المؤلفين الامتنان بالدعم المالي المقدم من مؤسسة البحوث الألمانية (DFG) عن طريق مركز البحوث التعاونية SFB 578 &quot;من جين إلى المنتج&quot; في الجامعة التقنية براونشفايغ، ألمانيا.

<ol>
	<li>Wucherpfennig, T., 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> Advances in Applied Microbiology. 72, 89-136 (2010).</li><li>Driouch, H., H&#228;nsch, R., Wucherpfennig, T., Krull, R., Wittmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+enzyme+production+by+bio-pellets+of+Aspergillus+niger:+Targeted+morphology+engineering+using+titanate+microparticles.">Improved enzyme production by bio-pellets of Aspergillus niger: Targeted morphology engineering using titanate microparticles.</a> Biotechnology and Bioengineering. 109, 462-471 (2012).</li><li>Driouch, H., Roth, A., Dersch, P., Wittmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+bioprocess+for+production+of+fructofuranosidase+by+recombinant+Aspergillus+niger.">Optimized bioprocess for production of fructofuranosidase by recombinant Aspergillus niger.</a> Applied Microbiology and Biotechnology. 87, 2011-2024 (2010).</li><li>Driouch, H., Roth, A., Dersch, P., Wittmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+fungi+in+good+shape:+Microparticles+for+tailor-made+fungal+morphology+and+enhanced+enzyme+production.">Filamentous fungi in good shape: Microparticles for tailor-made fungal morphology and enhanced enzyme production.</a> Bioengineered Bugs. 2, 100-104 (2011).</li><li>Driouch, H., Sommer, B., Wittmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+engineering+of+Aspergillus+niger+for+improved+enzyme+production.">Morphology engineering of Aspergillus niger for improved enzyme production.</a> Biotechnology and Bioengineering. 105, 1058-1068 (2010).</li><li>Kaup, B. -. A., Ehrich, K., Pescheck, M., Schrader, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticle-enhanced+cultivation+of+filamentous+microorganisms:+Increased+chloroperoxidase+formation+by+Caldariomyces+fumago+as+an+example.">Microparticle-enhanced cultivation of filamentous microorganisms: Increased chloroperoxidase formation by Caldariomyces fumago as an example.</a> Biotechnology and Bioengineering. 99, 491-498 (2008).</li><li>Hirayama, M., Sumi, N., Hidaka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+a+fructooligosaccharide-producing+beta-fructofuranosidase+from+Aspergillus+niger+ATCC+20611.">Purification and characterization of a fructooligosaccharide-producing beta-fructofuranosidase from Aspergillus niger ATCC 20611.</a> Agricultural and Biological Chemistry. 53, 667-673 (2006).</li><li>Rajoka, M. I., Yasmeen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+productivity+of+beta-fructofuranosidase+by+a+derepressed+mutant+of+Aspergillus+niger+form+conventional+and+non-conventional+substrates.">Improved productivity of beta-fructofuranosidase by a derepressed mutant of Aspergillus niger form conventional and non-conventional substrates.</a> World Journal of Microbiology and Biotechnology. 21, 471-478 (2005).</li><li>Zuccaro, A., G&#195;&#182;tze, S., Kneip, S., Dersch, P., Seibel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailor-made+fructooligosaccharides+by+a+combination+of+substrate+and+genetic+engineering.">Tailor-made fructooligosaccharides by a combination of substrate and genetic engineering.</a> ChemBioChem. 9, 143-149 (2008).</li><li>Huggett, A. S. G., Nixon, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+glucose+oxidase,+peroxidase,+and+o-dianisidin+in+determination+of+blood+and+urinary+glucose.">Use of glucose oxidase, peroxidase, and o-dianisidin in determination of blood and urinary glucose.</a> The Lancet. , 270-368 (1957).</li><li>Wucherpfenning, T., Hestler, T., Krull, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+engineering+-+Osmolality+and+its+effect+on+Aspergillus+niger+morphology+and+productivity.">Morphology engineering - Osmolality and its effect on Aspergillus niger morphology and productivity.</a> Microb. Cell Fact. 10, (2011).</li><li>Rasband, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (1997).</li><li>Papagianni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+fractal+nature+of+mycelial+aggregation+in+Aspergillus+niger+submerged+cultures.">Quantification of the fractal nature of mycelial aggregation in Aspergillus niger submerged cultures.</a> Microbial Cell Factories. 5, 5 (2006).</li></ol>

ليس لدي ما يكشف.

وكان التعديل من التشكل الفطرية من اهتمام في مجال التكنولوجيا الحيوية منذ عقود عديدة. وقد حاولت دراسات مختلفة لعملية اختيار تختلف المعلمات مثل قيمة درجة الحموضة، ومدخلات الطاقة، ودرجة الحرارة، والمغذيات متوسطة الحجم أو تركيز اللقاح 1، ولكن تعاني من سيطرة غير دقيقة وغير كاملة بدلا من التشكل، ارتفاع تكاليف الطاقة، والآثار تثبيط أو عدم الاستقرار الناتج، وفي المقابل، ومكملات من الجسيمات الدقيقة يسمح للهندسة دقيقة من التشكل الفطرية من خلال صقل تباين حجم الجسيمات وتركيز. هذا يفتح آفاقا جديدة لاستخدام جزيئات متناهية الصغر من أجل التحسين ومصممة خصيصا من تصميم التشكل إنتاج عالية في إنتاج التكنولوجيا الحيوية مع أ. النيجر والكائنات الدقيقة الخيطية الأخرى. تحليل الصور الرقمية هي وسيلة سهلة للتكرار لوصف الفطرية مورفولوجيا الماكرو. ومع ذلك، فإن العديد من المعلمات لقصص الأطفال الحجم والشكل والسطحيةثالثا البنى الصرفية وصفها في المؤلفات يجعل تقييم سريع للمورفولوجيا الفطرية معقدة. عدد الصرف كما عرضت مجموعة من المعايير ذات الصلة، والابتعاد عن هذا النقص، ويمكن استخدامها ليس فقط لتوصيف شامل لهياكل شكلية، بل أيضا لارتباط الرياضية مباشرة مع الإنتاجية. هذا يجعل من جديد تقدير من الإنتاجية عن طريق التشكل معين، وبالتالي التخصيص من التشكل للالعملية تحتاج ممكن. باستخدام عدد الصرف، فمن الممكن أن نميز بين مختلف بيليه وmorphologies أجمة 4،5. لمزيد من التطوير لعدد الصرف ومراعاة البعد كسورية ويبدو أن واعدة. والبعد كسورية يعطي قياس لمدى تعقيد والشامل ملء خصائص كائن 13 و predestinated وبالتالي لتوصيف شامل لمورفولوجيا فطر. على كرeation من مورفولوجية عالية فطر المنتجة، ومع ذلك، قد يؤدي إلى مشاكل مع أداء عملية خاصة في زراعة على نطاق واسع، لأنه قد تم على شكل نمو mycelial أظهرت في وقت سابق لعرض أكبر بكثير اللزوجة مرق ثقافة 2. هذا يؤدي إلى مشاكل مع انتقال الحرارة والكتلة وتشكيل مناطق راكدة غير مختلطة، والتي تتطلب مساهمة أكبر من الطاقة، وجعل زراعة أكثر تكلفة لتشغيل 1. ولذلك ينبغي النظر في العلاقة بين مورفولوجيا الفطرية وثقافة مرق اللزوجة عند تغيير الصرف وإدماجها في نماذج أخرى.

Table of Equipment:
<table border="1">
	<tbody>
		<tr>
			<td>
			Equipment</td>
			<td>
			Company</td>
			<td>
			Catalogue Number/model</td>
		</tr>
		<tr>
			<td>
			autoclave</td>
			<td>
			Systec</td>
			<td>
			V150</td>
		</tr>
		<tr>
			<td>
			B&uuml;chner funnel (plastic)</td>
			<td>
			VWR</td>
			<td>
			-</td>
		</tr>
		<tr>
			<td>
			cellulose filter (for biomass dry weight)</td>
			<td>
			Sartorius Stedim Biotech</td>
			<td>
			Filter Discs Grade 389</td>
		</tr>
		<tr>
			<td>
			cellulose acetate filter (for air filtration at reactor)</td>
			<td>
			Sartorius stedim biotech</td>
			<td>
			Midisart 200 PTFE</td>
		</tr>
		<tr>
			<td>
			cellulose acetate filter (for enzyme activity)</td>
			<td>
			Sartorius Stedim Biotech</td>
			<td>
			Midisart NML</td>
		</tr>
		<tr>
			<td>
			centrifuge</td>
			<td>
			Eppendorf</td>
			<td>
			Centrifuge 5415R</td>
		</tr>
		<tr>
			<td>
			centrifuge</td>
			<td>
			Heraeus</td>
			<td>
			Biofuge fresco</td>
		</tr>
		<tr>
			<td>
			centrifuge</td>
			<td>
			Heraeus sepatech</td>
			<td>
			Varifuge 3.0R</td>
		</tr>
		<tr>
			<td>
			compartment dryer (105 &deg;C)</td>
			<td>
			Heraeus</td>
			<td>
			Kelvitran t</td>
		</tr>
		<tr>
			<td>
			control unit (temperature)</td>
			<td>
			Jumo</td>
			<td>
			Jumo iTron 08</td>
		</tr>
		<tr>
			<td>
			control unit (pH-value)</td>
			<td>
			meredos</td>
			<td>
			pH Control 2</td>
		</tr>
		<tr>
			<td>
			desiccator</td>
			<td>
			Duran</td>
			<td>
			Vacuum stable</td>
		</tr>
		<tr>
			<td>
			Falcon tubes</td>
			<td>
			Omnilab</td>
			<td>
			FALC352070</td>
		</tr>
		<tr>
			<td>
			heating block 40 &deg;C</td>
			<td>
			Biometra</td>
			<td>
			TB1 Thermoblock</td>
		</tr>
		<tr>
			<td>
			heating block 95 &deg;C</td>
			<td>
			HLC</td>
			<td>
			HBT 130</td>
		</tr>
		<tr>
			<td>
			micro plate reader</td>
			<td>
			Tecan</td>
			<td>
			Sunrise-Microplate-Reader</td>
		</tr>
		<tr>
			<td>
			micro scales</td>
			<td>
			Sartorius</td>
			<td>
			CP 225</td>
		</tr>
		<tr>
			<td>
			microscope (digital inverted)</td>
			<td>
			AMG</td>
			<td>
			EVOS xl</td>
		</tr>
		<tr>
			<td>
			micro pipettes and tips (different sizes)</td>
			<td>
			Omnilab</td>
			<td>
			5283303
			5283298 
			5283299 
			5283300</td>
		</tr>
		<tr>
			<td>
			micro titer plate</td>
			<td>
			Nunc</td>
			<td>
			MaxiSorp</td>
		</tr>
		<tr>
			<td>
			multi pipette and tips</td>
			<td>
			Eppendorf/ Omnilab</td>
			<td>
			5283611/ 5283611</td>
		</tr>
		<tr>
			<td>
			pH-electrode</td>
			<td>
			Schott</td>
			<td>
			pH-Meter CG840</td>
		</tr>
		<tr>
			<td>
			reaction tubes</td>
			<td>
			Roth</td>
			<td>
			E518.1</td>
		</tr>
		<tr>
			<td>
			scale</td>
			<td>
			Sartorius</td>
			<td>
			CP 3202 S</td>
		</tr>
		<tr>
			<td>
			stirred tank bioreactor with equipment</td>
			<td>
			Applikon Biotechnology</td>
			<td>
			2L Bioreactor set</td>
		</tr>
		<tr>
			<td>
			syringe</td>
			<td>
			Eppendorf</td>
			<td>
			Combitips Plus 5 mL</td>
		</tr>
	</tbody>
</table>
Table of Reagents:
<table border="1">
	<tbody>
		<tr>
			<td>
			Name of the reagent</td>
			<td>
			Company </td>
		</tr>
		<tr>
			<td>
			Acetic acid</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Disodium hydrogen phosphate</td>
			<td>
			Merck</td>
		</tr>
		<tr>
			<td>
			Ethanol (95%)</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Glucose monohydrate, (&alpha;-D-)</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Glucose oxidase (Typ II from Aspergillus niger)</td>
			<td>
			Sigma</td>
		</tr>
		<tr>
			<td>
			Hydrochloride acid (37 % w/v)</td>
			<td>
			Fiedel-de Ha&euml;n</td>
		</tr>
		<tr>
			<td>
			Hydrous magnesium silicate</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Monopotassium phosphate</td>
			<td>
			Merck</td>
		</tr>
		<tr>
			<td>
			o-dianoisidine dihydrochloride</td>
			<td>
			Sigma</td>
		</tr>
		<tr>
			<td>
			Peroxidase (Typ II from horseradish)</td>
			<td>
			Sigma</td>
		</tr>
		<tr>
			<td>
			Sodium acetate</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Sodium hydroxide</td>
			<td>
			Merck</td>
		</tr>
		<tr>
			<td>
			Sucrose, D-(+)</td>
			<td>
			Fluka</td>
		</tr>
		<tr>
			<td>
			Water (deionized)</td>
			<td>
			-</td>
		</tr>
	</tbody>
</table>
Table of Solutions and Medium Composition:
<table border="1">
	<tbody>
		<tr>
			<td>
			Solution</td>
			<td>
			Components</td>
			<td>
			Amount</td>
		</tr>
		<tr>
			<td>
			50 mM sodium acetate buffer (pH 6.5)</td>
			<td>
			Sodium acetate
			Bring to volume with deionized water
			Adjust at pH 6.5 with acetic acid</td>
			<td>
			4.1 g L-1</td>
		</tr>
		<tr>
			<td>
			0.05 M monopotassium phosphate solution</td>
			<td>
			Monopotassium phosphate
			Bring to volume with deionized water</td>
			<td>
			6.805 g L-1</td>
		</tr>
		<tr>
			<td>
			0.05 M disodium hydrogen phosphate solution</td>
			<td>
			Disodium hydrogen phosphate
			Bring to volume with deionized water</td>
			<td>
			7.1 g L-1</td>
		</tr>
		<tr>
			<td>
			0.05 M phosphate buffer (pH 7.0)</td>
			<td>
			0.05 M disodium hydrogen phosphate solution
			Bring to volume with 0.05 M monopotassium phosphate solution</td>
			<td>
			61.2 mL</td>
		</tr>
		<tr>
			<td>
			0.05 M phosphate buffer (pH 5.4)</td>
			<td>
			0.05M disodium hydrogen phosphate solution
			Bring to volume with 0.05 M monopotassium phosphate solution</td>
			<td>
			3 mL</td>
		</tr>
		<tr>
			<td>
			1.65 M sucrose solution</td>
			<td>
			D-(+)-sucrose
			Bring to volume with phosphate buffer (pH 5.4)</td>
			<td>
			564.8 g L-1</td>
		</tr>
		<tr>
			<td>
			reagent solution</td>
			<td>
			o-Dianisidin-Dihydrochlorid
			Ethanol (95%)</td>
			<td>
			25 mg
			10 mL</td>
		</tr>
		<tr>
			<td>
			Glucose reagent solution</td>
			<td>
			Glucose oxidase
			Peroxidase
			Phosphate buffer (pH 7.0)
			reagent solution</td>
			<td>
			10.5 mg
			3 mg
			90 mL
			10 mL</td>
		</tr>
	</tbody>
</table>

customization of aspergillus niger morphology through addition of talc micro particles

أ. الفطريات الخيطية النيجر هو سلالة تستخدم على نطاق واسع في مجموعة واسعة من العمليات الصناعية من المواد الغذائية إلى صناعة المستحضرات الصيدلانية. واحدة من الخصائص الأكثر إثارة للاهتمام، وغالبا ما لا يمكن السيطرة عليها من هذا الكائن الحي هو الخيطية مورفولوجيا المعقدة. وهي تتراوح بين كريات كروية كثيفة على فطر لزج (الشكل 1). ومن المعروف المعلمات عملية مختلفة ومكونات للتأثير على التشكل الفطرية 1. منذ الإنتاجية المثلى يرتبط بقوة مع شكل المورفولوجية محددة، ومورفولوجيا الفطرية غالبا ما يمثل عنق الزجاجة للإنتاجية في الإنتاج الصناعي. وثمة نهج مستقيم إلى الأمام وأنيقة للسيطرة على وجه التحديد شكل الصرفي هو إضافة الجزيئات غير العضوية الصغيرة غير قابلة للذوبان (مثل سيليكات المغنيسيوم المائي، وأكسيد الألومنيوم أو أكسيد التيتانيوم السليكات) إلى الثقافة المتوسطة التي تسهم في انتاج الانزيم زادت 2-6. منذ وجود obv[إيووس] العلاقة بين مورفولوجيا الجسيمات الدقيقة تعتمد على إنتاج انزيم من المرغوب فيه لربط رياضيا الإنتاجية وظهور الصرفي. ولذلك يستهدف كمي وصف دقيق وشامل الصرفي. وبالتالي، فإننا نقدم وسيلة لتوليد وتوصيف الجسيمات الصغيرة التي تعتمد هياكل شكلية، وإلى ربط مورفولوجيا الفطرية مع إنتاجية (الشكل 1) الذي يساهم ربما التوصل إلى فهم أفضل للmorphogenesis من الكائنات الحية الدقيقة الخيطية. أ. سلالة المؤتلف يزرع النيجر SKAn1015 لمدة 72 ساعة في L 3 أثارت مفاعل حيوي للدبابات. بالإضافة إلى ذلك من قبل من الجسيمات الدقيقة التلك في تركيزات من 1 غرام / لتر، 3 غ / ل و 10 جرام / لتر قبل التلقيح يتم إنشاء بتكاثر مجموعة متنوعة من الهياكل الشكلية. وتؤخذ عينات معقمة بعد 24 ساعة و 48 و 72 لتقرير من التقدم والنمو ونشاط انزيم المنتجة. والمنتج شكلت هو انزيم ذات القيمة العالية β-فركتوفورانوزيداز، وحفاز بيولوجي هام لتشكيل الجدد سكر في المواد الغذائية أو صناعة الأدوية، والذي يحفز من بين أمور أخرى على رد فعل من السكروز إلى جلوكوز 7-9. ولذلك، فإن القياس الكمي للجلوكوز بعد إضافة السكروز ينطوي على قدر من إنتاج فركتوفورانوزيداز-β. يتم تقدير الجلوكوز من قبل الله / POD الفحص-10، والتي يتم تعديلها لتحليل الإنتاجية العالية في لوحات عيار 96-جيدا الصغرى. يتم فحص مورفولوجيا الفطرية بعد 72 ساعة من قبل المجهر وتتميز تحليل الصور الرقمية. في القيام بذلك، ويتم حساب عوامل شكل جسيمات عن التشكل الكلي الفطرية مثل قطر فيريه، ومنطقة المتوقعة، محيط، دائرية، نسبة الجانب، استدارة صلابة اوند مع صورة مفتوحة المصدر يماغيج برنامج معالجة. يتم الجمع بين المعايير ذات الصلة لعدد الصرف أبعاد (MN) 11، الذي تمكن من توصيف شاملمورفولوجيا الفطرية. وسلط الضوء على علاقة وثيقة من عدد الصرف والإنتاجية من خلال الانحدار الرياضية.

Watch this Scientific Journal Video about Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles at JoVE.com

Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

وهناك طريقة لتوليد وعلى وجه التحديد لتوصيف شامل التشكل من الفطريات الخيطية<em&gt; الرشاشيات النيجر</emيوصف&gt;، والذي يسمح للعلاقة الرياضية للظهور المورفولوجية والإنتاجية.

التخصيص الرشاشيات النيجر علم الصرف عن طريق إضافة من الجسيمات الدقيقة التلك

The filamentous fungus A. niger is a widely used strain in a broad range of industrial processes from food to pharmaceutical industry. One of the most ...

customization-aspergillus-niger-morphology-through-addition-talc

Research

JoVE Journal

Immunology and Infection

Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

18.8K Views.  Technische Universität Braunschweig. وهناك طريقة لتوليد وعلى وجه التحديد لتوصيف شامل التشكل من الفطريات الخيطية الرشاشيات النيجر، والذي يسمح للعلاقة الرياضية للظهور المورفولوجية والإنتاجية.

Video: Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

التعبئة والتغليف بفيروس نقص المناعة البشرية أو FIV المستندة إلى محددات التعبير Lentivector وتنبيغ من الجزيئات VSV-G الفيروسية Pseudotyped

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

الكشف عن جسيمات متناهية الصغر نيون التفاعل مع مجموعات سكانية فرعية خلية المناعة الأولية من قبل التدفق الخلوي

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

الاستفادة المثلى من الكمية الصغيرة تحييد الفحص

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

إنتاج جسيمات بسيودوتيبيد لدراسة كورونافيروس شديد العدوى في إعداد 2 مستوى السلامة الأحيائية

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

قياس كثرة الفندره من الرشاشيات تبخير كونيديا من قبل الكريات البيضاء البشرية باستخدام تدفق الخلوي

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

عدوى يرقات حمار وحشي مع جراثيم أسبيرغيلوس لتحليل التفاعلات المضيفة الممرضة

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular deoxyribonucleic acid (ecDNA) is released during different forms of cell death including apoptosis, necrosis, necroptosis, neutrophil extracellular traps (NETs) and pyroptosis. Measurement of this cell death is time consuming with several different biomarkers required to identify the different biochemical forms of cell death. Measurement of ecDNA is generally conducted in serum and urine as a surrogate for renal damage, not in the actual target organ where the pathological injury occurs. The current difficulty in investigating ecDNA in the kidney is the lack of methods for formalin fixed paraffin embedded tissue (FFPE) both experimentally and in archived human kidney biopsies. This protocol provides a summary of the steps required to stain for ecDNA in FFPE tissue (both human and murine), quench autofluorescence and measure the ecDNA in the resulting images using a machine learning tool from the publicly available open source ImageJ plugin trainable Weka segmentation. Trainable Weka segmentation is applied to ecDNA within the glomeruli where the program learns to classify ecDNA. This classifier is applied to subsequent acquired kidney images, reducing the need for manual annotations of each individual image. The adaptability of the trainable Weka segmentation is demonstrated further in kidney tissue from experimental murine anti-MPO glomerulonephritis (GN), to identify NETs and ecMPO, common pathological contributors to anti-MPO GN. This method provides objective analysis of ecDNA in kidney tissue that demonstrates clearly the efficacy in which the trainable Weka segmentation program can distinguish ecDNA between healthy normal kidney tissue and diseased kidney tissue. This protocol can easily be adapted to identify ecDNA, NETs and ecMPO in other organs.

Extracellular DNA (ecDNA) released during cell death is proinflammatory and contributes to inflammation. Measurement of ecDNA at the site of injury can determine the efficacy of therapeutic treatment in the target organ. This protocol describes the use of a machine learning tool to automate measurement of ecDNA in kidney tissue.

تحت إشراف التعلم الآلي من أجل تحديد شبه كمي للحمض النووي خارج الخلية في التهاب الجلوميرولونية

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular ...

Fecal (micro) RNA Isolation

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs from fecal samples of animal and human subjects. The aim is to isolate total RNA with high purity and quantity for downstream analyses such as RNA sequencing, RT-PCR, and micro-array. The advantages of this optimized protocol in the miRNA isolation are capabilities of isolating highly purified RNA products with additional washing steps described, increased quantity of RNA obtained with an improved method in the resuspension of sample, and important tips of decontamination. One limitation is the inability to process and purify larger sample of more than 200 mg as these sample sizes would cause a difficulty in the clear formation of the interphase. Consequently, the large sample size may contaminate the aqueous phase to be extracted as described in the protocol with organic matters that affect the quality of RNA isolated in the end. However, RNA isolates from a sample of up to 200 mg are sufficient for most of downstream analyses.

Fecal micro RNA Isolation

This protocol isolates high quality total RNA from fecal samples of animal and human subjects. A commercial miRNA isolation kit is used with significant adaption to isolate pure RNA with optimized quantity and quality. The RNA isolates are good for most downstream RNA assays such as sequencing, micro-array, and RT-PCR.

البراز (الصغير) عزل الحمض النووي الريبي

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs ...

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and immunological tolerance, while in immunocompromised patients, conidia can colonize airways and cause severe invasive respiratory disorders. Various cells in different airway compartments are involved in the immune response that prevents fungal invasion; however, the spatio-temporal aspects of pathogen elimination are still not completely understood. Three-dimensional (3D) imaging of optically cleared whole-mount organs, particularly the lungs of experimental mice, permits detection of fluorescently labeled pathogens in the airways at different time points after infection. In the present study, we describe an experimental setup to perform a quantitative analysis of A. fumigatus conidia distribution in the airways. Using fluorescent confocal laser scanning microscopy (CLSM), we traced the location of fluorescently labeled&#160;conidia in the bronchial branches and the alveolar compartment 6 hours after oropharyngeal application to mice. The approach described here was previously used for detection of the precise pathogen location and identification of the pathogen-interacting cells at different phases of the immune response. The experimental setup can be used to estimate the kinetics of the pathogen elimination in different pathological conditions.

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

We describe the method for quantitative analysis of the distribution of Aspergillus fumigatus conidia (3 &#181;m in size) in the airways of mice. The method also can be used for the analysis of microparticles and nanoparticle agglomerate distribution in the airways in various pathological condition models.

Confocal الليزر المسح المجهري القائم على التحليل الكمي من Aspergillus fumigatus كونيديا التوزيع في كامل جبل تطهيرها بصريا الرئة الماوس

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and ...

<meta charset="utf8"></head><body>تحليل SARS-CoV-2 باستخدام تقنية المراسل المزدوج والمتعدد

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

<meta charset="utf8"></head><body>تسليط الضوء على المؤلف: تطوير الاستراتيجيات المضادة للفيروسات من خلال تقنيات التقاط المناعة وقياس الطيف الكتلي الجديدة

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

تنميط حوائط البروتين السطحي على الجسيمات الفيروسية من خلال استراتيجية المراسل المزدوج المتعدد

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...