다음 프로토콜은 메릴랜드 대학 기관 동물 관리 및 사용 위원회로 기관 검토 위원회에서 허용 됩니다. 프로토콜에 따라 연방, 주 및 지역 표준, 그리고 Biosafety에 메릴랜드 대학 정책에 의해 밖으로 설정 하는 지침.
1입니다. 눈 샘플의 해 부
<ol><li>
로컬 도살 또는 조직 은행에서 눈 샘플을 가져옵니다. 전체 눈 글로브 얻은 경우은 즉시 렌즈, 연결 된 zonules 및 유리에 압축을 풉니다. 참고: 아래에서 설명 하는 구체적인 내용을 돼지와 인간의 눈에 관련이 있습니다.<ol>
<li>소독된 수술가 위, 집게를 사용 하 여 잘라내어는 sclera를 둘러싼 모든 과잉 조직을 제거.</li>
		<li>단단히의 측면에 눈을 보유 하 고, 눈 각 막에서 3 mm의 측면을 따라 작은 절 개를 만들 면도칼 블레이드를 사용 하 여. 눈 안의 유리 체에 도달 했습니다 컷된 깊은 충분히 확인 합니다.</li>
		<li>가 위를 사용 하 여 신중 하 게 눈의 둘레 주위 절 개를 따라 잘라. 렌즈를 puncturing 하지 마십시오. 대표 이미지 그림 2A에 표시 됩니다.</li>
		<li>일단 눈의 외부 둘레를 잘라 집게를 사용 하 여 눈의 후부 조직을 제거 합니다. 집게와 렌즈 zonules, 속눈썹, 몸과 연결 된 유리를 격리 합니다. 대표 이미지 그림 2B에 표시 됩니다.</li>
		<li>집게와가 위를 사용 하면, 그래서 렌즈는 MLS에 평평 하다 수 초과 유리 제거할. 참고: 각 막 이식의 경우, 각 막 버튼 사용 됩니다 수술에서 그리고 세계의 나머지는 연구 목적으로 사용할 수 있습니다. 그러나,이 부분 글로브 렌즈 들 것 설치 프로그램의 조직 준비에 여전히 사용할 수 있습니다. 뒷부분을 취득 하는 경우에 단계 1.1.4–1.1.5 수행할.</li>
	</ol>
</li>
	<li>30 분 동안 15%의 표 백제 솔루션에서 모든 장비를 사용된 후 해 부를 소독.</li>
</ol>2. 수동 렌즈 들 것의 재판 어셈블리
<ol><li>거기 남아 신발 들여쓰기의 뒤 벽과 신발 자체 사이의 5mm 간격 10mm 신발 바닥 및 해당 신발 상판 MLS의 바닥판에 삽입 합니다.</li>
	<li>위쪽 및 아래쪽 접시, 접시를 함께; 물리기 정렬 장치 스트레치 위치에 지금 있다입니다.</li>
	<li>플레이트 케이스와 아래 접시의 측면에 있는 구멍에 마 개 나사에 격판덮개를 삽입 합니다.</li>
	<li>접시 경우 베이스에 삽입 고 렌치 정렬된 들여쓰기 합니다.</li>
	<li>신발, 계약 하 고 다시 시계 반대 방향으로 스트레치 원위치에 신발을 반환할 중지 나사에 도달할 때까지 렌치를 시계 방향으로 트위스트.</li>
</ol>3. 렌즈의 장착
<ol><li>삽입 10 m m 신발 바닥 MLS에 바닥판에는 5mm 차이 남아 사이 신발 들여쓰기 및 신발 자체의 뒷면.</li>
	<li>곡선된 겸 자 사용 하 여, 장소 추출된 렌즈 얼굴 아래 접시의 중앙에 있도록 신발 중앙 구멍에 렌즈를 지원 하.</li>
	<li>신발 상단의 해당 클리핑만 zonules 및 유리 체에 스냅 합니다. 시각적으로 확인할 것 렌즈 중심으로 하단 플레이트에 가능한.</li>
	<li>2.3-2.4 단계를 반복 합니다.</li>
</ol>4입니다. 렌즈의 측정
<ol><li>기지개 과정의 사진과 비디오를 캡처하기 위해 바로 위에 장치 이미징 시스템을 배치. 후 처리에서 정확 하 게 크기와 스케일 이미지 그림의 프레임에 통치자를 포함 해야 합니다. 참고: 어떤 적합 한 이미징 시스템은이 단계;에 대 한 충분 한 여기 우리는 12 메가 픽셀, autofocusing 스마트폰 샘플에서 한 발을 사용합니다.</li>
	<li>단단히 아직 원활 하 게, 렌즈를 기지개 하는 시계 방향에 렌치를 회전 합니다. 그림 3 스트레치와 연장 된 상태에서 대표 이미지를 보여준다.</li>
	<li>뻗어 렌즈 촬영, 후 렌치 샘플 휴식 상태로 복원 하려면 시계 반대 방향에서 회전 합니다. 참고: 렌즈의 측정 렌즈의 탈수를 최소화 하기 위해 적시에 수행은 필수적 이다.</li>
	<li>명확 하 게 사진 렌즈의 최종 휴식 상태.</li>
</ol>5. 데이터 분석
<ol><li>ImageJ로 이미지를 업로드 하 고, "포인트" 기능을 사용 하 여 그림 4와 같이 렌즈의 둘레 40 포인트 이상 선택. 각 선택 된 포인트의 위치를 "분석" → "측정" 옵션을 사용 합니다.</li>
	<li>적합 (사용 하 여 소프트웨어 예: MATLAB) 위치 포인트 반경 및 카이 맞는 그림 4B의 순서. 픽셀 반경 및 오류 통계 촬영된 눈금자를 사용 하 여 변환.</li>
	<li>쌍을 이루는 두 꼬리 t-검정 MLS에서 스트레칭 전후 개별 렌즈 비교를 수행 합니다.</li>
</ol>

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AB은 Bioniko 컨설팅 LLC에 소유권 관심이 있다.

우리는 방법을 제공 하는 정확 하 고 효율적인 듀얼 조각 클램핑 메커니즘을 이용 하 여 렌즈의 숙박 능력을 공부 하는 샘플 들 것 새로운 방법을 고안 했다. 숙박 시설, 동안 렌즈 이완, 그리고 직경 감소 zonular 섬유1,2,,419의 이완에 대 한 응답. 메서드는 클램핑 및 zonular 섬유의 장력을 제어 하 여이 현...

숙박 시설에는 인간의 눈을 수 동적으로 날카로운 초점에서 멀리 또는 가까운 거리에서 개체를 참조 하는 크리스탈 렌즈의 모양을 조정 하는 프로세스입니다. 숙박 시설에는 본질적으로 biomechanical 과정이 있습니다. 신경 자극 시 속눈썹 근육 ciliary 시체와 zonule 섬유는 렌즈 캡슐1,2의 둘레에 연결 하는 힘을 생성 합니다. 숙박의 역학에 뒤에 여러 가지 이론이 있지만, 가장 널리 통용 되는 헬름홀츠 가설입니다. 가설에 따르면 렌즈에 해당 하는 먼 물체의 초점에 대 한 최적의 렌즈의 얇은 모양 자연 연장 된 상태에서입니다. Ciliary 근육 계약 가까이 개체에 포커스를 변경 하려면과 zonular 섬유 완화 됩니다. 앞쪽 및 후부 표면 곡률을 증가, 렌즈 복잡 합니다. 이 비전, 따라서, 짧은 초점 거리1근처에 필요한 시도 힘 증가에 해당 합니다.
능력에 맞게 노 라는 조건을 통해 시간이 지남에 따라 손상 됩니다. 에 영향을 미치는 모든 사람 나이 50에 의해, 노 안은 눈 거리3을 멀리에서 포커스를 동적으로 변경할 수 없습니다. 노 안 방지 하기 위해 현재 메서드는 수동 교정 렌즈와 촛점을 포함 하 여. 몇 가지 평면에 가까운 물체에 초점을 하나의 능력을 증가 하는 동안 이러한 수동 치료 렌즈4,5의 동적 초점 능력을 복원할 수 없습니다. 효율적으로 노 안을 치료 하거나 가능 하 게 그것을 방지 하는 것, 더 나은 숙박 시설을 이해 하는 지속적인 필요가 있다.
공부 하 고 렌즈 숙박, 장치 수 현상 비보 전4,6,7,,89를 시뮬레이션 하기 위해 개발 되었습니다. 회전 디스크 처음 원심력8통해 렌즈의 스트레칭 모니터를 소개 했다. 현상은으로 더 충실 하 게 복제 하기 위해 렌즈 스트레칭 장치 점차적으로 도입 되었고 혁신. 렌즈 들 것, Manns 외에 사용 하 여. 렌즈 전원 및 적도 직경9등을 상호 연결 하는 동안 렌즈를 수용 하는 데 필요한 힘이 특징. 현재 이해는 렌즈 자나 나이, 렌즈 모양 모양 바디3,10,,1112동등한 힘에 대 한 응답에서에서 감소 변경 결과입니다.
현재 렌즈 들 종종 전자 제품 및 프로그래밍 가능한 스트레칭 속도, 복잡 한 설치 프로그램을 포함 하며 전체 시신 안구6,7,10,13. 이 요구 사항은 실험 눈 당 $500.00 이상 당 비용을 증가 하 고 샘플 가용성을 감소. 여기 우리 후부 눈 합계 약 $200.00으로 저렴 한 비용으로 숙박 시설 렌즈를 복제 하는 방법을 제시. 동안 많은 장치 오늘, 보다 적게 정교한 기술은 이다 훨씬 더 비용 효과적이 고 adoptable 결과 없이. 이 메서드는 그림 1에 묘사 된 수동 렌즈 들 (MLS)를 중심 하 고 zonular 섬유 및 방사형 트위스트 방법에 독특한 클램핑 시스템을 사용 하 여 렌즈의 직경을 확장 하. 프로토콜의 생리 정확도 베 외의 결과 의해 유효성이 검사 됩니다., 누가 공부는 앞쪽에 및 사후 zonular 섬유 렌즈 캡슐14에 연결 하는 통로. 사용자 지정 신발만 렌즈, zonule, 및 ciliary 시체를 요구 하는 디자인을 사용 하 여, 우리 생리 숙박을 복제 하 여 렌즈 역학을 연구 목적.

<table border="0" cellpadding="0" cellspacing="0" style="width:856px;" width="856">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Manual Lens Stretcher</td>
			<td style="width:233px;">Bioniko</td>
			<td style="width:132px;">MLS</td>
			<td style="width:300px;">Different animal species will require different shoe sizes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Porcine Eye Samples</td>
			<td>George G. Ruppersberger; slaughterhouse</td>
			<td>N/A</td>
			<td>Whole eyeballs were obtained</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Eye Samples</td>
			<td>The National Disease Research Interchange</td>
			<td>N/A</td>
			<td>Posterior poles without corneas were ordered</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Scissors (5 1/2&#39;&#39; Straight)</td>
			<td>Electron Microsopy Sciences</td>
			<td>72960</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Forceps (4 1/2&#39;&#39;)</td>
			<td>Electron Microsopy Sciences</td>
			<td>72960</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iPhone 6s</td>
			<td>Apple</td>
			<td>N/A</td>
			<td>Any imaging system with ~0.1 mm resolution will work</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Hypochorite</td>
			<td>Clorox</td>
			<td>Clorox Regular-Bleach</td>
			<td>Any disinfectant will work</td>
		</tr>
	</tbody>
</table>

simulating the mechanics of lens accommodation via a manual lens stretcher

이 프로토콜의 목표 비용 효율적이 고 실용적인 방식으로 생리 숙박의 역학을 모방 하는 것입니다. 숙박 모양 체의 수축과 비전 근처에 필요한 렌즈의 두껍게 귀착되는 zonule 섬유의 이완을 통해 이루어집니다. 여기, 우리 소설, 숙박 긴장 풀어 zonules 수동 렌즈 들 것 (MLS)를 통해 렌즈 캡슐에 연결 하 여 복제 하는 간단한 방법을 제시. 이 메서드는 방사형 스트레칭 렌즈 때 일관 된 힘에 의해 달성 모니터링 하 고 렌즈, 비 수용 렌즈를 신축 할 수 없습니다를 기지개 할 수 있는 수용의 비교에 대 한 허용. 중요 한 것은, 들 것만 백업은 렌즈, zonules, 및 전체 세계 샘플 보다는 오히려 모양 몸, 눈의 공 막 아니라 직접 zonules에 커플. 이 차이가 크게 인수는 전체 세계에 비해 약 62%를 취득 하는 기증 시신 렌즈의 비용을 줄일 수 있습니다.

돼지 눈, 노 안 렌즈4,15, 스트레칭을 통해 공부에 대 한 일반적인 샘플 획득 했다 (n = 10) 로컬 도살이이 프로토콜에서 렌즈의 숙박 능력을 관찰 하기 위해 사용 되었다. 그림 5 MLS를 통해 스트레칭 전후 A 돼지 렌즈의 비교를 보여줍니다. 렌즈의 반경 기지개 하는 때에 평균 0.19 ± 0.07 m m 증가 (...

Watch this Scientific Journal Video about Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher at JoVE.com

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

수동 렌즈 들을 통해 렌즈 숙박의 역학을 시뮬레이션

우리는 수동 렌즈 들을 사용 하 여 렌즈 숙박을 공부 하는 효율적인 방법을 제시. 프로토콜 모방은 zonules를 당겨서 생리 숙박 연결 주위 렌즈 캡슐, 렌즈를 스트레칭 합니다.

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

simulating-mechanics-lens-accommodation-via-manual-lens

Research

JoVE Journal

Bioengineering

6.7K 조회수.  University of Maryland. 우리는 수동 렌즈 들을 사용 하 여 렌즈 숙박을 공부 하는 효율적인 방법을 제시. 프로토콜 모방은 zonules를 당겨서 생리 숙박 연결 주위 렌즈 캡슐, 렌즈를 스트레칭 합니다.

비디오: Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). We demonstrate in this video article how blast TBI-relevant impulsive pressurization is applied to the neuronal cells in vitro. This is achieved by using well-controlled pressure pulse created by a specialized Kolsky bar device, with complete pressure history within the cell pressurization chamber recorded. Pressurized neuronal cells are inspected immediately after pressurization, or further incubated to examine the long-term effects of impulsive pressurization on neurite/axonal outgrowth, neuronal gene expression, apoptosis, etc. We observed that impulsive pressurization at about 2 MPa induces distinct neurite loss relative to unpressurized cells. Our technique provides a novel method to investigate the molecular/cellular mechanisms of blast TBI, via impulsive pressurization of brain cells at well-controlled pressure magnitude and duration.

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate the molecular/cellular mechanisms of blast-induced traumatic brain injury.

외상성 뇌 손상 연구를위한 Neuronal 셀의 충동 가압

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). ...

연구

생체공학

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

조직 평가에 대한 자기 공명 Elastography 방법론은 구조 성장하는 기술

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

선박의 스텐 트를 배포하는 동안 벽 역학 모니터링

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

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 ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

조정 나노 기공을 통해 나노 입자 전좌 속도를 통해 제타 전위의 측정 : 예를 들어 DNA 변성 입자를 사용하여

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

조직 수준 역학을 제어하기 위해 준수 기판에 인간 배아 줄기 세포 식민지의 기하학적 패터닝

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

심근 경색의 뮤린 모델에서 3D 생체 인쇄 패치의 이식

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle contraction. To understand their force-generating mechanisms, myosin II has been investigated both at the single-molecule (SM) level and as teams of motors in vitro using biophysical methods such as optical trapping.
These studies showed that myosin force-generating behavior can differ greatly when moving from the single-molecule level in a three-bead arrangement to groups of motors working together on a rigid bead or coverslip surface in a gliding arrangement. However, these assay constructions do not permit evaluating the group dynamics of myosin within viscoelastic structural hierarchy as they would within a cell. We have developed a method using optical tweezers to investigate the mechanics of force generation by myosin ensembles interacting with multiple actin filaments.
These actomyosin bundles facilitate investigation in a hierarchical and compliant environment that captures motor communication and ensemble force output. The customizable nature of the assay allows for altering experimental conditions to understand how modifications to the myosin ensemble, actin filament bundle, or the surrounding environment result in differing force outputs.

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and discussed.

광학 핀셋을 사용하여 액틴 필라멘트 번들에서 미오신 앙상블 역학 조사

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle ...

Chemical Triphosphorylation of Oligonucleotides

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5&#8242;-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible.
This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

Oligonucleotide 5&#8242;-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5&#8242;-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

올리고뉴클레오티드의 화학적 트리포스포릴화

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of ...

Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

At present, cerebral organoid culture technology is still complicated to operate and difficult to apply on a large scale. It is necessary to find a simple and practical solution. Therefore, a more feasible cerebral organoid protocol is proposed in the present study. To solve the unavoidable inconvenience in medium change and organoid transfer in the early stage, the current research optimizes the operation technology by applying the engineering principle. A soft light lamp was adopted to laterally illuminate the embryoid body (EB) samples, allowing the EBs to be seen by the naked eye through the enhanced diffuse reflection effect. Using the principle of secondary flow generated by rotation, the organoids gather toward the center of the well, which facilitates the operation of medium change or organoid transfer. Compared to the dispersed cell, the embryoid body settles faster in the pipette. Using this phenomenon, most of the free cells and dead cell fragments can be effectively removed in a simple way, preventing EBs from incurring damage from centrifugation. This study facilitates the operation of cerebral organoid culture and helps to promote the application of brain organoids.

Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

Cerebral organoids provide unprecedented opportunities for studying organ development and human disease pathology. Although great success has been achieved with cerebral organoid culture systems, there are still operational difficulties in applying this technology. The present protocol describes a cerebral organoid procedure that facilitates medium change and organoid transfer.

측면 소프트 라이트 조명을 통한 대뇌 오가노이드 문화 촉진

At present, cerebral organoid culture technology is still complicated to operate and difficult to apply on a large scale. It is necessary to find a simple ...