우리는 FCU1 유전자의 순서와 함께 우리를 제공하는 닥터 필립 업스 감사하고 싶습니다. 이 작품은 NIH 부여 AI 26649에 의해 지원되었다.

1. Entamoeba histolytica에 FCU 부정적 선택 시스템 <ol><li> 벡터 제어 라인 (HM1 / pKT3M) 및 실험 라인 (HM1/pKT3M-FCU1)은 이전에 설명한 기법 1,2에 의해 생성되었습니다. 종자는 T - 25 flasks에 재고 튜브에서 연구하에 라인 TYI - S - 33 3 중간 적절한 항생제 (12 μg / ML G418)을 추가하여 선택을 유지합니다. flasks, 37에서 성장 16~18시간 후 70-80% 합류해야 ° C. </li><li> 따뜻한 TYI - S - 33 중간에 5 Fluorocytosine (5 - FC, 시그마)의 새로운 50mM 주식 솔루션을 준비합니다. 와동이 엄격하게 몇 분 동안 완전히 5 FC를 해산합니다. 필터 - 필터 소독 0.22 μm의를 통해 전달하여 주식 솔루션을. </li><li> 3~5분 위해 얼음에 flasks를 삽입하고 실온에서 5 분 200xg에 centrifuging하여 세포를 수집하여 수확 trophozoites. 신선한 TYI 매체에서 펠렛을 Resuspend하고 세포를 계산합니다. </li><li> 전형적인 실험 설계 1 ML의 최종 볼륨에 48 잘 접시 잘 당 0.5 X 10 4 세포를 사용하며, 각 시험 조건에 대한 세중의 반복. 또한, 형광 측정을 촉진하기 위하여, 별도의 접시는 시점마다 각 변형에 사용됩니다. </li><li> 각 시험 5 - FC 농도, 세중의에 대해 잘 당 종자 0.5 X 10 4 세포. TYI 매체에서 항생제 선택 압력을 유지합니다. 지금 5 - FC 주식 솔루션과 각 잘하는 trophozoites의 필요한 금액을 추가합니다. </li><li> 37 무산소 가방에 접시를 품어 ° C. </li></ol> 2. 선택 효율성의 결정 <ol><li> 2 MG / DMSO에 셀 추적 녹색 CMFDA (Invitrogen)의 ML 주식 솔루션을 준비합니다. 작업 솔루션 주식​​ 혈청 무료 TYI 매체 1000 배 희석하기 위해. </li><li> trophozoite 문화 접시에서 완전히 매체를 제거하고 150 μL 혈청 무료 TYI 포함 CMFDA로 바꿉니다. 37 배양을 계속 ° C 1 시간을 위해. </li><li> 우물에서 염료 솔루션을 제거하고 Spectramax M2 microplate의 spectrofluorometer에서 접시를 참조하십시오. 여기 파장은 492 nm의에서 설정하고 형광 방출 517 NM에서 읽을 수 있습니다. </li><li> 테스트 transfectants 대 제어 세포에 대한 형광 값을 비교합니다. </li><li> 각 시간 간격에 접시를 읽고 계속합니다. 각 플레이트에 적절한 양의 (TYI 전용)과 부정 (25 μg / ML hygromycin) 제어 우물을 사용합니다. </li></ol> 3. 대표 결과 E. histolytica의 transfectants는 코돈 최적화 재조합 FCU1 (그림 1)의 강력한 표현을 보여주었다.이 프로토콜은 E.의 선택 제거에서 결과가 제대로 따라하면 0.5 MM 5 fluorocytosine의 존재에 FCU1을 표현 histolytica 세포. 제어 세포는 반면 5 - FC 5 MM의 농도에서 정상적으로 자라서 72 96 시간 사이의 합류에 도달 계속 (그림 2 - A). 치료 우물이 현미경으로 볼 때 48 시간에 FCU1지고 세포가 완전히 lysed 있습니다. 샘플 다수 (그림 2 - B)를 포함하는 양적 연구에서 사용되는 중요한 염료 CMFDA, 물들일 때 이들은 또한 형광 감소 보여줍니다. FCU1/5-FC 시스템 E.위한 효과적이고 강력한 부정적인 선택 도구 histolytica은 trophozoites. <img src="/files/ftp_upload/2410/2410fig1.jpg" alt="그림 1" /> 그림 1. E. 생성 부정적인 선택 마커를 표현 histolytica HM1 transfectants (A) 재조합 단백질 FCU1는 안티 - C Myc 항체 (상단 패널)을 사용하여 서양 얼룩을 발견했다. 예상했던대로, 42kDa 밴드는 아니지만 혼자 벡터 제어 transfectants의 FCU의 transfectants에서 총 lysate에서 (화살표로 표시) 감지 수 있습니다. 하단 패널은 같은 얼룩이 로딩 제어로 안티 굴지 항체와 탐지 보여줍니다. (B) 정량 실시간 PCR의 결과 lgl4 제어하는 표준 FCU1 특정 mRNA의 표현을 보여주는. <img src="/files/ftp_upload/2410/2410fig2.jpg" alt="그림 2" /> E. 그림 2.에서는 체외 마약 감도 부정적인 선택 마커를 표현 histolytica의 transfectants (A) 제어 transfectants 및 (b) FCU1 표현 transfectants 48 - 잘 판에 교양의 생존 곡선. 세포의 증가 농도의 존재에 재배되었던 prodrug - 5 - fluorocytosine, X - 축에 표시된로 세포 생존을 각 시간 간격으로 CMFDA의 얼룩에 의해 계량했다. Y - 축 단위 형광을 나타내고 살아있는 세포 인구의 직접적인 반영이다. FCU1 표현 transfectants가 컨트롤에 비해 0.5mM prodrug 농도에 큰 감도를 보여주는 점에 유의하십시오. 대한 세포는 COM 이러한 우물에 120h 시점에서 관찰되지 않았습니다현미경 해당 컨트롤 우물 (데이터가 표시되지 않음)에서 본 합류 성장 앞에 서. Hygro는 대조군으로 사용 25μg / ML hygromycin를 나타냅니다.

<ol>
	<li>Olvera, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transfection+of+Entamoeba+histolytica+trophozoites+by+lipofection.">Stable transfection of Entamoeba histolytica trophozoites by lipofection.</a> Arch. Med. Res. , 49-51 (1997).</li><li>Saito-Nakano, Y., Yasuda, T., Nakada-Tsukui, K., Leippe, M., Nozaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab5-associated+vacuoles+play+a+unique+role+in+phagocytosis+of+the+enteric+protozoan+parasite+Entamoeba+histolytica.">Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica.</a> J. Biol. Chem. 279, 49497-49507 (2004).</li><li>Diamond, L. S., Harlow, D. R., Cunnick, C. 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이 비디오의 제작이 문서에서 사용되는 시약의 일부를 생산 시그마 알드리치, Inc의 후원했다.

Entamoeba 분자 생물 학적 도구에서 상당한 진보가 있었다고했지만 유전자 4 삭제하거나 혼란 사용할 수있는 현재의 방법은 없습니다. 유전자 특정 표현의 최저는 RNA 6 방해 작은 헤어핀 RNAS 5의 사용에 의해 달성하고 bacterially dsRNA 7 표현되었습니다. 각각의 대상 유전자에 대한 효과적인 동시에 그러나 이러한 방법은 비용이 많이 드는 화학 물질, 항생제에 대한 지속적인 선택과 시간이 지남에 따라 발생 복귀의 높은 발병률 (알리샤 린포드, 개인 통신) 8으로 인해 지속적인 검증에 대한 필요성의 사용 등 여러 가지 제한이 있습니다. DNA 복제 9 기원에 대한 엄격한 순서 요구 사항이 분명이 아니므로 모든 플라스미드는 Entamoeba에 복제할 수 있습니다, 그래서 한번 transfected, 그것은 보통 플라스미드 치료하기가 어렵습니다. 이것은 재조합과 유전자 녹아웃 실험에 그대로 plasmids의 가능한 사용을 제한합니다. 부정적인 선택 마커들은 재조합 subpopulations을 제거하는 데 사용할 수있는 방법을 대상으로 유전자의 핵심 구성 요소입니다. 우리는 성공적으로 코돈을 표명한 것은 Entamoeba histolytica에 FCU1 유전자를 최적화하고 부정적인 선택 마커로서의 가능성을 테스트했습니다. 이 유전자 융합은 인간의 종양 세포의 자살 유전자 치료에 사용되며 RNA 및 DNA 합성 억제에 10 결과 독성 하류 제품에 prodrug 5 - FC를 대사되었습니다. FCU1 최근 Plasmodium 다른 protozoan 기생충에 부정적인 선택 마커로 사용되고 있습니다. FCU1을 표현 Plasmodium berghei 세포는 5 - FC와 함께 체외 및 생체내 치료의 prodrug으로 거의 1000 배 더 민감한 것으로 발견된의 99.9 % 이상 죽였다 말라리아 11 erythrocytic 단계 마우스 모델에서 재조합 기생충 감염. E.가 FCU1을 표현 histolytica 전지는 P.에서 관찰 감도 달리 prodrug 5 FC에만 30 배 증가 감도를 보여주었다 berghei. 이런 경우에 대한 가능성이있는 이유는 독성 metabolites와 경쟁의 성장 매체에서 사용 가능한 세포핵의 대형 수영장 수 있습니다. P. 모두 berghei와 P. falciparum FCU1 마커는 타겟 유전자 장애에 사용되었습니다. 연구 11,12. 우리는 상동 재조합을 사용하여 Entamoeba의 게놈을 조작하는 것을 목표로하고 있습니다. FCU1/5-FC 부정적인 선택 시스템의 검증은 이러한 목표를 향한 중요한 단계입니다.

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comments</td>
</tr>
<tr>
<td>TYI-S-33 medium</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>&nbsp;</td>
<td>Reference included in the text</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F7129|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC">5-Fluorocytosine</a></td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F7129</td>
<td>&nbsp;</td>
<tr>
<td>Cell tracker green CMFDA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C2925</td>
<td>&nbsp;</td>
<tr>
<td>Spectra Max M2 plate-reader</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		
</table>

development of a negative selectable marker for entamoeba histolytica

Entamoeba histolytica은 amebiasis의 원인이되는 에이전트이며, 세계 인구의 10 %까지 감염. 유전자 표현의 위 - 아래로 규제를 활성화 분자 기술은 안정적으로 유지 plasmids의 transfection에 의존하고 있습니다. 이들은 Entamoeba의 독성 요인 우리의 이해를 증가하지만, 용량은 역방향 유전학 실험,이 기생충의 연구에 큰 이점 될 수있는 것이다 게놈로 외인성 DNA를 통합할 수 있습니다. 이 유기체에 의해 제공 도전 상동 재조합 이벤트와 transfected trophozoites에서 플라스미드 DNA episomal 치료 난이도에 대해 선택하는 무능력을 포함합니다. 외인성 DNA, 타고난 게놈 통합 이벤트가 발생하는 trophozoites의 식별의 주요 문제에 대한 높은 배경으로 나중에 발생합니다. 우리는 효모 사이 토신의 deaminase 및 유라실 prodrug 5 ​​- fluorocytosine (5 - FC)과 phosphoribosyl transferase 키메라 (FCU1)와 선택의 형질 표현에 따라 부정적인 선택 시스템의 개발을보고합니다. FCU1 효소는 독성 5 - fluorouracil, 5 - fluorouridine - 5&#39; - monophosphate. E.으로 무독성 5 FC로 변환 FCU1을 표현 histolytica 라인 제어 변형에 비해 prodrug에 더 민감한 30 배 이상으로 발견되었다.

Watch this Scientific Journal Video about Development of a Negative Selectable Marker for Entamoeba histolytica at JoVE.com

Development of a Negative Selectable Marker for Entamoeba histolytica

우리의 부정적인 선택 시스템의 개발을보고<em&gt; E. histolytica</em&gt; 형질 키메라 단백질의 표현 (FCU1)과 prodrug 5 ​​- fluorocytosine와 선택에 따라. FCU1 단백질은 효모 사이 토신의 deaminase와 phosphoribosyltransferase 유라실의 융합이다. FCU1 표현 증가의 결과<em&gt; E. histolytica</em5 fluorocytosine 향해&gt; 감성.

에 대한 부정적인 선택 마커의 개발 Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

development-of-a-negative-selectable-marker-for-entamoeba-histolytica

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Immunology and Infection

Development of a Negative Selectable Marker for Entamoeba histolytica

12.4K 조회수.  University of Virginia Health System. 우리의 부정적인 선택 시스템의 개발을보고 E. histolytica 형질 키메라 단백질의 표현 (FCU1)과 prodrug 5 ​​- fluorocytosine와 선택에 따라. FCU1 단백질은 효모 사이 토신의 deaminase와 phosphoribosyltransferase 유라실의 융합이다. FCU1 표현 증가의 결과 E. histolytica 감성.

비디오: Development of a Negative Selectable Marker for Entamoeba histolytica

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

Entamoeba histolytica DNA와 tRNA Methyltransferase Dnmt2 (Ehmeth) 효소를 체외 tRNA의 Methylation 분석에서

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

연구

면역학 및 감염병학

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

유동 세포 계측법에 의한 Thymic 긍정적이고 부정적인 선택의 시험

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

호스트 세포 RN​​A의 합성에 바이러스 감염의 효과를 측정하는 클릭 화학을 사용하여

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF 질량 분석기를 사용하여 혈액 배양액에서 그람 음성 박테리아의 빠른 확인

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

에 의해 제작 상피 손상의 분석<em&gt; Entamoeba 아메바</em&gt; 감염

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

라벨이없는 바이오 센서 기술을 활용 유방암 인간 백혈구 항원 바이오 마커의 검출

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

챔버의 coverglass 모델을 사용하여 바이오 필름 개발에 객담의 효과를 시각화

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

B 형 간염 바이러스 리포터 시스템 개발은 복제주기의 초기 단계를 모니터링하기

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Biocontainment 내 바이러스 샘플의 음성 염색을위한 캡슐의 활용

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

DFS70 autoantibodies mimic common disease-associated antinuclear antibody patterns making accurate interpretation challenging when using conventional HEp-2 substrates. The protocol describes advantages of novel engineered HEp-2 substrate over conventional HEp-2 in ANA screening and distinguishing DFS70 patterns with high confidence in both monospecific and mixed ANA positive cases.

Monospecific 및 소설 형 간염-2 엘리트/DFS70 녹아웃 기판으로 아나 심사 중 혼합된 DFS70 패턴의 동시 구별

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies ...