저자 맨디 Loebert 우수한 기술 지원을 티 나 Czajka를 인정 하 고 싶습니다. 우리는 편집 도움말 언어에 대 한 식물 루 오 (하버드의과 대학, 보스톤, 메사추세츠, 미국) 감사 합니다.

1. 준비의 돌연변이 pcDNA3.1/GLA와 pcDNA3.1/GAA 구문
참고: GLA 와 GAA 코딩 시퀀스 (cd)에 대 한 복제 전략 보고 이전15,18되었습니다.
<ol>
	<li>사이트 감독 Mutagenesis 사이트 지시 된 Mutagenesis를 사용 하 여
	<ol>
		<li>참조를 사용 하 여 시퀀스 NM_000169.2 및 NM_000152.4 mutagenesis GLA 와 GAA 유전자의에 대 한 템플릿으로 각각. 고 순도 소금 무료 뇌관 (25-37-mers) 개별적으로 돌연변이 소개 하는 그들의 길이 중앙 각각 시퀀스 수정 중 하나를 들고와 antisense 뇌관으로 상업적인 공급자에 의해 합성의 세트가 있다. 무료 뇌관 설계 도구를 사용 하 여 뇌관 디자인19를 지원 하기 위해.</li>
		<li>반응 혼합물 반응 솔루션에 대 한 표준 조건 및 제조업체에서 제공 하는 PCR 상태를 사용 하 여.<ol>
			<li>믹스 5 µ L 반응 버퍼, 10 x 10의 더블-좌초 템플릿 플라스 미드 DNA (pcDNA3.1/GLA 또는 pcDNA3.1/GAA)의 ng 125 각 뇌관의, 제공된 dNTP 혼합물의 1 µ L, 이온된 수 (최종 반응 볼륨의 적절 한 볼륨 시 약 DMSO의 3 µ L : 50 Μ L). 마지막으로, 2.5 U의 DNA 중 합 효소를 추가 하 고 아래로 pipetting으로 혼합. 부정적인 통제로 아니 뇌관 샘플 따라 수행.</li>
		</ol>
		</li>
		<li>다음 프로그램을 사용 하 여 PCR을 시작: 1 분 1: 95 ° C 단계, 단계 2: 95 ° C 50에 대 한 s, 단계 3시 60분 ° C 50에 대 한 s, 8 분 4: 68 ° C 단계, 18 번, 2-4 단계를 반복 하 고 단계 5: 68 ° C 10 분.<ol>
			<li>Dpn의 1 µ L 추가 PCR, 다음 나 제한 효소 (10 U / µ L)와 추가 1 시간 동안 37 ° C에서 반응 유리병을 품 어.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>변환 및 원하는 클론을 위한 심사
	<ol>
		<li>제조업체의 권장 사항 따라 ultracompetent 셀의 약 수를 변환 합니다. 사용 SOC 매체 (0.5% (w/v), NaCl 10 m m, KCl 2.5 m m를 추출, 121 ° c, 소독 및 살 균 필터링 솔루션 MgCl2 , 10 및 20 m m의 최종 농도까지 포도 당을 각각 추가 tryptone 2% (w/v), 효 모) 제조업체의 대신 중간입니다. 절차 후 샘플 mutagenesis는 파운드에의 플레이트 250 µ L 플레이트 포함 100 µ g/mL 암 피 실린 고 18 h 37 ° C에서 품 어.</li>
		<li>Transformants의 수는 보장 &#62; 10 반응 생성 적어도 3 배 많은 식민지는 no-뇌관 제어 반응으로, 예를 들면, 식민지 계산을 촉진 하기 위하여 발광 격판덮개를 사용 하 여. 3 식민지를 선택 그리고 파운드 국물 매체에서 하룻밤 문화 3 mL를 준비 합니다.</li>
		<li>다음 날, 플라스 미드 준비 표준 키트와 함께 수행 하 고 t 7을 사용 하 여 전체 시퀀스 분석 (5ʹ-TAA 전술 GAC TCA CTA 태그 GG-3ʹ) BGHr (5ʹ 태그 아 그 GCA CAG TCG AGG-3ʹ) 뇌관 통해 표준 생어 시퀀싱 및.</li>
		<li>분자 생물학 적당 한 도구를 사용 하 여 시퀀스를 분석 하. 원하는 돌연변이 검출 될 때 없고 더 이상 NM_000169.2 (α-galactosidase A)에 비해 시퀀스 또는 NM_000152.4 (산 성 α-glucosidase) 본, transfection 급 플라스 미드 정화에 대 한 복제를 선택 합니다.</li>
		<li>분 광 광도 계에서 흡 광도 측정 하 여 DNA의 순도 결정 합니다. 참고: 허용의 260/280 흡 광도 비율 플라스 미드 순도 항복 준비만 &#62; 셀 1.8 문화 실험.</li>
	</ol>
	</li>
</ol>
2입니다. HEK293H 셀의 재배
<ol>
	<li>높은 포도 당 (4.5 g/L) Dulbecco´s 수정이 글 매체 (DMEM) 10% 태아 둔감 한 혈 청 (FBS)와 1% 페니실린/스 보충에 HEK293H 세포를 유지 합니다. 셀 유지 한 5% CO2 분위기에서 37 ° C에서 인큐베이터 water-jacket.</li>
	<li>80-90%의 밀도를 세포 배양.</li>
	<li>매체를 발음 하 고 인산 염을 사용 하 여 버퍼링 없이 캘리포니아2 + 그리고 마그네슘2 +염 분 (PBS) 일단 씻어.</li>
	<li>0.05%를 추가 하 여 통로 트립 신-EDTA와 37 ° C, 5% CO25 분 동안 품 어.</li>
	<li>영구 문화를 유지 하기 위해 새로운 T75 플라스 크로 신선한 매체와 씨앗에 셀 1시 15분을 분할 한다. 25 이상 통행으로 셀을 사용 하지 마십시오.</li>
</ol>
3. pcDNA3.1/GLA와 pcDNA3.1/GAA 플라스 미드 Transfection 그리고 HEK293H의 치료
<ol>
	<li>transfection, 전에 24 시간 캘리포니아2 +, Mg2 +PBS 한번 T75 셀 문화 플라스 크에서 HEK293H 셀 세척. 위에 명시 된 트립 신-EDTA 0.05% 셀과 10% 보충 500 µ L DMEM 매체를 사용 하 여 24 잘 문화 판의 구멍에 씨앗 1.5 x 105 셀 수확 FBS 항생제 없이.</li>
	<li>제조업체의 설명서에 따라 transfection 프로토콜 수행 합니다. 일반적으로 혈 청 무료 DMEM의 100 µ L에 플라스 미드 DNA의 1 µ g의 transfection 시 약 2.5 µ L의 혼합물을 사용 합니다. 실 온에서 20 분 동안 incubate 고 그 후 drop-wise 방식으로 셀에 추가 합니다.</li>
	<li>37 °C/5% CO2 에서 4 h 기간 transfection 시 약을 포함 하는 매체를 제거 하 고 추가 10% 신선한 DMEM의 500 µ L FBS / 1% 페니실린/스. 참고:이 단계 1-Deoxygalactonojirimycin 염 산 염 (DGJ) 또는 1-Deoxynojirimycin 염 산 염 (DNJ) 추가할 수 있습니다 문화 매체에 의도 하는 곳 (20 µ M의 최종 농도 얻기 위해 10 mM의 수성 재고 솔루션을 사용 DGJ 및 DNJ ). 신선한 DGJ 또는 DNJ 플라스 미드 transfection 후 42 시간을 추가 되었습니다.</li>
</ol>
4. 세포 수확 및 α-galactosidase A 또는 산 성 α-glucosidase 활동 측정
<ol>
	<li>셀 수확
	<ol>
		<li>수확의 날, 인큐베이터에서 셀을 제거 하 고 매체를 발음 합니다. 조심 스럽게 셀 캘리포니아2 + 그리고 마그네슘2 +PBS로 2 회 세척. 참고:이 단계는 DGJ 및 DNJ는 α-galactosidase와 α-glucosidase의 강력한 억제제 각각, 어떤 남은 시험을 무효화 시키는 때문에 중요 한.</li>
		<li>셀 위에 직접 이온된 수의 200 µ L를 추가 합니다. 접시에서 셀 린스를 1.5 mL 반응 관에 그들을 전송 합니다.</li>
	</ol>
	</li>
	<li>중지 및 재개 하 여 균질 화
	<ol>
		<li>적절 한 거품 랙 5 소용돌이에 샘플을 넣어 s는 세포 보다 효율적으로 만들기를. 액체 질소 10에 번갈아 샘플 넣어 s 및 실내 온도 물 목욕까지 완료 (5 분)는 녹고.</li>
		<li>5 번이이 절차를 반복 하 고 스핀에 10000 x g 5 분에 대 한 샘플.</li></ol></li></ol>상쾌한 및 새로운 반응 관에 피 펫을 유지 합니다.
	
	
	<li>단백질 농도 결심 bicinchoninic 산 (BCA) 분석 결과 사용 하 여
	<ol>
		<li>이온된 H2O의 40 µ L을 포함 하는 각 샘플에 대 한 신선한 튜브를 준비 하 고 샘플의 10 µ L를 추가 합니다. 짧게 vortexing에 의해 솔루션을 믹스와 10 µ L 96 잘 접시 (3 중에서 각 샘플)의 캐비티에 전송. 이온 H2O 다음과 같이 표준 곡선을 얻기 위해 2 mg/mL 소 혈 청 알 부 민 (BSA) 재고 솔루션을 희석: 50 µ H L2O/50 µ L BSA; 60 µ L H2O/40 µ L BSA; 70 µ L H2O/30 µ L BSA; 80 µ L H2O/20 µ L BSA; 90 µ L H2O/10 µ L BSA; 100 µ L H2o.</li>
		<li>BCA 시 약의 200 µ L을 추가 하 여 반응을 시작 (시 약 A와 B 시 50: 1 비율로 혼합) 및 궤도 통 (300 rpm)에 약간의 동요에서 37 ° C에서 어둠 속에서 1 시간에 품 어. 560에서 흡 광도 측정 플레이트 리더에 nm. 참고: 샘플은 일반적으로 1 µ L 당 1.5 µ g 단백질 사이의 포함 되어 있습니다.</li>
	</ol>
	</li>
	<li>인공 4-Methylumbelliferyl 기판 (4-얼굴)와 효소 활동 측정
	<ol>
		<li>0.05 (대 한 α-galactosidase A) 또는 (산 성 α-glucosidase)에서 0.5 µ g 단백질 / µ L 솔루션을 얻기 위해 신선한 1.5 mL 반응 관에 각 샘플의 피펫으로 계산 된 금액을 희석. 와 동 5 다시 s를 피펫으로 10 µ L를 96으로이 희석의 샘플 잘 플레이트 (중복에서 각 샘플).</li>
		<li>반응이 각각 기판 솔루션의 20 µ L을 추가 하 여 시작: Α-galactosidase a: 2 mM 4-Methylumbelliferyl-α-D-galactopyranoside (4-뮤-gal) 0.06 M 인산 염 구 연산 염 버퍼, pH 4.7에서에서에 대 한. 산 성 α-glucosidase에 대 한: 2 mM 4 methylumbelliferyl α-D-glucopyranoside (4-뮤-glu) 0.025 M 아세트산 나트륨, pH 4.0에서에서.</li>
		<li>궤도 통 (300 rpm)에 약간의 동요에서 37 ° C에서 어둠 속에서 1 시간에 대 한 효소 반응을 품 어. 1.0 M, pH 10.5 조정된 글리신-NaOH 버퍼의 200 µ L의 추가 의해 반응 종료.</li>
		<li>다음과 같이 표준 곡선 4-methylumbelliferone (4-MU) 0.01 mg/mL 주식에서의 준비: 100 µ L H2O / 아니 4-무; 80 µ L H2O / 20 µ L 4-무; 60 µ L H2O / 40 µ L 4-무; 40 µ L H2O / 60 µ L 4-무; 20 µ L H2O / 80 µ L 4-무; 아니 H2O / 100 µ L 4-뮤, 피펫으로 10 µ L 96 잘으로 각 희석의 (중복)에 접시와 볼륨 및 pH를 조정 하기 위하여 각 잘 하 1.0 M 글리신-NaOH 버퍼의 200 µ L를 추가.</li>
		<li>측정 형광 판독기에 효소 활동 적절 한 필터 집합 있고 형광 판독기 장치에 대 한 적절 한 소프트웨어를 사용 하 여 데이터를 분석 합니다. 참고: 두 4-얼굴 기판 α-galactosidase A 또는 산 성 α-glucosidase 동안 4-무로 감소 된다. 출시 4-무 측정 될 수 있는 360와 465 nm에서 여기 및 방출 파장으로 각각 microplate 형광 판독기를 사용 하는 형광 색소 이다.</li>
	</ol>
	</li>

<ol>
	<li>Ishii, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+alpha-galactosidase+A+enzymes+identified+in+Fabry+disease+patients+with+residual+enzyme+activity:+biochemical+characterization+and+restoration+of+normal+intracellular+processing+by+1-deoxygalactonojirimycin.">Mutant alpha-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin.</a> Biochem J. 406, 285-295 (2007).</li><li>Tajima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+biochemical+studies+on+Pompe+disease+and+a+&amp;#34;pseudodeficiency+of+acid+alpha-glucosidase&amp;#34.">Structural and biochemical studies on Pompe disease and a &amp;#34;pseudodeficiency of acid alpha-glucosidase&amp;#34.</a> J Hum Genet. 52, 898-906 (2007).</li><li>Lukas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+Clinical+Consequences+of+Novel+&#945;-Galactosidase+A+Mutations+in+Fabry+Disease.">Functional and Clinical Consequences of Novel &#945;-Galactosidase A Mutations in Fabry Disease.</a> Hum Mutat. 37, 43-51 (2016).</li><li>Parenti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treating+lysosomal+storage+diseases+with+pharmacological+chaperones:+from+concept+to+clinics.">Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics.</a> EMBO Mol Med. 1, 268-279 (2009).</li><li>Yasuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabry+disease:+characterization+of+alpha-galactosidase+A+double+mutations+and+the+D313Y+plasma+enzyme+pseudodeficiency+allele.">Fabry disease: characterization of alpha-galactosidase A double mutations and the D313Y plasma enzyme pseudodeficiency allele.</a> Hum Mutat. 22, 486-492 (2003).</li><li>Shimotori, M., Maruyama, H., Nakamura, G., Suyama, T., Sakamoto, F., Itoh, M., Miyabayashi, S., Ohnishi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+mutations+of+the+GLA+gene+in+Japanese+patients+with+Fabry+disease+and+their+functional+characterization+by+active+site+specific+chaperone.">Novel mutations of the GLA gene in Japanese patients with Fabry disease and their functional characterization by active site specific chaperone.</a> Hum Mutat. 29, 331 (2008).</li><li>Andreotti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapy+of+Fabry+disease+with+pharmacological+chaperones:+from+in+silico+predictions+to+in+vitro+tests.">Therapy of Fabry disease with pharmacological chaperones: from in silico predictions to in vitro tests.</a> Orphanet J Rare Dis. 6, 66 (2011).</li><li>Khanna, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharmacological+chaperone+AT2220+increases+the+specific+activity+and+lysosomal+delivery+of+mutant+acid+alpha-glucosidase,+and+promotes+glycogen+reduction+in+a+transgenic+mouse+model+of+Pompe+disease.">The pharmacological chaperone AT2220 increases the specific activity and lysosomal delivery of mutant acid alpha-glucosidase, and promotes glycogen reduction in a transgenic mouse model of Pompe disease.</a> PLoS One. 9, e102092 (2014).</li><li>Siekierska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-Galactosidase+aggregation+is+a+determinant+of+pharmacological+chaperone+efficacy+on+Fabry+disease+mutants.">&#945;-Galactosidase aggregation is a determinant of pharmacological chaperone efficacy on Fabry disease mutants.</a> J Biol Chem. 287, 28386-28397 (2012).</li><li>Parenti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+enhancement+of+mutated+alpha-glucosidase+activity+in+fibroblasts+from+patients+with+Pompe+disease.">Pharmacological enhancement of mutated alpha-glucosidase activity in fibroblasts from patients with Pompe disease.</a> Mol Ther. 15, 508-514 (2007).</li><li>Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pharmacogenetic+approach+to+identify+mutant+forms+of+&#945;-galactosidase+A+that+respond+to+a+pharmacological+chaperone+for+Fabry+disease.">A pharmacogenetic approach to identify mutant forms of &#945;-galactosidase A that respond to a pharmacological chaperone for Fabry disease.</a> Hum Mutat. 32, 965-977 (2011).</li><li>Lukas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterisation+of+alpha-galactosidase+a+mutations+as+a+basis+for+a+new+classification+system+in+fabry+disease.">Functional characterisation of alpha-galactosidase a mutations as a basis for a new classification system in fabry disease.</a> PLoS Genet. 9, e1003632 (2013).</li><li>Andreotti, G., Citro, V., Correra, A., Cubellis, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thermodynamic+assay+to+test+pharmacological+chaperones+for+Fabry+disease.">A thermodynamic assay to test pharmacological chaperones for Fabry disease.</a> Biochim Biophys Acta. 1840, 1214-1224 (2014).</li><li>. Available from: <a target="_blank" href="https://www.google.ch/patents/US9095584">https://www.google.ch/patents/US9095584</a> (2017)</li><li>Lukas, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+enhancers+for+the+treatment+of+Fabry+and+Pompe+disease.">Enzyme enhancers for the treatment of Fabry and Pompe disease.</a> Mol Ther. 23, 456-4564 (2015).</li><li>Yam, G. H., Bosshard, N., Zuber, C., Steinmann, B., Roth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+chaperone+corrects+lysosomal+storage+in+Fabry+disease+caused+by+trafficking-incompetent+variants.">Pharmacological chaperone corrects lysosomal storage in Fabry disease caused by trafficking-incompetent variants.</a> Am J Physiol Cell Physiol. 290, C1076-C1082 (2006).</li><li>Shin, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+pharmacological+chaperones+in+Fabry+disease.">Screening for pharmacological chaperones in Fabry disease.</a> Biochem Biophys Res Commun. 359, 168-173 (2007).</li><li>Shin, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+response+of+mutated+alpha-galactosidase+A+to+a+pharmacological+chaperone.">Prediction of response of mutated alpha-galactosidase A to a pharmacological chaperone.</a> Pharmacogenet Genomics. 18, 773-7780 (2008).</li><li>Filoni, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+studies+of+new+GLA+gene+mutations+leading+to+conformational+Fabry+disease.">Functional studies of new GLA gene mutations leading to conformational Fabry disease.</a> Biochim Biophys Acta. 1802, 247-252 (2010).</li><li>Citro, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Large+Phenotypic+Spectrum+of+Fabry+Disease+Requires+Graduated+Diagnosis+and+Personalized+Therapy:+A+Meta-Analysis+Can+Help+to+Differentiate+Missense+Mutations.">The Large Phenotypic Spectrum of Fabry Disease Requires Graduated Diagnosis and Personalized Therapy: A Meta-Analysis Can Help to Differentiate Missense Mutations.</a> Int J Mol Sci. 17, (2016).</li><li>Cammisa, M., Correra, A., Andreotti, G., Cubellis, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabry_CEP:+a+tool+to+identify+Fabry+mutations+responsive+to+pharmacological+chaperones.">Fabry_CEP: a tool to identify Fabry mutations responsive to pharmacological chaperones.</a> Orphanet J Rare Dis. 8, 111 (2013).</li><li>Leinekugel, P., Michel, S., Conzelmann, E., Sandhoff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+correlation+between+the+residual+activity+of+beta-hexosaminidase+A+and+arylsulfatase+A+and+the+severity+of+the+resulting+lysosomal+storage+disease.">Quantitative correlation between the residual activity of beta-hexosaminidase A and arylsulfatase A and the severity of the resulting lysosomal storage disease.</a> Hum Genet. 88, 513-523 (1992).</li><li>Germain, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+pharmacodynamic+effects+of+a+pharmacological+chaperone+on+&#945;-galactosidase+A+activity+and+globotriaosylceramide+clearance+in+Fabry+disease:+report+from+two+phase+2+clinical+studies.">Safety and pharmacodynamic effects of a pharmacological chaperone on &#945;-galactosidase A activity and globotriaosylceramide clearance in Fabry disease: report from two phase 2 clinical studies.</a> Orphanet J Rare Dis. 7, 91 (2012).</li><li>Hughes, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+pharmacological+chaperone+migalastat+compared+with+enzyme+replacement+therapy+in+Fabry+disease:+18-month+results+from+the+randomised+phase+III+ATTRACT+study.">Oral pharmacological chaperone migalastat compared with enzyme replacement therapy in Fabry disease: 18-month results from the randomised phase III ATTRACT study.</a> J Med Genet. 54, 288-296 (2017).</li><li>Parenti, G., Andria, G., Valenzano, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Chaperone+Therapy:+Preclinical+Development,+Clinical+Translation,+and+Prospects+for+the+Treatment+of+Lysosomal+Storage+Disorders.">Pharmacological Chaperone Therapy: Preclinical Development, Clinical Translation, and Prospects for the Treatment of Lysosomal Storage Disorders.</a> Mol Ther. 23, 1138-1148 (2015).</li><li>Parenti, G., et al. <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+Enhances+Blood+&#945;-Glucosidase+Activity+in+Pompe+Disease+Patients+Treated+With+Enzyme+Replacement+Therapy.">A Chaperone Enhances Blood &#945;-Glucosidase Activity in Pompe Disease Patients Treated With Enzyme Replacement Therapy.</a> Mol Ther. 22, 2004-2012 (2014).</li></ol>

저자 들은 아무 경쟁 금융 관심사 선언 합니다.

여기에 설명 된 프로토콜 대사의 유전 lysosomal 질병의 효소 손상 평가 대 한 강력한 결과 제공 합니다. 이 원고는 프로토콜 개정 출판 이전15입니다. 가장 중요 한 수정 포함 엄중 (즉, 돌연변이 벡터 구축 준비 과정), 세포 문화 프로토콜 (즉, HEK293H 셀 유지 보수 및 transfection 조건), 그리고 높은의 표준화 결과의 재현성에 매우 공헌한 실험 반복 (적어도 5)의 수입니다. ?...

12 lysosomal 저장 장애 (LSDs) 기본 유전자 변이 결과로 glycosidase 장애에 관련 된 이상 있다. Fabry (OMIM #301500) 병과 Pompe (OMIM #232300),1,2,3 보고 되었습니다, 이상의 500와 200 missense 돌연변이에 각각,는 총 변이 수의 약 60%에 해당 합니다. 수많은 새로운 유전자 이체는 아직도 확인 되 고, 많은 알 수 없는 의미. 광범위 한 생 화 확 적인 연구 공개 완전 한 손실-의-함수에 GLA 유전자의 특정 genotypes 지도 하지 않는다 (OMIM * 300644) Fabry 질병에 발생 하지만 열역학으로 호의 보인 접는 상태가4 실패 해당 효소 . ER 보존과 달리 기능적인 효소의 조 저하 발생합니다. 유사한 결론 Pompe 질병5를 포함 하 여 다른 LSDs에 그려 왔다. 또한, 효소 이체의 분자 특성 진단6, LSD 진행은 돌연변이의 특성에 따라 개별 과정 제안 시 변이의 임상 해석을 촉진 수 있습니다. 따라서, 임상 상담 및 치료 결정을 합리화 하기 위해서는 일반적으로 2 ~ 3 가지 임상 유형으로 기존의 분류를 재평가 해야 합니다.
효소 대체 요법 (ERT)는 두 질병 수 있습니다. 그러나, ERT, 두뇌와 골격 근육 등 영향을 받는 조직/장기에서 효능을 제한 했다. 또한, ERT의 치료 혜택을 위태롭게 하는 면역성 응답을 유도 수 있습니다. 약리학 보호자 (Pc)는 소위 응답 돌연변이 가진 환자에 대 한 매력적인 치료 대안 이다. Pc는 올바른 단백질 폴딩 및 차례로 바인딩과 그물 (응급실) 보존 및 응급실 관련 효소의 저하를 방지 하는 안정화에 대 한 분자 비 계 역할을 합니다. 또한, Pc 구두로 관리 될 수 있습니다 그리고 잠재적으로 혈액 뇌 장벽을 교차 수 있습니다. 따라서, PCT에 특정 genotypes 가진 환자를 치료를 위한 더 실행 가능한 옵션이 있을 수 있습니다. LSDs 응용 프로그램 PC에 광범위 한 검토를 위해 Parenti7에 의해 우수한 검토를 참조 하십시오.
질병을 일으키는 돌연변이 체 대립 유전자의 수백의 발견 전 임상 약물 테스트에 도전 하 고 맞춤된 의학 접근에 대 한 의무가 환자가의 간단 하 고 빠른, 매우 표준화 된 평가 필요로. LSD 유전자 돌연변이의 해로운 효과 평가 하 고 예측 PCT에 대 한 의무가 환자 후보 돌연변이 테스트, 신속 하 고 신뢰할 수 있는 효소 활동 측정 허용 하는 HEK293H 세포에 매우 표준화 초과 식 시스템은 개발. 유사한 이상 식 시스템 이전 COS-78,,910,11, HeLa 세포12또는 HEK293 Fabry Pompe 질병에 대 한 설명 되었습니다13 ,14,,1516 셀 glycosidase 유전자에 대 한.
아주 유사한 방법도 약리 보호자 질병의 치료에 대 한 응답을 예측 하는 "방법"으로 특허 되었습니다17 셀의 관련성을 나타내는 문화 시스템 임상 연습으로 통합 되 고.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuikChange II XL Site-Directed Mutagenesis Kit&#160;</td>
			<td>Stratagene, La Jolla, CA, USA</td>
			<td>#200522</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[R301Q]-frw: 5&#180;- CTA ATG ACC TCC AAC ACA TCA GCC C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[R301Q]-rev: 5&#180;- GGG CTG ATG TGT TGG AGG TCA TTA G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-frw: 5&#180;-CTA CGA CAT TGA TGT CCA GAC CTT TGC TG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-rev: 5&#180;-CAG CAA AGG TCT GGA CAT CAA TGT CGT AG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-frw: 5&#180;-GGA AAT AAA ACC TGC ACA GGC TTC CCT GGG A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A143T]-rev: 5&#180;-TCC CAG GGA AGC CTG TGC AGG TTT TAT TTC C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[F455Y]-frw: 5&#180;-CTG CCG GGA GCT TCA GGC CCT ACG A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[F455Y]-rev: 5&#180;-TCG TAG GGC CTG AAG CTC CCG GCA G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-frw: 5&#180;-CAC CCT ACG TGC TTG GGG TGG TTG G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-rev: 5&#180;-CCA ACC ACC CCA AGC ACG TAG GGT G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[L552P]-frw: 5&#180;-TTG GGG GGA CCC CCC AGG CGG CCA C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-rev: 5&#180;-GTG GCC GCC TGG GGG GTC CCC CCA A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer T7: 5&#180;-TAA TAC GAC TCA CTA TAG GG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer BGHrev: 5&#180;-TAG AAG GCA CAG TCG AGG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>8952.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>103,753</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,064,041,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potasium chloride</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,049,360,500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 x 6H2O</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,058,330,250</td>
			<td></td>
		</tr>
		<tr>
			<td>D (+)-Glucose monohydrate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,083,422,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,064,980,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>3908.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X863.3</td>
			<td></td>
		</tr>
		<tr>
			<td>disodium hydrogen phosphate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,065,860,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,062,640,050</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X969.2</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Medium</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X968.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Zyppy Plasmid Miniprep Kit</td>
			<td>ZymoResearch, Freiburg, Germany</td>
			<td>D4020</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAfilter Plasmid Midi Kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>12245</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293H cell line</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11631-017&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Modified Eagle Medium&#160;</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>31966-047</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone fetal bovine serum&#160;</td>
			<td>GE Healthcare, South Logan, Utah, USA</td>
			<td>SV30160.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR Standard Cell Culture Flasks, Greiner Bio One</td>
			<td>VWR International GmbH, Hannover, Germany</td>
			<td>82050-854</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates (24 well)</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>831,836</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellstar 96 well plate</td>
			<td>Greiner bio one, Frickenhausen, Germany</td>
			<td>655185</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeSeal reaction tubes, 1,5mL&#160;</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>72,706</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxygalactonojirimycin Hydrochloride&#160;</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>D9641</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxygalactonojirimycin Hydrochloride&#160;</td>
			<td>Toronto Research Chemicals, Toronto, Canada</td>
			<td>D236500</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxynojirimycin&#160;</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>D9305</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Dulbecco w/o Calcium, w/o Magnesium</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>L 1825</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermo Scientific, Braunschweig, Germany</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific, Braunschweig, Germany</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferone</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M1381</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferyl &#945;-D-galactopyranoside</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M7633</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferyl &#945;-D-glucopyranoside</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M9766</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>incubator type T6</td>
			<td>Heraeus Instruments, Hanau, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminous Plate Gr. 2 E</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>P265.1</td>
			<td></td>
		</tr>
		<tr>
			<td>3130 xl Genetic Analyzer&#160;</td>
			<td>Applied Biosystems, Darmstadt, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>GFL-3032 bacterial shaker</td>
			<td>GFL, Burgwedel, Germany&#160;</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-25 centrifuge</td>
			<td>Beckman/Coulter, Krefeld, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraspec 3100 pro Spectrophotometer</td>
			<td>Amersham Biosciences, Buckinghamshire, United Kingdom</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>water-jacket incubator&#160;</td>
			<td>Binder, Tuttlingen, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie 1 touch mixer</td>
			<td>Scientific Industries, Bohemia, NY, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Z 233 MK-2 refrigerated microtube centrifuge</td>
			<td>Hermle, Tuttlingen, Germany&#160;</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>LaboStar ultrapure water device</td>
			<td>Siemens, Berlin, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Shaker PST-60HL-4 orbital shaker</td>
			<td>Biosan, Riga, Latvia</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan GENios Reader</td>
			<td>Tecan, M&#228;nnedorf, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>HI 223 Microprocessor pH Meter</td>
			<td>HANNA Instruments, Arvore, Portugal</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>CASY 
			Cell Counter + Analyser System 
			Model TT</td>
			<td>Innovatis AG, Cham/Zug, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magellan data analysis software v6.6</td>
			<td>Tecan, M&#228;nnedorf, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 5.04</td>
			<td>GraphPad Software, Inc., La Jolla, CA; USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Office 2010</td>
			<td>Microsoft Corporation, Redmond, WA, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>BioEdit Alignment Editor, V7.0.1</td>
			<td>http://www.mbio.ncsu.edu/bioedit/bioedit.html</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Abbreviations</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>frw = forward; rev = reverse</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>n.a. = not applicable</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro enzyme measurement to test pharmacological chaperone responsiveness in fabry and pompe disease

Lysosomal 저장 장애 (LSDs) 같은 희귀 monogenic 질병을 치료 하는 맞춤된 의학의 사용은 복잡 한 임상 시험 디자인, 높은 비용 및 낮은 환자 수에 의해 도전을 했습니다. 수백 개의 돌연변이 체 대립 유전자는 LSDs의 대부분에 연루 됩니다. 질병은 일반적으로 심각도 따라 2 ~ 3 가지 임상 유형으로 분류 됩니다. 또한, 유전자 형의 분자 특성 임상 결과 예측 하 고 환자 치료를 알릴 수 있습니다. 따라서, 우리는 heterologously-Fabry Pompe 질병에서 확인 된 돌연변이 표현 하는 HEK293H 세포에 따라 간단한 셀 문화 분석 결과를 개발 했다. 유사한 분석 결과 최근 Fabry 질병에 의무가 돌연변이 약리 보호자 치료 (에서 PCT)에 대 한 식별 하기 위해 전 임상 시험으로 도입 되었습니다. 이 원고는 PCT에 대 한 적격 환자 식별 Fabry Pompe 질병 유전자 이체의 신속한 phenotypic 평가 하 고 새로운 pharmacochaperones의 개발에 도움이 있습니다 개정된 셀 문화 분석 결과를 설명 합니다.

Mutagenesis 절차 GLA 유전자 mutagenesis의 효율성을 평가 하는 돌연변이 다음 범주 중 하나로 분류 되었다. 이 방법은 돌연변이 생성 하는 GLA 돌연변이의 66.5%에 대 한 첫 번째 시도에서 가져온 밝혔다. 약간 수정 된 두 번째 PCR 후 추가 25%를 얻을 수 있습니다.
카테고리 1:는 mutagenesis PCR 첫 번째 시도에서 ?...

Watch this Scientific Journal Video about In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease at JoVE.com

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

전 임상 재현, 빠르고, 효율적인 약리 보호자를 호출 하는 "고아" 약물의 소설 클래스에 대 한 테스트 있도록 수요가 있다. 우리 소설 약리 보호자 약물으로 서 적격 환자에 대 한 간단 하 고, 고도로 표준화, 다양 한 세포 문화 기반 분석 결과 화면을 개발 했다.

생체 외에서 Fabry에 Pompe 질병 테스트 약리 보호자 응답성을 효소 측정

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, ...

in-vitro-enzyme-measurement-to-test-pharmacological-chaperone

Research

JoVE Journal

Medicine

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

8.0K 조회수.  University Rostock Medical Center. 전 임상 재현, 빠르고, 효율적인 약리 보호자를 호출 하는 "고아" 약물의 소설 클래스에 대 한 테스트 있도록 수요가 있다. 우리 소설 약리 보호자 약물으로 서 적격 환자에 대 한 간단 하 고, 고도로 표준화, 다양 한 세포 문화 기반 분석 결과 화면을 개발 했다.

비디오: In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

IMAP 테스트 배터리 : 듣기의 감각을 만들기

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

연구

의학

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

고양이에서 급성 심근 경색

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

같은 자폐증과 정신 분열증과 같은 일반적인 장애에 드 노보 변이를 식별하는 전략

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

약시의 억제의 측정 및 치료

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

마우스 폐 삽관 법의 간단한 방법

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

신경 근육 질환의 마우스 모델에서 호흡 근육 활동 및 환기의 반복 측정

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

신생아 쥐의 달팽이관 Explant 기법으로<em&gt; 체외에서</em&gt; 청력 연구의 스크리닝 도구

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

이식 품질 평가를 위해 화학 생검 사용

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

메타볼로믹스 및 리피도믹스 분석을 위한 뇌종양의 현장 샘플링 및 추출

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

괴사 장염의 신생아 BALB/c 마우스 모델

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...