설명의 예제로 사용된 모든 NMR 스펙트럼은 재정적으로 비벼 대다가 지원되었습니다 이전에 출판 연구 7에서 파생됩니다.

1. 세균없는 동물과 샘플 컬렉션의 식민지 <ol><li> 컨트롤 (그림 1)로 될 것입니다 종래의 동물 앞에 필터를 갖춘 연습장에서 종래의 축산 룸에 isolators하고 집에서 그들로부터 세균없는 동물을 제거합니다. </li><li> 쓰레기의 혼합 절반 (옛 3 일) 세균없는 동물의 쓰레기와 컨트롤 기존의 케이지에서 찍은. 항상 더러운 일반 쓰레기의 1 / 3이 박테리아의 수준 (적어도 3 일 동안 그것을 보관) 유지하기 위해서는 그것을 갱신하는 데 필요한 때마다 유지. </li><li> 튜브 위에 마우스를 처리하여 1.5 ML의 microtube에 소변을 수집하고 부드럽게 마사지하여 대장 방뇨를 도와줍니다. 스냅인 동결 액화 질소에 즉시. NMR 분석까지 적어도 -40 ° C에서 보관하십시오. 20 μL의 최소 볼륨 5mm NMR 프로브와 함께 취득 필요하지만, 그것은 신진 대사 프로파일 링의 품질을 향상시키기 위해 30 μL를 사용하는 것이 좋습니다. </li><li> 동물 B해야E는 마취 화합물의 간장 대사 (예를 들어, 과다에 의해 죽음의 확인 다음 경추 탈구를 사용)로 인해 혼란함을 주죠 NMR의 resonances를 피하기 위해 어떤 마취를 사용하지 않고 euthanized </li></ol> 2. 간 생검의 수집에 대한 추천 <ol><li> 오염을 피하기 위해 알코올을 포함하는 모든 제품을 사용하지 마십시오. 물 전용 또는 생리 식염수를 사용하여 도구를 씻으십시오. </li><li> 담즙 방광 구멍하지 마십시오. 담즙 누출의 경우 물 또는 생리 식염수 즉시 조직을 씻는다. </li><li> 그림 2에 표시된 왼쪽 엽에서 간 biopsies을 (15-50 대해 MG)를 수집합니다. 재현성 biopsies 들어, 조직이 얇은있는 주변 지역을 피하는 왼쪽 엽의 중앙에 지속적으로 수집합니다. </li><li> 즉시 액화 질소에 냉동 biopsies를 스냅과 -80에 그들을 저장 ° C를 NMR 분석까지. </li></ol> 소변 microvolume 3 1. H NMR 획득 </P&gt; <ol><li> D2O (99.8 %), 1 ㎜ 3가 들어있는 산도 7.4의 0.2M 인산 나트륨 버퍼 솔루션을 준비 - (trimethylsilyl) 프로피 온산 - D 4 (TSP). </li><li> 인산 나트륨 완충액 30 μL로 소변을 30 μL를 섞습니다. </li><li> 금속 바늘 (OD 0.5 ㎜)을 갖춘 50 μL 유리 주사기를 사용하여 1.7 mm NMR 모세관 (그림 3 (2))에 혼합 용액 50 μL를 전송합니다. 거품을 피하기 위해주의하십시오. </li><li> 모세관 포함하는 소변 샘플의 상단에있는 모세관 어댑터 (그림 3 (3))에 맞게하고 5mm NMR 프로브 (그림 3 (1))에 2.5 mm의 NMR의 microtube에 넣습니다. 분광 취득 정규 5mm의 NMR 튜브로 튜브의 조합을 사용합니다. </li><li> 그것을 꺼내 부드럽게 모세관 어댑터를 속이고하여 2.5 mm의 NMR 튜브에서 모세를 제거하기 위해 추출로드 (그림 3 (4))를 사용합니다. </li></ol> 4. 간 조직의 1 H 인사 매스 NMR은 생검 : 샘플 준비 <ol><li> 로터 구성 요소와 도구가 MAS그림 4에 설명되어. </li><li> 삽입 지르코늄 로터에 생검 (15-50 대한 MG) (그림 4 (1)) 및 NMR 잠금에 대한 순수 D 2 O로 볼륨의 나머지 부분을 입력합니다. 이 데이터 수집의 후속 shimming 및 품질의 품질을 변경할 수 있기 때문에 모든 거품을하지 않도록주의한다. </li><li> 원통형 나사 (그림 4 (5))을 사용하여 50 μL 테플론 스페이서 (그림 4 (2))를 넣습니다. 나사가 빠지다 그것을하고 (그림 4 (8)) 짧은 측면에 깊이 게이지를 사용하여 보정합니다. 이 단계에서, 그것의 부분은 스페이서 구멍을 통해 누설 수 있기 때문에 샘플에 대한 구체적인 관심을 지불하는 것이 중요합니다. 이 경우 다음 생검의 일부가 파괴되고 샘플 무게는 더 이상 신뢰할 수 없습니다. 그것은 따라서 처음 샘플의 준비부터 다시 시작하는 것이 필요합니다. </li><li> thead 핀 (그림 4 (3)) 장소 및 드라이버 (그림 4 (6))로 부드럽게 그것을 나사. 조직의 조각으로 잔여 물을 건조. </li><li> 에 캡 (그림 4 (4)) 장소회전자의 상단과 회전자 포장 (그림 4 (6))에 넣습니다. 뚜껑이 곳에 때까지 단단히 누르십시오. 회전자와 뚜껑 사이에 남아있는 공간이 없습니다. </li><li> 광학 스핀 속도 감지를 허용하도록 검은 마커 펜을 사용하여 회전자의 바닥의 절반을 표시합니다. </li><li> NMR 분광계 내부의 회전자를 놓고 5 kHz에서에서 회전 시작합니다. 제조 업체의 지침에 따라 CPMG 펄스 순서 13를 사용하여 1 H NMR 스펙트럼을 취득. </li><li> NMR 스펙트럼을 교정하기 위해 5.22 PPM (이중어)에서 α anomeric 포도당 공진을 사용합니다. </li><li> 날개를 풀고하려면 캡 제거합니다 (그림 4 (9))를 사용하여 뚜껑을 제거하여 진행합니다. 나사가 빠지다 thead 핀과 원통형 나사를 사용하여 제거 테플론 스페이서. 철저하게 물과 세제를 사용하여 씻으십시오. </li></ol> 5. 대표 결과 내장 미생물 활동이 요도 신진 대사 프로파일을 사용하여 모니터링할 수 있습니다. 요도 미생물의 대량1 H NMR에 의해 식별 공동 metabolites은 문학 7,14-17에 설명되어있다. 이러한 미생물 공동 metabolites들은 새로 설립된 생태계가 안정되었을 때 추정하기 위해 신속하고 비침 투 방법을 제공으로 식민지 과정을 모니터에 특히 유용합니다. 그림 5A 명확하게 식민지 과정을 통해 내장 미생물의 공동 metabolites의 모양을 보여줍니다. 이 그림은 동물에 대한 2 단계에서 설명한 절차를 수행하여 얻은 요도 신진 대사 프로필 1 단계에서 설명한 절차를 사용 이십일 식민지 보여줍니다. 이 동물은 indoxyl 황산 나물을하고 phenylacetylglycine (PAG)과 세균이없는 상태에서 P - 크레졸 황산 (매일 0 - 파란색)의 아주 작은 양의하지 않았다. 식민이 진행되는 창자의 microbiota하여 단백질 대사의 이러한 세 마커는 하루에 20 (적색)에서 평형에 도달하는 상당히 향상시킬 수 있습니다. 이것은 PAG를 사용하여 그림 5B에서 그림과 같이 동물의 그룹에 대한 모니터 특히 쉽습니다공명. 이 그림 7 동물의 그룹의 PAG의 특정 공명 (삼인승)에 해당하는 그림 5A에서 회색 (δ 7.40-7.43)에 강조 resonances, 아래 지역을 통합하여 얻은 것입니다. 1 H 고해상도 매직 앵글 스피닝 (HR의 MAS) NMR 분광법은 생검 18 어떤 종류의 대사 프로파일을 빠르고 재현성 인수를 허용하지 않는 파괴적인 기술입니다. 이 프로토콜에서는, 우리는 전 (파란색)과 (적색) 식민지 (그림 6) 후 2 마우스의 간장 대사 프로필을 얻으려면이 강력한 기술을 사용합니다. 이 그림은 잘 NMR 기반의 신진 대사 프로필 MAS에서 파생 될 수있는 정보를 보여줍니다. 수많은 아미노산뿐만 아니라 포도당, 글리코겐, 락테이트, 트리 글리세 리드, (D) - 3 - 하이드 록시 및 nicotinurate 같은 에너지 대사에서 파생된 metabolites는 시각하실 수 있습니다. 이 프로파일은 또한 산화 스트레스 관련 정보 (예 ascorbic 포함되어 있습니다CID, 글루타티온), 뉴클레오 티드 대사 (예 : 이노신, 유리딘)와 methylamine 대사 (즉, 콜린, 트리 메틸 아민 - N - 산화물). 이 예제에서는, 그것은 세균없는 마우스가 거의 글리코겐과 포도당과 트리 글리세 리드가 매우 낮은 금액으로 이전 7로 출판 되었음를 표시하지 않는 매우 분명하다. <img src="/files/ftp_upload/3642/3642fig1.jpg" alt="그림 1."/> 그림 1. 식민 프로토콜의 개요. 세균없는 일반 동물은 측면과 그 새끼는 기존의 내장 microbiota (1)에서 진보적인 식민지를 허용 교환 의해 필터 사이드 갖춘 케이지 안에 보관되어있다. 내장 미생물 활동은 1 H NMR 기반의 신진 대사 프로 파일링 (2-3)를 사용하여 모니터링할 수 있습니다. 간장 대사는 1 H 인사 MAS NMR 기반의 신진 대사 프로 파일링 (4-5)에 의해 평가됩니다. <img src="/files/ftp_upload/3642/3642fig2.jpg" alt="그림 2."/> 그림 2. 마우스 살고R의 해부학. 간이 같은 장기의 플랫 사이드 테이블을 얼굴로 표시됩니다. 재현성 biopsies 위해, 그것은 언제나처럼 점선 사각형으로 표시된 좌측 엽의 중심에서 샘플을 수집하는 것이 좋습니다. <img src="/files/ftp_upload/3642/3642fig3.jpg" alt="그림 3."/> 그림 3 microvolumes 작동하도록 1.7 mm NMR 모세관 키트 키 :.. 1 : 2.5 mm의 NMR의 microtube, 2 : 1.7 mm NMR 모세관, 3 : 모세관 어댑터, 4 추출로드. <img src="/files/ftp_upload/3642/3642fig4.jpg" alt="그림 4."/> . 그림 4 MAS 로터 장비 키 : 1. : MAS의 회전자, 2 : 50 μL 테플론 스페이서, 3 : Thead 핀, 4 : 모자, 5 : 원통형 나사, 6 : 드라이버, 7 : 로터 포장, 8 : 깊이 게이지. <img src="/files/ftp_upload/3642/3642fig5.jpg" alt="그림 5."/> 그림 5. colonizatio 동안 요도 신진 대사 프로파일의 진화N. <ol class="ualpha"><li> 공동 metabolites은 시각 1. H NMR 스펙트럼 수있는 미생물 6.8-7.8 PPM 사이의 스펙트럼의 방향족 지역에 확대는 일 0 단일 개인 (파란색), 4 (녹색), 15 (오렌지) 20에서 파생되었습니다 (적색) 이후 식민지. Indoxyl 황산, 그의 : 히스티딘, P - 크레졸 - S :; : 1 MeHistamine : Indoxyl - S 1 - methylhistamine 회색 영역 B. 키의 다이어그램을 만들기 위해 통합되었습니다 영역에 해당하는 P - 크레졸 황산, PAG : Phenylacetylglycine. </li><li> 식민 기간 동안 평균 PAG 농도 (n은 = 7). 학생의 T - 테스트는 여러 시간 지점에서 PAG 농도의 차이를 비교하는 데 사용되었습니다 : P 일 0으로 비교하여 &lt;0.05, B : P &lt;0.01 하루 10 비교했다. </li></ol><img src="/files/ftp_upload/3642/3642fig6.jpg" alt="그림 6."/> 그림 6. 세균 무료 (파란색)과 전 세균 무료 (재에서 파생 간 biopsies의 전형적인 600 MHz의 1 H 인사 MAS NMR의 스펙트럼D) 마우스. 볼드 양자는 트리 글리세 라이드의 공명에 대한 책임은 키 :. 3 - HB : 3 - 하이드 록시, GSH : 감소 글루타티온, TGS : 트리 글리세 리드, TMAO : 트리 메틸 아민 - N - 산화물.

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우리는 공개 아무것도 없어.

이 프로토콜에서는, 우리는 더 이상 그대로 생검의 1 H 인사 MAS NMR의 프로 파일링에 의해 평가 간장 대사에 내장 microbiota의 영향을 조사하기 위해 열린 환경에서 진보 식민지 절차를 설명했다. 식민의 다양한 방법은 문헌에 설명되어있다. 정의된 microbiota과 동물을 식민지하는 가장 일반적인 방법은 구두 gavage이나 오염된 식수 19,20입니다. 이전 21 설명된대로 방패 접종도 사...

특정 시약 및 장비의 테이블 : <table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> 2.5 mm의 microtube </td><td> 새로운 시대 </td><td> NE-H5/2.5-V-Br </td><td></td></tr><tr><td> 1.7 mm 모세관 </td><td> 시그마 - 올드 리치 </td><td> NORS175001 </td><td></td></tr><tr><td> 모세관 어댑터 </td><td> 새로운 시대 </td><td> NE-325-5/1.7 </td><td></td></tr><tr><td> 추출로드 </td><td> 새로운 시대 </td><td> NE - 341-5 </td><td></td></tr><tr><td> 50 μL와 HR - MAS 로터 BL4 구면 테플론 스페이서 키트 </td><td> Bruker </td><td> HZ07213 </td><td></td></tr><tr><td> 50 μL 삽입을위한 도구 키트 </td><td> Bruker </td><td> B2950 </td><td></td></tr><tr><td> 사전 III 600MHz의 NMR </td><td> Bruker </td><td></td><td></td></tr><tr><td> 1 H 인사 MAS NMR 견고한 프로브 </td><td> Bruker </td><td></td><td></td></tr><tr><td> 중수소 산화 99.9 % </td><td> 시그마 - 올드 리치 </td><td> 530867 - 1L </td><td></td></tr><tr><td> 3 - (trimethylsilyl) 프로피 산성 - D 4 (TSP) </td><td> 시그마 - 올드 리치 </td><td> 269​​,913 </td><td></td></tr></tbody></table>

assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1h nmr spectroscopy

그것은 내장 박테리아가 이러한 면역 보호와 비타민 합성과 같은 혜택의 범위를 제공하는 호스트 항상성에 크게 기여 잘 알려져있다. 그들은 또한 생태계 필수적인 대사 오르간 제작, 영양소의 상당한로 호스트를 제공합니다. 호스트와 내장 microbiota 사이의 신진 대사 상호 작용을 이해하는 창자의 식물과 대사 증후군 사이의 링크의 증가 증거의 맥락에서 현대 생물학의 중요한 과제가되고있다. 1-4 식민지는 (또한 정상화 과정으로 함) 전 세균없는 동물의 미생물의 설립을 지정합니다. 그것이 출생시 발생하는 자연적인 과정이지만, 그것은 또한 창자 꽃 생태계를 관리하고 추가로 호스트 신진 대사에 미치는 영향을 결정하기 위해 성인 세균없는 동물에 사용됩니다. 식민 프로세스를 제어하는​​ 일반적인 절차는 singl로 gavage 방법을 사용하는 것입니다E 또는 미생물의 혼합물. 이 방법은 매우 빠른 식민지의 결과와 5 매우 스트레스가되는 단점을 선물한다. 그것은 스트레스를 최소화하고 점차 호스트 신진 대사에 세균 설립의 영향을 관찰하기 위해 느린 식민지 과정을 취득하는 것이 유용합니다. 이 원고에서는, 우리는 비파괴 신진 대사 프로 파일링 기법을 사용하여 점차 식민지 과정에서 간장 대사의 수정을 평가하는 절차를 설명합니다. 우리는 1 H NMR 기반의 신진 대사 프로 파일링 공동 metabolites 미생물의 비뇨기 배설에 의해 반영 내장 미생물의 신진 대사 활동을 평가하여 내장 미생물의 식민지를 모니터링 제안합니다. 이것은 일반적으로 DGGE (기울기 겔 전기 영동을 denaturing)에 의해 배설물 박테리아를 모니터링하여 평가 직감의 미생물 생태계의 안정 설립 넘어 직감의 미생물 활동의 안정성을 감사하실 수 있습니다. 6식민지는 종래의 열린 환경에서 열립니다하고 더러운 쓰레기 컨트롤로 될 것입니다 기존의 동물에 의해 더러워진에 의해 시작됩니다. coprophagous 동물 설치류되고, 이쪽은 이전에 설명한 것처럼 동질적인 식민지를 보장합니다. 7 간장 대사 프로 파일링은 NMR 분광법을 치는것 1 H 고해상도 매직 각도를 사용하여 손상 간 생검에서 직접 측정됩니다. 이 세미 양적 기술은 세포 구조를 손상시키지 않고, 평가하는 빠른 방법을 제공, 그러한 트리 글리세 리드, 포도당과 글리코겐으로 주요 metabolites 추가하기 위해서는 식민지 과정과 70-10 간장 신진 대사 사이의 복잡한 상호 작용을 예상하고있다. 이 방법은 모든 조직 생검 11,12에도 적용할 수 있습니다.

Watch this Scientific Journal Video about Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy at JoVE.com

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

진보 식민 절차는 더욱 호스트 간장 대사에 미치는 영향을 평가하기 위해 설명되어 있습니다. 간장 신진 대사가 고해상도 매직 앵글 스피닝 (HR의 MAS) 그대로 생검의 NMR 프로 파일링에 의해 평가하는 동안 식민지는 NMR 기반의 신진 대사 프로 파일링 공동 metabolites 미생물의 비뇨기 배설을 평가하여 invasively 아닌 모니터입니다.

하여 세균없는 마우스 프로 그레시브 콜로니 동안 간장 신진 대사 변경 사항 평가 1 H NMR 분광학

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

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Immunology and Infection

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

13.1K 조회수.  The University of Reading. 진보 식민 절차는 더욱 호스트 간장 대사에 미치는 영향을 평가하기 위해 설명되어 있습니다. 간장 신진 대사가 고해상도 매직 앵글 스피닝 (HR의 MAS) 그대로 생검의 NMR 프로 파일링에 의해 평가하는 동안 식민지는 NMR 기반의 신진 대사 프로 파일링 공동 metabolites 미생물의 비뇨기 배설을 평가하여 invasively 아닌 모니터입니다.

비디오: Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

커널 Bioassays를 사용하여 곰팡이 콜로니, Sporogenesis 및 Mycotoxins 생산의 부량

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

연구

면역학 및 감염병학

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.

의 식민지 Euprymna scolopes 오징어에 의한 장염 fischeri</em

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

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

일반 CNS의 실시간 PCR에 의한과 Neuroinflammation 동안 Microglia에 MicroRNAs의 탐지

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

연쇄상 구균 폐렴으로 비강 내 식민지 화 중 염증 반응의 특성화

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

공 초점 현미경으로 고립 된 폐포 대 식세포의 반대로 곰팡이 활동을 평가

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

리스테리아 단세포 유전자를 사용하여 감염된 마우스에 있는 문화와 심혼 식민지에 있는 심장 세포의 세균성 침략 평가

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-간 백혈구의 선택적 수확

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

그룹 B의 쥐 모델<em&gt; 연쇄상 구균</em&gt; 질 식민

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

형광 상관 분광학에 의해 측정 된 차 림프구의 플라즈마 막 분자 확산

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

생물 발광 시스템 비-침략 적 비보에 의해 Murine 호스트에서 Enterohemorrhagic 대장균 식민지의 검출

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

하여 세균없는 마우스 프로 그레시브 콜로니 동안 간장 신진 대사 변경 사항 평가¹ H NMR 분광학

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