우리 장 Roch Mouret, 실 비 Dequin 로봇 기반 발효 시스템의 개념에 기여에 대 한 장-마리 Sabalyrolles 마틴 Pradal, 니콜라 부 비 그리고 Pascale Brial 그들의 기술 지원에 대 한 감사 합니다. 이 프로젝트에서 제공한 Ministère 드 l'Education 회, 드 라 검색 외에 대 한 자금 드 라 Technologie.

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+metabolism+in+Saccharomyces+cerevisiae.">Nitrogen metabolism in Saccharomyces cerevisiae.</a> The molecular biology of the yeast Saccharomyces: Metabolism and gene expression. , 39-99 (1982).</li><li>Jones, E. W., Fink, G. R., Strathern, J. N., Jones, E. W., Broach, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+amino+acid+and+nucleotide+biosynthesis+in+yeast.">Regulation of amino acid and nucleotide biosynthesis in yeast.</a> The molecular biology of the yeast Saccharomyces: Metabolism and gene expression. , 182-299 (1982).</li><li>Hazelwood, L. A., Daran, J. -. M., van Maris, A. J. A., Pronk, J. T., Dickinson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ehrlich+pathway+for+fusel+alcohol+production:+a+century+of+research+on+Saccharomyces+cerevisiae+metabolism.">The Ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism.</a> Appl Environ Microbiol. 74, 2259-2266 (2008).</li><li>Bely, M., Sablayrolles, J. M., Barre, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+detection+of+assimilable+nitrogen+deficiencies+during+alcoholic+fermentation+in+oenological+conditions.">Automatic detection of assimilable nitrogen deficiencies during alcoholic fermentation in oenological conditions.</a> J Ferment Bioeng. 70, 246-252 (1990).</li><li>Forster, J., Famili, I., Fu, P., Palsson, B. 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저자는 공개 없다.

동위 원소 추적 실험을 사용 하 여 신진 대사 네트워크를 통해 화합물의 분할을 측정 하는 것은 미생물 물질 대사의 동작을 이해 하기 위한 유망한 접근 이다. 이 방법론, 하나 또는 두 개의 레이블된 기판, 성공적으로 적용 하는 동안 수 없습니다 현재 구현 여러 레이블이 원소 동위 원소 (즉, 두 개 이상의 기판)를 사용 하 여 다양 한 소스의 대사를 공부. 실제로, 사용할 수 있는 분석 기법 ?...

미생물 물질 대사 작동 하는 방법 이해는 발효 프로세스를 개선 하 고 발효 화합물의 생산을 조절 하는 효율적인 전략의 디자인에 대 한 중요 한 문제 이다. 유전체학 및 기능 유전체학이 마지막이 십년에서 크게 지식의 많은 미생물 대사 네트워크의 토폴로지를 확장에 기여. 이 정보에 액세스할 세포질 기능1의 포괄적인 개요를 목표로 하는 방식의 개발을 주도. 이러한 방법론은 자주 측정 매개 변수 모델 기반 해석에 의존합니다. 이 실험 데이터 포함, 한 손으로, 대사 산물 통풍 관 및 생산 속도 하 고, 다른 한편으로, 양적 세포내 얻은 정보를 동위 원소 추적 프로그램에서 실험. 이러한 데이터는 정의 된 대사 네트워크2,,34다른 통로의 vivo에서 활동의 공제에 대 한 필수 정보를 제공합니다. 현재, 사용할 수 있는 분석 기법만 단일 요소 동위 원소를 사용 하 여 분자의 패턴을 라벨의 정확한 검색을 사용 가능 하 게 하 고 공동 두 동위 요소와 라벨 때. 또한, 대부분의 성장 조건에서 탄소 소스만 구성 되어 하나 또는 두 화합물의 있습니다. 따라서, 탄소 기판에서 13C 동위 원소 예 광 탄에 따라 접근 넓게 그리고 성공적으로 적용 된 탄소 대사 네트워크 작업5,6,7의 완전 한 이해를 개발 하 ,8.
대조적으로, 많은 자연 및 산업 환경에서 미생물 성장을 지 원하는 사용할 수 있는 질소 리소스는 다양 한 분자의 구성 자주 됩니다. 예를 들어 와인 또는 맥주 발효 하는 동안 질소 18 아미노산 및 가변 농도9암모늄의 혼합물으로 제공 됩니다. Anabolism 액세스할 수 있는 N 화합물의이 만드는이 복잡 한 미디어 조건 크게 다른 생리 적 연구에 대 한 일반적으로 사용 되는 후자 질소, 일반적으로 암모늄의 독특한 소스를 사용 하 여 달성 하는.
전반적으로, 질소 화합물 직접 단백질에 통합 되거나 catabolized 내 면. 효 모 Saccharomyces cerevisiae를 포함 하 여 많은 미생물 물질 대사 질소의 네트워크 구조는 매우 복잡 한 기판의 다양성에 따라. 개요로,이 시스템은 글루타민, 조미료, α ketoglutarate10,11, transaminases와 deaminases의 interconversion를 catalyzes 질소 물질 대사의 중앙 코어의 조합을 기반으로 합니다. 이 네트워크를 통해 암모늄 또는 다른 아미노산에서 아민 그룹 수집 하 고 α-keto 산 발표 했다. 이 중간체는 또한 중앙 탄소 대사 (CCM)12,13synthetized. 이 많은 수의 분기 반응 및 중간체, 외 인 질소 소스의 놓을 proteinogenic 아미노산의 anabolism에 관련 된 세포의 근육 요구 충족. 이러한 다른 상호 경로 통해 활동 대사 산물의 배설에도 발생합니다. 특히, α-keto 산 제품의 감각 프로필에 필수적인 참여자는 더 높은 알콜 및 그들의 아세테이트 에스테 르 유도체14, Ehrlich 통로 통해 리디렉션할 수 있습니다. 그 후, 질소 대사 작동 하는 방법 바이오 매스 생산 및 휘발성 분자를 (향기)의 형성에 중요 한 역할을 재생 합니다.
반응, 효소, 및 질소 대사에 관여 하는 유전자는 문학에서 광대 하 게 기술 된다. 그러나, 대사 네트워크를 통해 여러 질소 소스의 배포의 문제는 아니라 아직 해결 되었습니다. 정보의이 부족을 설명 하는 두 가지 주요 이유가 있습니다. 첫째, 질소 대사 네트워크의 중요 한 복잡성의 관점에서 정량적 데이터의 큰 금액은 사용할 수 작업의 완전 한 이해에 필요한 지금까지. 두 번째, 많은 실험의 제약 조건 및 분석 방법의 한계 방지 이전 CCM 함수 설명에 사용 된 방법의 구현.
이러한 문제를 극복 하기 위해 우리는 일련의 동위 원소 추적 실험에서 데이터의 화해를 기반으로 하는 시스템 수준 접근 방법을 개발 하기로 결정 했다. 워크플로가 포함 되어 있습니다. -다른 선택한 영양 소스 (기판) 때마다 표시 하는 동안 발효 된 동일한 환경 조건 하에서 실시. -레이블된 기판 및 농도의 잔류 농도 및에서 파생 된 화합물의 동위 원소 농축 발효의 여러 단계에서 정확한 결정을 위한 분석 절차 (HPLC, GC-MS)의 조합 파생 된 바이오 매스를 포함 하 여 레이블이 지정 된 분자의 놓을. -각 질량과 동위 균형의 계산 소비 레이블이 분자 및 유동 비율의 결정을 통해 미생물에 의해 여러 영양소 소스 관리에 대 한 글로벌 개요를 얻기 위해 데이터 집합의 추가 통합된 분석 .
이 방법론을 적용 하기 위해 주의 문화 사이의 긴장/미생물의 재현 동작에 지불 되어야 합니다. 또한, 다른 문화에서 샘플 같은 잘 정의 된 발효 진행 중 촬영 해야 합니다. 이 원고에 보고 하는 실험적인 작품에서 로봇 기반 시스템 이러한 제약 조건에 대 한 계정에 발효의 온라인 모니터링에 대 한 사용 됩니다.
또한, 연구의 과학적 문제를 해결 하기 위해 적절 한 레이블이 기판 (화합물, 자연, 그리고 라벨의 위치)의 집합을 선택 하는 것이 필수적입니다. 여기, 15N 표시 된 암모늄, 글루타민, 아르기닌 포도 주스에서 발견 세 가지 주요 질소 근원으로 선정 됐다. 이 proteinogenic 아미노산에 소비 화합물에서 질소 재배포의 패턴을 평가 허용. 우리는 또한 소비 아미노산 및 휘발성 분자의 생산에 그들의 공헌의 탄소 등뼈의 운명을 조사 목적. 충족 시키기 위해이 목표, 균일 하 게 13C 라는 신, 이소류신, 트레오닌, valine 포함 되었다 연구에 Ehrlich 통로의 주요 중간체에서 파생 된 아미노산으로.
전반적으로, 우리 양적 효 모 또한으로 탄소 선구자의 초과 제거 하는 동안 발효를 통해 근육의 요구 사항을 충족 하는 외 인 질소 소스를 재배포 하 여 복잡 한 질소 리소스를 관리 하는 방법을 탐구 휘발성 분자입니다. 이 논문에 보고 된 실험 절차 다른 미생물에 의해 사용 된 다른 여러 영양소 소스 조사에 적용할 수 있습니다. 그것은 미생물의 대사 행동에 유전 배경이 나 환경 조건 영향의 분석에 대 한 적절 한 접근을 것 처럼 보인다.

<table>
	<tbody>
		<tr>
			<td>D-Glucose</td>
			<td>PanReac</td>
			<td>141341.0416</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Fructose</td>
			<td>PanReac</td>
			<td>142728.0416</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Malic acid</td>
			<td>Sigma Aldrich</td>
			<td>M0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid monohydrate</td>
			<td>Sigma Aldrich</td>
			<td>C7129</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>P5379</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium sulfate</td>
			<td>Sigma Aldrich</td>
			<td>P0772</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma Aldrich</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma Aldrich</td>
			<td>A4514</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>71690</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese sulfate monohydrate</td>
			<td>Sigma Aldrich</td>
			<td>M7634</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc sulfate heptahydrate</td>
			<td>Sigma Aldrich</td>
			<td>Z4750</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper (II) sulfate pentahydrate</td>
			<td>Sigma Aldrich</td>
			<td>C7631</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium iodine</td>
			<td>Sigma Aldrich</td>
			<td>P4286</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt (II) chloride hexahydrate</td>
			<td>Sigma Aldrich</td>
			<td>C3169</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma Aldrich</td>
			<td>B7660</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium heptamolybdate</td>
			<td>Sigma Aldrich</td>
			<td>A7302</td>
			<td></td>
		</tr>
		<tr>
			<td>Myo-inositol</td>
			<td>Sigma Aldrich</td>
			<td>I5125</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Pantothenic acid hemicalcium salt</td>
			<td>Sigma Aldrich</td>
			<td>21210</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine, hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>T4625</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinic acid</td>
			<td>Sigma Aldrich</td>
			<td>N4126</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxine</td>
			<td>Sigma Aldrich</td>
			<td>P5669</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotine</td>
			<td>Sigma Aldrich</td>
			<td>B4501</td>
			<td></td>
		</tr>
		<tr>
			<td>Ergost&#233;rol</td>
			<td>Sigma Aldrich</td>
			<td>E6510</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma Aldrich</td>
			<td>P1754</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>VWR Chemicals</td>
			<td>101074F</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (III) chloride hexahydrate</td>
			<td>Sigma Aldrich</td>
			<td>236489</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspartic acid</td>
			<td>Sigma Aldrich</td>
			<td>A9256</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic acid</td>
			<td>Sigma Aldrich</td>
			<td>G1251</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Alanine</td>
			<td>Sigma Aldrich</td>
			<td>A7627</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine</td>
			<td>Sigma Aldrich</td>
			<td>A5006</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma Aldrich</td>
			<td>G3126</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma Aldrich</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine</td>
			<td>Sigma Aldrich</td>
			<td>H8000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Isoleucine</td>
			<td>Sigma Aldrich</td>
			<td>I2752</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Leucine</td>
			<td>Sigma Aldrich</td>
			<td>L8000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Sigma Aldrich</td>
			<td>L5501</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Sigma Aldrich</td>
			<td>M9625</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Phenylalanine</td>
			<td>Sigma Aldrich</td>
			<td>P2126</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Sigma Aldrich</td>
			<td>P0380</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Serine</td>
			<td>Sigma Aldrich</td>
			<td>S4500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Threonine</td>
			<td>Sigma Aldrich</td>
			<td>T8625</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tryptophane</td>
			<td>Sigma Aldrich</td>
			<td>T0254</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine</td>
			<td>Sigma Aldrich</td>
			<td>T3754</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Valine</td>
			<td>Sigma Aldrich</td>
			<td>V0500</td>
			<td></td>
		</tr>
		<tr>
			<td>13C5-L-Valine</td>
			<td>Eurisotop</td>
			<td>CLM-2249-H-0.25</td>
			<td></td>
		</tr>
		<tr>
			<td>13C6-L-Leucine</td>
			<td>Eurisotop</td>
			<td>CLM-2262-H-0.25</td>
			<td></td>
		</tr>
		<tr>
			<td>15N-Ammonium chloride</td>
			<td>Eurisotop</td>
			<td>NLM-467-1</td>
			<td></td>
		</tr>
		<tr>
			<td>ALPHA-15N-L-Glutamine</td>
			<td>Eurisotop</td>
			<td>NLM-1016-1</td>
			<td></td>
		</tr>
		<tr>
			<td>U-15N4-L-Arginine</td>
			<td>Eurisotop</td>
			<td>NLM-396-PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>Sigma Aldrich</td>
			<td>270989</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl propanoate</td>
			<td>Sigma Aldrich</td>
			<td>112305</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl 2-methylpropanoate</td>
			<td>Sigma Aldrich</td>
			<td>246085</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl butanoate</td>
			<td>Sigma Aldrich</td>
			<td>E15701</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl 2-methylbutanoate</td>
			<td>Sigma Aldrich</td>
			<td>306886</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl 3-methylbutanoate</td>
			<td>Sigma Aldrich</td>
			<td>8.08541.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl hexanoate</td>
			<td>Sigma Aldrich</td>
			<td>148962</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl octanoate</td>
			<td>Sigma Aldrich</td>
			<td>W244910</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl decanoate</td>
			<td>Sigma Aldrich</td>
			<td>W243205</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl dodecanoate</td>
			<td>Sigma Aldrich</td>
			<td>W244112</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl lactate</td>
			<td>Sigma Aldrich</td>
			<td>W244015</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl succinate</td>
			<td>Sigma Aldrich</td>
			<td>W237701</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methylpropyl acetate</td>
			<td>Sigma Aldrich</td>
			<td>W217514</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methylbutyl acetate</td>
			<td>Sigma Aldrich</td>
			<td>W364401</td>
			<td></td>
		</tr>
		<tr>
			<td>3-methyl butyl acetate</td>
			<td>Sigma Aldrich</td>
			<td>287725</td>
			<td></td>
		</tr>
		<tr>
			<td>2-phenylethyl acetate</td>
			<td>Sigma Aldrich</td>
			<td>290580</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methylpropanol</td>
			<td>Sigma Aldrich</td>
			<td>294829</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methylbutanol</td>
			<td>Sigma Aldrich</td>
			<td>133051</td>
			<td></td>
		</tr>
		<tr>
			<td>3-methylbutanol</td>
			<td>Sigma Aldrich</td>
			<td>309435</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexanol</td>
			<td>Sigma Aldrich</td>
			<td>128570</td>
			<td></td>
		</tr>
		<tr>
			<td>2-phenylethanol</td>
			<td>Sigma Aldrich</td>
			<td>77861</td>
			<td></td>
		</tr>
		<tr>
			<td>Propanoic acid</td>
			<td>Sigma Aldrich</td>
			<td>94425</td>
			<td></td>
		</tr>
		<tr>
			<td>Butanoic acid</td>
			<td>Sigma Aldrich</td>
			<td>19215</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methylpropanoic acid</td>
			<td>Sigma Aldrich</td>
			<td>58360</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methylbutanoic acid</td>
			<td>Sigma Aldrich</td>
			<td>193070</td>
			<td></td>
		</tr>
		<tr>
			<td>3-methylbutanoic acid</td>
			<td>Sigma Aldrich</td>
			<td>W310212</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexanoic acid</td>
			<td>Sigma Aldrich</td>
			<td>153745</td>
			<td></td>
		</tr>
		<tr>
			<td>Octanoic acid</td>
			<td>Sigma Aldrich</td>
			<td>W279900</td>
			<td></td>
		</tr>
		<tr>
			<td>Decanoic acid</td>
			<td>Sigma Aldrich</td>
			<td>W236403</td>
			<td></td>
		</tr>
		<tr>
			<td>Dodecanoic acid</td>
			<td>Sigma Aldrich</td>
			<td>L556</td>
			<td></td>
		</tr>
		<tr>
			<td>Fermentor 1L</td>
			<td>Legallais</td>
			<td>AT1357</td>
			<td>Fermenter handmade for fermentation</td>
		</tr>
		<tr>
			<td>Disposable vacuum filtration system</td>
			<td>Dominique Deutscher</td>
			<td>029311</td>
			<td></td>
		</tr>
		<tr>
			<td>Fermenters (250 ml)</td>
			<td>Legallais</td>
			<td>AT1352</td>
			<td>Fermenter handmade for fermentation</td>
		</tr>
		<tr>
			<td>Sterile tubes</td>
			<td>Sarstedt</td>
			<td>62.554.502</td>
			<td></td>
		</tr>
		<tr>
			<td>Fermentation locks</td>
			<td>Legallais</td>
			<td>AT1356</td>
			<td>Fermetation locks handmade for fermentation</td>
		</tr>
		<tr>
			<td>BactoYeast Extract</td>
			<td>Becton, Dickinson and Company</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>BactoPeptone</td>
			<td>Becton, Dickinson and Company</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator shaker</td>
			<td>Infors HT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Particle Counter</td>
			<td>Beckman Coulter</td>
			<td>6605697</td>
			<td>Multisizer 3 Coulter Counter</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Jouan</td>
			<td></td>
			<td>GR412</td>
		</tr>
		<tr>
			<td>Plate Butler Robotic system</td>
			<td>Lab Services BV</td>
			<td>PF0X-MA</td>
			<td>Automatic instrument</td>
		</tr>
		<tr>
			<td>Plate Butler Software</td>
			<td>Lab Services BV</td>
			<td></td>
			<td>Robot monitor software</td>
		</tr>
		<tr>
			<td>RobView</td>
			<td></td>
			<td></td>
			<td>In-house developed calculation software</td>
		</tr>
		<tr>
			<td>My SQL</td>
			<td></td>
			<td></td>
			<td>International source database</td>
		</tr>
		<tr>
			<td>Cimarec i Telesystem Multipoint Stirrers</td>
			<td>Thermo Fisher Scientific</td>
			<td>50088009</td>
			<td>String Drive 60</td>
		</tr>
		<tr>
			<td>BenchBlotter platform rocker</td>
			<td>Dutscher</td>
			<td>60903</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonia enzymatic kit</td>
			<td>R-Biopharm AG</td>
			<td>5390</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer cuvettes</td>
			<td>VWR</td>
			<td>634-0678</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer UviLine 9400</td>
			<td>Secomam</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amino acids standards physiological - acidics and neutrals</td>
			<td>Sigma Aldrich</td>
			<td>A6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Amino acids standards physiological - basics</td>
			<td>Sigma Aldrich</td>
			<td>A6282</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate lithium buffers - Ultra ninhydrin reagent</td>
			<td>Biochrom</td>
			<td>BC80-6000-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfosalycilic acid</td>
			<td>Sigma Aldrich</td>
			<td>S2130</td>
			<td></td>
		</tr>
		<tr>
			<td>Norleucine</td>
			<td>Sigma Aldrich</td>
			<td>N1398</td>
			<td></td>
		</tr>
		<tr>
			<td>Biochrom 30 AAA</td>
			<td>Biochrom</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EZChrom Elite</td>
			<td>Biochrom</td>
			<td></td>
			<td>Instrument control and Data analysis software</td>
		</tr>
		<tr>
			<td>Ultropac 8 resin Lithium</td>
			<td>Biochrom</td>
			<td>BC80-6002-47</td>
			<td>Lithium High Resolution Physiological Column</td>
		</tr>
		<tr>
			<td>Filter Millex GV</td>
			<td>Merck Millipore</td>
			<td>SLGVX13NL</td>
			<td>Millex GV 13mm (pore size 0.22 &#181;m)</td>
		</tr>
		<tr>
			<td>Membrane filter PALL</td>
			<td>VWR</td>
			<td>514-4157</td>
			<td>Supor-450 47mm 0.45&#181;m</td>
		</tr>
		<tr>
			<td>Vacuum pump Millivac Mini</td>
			<td>Millipore</td>
			<td>XF5423050</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium smooth weigh dish 70mm</td>
			<td>VWR</td>
			<td>611-1380</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision balance</td>
			<td>Mettler</td>
			<td></td>
			<td>Specifications AE163</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxid dried</td>
			<td>Merck</td>
			<td>1029310161</td>
			<td>(max. 0.025% H2O) SeccoSolv</td>
		</tr>
		<tr>
			<td>Combustion oven</td>
			<td>Legallais</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA protein assay kit</td>
			<td>Interchim</td>
			<td>UP40840A</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Fluka</td>
			<td>94318</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Sigma Aldrich</td>
			<td>H1009</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid Fuming 37% Emsure</td>
			<td>Merck</td>
			<td>1003171000</td>
			<td>Grade ACS,ISO,Reag. Ph Eur</td>
		</tr>
		<tr>
			<td>Lithium acetate buffer</td>
			<td>Biochrom</td>
			<td>80-2038-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial solution of hydrolyzed amino acids</td>
			<td>Sigma Aldrich</td>
			<td>AAS18</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine sulfone</td>
			<td>Sigma Aldrich</td>
			<td>M0876</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteic acid monohydrate</td>
			<td>Sigma Aldrich</td>
			<td>30170</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex glass culture tubes</td>
			<td>Sigma Aldrich</td>
			<td>Z653586</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridine</td>
			<td>Acros Organics</td>
			<td>131780500</td>
			<td>99% Extrapure</td>
		</tr>
		<tr>
			<td>Ethyl chloroformate</td>
			<td>Sigma Aldrich</td>
			<td>23131</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>32222</td>
			<td></td>
		</tr>
		<tr>
			<td>Vials</td>
			<td>Sigma Aldrich</td>
			<td>854165</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinserts for 1.5ml vials</td>
			<td>Sigma Aldrich</td>
			<td>SU860066</td>
			<td></td>
		</tr>
		<tr>
			<td>GC/MS</td>
			<td>Agilent Technologies</td>
			<td>5890 GC/5973 MS</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemstation</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td>Instrument control and data analysis software</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich</td>
			<td>34860</td>
			<td>Chromasolv, for HPLC</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma Aldrich</td>
			<td>34998</td>
			<td>ChromasolvPlus, for HPLC</td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide dimethyl acetal</td>
			<td>Sigma Aldrich</td>
			<td>394963</td>
			<td></td>
		</tr>
		<tr>
			<td>BSTFA</td>
			<td>Sigma Aldrich</td>
			<td>33024</td>
			<td></td>
		</tr>
		<tr>
			<td>DB-17MS column</td>
			<td>Agilent Technologies</td>
			<td>122-4731</td>
			<td>30m*0.25mm*0.15&#181;m</td>
		</tr>
		<tr>
			<td>Sodium sulfate, anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>238597</td>
			<td></td>
		</tr>
		<tr>
			<td>Technical nitrogen</td>
			<td>Air products</td>
			<td>14629</td>
			<td></td>
		</tr>
		<tr>
			<td>Zebron ZB-WAX column</td>
			<td>Phenomenex</td>
			<td>7HG-G007-11</td>
			<td>30m*0.25mm*0.25&#181;m</td>
		</tr>
		<tr>
			<td>Helium BIP</td>
			<td>Air products</td>
			<td>26699</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipettes</td>
			<td>VWR</td>
			<td>612-1702</td>
			<td></td>
		</tr>
	</tbody>
</table>

workflow based on the combination of isotopic tracer experiments to investigate microbial metabolism of multiple nutrient sources

미생물학의 분야에 있는 연구는 다양 한 방법론의 구현에 의존합니다. 특히, 적절 한 방법의 개발은 실질적으로 독특한 질소와 탄소 소스를 포함 하는 화학적으로 정의 된 미디어에서 성장 하는 미생물의 물질 대사의 광범위 한 지식을 제공에 기여 한다. 반면, 여러 소스의 영양소, 자연 또는 산업 환경에 광범위 한 그들의 존재에도 불구 하 고 신진 대사를 통해 관리 거의 미개척 남아 있습니다. 이 상황은 조사 방해 적합 한 방법론의 부족 때문에 주로 이다.
우리는 양적 및 포괄적 대사 영양소는 다른 분자, 즉, 복잡 한 리소스의 혼합물으로 제공 하는 때 작동 하는 방법을 탐구 하는 실험적인 전략을 보고 합니다. 여기, 우리는 효 모 대사 네트워크를 통해 여러 질소 소스의 분할을 평가 하기 위한 응용 프로그램을 설명 합니다. 워크플로 안정 동위 원소 추적 실험 선택한 13C-또는 15N 표시 기판 사용 하는 동안 얻은 정보를 결합 합니다. 그것은 먼저 이루어져 있다 N 포함 된 분자;의 혼합물을 포함 하는 동일한 매체에 병렬 및 재현성 발효 그러나, 선택 된 질소 소스 때마다 표시 됩니다. 대상된 화합물의 라벨 패턴을 평가 하 고 소비 및 다른 대사 산물에 기판의 복구를 계량 분석 절차 (HPLC, GC-MS)의 조합을 구현 됩니다. 완전 한 데이터 집합의 통합된 분석 셀 내에서 사용 된 기판의 운명에 대 한 개요를 제공합니다. 이 방법은 발효의 온라인 모니터링을 위한 로봇 기반 시스템에 의해 샘플-촉진의 컬렉션에 대 한 정확한 프로토콜을 필요-그리고 수많은 시간이 걸리는 분석의 공적. 이러한 제약에도 불구 하 고 그것은 이해 하 고, 처음으로, 효 모 대사 네트워크를 통해 여러 질소 소스의 분할 허용. 우리는 다른 N 화합물 쪽으로 더 풍부한 소스에서 질소의 재분배를 해명 하 고 휘발성 분자와 proteinogenic 아미노산의 대사 기원 결정.

그림 3 워크플로 여러 질소 소스 와인 발효 하는 동안 발견 되는 효 모에 의해 관리를 조사 하기 위해 구현 된의 회로도를 선물 한다. 샘플링, 생물 학적 매개 변수-성장 특성, 질소 소비 패턴 및 proteinogenic 아미노산-쇼 발효 (그림 4) 중 높은 재현성의 프로필의 다른 지점에 대 한 이 일관성 세트 14 독립 발효 중 생성 된 데이터?...

Watch this Scientific Journal Video about Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources at JoVE.com

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

이 프로토콜 양적이 고 포괄적으로 여러 영양소 소스의 신진 대사를 조사 하는 실험 절차를 설명 합니다. 이 워크플로, 동위 원소 추적 실험 및 분석 절차의 조합에 따라 결정 될 미생물 소비 영양분의 운명과 분자 synthetized의 대사 기원 수 있습니다.

여러 영양소 소스의 미생물 대사를 조사 하기 위해 동위 원소 추적 실험의 조합에 따라 워크플로

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

workflow-based-on-combination-isotopic-tracer-experiments-to

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.2K 조회수.  Université de Montpellier. 이 프로토콜 양적이 고 포괄적으로 여러 영양소 소스의 신진 대사를 조사 하는 실험 절차를 설명 합니다. 이 워크플로, 동위 원소 추적 실험 및 분석 절차의 조합에 따라 결정 될 미생물 소비 영양분의 운명과 분자 synthetized의 대사 기원 수 있습니다.

비디오: Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

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생체공학

Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules in the pathogenesis of osteoarthritis (OA) in vivo. However, the detailed, especially the visualized protocol for establishing this complicated model in mice, is not available. Herein we took advantage of wildtype and progranulin (PGRN)-/- mice as examples to introduce a protocol for inducing DMM model in mice, and compared the onset of OA following establishment of this surgically induced model. The operations performed on mice were either sham operation, which just opened joint capsule, or DMM operation, which cut the menisco-tibial ligament and caused destabilization of medial meniscus. Osteoarthritis severity was evaluated using histological assay (e.g. Safranin&#160;O staining), expressions of OA-associated genes, degradation of cartilage extracellular matrix molecules, and osteophyte formation. DMM operation successfully induced OA initiation and progression in both wildtype and PGRN-/- mice, and loss of PGNR growth factor led to a more severe OA phenotype in this surgically induced model.

We describe a protocol for the destabilization of the medial meniscus (DMM) model in mice, an effective tool for osteoarthritis (OA) research. In addition, we have demonstrated that deficiency of progranulincan exaggerate OA development and progression by using this model, indicating that progranulin plays a protective role in the pathogenesis of OA.

관절염에 Progranulin의 보호 역할을 조사하기 위해 마우스의 수술로 유발 된 모델 구축

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

의 리그닌 아래로 규제<em&gt; ZEA 메이스</em&gt; dsRNAi 및 Klason 리그닌 분석을 통해

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ultrasound (US) probes. Particularly, a more realistic analysis with application-specific imaging targets can be extremely valuable to assess the expected performance of US probes in their potential clinical field of application.
The experimental protocol presented in this work was purposely designed to provide an application-specific assessment procedure for newly-developed US probe prototypes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology in relation to brain imaging.
The protocol combines the use of a bovine brain fixed in formalin as the imaging target, which ensures both realism and repeatability of the described procedures, and of neuronavigation techniques borrowed from neurosurgery. The US probe is in fact connected to a motion tracking system which acquires position data and enables the superposition of US images to reference Magnetic Resonance (MR) images of the brain. This provides a means for human experts to perform a visual qualitative assessment of the US probe imaging performance and to compare acquisitions made with different probes. Moreover, the protocol relies on the use of a complete and open research and development system for US image acquisition, i.e. the Ultrasound Advanced Open Platform (ULA-OP) scanner.
The manuscript describes in detail the instruments and procedures involved in the protocol, in particular for&#160;the calibration, image acquisition and registration of US and MR images. The obtained results prove the effectiveness of the overall protocol presented, which is entirely open (within the limits of the instrumentation involved), repeatable, and covers the entire set of acquisition and processing activities for US images.

The development of new ultrasound (US) probes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology requires an early realistic assessment of imaging capabilities. We describe a repeatable experimental protocol for US image acquisition and comparison with magnetic resonance images, using an ex vivo bovine brain as an imaging target.

뇌 이미징 응용 프로그램에서 CMUT 기술에 기반 하는 새로운 초음파 프로브의 성능을 평가 하기 위한 실험 프로토콜

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry:에 대 한 강력 하 고, 비 침 투 적인 접근 세포 에너지 대사를 조사

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

실시 한 형광 현미경으로 여러 영상 모드

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

가 여러 명의 병렬 접시 흐름 챔버를 사용 하 여 스트레스에 노출 된 내 피 세포의 유전자 표정 분석

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

미생물 행동을 연구하기 위한 제어된 동적 화학 적 경관 생성

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as an&#160;innovative source of such new compounds. One bottleneck in the process is typically the elucidation of their mode-of-action. We recently established isothermal microcalorimetry as part of a routine profiling pipeline. This technology allows for investigating the effect of antibiotic exposure on the total bacterial metabolic response, including processes that are decoupled from biomass formation. Importantly, bacteriostatic and bactericidal effects are easily distinguishable without any user intervention during the measurements. However, isothermal microcalorimetry is a rather new approach and applying this method to different bacterial species usually requires pre-evaluation of suitable measurement conditions. There are some reference thermograms available of certain bacteria, greatly facilitating interpretation of results. As the pool of reference data is steadily growing, we expect the methodology to have increasing impact in the future and expect it to allow for in-depth fingerprint analyses enabling the differentiation of antibiotic classes.

The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe interactions.

이더말 미세칼로리메트리를 사용하여 새로운 천연 제품의 박테리시달 또는 박테리오정성 효과를 결정하는 대사 프로파일링

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as ...

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and reagents. Moreover, microplate-based high-throughput cultivation methods developed in recent years have poor microbial growth status and experiment parallelization because of their low dissolved oxygen, poor mixture, and severe evaporation and thermal effect. Due to many advantages of micro-droplets, such as small volume, high throughput, and strong controllability, the droplet-based microfluidic technology can overcome these problems, which has been used in many kinds of research of high-throughput microbial cultivation, screening, and evolution. However, most prior studies remain&#160;at the stage of laboratory construction and application. Some key issues, such as high operational requirements, high construction difficulty, and lack of automated integration technology, restrict the wide application of droplet microfluidic technology in microbial research. Here, an automated Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology, achieving the integration of functions such as inoculation, cultivation, online monitoring, sub-cultivation, sorting, and sampling required by the process of microbial droplet cultivation. In this protocol, wild-type Escherichia coli (E. coli) MG1655 and a methanol-essential E. coli strain (MeSV2.2) were taken as examples to introduce how to use the MMC to conduct automated and relatively high-throughput microbial cultivation and adaptive evolution in detail. This method is easy to operate, consumes less labor and reagents, and has high experimental throughput and good data parallelity, which has great advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and result-reliable experimental platform for scientific researchers to conduct related microbial research.

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System MMC

This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.

여러 영양소 소스의 미생물 대사를 조사 하기 위해 동위 원소 추적 실험의 조합에 따라 워크플로

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의 순환 압력 생물 반응기 설계 예 VIVO 학습