Eu gostaria de agradecer Olga de Souza Silva para assistência técnica. Este trabalho foi financiado pela Swiss National Science Foundation (SNSF) Grant 31003A_127029.

O método TransFLP (Figura 1) é exemplificado por três abordagens diferentes: I) a eliminação de dois genes adjacentes que codificam determinantes de virulência de V. cholerae (por exemplo, ΔctxAB), II) a remoção de uma ilha de patogenicidade (por exemplo, ΔVPI-1), e III) a integração de uma polimerase de RNA dependente de sequência do promotor de T7 a montante de um gene de interesse, o que permite a sua subsequentemente expressão artificial no fundo estirpe respectivo (por exemplo, T7-TFox) (Figura 2). 1. Preparação de Quitina Flakes <ol><li> 50-80 mg de peso de flocos de quitina de um padrão de tubo de plástico de 1,5 ml. Isto pode ser preparado em grandes quantidades. Quitina flocos estão comercialmente disponíveis a partir de Sigma (cat. número C9213). </li><li> Autoclavar os flocos mantendo as tampas dos tubos aberto. </li><li> Imediatamente fechar as pálpebras após a autoclave tenha arrefecido. </li><li> Loja autoclavado quitina flocosà temperatura ambiente. </li></ol> 2. Opcional: Transformação Artificial de V. cholerae com Flp Recombinase-encoding Plasmid <ol><li> Prepare electrocompetentes V. cholerae células utilizando métodos padrão 11. Armazenar aliquotas de células competentes a -80 ° C. </li><li> Adicionar 1-2 uL de uma regularidade de mini-preparação de plasmídeo pBR-flp 5 ao electrocompetentes V. células cholerae e transferir a mistura para uma cuvete de electroporação (0,2 largura da abertura cm). </li><li> Aplicar um pulso de 1,6 kV. </li><li> Adicionar 0,9 ml de meio SOC, gentilmente transferir células de um tubo de ml padrão 14. Incubar sem movimento durante 2,5 a 3 horas a 30 ° C. </li><li> Ul placa 100 e 300 ul em placas LB contendo 100 ug / ml de ampicilina e incubado a 30 ° C durante a noite. </li><li> Purificar único clone e armazenar como estoque de glicerol para experimentos TransFLP futuras. </li></ol> 3. Desenho oligonucleótido e Reacção em Cadeia da Polimerase <ol><li> Pelo menos seis oligonucleótidos são necessários para amplificar a região de ADN (s) de interesse, bem como o cassete de FRT-flanqueado antibiótico por PCR (Figura 3). Recomenda-se também incluir um par de oligonucleótidos escorva fora do fragmento de PCR inserido. Isto permite a verificação de integração e a excisão correcta da cassete de resistência FRT-flanqueado antibiótico (por exemplo, descrito como &quot;chk-up&quot; e primers &quot;chk-down&quot; nas Figuras 3 a 5). </li><li> Projetar os oligonucleótidos # 2, # 3, # 4 e # 5 com cuidado. Eles devem ser suficientemente capazes de hibridar (por exemplo, ~ 28 pb) com o DNA molde. O molde de ADN é ADN genómico (gDNA), para os iniciadores # 2 e # 5, e FRT-Kan-FRT contendo DNA de plasmídeo como pROD17 12 e pBR-FRT-KAN-FRT (neste estudo) para os iniciadores # 3 e # 4 ( Figura 3A). </li><li> Conceber os iniciadores # 2 a # 5 permitindo extensa de emparelhamento de bases na sua extremidade 5 &#39;entre # 2 / # 3 e # 4 / # 5, respectivamente (Figura 3A </strong&gt; inset). Duas rodadas de PCR seguir. </li><li> Realizar três reações independentes de PCR como uma rodada 1 º de PCR. A primeira vai amplificar a região a montante do gene / ADN da região-de-interesse com a ajuda de oligonucleótidos # 1 e # 2. A PCR deve resultar num fragmento de pelo menos 500 pb de comprimento que permitem a recombinação homóloga dentro das células. Fragmentos mais curtos (~ 250 pb) pode ser de 8 suficiente mas não são recomendados. </li><li> Em paralelo realizar a segunda PCR, que amplifica a cassete de FRT-flanqueado antibiótico. Para os exemplos fornecidos aqui nós usamos principalmente aph (kan R). Oligonucleotídeos uso # 3 e # 4 para essa reação (Figura 3A). </li><li> Concomitantemente, a amplificação da região de ADN a jusante através de PCR (amostra III). O fragmento de PCR também deve ser de pelo menos 500 pb de comprimento. Realizar a PCR com gDNA como molde e os oligonucleotídeos # 5 e # 6 (Figura 3A). </li><li> Após a purificação de todos os três PCRfragmentos, realizar a 2 ª rodada de PCR. Use uma mistura igual de todos os três fragmentos obtidos no primeiro turno como modelo. A amplificação é catalisada por oligonucleótidos # 1 e # 6. O fragmento de PCR resultante (Figura 3B), que serve como a transformação de ADN na experiência de transformação natural (Figura 1). </li></ol> 4. Quitina induzida Transformação Natural <ol><li> Crescer V. células cholerae aerobicamente em meio rico a 30 ° C até atingirem uma densidade óptica a 600 nm de aproximadamente 0,5. </li><li> Colheita das bactérias por centrifugação. Lavar pelota uma vez em meio artificial definido (DASW 6) antes de ressuspender as células em 2 volumes de DASW. Adicionar 1 ml da cultura para 50-80 mg de estéril quitina flocos (explicado no ponto 1). Se as bactérias porto plasmídeo pBR-flp (opcional ponto 2), adicionar ampicilina (50 ug / ml) à cultura. Incubar a 30 ° C durante 16-24 h sem movimento. </li><li> Adicionar &amp; ge; 200 ng do fragmento 2 ª rodada de PCR-derivada (explicado no ponto 3). Misturar cuidadosamente, sem extensivamente destacando as bactérias da superfície da quitina. Incubar a 30 ° C durante 24 horas sem movimento. </li><li> Vortex a cultura extensivamente para ≥ 30 seg. Espalhe 100-300 ul em placas LB seletivos médio (por exemplo, kanamicina ou gentamicina placas contendo LB para exemplos fornecidos aqui). Utilizar placas selectivas duas vezes se as bactérias abrigam o plasmídeo pBR-flp adicionando ampicilina concomitantemente com outros antibióticos. Incubar as placas a 30 ° C durante 16-24 horas ou até que as colónias são visíveis. </li><li> Isolar transformantes individuais de placas selectivas. Tais transformantes ter substituído o locus cromossómico original, pelo fragmento de PCR devido a um evento de crossover duplo. Estas estirpes podem ser directamente utilizados para experiências posteriores, no caso da remoção da cassete de antibiótico não é necessário. </li></ol> 5. Remoção de Cassete Seletivo (s) por FlpA recombinação <ol><li> Artificialmente transformar seus transformantes com plasmídeo pBR flp, conforme descrito no ponto 2 se não for feito antes do ensaio de transformação natural. </li><li> Crescer as bactérias em placas de agar LB contendo ampicilina a 37 ° C durante 16-24 hr. Opções: mudar a temperatura entre 2-3 horas para a 40 ° C, como expressão de flp de plasmídeo pBR-flp é de-reprimida em 5,10 altas temperaturas, bactérias transferência para placas frescas após aprox. 8 horas de incubação. </li><li> Ensaio de sensibilidade aos antibióticos (demonstrado aqui para canamicina; Figura 1) por restreaking os clones em paralelo em placas de agar contendo antibióticos e livre de antibióticos. Isolar o único colônias sensíveis. Congelar ampicilina resistente clone como estoque de glicerol se você pretende modificar ainda mais a tensão com a supressão outro / inserção usando TransFLP. </li></ol> 6. Cura plasmídeo <ol><li> Crescer a cultura durante a noite sob condições aeróbias ea 30 ° C. Use meio rico sem a adição de qualquer antibiótico. </li><li> Opcional: Crescer cultura fresco por 3-6 horas, diluindo a 1:100 cultura durante a noite em meio livre de antibióticos fresco. </li><li> Placa raia ou diluição (s) de cultura em placa de ágar LB simples (s) e incubar a 30 ° C durante 8-16 horas (até que as colónias visíveis). </li><li> Teste para ampicilina sensibilidade dos clones por restreaking os clones em paralelo em placas de ágar contendo antibiótico e antibiótico-livre (Figura 1). Congelar ampicilina cepa sensíveis como o estoque de glicerol. </li></ol>

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Não há conflitos de interesse declarados.

O método TransFLP descrito acima e noutros locais 5 tem sido extensivamente utilizada no nosso laboratório. O tipo de manipulações genéticas que são viáveis ​​incluem, entre outros: a eliminação de genes individuais e aglomerados de genes, a deleção de ilhas genómicas (por exemplo, VPI-1), a inserção de sequências a montante de um gene de interesse (por exemplo, sequências do promotor) e da inserção sequências na extremidade de um gene específico (por exemplo,</em...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Quitina flocos </td><td> Sigma </td><td> C9213 </td><td> Autoclavagem exigido </td></tr><tr><td> OPCIONAL: Micropulser (no caso cassete antibiótico deve ser extirpado por Flp recombinase codificado em pBR-flp) </td><td> Biorad </td><td> 165-2100 </td><td> Ou electroporators comparáveis </td></tr></tbody></table>

transflp &#x2014; a method to genetically modify vibrio cholerae based on natural transformation and flp-recombination

Vários métodos estão disponíveis para manipular cromossomas bacterianos 1-3. A maior parte destes protocolos contar com a inserção de plasmídeos replicativos condicionalmente (por exemplo, abrigando replicões pir-dependentes ou sensíveis à temperatura 1,2). Estes plasmídeos são integrados nos cromossomas bacterianos com base na homologia mediada por recombinação. Esses mutantes de inserção são muitas vezes usado diretamente em ambientes experimentais. Em alternativa, a selecção para a excisão do plasmídeo seguido por sua perda pode ser realizada, que para bactérias gram-negativas depende frequentemente da enzima sucrase contra-seleccionável de levana, codificada pelo gene sacB 4. A excisão pode restaurar o genótipo de pré-inserção, ou em resultado de uma troca entre o cromossoma e a cópia mediada por plasmídeos do gene modificado. Uma desvantagem desta técnica é que é demorado. O plasmídeo tem que ser clonado primeiro, que exige a transferência horizontal em V. cholerae (mais n<html> otably por acasalamento com um E. coli estirpe dadora) ou transformação artificial deste último, e da excisão do plasmídeo é aleatório e pode restaurar o genótipo inicial ou criar a modificação desejada se nenhuma selecção positiva é exercida. A seguir, apresentamos um processo para a manipulação rápida da V. cholerae cromossoma (s) 5 (Figura 1). Este método TransFLP baseia-se na descoberta recentemente indução mediada por quitina de competência natural nesse organismo representativo 6 e outros do género, tais como Vibrio V. fischeri 7. Competência natural permite a captação de ADN livre, incluindo fragmentos de PCR gerados pelo DNA. Uma vez absorvido, o ADN recombina com o cromossoma, dada a presença de um mínimo de 250-500 pb da região flanqueadora homólogo 8. Incluindo um marcador de selecção de entre estas regiões flanqueadoras permite a fácil detecção de transformantes que ocorrem com frequência. t &quot;&gt; Este método pode ser usado para diferentes manipulações genéticas de V. cholerae e potencialmente também outras bactérias naturalmente competentes. Fornecemos três exemplos novos do que pode ser conseguida por este método, além do nosso estudo publicado anteriormente em deleções de um único gene e do Além da afinidade tag-seqüências 5. passos de otimização Vários sobre o protocolo inicial de quitina induzida por transformação natural 6 são incorporados neste protocolo TransFLP. Estes incluem, entre outros, a substituição de fragmentos de conchas de caranguejos por disponível comercialmente quitina flocos 8, a doação de PCR locais derivado de ADN como a transformação de material 9, e a adição de sítios de recombinação FLP-alvo FRT () 5. DRF permitir a excisão sítio-dirigida do marcador de selecção mediada pela recombinase Flp 10.

Os resultados representativos das três exemplos são mostrados nas Figuras 4 a 6. A primeira abordagem, que visa suprimir os genes vizinhos ctxA e CtxB. Juntos, eles codificar o principal fator de virulência de V. cholerae, a toxina da cólera. Concebemos oligonucleótidos para o método TransFLP como descrito acima e de acordo com a Figura 3. A estirpe parental, a estirpe portadora do intermediário FRT-flanqueado cassete de resistência a antibióticos no ...

Watch this Scientific Journal Video about TransFLP â€” A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination at JoVE.com

TransFLP &amp;#x2014; A Method to Genetically Modify &lt;em&gt;Vibrio cholerae&lt;/em&gt; Based on Natural Transformation and FLP-recombination

Um método rápido para modificar o genoma de<em&gt; V. cholerae</em&gt; É descrito. Essas modificações incluem a eliminação de genes individuais, grupos de genes e ilhas genômicas, bem como a integração de seqüências curtas (por exemplo, elementos promotor ou afinidade tag-seqüências). O método baseia-se na transformação de recombinação natural e FLP.

TransFLP - um método para modificar geneticamente<em&gt; Vibrio cholerae</em&gt; Com base Transformação Natural e recombinação FLP-

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

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Immunology and Infection

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

14.5K visualizações.  Ecole Polytechnique Fédérale de Lausanne (EPFL). Um método rápido para modificar o genoma de V. cholerae É descrito. Essas modificações incluem a eliminação de genes individuais, grupos de genes e ilhas genômicas, bem como a integração de seqüências curtas (por exemplo, elementos promotor ou afinidade tag-seqüências). O método baseia-se na transformação de recombinação natural e FLP.

Vídeo: TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

Colonização de Euprymna scolopes Squid por Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Imunologia e Infecção

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Um parasita Ensaio de Resgate e Transformação de Triagem leishmanicida contra intracelular<em&gt; Leishmania donovani</em&gt; Amastigotas em THP1 linha celular humana aguda Monocytic Leucemia

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Aplicação Clínica de<em&gt; A Bela Adormecida</em&gt; Artificial e Antigen Presenting Cells para modificar geneticamente células T de Sangue do Cordão Umbilical e periférica

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

Super-Resolution Imaging da Célula Natural Killer imunológica Synapse em um vidro com suporte Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Implementação de um Sistema de Infecção baseado em Inserir Permeável Membrana para estudar os efeitos de secretada bacterianas toxinas em células hospedeiras de mamíferos

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Desenvolvimento de uma mais sensível e específica Cromogênico Agar para a detecção de<em&gt; Vibrio parahaemolyticus</em&gt; E outro<em&gt; Vibrio</em&gt; Espécies

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

A Plataforma de Ensaios Anti-biofilme adequado para a Exploração de bibliotecas de compostos naturais

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

-Fluorescência baseado Neuraminidase Ensaio de Inibição Para avaliar a susceptibilidade do vírus influenza A de neuraminidase Inibidor de Classe Antivirais

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

Técnicas de laboratório usadas para manter e diferenciar biótipos de<em&gt; Vibrio cholerae</em&gt; Isolados clínicos e ambientais

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Um novo sistema alimentador-livre para produção em massa de murino Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...