우리는 티베트 의과대학 제14차 5개년 계획의 함축 건설 프로젝트(2022ZYYGH12), 티베트 의과대학 교육부 티베트 의학 및 기초 교육의 핵심 실험실 및 기본 교육의 2022년 공개 과목, 닝샤 의과대학 핵심 연구 개발 프로그램(2023BEG02012)에서 받은 재정적 지원에 대해 진심으로 감사를 표합니다. Chengdu University of Traditional Chinese Medicine (XKTD2022013)의 Xinglin Scholar Research Promotion Project.

화합물 용액, 단백질 용액 및 염료를 PBS 용액에 도입했습니다. 그 후, 샘플은 디지털 가열 진탕 건식 수조를 사용하여 점진적으로 가열하고, 복잡한 시스템 내에서 형광 강도의 변화를 측정하여 단백질의 열적 안정성을 평가했습니다. 자세한 단계는 아래에 설명되어 있으며 그림 1 은 프로토콜의 개요를 보여줍니다.
1. 솔루션 준비
<ol><li>0.1mg의 umbelliferone을 616.75μL의 DMSO 용액에 첨가하여 1mM umbelliferone 용액을 준비합니다( 재료 표 참조).</li>
	<li>0.1mg의 CD40 내지 500 μL의ddH2O용액을 첨가하여 200 μg/mL CD40 단백질 용액을 준비한다( 재료 표 참조).</li>
	<li>1μL의 5000x 주황색 염료를 9μL의 인산염 완충 식염수(PBS)에 첨가하여 500x 주황색 염료 용액을 준비합니다( 재료 표 참조). 참고: 얼음에 대해 위의 작업을 수행하고 피펫의 최소 범위는 0.1μL이므로 처음에 주황색 단백질 염료를 10배로 희석하여 후속 사용을 용이하게 합니다.</li>
</ol>2. 염료 성능 테스트
<ol><li>500x 주황색 염료를 PBS 용액에 넣고 희석하여 염료(PBS 비율이 1:500, 1:1000, 1:2000 및 1:4000)가 되도록 합니다. 알림: DMSO의 최종 농도가 2%(v/v)를 초과하지 않는지 확인하십시오.</li>
	<li>형광 마이크로플레이트 리더를 켜고 10분 동안 예열합니다.</li>
	<li>컴퓨터와 데이터 수집 소프트웨어를 순서대로 엽니다( 재료 표 참조).</li>
	<li>Instrument를 클릭하고 해당 형광 마이크로플레이트 모델을 선택한 다음 데이터 수집 소프트웨어에서 OK를 선택합니다. 참고: 형광 마이크로플레이트가 예열을 완료하고 패널에 온도를 표시할 때까지 기다렸다가 데이터 수집 소프트웨어를 열어 기기와 소프트웨어 간의 성공적인 연결을 확인하십시오.</li>
	<li> Acquisition Settings(획득 설정)를 클릭하고 데이터 수집 소프트웨어에서 Fluorescence(형광)를 선택합니다.</li>
	<li>파장 인터페이스에서 프로그램을 편집합니다: Lm1 = 470 nm, 570 nm를 선택한 다음 확인을 선택합니다. 참고: 주황색 염료는 300nm의 UV 광 또는 470nm의 가시광선으로 여기될 수 있으며 방출은 570nm에서 측정할 수 있습니다.</li>
	<li>다양한 농도의 주황색 염료(1:500, 1:1000, 1:2000 및 1:4000)와 PBS를 100μL/웰의 96웰 플레이트에 추가합니다. 참고: PBS에 첨가하기 전에 DMSO에 용액을 용해시키고 염료의 침전 경향으로 인해 완전히 용해되도록 준비 과정에서 2000 x g 에서 완전히 소용돌이치십시오.</li>
	<li>96웰 플레이트를 기기의 검출 단계에 놓고, 데이터 수집 소프트웨어에서 읽기 버튼을 클릭하고, 각 염료 농도의 형광을 실온에서 매번 2분 간격으로 13회 측정합니다.</li>
	<li>각 결정 후 내보내기 버튼을 클릭하고, XML XLS TXT로 내보내기를 선택하고, 모든 플레이트를 선택한 다음, 출력 형식의 플레이트를 선택하고, 마지막으로 확인을 클릭하여 추가 분석을 위해 "XLS" 형식의 폴더에 데이터를 저장합니다. 참고: 이 단계는 실온에서 오렌지 염료의 자가형광에 대한 연속 측정의 영향을 결정하고 최적의 염색 농도를 식별하는 데 도움이 됩니다.</li>
	<li>디지털 가열 진탕 건욕을 켜고 가열 온도를 35°C, 가열 시간을 2분으로 설정합니다.</li>
	<li>1.5mL 미세 원심분리기 튜브에서 염료의 최적 희석 비율을 준비하고 디지털 가열 진탕 건욕에 2분 동안 넣습니다.</li>
	<li>PBS 용액과 35°C에서 가열된 염색 용액을 웰당 100μL의 부피로 96웰 플레이트에 추가합니다.</li>
	<li>96웰 플레이트를 기기의 검출 단계에 놓고 데이터 수집 소프트웨어에서 Read 버튼을 클릭합니다.</li>
	<li>가열 온도를 40°C로, 가열 시간을 2분으로 설정합니다.</li>
	<li>96웰 플레이트의 염료 용액을 1.5mL 미세 원심분리기 튜브에 다시 피펫팅하고 기기에서 2분 동안 가열합니다.</li>
	<li>2.12-2.15 단계 및 2.9 단계의 작업을 반복하여 각각 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C 및 95 °C에서 염료 용액의 흡광도를 테스트합니다. 참고: 이 단계는 35 °C에서 95 °C까지의 구배에서 연속 가열 하에서 주황색 염료의 자가형광 안정성을 결정하는 데 도움이 됩니다. 급격한 온도 강하나 실험 오류로 이어질 수 있는 잘못된 액체를 추가하지 않도록 신속하게 작업하는 것이 중요합니다.</li>
</ol>3. 단백질의 온도 용융(Tm) 감지
<ol><li>CD40 단백질과 오렌지 염료를 PBS 용액과 결합하여 각각 5μg/mL, 10μg/mL, 15μg/mL, 20μg/mL 및 1:500의 최종 농도를 달성합니다.</li>
	<li>2.2-2.6단계 및 2.10-2.16단계에 설명된 작업을 반복하여 각각 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C 및 95°C의 온도에서 CD40 단백질 용액의 흡광도를 평가합니다.</li>
	<li>2.9단계를 반복하여 데이터를 저장 및 분석한 다음 데이터 분석 소프트웨어를 사용하여 Tm 값을 계산합니다( 재료 표 참조).</li>
	<li>CD40 단백질, 움벨리페론 및 오렌지 염료를 PBS 용액에 첨가하여 각각 5μg/mL, 10μg/mL, 15μg/mL, 20μg/mL, 10μM 및 1:500의 최종 농도를 얻습니다. 참고: 용액 준비 중 격렬한 와류는 PBS에서 염료, CD40 단백질 및 umbelliferone의 균일한 분포를 보장하는 데 필수적입니다.</li>
	<li>3.2-3.3 단계의 작업을 반복하여 각각 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C 및 95 °C의 온도에서 CD40-umbelliferone 복합체 용액의 흡광도를 측정합니다. 참고: 이 단계는 CD40 단백질 및 CD40-umbelliferone 복합체의 Tm 값을 결정하기 위해 수행되었으며, umbelliferone에 대한 최적의 CD40 단백질 결합 농도를 식별하는 것을 목표로 합니다.</li>
</ol>

형광을 이용한 화합물 단백질 결합의 고감도 검출

<ol>
	<li>Cairang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+the+standardization+of+clinical+application+of+Tibetan+medicine+&amp;#34;five+roots&amp;#34.">Study on the standardization of clinical application of Tibetan medicine &amp;#34;five roots&amp;#34.</a> Guid J Trad Chin Med Pharm. 28 (11), 133136 (2022).</li><li>Li, L., 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=Effects+of+Xigui+extract+on+immunosuppressive+mice+induced+by+cyclophosphamide.">Effects of Xigui extract on immunosuppressive mice induced by cyclophosphamide.</a> Chin J Hosp Pharm. 37 (03), 244-247 (2017).</li><li>Lu, 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=Effects+of+Vicatia+thibertica+de+boiss+on+immunologic+and+hematopoietic+function+of+mice.">Effects of Vicatia thibertica de boiss on immunologic and hematopoietic function of mice.</a> J Dali Univ. 2 (02), 6-10 (2017).</li><li>Dong, S., Zhang, X., Hu, Y., Gong, X., Yang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+constituents,+quality+control+and+pharmacology+research+progress+of+Xigui.">Chemical constituents, quality control and pharmacology research progress of Xigui.</a> Chin J Ethnomed Ethnopharm. 27 (13), 40-42 (2018).</li><li>Lu, Z., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+total+flavone+extract+from+Shuiqin+(Oenanthe+Javanica)+on+immune+function+of+immunosuppression+mice.">Effects of total flavone extract from Shuiqin (Oenanthe Javanica) on immune function of immunosuppression mice.</a> Chin J Trad Med Sci Technol. 23 (04), 423-425 (2016).</li><li>Vonderheide, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+agonist+antibodies+in+cancer+immunotherapy.">CD40 agonist antibodies in cancer immunotherapy.</a> Annu Rev Med. 71, 47-58 (2020).</li><li>Grewal, I. S., Flavell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+and+CD154+in+cell-mediated+immunity.">CD40 and CD154 in cell-mediated immunity.</a> Annu Rev Immunol. 16, 111-135 (1998).</li><li>Long, K. B., 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=IFN&#947;+and+CCL2+cooperate+to+redirect+tumor-infiltrating+monocytes+to+degrade+fibrosis+and+enhance+chemotherapy+efficacy+in+pancreatic+carcinoma.">IFN&#947; and CCL2 cooperate to redirect tumor-infiltrating monocytes to degrade fibrosis and enhance chemotherapy efficacy in pancreatic carcinoma.</a> Cancer Discov. 6 (4), 400-413 (2016).</li><li>He, Y., 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=The+roles+of+regulatory+B+cells+in+cancer.">The roles of regulatory B cells in cancer.</a> J Immunol Res. 2014, 215471 (2014).</li><li>Eliopoulos, A. 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=CD40+induces+apoptosis+in+carcinoma+cells+through+activation+of+cytotoxic+ligands+of+the+tumor+necrosis+factor+superfamily.">CD40 induces apoptosis in carcinoma cells through activation of cytotoxic ligands of the tumor necrosis factor superfamily.</a> Mol Cell Biol. 20 (15), 5503-5515 (2000).</li><li>Niesen, F. H., Berglund, H., Vedadi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+differential+scanning+fluorimetry+to+detect+ligand+interactions+that+promote+protein+stability.">The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.</a> Nat Protoc. 2 (9), 2212-2221 (2007).</li><li>Rosa, N., 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=Meltdown:+A+Tool+to+help+in+the+interpretation+of+thermal+melt+curves+acquired+by+differential+scanning+fluorimetry.">Meltdown: A Tool to help in the interpretation of thermal melt curves acquired by differential scanning fluorimetry.</a> J Biomol Screen. 20 (7), 898-905 (2015).</li><li>Hellman, L. M., 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=Differential+scanning+fluorimetry+based+assessments+of+the+thermal+and+kinetic+stability+of+peptide-MHC+complexes.">Differential scanning fluorimetry based assessments of the thermal and kinetic stability of peptide-MHC complexes.</a> J Immunol Methods. 432, 95-101 (2016).</li><li>Pantoliano, M. W., 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=High-density+miniaturized+thermal+shift+assays+as+a+general+strategy+for+drug+discovery.">High-density miniaturized thermal shift assays as a general strategy for drug discovery.</a> J Biomol Screen. 6 (6), 429-440 (2001).</li><li>Gorny, 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=Combining+nano-differential+scanning+fluorimetry+and+microscale+thermophoresis+to+investigate+VDAC1+interaction+with+small+molecules.">Combining nano-differential scanning fluorimetry and microscale thermophoresis to investigate VDAC1 interaction with small molecules.</a> J EnzymeInhib Med Chem. 38 (1), 2121821 (2023).</li><li>Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+denaturation+methods+in+the+study+of+protein+folding.">Thermal denaturation methods in the study of protein folding.</a> Methods Enzymol. 259, 144-168 (1995).</li><li>Phiri, M. J., Mofokeng, J. P., Phiri, M. M., Mngomezulu, M., Tywabi-Ngeva, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical,+thermal+and+morphological+properties+of+polybutylene+succinate-waste+pineapple+leaf+fibres+composites.">Chemical, thermal and morphological properties of polybutylene succinate-waste pineapple leaf fibres composites.</a> Heliyon. 9 (11), 21238 (2023).</li><li>Zhou, C., Yu, M., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparation+of+three+measuring+methods+for+thermodynamic+stability+of+protein.">Comparation of three measuring methods for thermodynamic stability of protein.</a> Anal Test Technol Instruments. 27 (04), 252-259 (2021).</li><li>Hou, Y., 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=Salidroside+intensifies+mitochondrial+function+of+CoCl2-damaged+HT22+cells+by+stimulating+PI3K-AKT-MAPK+signaling+pathway.">Salidroside intensifies mitochondrial function of CoCl2-damaged HT22 cells by stimulating PI3K-AKT-MAPK signaling pathway.</a> Phytomedicine. 109, 154568 (2023).</li><li>Wang, X., 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=Salidroside,+a+phenyl+ethanol+glycoside+from+Rhodiola+crenulata,+orchestrates+hypoxic+mitochondrial+dynamics+homeostasis+by+stimulating+Sirt1/p53/Drp1+signaling.">Salidroside, a phenyl ethanol glycoside from Rhodiola crenulata, orchestrates hypoxic mitochondrial dynamics homeostasis by stimulating Sirt1/p53/Drp1 signaling.</a> J Ethnopharmacol. 293, 115278 (2022).</li><li>Magnez, R., Bailly, C., Thuru, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+thermophoresis+as+a+tool+to+study+protein+interactions+and+their+implication+in+human+diseases.">Microscale thermophoresis as a tool to study protein interactions and their implication in human diseases.</a> Int J Mol Sci. 23 (14), 7672 (2022).</li><li>Kamat, V., Rafique, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+binding+kinetic+assay+on+the+bio-layer+interferometry+(BLI)+biosensor+to+characterize+antibody-antigen+interactions.">Designing binding kinetic assay on the bio-layer interferometry (BLI) biosensor to characterize antibody-antigen interactions.</a> Anal Biochem. 536, 16-31 (2017).</li><li>Freyer, M. W., Lewis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+titration+calorimetry:+experimental+design,+data+analysis,+and+probing+macromolecule/ligand+binding+and+kinetic+interactions.">Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions.</a> Methods Cell Biol. 84, 79-113 (2008).</li><li>Sharma, R., 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=Atosiban+and+Rutin+exhibit+anti-mycobacterial+activity+-+An+integrated+computational+and+biophysical+insight+toward+drug+repurposing+strategy+against+Mycobacterium+tuberculosis+targeting+its+essential+enzyme+HemD.">Atosiban and Rutin exhibit anti-mycobacterial activity - An integrated computational and biophysical insight toward drug repurposing strategy against Mycobacterium tuberculosis targeting its essential enzyme HemD.</a> Int J Biol Macromol. 253, 127208 (2023).</li><li>Gr&#228;dler, U., 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=Biophysical+and+structural+characterization+of+the+impacts+of+MET+phosphorylation+on+tepotinib+binding.">Biophysical and structural characterization of the impacts of MET phosphorylation on tepotinib binding.</a> J Biol Chem. 299 (11), 105328 (2023).</li><li>Xu, Y., 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=Differential+scanning+fluorimetry+and+its+application+in+the+study+of+protein.">Differential scanning fluorimetry and its application in the study of protein.</a> Chemistry of Life. 38 (03), 351-357 (2018).</li></ol>

저자는 밝힐 것이 없습니다.

열 이동 분석 또는 열 형광 분석으로도 알려진 DSF는 느리고 프로그래밍된 온도 상승 동안 테스트 샘플 또는 염료의 형광 신호 변화를 모니터링하여 샘플에서 단백질의 열 변성 과정을 감지하는 데 사용되는 기술입니다. Pantoliano14에 의해 처음 확립된 DSF는 고처리량 방법 역할을 합니다. 주요 절차에는 컴퓨터로 제어되는 가열판의 온도를 높이고, 장파장 자외선 램프를 사용하여 ?...

비카티아 티베티카(Vicatia thibetica ) H. Boissieu는 티베트 의학에서 일반적으로 사용되며, 5가지 기본 성분(Polygonatum sibiricum Delar. ex Redoute, Asparagus cochinchinensis (Lour.) Merr, Vicatia thibetica H. Boissieu, Oxybaphus himalaicus Edgew. 및 Gymnadenia conopsea (L.) R. Br.) 1. 주로 윈난성 북서부, 쓰촨성 서부, 티베트 및 기타 지역과 같은 중국 남서부에 분포하며 말린 뿌리는 Angelica1의 현지 대체품 역할을합니다. 향기로 유명한 뿌리는 종종 스튜 조미료로 사용되며, 티베트 셀러리라고 불리는 잎은 맛있는 요리에 기여합니다. 따라서 Vicatia thibetica 는 독특한 약용 식물뿐만 아니라 티베트의 식품 공급원으로도 중요합니다.
국내외 연구에 따르면 Vicatia thibetica는 혈액 보충 및 기(힘 또는 에너지) 활력을 주는 특성을 가지고 있어 월경 조절에 도움이 됩니다. 두근거림항진과 혈액 결핍으로 인한 불규칙한 월경통의 증상을 해결하기 위해 사용되며, 신체 면역의 항산화 조절과 같은 약리학적 효과를 나타낸다2. 비카티아 티베티카(Vicatia thibetica)의 알코올 추출물은 사이클로포스파마이드(cyclophosphamide)에 의해 유발된 면역력이 저하된 쥐의 체질량과 장기 지수를 회복하는 능력을 입증했습니다. 또한 혈청 내 슈퍼옥사이드 디스뮤타아제의 활성을 증가시키고 말론디알데히드의 함량을 감소시켜 항산화 능력의 향상과 지질 과산화 2,3에 대한 균형 효과를 시사합니다. 동시에 혈액 세포와 헤모글로빈의 수를 증가시켜 신체의 조혈 기능을 객관적으로 반영할 뿐만 아니라 면역 체계에 중요한 역할을 합니다 2,3.
침상 결정과 쓴 맛이 특징인 Umbelliferone은 분자량이 작고 휘발성이며 증기로 증류할 수 있습니다. 그것은 쉽게 승화하고 물에 대한 용해도가 낮고 유기물에 대한 용해도가 높습니다. Vicatia thibetica의 주요 화학 성분 중 하나인 umbelliferone은 하이드로코르티손 유도 면역억제 마우스 모델 4,5에서 세포 면역, 체액성 면역 및 비특이적 면역에 대한 면역조절 효과를 나타냅니다.
종양괴사인자 수용체 슈퍼패밀리(tumor necrosis factor receptor superfamily)의 구성원인 세포 표면 분자 CD40은 면역 세포에서 널리 발현됩니다6. CD40L이라고도 하는 상동 리간드인 CD154는 활성화된 T 림프구에 의해 발현되는 II형 막관통 단백질입니다. CD40 활성화는 수지상세포(DC)와 단핵구의 표면에서 공동 자극 분자의 발현을 상향 조절할 수 있는 능력을 가지고 있습니다. 이 과정은 주요 조직적합성 복합체(MHC) 분자의 항원 제시 기능을 촉진하고 CD8+ T 세포를 더욱 활성화시킨다7.
대식세포는 종양 미세환경의 형성과 조절에 중요한 역할을 하며, CD40 활성화는 대식세포에 의한 종양 미세환경의 리모델링을 향상시킬 수 있다8. CD40 신호 활성화는 B 세포의 증식과 활성화에 큰 영향을 미칩니다. B 세포는 CD40에 의해 활성화될 때 효과적인 항원 제시 세포로 기능할 수 있습니다. 이들은 항원을 제시하고, effector T cell 활성을 생성하며, 이에 따라 항종양 효과에 기여한다9. 또한, 종양 세포에서 CD40 활성화는 세포사멸을 유도하고 종양 성장을 억제할 수 있다10. CD40은 주로 Lyn, Fyn, Syk 등을 포함한 비수용체 티로신 단백질 키나아제의 활성을 조절하여 신호를 전달합니다. 또한 Bcl-xL, Cdk4 및 Cdk6 단백질을 자극하고, Rel/NF-kB 전사 인자를 활성화하고, CG-2 및 PI3K10을 인산화하는 능력이 있습니다.
DSF(Differential Scanning Fluorescence) 방법은 완충액 조성, 온도 및 저분자 리간드와 같은 다양한 환경 조건이 단백질 구조의 열적 안정성에 미치는 영향을 평가하는 데 널리 사용됩니다. DSF에 일반적으로 사용되는 염료는 주황색, 환경에 민감한 소수성 염료입니다. 정상적인 조건에서는 단백질 구조가 접혀 내부적으로 소수성 세그먼트가 숨겨집니다. 온도가 상승함에 따라 단백질의 소수성 영역이 더 많이 노출되어 자연 단백질 구조가 점진적으로 파괴됩니다. 염료는 이 노출된 단백질 부분에 선택적으로 결합하여 형광을 증폭합니다. 그런 다음 형광 신호 검출(11)의 변화를 모니터링하여 Tm 값을 계산합니다. 어느 정도까지, Tm 값의 변화는 돌연변이, 완충액의 변화 또는 리간드 결합으로 인한 단백질 안정성의 변화를 측정할 수 있습니다. 또한, 단백질 접힘 과정(protein folding process) 동안의 구조적 변화를 나타낼 수 있다12. 이 접근법은 정확한 데이터, 넓은 온도 범위, 높은 감도 및 최소한의 단백질 샘플 손실을 제공합니다13.
이 연구에서는 형광 정량 중합효소 연쇄 반응(PCR) 장치 대신 형광 마이크로플레이트 리더를 사용하여 형광 측정을 수행했습니다. 이러한 수정을 통해 형광 정량 PCR 기기가 없는 실험실에서 DSF를 검출할 수 있으므로 분석법이 덜 복잡해지고 기기 설정에 필요한 단계가 줄어들어 실험 프로세스가 간소화됩니다. 그러나 이 방법에는 몇 가지 단점이 있습니다. 복잡성은 줄어들지만 절차는 더 번거로워집니다. 다양한 온도에서 수동 형광 검출이 필요하며, 시스템에서 형광을 자동적이고 지속적으로 수집할 수 없습니다. 따라서 이 연구는 DSF 기술을 활용하여 CD40 단백질과 움벨리페론 사이의 상호 작용을 탐구하여 티베트 의학의 분자 메커니즘에 대한 새로운 통찰력을 제공했습니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CD40 protein</td>
			<td>MedChemExpress</td>
			<td>HY-P75408</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0040</td>
			<td></td>
		</tr>
		<tr>
			<td>FlexStation 3 multifunctional microplate reader</td>
			<td>Shanghai Meigu Molecular Instruments Co., LTD</td>
			<td>FlexStation 3</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro 8 software</td>
			<td>OriginLab Corporation</td>
			<td>v8.0724(B724)</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (1x)</td>
			<td>Gibco</td>
			<td>8120485</td>
			<td></td>
		</tr>
		<tr>
			<td>SoftMax Pro 7.1</td>
			<td>Shanghai Meigu Molecular Instruments Co., LTD</td>
			<td>SoftMax Pro 7.1</td>
			<td></td>
		</tr>
		<tr>
			<td>SSYPRO orange dye</td>
			<td>Sigma</td>
			<td>S5942</td>
			<td></td>
		</tr>
		<tr>
			<td>Umbelliferone</td>
			<td>Shanghai Yuanye Biotechnology Co.</td>
			<td>B21854</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of cd40 protein-umbelliferone interaction via differential scanning fluorescence

서로 다른 분자 간의 상호 작용에 대한 조사는 질병 발병 기전을 이해하고 약물 표적을 스크리닝하는 데 중요한 측면입니다. 티베트 의학 Vicatia thibetica의 활성 성분인 Umbelliferone은 메커니즘을 알 수 없는 면역 조절 효과를 나타냅니다. CD40 단백질은 면역 반응의 핵심 표적입니다. 따라서 본 연구는 시차주사형광기술의 원리를 이용하여 형광효소마커를 이용하여 CD40 단백질과 움벨리페론의 상호작용을 분석한다. 초기에 단백질 형광 오렌지 염료의 안정성을 실험적으로 검증하고 1:500의 최적 희석 비율을 결정했습니다. 그 결과, CD40 단백질의 온도 용융(Tm) 값이 농도가 증가함에 따라 감소하는 경향이 있음을 관찰했습니다. 흥미롭게도, CD40 단백질과 umbelliferone 사이의 상호 작용은 CD40 단백질의 열 안정성을 향상시키는 것으로 밝혀졌습니다. 이 연구는 형광 마이크로플레이트와 형광 염료를 사용하여 저분자 화합물 및 단백질의 결합 잠재력을 감지하려는 최초의 시도입니다. 이 기술은 높은 감도와 정확성이 특징이며 단백질 안정성, 단백질 구조 및 단백질-리간드 상호 작용 분야에서 유망한 발전을 약속하여 추가 연구 및 탐색을 용이하게 합니다.

주황색 염료는 실온 및 고온 모두에서 Ex = 470 nm 및 Em = 570 nm에서 안정적인 형광 여기를 일관되게 나타냈습니다. 1:500의 최적 희석 비율이 결정되었습니다(그림 2A,B). CD40 단백질의 농도가 15μg/mL 미만일 때는 Tm 값을 검출하기가 어려웠습니다(그림 3A,B). 그러나 15μg/mL의 농도에서는 51.82°C의 안정적인 Tm 값이 검출되었으며, CD40 단...

Watch this Scientific Journal Video about Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency at JoVE.com

저자 스포트라이트: 접근성 및 효율성 향상을 위한 PCR 방법 간소화

이 프로토콜은 화합물과 단백질 분자 간의 결합을 검출하는 고유한 방법을 설명하며, 단백질 샘플 손실을 최소화하고 데이터 정확도가 높다는 이점을 제공합니다.

Differential Scanning Fluorescence를 통한 CD40 단백질-Umbelliferone 상호작용 검출

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

당사의 프로토콜은 특수 장비 없이 사용할 수 있도록 함으로써 상당한 이점을 제공합니다. 기존의 형광 정량 PCR 방법과 달리 실험 과정을 단순화하여 더 적은 단계를 필요로하고 전반적인 복잡성을 줄여 더 안전하고 쉽게 수행 할 수 있습니다. 이 실험을 위해서는 작업자가 실험 절차와 형광 마이크로플레이트 리더에 매우 익숙해야 하며 용액 가열 및 형광 검출 중에 너무 많은 시간을 낭비하지 않아야 합니다.우리는 umbelliferone이 CD14 단백질에 결합하여 면역 효과를 일으키는 분자 메커니즘에 초점을 맞출 것입니다.

detection-cd40-protein-umbelliferone-interaction-via-differential

Research

JoVE Journal

Medicine

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

371 조회수.  University of Tibetan Medicine. 당사의 프로토콜은 특수 장비 없이 사용할 수 있도록 함으로써 상당한 이점을 제공합니다. 기존의 형광 정량 PCR 방법과 달리 실험 과정을 단순화하여 더 적은 단계를 필요로하고 전반적인 복잡성을 줄여 더 안전하고 쉽게 수행 할 수 있습니다. 이 실험을 위해서는 작업자가 실험 절차와 형광 마이크로플레이트 리더에 매우 익숙해야 하며 용액 가열 및 형광 검출 중에 너무 많...

비디오: 저자 스포트라이트: 접근성 및 효율성 향상을 위한 PCR 방법 간소화

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Gynecologic 종양학의 센티널 림프절의 Intraoperative 감지 Multispectral 실시간 형광 이미징

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

연구

의학

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

형광에 원위치 하이브리드화 및 공촛점 레이저 스캐닝 현미경에 의해 맛이 좋은 확장기의 사정 Biofilm 분석

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

인간 Mammary 섬유아 세포에서 암종 - 관련 섬유아 세포의 실험 세대 (CAFs)

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

암 치료 모니터링을위한 Cerenkov 발광 이미징 (CLI)

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns

We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of breast tissue patterns in mammograms. We hypothesize that architectural distortion modifies the normal orientation of breast tissue patterns in mammographic images before the formation of masses or tumors. In the initial steps of our methods, the oriented structures in a given mammogram are analyzed using Gabor filters and phase portraits to detect node-like sites of radiating or intersecting tissue patterns. Each detected site is then characterized using the node value, fractal dimension, and a measure of angular dispersion specifically designed to represent spiculating patterns associated with architectural distortion.
Our methods were tested with a database of 106 prior mammograms of 56 interval-cancer cases and 52 mammograms of 13 normal cases using the features developed for the characterization of architectural distortion, pattern classification via quadratic discriminant analysis, and validation with the leave-one-patient out procedure. According to the results of free-response receiver operating characteristic analysis, our methods have demonstrated the capability to detect architectural distortion in prior mammograms, taken 15 months (on the average) before clinical diagnosis of breast cancer, with a sensitivity of 80% at about five false positives per patient.

Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns

We demonstrate methods for the detection of architectural distortion in prior mammograms. Oriented structures are analyzed using Gabor filters and phase portraits to detect sites of radiating tissue patterns. Each site is characterized and classified using measures to represent spiculating patterns. The methods should assist in the detection of breast cancer.

이전 유방 X 선 사진에있는 건축 왜곡의 검출<em&gt;를 통해</em지향 패턴의&gt; 분석

We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

검색 및 대 식 세포 관련 Murine 창 자 염증의 정량화에 대 한 단층 형광 중재

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

콜레스테롤 입자의 형태를 변조에 지질 저하 약물의 차동 효과

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts

The purpose of this paper is to introduce a promising, novel method to aid in the assessment of bone quality in forensically relevant skeletal remains. BMD is an important component of bone&#39;s nutritional status and in skeletal remains of both juveniles and adults, and it can provide information about bone quality. For adults remains, it can provide information on pathological conditions or when bone insufficiency may have occurred. In juveniles, it provides a useful metric to elucidate cases of fatal starvation or neglect, which are generally difficult to identify. This paper provides a protocol for the anatomical orientation and analysis of skeletal remains for scanning via dual-energy X-ray absorptiometry (DXA). Three case studies are presented to illustrate when DXA scans can be informative to the forensic practitioner. The first case study presents an individual with observed longitudinal fractures in the weight bearing bones and DXA is used to assess bone insufficiency. BMD is found to be normal suggesting another etiology for the fracture pattern present. The second case study employed DXA to investigate suspected chronic malnutrition. The BMD results are consistent with results from long bone lengths and suggest the juvenile had suffered from chronic malnutrition. The final case study provides an example where fatal starvation in a fourteen-month infant is suspected, which supports autopsy findings of fatal starvation. DXA scans showed low bone mineral density for chronological age and is substantiated by traditional assessments of infant health. However, when dealing with skeletal remains taphonomic alterations should be considered before applying this method.

Bone mineral density (BMD) is an important factor in understanding nutritional intake. For human skeletal remains, it is a useful metric to assess quality of life in both juveniles and adults, particularly in fatal starvation and neglect cases. This paper provides guidelines for scanning human skeletal remains for forensic purposes.

법정 컨텍스트에서 뼈 미네랄 밀도 대 한 남아 골격 검사

The purpose of this paper is to introduce a promising, novel method to aid in the assessment of bone quality in forensically relevant skeletal remains. ...

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

주사 전자 현미경을 이용한 멤브레인 러플 형성 시각화

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...

Detection and Removal of Tooth-Colored Composite Resin Using the Fluorescence-Aided Identification Technique

The detection and removal of tooth-colored filling materials is a major challenge for every dentist. The Fluorescence-aided Identification Technique (FIT) is a noninvasive tool to facilitate the distinction of composite resin material from sound tooth substance. Compared to conventional illumination, FIT is a very accurate, reliable, and fast diagnostic method. When composite resin is illuminated with a wavelength of approximately 398 &plusmn; 5 nm, certain fluorescent components make the composite resin appear brighter than the tooth structure. Any fluorescence-inducing light source with the appropriate wavelength can be used for this method. Optimally, this technique is used without additional natural or artificial lighting. The application of FIT can be used for diagnostic purposes, for example, dental charts, and additionally for the complete and minimally invasive removal of composite resin restorations, bracket debonding, and trauma splint removal. The assessment of volumetric changes after composite removal can be provided by overlapping pre- and postoperative scans and subsequent calculation using suitable software.

The Fluorescence-aided Identification Technique is a practicable, fast, and reliable approach for the differentiation of composite resin restorations from tooth substance, and facilitates the minimally invasive and complete removal of composite resin restorations and composite bonded trauma splints.

형광 보조 식별 기술을 사용한 치아색 복합 수지의 검출 및 제거

The detection and removal of tooth-colored filling materials is a major challenge for every dentist. The Fluorescence-aided Identification Technique (FIT) ...