이 프로젝트는 E. 졸리에게 제공되는 케이스 웨스턴 리저브 대학 시작 기금에 의해 투자되었다. 감염된 달팽이는 Schistosome 리소스 센터, NIH - NIAID (NIAID 계약 번호 HHSN272201000009I)에 의해 제공되었다. 우리는 또한 감염된 schistosome 달팽이 확보의 지원과 도움이 토론을 위해 크리스 킹과 로널드 블랜턴 감사드립니다.

안전 대책 : Schistosome cercariae 직접 절단이나 모공의 필요없이 피부를 침투하고, 만성 질병 주혈흡충증 (기생충병)가 발생할 수 있습니다. S.은 mansoni는 biosafety 레벨 2 생물, 따라서 적절한 개인 보호 장비이며, infective의 cercaria 작업을 할 때 적절한 안전 조치가 따라야한다. schistosomes을 성장하고 변화하는 데 필요한 장비의 대부분은 프로토콜의 끝에있는 테이블에 명시된 바와 같이 몇 가지 중요한 예외와 함께, 표준입니다. 모든 cercarial 작품은 외딴 방에서 수행되어야합니다. Schistosomules는 살균, 승인 biosafety 후드에 교양하여야한다. nitrile 또는 라텍스 장갑 두 레이어는 착용한다로 실험실 외투, 신발의 방수 쌍, 손목과 팔을 구멍이나 눈물, 그리고 방수 슬리브 보호 장치와 함께 바지를해야합니다. 사고 유출의 경우, 즉시 적극적으로 당신의 사람에 경우 바르고, 70 % 에탄올 또는 10 %의 표백제로 오염된 지역을 스프레이. 이것은 기생충을 해제 충분하지만, 가능한 모든 감염은보고 심각하게 이동해야합니다. schistosomules가 innately infective 아니지만, 제도적 지침 다양하고, BSL2주의가 noninfectious 생활 단계 동안 필요한지 여부를 확인할 수 협의해야합니다. 실험이 승인된 생물 학적 컨테이너에서 수행하고 폐기물을 폐기 후 모든 잠재적으로 전염성 물질과 접촉에 와서 모든 표면을 소독. 참고 : 표지 달팽이 호스트 용기 1-2일 사전 흘리기와 빛으로부터 보호하기 위해 1. 달팽이 호스트에서 cercariae 수확 <ol><li> 실내 온도 equilibrated 증류수와 중간 전체 세제 무료로 깨끗하고, 500mL 비커를 입력합니다. 다른 날짜에 miracidia 감염된 호스트 달팽이와 함께 일하고있다면, (달팽이가 이후의 실험에 대한 재사용 될 경우 이것은 특히 중요합니다) 각 감염 기간에 대한 별도의 비이커를 사용합니다. 비커의 크기는 또한 사용자의 경험에 따라 작은 수 있습니다. </li><li> 당신은 필요에 따라 네트워크 표준 수족관 물고기, 일반 목적의 집게를 사용하여 비이커에서 그들을 창고 조심스럽게 입금하고자하는 감염된 달팽이 호스트를 수집합니다. </li><li> 비상 노출에 대한 플레이스 비커는 백열 광원 아래 약 8-12인치을 트레이. 불빛 아래 1-2시간에 대한있게 해줘요. 밝은 빛에 노출되면 Cercariae는 달팽이 호스트를 종료합니다. </li><li> 조심스럽게 cercariae 혼합물에서 달팽이를 필터링합니다. 혼합물은 매우 전염성이므로, 별도의 안전 조치를 가져가라. 기억 cercariae 직접 인간의 피부를 뚫을 수 있습니다! </li><li> 어떤 입자의 쓰레기 문제 해결 수 있도록 약 15 분 동안 광원에 따라 혼합물을 교체하십시오. </li><li> 비커의 하단에있는 파편을 피하기 위해, pipet을 사용하여 깨끗한 Erlenmeyer 플라스크에 cercariae을 전송합니다. 60 분 cercariae 한 구석에 정착 있도록 - 45 어둠 속에 얼음에 약 45 ° 각도로 술병를 놓습니다. </li><li> 뜨는의 75-90%를 제거하고 10 % 표백 제나 70 % 에탄올을 포함하는 비커에 입금, 물 방울이 pipet에서 cercariae의 드립을 포함하도록하지 간병하기 위해 50 ML의 pipet을 사용합니다. </li><li> 소용돌이 Erlenmeyer 플라스크는 솔루션 cercariae을 resuspend을 부드럽게 cercariae을 포함. 무균 50 ML 원뿔 튜브에 샘플을 전송하는 pipet을 사용합니다. 어둠 속에서 20 분간 얼음에 다시 튜브를 삽입합니다. Cercariae는 어둠 속에서 튜브의 아래쪽으로 해결됩니다. </li></ol> 2. schistosomules에 cercariae의 수동 변환 당신이 시작하기 전에 : RPMI 1640 완료 (RPMI 5 % 태아 소 혈청, 1X 펜 / Strep)와 37 ° C.에 따뜻한 확인 (그것은 사용되는 태아 소 혈청이 배양에 의해 inactivated했다는 중요합니다 56 ° C schistosomes가 보완에 민감 수 있으므로 1 시간). <ol><li> 생물 학적 후드에서 pipettor 또는 진공 장치를 사용하여 각 50 ML 원뿔의 솔루션 5-10 ML로 뜨는 제거합니다. </li><li> luer 록이 적절하게 크기 주사기에 바늘을 emulsifying 두 번 끝난 22 게이지를 부착합니다. </li><li> 천천히 주사기에 혼합을 그립니다. 주사기 50 ML 튜브에서 cercariae를 수집합니다. 흘리지의 세심한한다. </li><li> 조심스럽게 무소속 바늘 끝가있다 그러한 이상의 주사기를 켜십시오. emulsifying 바늘이 측면에 다른 주사기를 첨부하십시오. </li><li> cercariae를 변환하기 위해서 주사 바늘을 통해 40 회 (앞뒤로 20 회)의 총 혼합물을 전달합니다. </li><li> 혼합물이 아래의 주사기에 포함되어 상단 주사기가 비어 있는지 세로로 같은 주사기를 놓습니다. 최고 주사기를 제거하고 70 % 에탄올로 소독. </li><li> 깨끗한 높은 벽으로 둘러싸인 100mm X 50mm pyrex 요리 또는 crystallizing 그릇에 드롭하여 보증금 혼합 놓습니다. 70 % 에탄올에 바늘과 주사기를 소독. </li><li> 37 ° C RPMI 1640 전체 미디어와 같은 추가되는Luria의 국물 플레이트를 따르고처럼 배양 접시의 바닥이 완전히 덮여 있습니다. </li><li> 페트리 접시의 중앙에 schistosomules를 수집하는 부드럽게 소용돌이. 물 튀기지 마십시오. 참고 : 더 적절하게 중앙에 schistosomules을 시각화하기 위해이 단계에 대한 후드 라이트를 해제하는 것이 유용할 수 있습니다. </li><li> 즉시 멸균 스포이트를 사용하여 잘 신선한 12 웰 세포 배양 플레이트에 집중 schistosomules를 입금. </li><li> schistosomules 더 이상 접시 (약 10 배)의 중심에 존재하지 않습니다 때까지 2.9와 2.10를 반복합니다. 신선한 잘 각 후속 컬렉션을 입금. </li><li> 물론 최대 중간 (~ 1.5 ML) prewarmed RPMI 전체 미디어 각각 입력합니다. </li><li> schistosomules 및 cercariae 부재의 존재를 확인하기 위해 거꾸로 현미경을 시각화. </li><li> 성장 CO 2 배양기에서 schistosomules 37으로 설정되어 ° C와 5% CO 2. </li></ol> 3. 대표 결과 : 문화 미디어로 변모 schistosomules의 보류중인 전송, 거꾸로 밝은 - 필드 현미경 (예를 들어, 장비 표 참조) 아래의 시각화는 변형 schistosomules가 우물에 존재하는 1, 그림과 비슷한 이미지를 얻을 수 있습니다. 잠재적인 오염 물질은 그대로 cercariae, 절단 cercarial 꼬리, 또는 기타 이물질을 포함 수 있습니다. 더 거꾸로 현미경을 사용할 수 없다면, 표준 현미경 또는 해부 범위는 schistosomules을 시각화하기에 충분합니다. 이 절차는 항상 cercariae의 100 %를 변환하지 않습니다. <img src="/files/ftp_upload/3191/3191fig1.jpg" alt="그림 1"/> 그림 1 : 변환 Schistosomulum <img src="/files/ftp_upload/3191/3191fig2.jpg" alt="그림 2"/> 그림 2 : Cercariae 및 잘린 꼬리

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+ultrastructure+of+early+transformation+of+skin-+and+shear-pressure-derived+schistosomules.">Schistosoma mansoni: ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules.</a> Exp Parasitol. 51, 341-365 (1981).</li><li>Lewis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Curr Protoc Immunol. Chapter 19, 1-1 (2001).</li></ol>

관심 없음 충돌 선언하지 않습니다.

몇 가지 잠재적인 문제는 변환 과정에서 발생할 수 있지만,이 프로토콜은 이러한 문제의 대부분을 방지하도록 설계되었습니다. 한 잠재적인 위험은 일반적으로 단계 1.3 수확의 남은 잔해로 인해 발생 emulsifying 바늘의 막히는입니다. 단계 1.5에서 정착하고 단계 1.6에서 신중하게 cercariae를 전송하는 모든 미립자 물질이 문제를 방지하도록해야합니다. 또한, 생물 학적 안전 캐비닛 내부 작업 전에 설명, 개인 보호 장비의 사용 (심지어 얼굴 방패)와 주사기에 과도한 압력의 소지를 없애기는 것이 좋습니다. 분리된 cercarial 꼬리 단계 2.9 동안 가난한 분리 지표는 미디어에 시각하실 수 있습니다. 꼬리는 경련 같은 운동을 전시하고 독특한 모양을 (그림 2 참조), 그리고 표현할수 schistosomules에서 쉽게 구별할 수 있습니다. 꼬리는 schistosomules가 정착 있도록 전체 RPMI 초과 schistosomules을 세척하여 제거할 수 있습니다. 꼬리가 떠있는 상태로 유지됩니다 끌 진공 청소기로 청소를하실 수 있습니다. 그대로 cercariae는 미디어 존재하는 경우 단계 2.5의 변화가 제대로 실행되지 않았습니다. 손상 cercaria의 이미지를 참조 그림 3B에 그려져 있습니다. 이 문제가 발생하면, 사용중인 emulsifying 바늘이 적당한 크기입니다 확보. 40 번정도는 일반적으로 충분히 cercarial 꼬리의 100 %의 전단에 접근하는 것보다 훨씬 더 있지만 또한, cercarial 혼합물이 바늘 통과 횟수가 증가 수 있습니다. schistosomules에 cercaria의 변화가 먼저 콜리와 Wikel (13)에 의해 1974 년에 설명했다. 이후 schistosomules의 준비는 본 논문에서 입증이 프로토콜의 설명, 또는 vortexing 접근법을 사용하고 percoll의 그라디언트로 구분 (14, 15) 또는 소용돌이로 바늘과 주사기와 어느 전단 전략을 사용하여 수행되었습니다. 준비 schistosomules에 대한이 두 기술은 아름답게 프레드 루이스 (16)보다 장기적인 성장을위한 schistosomules의 자세한 culturing에 대한 정보 NIAID Schistosome 리소스 센터 (에서 찾을 수에 의해 설명되어 있습니다 <a href="http://www.schisto-resource.org/">www.schisto - resource.org</a> ).

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> 22g X 2-7/8 &quot;Emulsifying 바늘 </td><td> 피셔 사이 언티픽 </td><td> 14-825 - 17G </td></tr><tr><td> RPMI 1640 중간 1X (500ml) </td><td> Invitrogen </td><td> 11835030 </td></tr><tr><td> 태아 소 혈청 (FBS) 열 inactivated </td><td> Invitrogen </td><td> 10082139 </td></tr><tr><td> 페니실린은 / 스트렙토 마이신 (100X) </td><td> Invitrogen </td><td> 15070063 </td></tr><tr><td> Crystallizing 접시 (100mm) </td><td> 피셔 사이 언티픽 </td><td> 08 - 741D </td></tr><tr><td> 범용 족집게 </td><td> 피셔 사이 언티픽 </td><td> 10 - 316C </td></tr><tr><td> 염수 새우에 대한 페트코 수족관 NET </td><td> 페트코 </td><td> SKU : 1190954 </td></tr></tbody></table>

cercarial transformation and in vitro cultivation of schistosoma mansoni schistosomules

Schistosome 기생충 주혈흡충증 (기생충병), 전세계 2 억 명 이상에 영향을하고 공중 보건 및 사회 경제적 영향 (1-4)의 관점에서 기생 질병 사이에 말라리아의 두번째 계급 만성 쇠약 질병의 원인이되는 에이전트입니다. Schistosome 기생충이 복잡한 라이프 사이클은 자유 수영 단계를 개입과 연체 동물과 포유류의 호스트에서 기생 생활 사이 interchanging와 흡충의 벌레입니다. 간단히, 자유 수영 cercariae는 schistosomules로 변모, cercariae들이 꼬리를 잃게되는 시간 동안, 분비 프로 테아제의 도움으로 피부를 관통하여 포유류의 호스트를 감염. schistosomules 이제 호스트 면역 체계를 회피 적혈구의 소화에 대한 직감을 개발하고, 마이 그 레이션해야하지만 폐, 간장 포털 시스템에서 자신의 최종 목적지로가는 도중 포털 순환 결국 mesenteric 정맥 (S. mansoni에 대한) 남성과 여성 웜 쌍 및 친구는, 매일 계란의 수백 생산 어디에. 계란의 일부는 계란은 자유 수영 miracidia (5-10)에 해치 신선한 물로 신체에서 배설됩니다. miracidia 특정 달팽이 종의 감염과 차례로, 생활주기를 완료, infective의 cercariae를 만들어 엄마와 딸 sporocysts로 변환. 불행하게도, 전체 schistosome 생활주기는 체외에서 배양해 수 없다지만, infective의 cercariae는 schistosomules로 변환 될 수 있으며, schistosomules는 체외 또는 microarray 분석 schistosome 개발의 분석을 위해 주 동안 교양 수 있습니다. 이 프로토콜에서는, 우리는 cercarial 변화와 schistosomules의 체외의 culturing의 시각적 설명을 제공합니다. 우리는 달팽이를 호스트 Biomphalaria의 glabrata에서 전염성 cercariae를 버리고 수동 emulsifying 두 종단 바늘을 사용하여 꼬리를 분리하여 schistosomules로 그들을 변형. 체외 cercarial 변화와 schistosomules 문화 기술의는 schistosomes의 시각화를 단순화하고 실험 분석에 대한 기생충의 수집을 용이하게합니다. 체외 변환 및 schistosomes의 culturing 기술의 년간 수행되었습니다 포유류의 호스트 (11의 사용을 피하 설명 12),하지만 시각적인 프로토콜은 전체 커뮤니티에 사용할 수 있습니다 개발되지 않았습니다.

Watch this Scientific Journal Video about Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules at JoVE.com

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

우리는 설명을<em&gt; 체외에서</emflatworm 기생충의 culturing의 schistosomula을위한&gt; 방법<em&gt; Schistosoma mansoni</em&gt; 담수 달팽이 중간 호스트 Biomphalaria의 glabrata에서 infective의 cercariae의 수확과 변화를 통해.

Cercarial 변화와 체외에서</em의> 양성 Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

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JoVE Journal

Immunology and Infection

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

17.9K 조회수.  Case Western Reserve University. 우리는 설명을 체외에서 방법 Schistosoma mansoni 담수 달팽이 중간 호스트 Biomphalaria의 glabrata에서 infective의 cercariae의 수확과 변화를 통해.

비디오: Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

HIV - 1 코어의 시험 관내 Uncoating에서

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

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면역학 및 감염병학

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

에서 계란을 사용하여<em&gt; Schistosoma mansoni</em&gt; 등<em&gt; 생체내에서</em기생충 유도된 폐 염증의&gt; 모델

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

정량<em&gt; 체외</em정적 조건에서 활성화 된 기본 인간의 미세 혈관 내피 세포에 호중구 부착을 측정하는&gt; 분석

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

재배<em&gt; Heligmosomoides Polygyrus :</em&gt; 면역 선충 기생충과 은닉하는 제품

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

편의적 곰팡이 병원균에 형광 태그 유전자의과 유전자 변형<em&gt; 크립토 콕 쿠스 네오 포르</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

마우스의 구강 투여에 따라 생균제 효모의 변환 및 위장 면역 조직에서 그들의 복구

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

체외 방법에서 대상 바인딩 및 치료 항체 사이 CDC 유도 비교 : 응용 프로그램 생물학적 동등성 분석에

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

절연 및 Extracellular 소포 특성 성인 Schistosoma 나무 에서

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

체외 목욕 발효 시스템을 사용하여 내분비병제와 인간 장내 미생물 간의 상호 작용 분석

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

체외 전사 mRNA를 가진 1 차적인 대식세포의 고효율 형질감염

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

Cercarial 변화와 체외에서 양성 Schistosoma mansoni Schistosomules

Cercarial 변화와 체외에서 양성 Schistosoma mansoni* Schistosomules*