We thank Dr&#8217;s. Douglas Pittman and Michael Wyatt for their assistance with cell culture and Dr Wyatt and Ms. Erin Anderson for help in development of the binding assays. We acknowledge Mike Walla and Bill Cotham in the Department of Chemistry and Biochemistry at the University of South Carolina for assistance with Mass Spectrometry, Helga Cohen and Dr. Perry Pellechia for NMR spectrometry. This work was funded by the National Institutes of Health through the research project grant, 5R01CA131368.

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The authors declare that they have no competing financial interests.

Targeting protein-protein interactions (PPI) in drug discovery is highly challenging as these typically involve a large shallow contact interface comprised of numerous and diffuse contacts19. Furthermore, peptidic compounds which inhibit PPI&#8217;s that are amenable to drug discovery are problematic due to their higher molecular mass, metabolic instability and poor bioavailability20. Current strategies that have been applied for the development of PPI inhibitors include design of proteomimetics and...

في هذه المقالة، وصفت دراسة حالة لتطبيق استبدال (استبدال ببدائل يجند جزئية باستخدام الحسابية التخصيب) استراتيجية لتحويل مثبطات ببتيدية من تفاعلات البروتين البروتين إلى مزيد من صيدليا الجزيئات ذات الصلة 1-3. بينما تمثل مثبطات مضخة البروتون مصدرا غنيا لكن مستغلة من الأهداف المحتملة المخدرات والمنهجيات الحالية غير كافية إلى حد كبير لجعل هذه يمكن الوصول إليها على نطاق واسع. الاستراتيجيات الحالية بما في ذلك جزء يستند تصميم 4، الإنتاجية العالية فحص 5 والببتيدات تدبيس وفرت 6 التقدم، ولكن هذه هي في كثير من الحالات غير فعالة. ونتيجة لذلك، يطلب المزيد من التقدم ونهج أكثر كفاءة. استبدال تم التحقق من صحة تماما في تطوير مثبطات كيناز التي أدت إلى تحسين الخصائص مثل المخدرات ولها إمكانات لمزيد من التطوير كما العلاجات المضادة للورم. ويتمثل هذه الاستراتيجية في تطوير مثبطات غير ATP من الخلاياCDKs دورة وينطوي على النحو التالي: 1) الحصول على المعلومات الهيكلية 3D على تفاعلات HAKRRLIF / RRLIF مع السيكلين الأخدود ملزمة. 2) تحديد محددات ملزمة هامة للتفاعل الببتيد. 3) الاقتطاع من الببتيد N-محطة تحتوي على واحد أو أكثر من محددات ملزمة؛ 4) تحديد الحسابية بدائل جزيء صغير المحتملة (بدائل يجند جزئية، بلاس) للجزء المقتطعة من الببتيد والتي تحتفظ التفاعلات الرئيسية للالببتيد الأم. 5) توقع توليف أو مصادر تجارية البلاس لربط بشوق مع موقع فرعي المحتلة من قبل بقايا الببتيد حذف (ق)؛ 6) توليف تقلب من خلال ربط من أفضل بلاس لالببتيد اقتطاع باستخدام تركيب الحالة الصلبة. 7) اختبار تقلب في في المختبر ملزمة أو فحص وظيفي (الاستقطاب مضان في سياق / السيكلين معلمين) تليها مزيد من التوصيف في مقايسة بقاء الخلية. تمثيل تخطيطي لاستبدال الاستراتيجيههو مبين في الشكل 1 ذ. في هذه المقالة، وتناقش تكرار لاستراتيجية استبدال وتطبيق لCDK2 / السيكلين A وصف بالتفصيل. ويعتقد CDKs أن حررت مباشرة أو غير مباشرة في معظم الأورام وبالتالي تعتبر أهدافا المخدرات سرطان المناسب 7. CDKs تتطلب بالتعاون مع الحلقيات لتفعيل كامل وبعد ذلك الفوسفات البروتينات الرئيسية المشاركة في تنظيم دورة الخلية 8. الفئات الرئيسية اثنين من CDKs هي isotypes التي تتحكم في نقاط التفتيش دورة الخلية [G1 / S (CDK4 / السيكلين D، CDK6 / السيكلين D وCDK4 / السيكلين E)، مرحلة S (CDK2 / السيكلين A) وG2 / M (CDK1 / السيكلين B)] والمنظمين من RNA البلمرة من خلال الفسفرة (CDK7 / السيكلين H، CDK8 / السيكلين C، CDK9 / السيكلين T). خطوة رئيسية في S مرحلة تطور يحدث عندما يشكل عامل E2F1 النسخ مجمع مع البروتين الذي يربط موانئ دبي ثم إلى DNA ويبدأ النسخ الجيني. مطلوب CDK2 / السيكلين A لتحييد E2F1 النسخيالنشاط خلال الفسفرة مما يؤدي إلى الإفراج عن مجمع E2F1-DP وتدهور لاحقا. ويعتقد تثبيط CDK2 / السيكلين A للحفاظ على E2F1 في DNA حالته ملزمة مما يؤدي إلى تنشيط دائم. فإن مستوى الناتجة من E2F-1 النشاط تتجاوز الحد الأدنى المطلوب للحث على البروتين p53 الخلايا المستقلة بالتالي يشير إلى استراتيجية علاجية. بسبب البروتين p53 حررت ومسارات PRB، ومستويات عالية من E2F-1 تحدث بشكل متكرر في الخلايا السرطانية وتثبيط CDK2 / السيكلين A ينبغي أن يؤدي إلى موت الخلايا المبرمج انتقائية في الأورام ويمكن اعتباره هدفا سرطان صادق 7. سريريا مثبطات معلمين التحقيق تستهدف موقع ATP الحفظ جدا ملزمة مما يؤدي إلى عبور التفاعل بين تحركات البروتينات أكبر من 500 في kinome الإنسان ويحتمل أن تكون مما أدى إلى آثار جانبية وسمية 9. نهج بديل غير قابلة للATP تثبيط المنافسة من خلال استهداف توظيف الركيزة خلال CBGموجودة على السيكلين فرعية التنظيمية الإيجابية والتي هي بالتالي متميزة وبعيدة عن موقع ATP 10،11 ملزمة. وCBG هو في المقام الأول الأخدود مسعور الحالي في السيكلين A، D السيكلين والسيكلين E ولقد ثبت للتعرف على تسلسل إجماع وجدت في ركائز والمكثفات الورم. كما الببتيد معزولة، والسيكلين ملزمة عزر (CBM) بربط CBG ولقد ثبت لكبح النشاط كيناز من CDKs دورة الخلية. وقد تم تحسين CBM إلى ثماني الببتيد (HAKRRLIF، CDK2 / السيكلين A IC 50 0.07 ± 0.02 ميكرومتر، CDK4 / السيكلين D، IC 50 0.88 ± 0.34 ميكرومتر) واقتطاع علاوة على ذلك لخماسي الببتيد تمثل حلا وسطا جيدا بين الوزن الجزيئي للبالمخدرات الشبه ورجولية (RRLIF، CDK2 / السيكلين A IC 50 1.01 ± 0.17 ميكرومتر، CDK4 / السيكلين D، IC 50 25.12 ± 2.97 ميكرومتر) 12،13. تتألف CBGs من الانتخابات التمهيدية كبير وجيب مسعور الثانوي أصغر التي يتم سدها من قبل الحمضةج المنطقة (تشمل Glu220، Glu224 وAsp283). وتشمل محددات ملزمة الرئيسية HAKRRLIF تفاعل Ala2 مع السندات جيب مسعور الثانوي، الاقتران أيون الهيدروجين وللLys3، الارجنتين 4 و Arg5 مع المنطقة الحمضية وعلى درجة عالية من التكامل بين Leu6 وPhe8 مع موقع دهون الأساسي. بالإضافة إلى ذلك، ساهم السندات الهيدروجين العديد من العمود الفقري الببتيد في حين يعمل Ile7 كبقايا هل يسمح اتصال الأمثل مع جيب الأساسي. يظهر وضع ملزمة وتفاعلات HAKRRLIF مع CBG في الشكل 2. سوف تستهدف CBM / CBG تفاعل البروتين البروتين تمنع النشاط كيناز من CDK2 / السيكلين A، CDK2 / السيكلين E &amp; CDK4 / السيكلين D وهذا ينبغي أن يؤدي E2F1 الخلايا بوساطة الخلايا السرطانية في حين لا تؤثر الخلايا الطبيعية 7. على الرغم من أن CBM المستمدة الببتيدات هي مثبطات فعالة من CDKs دورة الخلية، فمن غير المحتمل أن يكون مفيدا كدواء بسبب الايضعدم الاستقرار ونقص عام في نفاذية الخلية. تحقيقا لهذه الغاية، قمنا بتطبيق استراتيجية استبدالها من أجل تحويل هذه المثبطات ببتيدية واسعا إلى مزيد من مركبات شبيهة المخدرات للحصول على مزيد من العلاجات المضادة للورم استغلال E2F1 حررت خلال CDK2 / السيكلين A تثبيط التنمية. بروتوكول التالية يلخص العمل الذي تم الانتهاء منه في تطبيق استبدال لالأخدود السيكلين. في المقام الأول، تم تحديد بدائل فريق السد مثل المخدرات للرباعي البيبتيد N-محطة من HAKRRLIF. وعلاوة على ذلك تم التحقيق التحسينات في هذه المجموعات في دراسة التحقق من صحة إضافية لاستبدال. وتعرض أيضا ممثل النتائج من هذه الدراسات.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Computational Chemistry</td>
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		<tr>
			<td>Accelyrs Discovery studio 3.0</td>
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			<td></td>
		</tr>
		<tr>
			<td>Dell Optiplex Workstations</td>
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			<td>Synthetic Organic Chemistry</td>
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			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel (GF-254 plates) for TLC, Biotage (Uppsala, Sweden) for flash chromatography, Waters Alliance 2695 HPLC with a 2996 diode-array detector and equipped with a C18 (2) 100 A, 250 x 4.6mm, 5&#956;m column (Phenomenox Luna) for purity determination, 1H NMR and 13C NMR spectra were recorded with a Varian Mercury 300 and 400 Spectrometer, respectively. Mass spectra were measured with a Micromass QTOF (Tandem quadruple-1 time of flight mass spectrometer), electrospray ionization (ESI) and VG 70S (Double-focusing magnetic sector mass spectrometer, EI).</td>
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			<td>Flourescence Polarization Assay</td>
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			<td></td>
			<td></td>
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		<tr>
			<td>384 micro well plates, ,&#160; Micro pipets,&#160;</td>
			<td>Grenier Bio-one</td>
			<td>110256602</td>
			<td></td>
		</tr>
		<tr>
			<td>CDK4D1 and CDK2CA (well purified recombinant human kinase complex)</td>
			<td>BPS Bio Sciences</td>
			<td>40094(CDK4/Cyclin D), 41101(CDK2/Cyclin A)</td>
			<td></td>
		</tr>
		<tr>
			<td>assay buffer (25 nM HEPES pH 7, 10 mM NaCl, 0.01% Nonidet P-40, 1mM dithiothretiol (DTT),</td>
			<td></td>
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			<td></td>
		</tr>
		<tr>
			<td>25 nM HEPES</td>
			<td>CALBIOCHEM</td>
			<td>375368</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>127838</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P-40</td>
			<td>US Biological</td>
			<td>N3500</td>
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		</tr>
		<tr>
			<td>DTT</td>
			<td>Aldrich</td>
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		<tr>
			<td>-70c freezer</td>
			<td>Revco (Ultima II)</td>
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			<td></td>
		</tr>
		<tr>
			<td>DTX880 multimode detector&#160; fitted with 485 nm/535 nm excitation/emission filters and a dichroic mirror suitable for fluorescein</td>
			<td>Beckman Coulter, Brea, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
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		</tr>
		<tr>
			<td>Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates, &#160;&#160;</td>
			<td>Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Frozen stocks of U2OS (osteosarcoma) and DU145 (prostate cancer) cell lines</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NU serum, DMEM media, trypsin, PEN/STRIP,&#160; MTT reagent,</td>
			<td colspan="2">Fisher, Life technology, Alfa Aesar</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Heamocytometer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-70c freezer</td>
			<td>Revco (Ultima II)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo electron corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf&#160;</td>
			<td>5804 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator 4-8C</td>
			<td>Isotemp Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DTX880 multimode detector fitted with 595nm filter.</td>
			<td>Beckman Coulter, Brea, CA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of inhibitors of protein-protein interactions through replace: application to the design and development non-atp competitive cdk inhibitors

استبدال هو استراتيجية فريدة من نوعها وضعت لاستهداف أكثر فعالية البروتين البروتين التفاعلات (مثبطات مضخة البروتون). انها تهدف الى توسيع المساحة المتوفرة على الهدف المخدرات من خلال توفير منهجية محسنة لتحديد مثبطات لمثل هذه المواقع الملزمة والتي تمثل غالبية الأهداف المحتملة المخدرات. والهدف الرئيسي من هذه الورقة هو تقديم لمحة المنهجية لاستخدام وتطبيق استراتيجية استبدال الذي ينطوي على النهج الكيمياء الحسابية والاصطناعية. استبدل يتجلى من خلال تطبيقها لتطوير تحركات غير ATP السيكلين تنافسية تعتمد (للمعلمين) مثبطات كما العلاجات المضادة للورم. وكثيرا ما حررت CDKs في السرطان، وبالتالي تعتبر أهدافا هامة للتنمية المخدرات. تم الإبلاغ عن تثبيط CDK2 / السيكلين A في المرحلة S لتعزيز موت الخلايا المبرمج انتقائية من الخلايا السرطانية بطريقة مستقلة البروتين p53 من خلال مسار E2F1. استهداف تفاعل البروتين البروتين في المربوطة السيكلينز الأخدود (CBG) هو النهج الذي سيسمح للتثبيط محددة من دورة الخلية على CDKs النسخي. يتم التعرف على CBG عن طريق سلسلة إجماع المستمدة من ركائز معلمين وورم البروتينات الكابتة يسمى السيكلين عزر (CBM) ملزمة. وقد سبق الأمثل CBM إلى ثماني الببتيد من p21Waf (HAKRRIF) ثم اقتطاع المزيد لخماسي الببتيد الاحتفاظ النشاط الكافي (RRLIF). الببتيدات بصفة عامة ليست قابلة للاختراق الخلية، غير مستقرة عملية الأيض وبالتالي تم تطبيق استبدال (مع استبدال الجزئي يجند البدائل من خلال الحاسوبية التخصيب) استراتيجية من أجل توليد المزيد من مثبطات مثل المخدرات. تبدأ استراتيجية مع تصميم جزء ligated الببتيد المثبطة (تقلب) التي تحول دون انتقائية دورة الخلية مجمعات معلمين / السيكلين. تم إنشاء تقلب عن طريق استبدال تكرارا مخلفات HAKRRLIF / RRLIF مع شظية مثل الجزيئات الصغيرة (متوجا مجموعات)، بدءا من N-محطة (Ncaps)، تليها بديل عنمحطة سي. هذه المركبات هي نقطة الانطلاق لتوليد مثبطات معلمين تنافسية غير ATP كما العلاجات المضادة للورم.

وتظهر تفاعلات HAKRRLIF مع الأخدود السيكلين في الشكل 2، وبقايا الببتيد التي تمثل محددات ملزمة رئيسية تشمل Ala2، Arg4، Leu6 وPhe8 مع المخلفات الأخرى التي تقدم مساهمات أصغر 12،13،18. في دراسة الحالة هذه قد استخدمت استراتيجية استبدالها من أجل إيجاد بدائل شظية م...

Watch this Scientific Journal Video about Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors at JoV

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

تطوير مثبطات البروتين البروتين التفاعلات استبدال من خلال: تطبيق لتصميم وتطوير غير ATP مثبطات معلمين تنافسي

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

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Biology

11.2K Views.  University of South Carolina. We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

Video: Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

استخدام SC1 (Pluripotin) لدعم MESC التجديد الذاتي في غياب LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

التصوير البروتين البروتين التفاعلات في الجسم الحي</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

إإكسبلنتس قسيم أرومي لاختبار الالتزام مصير الخلايا أثناء التطور الجنيني

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

التحقيق عالية الكثافة ميكروأرس البروتين وظيفية لكشف البروتين البروتين التفاعلات

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

كفاءة الجذع الجيل خلية من الدم عن طريق Episomes وHDAC المانعون

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

تصميم وتطوير أبتمر الذهب الجسيمات النانوية وبناء اللونية فحوصات للتطبيقات الموجودة على الشبكة العالمية الحقل

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

إزالة المواد من مصادر خارجية من الجزء الخارجي من النوى المجمدة للتحقيق في المجتمعات القديمة البيولوجية آوى داخل

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

دراسة لتفاعلات البروتين البروتين في البحوث أوتوفاجي

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

تقنيات الفحص المجهري [كنفوكل] لدراسة تفاعلات البروتين البروتين وحركية في الحمض النووي آفات متقدمة

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Application of Ultrasound and Shear Wave Elastography Imaging in a Rat Model of NAFLD/NASH

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat accumulation (steatosis) and inflammation leading to fibrosis. Preclinical models closely recapitulating human NASH/NAFLD are essential in drug development. While liver biopsy is currently the gold standard for measuring NAFLD/NASH progression and diagnosis in the clinic, in the preclinical space, either collection of whole liver samples at multiple timepoints during a study or biopsy of liver is needed for histological analysis to assess the disease stage.
Conducting a liver biopsy mid-study is an invasive and labor-intensive procedure, and collecting liver samples to assess disease level increases the number of research animals needed for a study. Thus, there is a need for a reliable, translatable, non-invasive imaging biomarker to detect NASH/NAFLD in these preclinical models. Non-invasive ultrasound-based B-mode images and Shear Wave Elastography (SWE) can be used to measure steatosis as well as liver fibrosis. To assess the utility of SWE in preclinical rodent models of NASH, animals were placed on a pro-NASH diet&#160;and underwent non-invasive ultrasound B-mode and shear wave elastography imaging to measure hepatorenal (HR) index and liver elasticity, measuring progression of both liver fat accumulation and tissue stiffness, respectively, at multiple time points over the course of a given NAFLD/NASH study.
The HR index and elasticity numbers were compared to histological markers of steatosis and fibrosis. The results showed strong correlation between the HR index and percentage of Oil Red O (ORO) staining, as well as between elasticity and Picro-Sirius Red (PSR) staining of livers. The strong correlation between classic ex vivo methods and in vivo imaging results provides evidence that shear wave elastography/ultrasound-based imaging can be used to assess disease phenotype and progression in a preclinical model of NAFLD/NASH.

This protocol describes the use of an enhanced ultrasound technique to non-invasively observe and quantify liver tissue changes in rodent models of nonalcoholic fatty liver disease.

تطبيق التصوير بالموجات فوق الصوتية والقص موجة التصوير في نموذج الفئران من NAFLD / ناش

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat ...