infection and immune response of the disease deserve, therefore, attention. In summary, the approaches and techniques used in this thesis are innovative and have allowed the identifcation of new peptides and molecules that may be useful for the development of new diagnostic and therapeutic methods for Chagas disease

Proteomic analysis for finding serum pathogenic factors and potential biomarkers in multiple myeloma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Multiple myeloma (MM) is a malignant tumor, which takes the second place in malignant blood disease. The clinical symptoms are complicated that make more difficult to diagnose and therapy. Lots of researches focus on the proteins about MM in order to solve those problems. We used proteomic methods to find potential biomarkers in MM patients.</p><p><b>METHODS</b>We applied the peptide ligand library beads (PLLBs) to deplete high abundance proteins in serum for finding potential pathogenic factors and biomarkers of MM. Using 1D-Gel-liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 789 and 849 unique serum proteins in MM patients and in healthy controls, respectively.</p><p><b>RESULTS</b>Twenty-two proteins were found differentially expressed between the two groups including serum amyloid A protein, vitamin D-binding protein isoform-1 precursor, plasma kallikrein, and apolipoprotein A-I. Changes of integrin alpha-11 and isoform-1 of multimerin-1 were validated with Western blotting. The linkage of the differentially expressed proteins and the pathogenesis pathways of MM were discussed.</p><p><b>CONCLUSIONS</b>PLLB combined with 1D-gel-LC-MS/MS analysis is an efficient method to identify differentially expressed proteins in serum from patients with MM.</p>

Virtual screening and molecular simulations of antisense peptides targeting MT1-MMP / 生物工程学报

Membrane type-1 matrix metalloproteinase (MT1-MMP or MMP14) plays the pivotal role in tumor development and metastasis, so it is a promising drug target in malignancy. To acquire MT1-MMP specific binding peptides, we first analyzed MMPs sequences to find the divergent and specific sequence of MT1-MMP by bioinformatics approach, then set the specific sequence as the sense peptide target and designed antisense peptide library. Finally, by means of molecular docking, molecular dynamics simulation and in vitro cell assays, we screened the antisense peptide library against MT1-MMP and further studied the obtained specific peptides. Here, we identified the divergent and specific sequence of AYIREGHE (Named MT1-loop) located in MT1-MMP loop by multiple sequence alignment and established the antisense peptides library with capacity of 1 536 sequences. After two rounds of virtual screening, we obtained five antisense peptides with Rerankscores in the top for further screening. They all interacted with MT1-MMP, and docked well at the active site composed of MT1-loop sequence. Analysis of the affinities of these five antisense peptides to other MMPs (MMP1-3, MMP7-13, MMP14 HPX, MMP16) revealed that the peptide FVTFPYIR was more specific to MT1-MMP. Molecular dynamics simulation showed that the peptide FVTFPYIR might affect the stability of MT1-MMP and thus have effects on its activities. Meanwhile, the peptide FVTFPYIR could specifically inhibit the growth of MG63 and MDA-MB-231 tumor cells both of which expressed MT1-MMP. The work provides a new insight and way for the development of antitumor lead peptides targeting MT1-MMP.

Identification of high-affinity VEGFR3-binding peptides through a phage-displayed random peptide library / 부인종양

OBJECTIVE: Vascular endothelial growth factor (VEGF) interaction with its receptor, VEGFR-3/Flt-4, regulates lymphangiogenesis. VEGFR-3/Flt-4 expression in cancer cells has been correlated with clinical stage, lymph node metastasis, and lymphatic invasion. The objective of this study is to identify a VEGFR-3/Flt-4-interacting peptide that could be used to inhibit VEGFR-3 for ovarian cancer therapy. METHODS: The extracellular fragment of recombinant human VEGFR-3/Flt-4 (rhVEGFR-3/Flt-4) fused with coat protein pIII was screened against a phage-displayed random peptide library. Using affinity enrichment and enzyme-linked immunosorbent assay (ELISA) screening, positive clones of phages were amplified. Three phage clones were selected after four rounds of biopanning, and the specific binding of the peptides to rhVEGFR-3 was detected by ELISA and compared with that of VEGF-D. Immunohistochemistry and immunofluorescence analyses of ovarian cancer tissue sections was undertaken to demonstrate the specificity of the peptides. RESULTS: After four rounds of biopanning, ELISA confirmed the specificity of the enriched bound phage clones for rhVEGFR-3. Sequencing and translation identified three different peptides. Non-competitive ELISA revealed that peptides I, II, and III had binding affinities for VEGFR-3 with Kaff (affinity constant) of 16.4+/-8.6 microg/mL (n=3), 9.2+/-2.1 microg/mL (n=3), and 174.8+/-31.1 microg/mL (n=3), respectively. In ovarian carcinoma tissue sections, peptide III (WHWLPNLRHYAS), which had the greatest binding affinity, also co-localized with VEGFR-3 in endothelial cells lining lymphatic vessels; its labeling of ovarian tumors in vivo was also confirmed. CONCLUSION: These finding showed that peptide III has high specificity and activity and, therefore, may represent a potential therapeutic approach to target VEGF-VEGFR-3 signaling for the treatment or diagnosis of ovarian cancer.

Phage display peptide library for screening the peptides that specifically bind to osteoblasts cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To explore the experimental methods that the phage peptide library technology screening human osteoblast specificity polypeptide, which will provide the basis of the experiment of the Ti surface biolization modification.</p><p><b>METHODS</b>Human calvarial osteoblasts were used as the target cells for whole-cell biopanning from a 12-mer peptide phage-display library. Cell eluent and cell lysis buffer were cultivate and count respectively after washing. Then the target cells were analyzed by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence detection to authenticate the positive phage clones by human gingival fibroblast as the absorber cells. The positive phage clones were deduced by DNA sequencing.</p><p><b>RESULTS</b>After four rounds of screening, twenty-two positive phage clones were found out from randomly selected phage monoclonals, whose single-strand DNA were extracted and sequenced. Amino acid sequence of the highest frequency peptide was MGWSWWPETWPM.</p><p><b>CONCLUSIONS</b>The specific peptide against human osteoblasts can be obtained from a phage-display peptide library for use as a new research approach and experimental basis of the biolization modification of the titanium surface.</p>

Identificação de peptídeos de Escherichia coli capazes de inibir a própria fagocitose em sepse / Identification of Escherichia coli peptides that can inhibit its own phagocytosis in sepsis

Introdução: Sepse é uma síndrome complexa definida por resposta inflamatória sistêmica, de origem infecciosa e caracterizada por manifestações múltiplas que podem determinar disfunção ou falência de um ou mais órgãos ou sistemas. É a principal causa de morte em unidades de terapia intensiva em pacientes críticos e tem representado uma fonte constante de preocupação para os sistemas de saúde em todo o mundo, devido, principalmente, às taxas elevadas de morbimortalidade. O tratamento da sepse é um desafio e continua a ser uma tarefa difícil devido a inúmeros fatores interferentes. Um estudo do nosso grupo demonstrou que a Escherichia coli (E. coli) é capaz de se ligar CD16 de um modo independente de opsonina, levando a um aumento na resposta inflamatória e a inibição da sua própria fagocitose, por conseguinte, procurou-se identificar os peptídeos no proteoma da E. coli envolvidos neste cenário. Metodologia: Utilizando a metodologia de Phage Display, que consiste numa técnica de clonagem, que permite a expressão de diversas sequências de peptídeos na superfície de bacteriófagos, nós identificamos 2 peptídeos que obtiveram interação com CD16. Após a seleção dos peptídeos identificamos uma proteína de membrana de E.coli que possui alta similaridade com um de nossos peptídeos selecionados. Nós acreditamos que esta proteína de membrana possa estar envolvida no processo de evasão imune desenvolvida pela E.coli e parece ser um forte candidato como uma nova opção terapêutica para controlar infecções por E. coli. Conclusão: A identificação de proteínas capazes de induzir inibição de fagocitose, através do receptor CD16, pode ser usada como uma nova forma de tratamento da sepse, assim como explorada no tratamento de doenças autoimunes.

Introduction: Sepsis is a complex syndrome defined by a systemic inflammatory response of infectious origin and characterized by multiple manifestations that can determine dysfunction/failure of one or more organs and systems. It is the leading cause of death in intensive care units and represents a major health problem around the world, mainly due to its high mortality and morbidity rates. The treatment of sepsis is challenging and remains a difficult task due to numerous interfering factors. A study from our group demonstrated that Escherichia coli (E. coli) is able to bind CD16 in an opsoninindependent manner, leading to an increase in the inflammatory response and inhibition of its own phagocytosis, therefore we sought to identify the peptides in the E. coli proteome involved in this scenario. Methods and Results: Using the Phage Display technique, which is a cloning technique that allows the expression of various peptide sequences on the surface of bacteriophages (phages) and selecting these on the basis of affinity for a target molecule, we identified two peptides that interact with CD16. Next, using bioinformatic tools, we found an E. coli membrane protein that has high similarity with one of our selected peptides. We believe this membrane protein is involved in the process of immune evasion developed by E. coli and it is a strong candidate as a new therapeutic option to control E. coli infections. Conclusion: The identification of proteins capable of inducing inhibition of phagocytosis through the CD16 receptor, can be used as a new treatment of sepsis, as well as exploited in the treatment of autoimmune diseases.

Comprehensiveness and programmatic vulnerability to stds/hiv/aids in primary care / La integralidad y vulnerabilidad programática de las ets/vih/sida en la atención básica / A integralidade e a vulnerabilidade programática às DST/HIV/AIDS na Atenção Básica




This study aimed to identify programmatic vulnerability to STDs/HIV/AIDS in primary health centers (PHCs). This is a descrip - tive and quantitative study carried out in the city of São Paulo. An online survey was applied (FormSUS platform), involving administrators from 442 PHCs in the city, with responses received from 328 of them (74.2%), of which 53.6% were nurses. At - tention was raised in relation to program - matic vulnerability in the PHCs regarding certain items of infrastructure, prevention, treatment, prenatal care and integration among services on STDs/HIV/AIDS care. It was concluded that in order to reach comprehensiveness of actions for HIV/ AIDS in primary health care, it is necessary to consider programmatic vulnerability, in addition to more investment and reor - ganization of services in a dialogue with the stakeholders (users, multidisciplinary teams, and managers, among others).


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Objetivo Fue identificar la vulnerabilidad programática de las Unidades Básicas de Salud con la atención a las ETS/VIH/SIDA. Método Es un estudio descriptivo con un abordaje cuantitativo llevado a cabo en el Municipio de San Pablo. Fue utilizado un formulario online (el FormSUS) con los gerentes de las 442 Unidades Básicas de Salud del Municipio de San Pablo. Participaran en el estudio 74.2% de los gerentes, estos 53.6% eran enfermeros. Resultados Se destaca la vulnerabilidad programática de las Unidades Básicas de Salud en relación a algunos elementos de la infraestructura, acciones de prevención, tratamiento, prenatal y la integración entre los servicios en la atención a las ETS/VIH/SIDA. Conclusión La construcción de tales marcadores constituye un instrumento, presentado en otro artículo, el cual puede ayudar a apoyar la captura de vulnerabilidades de las mujeres en relación a las ETS/VIH en el contexto de los servicios de Atención Primaria de Salud. Los marcadores constituyen importante herramienta para operacionalizar el concepto de vulnerabilidad en la Atención Primaria. Además, promueven procesos de trabajo inter e multidisciplinar e inter e multisectorial. La propuesta de un instrumento basado en dichos marcadores puede apoyar la captura de la vulnerabilidad de las mujeres en relación a las ETS/VIH. .

Objetivo Identificar a vulnerabilidade programática às DST/HIV/aids na Atenção Básica para o enfrentamento do HIV/Aids. Método Estudo descritivo, com abordagem quantitativa, realizado no Município de São Paulo (MSP). Utilizou-se formulário online (FormSUS), com gerentes das 442 Unidades Básicas de Saúde (UBS) do MSP. Participaram do estudo 74,2% gerentes, dos quais 53,6% eram enfermeiros. Resultados Destaca-se a vulnerabilidade programática nas UBS com relação a alguns itens de infraestrutura, ações de prevenção, de tratamento, no pré-natal e de integração entre os serviços na atenção às DST/HIV/aids. Conclusão Para a efetivação da integralidade no enfrentamento do HIV/aids na Atenção Básica é necessário atentar para a vulnerabilidade programática, além de mais investimentos e reorganização dos serviços, num diálogo com os atores sociais envolvidos (usuários, equipe multiprofissional, gerentes, gestores, entre outros).


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Characterization of Leiurus abdullahbayrami (Scorpiones: Buthidae) venom: peptide profile, cytotoxicity and antimicrobial activity

Background Scorpion venoms are rich bioactive peptide libraries that offer promising molecules that may lead to the discovery and development of new drugs.Leiurus abdullahbayrami produces one of the most potent venoms among Turkish scorpions that provokes severe symptoms in envenomated victims.Methods In the present study, the peptide profile of the venom was investigated by electrophoretic methods, size-exclusion and reversed-phase chromatography and mass spectroscopy. Cytotoxic and antimicrobial effects were evaluated on a breast cancer cell line (MCF-7) and various bacterial and fungal species.Results Proteins make up approximately half of the dry weight of L. abdullahbayrami crude venom. Microfluidic capillary electrophoresis indicated the presence of 6 to 7 kDa peptides and proved to be a highly practical peptidomics tool with better resolution when compared to conventional polyacrylamide gel electrophoresis. Mass spectroscopy analysis helped us to identify 45 unique peptide masses between 1 to 7 kDa with a bimodal mass distribution peaking between molecular weights of 1 to 2 kDa (29%) and 3 to 4 kDa (31%). L. abdullahbayrami crude venom had a proliferative effect on MCF-7 cells, which may be explained by the high concentration of polyamines as well as potassium and calcium ions in the arachnid venoms. Antimicrobial effect was stronger on gram-negative bacteria.Conclusions This work represents the first peptidomic characterization of L. abdullahbayrami venom. Considering the molecular weight-function relationship of previously identified venom peptides, future bioactivity studies may lead to the discovery of novel potassium and chloride ion channel inhibitors as well as new antimicrobial peptides fromL. abdullahbayrami venom.

Characterization of Leiurus abdullahbayrami (Scorpiones: Buthidae) venom: peptide profile, cytotoxicity and antimicrobial activity

Background Scorpion venoms are rich bioactive peptide libraries that offer promising molecules that may lead to the discovery and development of new drugs.Leiurus abdullahbayrami produces one of the most potent venoms among Turkish scorpions that provokes severe symptoms in envenomated victims.Methods In the present study, the peptide profile of the venom was investigated by electrophoretic methods, size-exclusion and reversed-phase chromatography and mass spectroscopy. Cytotoxic and antimicrobial effects were evaluated on a breast cancer cell line (MCF-7) and various bacterial and fungal species.Results Proteins make up approximately half of the dry weight of L. abdullahbayrami crude venom. Microfluidic capillary electrophoresis indicated the presence of 6 to 7 kDa peptides and proved to be a highly practical peptidomics tool with better resolution when compared to conventional polyacrylamide gel electrophoresis. Mass spectroscopy analysis helped us to identify 45 unique peptide masses between 1 to 7 kDa with a bimodal mass distribution peaking between molecular weights of 1 to 2 kDa (29%) and 3 to 4 kDa (31%). L. abdullahbayrami crude venom had a proliferative effect on MCF-7 cells, which may be explained by the high concentration of polyamines as well as potassium and calcium ions in the arachnid venoms. Antimicrobial effect was stronger on gram-negative bacteria.Conclusions This work represents the first peptidomic characterization of L. abdullahbayrami venom. Considering the molecular weight-function relationship of previously identified venom peptides, future bioactivity studies may lead to the discovery of novel potassium and chloride ion channel inhibitors as well as new antimicrobial peptides fromL. abdullahbayrami venom.(AU)

[Screening phage display library for isolation of human colon cancer cell-binding peptide ligands]

Recently, phage display libraries have received enormous attention for identification and isolation of pharmaceutical molecules with diagnostic and therapeutic properties. Peptide libraries are known as one of the most important and widely used types of phage display libraries. In the current study, we aimed to screen the Ph.D.[TM]-7 phage display peptide library through biopanning for the identification of human colon adenocarcinoma-binding peptide ligands. Three rounds of biopanning were performed on SW480 as the target cell and fibroblast [HF-SF-PI3], AGS, KYSE-30 and Huh-7 as control cells. The displayed peptide-encoding regions in the genome of SW480-binding phages obtained from the final round of panning were amplified by plaque-PCR and subsequently sequenced. Bioinformatic tools were used to determine the sequence of target cell-binding peptides and further characterization of these peptides. Biopanning of the phage library led to the enrichment of several peptides among which the peptide with sequence "HAMRAQP" was the most dominant. Bioinformatic analysis of the isolated peptides indicated that they are not target unrelated peptides [TUP]. The peptides, in particular those with the highest frequency, due to having the capability of specific binding to SW480 cells represent the potential for use in targeting of therapeutic genes and drugs to colon cancer cells

Identification of Antimicrobial Peptide Hexamers against Oral Pathogens through Rapid Screening of a Synthetic Combinatorial Peptide Library

A positional scanning synthetic peptide combinatorial library (PS-SCL) was screened in order to identify antimicrobial peptides against the cariogenic oral bacteria, Streptococcus mutans. Activity against Streptococcus gordonii and Aggregatibacter actinomycetemcomitans was also examined. The library was comprised of six sub-libraries with the format O(1-6)XXXXX-NH2, where O represents one of 19 amino acids (excluding cysteine) and X represents equimolar mixture of these. Each sub-library was tested for antimicrobial activity against S. mutans and evaluated for antimicrobial activity against S. gordonii and A. actinomycetemcomitans. The effect of peptides was observed using transmission electron microscopy (TEM). Two semi-mixture peptides, RXXXXN-NH2 (pep-1) and WXXXXN-NH2 (pep-2), and one positioned peptide, RRRWRN-NH2 (pep-3), were identified. Pep-1 and pep-2 showed significant antimicrobial activity against Gram positive bacteria (S. mutans and S. gordonii), but not against Gram negative bacteria (A. actinomycetemcomitans). However, pep-3 showed very low antimicrobial activity against all three bacteria. Pep-3 did not form an amphiphilic alpha-helix, which is a required structure for most antimicrobial peptides. Pep-1 and pep-2 were able to disrupt the membrane of S. mutans. Small libraries of biochemically-constrained peptides can be used to generate antimicrobial peptides against S. mutans and other oral microbes. Peptides derived from such libraries may be candidate antimicrobial agents for the treatment of oral microorganisms.

Screening and identification of C7C peptide ligands of Mycobacterium tuberculosis / 中华预防医学杂志

<p><b>OBJECTIVE</b>To obtain the C7C peptide ligands of Mycobacterium tuberculosis by affinity screening based on the phage-displayed random C7C peptide library, and preliminarily identify the binding capacity of the peptide to Mycobacterium.</p><p><b>METHODS</b>Inactive Mycobacterium tuberculosis reference strain H37Rv was used as the target molecule to screen the Ph. D.-C7C peptide library, and Mycobacterium bovis, BCG was used for reverse screening. After 4 rounds of affinity screening, single phages eluted by H37Rv and BCG were selected for DNA sequencing. ELISA was used to detect the binding affinities of different single phage clones. The cyclic peptides displayed by the phage clones showing the highest appetency were synthesized in vitro with fluorescent markers. Fluorescence microscopy and flow cytometer was used to detect the binding affinities of synthesized cyclic peptides, comparing with linear binding peptides obtained before.</p><p><b>RESULTS</b>After 4 rounds of biopanning, phages that could bind with target molecules were remarkable enriched. 16 common sequences were obtained by sequencing analysis of single phages. With ELISA, phage SB1, SB5, SB8 and SB26 all showed higher affinity with H37Rv and BCG, the ratio to negative control of which were ≥ 2.1, but could not bind to the 3 nonmycobacteria, which were identified as the positive clones. Based on the results of flow cytometer detection, the affinities to H37Rv of 4 cyclic peptides SB1, SB5, SB24, SB26 were (73.2 ± 6.3)%, (63.2 ± 5.3)%, (32.9 ± 3.1)%, (89.4 ± 7.0)%, and to BCG were (65.6 ± 6.1)%, (48.6 ± 4.5)%, (10.3 ± 1.8)%, (86.6 ± 7.9)%, separately, which were all higher than H8 ((4.0 ± 1.0)%, (5.5 ± 1.2)%) . From the results of fluorescence microscopy observation, all of the fluorescent labeled cyclic peptides SB1, SB5, SB24, SB26 could bind to H37Rv and showed higher fluorescence intensities, which also had certain affinities to other 18 mycobacteria, but the fluorescence intensities were lower than H37Rv, and didn't bind to 3 non-mycobacteria.</p><p><b>CONCLUSION</b>Based on the replacement of linear 7 peptide library with C7C peptide library, new ligands of Mycobacterium tuberculosis were achieved successfully, which showed significantly higher binding affinities to mycobacteria.</p>

Construction and identification of anti-HER2 phage display single chain fragment of variable region library in human breast cancer / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct human phage single-chain antibody (scFv) library against breast cancer, and to identify anti-HER2 specific antibodies from the human phage display scFv library to offer a stronger affinity sequence targeting HER2 for fusion protein targeting HER2 and CXCR4.</p><p><b>METHODS</b>Total RNA was extracted from the adjacent lymphatic tissue harvested from breast cancer patients. The variable regions of the whole antibody were amplified by using RT-PCR and were cloned into the vector pCANTAB-5E through a linker. The products were electroporated into competent E.coli TG1 cells. Recombinant phages specific for breast cancer cells were enriched in SKBR-3 after four rounds. The antigen-positive clones were selected by ELISA and immunohistochemistry.</p><p><b>RESULTS</b>The fragment of VH and VL were about 375 and 330 bp and were linked in vitro to form scFv of 750 bp that was resistant to the breast cancer HER2 single strand. A fusion phage display library that contained total of 2.48×10(8) pfu /ml was established. ELISA and immunohistochemical results confirmed that the antibody has a strong affinity with HER2 antigen in breast cancer tissue. Compared to human IgG antibody, a scFv phage library against human breast cancer was successfully constructed with high capacity. The scFv was highly specific to HER2 antigen and the sequencing results indicated that VL and VH genes were highly homologous with the variable region of human antibody.</p><p><b>CONCLUSION</b>This strategy may achieve new targeted antibody resistant to the breast cancer for clinical treatment and provide a carrier that uses HER2 as a target of the fusion protein for anti-tumor therapy.</p>