Duennwald 연구소의 작품은 노화 연구 (멀리), 유전성 질병 재단 (HDF)와 윌리엄 우드 재단에 대한 미국 연방의 보조금에 의해 지원됩니다.

1. 효모의 독성 PolyQ - 확장 단백질의 표현 체계적인 분석은 효모 7 독성을 생산하는 데 필요한 polyQ - 확장 단백질의 정확한 아미노산 서열을 확립했습니다. 이 독성 polyQ - 확장 단백질 그림을 참조 huntingtin 단백질, polyQ 지역 및 카르복시 - 터미널 융합의 원래 순서에서 형광 단백질 (GFP 또는 CFP 중 17 아미노산 뒤에 아미노 터미널 깃발 태그가 있습니다 1). inducible와 상대 강한 GAL1 발기인 7, 9의 통제하에 표현하면 46 glutamines 이상의 polyQ 확장과 단백질의 표정은 (72과 103 glutamines를 예) 효모의 독성을 생산하고 있습니다. 이전에 설명한 바와 같이, 효모의 polyQ의 독성는 prion의 형태로 단백질 Rnq1p 가지고 세포에만 없었던 이유는, [RNQ +], 예를 들어 효모 변형 W303 9. 독성 polyQ - 확장 단백질 R이러한 polyQ 길이 종속적인 독성 (아래 참조)과 집계 (그림 1 B)와 같은 효모에 polyQ - 생물학 ecapitulates 중앙 측면. 특히, 독성​​ polyQ - 확장 단백질은 즉시 효모 세포를 죽이지 않는, 그들은 오히려 손상 또는 세포주기와 세포 devision을 체포하고 따라서 번호판 또는 액체의 문화 (우리의 게시되지 않은 데이터)에 효모 콜로니의 성장을 둔화 또는 억제. 2. 효모 세포가있는 잠재적인 문제는 독성 PolyQ - 확장 단백질을 표현 달리 독성 polyQ 확장 단백질을 표현 효모 세포는 성장 결함 9 표시되지 않을 수 있습니다. polyQ - 독성 이러한 진압의 유전적 성격은 단순 Mendelian 변이에 따라 결정될 나타나지 않습니다하기 때문에 상대적으로 높은 빈도 (우리의 미발표 결과)와 발생할 수 있습니다. 우리는이 자연 진압이 비슷하게를 [RNQ +] polyQ toxicity.Thes을 결정하는데 작동 미확인 prions의 경화에 의해 발생되는 추측polyQ의 독성의 전자 자발적인 진압 장치가 polyQ의 독성을 특성화하거나 식별하고 polyQ의 독성의 수정자를 특성화하는 것을 목표로하는 실험의 성공적인 결과를 위태롭게 만들 수 있습니다. 이러한 자발적인 진압의 빈번한 발생을 방지하기 위해, 매우 효과적일 수있다 아래에 설명된 예방 조치를 따르십시오 <ol><li> 독성 실험에 신선한 효모 세포를 사용합니다. 장시간 동안 누룩을 보관하지 마십시오. 자주 냉동 주식 신선한 효모 식민지를 검색합니다. </li><li> 자주 형광 현미경에 의한 표현과 독성 polyQ - 확장 단백질의 집합을 모니터링합니다. </li><li> 어떠한 독성 측정을 시작하기 전에 항상 독성 polyQ의 독성 (단독 탄소 소스로 포도당을 포함하는 선택적 미디어 IE)의 표현을 주체할 매체 효모 세포 보관. </li><li> 각 polyQ - 독성 실험을 위해 적어도 세 독립 transformants을 사용합니다. &lt;/ 리&gt; </ol> 3. 성장 Assays <ol><li> 각 실험 조건의 경우 단독 탄소 소스로 포도당과 선택적 효모 미디어 3 ML의 독성 polyQ - 확장 단백질을 품고 효모 세포의 세 독립 transformants에서 각각 식민지의 예방. </li><li> 30에서 하룻밤 이러한 문화를 품어 ° C. 매체에서 포도당 그들의 표현을 represses 때문에 이러한 조건에서 세포 polyQ - 확장 단백질을 표현하지 않습니다. 이러한 문화의 자라다 못하게 해, 1 아래의 하룻밤 문화 OD 600 (600 nm의 파장의 빛을 흡수도) 보관. </li><li> 우리는 일상적으로 독성 polyQ - 확장 단백질을 표현 효모 세포의 성장 결함을 모니터링하는 세 가지 다른 assays를 사용 </li></ol> 접시에 3.1 성장 <ol><li> 나를 선택에 0.0005의 OD 600 (예 : OD600 = 0.5 문화 1:1000 희석)에 하룻밤 효모 문화를 (220 RPM의 진동과 함께 재배) 희석디아 7 함유 포도당. </li><li> 균등 선택적 유일한 탄소 소스로 포도당을 포함하는 매체와 단독 탄소 소스로 갈락토 오스를 포함하는 선택적 매체 한 접시에 하나의 플레이트 (10cm 직경)에 각 희석 문화의 50 μl를 (CA 결과 판 당. 700 식민지) 확산. </li><li> 30 ° C.에 3~4일에 대한 번호판을 품어 부화 후, 각각의 접시의 사진을 가지고 갈락토 오스 접시에 포도당과 식민지의 수에 콜로니의 수를 계산합니다. 높은 독성 polyQ - 확장 단백질 (103Q, 그림 2A) 표현 효모 세포를 사용할 때 이상적인 조건에서, 갈락토 오스 접시에 아무런이나에만 거의 식민지가 없습니다. </li></ol> 3.2 구경 assays 이 검정은 도금 분석은 위에서 설명한보다 양적이고 따라서 같은 접시 같은 실험과 polyQ 독성이 훨씬 미묘한 차이를 보여줄 수 있습니다. <ol><li> overn를 희석항공 문화 0.1의 OD 600 ~ 포도당과 매체에 재배. </li><li> 피펫 200 멸균 96 - 웰 플레이트에 이러한 희석 문화 μl와 멀티 채널 피펫을 사용하여 멸균 물 다섯개의 요소가있는 시리얼 dilutions를 준비합니다. </li><li> frogger을 (또한 세포 현탁액의 전달 8 X 6 핀으로 망보고라고도 함)에 사용하는 것은 전적으로 탄소 소스와 단독 탄소 소스로 갈락토 오스와 선택적 미디어를 포함하는 접시와 같은 포도당과 선택적 매체가 들어있는 접시에 세포 현탁액을 전송. </li><li> 접시 30에서 그들을 잠복기 ° C를 3~4일 동안 전에 건조하도록 허용합니다. </li><li> 부화 후, 각각의 접시의 사진 (그림 2B)을 가져가라. </li></ol> 3.3. 액체 배양의 성장에 의해 효모의 polyQ의 독성 모니터링 이 프로토콜은 여기에서 설명한 세 assays의 (OD600 숫자)가 가장 높은 양적이며 심지어 polyQ의 독성에 매우 미묘한 차이를 감지할 수 있습니다. 상기 OC자발적인 진압의 currence 그러나 잠재적으로 오해의 소지가있는 결과를 얻을 수 있습니다. 나는 그러므로 위에서 설명한 두 가지 도금 assays 중 하나 이상으로이 분석을 결합하는 것이 좋습니다. 우리는이 실험을 위해 BioscreenC 악기를 사용하도록 제안합니다. BioscreenC은 정의된 교반과 정의 온도에서 incubated 동안 자동으로 100도 접시에 효모 문화의 광학 밀도를 측정하는 계기이다. 효모 세포의 성장과 그 광학 밀도를 측정하는 다른 방법도 적용될 수 있습니다. <ol><li> 3 ML 멸균 물에 단독 탄소 소스로 세 번 포도당을 포함하는 최소한의 미디어에서 효모 세포를 씻으십시오. </li><li> 0.1의 OD 600 ~ 갈락토 오스와 매체 재배 야간 문화를 희석. </li><li> 효모 배양 300 μl로 100 잘 BioscreenC 판의 각 우물을 채웁니다. </li><li> 쉬운 Bioscreen 실험 프로그램을 엽니다. 당신이 (공백 중간만을 포함한 모니터링하고자하는 샘플의 수를 결정제어), 30 ° C로 온도를 설정할 3 일이 실험의 길이를 설정, 15 분 측정 간격을 설정, 600 nm의 필터 / 브라운 설정하고에서 각 측정 전에 15초에 흔들림 모드를 설정 중간 강도. </li><li> BioscreenC 악기 및 부속 소프트웨어는 실험 도중 찍은 각 데이터 포인트의 Excel 스프레드 시트를 생성합니다. </li><li> 각 샘플에 대해 엑셀과 함께 성장 곡선을 준비하고 다른 샘플 (그림 2C)의 성장을 비교합니다. BioscreenC 실험에 의해 생산되는 데이터의 분석에 대한 자세한 설명은 10시 이전까지 제공되었습니다. </li></ol> 4. 대표 결과 <img alt="그림 1" src="/files/ftp_upload/3461/3461fig1.jpg" /> 1 그림. 효모 polyQ 모델입니다. 짧은, 무독성 polyQ을 표현 효모 세포를 보여주는 독성 polyQ - 확장 단백질. B의) 도식 표현) 형광 현미경확장 단백질 (25Q, 왼쪽 패널)와 효모 세포는 긴 독성 polyQ - 확장 단백질 (103Q, 오른쪽 패널) 표현. <img alt="그림 2" src="/files/ftp_upload/3461/3461fig2.jpg" /> 그림 2. polyQ - 확장 단백질을 표현 효모 세포의 성장 assays 대표 결과입니다. ) 도금 분석. 약 700 효모 세포는 30 ° C.에 사흘 동안 접시에 펴고 incubated되었습니다 위쪽 패널은 포도당 매체를 포함하는 접시를 보여주고, 독성 polyQ - 확장 단백질 (103Q)의 표현 즉 것은 유도하지 않습니다. 하단 패널은 갈락토 오스 매체를 포함하는 효모 플레이트를 보여줍니다, 독성 polyQ - 확장 단백질의 표현 즉 것은 유도하고 있습니다. 실험로 표시되어 있습니다, 아무 자발적인 억제가 발생했습니다 없습니다. 나) 분석을 구경하기. 효모 세포의 다섯 직렬 5 배는 dilutions도 무독성 polyQ 단백질 (25Q) 또는 독성 polyQ - 확장 protei를 숨겨주N (103Q)은 단백질 (왼쪽 패널) 또는 그들의 표현을 유도 갈락토 오스 접시에있는 표현을 주체할 수 포도당 플레이트에서 목격됐다. 접시 그러면 ° C. C) BioscreenC의 실험. 중 무독성 polyQ 단백질 (25Q) 또는 독성 polyQ - 확장 단백질 (103Q)를 표현하는 효모 세포의 문화의 성장에 의해 감시되었다 30에서 사흘 동안 incubated되었습니다 BioscreenC 악기. 실험 조건 및 BioscreenC 실험 분석은 메인 텍스트에 설명되어 있습니다.

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chaperone+pathway+in+protein+disaggregation.+Hsp26+alters+the+nature+of+protein+aggregates+to+facilitate+reactivation+by+Hsp104.">A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104.</a> J. Biol. Chem. 280, 23869-2375 (2005).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

본 원고는 독성 polyQ - 확장 단백질을 표현 효모 세포의 감소 성장을 바탕으로 모델 유기체 효모에 polyQ - 독성을 측정하는 세 보완적인 실험 방법을 설명합니다. 효모의 작업 polyQ - 확장 단백질 9,11,12의 misfolding과 독성을 포함한 단백질 misfolding의 기본 세포와 분자 메커니즘과 그 다음의 독성,로 심오한 통찰력을 제공하고있다. 여기에 제시된 프로토콜을 기반으로 실험은 이미 polyQ 독성 <sup...

<table border="1"><tbody><tr><td> 악기의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> Frogger (6x8 핀) </td><td> V &amp; P 과학, 샌디에고 </td><td> VP 407 AH </td></tr><tr><td> BioscreenC </td><td> Growthcurves 미국 </td><td> 5,101,370 </td></tr><tr><td> 100 잘 허니컴 접시 </td><td> Growthcurves 미국 </td><td> 9,602,550 </td></tr></tbody></table>

growth assays to assess polyglutamine toxicity in yeast

단백질 misfolding 같은 알츠하이머 질환, 파킨슨병과 헌팅턴의 질병과 한 많은 인간의 질병, 특히 neurodegenerative 질병과 연관됩니다. 헌팅턴의 질병 (HD)은 단백질 huntingtin 이내 polyglutamine (polyQ) 지역의 비정상적인 확대로 인해 발생합니다. polyQ - 확장된 huntingtin 단백질이 비정상적 형태 (즉, 그것이 misfolds)을 attains 및 세포 독성에게 2를 일으 킵니다. 적어도 8 이상 neurodegenerative 질병은 Spinocerebellar Ataxias과 케네디의 질병 3를 포함해 polyQ - 확장에 의해 발생합니다. 모델 생물 효모는 polyQ - 확장을 표현 polyQ - 독성의 내부와 남북 분자 요인의 영향, 그리고 세포에 손상되는 세포 경로의 식별을 포함하여 polyQ - 독성의 세포 및 분자 기초에 중대한 통찰력을 촉진하고있다 단백질 3-8. 중요단조는 효모에서 발견된 polyQ - 독성의 여러 측면 따라서 polyQ - 독성 underpinning 기본적인 메커니즘의 발견을위한 효모 모델의 중요성을 보여주는 다른 실험적인 시스템에서와 HD 환자에서 샘플에 어느 정도까지 재현했다. 효모에 polyQ - 독성을 결정하는 직접적이고 비교적 간단한 방법 polyQ - 확장 단백질을 표현 효모 세포의 성장 결함을 측정하는 것입니다. 본 원고는 polyQ - 확장 단백질을 표현 효모 세포의 성장을 측정하여 효모에 polyQ - 독성을 결정하는 세 보완 실험적인 접근 방법을 설명합니다. 처음 두 실험 방법은 접시에 효모의 성장을 모니터링, 세 번째 접근법은 BioscreenC 악기를 사용하여 액체 효모 문화의 성장을 모니터링합니다. 또한이 원고는 효모 polyQ 모델을 다룰 때 발생할 수있는 실험적인 어려움을 설명하고 또는 피하기 위해 도움이 될 전략을 보여줍니다이러한 어려움을 최소화합니다. 여기에 설명된 프로토콜 식별 및 유전 경로 및 polyQ - 독성을 조절 작은 분자를 특성화하는 데 사용할 수 있습니다. 또한, 설명 assays는 효모 모델의 다른 질병 관련 misfolded 단백질에 의한 독성의 정확한 분석을위한 템플릿으로 사용될 수 있습니다.

Watch this Scientific Journal Video about Growth Assays to Assess Polyglutamine Toxicity in Yeast at JoVE.com

Growth Assays to Assess Polyglutamine Toxicity in Yeast

본 원고는 효모에 polyglutamine의 독성 (polyQ) - 확장 단백질을 평가 삼 보완적인 프로토콜을 설명합니다<em&gt; Saccharomyces cerevisiae</em&gt;. 이러한 프로토콜은 쉽게 효모의 다른 misfolded 단백질의 독성을 모니터링하기 위해 수정할 수 있습니다.

성장 Assays은 효모에서 Polyglutamine 독성을 평가하기 위해

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

growth-assays-to-assess-polyglutamine-toxicity-in-yeast

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Biology

13.5K 조회수.  Boston Biomedical Research Institute. 본 원고는 효모에 polyglutamine의 독성 (polyQ) - 확장 단백질을 평가 삼 보완적인 프로토콜을 설명합니다 Saccharomyces cerevisiae. 이러한 프로토콜은 쉽게 효모의 다른 misfolded 단백질의 독성을 모니터링하기 위해 수정할 수 있습니다.

비디오: Growth Assays to Assess Polyglutamine Toxicity in Yeast

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

성장 저해 Phenotypes의 확인은 효모에서 세균의 종류 III Effectors 표현에 의해 유도된

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

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생물학

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

성장 팩터 Progranulin와 상호 작용하는 단백질을 식별하기 위해 수정된 효모 - 2 하이브리드 시스템

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

갑각류와 장기 치명적인 독성 시험 Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

방법은 근육의 세포 내 구획을 평가하기 위해<em&gt; C. 엘레</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

SPRI, 나노 SPRI 및 ELISA 분석 실험을 사용하여 혈청에있는 인간 성장 호르몬의 비교 분석

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY : 효모 성장의 다 변수 정량화를위한 ​​대규모 방법

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

효 모에 Polyglutamine 독성 분석 실험을 사용 하 여 퓨전 파트너에 형광 단백질의 영향

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

매체-처리량에 캘리포니아2 +효과의 평가 대 한 분석 실험을 심사-신호 및 인간의 정자에서 Acrosome 반응

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C. 예쁜꼬마선충에서 RAN 펩타이드 독성 측정

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

원자력 현미경을 사용한 살아있는 식물 장기의 일차 세포벽의 기계적 특성 특성 분석

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...