وتمول وJMD MKB من خلال العلوم لطب النانوي IGERT وبرنامج التكنولوجيا في جامعة نورث إيسترن (بتمويل من لجنة التحقيق الوطنية وجبهة الخلاص الوطني منحة DGE - 0504331). كما تم تمويل هذا العمل من قبل المعاهد الوطنية للصحة والمعهد الوطني الطبي العام R01 المنح العلوم GM084366. ونحن نشكر أيضا Saumya داس وانطوني روزنزويج من BIDMC في بوسطن لتوفير myocytes القلب والنقدي كيفن لمساهمته في تطوير بروتوكول الحيوان.

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	<li>Adrogue, H. J., Madias, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyponatremia.">Hyponatremia.</a> N Engl J Med. 342, 1581-1589 (2000).</li><li>Kim, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BACE1+regulates+voltage-gated+sodium+channels+and+neuronal+activity.">BACE1 regulates voltage-gated sodium channels and neuronal activity.</a> Nat Cell Biol. 9, 755-764 (2007).</li><li>Dubach, J. M., Lim, E., Zhang, N., Francis, K. P., Clark, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+sodium+concentration+continuously+monitored+with+fluorescent+sensors.">in vivo sodium concentration continuously monitored with fluorescent sensors.</a> Integr Biol (Camb). , (2010).</li><li>Minta, A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+cytosolic+sodium.">Fluorescent indicators for cytosolic sodium.</a> J Biol Chem. 264, 19449-19457 (1989).</li><li>Dubach, J. M., Harjes, D. I., Clark, H. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+ion-selective+nanosensors+for+intracellular+analysis+with+improved+lifetime+and+size.">Fluorescent ion-selective nanosensors for intracellular analysis with improved lifetime and size.</a> Nano Letters. 7, 1827-1831 (1021).</li><li>Schaffar, B., Wolfbeis, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sodium-selective+optrode.">A sodium-selective optrode.</a> Mikrochim Acta III. III, 109-109 (1989).</li><li>Seiler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+sodium-selective+optode+membranes+based+on+neutral+ionophores+and+assay+of+sodium+in+plasma.">Characterization of sodium-selective optode membranes based on neutral ionophores and assay of sodium in plasma.</a> Clin Chem. 37, 1350-1355 (1991).</li><li>Zhujun, Z., Mullin, J., Seitz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sensor+for+sodium+based+on+ion-pair+extraction+and+fluorescence.">Optical sensor for sodium based on ion-pair extraction and fluorescence.</a> Anal Chim Acta. 184, 251-251 (1986).</li><li>Bakker, E., Buhlmann, P., Pretsch, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carrier-Based+Ion-Selective+Electrodes+and+Bulk+Optodes.+1.+General+Characteristics.">Carrier-Based Ion-Selective Electrodes and Bulk Optodes. 1. General Characteristics.</a> Chem Rev. 97, 3083-3132 (1997).</li><li>Dubach, J. M., Das, S., Rosenzweig, A., Clark, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+sodium+dynamics+in+isolated+cardiomyocytes+using+fluorescent+nanosensors.">Visualizing sodium dynamics in isolated cardiomyocytes using fluorescent nanosensors.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 16145-16150 (2009).</li><li>Harjes, D. I., Dubach, J. M., Rosenzweig, A., Das, S., Clark, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-Selective+Optodes+Measure+Extracellular+Potassium+Flux+in+Excitable+Cells.">Ion-Selective Optodes Measure Extracellular Potassium Flux in Excitable Cells.</a> Macromolecular Rapid Communications. 31, 217-221 (2010).</li><li>Balaconis, M. K., Billingsley, K. L., Dubach, J. M., Clark, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , .</li><li>Park, E. J., Brasuel, M., Behrend, C., Philbert, M. 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الإعلان عن أي تضارب في المصالح.

وينبغي تشكيل nanosensors اتخاذ أي أطول من 10 دقيقة مرة واحدة أحرز الحل optode وتجفف الدهون PEG. يمكن Optode ليس فقط عندما تكون هناك حاجة بسرعة ، ولكن عند تخزينها في 4 درجات مئوية ، وأنها يمكن أن تكون مستقرة لمدة شهور. أظهرنا أن nansensors ، التي تشكلت مرة واحدة ، هي مستقرة في محلول لمدة أسبوع على الأقل ، إلا أنه يجب توخي الحذر لمنع photobleaching خلال هذا الوقت من خلال منع nanosensors 6 من الضوء وبينما تظاهر nanosensors الصوديوم هنا ، تم optodes خلقت لأيونات بيولوجيا معظم ذات الصلة ، مثل البوتاسيوم وكلوريد. 10،12 لدينا أيضا تمديد هذه التكنولوجيا لجزيئات صغيرة مثل الجلوكوز. 13 بالإضافة إلى توليد nanosensors لرصد analytes مختلفة ، وهذه nanosensors قابلة للتغييرات أخرى من شأنها أن تجعل من المفيد لهم في معظم التجارب البيولوجية. على سبيل المثال ، طلاء السطح ، ويمكن في هذه الحالة بسهولة بولي (جلايكول الإثيلين) ، يمكن تغييرها باستخدام أي جزيء amphiphilic أن ذوبان في الماء. وهذا يتيح إمكانية functionalizing هذه الجسيمات النانوية لتطبيقات مختلفة أو الأهداف. ويمكن أيضا حجم جزيئات أن تعدل عن طريق تغيير طلاء السطح ، وشدة صوتنة أو استخدام المذيبات. كانت مؤشرات الكالسيوم الفلورسنت لا تقدر بثمن في تحديد الخلايا إشارات الكالسيوم ، ولكن لا الأصباغ الصوديوم الحساسة المتوفرة لديهم نفس الخصائص المثالية. النانوية تهدف إلى توفير Optode طريقة بديلة لأيونات intracellularly التصوير تم في مجال التنمية لسنة 14 ونحن بنينا على هذه الأبحاث السابقة لإنشاء nanosensors محددة للتصوير الصوديوم داخل الخلية والذي نأمل أن توفر وسيلة لفهم الكيفية التي تؤثر إشارات الصوديوم الخلوية وظيفة تعديلات في الإشارة التي تؤدي إلى أمراض معينة. استخدام nanosensors الفلورسنت في الجسم الحي يوفر الوقت الحقيقي بديل عن الحد الأدنى الغازية analytes الرقابة على وسائل أخرى مثل الدم تلفت ، وقد أظهرت 3 الصوديوم والجلوكوز nanosensors على أساس التكنولوجيا أعلاه لتعقب التغييرات في الصوديوم والجلوكوز ، على التوالي في فيفو. 3،13 ومع ذلك ، توجد حاليا قيود على هذا الأسلوب كأداة للرصد. على سبيل المثال ، يجب أن تحتوي على أجهزة استشعار fluorophore مع طائفة تحول نحو كاف الحمراء الأشعة تحت الحمراء بالقرب من أجل تقليل تألق ذاتي خلفية من الجلد 15 ثانيا ، تقنية الحقن الحالية لا تنتج حقن الموحد للnanosensors التي قد تنجم عن مثل هذه أخطاء والاختلاف في عمق الحقن. على الرغم من هذه القيود ، ويمكن تطوير هذه nanosensors الفلورسنت ومظاهرتهم ناجحة جعل هذه المجسات أداة بحث لا تقدر بثمن وطريقة لرصد صحة المريض.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم المنتج </th><th> شركة </th><th> رقم كتالوج </th><th> تعليقات </th></tr><tr><td> IVIS الصك الثاني لومينا حزمة </td><td> الفرجار العلوم الحياتية </td><td> 126273 </td><td></td></tr><tr><td> CD - 1 الفئران عارية </td><td> نهر تشارلز </td><td> قانون سلالة : 086 </td><td> العوز المناعي </td></tr><tr><td> محاقن الأنسولين </td><td> دينار بحريني </td><td> 328438 </td><td> 10/03 سم مل ، 8mm و 31G </td></tr><tr><td> الوزن الجزيئي عالية PVC </td><td> سيغما </td><td></td><td></td></tr><tr><td> DOS </td><td> سيغما </td><td></td><td></td></tr><tr><td> CHIII </td><td> سيغما </td><td></td><td></td></tr><tr><td> NaTFPB </td><td> سيغما </td><td></td><td></td></tr><tr><td> NaIX </td><td> سيغما </td><td></td><td></td></tr><tr><td> رقيقة الجدران الزجاجية الشعرية </td><td> سوتر </td><td></td><td></td></tr><tr><td> PEG الدهون </td><td> أفانتي الدهون القطبية </td><td></td><td></td></tr></tbody></table> الجدول 1. يوفر قائمة من المواد الكيميائية والمعدات المستخدمة في هذا الإجراء. وقد تم شراء جميع المواد الكيميائية غير المدرجة مشتركة من سيغما.

fluorescent nanoparticles for the measurement of ion concentration in biological systems

تنظيم محكم التوازن الأيوني في جميع أنحاء الجسم أمر ضروري للوقاية من الدول المنهكة مثل الجفاف. 1 وفي المقابل ، يتعين على تدفقات ايون السريع على المستوى الخلوي لبدء العمل في إمكانات الخلايا منفعل. تنظيم الصوديوم 2 يلعب دورا هاما في كل من هذه الحالات ، ولكن لا توجد حاليا طريقة لرصد مستمر مستويات الصوديوم في الجسم الحي 3 و 4 تحقيقات الصوديوم داخل الخلايا لا تعطي نتائج مماثلة لتحقيقات مفصلة الكالسيوم. في محاولة لملء كل من هذه الفراغات ، وضعت nanosensors الفلورسنت التي يمكن رصد تركيزات الصوديوم في المختبر والمجراة. 5،6 هذه المجسات تقوم على التكنولوجيا optode ايون انتقائية ، وتتألف من جزيئات البوليمر التي الملدن الاعتراف الصوديوم محددة هي جزء لا يتجزأ العناصر ، fluorophores الحموضة حساسة ، والمواد المضافة. 7-9 ميكانيكيا ، والاعتراف عنصر الصوديوم مقتطفات الصوديوم في الاستشعار. استخراج 10 هذا يسبب الحموضة fluorophore حساسة للافراج عن أيون الهيدروجين للحفاظ على الحياد في إطار استشعار المسؤول الذي يسبب التغيير في مضان. أجهزة الاستشعار الصوديوم هي انعكاس للانتقائية وأكثر من البوتاسيوم الصوديوم حتى بتركيزات عالية داخل الخلايا. 6 وهي مغلفة ما يقرب من 120 نانومتر في القطر مع والبولي ايثيلين جلايكول لنقل توافق مع الحياة. باستخدام تقنيات microinjection ، يمكن تسليم أجهزة الاستشعار في سيتوبلازم الخلايا حيث ثبت أنها لرصد ديناميات الصوديوم الزمانية والمكانية للضرب myocytes القلب. 11 بالإضافة إلى ذلك ، كما أنها تتبع في الوقت الحقيقي والتغيرات في تركيزات الصوديوم في الجسم الحي عند حقنه 3 تحت الجلد في الفئران وهنا ، يمكننا أن نفسر بالتفصيل وشرح منهجية لافتعال nanosensors الصوديوم الفلورسنت وشرح لفترة وجيزة من التطبيقات البيولوجية مختبرنا يستخدم nanosensors عن : لmicroinjection من أجهزة الاستشعار في الخلايا ، والحقن تحت الجلد من أجهزة الاستشعار في الفئران .

Watch this Scientific Journal Video about Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems at JoVE.com

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

وتستخدم جزيئات الفلورسنت المنتجة في المختبر للتركيز ايون ايون التصوير والتدفقات في النظم البيولوجية مثل خلايا السائل خلال الإشارات وخلالي خلال التوازن الفيزيولوجي.

الفلورسنت النانوية لقياس تركيز ايون في النظم البيولوجية

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

fluorescent-nanoparticles-for-measurement-ion-concentration

Research

JoVE Journal

Bioengineering

15.2K Views.  Northeastern University. وتستخدم جزيئات الفلورسنت المنتجة في المختبر للتركيز ايون ايون التصوير والتدفقات في النظم البيولوجية مثل خلايا السائل خلال الإشارات وخلالي خلال التوازن الفيزيولوجي.

Video: Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

المغناطيسي منهجية Elastography الرنين لتقييم الأنسجة المهندسة النمو كونستركت

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

الصلبة الدهون النانوية (SLNs) لاستهداف التطبيقات بين الخلايا

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. The instrument is optimized for the exploration of the wide momentum transfer Q range between 1x10-4 and 0.5 &#197;-1 by combining classical pinhole, focusing (with lenses), and time-of-flight (with chopper) methods, while simultaneously providing high-neutron intensities with an adjustable resolution. Because of its ability to adjust the intensity and the resolution within wide limits during the experiment, combined with the possibility to equip specific sample environments and ancillary devices, the KWS-2 shows a high versatility in addressing the broad range of structural and morphological studies in the field. Equilibrium structures can be studied in static measurements, while dynamic and kinetic processes can be investigated over time scales between minutes to tens of milliseconds with time-resolved approaches. Typical systems that are investigated with the KWS-2 cover the range from complex, hierarchical systems that exhibit multiple structural levels (e.g., gels, networks, or macro-aggregates) to small and poorly-scattering systems (e.g., single polymers or proteins in solution). The recent upgrade of the detection system, which enables the detection of count rates in the MHz range, opens new opportunities to study even very small biological morphologies in buffer solution with weak scattering signals close to the buffer scattering level at high Q.
In this paper, we provide a protocol to investigate samples with characteristic size levels spanning a wide length scale and exhibiting ordering in the mesoscale structure using KWS-2. We present in detail how to use the multiple working modes that are offered by the instrument and the level of performance that is achieved.

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

دراسة لينة المسألة والأنظمة البيولوجية على نطاق واسع طول مقياس من نانومتر وميكرومتر مقاسات صغيرة في الزاوية النيوترون حيود الهئية-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP induces a noninvasive region for charged biomolecules (i.e., the ion depletion zone), and targets can be preconcentrated on this region boundary. Despite the high preconcentration performances with ICP, it is difficult to find the operating conditions of non-propagating ion depletion zones. To overcome this narrow operating window, we recently developed a new platform for spatiotemporally fixed preconcentration. Unlike preceding methods that only use ion depletion, this platform also uses the opposite polarity of the ICP (i.e., ion enrichment) to stop the propagation of the ion depletion zone. By confronting the enrichment zone with the depletion zone, the two zones merge together and stop. In this paper, we describe a detailed experimental protocol to build this spatiotemporally defined ICP platform and characterize the preconcentration dynamics of the new platform by comparing them with those of the conventional device. Qualitative ion concentration profiles and current-time responses successfully capture the different dynamics between the merged ICP and the stand-alone ICP. In contrast to the conventional one that can fix the preconcentration location at only ~5 V, the new platform can produce a target-condensed plug at a specific location in the broad ranges of operating conditions: voltage (0.5-100 V), ionic strength (1-100 mM), and pH (3.7-10.3).

The protocol for a novel ion concentration polarization (ICP) platform that can stop the propagation of the ICP zone, regardless of the operating conditions is described. This unique ability of the platform lies in the use of merging ion depletion and enrichment, which are two polarities of the ICP phenomenon.

دمج ايون الاستقطاب التركيز بين جنبا إلى جنب التبادل الأيوني الأغشية لمنع الانتشار من منطقة الاستقطاب

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

إنتاج الجسيمات النانوية المعدنية عن طريق نابض بالليزر في الاجتثاث في السوائل: أداة لدراسة خصائص مضادة للجراثيم من الجسيمات النانوية

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

فلاش نانوبريسيبيتيشن لتغليف مركبات مسعور وماء في الجسيمات النانوية البوليمرية

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

القياس المستمر للضوضاء البيولوجية في الإشريكية القولونية باستخدام المجهر الفاصل الزمني

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli such as physical movement, activation of tactile sensors in the skin, and viewing images. Neural activation is inferred from an increase in the cerebral blood flow velocity (CBFV) supplying the region of the brain involved in processing sensory input. For example, viewing bright light causes increased neural activity in the occipital lobe of the cerebral cortex, leading to increased blood flow in the posterior cerebral artery, which supplies the occipital lobe. In fTCD, changes in CBFV are used to estimate changes in cerebral blood flow (CBF).
With its high temporal resolution measurement of blood flow velocities in the major cerebral arteries, fTCD complements other established functional imaging techniques. The goal of this Methods paper is to give step-by-step instructions for using fTCD to perform a functional imaging experiment. First, the basic steps for identifying the middle cerebral artery (MCA) and optimizing the signal will be described. Next, placement of a fixation device for holding the TCD probe in place during the experiment will be described. Finally, the breath-holding experiment, which is a specific example of a functional imaging experiment using fTCD, will be demonstrated.

Functional transcranial Doppler ultrasound complements other functional imaging modalities, with its high temporal resolution measurement of stimulus-induced changes in cerebral blood flow within the basal cerebral arteries. This Methods paper gives step-by-step instructions for using functional transcranial Doppler ultrasound to perform a functional imaging experiment.&#160;

وظيفية عبر الجمجمة دوبلر الموجات فوق الصوتية لرصد تدفق الدم الدماغي

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

أجهزة المجال الكهربائي والمغناطيسي لتحفيز الأنسجة البيولوجية

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 &#181;g of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

The study describes a protocol for creating large (&#181;g-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

طرق سريعة انزيمية لتضخيم الحد الأدنى من القوالب الخطية لنماذج البروتين باستخدام أنظمة خالية من الخلايا

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins ...