These experiments have been supported by research grants from Merck and Co. and Summit, PLC.

1. 다른 환경 적 또는 임상 적 출처로부터 C. difficile의 분리 <ol><li> 환경 분리 물 : 사전 멸균 된 면화 게이지 (0.85 % NaCl로 가볍게 적신)를 사용하여 관심 영역 (바닥, 문, 손잡이, 선반 등 )의 표면을 닦습니다. 8 멸균 된 장갑을 착용하고 완료된 후 멸균 된 튜브에 면봉을 놓습니다. </li><li> 임상 분리 물 (대변) : 접종 루프를 사용하여 cefoxitin-cycloserine-fructose 한천 (CCFA)에 임상 대변 샘플 10 ~ 100 mg을 넣고 48 ~ 72 시간 동안 엄격한 혐기 조건 하에서 배양합니다. 추가 분석을 위해 -80 ° C에서 cryovials에 C. difficile 스톡의 격리 된 식민지를 저장하십시오. 7 , 9 , 10 </li><li> 0.05 % sodium taurocholate가 포함 된 BHI (brain heart infusion) 배지에서 환경 면봉 샘플을 풍부하게 만들고 37 ° C의 혐기성 챔버에 두십시오5 일 동안. 10,000 XG에서 문화 1 ML 원심 분리기와 에탄올 100 μL에 펠렛을 resuspend. </li><li> cycloserinecefoxitin fructose 한천 (CCFA) 플레이트 위에 resuspended 세포 (50 μL)를 플레이트하고 40에서 48 시간 37 ° C에서 혐기성 챔버에서 알을 품다. 추가 분석을 위해 -80 ° C에서 cryovials에 C. difficile 스톡의 격리 된 식민지를 저장하십시오. </li><li> 라텍스 응집제 또는 PCR을 사용하여 의심되는 C. difficile 콜로니를 시험합니다. </li></ol> 2. C. difficile의 배양 과 Kinetic Procedures의 사멸 <ol><li> 37 ℃에서 48 시간 동안 혐기성 챔버에서 혈액 한천 평판에서 정제 된 환경 또는 임상 C. difficile 균주를 재배한다. </li><li> 접종 루프로 격리 식민지 하나를 취해 15 mL 튜브에 5 mL BHI 배지로 옮기고 37 ℃에서 혐기성 챔버에서 24 시간 동안 성장시킵니다. </li><li> 전 배양 물 1 : 100을 약 10 6 콜로니 형성 단위0.1 % 나트륨 타우로 콜레이트 및 항생제 (T0)의 적절한 농도로 보충 된 신선한 미리 감소 된 BHIS (BHI 플러스 5g / L 효모 추출물 및 1 % L- 시스테인)에서의 s (CFU) / mL. </li><li> 각 시점 (T0, T6, T24, T48)에서 피펫으로 1 ML 샘플을 수집하고 접시 / 혈액 한천 접시에 작은 나누어 (100 μL 시리얼 희석으로) 확산. 37 ° C에서 혐기성 챔버에서 48 시간 동안 혈액 한천 플레이트에서 세포가 자라도록하고 결과 CFU 수를 계산합니다. </li></ol> 3. 전자 현미경 검사 용 샘플 준비 <ol><li> microcentrifuge 튜브와 10 분 10,000 XG에서 원심 분리기의 각 시점에서 세포 1 ML을 수집합니다. 상등액을 버리고 PBS로 세포를 씻는다. </li><li> 다시 10 분 동안 10,000 xg에서 샘플을 원심 분리하고 상등액을 버린다. 4 % paraformaldehyde 1 ML에 세포를 다시 희석하고 실온에서 1 시간 품어. </li><li> 1시 10 분 동안 다시 원심 시료0,000 xg로 상등액을 버린다. 증류수로 세포를 두 번 씻고 증류수 100 μL로 다시 희석합니다. 솔루션의 탁도에 따라 볼륨을 조정합니다. 참고 : 여러 번 연속 희석을하는 것이 좋습니다. </li><li> coverslips 레이블을 추가하고 그것에 샘플의 40 μL을 추가합니다. 흐름 후드에서 15 분 동안 품어 액체를 증발시키고 세포가 coverslip에 고착하도록합니다. 액체가 여전히 남아 있으면 송풍기를 사용하여 액체를 제거하십시오. </li><li> 탁상용 스퍼터링 기계 및 테이프에 셀을 넣은 커버 슬립을 배치하십시오. 스퍼터링 기계에 순금을 확보하십시오. 기계를 켜고 저압 (50 mTorr)에서 스퍼터링을 시작하십시오. 80 nm에서 30 초 동안 코팅 세포를 코팅하면 20 nm의 금 코팅이됩니다. </li><li> 주사 전자 현미경으로 코팅 된 세포를 옮깁니다. </li></ol> 4. 주사 전자 현미경에서 영상 C. difficile 세포 <ol><li> 주사 전자 현미경 (주사 전자 현미경)을 적절히 환기 시키십시오.컴퓨터 소프트웨어의 배출 버튼을 누르십시오. </li><li> 카본 테이프를 사용하여 코팅 된 커버 슬립을 금속 스테이지에 고정시킵니다. SEM이 배출되면 문을 쉽게 열어야합니다. 금속 스테이지를 나사로 죄어 SEM 챔버에 잠급니다. </li><li> 컴퓨터 소프트웨어에서 PUMP 버튼을 클릭하십시오. 시스템이 &quot;Vac OK&quot;라고 표시되면 SEM을 사용할 수 있습니다. </li><li> Detector 탭에서 SE 탐지기를 클릭하십시오. 전압을 표시하는 버튼을 클릭하여 보를 켭니다. 전압을 높이기 전에 낮은 전압 (5 kV)에서 이미징을 시작하십시오 (최대 15 kV). 빔이 켜지면 이미지가 나타납니다. </li><li> 추적 기능을 사용하여 코팅 된 커버 슬립에서 이미지를 볼 수있는 영역을 찾습니다. 지역을 확대하고 막대 모양의 구조를 찾습니다. 이들은 클로스 트리 디움 디피 실 입니다. </li><li> 이미지를 확대하고 초점을 맞춰 시스템을 보정하십시오. 이 작업 거리는 15, 9 및 5 mm의 여러 작업 거리에서 수행해야합니다. 5mm의 작동 거리에서 이미지. </li><li> 너를 켜라.ltra 고해상도 이미징 모드로 초점을 맞추고 난시를 최적화합니다. </li><li> 높은 배율에서 초점을 맞추기 시작하십시오. 이를 위해 거친 초점과 미세한 초점을 사용하십시오. 더 명확한 이미지를 얻으려면 난시도 조정하십시오. <ol><li> 높은 배율로 난시를 조정하십시오. 이렇게하려면 비점 수식 토글 (미세한 / 거친 포커스 토글과 유사하게 보임)을 조정하고 컴퓨터 소프트웨어를 디지털 확대하여 명확하게 이미지를 확인하십시오. </li></ol></li><li> 고화질 이미지를 수집하는 느린 스캔 기능. 수집 된 이미지를 .TIFF 파일로 저장하면 분석에 사용됩니다. 분석 중에 측정이 이루어지면 데이터 막대가 선택되었는지 확인하십시오. </li><li> SEM에서 직접 각도를 수동으로 돌리면 서로 다른 각도 (최대 52 °)에서 이미지를 수집 할 수 있습니다. 각 이미지는 깊이 정보를 더 많이 나타내는 경향이 있습니다. </li><li> 이미징되는 셀에 따라 빔 전압을 변경하십시오. 이것을 모든 실험에서 5 - 15 kV로 유지하십시오. </li&gt; <li> 이미징이 완료되면 빔을 끄고 작동 거리를 20 mm로 올립니다. 그런 다음 챔버를 배출하고 스테이지를 제거 할 수 있습니다. 추가 분석을 위해 모든 이미지를 드라이브에 복사하십시오. </li></ol> 5. 이미지 처리 및 분석 <ol><li> 자유롭게 사용할 수있는 소프트웨어 인 FIJI (http://fiji.sc)를 다운로드하여 컴퓨터에 설치하십시오. </li><li> FIJI에서 이미지 파일을 엽니 다. </li><li> 선 기능을 사용하여 스케일 바를 정확하게 추적하십시오. </li><li> FIJI 프로그램에서 Analyze 탭을 클릭하고 Select Scale 기능을 선택하십시오. 눈금 막대를 기준으로 알려진 거리를 설정해야하는 창이 나타납니다. 길이 단위도 변경하고 확인을 클릭하십시오. </li><li> 이제 프로그램이 거리에 대해 보정되었으므로 라인 함수를 사용하여 셀 길이를 측정합니다. 길이를 얻으려면 line 함수를 사용하여 셀 전체를 추적하십시오. 분석 탭을 다시 선택한 다음 측정을 클릭하십시오. 길이는 app이어야합니다.귀는 이전에 표시된 단위로. </li></ol>

<ol>
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KWG has received past and current research support from Merck &amp; Co. and Summit, PLC.

현재 연구의 목표는 C. difficile을 분리 하고 항생제의 약리 작용을보다 철저히 특성화하기위한 수단으로 주사 전자 현미경 (SEM)을 사용하여 항생제 감수성을 검사하는 높은 처리량 방법을 만드는 것이 었습니다. 여기에 설명 된 프로토콜을 사용하여 우리는 항생제 치료에 대한 세포의 표현형 반응을 이미지화하면 약물의 약리 작용에 대한 통찰력을 확인할 수 있음을 입증했습니다. 전체적?...

Clostridium difficile 은 그람 양성, 포자 형성 세균으로 매년 미국에서 약 50 만 건의 감염을 일으키며 질병 위험 예방 센터 (CDC)에서 위협 수준의 긴급 병원체로 간주됩니다. 1 지난 10 년 동안 C. difficile 에 대한 항균제 개발에 상당한 약물 개발이 있었습니다 . 2 , 3 시험 관내 연구는 약물 개발 과정의 필수 구성 요소입니다. 전통적으로 in vitro 감수성 및 시간 사멸 연구는 미래의 동물 및 기타 생체 내 연구를 검증하는 데 사용됩니다. 이 방법은 살상 행동을 평가하는 데 중요한 역할을하지만, 약리학 적 치료에 대한 세포의 표현형 반응을 포착하지는 않습니다. 표준 전자 현미경 (SEM)을 통합하여동역학 연구를 중단하면 항생제 직접 효과에 대한보다 철저한 특성 분석이 가능하다. 5 , 6 , 7 여기서 우리는 SEM을 항생제 치료의 효능을 설명하는 수단으로 사용하는 방법을 제시한다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>cotton gauze&#160;</td>
			<td>Caring</td>
			<td>PRM21408C</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Macron</td>
			<td>7532</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion (BHI)&#160;</td>
			<td>Criterion</td>
			<td>C5141</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Alfa Aesar</td>
			<td>A10389</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>Criterion</td>
			<td>C741</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium taurocholate</td>
			<td>Alfa Aesar</td>
			<td>A18346</td>
			<td></td>
		</tr>
		<tr>
			<td>anaerobic chamber</td>
			<td>Coy</td>
			<td></td>
			<td>vinyl anaerobic chamber</td>
		</tr>
		<tr>
			<td>cycloserinecefoxitin fructose agar (CCFA) plates</td>
			<td>Anaerobe systems</td>
			<td>AS-213</td>
			<td></td>
		</tr>
		<tr>
			<td>blood agar plates</td>
			<td>Hardy diagnostics</td>
			<td>A-10</td>
			<td></td>
		</tr>
		<tr>
			<td>latex agglutination reagent</td>
			<td>Oxoid</td>
			<td>DR1107A</td>
			<td>C. diff test kit</td>
		</tr>
		<tr>
			<td>microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>222363204</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>50-259-98</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>J. Melvin freed brand</td>
			<td>7525M</td>
			<td>75x25mm</td>
		</tr>
		<tr>
			<td>flow hood</td>
			<td>Labconco</td>
			<td>Class II type A2&#160;</td>
			<td>biosafety cabinet</td>
		</tr>
		<tr>
			<td>desk sputtering machine</td>
			<td>Denton Vacuum</td>
			<td>Desk II</td>
			<td></td>
		</tr>
		<tr>
			<td>tape</td>
			<td>Plastic Core</td>
			<td>05072-AB</td>
			<td>SPI Double Sided Adhesive Carbon Tape</td>
		</tr>
		<tr>
			<td>gold</td>
			<td>Denton Vacuum</td>
			<td>TAR001-0158</td>
			<td>2.375&#8221; Diameter x .002&#8221; Thick Gold foil</td>
		</tr>
		<tr>
			<td>scanning electron microscope</td>
			<td>FEI</td>
			<td>XL-30</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol to characterize the morphological changes of clostridium difficile in response to antibiotic treatment

혐기성 박테리아에 대한 신약 개발로 항생제 작용을 평가하는 것은 어렵고 기술적으로 까다 롭습니다. 가능한 MOA에 대한 통찰력을 얻으려면 항생제 노출과 관련된 형태 학적 변화를 주사 전자 현미경 (SEM)을 사용하여 시각화 할 수 있습니다. 전통적인 살인 곡선과 SEM 이미징을 통합하면 약물 작용에 대한 우리의 통찰력을 향상시키고 약물 개발 프로세스를 향상시킬 수 있습니다. 이 전제를 테스트하기 위해 알려진 MOA (vancomycin 및 metronidazole)를 가진 약물을 사용하여 곡선을 죽이고 SEM 연구를 수행했습니다. C. difficile 세포 (R20291)는 항생제의 존재 또는 부재하에 48 시간까지 성장시켰다. 48 시간 간격으로 항생제 유효성을 결정하고 SEM에서 영상을 얻기 위해 여러 시점에서 세포를 수집했습니다. 이전 보고서와 일치하여, vancomycin과 metronidazole은 식민지 형성 단위 (colony-forming unit, CFU)에서 측정 한 24 시간 처리 후에도 상당한 살균 활성을 보였다.팅. SEM 이미징을 사용하여 metronidazole은 대조군과 vancomycin에 비해 세포 길이에 유의 한 영향을 주었다 (각 항생제의 세포 길이가 50 % 이상 감소, P &lt;0.05). 약물 치료에 대한 표현형 반응은 이전에 이러한 방식으로 문서화되지 않았지만, 약물의 MOA와 일관성이 있으며, 이미징 및 측정의 다양성과 신뢰성 및 다른 실험 화합물에 대한이 기술의 적용을 입증합니다.

Clostridium difficile 은 포자 형성 세균이며 기능 분석 전에 식물과 포자 세포 사이의 형태 학적 차이를 결정하는 것이 필수적입니다. 그림 1 은 성장 곡선과 포자 세포의 지수 단계에서 캡처 한 식물 세포의 대표 이미지를 보여줍니다. 묘사 된 바와 같이 식물 세포는 길고 매끄 럽고 막대 모양의 구조이며 포자는 거칠고 외형이 작은 타원형 구조입니다. 기?...

Watch this Scientific Journal Video about A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment at JoVE.com

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

형태 변화를 규명하기위한 프로토콜<em&gt; 클로스 트리 디움 디피 실</em&gt; 항생제 치료에 대한 반응

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

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Research

JoVE Journal

Immunology and Infection

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

8.9K 조회수.  University of Houston College of Pharmacy. Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

비디오: A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

위반 항생제 효능을 테스트하는 인공 가래 매체의 사용<em&gt; 녹농균</em&gt; 낭성 섬유증의 폐 관련성이 높은 조건에서

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

연구

면역학 및 감염병학

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

호스트 병원체 단백질 - 단백질 상호 작용의 풍경의 특성을 비교 접근

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

배양 및 유지<em&gt; 클로 스트 리듐 디피</em&gt; 혐기성 환경에서

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

웨스트 나일 바이러스 돌연변이 균주의 감염에에서 MyD88 매개 세포 면역 반응의 생체 외 분석

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

파지 Phenomics : 생리 학적 접근 방법은 소설 바이러스 성 단백질의 특성을

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

박테리아 - 호스트 상호 작용의 특성을 생체 모방 재료

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

의 Cefoperazone 처리 된 마우스 모델 임상 관련<em&gt; 클로스 트리 디움 디피</em&gt; 스트레인 R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

호스트 표현형의 시험에<em&gt; Gambusia의 affinis입니다</em&gt; 다음은 항생제 치료

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

심사 분석 실험 소설 내 피 레 귤 레이 터 염증 반응에 관여 하

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

인간의 과발현과 정제<em&gt; 시스</em&gt; - 프레 닐 트랜스퍼 라제<em&gt; 대장균</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

형태 변화를 규명하기위한 프로토콜

요약

더 많은 비디오 탐색

이 비디오의 챕터

위반 항생제 효능을 테스트하는 인공 가래 매체의 사용

호스트 병원체 단백질 - 단백질 상호 작용의 풍경의 특성을 비교 접근

배양 및 유지

웨스트 나일 바이러스 돌연변이 균주의 감염에에서 MyD88 매개 세포 면역 반응의 생체 외 분석

파지 Phenomics : 생리 학적 접근 방법은 소설 바이러스 성 단백질의 특성을

박테리아 - 호스트 상호 작용의 특성을 생체 모방 재료

의 Cefoperazone 처리 된 마우스 모델 임상 관련

호스트 표현형의 시험에

심사 분석 실험 소설 내 피 레 귤 레이 터 염증 반응에 관여 하

인간의 과발현과 정제