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Evaluation of suitable reference genes for gene expression studies in th. developing mouse cortex using RT-qPCR

BMC NEUROSCIENCE 2025年第1期26卷 1-11页

作者： Uppalapati, Ananya Wang, Timoth. nguyen, lena h. Univ Texas Dallas Sch Behav & Brain Sci Dept Neurosci Richardson TX 75080 USA

BackgroundReal-time quantitative PCR (RT-qPCR) is a widely used meth.d to investigate gene expression in neuroscience studies. Accurate relative quantification of RT-qPCR requires th. selection of reference genes th.t are stably expressed across th. experimental conditions and tissues of interest. Wh.le RT-qPCR is often performed to investigate gene expression ch.nges during neurodevelopment, few studies h.ve examined th. expression stability of commonly used reference genes in th. developing mouse ***, we evaluated th. stability of five h.usekeeping genes, Actb, Gapdh. B2m, Rpl13a, and h.rt, in cortical tissue from mice at embryonic day 15 to postnatal day 0 to identify optimal reference genes with.stable expression during late corticogenesis. Th. expression stability was assessed using five computational algorith.s: BestKeeper, geNorm, NormFinder, DeltaCt, and RefFinder. Our results sh.wed th.t B2m, Gapdh. and h.rt, or a combination of B2m/Gapdh.and B2m/h.rt, were th. most stably expressed genes or gene pairs. In contrast, Actb and Rpl13a were th. least stably *** study identifies B2m, Gapdh. and h.rt as suitable reference genes for relative quantification in RT-qPCR-based cortical development studies spanning th. period of embryonic day 15 to postnatal day 0.

关键词： Cerebral cortex Neurodevelopment Mouse RT-qPCR Gene expression Reference genes h.usekeeping genes

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Expression of 4E-BP1 in juvenile mice alleviates mTOR-induced neuronal dysfunction and epilepsy

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BRAIN 2022年第4期145卷 1310-1325页

作者： nguyen, lena h. Xu, Youfen Mah.deo, Travorn Zh.ng, Longbo Lin, Tiffany, V Born, h.ath.r A. Anderson, Anne E. Bordey, Angelique Yale Univ Dept Neurosurg Sch Med 310 Cedar StLH403 New Haven CT 06510 USA Yale Univ Dept Cellular & Mol Physiol Sch Med New Haven CT 06510 USA Texas Childrens Hosp Cain Fdn Labs Jan & Duncan Neurol Res Inst Houston TX 77030 USA Baylor Coll Med Dept Pediat Houston TX 77030 USA

h.peractivation of th. mTOR path.ay during foetal neurodevelopment alters neuron structure and function, leading to focal malformation of cortical development and intractable epilepsy. Recent evidence suggests a role for dysregulated cap-dependent translation downstream of mTOR signalling in th. formation of focal malformation of cortical development and seizures. h.wever, it is unknown wh.th.r modifying translation once th. developmental path.logies are establish.d can reverse neuronal abnormalities and seizures. Addressing th.se issues is crucial with.regards to th.rapeutics because th.se neurodevelopmental disorders are predominantly diagnosed during ch.ldh.od, wh.n patients present with.symptoms. h.re, we report increased ph.sph.rylation of th. mTOR effector and translational repressor, 4E-BP1, in patient focal malformation of cortical development tissue and in a mouse model of focal malformation of cortical development. Using temporally regulated conditional gene expression systems, we found th.t expression of a constitutively active form of 4E-BP1 th.t resists ph.sph.rylation by focal malformation of cortical development in juvenile mice reduced neuronal cytomegaly and corrected several neuronal electroph.siological alterations, including depolarized resting membrane potential, irregular firing pattern and aberrant expression of h.N4 ion ch.nnels. Furth.r, 4E-BP1 expression in juvenile focal malformation of cortical development mice after epilepsy onset resulted in improved cortical spectral activity and decreased spontaneous seizure frequency in adults. Overall, our study uncovered a remarkable plasticity of th. juvenile brain th.t facilitates novel th.rapeutic opportunities to treat focal malformation of cortical development-related epilepsy during ch.ldh.od with.potentially long-lasting effects in adults.

关键词： malformation of cortical development seizures cap-dependent translation h.perpolarization-activated cyclic nucleotide-gated (h.N) ch.nnels in utero electroporation

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Current Review in Basic Science: Animal Models of Focal Cortical Dysplasia and Epilepsy

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EPILEPSY CURRENTS 2022年第4期22卷 234-240页

作者： nguyen, lena h. Bordey, Angelique Yale Univ Sch Med Dept Neurosurg 333 Cedar St New Haven CT 06520 USA Yale Univ Sch Med Dept Cellular & Mol Physiol 333 Cedar St New Haven CT 06520 USA

Focal cortical dysplasia (FCD) is a malformation of cortical development th.t is a prevalent cause of intractable epilepsy in ch.ldren. Of th. th.ee FCD subtypes, understanding th. etiology and path.genesis of FCD type II h.s seen th. most progress owing to th. recent advances in identifying gene mutations along th. mTOR signaling path.ay as a frequent cause of th.s disorder. Accordingly, numerous animal models of FCD type II based on genetic manipulation of th. mTOR signaling path.ay h.ve emerged to investigate th. mech.nisms of epileptogenesis and novel th.rapeutics for epilepsy. Th.se include transgenic and in utero electroporation-based animal models. h.re, we review th. h.stopath.logical and electroclinical features of existing FCD type II animal models and discuss th. scientific and tech.ical considerations, clinical applications, and limitations of current models. We also h.gh.igh. oth.r models of FCD based on early life acquired factors.

关键词： malformation of cortical development cortical development cortical lesion transgenic mice in utero electroporation seizure pyramidal neurons astrocytes CRISPR Cas9

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In utero electroporation-based translating ribosome affinity purification identifies age-dependent mRNA expression in cortical pyramidal neurons

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NEUROSCIENCE RESEARCh 2019年第0期143卷 44-52页

作者： h.ang, Tianxiang nguyen, lena h. Lin, Tiffany, V Gong, Xuan Zh.ng, Longbo Kim, Gi Bum Sarkisian, Matth.w R. Breunig, Josh.a J. Bordey, Angelique Cent S Univ Xiangya Hosp Dept Neurosurg 85 Xiangya St Changsha 410008 Hunan Peoples R China Yale Univ Sch Med Dept Neurosurg 333 Cedar StFMB 422 New Haven CT 06520 USA Yale Univ Sch Med Dept Cellular & Mol Physiol 333 Cedar St New Haven CT 06520 USA Cedars Sinai Med Ctr Ctr Neural Sci & Med Board Governors Regenerat Med Inst Los Angeles CA 90048 USA Cedars Sinai Med Ctr Dept Biomed Sci Los Angeles CA 90048 USA Univ Florida Dept Neurosci McKnight Brain Inst Gainesville FL 32610 USA Univ Calif Los Angeles David Geffen Sch Med Dept Med Los Angeles CA 90095 USA

We combined translating ribosome affinity purification (TRAP) with.in utero electroporation (IUE), called iTRAP to identify th. molecular profile of specific neuronal populations during neonatal development with.ut th. need for viral approach.s and FACS sorting. We electroporated a plasmid encoding EGFP-tagged ribosomal protein L10a at embryonic day (E) 14-15 to target layer 2-4 cortical neurons of th. somatosensory cortex. At th.ee postnatal (P) ages-P0, P7, and P14-wh.n morph.genesis occurs and synapses are forming, TRAP and molecular profiling was performed from electroporated regions. We found th.t ribosome bound (Ribo)-mRNAs from similar to 7300 genes were significantly altered over time and included classical neuronal genes known to decrease (e.g., Tbr1, Dcx) or increase (e.g., Eno2, Camk2a, Syn1) as neurons mature. Th.s approach.led to th. identification of specific developmental patterns for Ribo-mRNAs not previously reported to be developmentally regulated in neurons, providing rationale for future examination of th.ir role in selective biological processes. Th.se include upregulation of Lynx1, Nrn1, Cntnap1 over time;downregulation of St8sia2 and Draxin;and bidirectional ch.nges to Fkbp1b. iTRAP is a versatile approach.th.t allows research.rs to easily assess th. molecular profile of specific neuronal populations in selective brain regions under various conditions, including overexpression and knockdown of target genes, and in disease settings. (C) 2018 Elsevier B.V. and Japan Neuroscience Society. All righ.s reserved.

关键词： TRAP Corticogenesis Migration Spine Glutamatergic neurons

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mTOR-dependent alterations of Kv1.1 subunit expression in th. neuronal subset-specific Pten knockout mouse model of cortical dysplasia with.epilepsy

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SCIENTIFIC REPORTS 2018年第1期8卷 3568-3568页

作者： nguyen, lena h. Anderson, Anne E. Baylor Coll Med Dept Neurosci Houston TX 77030 USA Texas Childrens Hosp Jan & Dan Duncan Neurol Res Inst Houston TX 77030 USA Texas Childrens Hosp Gordon & Mary Cain Pediat Neurol Res Fdn Labs Houston TX 77030 USA Baylor Coll Med Dept Pediat Houston TX 77030 USA Baylor Coll Med Dept Neurol Houston TX 77030 USA

Cortical dysplasia (CD) is a common cause for intractable epilepsy. h.peractivation of th. mech.nistic target of rapamycin (mTOR) path.ay h.s been implicated in CD;h.wever, th. mech.nisms by wh.ch.mTOR h.peractivation contribute to th. epilepsy ph.notype remain elusive. h.re, we investigated wh.th.r constitutive mTOR h.peractivation in th. h.ppocampus is associated with.altered voltage-gated ion ch.nnel expression in th. neuronal subset-specific Pten knockout (NS-Pten KO) mouse model of CD with.epilepsy. We found th.t th. protein levels of Kv1.1, but not Kv1.2, Kv1.4, or Kv beta 2, potassium ch.nnel subunits were increased, along with.altered Kv1.1 distribution, with.n th. h.ppocampus of NS-Pten KO mice. Th. aberrant Kv1.1 protein levels were present in young adult (>= postnatal week 6) but not juvenile (<= postnatal week 4) NS-Pten KO mice. No ch.nges in h.ppocampal Kv1.1 mRNA levels were found between NS-Pten KO and WT mice. Interestingly, mTOR inh.bition with.rapamycin treatment at early and late stages of th. path.logy normalized Kv1.1 protein levels in NS-Pten KO mice to WT levels. Togeth.r, th.se studies demonstrate altered Kv1.1 protein expression in association with.mTOR h.peractivation in NS-Pten KO mice and suggest a role for mTOR signaling in th. modulation of voltage-gated ion ch.nnel expression in th.s model.

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mTOR inh.bition suppresses establish.d epilepsy in a mouse model of cortical dysplasia

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EPILEPSIA 2015年第4期56卷 636-646页

作者： nguyen, lena h. Brewster, Amy L. Clark, Madeline E. Regnier-Golanov, Angelique Sunnen, C. Nicole Patil, Vinit V. D'Arcangelo, Gabriella Anderson, Anne E. Baylor Coll Med Dept Neurosci Houston TX 77030 USA Texas Childrens Hosp Jan & Dan Duncan Neurol Res Inst Houston TX 77030 USA Texas Childrens Hosp Gordon & Mary Cain Pediat Neurol Res Fdn Labs Houston TX 77030 USA Purdue Univ Dept Psychol Sci W Lafayette IN 47907 USA Baylor Coll Med Dept Pediat Houston TX 77030 USA Univ Sci Dept Biol Sci Philadelphia PA USA Baylor Coll Med Program Translat Biol & Mol Med Houston TX 77030 USA Rutgers State Univ Dept Cell Biol & Neurosci Piscataway NJ USA Baylor Coll Med Dept Neurol Houston TX 77030 USA

Objectiveh.peractivation of th. mech.nistic target of rapamycin (mTOR;also known as mammalian target of rapamycin) path.ay h.s been demonstrated in h.man cortical dysplasia (CD) as well as in animal models of epilepsy. Alth.ugh.inh.bition of mTOR signaling early in epileptogenesis suppressed epileptiform activity in th. neuron subset-specific Pten knockout (NS-Pten KO) mouse model of CD, th. effects of mTOR inh.bition after epilepsy is fully establish.d were not previously examined in th.s model. h.re, we investigated wh.th.r mTOR inh.bition suppresses epileptiform activity and oth.r neuropath.logical correlates in adult NS-Pten KO mice with.severe and well-establish.d epilepsy. Meth.dsTh. progression of epileptiform activity, mTOR path.ay dysregulation, and associated neuropath.logy with.age in NS-Pten KO mice were evaluated using video-electroenceph.lograph. (EEG) recordings, Western blotting, and immunoh.stoch.mistry. A coh.rt of NS-Pten KO mice was treated with.th. mTOR inh.bitor rapamycin (10mg/kg i.p., 5days/week) starting at postnatal week 9 and video-EEG monitored for epileptiform activity. Western blotting and immunoh.stoch.mistry were performed to evaluate th. effects of rapamycin on th. associated path.logy. ResultsEpileptiform activity worsened with.age in NS-Pten KO mice, with.parallel increases in th. extent of h.ppocampal mTOR complex 1 and 2 (mTORC1 and mTORC2, respectively) dysregulation and progressive astrogliosis and microgliosis. Rapamycin treatment suppressed epileptiform activity, improved baseline EEG activity, and increased survival in severely epileptic NS-Pten KO mice. At th. molecular level, rapamycin treatment was associated with.a reduction in both.mTORC1 and mTORC2 signaling and decreased astrogliosis and microgliosis. SignificanceTh.se findings reveal a wide temporal window for successful th.rapeutic intervention with.rapamycin in th. NS-Pten KO mouse model, and th.y support mTOR inh.bition as a candidate th.rapy for establish.d, late

关键词： Malformation of cortical development Seizures Rapamycin Astrogliosis Microgliosis

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Filamin A inh.bition reduces seizure activity in a mouse model of focal cortical malformations

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SCIENCE TRANSLATIONAL MEDICINE 2020年第531期12卷 eaay0289页

作者： Zh.ng, Longbo h.ang, Tianxiang Teaw, Sh.nnon nguyen, lena h. h.ieh. Lawrence S. Gong, Xuan Burns, Lindsay h. Bordey, Angelique Yale Univ Sch Med Dept Neurosurg New Haven CT 06520 USA Yale Univ Sch Med Dept Cellular & Mol Physiol New Haven CT 06520 USA Cent S Univ Xiangya Hosp Dept Neurosurg 87 Xiangya St Changsha 410008 Hunan Peoples R China Cassava Sci Inc Austin TX 78731 USA

Epilepsy treatments for patients with.mech.nistic target of rapamycin (mTOR) disorders, such.as tuberous sclerosis complex (TSC) or focal cortical dysplasia type II (FCDII), are urgently needed. In th.se patients, th. presence of focal cortical malformations is associated with.th. occurrence of lifelong epilepsy, leading to severe neurological comorbidities. h.re, we sh.w th.t th. expression of th. actin cross-linking protein filamin A (FLNA) is increased in resected cortical tissue th.t is responsible for seizures in patients with.FCDII and in mice modeling TSC and FCDII with.mutations in ph.sph.inositide 3-kinase (PI3K)-ras h.molog enrich.d in brain (Rh.b) path.ay genes. Normalizing FLNA expression in th.se mice th.ough.genetic knockdown limited cell misplacement and neuronal dysmorph.genesis, two h.llmarks of focal cortical malformations. In addition, Flna knockdown reduced seizure frequency independently of mTOR signaling. Treating mice with.a small molecule targeting FLNA, PTI-125, before th. onset of seizures alleviated neuronal abnormalities and reduced seizure frequency compared to veh.cle-treated mice. In addition, th. treatment was also effective wh.n injected after seizure onset in juvenile and adult mice. Th.se data suggest th.t targeting FLNA with.eith.r sh.rt h.irpin RNAs or th. small molecule PTI-125 migh. be effective in reducing seizures in patients with.TSC and FCDII bearing mutations in PI3K-Rh.b path.ay genes.

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Inh.bition of MEK-ERK signaling reduces seizures in two mouse models of tuberous sclerosis complex

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EPILEPSY RESEARCh 2022年第0期181卷 106890-106890页

作者： nguyen, lena h. Leiser, Steven C. Song, Dekun Brunner, Daniela Roberds, Steven L. Wong, Mich.el Bordey, Angelique Yale Univ Sch Med Dept Neurosurg 333 Cedar StLH403 New Haven CT 06520 USA Yale Univ Sch Med Dept Cellular & Mol Physiol New Haven CT USA PsychoGenics Inc Dept Translat EEG Paramus NJ USA Tuberous Sclerosis Alliance Silver Spring MD USA Washington Univ Sch Med Dept Neurol St Louis MO USA

Tuberous sclerosis complex (TSC) is a monogenic disorder ch.racterized by h.peractivation of th. mTOR signaling path.ay and developmental brain malformations leading to intractable epilepsy. Alth.ugh.treatment with.th. recently approved mTOR inh.bitor, everolimus, results in clinically relevant seizure suppression in up to 40% of TSC patients, seizures remain uncontrolled in a large number of cases, underscoring th. need to identify novel treatment targets. Th. MEK-ERK signaling path.ay h.s been found to be aberrantly activated in TSC and inh.bition of MEK-ERK activity independently of mTOR rescued neuronal dendrite overgrowth.in mice modeling TSC neuropath.logy. h.re, we evaluated th. efficacy of MEK-ERK inh.bition on seizures in two mouse models of TSC. We found th.t treatment with.th. MEK inh.bitor PD0325901 (mirdametinib) significantly reduced seizure activity in both.TSC mouse models. Th.se findings support inh.biting MEK-ERK activity as a potential alternative strategy to treat seizures in TSC.

关键词： Epilepsy Seizures Tuberous sclerosis complex MEK inh.bitor MEK-ERK signaling MAPK

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Ectopic h.N4 expression drives mTOR-dependent epilepsy in mice

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SCIENCE TRANSLATIONAL MEDICINE 2020年第570期12卷 eabc1492页

作者： h.ieh. Lawrence S. Wen, Joh. h. nguyen, lena h. Zh.ng, Longbo Getz, Steph.nie A. Torres-Reveron, Juan Wang, Ying Spencer, Dennis D. Bordey, Angelique Yale Univ Sch Med Dept Neurosurg 333 Cedar St New Haven CT 06520 USA Cent South Univ Xiangya Hosp Dept Neurosurg 87 Xiangya St Changsha 410008 Hunan Peoples R China Cent South Univ Xiangya Hosp Emergency Dept 87 Xiangya St Changsha 410008 Hunan Peoples R China Yale Univ Sch Med Dept Cellular & Mol Physiol 333 Cedar St New Haven CT 06520 USA Univ Cincinnati Acad Hlth Ctr Dept Neurosurg 222 Piedmont AveSuite 6200 Cincinnati OH 45219 USA

Th. causative link between focal cortical malformations (FCMs) and epilepsy is well accepted, especially among patients with.focal cortical dysplasia type II (FCDII) and tuberous sclerosis complex (TSC). h.wever, th. mech.nisms underlying seizures remain unclear. Using a mouse model of TSC- and FCDII-associated FCM, we sh.wed th.t FCM neurons were responsible for seizure activity via th.ir unexpected abnormal expression of th. h.perpolarization-activated cyclic nucleotide-gated potassium ch.nnel isoform 4 (h.N4), wh.ch.is normally not present in cortical pyramidal neurons after birth. Increasing intracellular cAMP concentrations, wh.ch.preferentially affects h.N4 gating relative to th. oth.r isoforms, drove repetitive firing of FCM neurons but not control pyramidal neurons. Ectopic h.N4 expression was dependent on th. mech.nistic target of rapamycin (mTOR), preceded th. onset of seizures, and was also found in diseased neurons in tissue resected from patients with.TSC and FCDII. Last, blocking h.N4 ch.nnel activity in FCM neurons prevented epilepsy in th. mouse model. Th.se findings suggest th.t h.N4 play a main role in seizure and identify a cAMP-dependent seizure mech.nism in TSC and FCDII. Furth.rmore, th. unique expression of h.N4 exclusively in FCM neurons suggests th.t gene th.rapy targeting h.N4 migh. be effective in reducing seizures in FCDII or TSC.

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Biliary Tubes in Liver Transplantation

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JOURNAL OF GASTROINTESTINAL SURGERY 2011年第1期15卷 232-233页

作者： Sibulesky, lena nguyen, Justin h. Mayo Clin Div Transplant Surg Dept Transplantat Jacksonville FL 32224 USA

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