الكتاب نود أن نعترف الدكتور بول دراسة Witting والسيدة جينيفيف فونغ على المساعدة التي قدموها مع استخراج مرنا وPCR، وغارنيت موديل قديم ومؤسسة رودني ويليامز التذكاري للحصول على الدعم.

بيان الأخلاق: جميع الإجراءات المبينة أدناه تمت الموافقة من قبل جامعة سيدني جنة الأخلاقيات الحيوانية. 1. الحيوانات ملاحظة: 1 و 8 - الفئران من العمر 9 أشهر (C57 / BL6) تم الحصول عليها من مركز الثروة الحيوانية (بيرث، أستراليا). وتم إيواء الفئران في مرفق بوش القوارض في جامعة سيدني. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بيت الفئران في أقفاص الماوس القياسية على ضوء 12/12 ساعة / دورة الظلام مع الحصول على الغذاء والماء بالمال وبالشهرة أيضا الإعلانية. </li><li style=";text-align:right;direction:rtl"> الفئران الفجوة في كل فئة عمرية في القريب الأشعة تحت الحمراء (نير) معاملة، أو صورية معاملة (مراقبة) مجموعات للمقارنة. </li></ol> 2. الأدنى الأشعة تحت الحمراء (نير) الإشعاع والعلاج الشام <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حلق الفراء على منطقة الرأس والرقبة من الفأرة مع الحلاقة الكهربائية قدر الإمكان لمنع إعادة نمو سريع للشعر قبل الانتهاء من نظام العلاج. من أجل الحفاظ على الوضع خالية من مسببات الأمراض من الفئران، واستخدام الايثانول 70٪لتنظيف أدوات الحلاقة بين الحيوانات ومطهر F10 البيطرية بين الحيوانات من أقفاص منفصلة. القيام بذلك 2 - 3 أيام قبل بداية العلاج حتى الحيوانات ليست أوفر التعامل معها. </li><li style=";text-align:right;direction:rtl"> كبح الماوس من خلال عقد نهاية القريبة من الذيل والسماح لها للاسترخاء في راحة اليد أو مقعد واحد أعلى وذلك لتقليل التوتر التي يمكن أن تؤدي إلى نتائج خاطئة. </li><li style=";text-align:right;direction:rtl"> عقد نير (670 نانومتر) LED (الصمام الثنائي الباعثة للضوء) جهاز 1 - 2 سم بعيدا عن يتعرض (حليق) منطقة والتبديل على الجهاز لمدة 90 ثانية (الشكل 1A). وقد تم قياس التغير في درجة الحرارة نتيجة للتعرض 90 ثانية ك &lt;0.2 درجة مئوية في 100 مل من الماء: ملاحظة. </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من 2،2-2،3 لمجموعة العلاج صورية من الحيوانات ولكن ترك فيها تشغيل الجهاز (الشكل 1B). </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من 2،2-2،3 لمجموعة العلاج نير منعت من الحيوانات ولكن تغطي الجهاز مع رقائق الألومنيوم. </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من 2،2-2،5 يوميا في approximatه 24 فترات ساعة لمدة 5 أيام متتالية. </li><li style=";text-align:right;direction:rtl"> استخراج ظهارة الدهليزي الحسية (3 أعراف و1 البقعة القريبي) من كلتا الأذنين في اليوم الخامس بعد المعالجة (انظر القسم 3 أدناه). </li></ol> 3. الأنسجة استخراج 20 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد 300 مل من القائم الجلسرين السائل النخاعي الاصطناعي (ACSF) تتكون من (مم): 26 NaHCO 3، 11 الجلوكوز، 250 الجلسرين، و 2.5 بوكل، 1.2 ناه 2 PO 4، 1.2 MgCl 2، و 2.5 CaCl 2. قبل إضافة CaCl 2، والغاز الحل مع كربوجين (95٪ O 2 و 5٪ CO 2) لتأسيس الرقم الهيدروجيني من 7.4 وتجنب هطول الأمطار الكالسيوم (الغيوم). هدئ الحل في الفريزر -80 درجة مئوية لمدة 45 دقيقة حتى يتم تشكيل الطين الجليد. </li><li style=";text-align:right;direction:rtl"> إعداد العازلة تحلل عزل الحمض النووي الريبي في المسمى أعلى microtubes المسمار (توقف ظهرت من الأغطية عندما تغيرات الضغط أنبوب بين التجميد والذوبان في النيتروجين السائل) وفقالتعليمات الشركة المصنعة أو الممارسات المخبرية المعتادة. لدينا النيتروجين السائل جاهزة في قارورة ديوار الألومنيوم. ملاحظة: استخدام ملابس واقية وeyeware عند التعامل مع النيتروجين السائل. تأكد من أن النيتروجين السائل يستخدم في غرفة جيدة التهوية، حيث بلغ حجم شكل الغاز ما يقرب من 700 مرة من شكله السائل ويمكن أن يسبب الاختناق. </li><li style=";text-align:right;direction:rtl"> بعمق تخدير الفئران مع الكيتامين (400 ملغ / كلغ) عن طريق الحقن داخل البريتوني. السماح للالمنعكس القائمة الخلفية لتهدأ تماما كما يشير إلى أن الفئران هي تخدير كامل. </li><li style=";text-align:right;direction:rtl"> قطع رأس الفئران مع حادة مقص الفولاذ المقاوم للصدأ وإجراء شق على طول الجلد سهمي من الجمجمة باستخدام شفرة حلاقة (تقريب رقم 22). عند هذه النقطة والخطوات جميع أنحاء 3،5-3،9، والحفاظ على قبو الجمجمة والدماغ، والجهاز الدهليزي الأساسي بارد ممكن عن طريق تطبيق المنتظم للACSF الجليد الباردة على الأنسجة. </li><li style=";text-align:right;direction:rtl"> باستخدام ذراع أشار من مقص نمط القياسية جعل األصغرل شق في الجمجمة في امدا وقطع على طول الدرز السهمي. </li><li style=";text-align:right;direction:rtl"> الشريحة بلطف ذراع واحدة من رينجرز الضحلة تحت العظم الجداري الحفاظ على شفرة أقرب وقت ممكن إلى السطح السفلي للعظم بدون سحب الدماغ. تأمين وسحب بعيدا العظم الجداري أفقيا وعظم القذالي الخلف حتى يتعرض الدماغ. </li><li style=";text-align:right;direction:rtl"> استخدام صغيرة غير القابل للصدأ ملعقة الصلب ورفع الدماغ بعيدا عن الحفرة القحفية الأمامية والوسطى لفضح العصب الدهليزي القوقعي (CN الثامن). القطع العصب لمنع التوتر لا داعي لها على محاور عصبية وارد الأولية التي يعصب بشكل مباشر على خلايا الشعر الدهليزي. </li><li style=";text-align:right;direction:rtl"> إزالة الدماغ جملة وتفصيلا بعد transection من CN الثامن. </li><li style=";text-align:right;direction:rtl"> مراقبة متاهة العظمية التي تحتوي على القوقعة والأجهزة الطرفية الدهليزي في الحفرة القحفية الوسطى. جعل اثنين من الشقوق الصغيرة بجانب كل متاهة العظمية واستئصال هيكل كامل من خلال عقد نصف دائري القناة الأمامية وسحب في وقت لاحقحليف. </li><li style=";text-align:right;direction:rtl"> تزج فورا المتاهات رفعه في طبق تشريح تحتوي على حل ACSF المثلج (كما هو موضح في الخطوة 3.1)، في حين perfusing باستمرار مع كربوجين. </li><li style=";text-align:right;direction:rtl"> تحت stereomicroscope، باستمرار متاهة من القوقعة وضمان الحصول عليها إلى أسفل الطبق مع ملقط. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام ملقط غرامة مباشرة الى نقطة الصفر فتحة صغيرة في العظم فوق الأمامية القناة الهلالية (SSC) أمبولة. </li><li style=";text-align:right;direction:rtl"> بلطف تكبير هذا الانفتاح من خلال الوصول مباشرة أسفل العظم والتحريك إلى الخارج بعيدا عن أمبولة. توخي الحذر هنا ليست دفع ملقط في افتتاح وتلف التيه الغشائي الهشة أدناه. الاستمرار في هذا الشكل حتى يتعرض قريبة، الأمامية والجانبية الأمبولات جميع. إذا كان ذلك ممكنا إزالة أمبولة الخلفي أيضا. </li></ol></li><li style=";text-align:right;direction:rtl"> باستخدام ملقط غرامة، ورفع بلطف قريبة والأمبولات بعيدا عن متاهة العظمية حتى يتم فصل أنها تماما. حيثما كان ذلك ممكنا، وعقد لهمعن طريق القنوات نصف دائري المرتبطة بها لتجنب إتلاف الظهارة الحسية. في بعض الحالات، والجزء القريب من القناة غشائي الهلالية قد تحتاج إلى قطع مع مقص القزحية للافراج عن الأمبولات من العظام. </li><li style=";text-align:right;direction:rtl"> آمن فهم أجهزة الدهليزي بين غيض من الملقط ومكان في المخزن المؤقت تحلل أعدت في وقت سابق في الخطوة 3.2. دوامة بلطف ملقط حولها في المخزن المؤقت لضمان وفصل أجهزة الدهليزي من الملقط. تحقق هذا هو الحال من خلال جلب ملقط تحت stereomicroscope. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> المسمار على غطاء أنيبيب وتجميد العينة في النيتروجين السائل على الفور. </li></ol></li></ol> 4. استخراج الحمض النووي الريبي وRT-PCR <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> اتبع الطرق المعيارية الرنا المرسال (مرنا) استخراج وفقا لتعليمات الشركة الصانعة أو بروتوكولات مختبر المفضل. ملاحظة: هناك عدة التجارية التي تستخدم الرنا الناقل للمساعدة في عزل غلة صغيرة من مرنا واستخدم في هذاالبروتوكول. ويمكن استخدام كميات أقل شطف إلى زيادة تركيزات النهائي من مرنا. ملاحظة: السلبية الضوابط &quot;لا إنزيم&quot; (NECS) والضوابط &quot;لا قالب DNA&quot; (NTCS) يجب أيضا أن تكتمل لضمان صحة الآثار الملاحظة. </li><li style=";text-align:right;direction:rtl"> تطبيق الطرق المعيارية لعكس النسخ من مرنا لDNA مكملة ([كدنا]) والتضخيم من الجينات المستهدفة 21-23. </li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoglobin+induces+oxidative+stress+and+decreases+endocytosis+and+monolayer+permissiveness+in+cultured+kidney+epithelial+cells+without+affecting+viability.">Myoglobin induces oxidative stress and decreases endocytosis and monolayer permissiveness in cultured kidney epithelial cells without affecting viability.</a> Kidney and Blood Pressure Research. 31, 16-28 (2008).</li><li>Saee-Rad, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+superoxide+dismutase+1,+dual-specificity+phosphatase+1,+and+transforming+growth+factor,+beta+1+genes+expression+in+keratoconic+and+non-keratoconic+corneas.">Analysis of superoxide dismutase 1, dual-specificity phosphatase 1, and transforming growth factor, beta 1 genes expression in keratoconic and non-keratoconic corneas.</a> Molecular vision. 19, 2501-2507 (2013).</li><li>Albert, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPV4+channels+mediate+the+infrared+laser-evoked+response+in+sensory+neurons.">TRPV4 channels mediate the infrared laser-evoked response in sensory neurons.</a> Journal of neurophysiology. 107, 3227-3234 (2012).</li><li>Moro, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobiomodulation+inside+the+brain:+a+novel+method+of+applying+near-infrared+light+intracranially+and+its+impact+on+dopaminergic+cell+survival+in+MPTP-treated+mice.">Photobiomodulation inside the brain: a novel method of applying near-infrared light intracranially and its impact on dopaminergic cell survival in MPTP-treated mice.</a> Journal of neuroscience. 120, 670-683 (2014).</li><li>Moreno, L. E., 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=Infrared+neural+stimulation:+beam+path+in+the+guinea+pig+cochlea.">Infrared neural stimulation: beam path in the guinea pig cochlea.</a> Hearing research. 282, 289-302 (2011).</li><li>Curthoys, I. 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+red+thread+as+a+guide+in+the+vestibular+labyrinth.">A red thread as a guide in the vestibular labyrinth.</a> The Journal of physiology. 589, 1241-1241 (2011).</li><li>Chakrabarti, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Dysfunction+during+Brain+Aging:+Role+of+Oxidative+Stress+and+Modulation+by+Antioxidant+Supplementation.">Mitochondrial Dysfunction during Brain Aging: Role of Oxidative Stress and Modulation by Antioxidant Supplementation.</a> Aging and disease. 2, 242-256 (2011).</li><li>Petrosillo, G., De Benedictis, V., Ruggiero, F. M., Paradies, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decline+in+cytochrome+c+oxidase+activity+in+rat-brain+mitochondria+with+aging.+Role+of+peroxidized+cardiolipin+and+beneficial+effect+of+melatonin.">Decline in cytochrome c oxidase activity in rat-brain mitochondria with aging. Role of peroxidized cardiolipin and beneficial effect of melatonin.</a> Journal of bioenergetics and biomembranes. 45, 431-440 (2013).</li><li>Zhu, H., Sun, A., Zou, Y., Ge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+metabolic+adaptation+promotes+mesenchymal+stem+cell+therapy+for+ischemia:+a+hypoxia-induced+and+glycogen-based+energy+prestorage+strategy.">Inducible metabolic adaptation promotes mesenchymal stem cell therapy for ischemia: a hypoxia-induced and glycogen-based energy prestorage strategy.</a> Arteriosclerosis, thrombosis, and vascular biology. 34, 870-876 (2014).</li></ol>

تعلن المؤلفين ليس لديهم المصالح المالية المتنافسة.

نتائج تمثيلية الموصوفة هنا تشير إلى أن تسليم عبر الجمجمة وجيزة من نير ضوء (90 ثانية / يوم لمدة 5 أيام) كافية لرفع مستويات التعبير المضادة للأكسدة في الفئران الأكبر سنا بالمقارنة مع الفئران المعالجة الصورية. في حين الحرارة المنبعثة يمكن أن تمثل مصدرا للالميتوكوندريا و / ...

تراجع أداء التوازن والسقوط اللاحقة شائعة، وميزات للأسف في كثير من الأحيان تحديد الشيخوخة الطبيعية 1. تأثير هذا الانخفاض يمكن أن تكون المادي والاجتماعي، ويقلل بشكل ملحوظ نوعية الحياة لكبار السن. وردا على ذلك، كانت العلاجات الفيزيائية وإعادة تأهيل محور البحث في السقوط ولكن لم تترافق مع تخفيض ثابت في انتشار السقوط المتكرر. في نفس الوقت، والعمل يحقق تغييرات في نظام المحيطي أو المركزي الدهليزي (النظام مسؤول عن الحفاظ على التوازن) هي نادرة، والاستراتيجيات العلاجية المحتملة التي تستهدف هذه النظم والأسباب الكامنة وراء الخلل محدود. وقد أظهرت الأعمال الأخيرة على اضطرابات الاعصاب المرتبط بالعمر بما في ذلك المتعلقة بالعمر تحلل البقعة الصفراء 2-4، ونماذج مرض الزهايمر 5-8، ومرض باركنسون 9-12 آثار اعصاب سيmple تطبيق غير الغازية القريب من الأشعة تحت الحمراء (نير) الضوء. وعلاوة على ذلك، في النظام الدهليزي، وقد استخدمت نير إلى زيادة نشاط الخلايا العصبية الدهليزي وارد الابتدائية في المختبر 13. ومع أن آلية نير ضوء ليست مفهومة جيدا، وقد اقترح معظم الدراسات باستخدام نير نير أن يحفز الميتوكوندريا IV المعقدة (ج السيتوكروم أوكسيديز) 14-17 لتسهيل عملية الأيض الخلوي. في الظهارة الحسية الدهليزي لوحة تحت البشرة من نوع خلايا I الشعر كثيفة في الميتوكوندريا 18 وعلى هذا النحو قد تمثل موقع العمل لتلقي العلاج نير العلاجية. هنا، نظام العلاج وجيزة، غير الغازية من transcranially تطبيق نير التي يمكن استخدامها لقياس الأيض الخلوي (وضمنا، وظيفة الميتوكوندريا) في الماوس الدهليزي ظهارة حسية يوصف. ناقش أيضا هو إعداد الظهارة الحسية الدهليزي ويتبين أن نير يزيد من التعبير عن ubiquitoلنا مضادة للأكسدة (الفائق ديسموتاز 1) في الظهارة الحسية - أظهرت في وقت سابق إلى أن تكون مهمة لبقاء الخلية الشعر القوقعة 19.

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near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium

استراتيجيات لتخفيف انخفاض في وظيفة التوازن مع زيادة العمر هي مركزة بشكل رئيسي على العلاجات الجسدية بما في ذلك المهام التوازن وممارسة الرياضة. ومع ذلك، هذه الأساليب لا تعالج الأسباب الكامنة وراء تراجع التوازن. باستخدام الفئران، تم تقييم تأثير ضوء الأشعة تحت الحمراء القريب (نير) على التمثيل الغذائي للخلايا في الظهارة الحسية الدهليزي. البيانات التي تم جمعها تبين أن هذا التدخل بسيطة وآمنة قد تحمي هذه الخلايا الضعيفة من الآثار الضارة للشيخوخة الطبيعية. تم استخراج مرنا من الطرفية ظهارة معزولة الدهليزي الحسية (الأمبولي عرف والبقعة القريبي) وكتب لاحقا في مكتبة [كدنا]. وبعد ذلك تم بحث هذه المكتبة للتعبير عن مضادات الأكسدة في كل مكان (SOD-1). وبعد ذلك تستخدم مضادات الأكسدة التعبير الجيني لتحديد الأيض الخلوي. عن طريق تسليم عبر الجمجمة من نير في الشباب (4 أسابيع) وكبار السن (8-9 أشهر) الفئران، ونظام المعالجة قصيرة (90 ثانية / يوم لمدة 5 دآيس)، يشير هذا العمل نير وحدها قد تكون كافية لتحسين وظيفة الميتوكوندريا في الظهارة الحسية الدهليزي. وبما أن هناك حاليا أي الطرق المتاحة بأسعار معقولة، وغير الغازية العلاج لتحسين الدهليزي وظيفة خلايا الشعر، وتطبيق الإشعاع نير الخارجي يوفر استراتيجية محتملة لمواجهة تأثير الشيخوخة على الأيض الخلوي تي إتش الظهارة الحسية الدهليزي.

لمقارنة تأثير العلاج نير في الشباب (4 أسابيع) وكبار السن (8-9 أشهر) الفئران قمنا بقياس التعبير عن مضادات الأكسدة الفائق ديسموتاز 1 (SOD-1) في الشباب (ن = 16) وكبار السن (ن = 20) الفئران التي كانت نير المعالجة، وتعامل الشام، أو نير منعت-الشكل 2 يوضح زيادة كبيرة في β أكتين ت...

Watch this Scientific Journal Video about Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium at JoVE.com

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

ضعف الميتوكوندريا هو السمة المميزة لالشيخوخة الخلوية. تستخدم هذه الورقة غير الغازية بالقرب من الأشعة تحت الحمراء (نير) العلاج لتحسين وظيفة الميتوكوندريا في الشيخوخة الماوس الدهليزي ظهارة حسية.

بالقرب الأشعة تحت الحمراء (نير) ضوء يزيد من التعبير عن علامة من الميتوكوندريا وظيفة في ماوس الدهليزي الحسية الظهارة

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

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Research

JoVE Journal

Biology

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

9.8K Views.  University of Sydney.  ضعف الميتوكوندريا هو السمة المميزة لالشيخوخة الخلوية. تستخدم هذه الورقة غير الغازية بالقرب من الأشعة تحت الحمراء (نير) العلاج لتحسين وظيفة الميتوكوندريا في الشيخوخة الماوس الدهليزي ظهارة حسية. 

Video: Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

استكشاف الوظائف المعرفية لدى الأطفال والرضع والكبار مع التحليل الطيفي بالأشعة تحت الحمراء القريبة

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

باستخدام الجينات Electroporation MXcell بولسير النظام لTransfect الخلايا الأولية ذات الكفاءة العالية

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

مرحبا - C : طريقة لدراسة الهندسة المعمارية ثلاثي الأبعاد للخريطة جينوم.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

باستخدام خلية مكافحة الآلي لتبسيط دراسات التعبير الجيني : ضربة قاضية سيرنا من IL - 4 التعبير الجيني في الخلايا التابعة Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

إعادة إعماره في المختبر من أحدث T. castaneum تيلوميراز

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

باستخدام التصوير المقطعي عالي الدقة لتصور البنية الثلاثية وظيفة الأوعية الدموية النباتية

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

ملامح التعبير microRNA من خلايا الجذع الإنسان، الشبكية المشتقة من الظهارة الصبغية المحفزة، والجنين الظهارة الصبغية الشبكية

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

طريقة لتقييم الآثار المترتبة على مجموعة من موجات وشدة الأحمر / الأشعة تحت الحمراء القريبة العلاج بالضوء على الإجهاد التأكسدي<em&gt; في المختبر</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

دراسة الميتوكوندريا هيكل وظيفة في<em&gt; ذبابة الفاكهة</em&gt; المبايض

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

الثقافات الخلية الأولية من ظهارة صباغ الشبكية الماوس

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...