본 연구에 의해 부분적으로 지원되었다 보조금-원조 교육, 문화, 스포츠, 과학 기술부에서 답사의 연구 (JK) 및 학술 연구 (A) (JK 및 SM)에 도전을위한 과학 연구, 기술, 일본 . RIKEN FPR 교제 (경찰과)이 추가 지원을 제공했습니다. 저자는 Drs들에게 감사드립니다. Eisuke Chikayama, NMR 및 통계 분석과 기술 지원을 Yasuyo Sekiyama와 마미 오카모토.

1. Extractables를 제​​거하는 필터 준비 <ol><li> 25-mm 직경 0.22 μm의 기공 크기 Durapore PVDF 친수성 ​​필터 (Millipore)를 사용합니다. 깨끗한 500 ML Pyrex의 비커에 장소 필터 핀셋을 사용합니다. 증류수로 세 번 사전 린스. 당신은 서로 고집에서 필터를 방지하기 위해 린스로서 소용돌이. 300 ML 밀리 Q (Millipore) 또는 이에 상응하는 양질의 물을 추가합니다. 필터에서 extractables의 완전한 제거를 용이하게하기 위해 압력솥. </li><li> 밀리 Q를 붓고 다시 트리플 밀리 Q와 함께, 이번에는 필터를 린스. 깨끗하고 건조한 표면 (예 : 알루미늄 호일 같은)과 합당한 온도 (예, 37는 ° C) 또는 공기 건조시 건조 중의 족집게 장소 개별 필터를 사용합니다. 필터를 사용할 수 있습니다. </li></ol> 2. 샘플 재질의 여과 <ol><li> 유리가 지원과 함께 25 mm microanalysis 필터 컬럼으로이 프로토콜, Millipore 스테인리스 3 곳 필터 매니폴드를 입증할그리고 기계식 펌프가 사용됩니다. 무균 기술을 사용하여 필터를 열베이스에 단일 25-mm 필터를 추가, 컬럼을 적용하고 함께 클램프. </li><li> , 컬럼에 시료의 15 ML을 로드할 필터 매니폴드에 정지 밸브를 열고 펌프를 켜십시오. 세포 파손 (&lt;5 kPa)을 최소화하기 위해 부드러운 압력을 받고 필터링합니다. 그러한 손 또는 연동 펌프와 같은 다른 펌프는,이 프로토콜에 적용할 수 있습니다. 낮은 밀도의 샘플의 경우 물을 연속적으로 추가가 필요할 수 있습니다, 필터가 물을 첨가 사이의 오랜 기간 동안 건조한 놓지 않습니다. </li><li> 당신의 시료를 여과 후 해양 샘플 들어, 선택적 담수가 샘플과 필터에 잔류 상자 성체의 이온을 줄이기 위해 린스 수행할 수 있습니다. 이것은 분광계 자석보다 정확한 튜닝에 도움이 될 수 있습니다. 간단히 부드럽게 물을 작은 볼륨을 추가하고 마지막 부분에서 수집된 샘플을 통해 필터링합니다. </li><li> 일단 필터링이 완료되면 펌프를 끄고 밸브 ope를 떠나N 그래서 필터 부정적인 압력은 여전히​​있다. 클램프 및 필터 열을 제거합니다. </li><li> 한손으로, 필터의 보류를 데리고 깨끗한 핀셋을 사용합니다. 그 자체를 통해 필터를 접어하지만 포복 절도하지. 다른 손으로 필터를 누르고 멸균 2-ML microcentrifuge 튜브의 입술을 사용합니다. 필터의 양쪽 가장자리를 다시 놓치 않으이를 사용 후 족집게를 놓습니다. 접이식으로 45 ° 각도에서 Regrip. </li><li> 그것이 안쪽을 향하게 샘플 측면과 함께 열립니다 있도록 2-ML 관과 버전에 필터를 놓습니다. 당신은 멸균이 ML의 microcentrifuge 튜브 이렇게 둘이 필터를 넣을 수 있습니다. 이 필터를 사용하는 경우, 그들은 가능​​한 한 조금은 중복 보장합니다. 즉시 (최소 -30 ° C)에 고정. </li></ol> 3. 수성 - 수용성 Metabolites의 추출 <ol><li> 샘플의 하룻밤 혹은 적어도 10 시간을 Lyophilize. </li><li> lyophilization 후, 각 튜브 (Tokken)에 스테인레스 스틸 분쇄기를 추가합니다. 750 μl 표준화된 칼륨 추가2,2 - 디메틸 -2 - silapentane-5-산염 (DSS) 표준 (KPI와 중수소 산화 (2 H&gt; 90 %)의 인산염 NMR 버퍼, 38.3 MM KH 2 PO 4, 61.7 MM K 2 HPO 4, DSS 0.1 MM, 산도 7.0, 90 % D 2 O) 2. </li><li> 물 sonicator에서 ° C (Bioruptor, Diagenode)이 필터에서 세포 물질을 제거하려면 4에서 5 분 샘플을 Sonicate. 깨끗한 핀셋으로 필터를 제거합니다. </li><li> 5 분 밀 분쇄기 (1600 RPM)를 사용하여 세포를 중단. </li><li> 15 분 동안 벤치 위로 흔드는 (Eppendorf)에 (1,400 RPM) 흔들림으로 65 ° C에서 품어. </li><li> 깨끗한 핀셋으로 금속 돼지를 제거하고 5 분 13,000그램에서 샘플을 원심 분리기. </li><li> NMR 분광법을위한 NMR 튜브에 직접 뜨는을 분산. </li></ol> 4. NMR 분광학 및 데이터 분석 <ol><li> (여기서, Bruker DRX-500 분광계가 장착된 NMR 분광계로 샘플을로드XWIN-NMR을 실행하는 컴퓨터)에 의해 제어 트리플 축 기울기와 TXI 프로브. </li><li> XWIN-NMR 인터페이스에서 적절한 이전에 다음 ID로 출판 방법에게 2,15를 사용하여 1D 1 H NMR의 스펙트럼을 구합니다. 현재 연구 1 H NMR 스펙트럼은 1.2 s의 반복 시간과 함께 워터 게이트 펄스 시퀀스에 의해 억압 받고 있었다 298 K. 잔여 물이 신호에서 500.03 MHz로 DRX-500 분광계 운영에 기록되었다. 128 과도는 스펙트럼 당 32,000 데이터 포인트를 얻기 위해 수집되었다. </li><li> NMRPipe 소프트웨어 16 설치된 PC에 NMR 데이터 디렉토리를 전송합니다. 0 PPM 참조 그런 다음 수동으로 스펙트럼을 단계적으로 폐지로 원시 데이터와 설정 DSS를 처리합니다. 디지털화 통합 또는 &#39;binning&#39;그들을 같은 rNMR, Automics와 같은 소프트웨어와 함께, 또는 ECOMICS 웹 사이트 (에서 공개 FT2B 패키지를 사용하여 개별 값의 집합으로 스펙트럼 데이터 <a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a> ) 3,17. 일 년, 스펙트럼은 0.032 PPM 통합 ECOMICS를 사용하는 지역 및 DSS 또는 전체 신호 강도 중 하나에 대한 표준을 통해 0.5 및 10.5 PPM 사이에 예를 들어 통합되었다 있습니다. 출력 데이터는 이제 이러한 연구 18 같은 무료 소프트웨어 패키지를 사용하는 주요 구성 요소 분석 (PCA)와 같은 다운 스트림 통계 분석을 위해 사용될 수 있습니다. </li></ol> 5. 대표 결과 1 H NMR의 스펙트럼의 예제는 위의 방법은 그림 1에 표시된 사용하여 획득하였습니다. 이러한 샘플은 소우주 실험의 두 시간이 지점에서 algal 신진 대사 활동으로 인해 명확한 차이점을 보여줍니다. 4 일 스펙트럼은 1 일 샘플에 비해 특히 3-4 PPM 범위에서 피크의 상당한 풍요로움을 보여줍니다. 이 봉우리는 소우주 내에서 규조류는을 피는 제작한 설탕에 의한 것이 있습니다. 인공 또는 자연 해수의 천연 플랑크톤 커뮤니티의 성장을 비교 유사한 실험에서 통계적 접근 방식의로딩 플롯은 스펙트럼 내에서 봉우리를 식별할 수있는 반면 binned NMR의 스펙트럼에서 파생된 주요 구성 요소 분석 (PCA) 점수 줄거리 등 uch는 두 개의 트리 트먼트 (그림 2) 사이의 명확한 대사의 차이를 표시하는 데 사용할 수있는 형상 데이터의 분포 . 이러한 결과는 같은 게놈 지문 채취 기법에서 같은 다른 omics 수준, (그림 3)에서 데이터와 비교할 수 있습니다. ; 이러한 NMR의 피크는 (BMRB에서 예 개별적으로 질의를 할수 <a href="http://www.bmrb.wisc.edu/">http://www.bmrb.wisc.edu/</a> 19 일, 또는 전체 스펙트럼은 (시 SpinAssign있는 등 통계적으로 분석할 수) <a href="http://prime.psc.riken.jp/?action=nmr_search">http://prime.psc.riken. JP /? 조치 = nmr_search</a> ) 2. 이 예제에서는 트리 트먼트의 차이점은 천연 플랑크톤 커뮤니티 metabolites의 스펙트럼의 설탕 지역의 봉우리 풍부한 (3.39 PPM으로 4.04 PPM)에 의한 것이라하고, 인공 해수 사회에 특징적인 여러 봉우리가 미정 IDE 있었다락테이트 및 SpinAssign를 사용하여 편대로 ntified. <img src="/files/ftp_upload/3163/3163fig1.jpg" alt="그림 1" /> 그림 1.이 절차를 사용하여 처리 표본에서 얻은 대표 1 H NMR의 스펙트럼. 소우주 샘플은 이전에 (매일 1) 및 (4 일) 강렬한 diatom의 개화 중에 찍은 사진. NMR 실험은 내부 표준 피크의 높이 (; 0 PPM DSS)으로 정규화 신호로 Bruker DRX-500에서 수행되었다. <img src="/files/ftp_upload/3163/3163fig2.jpg" alt="그림 2" /> 그림 2. 자연과 함께 microcosms 재배 자연스럽게 - 파생 미생물의 planktonic 커뮤니티 (오픈 다이아몬드) 또는 인공 (블랙 서클) 해수의 metabolomes에서 binned NMR의 스펙트럼을위한 주요 구성 요소 분석 (PCA) 점수 플롯. 클리어 신진 대사의 차이는 scatterplot에서 볼 수 있습니다. 이러한 분석에서 로딩 음모 그 다음에 중요성을 별개의 봉우리를 식별하는 데 사용될 수시스템은, 필요에 따라 이들 봉우리들은 더 이상 분석할 수 있습니다. <img src="/files/ftp_upload/3163/3163fig3.jpg" alt="그림 3" /> 그림 3. 멀티 omics 분석의 예제는 게놈 데이터와 NMR을 결합. 커뮤니티 조성은 denaturing 기울기 겔 18S의 전기 영동 (왼쪽)와 그림 2에서 분석하는 것과 같은 샘플에서 16 (오른쪽) rRNA의 유전자를 기반으로 또한 자연 (오픈 다이아몬드)과 인공 (검은색 원) 해수 microcosms 사이에 별개의 미생물 커뮤니티의 패턴을 보여줍니다. 자연 시스템의 metabolome과 게놈 사이의 이러한 대응은이 접근법의 유용성을 보여줍니다.

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NMR. 6, 277-293 (1995).</li><li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automics:+an+integrated+platform+for+NMR-based+metabonomics+spectral+processing+and+data+analysis.">Automics: an integrated platform for NMR-based metabonomics spectral processing and data analysis.</a> BMC Bioinformatics. 10, 83 (2009).</li><li>Eldon, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BioMagResBank.">BioMagResBank.</a> Nucleic Acids Res. 36, D402-D408 (2007).</li><li>Sekiyama, Y., Chikayama, E., Kikuchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+polar+and+semipolar+plant+metabolites+throughout+extraction+processes+using+a+combined+solution-state+and+high-resolution+magic+angle+spinning+NMR+approach.">Profiling polar and semipolar plant metabolites throughout extraction processes using a combined solution-state and high-resolution magic angle spinning NMR approach.</a> Anal. Chem. 82, 1643-1652 (2011).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

여기서 보여준 여과 및 신진 대사 추출 방법은 NMR의 metabolomics에 충분한 금액을 회수하기 위해 미생물 planktonic의 바이오 매스를 얻을 수 있습니다. KPI 및 1D 1 H NMR을 사용하여 수성 - 용해 metabolites만을 추출을 시연하는 동안 다른 추출 용매와 spectroscopic 방법을 사용할 수 있습니다. 하나의 유용한 예제는 이기종 샘플에서 우수한 NMR의 스펙트럼을 생산하기 위해 그림과 상자 성체의 이온에 ?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 0.22 μm의 친수성 ​​PVDF Durapore 필터, 25mm </td><td> Millipore </td><td> GVWP02500 </td><td></td></tr><tr><td> Microanalysis 필터 홀더, 25mm가 유리를 지원 fritted </td><td> Millipore </td><td> XX1002500 </td><td></td></tr><tr><td> 3 - 장소 매니폴드, 47mm, 스테인레스 스틸 </td><td> Millipore </td><td> XX2504735 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td> 와코 </td><td> 169-04245 </td><td></td></tr><tr><td> K 2 HPO 4 </td><td> 와코 </td><td> 164-04295 </td><td></td></tr><tr><td> 중수소 산화, 2 H&gt; 90% </td><td> Campridge 동위 원소 Laboratoties </td><td> DLM-4 </td><td></td<html> &gt; </tr><tr><td> DSS </td><td> Fluka </td><td> 92,754 </td><td></td></tr><tr><td> Automill </td><td> Tokken </td><td> TK-AM4 </td><td> 스테인레스 스틸 crushers 포함 </td></tr><tr><td> Thermomixer 편안함 </td><td> Eppendorf </td><td> 5355 000.011 </td><td></td></tr><tr><td> Bioruptor </td><td> Diagenode </td><td> UCD-200 </td><td></td></tr><tr><td> 진공 증발기 </td><td> EYELA </td><td> CVE-3100 </td><td></td></tr><tr><td> NMR </td><td> Bruker </td><td> 5mm-TXI 프로브와 DRX-500 </td><td></td></tr><tr><td> 스펙트럼 binning 도구 </td><td> 원래 개발 </td><td> FT2DB </td><td> <a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a> </td></tr><tr><td> 신진 대사 주석 도구 및 데이터베이스 </td><td> 원래 개발 </td><td> SpinAssign </td><td>= nmr_search &quot;&gt; http://prime.psc.riken.jp/?action=nmr_search </td></tr></tbody></table>

concentration of metabolites from low-density planktonic communities for environmental metabolomics using nuclear magnetic resonance spectroscopy

환경 metabolomics는 유기체가 반응 및 생화 학적 수준 1의 환경과 서로 상호 작용하는 방법에 새로운 이해를 추진하고 신흥 분야이다. 핵 자기 공명 (NMR) 분광법은 공부에 대한 상당한 약속과 함께, 가스 크로마 토그래피 - 질량 분석계 (GC-MS) 등 여러 기술 중 하나입니다. NMR의 장점은, 타겟이 분명하지 않은 분석에 적합한 구조 정보를 제공하며 스펙트럼은 각각의 신진 대사 스펙트럼 2,3 최근에 사용 가능한 데이터베이스에 대한 양적 및 통계 매너에 쿼리할 수 있습니다. 또한, NMR 분광 데이터는 서로 taxa의 생리적 반응과 환경 4,5,6의보다 포괄적인 이해를 제공하기 위해 다른 omics 수준 (예 : transcriptomics, 유전체학)의 데이터와 조합하여 사용할 수 있습니다. 그러나 NMR은 어려운에게주고, 다른 metabolomic 기법 미만 구분 AP샘플 인구가 낮은 잘 정의와 같은 전체 조직, biofluids 또는 셀 - 문화 등 쉽게 추출물 소스.에서 metabolites에 비해 낮은 밀도와 신진 대사 농도가 될 수있는 천연 미생물 시스템에 플라이 따라서 날짜에 수행된 미생물의 몇 가지 직접적인 환경 metabolomic 연구는 이러한 호스트 symbiont 시스템과 같은 문화 기반 또는 쉽게 정의 고밀도 생태계, 안정 동위 원소 라벨이 될 수 직감 환경의 공동 문화 또는 조작 구축으로 제한되었습니다 또한 NMR 신호에게 7,8,9,10,11,12을 향상시키기 위해 사용됩니다. NMR에 적합한 농도로 환경 metabolites의 농도와 컬렉션을 촉진 방법은 부족합니다. 최근 관심이 많은 에너지와 물질의 흐름은 planktonic 커뮤니티 13,14에 의해 매개되는 수생 환경 내에서 생물의 환경 metabolomics 부여 되었기 때문에, 우리는 집중하는 방법을 개발했습니다기 및 여과에 의한 planktonic의 미생물 시스템과 전체 - 커뮤니티 metabolites의 추출. 상용 친수성 ​​폴리-1 ,1-difluoroethene (PVDF) 필터는 특별히 완전히 달리 후속 분석에서 오염 물질이 나타날 수 extractables를 제​​거하는 처리됩니다. 이러한 처리 필터는 다음 관심있는 환경 또는 실험용 샘플을 필터링하는 데 사용됩니다. 젖은 샘플 자료가있는 필터는 동결 건조된이며 수성 - 용해 metabolites는 표준화된 칼륨 인산 추출 버퍼 2를 사용하여 기존의 NMR 분광법에 대해 직접적으로 압축이 풀립니다. 이러한 방법에서 파생된 데이터는 통계적으로 의미있는 패턴을 식별하는 분석, 또는 지역 사회와 생태계 기능의 포괄적인 이해를위한 다른 omics 수준과 통합 할 수 있습니다.

Watch this Scientific Journal Video about Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy at JoVE.com

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

미생물 planktonic 커뮤니티에서 신진 대사 추출을위한 방법을 설명하고있다. 전체 지역 사회의 샘플링 특별히 준비 필터로 여과에 의해 이루어진다. lyophilization 후 수성 - 용해 metabolites가 추출됩니다. 이 접근법은 자연이나 실험적인 미생물 커뮤니티의 횡단 omics 조사에 환경 metabolomics의 적용을 허용합니다.

핵 자기 공명 분광학을 사용하여 환경 Metabolomics를위한 낮은 밀도 Planktonic 커뮤니티에서 Metabolites의 농도

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

concentration-metabolites-from-low-density-planktonic-communities-for

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Biology

12.7K 조회수.  RIKEN Advanced Science Institute. 미생물 planktonic 커뮤니티에서 신진 대사 추출을위한 방법을 설명하고있다. 전체 지역 사회의 샘플링 특별히 준비 필터로 여과에 의해 이루어진다. lyophilization 후 수성 - 용해 metabolites가 추출됩니다. 이 접근법은 자연이나 실험적인 미생물 커뮤니티의 횡단 omics 조사에 환경 metabolomics의 적용을 허용합니다.

비디오: Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Microbial Communities in Nature and Laboratory - Interview

자연과 실험실에서 미생물 커뮤니티 - 인터뷰

연구

생물학

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

자기 공명 영상을위한 인간 배아 줄기 세포의 체외 라벨에

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage without prior knowledge of what genes the community might be expressing. Thus it provides the most unbiased perspective on community gene expression in situ. Environmental transcriptomics protocols are technically difficult since prokaryotic mRNAs generally lack the poly(A) tails that make isolation of eukaryotic messages relatively straightforward 1 and because of the relatively short half lives of mRNAs 2. In addition, mRNAs are much less abundant than rRNAs in total RNA extracts, thus an rRNA background often overwhelms mRNA signals. However, techniques for overcoming some of these difficulties have recently been developed. A procedure for analyzing environmental transcriptomes by creating clone libraries using random primers to reverse-transcribe and amplify environmental mRNAs was recently described was successful in two different natural environments, but results were biased by selection of the random primers used to initiate cDNA synthesis 3. Advances in linear amplification of mRNA obviate the need for random primers in the amplification step and make it possible to use less starting material decreasing the collection and processing time of samples and thereby minimizing RNA degradation 4. In vitro transcription methods for amplifying mRNA involve polyadenylating the mRNA and incorporating a T7 promoter onto the 3 end of the transcript. Amplified RNA (aRNA) can then be converted to double stranded cDNA using random hexamers and directly sequenced by pyrosequencing 5. A first use of this method at Station ALOHA demonstrated its utility for characterizing microbial community gene expression 6.

We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.

환경 Transcriptomics를 사용하여 해양 미생물 커뮤니티의 유전자 발현 분석

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

질소 산화물과 세포로부터 전자 상자 성체의 프로그램 공명 분광학에 의한 초과 산화물 음이온 급진적가 탐지 스핀 트랩을 사용하여

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

자기 공명 영상을 사용하여 혼합 Quantifying

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

성적 증명서와 Metabolites에서의 정화<em&gt; Drosophila</em&gt; 헤드

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

고대 생물 커뮤니티 숨겨 내부를 조사하기 위해 냉동 코어의 바깥 부분에서 외인성 물질의 제거

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

무척 추 동물 모델 시스템으로 독성 테스트 실험실에서 초파리 를 사용 하 여 실험 프로토콜

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

이중 양전자 방출 단층 촬영에 대 한 68가 코어 첨가 산화 철 나노 입자의 합성 / (T1) 자기 공명 이미징

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;