이 작품은 젊은 학자 (No. 81300810), 상하이 젊은 의사 훈련 프로그램 (No. 20141057), 및 자연 과학 재단의 중국 국가 (81671982, 81271791 및 81571955)에 대 한 자연 과학 재단의 중국 국가 의해 투자 되었다. 우리는이 원고 준비 기간 동안 언어 지원을 제공 하기 위한 LetPub (www.letpub.com)를 감사 하 고 싶습니다.

1. Biofilm 형성
<ol><li>
96-잘 microplates에 Biofilm 형성
	<ol>
<li>S. 구 균 긴장 USA300 및 3 biofilm 형성 임상 긴장 (C1-C3)-80 ° c.에 저장 된 검색 참고: 양식에 biofilms 결정 플레이트 분석 결과 의해 결정 되었다 임상 긴장의 능력15이전 설명.</li>
		<li>5 mL tryptone 콩 국물 (TSB) 매체에 박테리아를 접종 하 고 하룻밤 고정 단계를 37 ° C에서 흔들어와 인큐베이터에서 배양.</li>
		<li>원심 4000 x g 25 ° C에서 10 분에서 하룻밤 세균성 문화 하 고는 상쾌한 다음 삭제. 알 약 2.0 x 10의 최종 농도에 버퍼링 하는 인산 염 (PBS)에 resuspend9 CFU/mL. 참고:는 박테리아의 농도 광학 밀도 측정 하 여 예상 되었고 더 판에 의해 결정 수16을 포함 된 서 스 펜 션의 그 1 OD600 공개 1.5 x 108 CFU/mL.</li>
		<li>1: 200 세균 현 탁 액 희석 (1.0 x 107 CFU/mL)에 TSB 매체 0.5% 포도 당을 포함 하. 셀 문화 취급 하는 폴리스 티 렌 96 잘 microplate의 각 음에 세균 현 탁 액의 200 µ L을 접종.</li>
		<li>잘 산소 환경에서 24 h 동안 37 ° C에서 정적 microplate 품 어. 참고: 성숙한 biofilm 형성을 위한 보육 시간 따라 달라질 수 있습니다 다른 세균성 긴장; 이 PDT15실험 하기 전에 결정 되어야 합니다.</li>
		<li>우물에서 미디어를 삭제 하 고 PBS와 부드럽게 microplate 우물 세 번 세척 하 고는 상쾌한 다음 삭제. 참고: 단계 실행 되어야 한다 밖으로 매우 부드럽게 형성된 된 biofilm을 방해 하지 않으려면.</li>
	</ol>
</li>
	<li>
요리에 Biofilm 형성
	<ol>
<li>TSB 매체의 5 mL로 S. 구 균 긴장 USA300를 inoculate 하 고 고정 단계로 하룻밤 37 ° C에서 흔들어와 인큐베이터에서 배양.</li>
		<li>원심 4000 x g 25 ° C에서 10 분에서 하룻밤 세균성 문화 하 고는 상쾌한 다음 삭제. 2.0 x 10의 최종 농도에 PBS에 산 탄을 resuspend9 CFU/mL. 다음, 1: 200 세균 현 탁 액 희석 (1.0 x 107 CFU/mL)에 TSB 매체 0.5% 포도 당을 포함 하. 35 m m 광학 품질 유리 아래 셀 문화 접시에, 세균 현 탁 액 2 mL를 접종 하 고 정적으로 24 시간 동안 37 ° C에서 품 어. 참고:는 박테리아의 농도 광학 밀도 측정 하 여 추정 되었다.</li>
		<li>피 펫와 함께 미디어를 발음 하 고 린스는 biofilms에 부드럽게 PBS 가진 접시 3 번 후, 신중 하 게는 상쾌한을 삭제. 참고: 접시의 바닥에 피 펫 팁을 만지지 마십시오. 2.2 단계 형성된 된 biofilm의 건조를 방지 하기 위해이 단계 후에 즉시 수행 되어야 한다.</li>
	</ol>
</li>
</ol>2. 가벼운 방사선 조사
<ol><li>4 ° C 냉장고에 5-ALA를 저장 합니다. 실험을 하기 전에 10 m m PBS 가진 5-ALA 희석. 참고: 5-ALA 솔루션 준비 되어야 한다 갓 실험 전에.</li>
	<li>실험적인 그룹에 미 판 또는 문화 접시 2 mL의 각 음에 10mm ALA의 200 µ L를 추가 합니다. 커버 플레이트/접시 알루미늄 호 일, 그리고 1 시간에 품 어. 다음, 1 h 633 ± 10 nm17의 주요 파장에서 360 J/c m2 의 가벼운 복용량을 달성 하기 위한 100 mW/cm2 의 빛 강도 발광 다이오드 (LED)와 플레이트/접시를 비추는. 참고: 웰 스/요리의 모든 biofilm에 동등 하 게 효과적으로 배달할 빛 에너지를 하자, 6.0 cm에서 잘/요리 하는 광원의 절정에서 거리를 해결 하 고 중앙 조사 면적 (10 cm x 8을 실험 지역 제한 cm). 하려면 결과 재현성 실험 같은 실내 온도에서 수행 되어야 한다. LED 조사 단계에서 접시의 햇빛, 실내 조명 등 등불, 다른 광원에 직접 노출을 피하기 위해 LED는 방사선 조사 영역에 접시/접시를 이동 하기 전에 돌아서 빛 충분히 밝은 작업을 마무리 했다.</li>
	<li>제어 그룹 (3 개의 제어 그룹 설치 되었다 우리의 실험)을 설정 합니다.<ol>
<li>첫 번째 컨트롤에 대 한 그룹 (-LED-ALA)는 미 판 또는 문화 접시 2 mL 각 우물에 PBS의 200 µ L를 추가 합니다. 커버 플레이트/접시 알루미늄 호 일, 그리고 2 시간을 위해 그것을 품 어.</li>
		<li>두 번째 컨트롤 그룹에 대 한 (ALA + LED-), 각 잘은 미 판 또는 문화 접시 2 mL에 10 mM ALA의 200 µ L를 추가. 커버 플레이트/접시 알루미늄 호 일, 그리고 2 시간을 위해 그것을 품 어.</li>
		<li>세 번째 컨트롤 그룹에 대 한 (ALA-LED +), 각 잘은 미 판 또는 문화 접시 2 mL PBS의 200 µ L 추가. 커버 플레이트/접시 알루미늄 호 일, 그리고 1 시간에 품 어. 다음 접시 360 J/c m2 빛 주요 파장에서의 방사선 633 ± 10 nm17와 LED 노출 합니다. 참고: LED 조사 단계에서 접시의 햇빛, 실내 조명, 등불 등 다른 광원에 직접 노출을 하지 마십시오.</li>
	</ol>
</li>
</ol>3. PDT 치료의 효과의 결정
참고: S. 구 균 biofilms에 PDT ALA의 효과 확인 하려면 또는 PDT ALA 없이 세포의 생존 생존 얼룩 뿐만 아니라 CFU 계산 하 여 평가 되었습니다.
<ol><li>
나머지 실행 가능한 박테리아 세포의 결정
	<ol>
<li>PDT ALA 치료 후 우물에서 미디어를 삭제 하 고 모두 실험에 대 한 모든 비 부착 한 세포를 제거 하 여 그룹 제어 PBS와 웰 스 세 번을 씻어. 참고:이 단계가 수행 되어야 합니다 밖으로 아주 부드럽게.</li>
		<li>피 펫 팁 우물에서 부착 박테리아 세포를 철저 하 게 긁 고 원뿔 튜브에 세포를 수집 합니다.</li>
		<li>4 ° C에서 10 분 동안 4000 x g에서 세균 현 탁 액을 원심 다음 삭제는 상쾌한.</li>
		<li>PBS에서 0.25 %pancreatin 효소의 1 mL에 박테리아 resuspend와 1.5 h 37 ° C에서 품 어.</li>
		<li>10 분;에 대 한 4000 x g에서 원심 상쾌한, 삭제 후 PBS의 200 µ L에 펠 릿을 resuspend.</li>
		<li>확인 1시 10분; PBS와 셀 솔루션의 직렬 희석 그런 다음, 5 µ L tryptone 콩 agar (TSA) 접시에 각 직렬 희석 샘플의 추가. 16 h; 37 ° C에서 TSA 격판덮개를 품 어 그런 다음, 수를 셀 (맨 눈)에 의해 세균성 식민지 (CFU/mL)의.</li>
	</ol>
</li>
	<li>
CLSM으로 S. 구 균 biofilms의 관찰
	<ol>
<li>가벼운 방사선 조사 후 PBS로 문화 접시에 있는 biofilms 세 번 씻어. 참고:이 단계가 수행 되어야 합니다 밖으로 아주 부드럽게.</li>
		<li>1 µ M 녹색 형광 핵은 prokaryotic 세포 막 (예, SYTO9) 하 고 1 µ M propidium 요오드 화물 (PI) 죽은 세포 뿐 아니라 biofilm 얼룩을 20 분의 1 mL 침투성 이다 염색체 얼룩의 1 mL를 추가 합니다.</li>
		<li>관찰 가능한 셀 (녹색 형광, 전 / Em 485 nm/530 nm) 및 죽은 세포 (빨간 형광, 전 / Em 485 nm/630nm)는 63 X 1.4 나 기름 침수 렌즈와 CLSM 아래.</li>
		<li>현미경 소프트웨어를 사용 하 여 이미지를 생성 합니다.</li>
	</ol>
</li>
</ol>

<ol>
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저자 공개할 게 없다

초 암 치료에 대 한 잘 공부 치료 이후 100 여 년 전 발명 했다18. 지난 10 년간 초 항균 전략으로 적용 하 고12일부 항생제 내성 병원 성 박테리아 대 한 효과 보이고 있다. Planktonic 상태에 비해 박테리아 biofilms biofilms에 PDT ALA의 효과 없습니다 되어 완전히 아직 조사 하는 동안 항생제 치료3, 더 강한 것 처럼.
이 문서에서는, <em...

S. 구 균 은 피부와 점 막의 인간의 호스트 colonizes는 중요 한 그람 양성 병원 체 이다. 양식 biofilms 능력 그것의 병 인1의 중요 한 측면으로 간주 됩니다. 박테리아 biofilms는 박테리아 세포 외 고분자 물질, 다 당 류, 단백질, DNA 등의 구성 되는 자체 제작된 매트릭스에 포함 된의 커뮤니티입니다. 이 행렬은 인간의 면역 체계와 현재 항균 요법2의 높은 수준에 기여 하는 세균성 감염의 지 속성에 중요 한 역할을 한다. 항생제는 여전히 biofilm 감염에 대 한 주요 치료 biofilms에 항생제의 효과 제한. 그것은 표시 되었습니다 이전 biofilms에 셀 10-1000 배 더 그들의 planktonic 대응3에 비해 항생제에 저항력이 있다. 따라서,이 문제를 정복 하기 위해 대체 전략 필요 합니다.
PDT, 세균 감염에 대 한 대안 치료는 적절 한 파장의 빛을 사용 하 여 photosensitizers를 활성화. 세포 벽을 방해, 효소, 비활성화 및 손상 DNA4대상 세포에 치명적인는 반응성 산소 종 (선생님)의 생산을이 끈다. 이 다중 대상 특성 PDT 치료에 저항을 개발 하는 박테리아에 대 한 어려운 있습니다.
이전 보고서5,6,에서에서 공부 하고있다 톨루이 블루 등 여러 photosensitizers와 세균과 곰 팡이 biofilms, 말라 카이트 그린, 메 틸 렌 블루, 염소 e6 및 porphyrins, PDT의 항균 효과 7,,89,10,11,,1213. 5-ALA, 실제 photosensitizer PpIX, prodrug 작은 분자량 및 신속한 허가12,14에 의해 특징입니다. 이러한 장점은 치료 응용 프로그램으로 PDT ALA 주요 잠재력을 제공합니다. Planktonic 박테리아에 PDT ALA의 효과 많은 그룹12공부 하고있다, 비록 박테리아 biofilms에 PDT ALA의 항균 효과 하지 아직 고 해명 했습니다. 한편, 이전 연구 사이 결과 비교 하기가 어렵습니다. 이유 중 하나는 다양 한 그룹에서 다른 프로토콜을 사용 하는. 따라서,이 프로토콜 이전 작업15에 따라 PDT ALA 시스템의 생체 외에서 모델을 설명 합니다. 이 모델의 효과 CFU 계산 및 생존 CLSM과 얼룩에 의해 확인 되었다.

<table>
	<tbody>
		<tr>
			<td>Tryptone Soya Broth (TSB)</td>
			<td>OXOID</td>
			<td>CM0129B</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone Soya Agar (TSA)</td>
			<td>OXOID</td>
			<td>CM0131</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTO9</td>
			<td>Thermo Fisher Scientific</td>
			<td>L7012</td>
			<td>The LIVE/DEAD BacLight Bacterial Viability Kits&#160;</td>
		</tr>
		<tr>
			<td>Propidium iodide (PI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>L7012</td>
			<td>The LIVE/DEAD BacLight Bacterial Viability Kits&#160;</td>
		</tr>
		<tr>
			<td>Pancreatin</td>
			<td>Sigma-Aldrich</td>
			<td>P3292</td>
			<td></td>
		</tr>
		<tr>
			<td>5-aminolevulinic acid (ALA)</td>
			<td>Fudan Zhangjiang Bio-Pharm</td>
			<td>3.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus strain USA300</td>
			<td>/</td>
			<td>/</td>
			<td>The source of USA 300 references &#8220;Tenover FC, Goering RV. J Antimicrob Chemother. 2009 Sep; 64(3):441-6&#8221;.</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus clinical strains (C1-C3)</td>
			<td>/</td>
			<td>/</td>
			<td>All clinical strains were isolated from patients with chronic rhinosinusitis in the Department of Otorhinolaryngology-Head and Neck Surgery, Eye and ENT Hospital of Fudan University [Zhang QZ, Zhao KQ, Wu Y, et al. PLoS One. 2017 Mar; 12(3): e0174627].</td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>Corning Inc</td>
			<td>3599</td>
			<td>Clear Flat Bottom Polystyrene TC-Treated Microplates, Individually Wrapped, with Lid, Sterile</td>
		</tr>
		<tr>
			<td>Fluorodish</td>
			<td>NEST Biotechnology</td>
			<td>801001</td>
			<td>Glass bottom, Non-pyrogenic</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microcentrifuge 5417</td>
			<td>Eppendorf</td>
			<td>Z365998 |&#160;SIGMA</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>SHKE4000</td>
			<td>MaxQ 4000 Benchtop Orbital Shakers</td>
		</tr>
		<tr>
			<td>Light emitting diode (LED)</td>
			<td>Wuhan Yage Optic and Electronic Technique CO</td>
			<td>LED-IB</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP8 confocal laser-scanning microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica LAS AF software</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMARIS software</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro model to study the effect of 5-aminolevulinic acid-mediated photodynamic therapy on staphylococcus aureus biofilm

황색 포도상구균 (S. 구 균)은 pyogenic 및 조직의 감염을 일으키는 원인이 되는 일반적인 인간 병원 체. S. 구 균 감염은 항생제-저항 하는 긴장의 출현 뿐만 아니라 양식 biofilms 능력 때문이 아니라 근절 어렵다. 최근, photodynamic 치료 (태평양 서머 타임) biofilm 감염을 제어 하기 위한 잠재적인 치료의 하나로 표시 되었습니다 했습니다. 그러나, 더 연구 해야 우리의 지식의 기본 메커니즘 뿐 아니라 박테리아 biofilms에 미치는 영향을 개선 합니다. 이 원고는 5-aminolevulinic 산 (5-ALA), 실제 photosensitizer protoporphyrin IX (PpIX)의 선구자 PDT의 에 체 외 모델을 설명합니다. 간단히, 성숙 미 균 biofilms ALA와 incubated 되었고 그 후 빛에 노출. 그 후, 미 균 biofilm에 PDT ALA의 항균 효과 단위 (CFUs)를 형성 하는 식민지를 계산 하 여 정량 되었고 생존 confocal 레이저 스캐닝 현미경 검사 법 (CLSM) 통해 얼룩이 지기 형광에 의해 시각. 대표 결과 미 균 biofilms에 PDT ALA의 강한 항균 효과 설명 했다. 이 프로토콜은 간단 하 고 PDT ALA와 S. 구 균 biofilms의 치료 연구를 생체 외에서 모델을 개발 하기 위해 사용할 수 있습니다. 미래에, 그것은 또한 최소한의 조정으로 다른 세균성 긴장에 대 한 다른 photosensitizers를 이용 하 여 초 연구에서 참조할 수 수 있습니다.

biofilms에 있는 박테리아의 생존 컨트롤에 비해 PDT ALA 치료 후 감소 했다 (ALA-LED-, ALA + LED-, 그리고 살라-LED +) USA300에 3 개의 임상 변종 (그림 1).
CFU에서 결과 분석 결과 및 관찰 하는 항균 S. 구 균 biofilm에는 제자리에서USA300 biofilms에 PDT ALA의 효과 생존 얼룩과 CLSM으로 시각화 했다. 실용적이 고 ?...

Watch this Scientific Journal Video about An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm at JoVE.com

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

이 원고는 황색 포도상구균 biofilm에 5-aminolevulinic 산-중재 photodynamic 치료 (PDT ALA)의 항균 효과 연구 하는 프로토콜을 설명 합니다. 미래에 PDT와 박테리아 biofilms의 치료 연구를 생체 외에서 모델을 개발 하는 것이 프로토콜을 사용할 수 있습니다.

황색 포도상구균 Biofilm에 5-Aminolevulinic 산-중재 Photodynamic 치료의 효과 연구 하는 체 외에서 모델

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

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An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

7.8K 조회수.  Fudan University. 이 원고는 황색 포도상구균 biofilm에 5-aminolevulinic 산-중재 photodynamic 치료 (PDT ALA)의 항균 효과 연구 하는 프로토콜을 설명 합니다. 미래에 PDT와 박테리아 biofilms의 치료 연구를 생체 외에서 모델을 개발 하는 것이 프로토콜을 사용할 수 있습니다.

비디오: An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

연구

생물학

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

크리스토퍼 휴즈 : Angiogenesis 연구를위한 체외 모델 (인터뷰)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

컴퓨터가 만든 동물 모델 자극

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

마우스의 전통 NASA Hindlimb 하역 모델 Alternant 방법

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

파장과 산화 스트레스에 레드 / 근적외선 치료의 강렬의 범위의 효과의 평가를위한 방법<em&gt; 체외</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

공부 하는 Kaposi 육 종 Herpesvirus에 의해 세포 변환에 대 한 생체 외에서 모델

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

아디포세포 기능에 대한 단백질 및 마이크로 RNA 변조의 영향을 연구하는 세포 배양 모델

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.

This paper details methods that demonstrate three common biofilm harvesting and disaggregation techniques on two surface types, ruggedness testing of a harvesting method and minimum information to consider when choosing and optimizing harvesting and disaggregation techniques to increase reproducibility.

수확 및 분해 : 생물막 방법 연구에서 간과 된 단계

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the ...