A unified solution for free vibration of orthotropic circular, annular and sector plates with general boundary conditions

This paper adopts a modified Fourier-Ritz approach to study the free vibration characteristics of orthotropic circular, annular and sector thin plates subjected to general boundary conditions. For the arbitrary plate forms and the boundary conditions, the displacements can be written in the form of a standard Fourier cosine series supplemented with several auxiliary functions. The auxiliary functions, which are closed-form and introduced to remove all the potential discontinuities of the original displacement function and its derivatives in the whole domain, can be usefully employed in improving the convergence of the results. The artificial boundary spring technique and artificial coupling spring technique are adopted to simulate the arbitrary boundary conditions and to ensure appropriate continuity conditions along the radial edges, respectively. Because the displacement field is sufficiently smooth in the whole solution domain, the accurate solution can be obtained by using the Ritz procedure on the basis of the energy functions. The accuracy, reliability and versatility of the current method are fully demonstrated and verified through numerical examples involving plates with various shapes and boundary conditions

Multimodal investigations of trans-endothelial cell trafficking under condition of disrupted blood-brain barrier integrity

<p>Abstract</p> <p>Background</p> <p>Stem cells or immune cells targeting the central nervous system (CNS) bear significant promises for patients affected by CNS disorders. Brain or spinal cord delivery of therapeutic cells is limited by the blood-brain barrier (BBB) which remains one of the recognized rate-limiting steps. Osmotic BBB disruption (BBBD) has been shown to improve small molecule chemotherapy for brain tumors, but successful delivery of cells in conjunction with BBBD has never been reported.</p> <p>We have used a clinically relevant model (pig) of BBBD to attempt brain delivery of TALL-104, a human leukemic T cell line. TALL-104 cells are potent tumor killers and have demonstrated potential for systemic tumor therapy. The pig model used is analogous to the clinical BBBD procedure. Cells were injected in the carotid artery after labeling with the MRI T1 contrast agent GdHPDO3A. Contrast CT scans were used to quantify BBBD and MRI was used to detect Gd⁺⁺-loaded cells in the brain. Transcranial Doppler was used to monitor cerebral blood flow. EEG recordings were used to detect seizures. Immunocytochemical detection (Cresyl Violet, anti-human CD8 for TALL-104, Evans Blue for BBB damage, GFAP and NEUN) was performed.</p> <p>Results</p> <p>At the concentration used TALL-104 cells were tolerated. Incomplete BBBD did not allow cell entry into the brain. MRI scans at 24 and 48 hours post-injection allowed visualization of topographically segregated cells in the hemisphere that underwent successful BBBD. Perivascular location of TALL-104 was confirmed in the BBBD hemisphere by Cresyl violet and CD8 immunocytochemistry. No significant alteration in CBF or EEG activity was recorded during cell injections.</p> <p>Conclusions</p> <p>Our data show that targeted CNS cell therapy requires blood-brain barrier disruption. MRI-detectable cytotoxic anti-neoplastic cells can be forced to transverse the BBB and accumulate in the perivascular space. The virtual absence of toxicity, the high anti-tumor activity of TALL-104, and the clinical feasibility of human osmotic BBBD suggest that this approach may be adopted to treat brain or spinal cord tumors. In addition, BBBD may favor CNS entry of other cells that normally lack CNS tropism.</p

Synergy between vinorelbine and afatinib in the inhibition of non-small cell lung cancer progression by EGFR and p53 signaling pathways

Currently, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) were approved for the treatment of non-small cell lung cancer (NSCLC) patients harboring EGFR mutation. However, some lung cancer patients fail to respond and eventually develop drug resistance. Therefore, new therapeutic strategies are needed to improve the outcomes for substantial clinical benefit. Here we aimed to explore the combination of vinorelbine with the second EGFR-TKI afatinib in NSCLC cells with or without EGFR mutation. The three cells of H1975, HCC827, and H460 were assessed for the combination of vinorelbine and afatinib. Vinorelbine combined with afatinib synergistically inhibited the three lung cancer cells growth without aggravating adverse effect on the normal lung cells. The combination of low doses of vinorelbine and afatinib suppressed the cancer cell proliferation by cell colony formation assay and significantly induced cell apoptosis. The anti-apoptotic proteins Bcl-xL and Bcl-2 showed significant reduction after the drug combination treatment, while the pro-apoptotic protein Bax as well as apoptosis indicators cytochrome C and cleaved PARP were observed a notable increasing. EGFR downstream pathways including AKT, ERK, JNK, and p38 were highly active and p53 was inactive in the three lung cancer cells, favoring tumor growth. The low doses of vinorelbine plus afatinib blocked the phosphorylation of AKT, ERK, JNK, and p38, but restored the expression of p53. Our findings suggested that the combination of vinorelbine and afatinib could be recommended as a therapeutic regimen for treatment of NSCLC with or without EGFR mutation

Extracranial sources of S100B do not affect serum levels.

S100B, established as prevalent protein of the central nervous system, is a peripheral biomarker for blood-brain barrier disruption and often also a marker of brain injury. However, reports of extracranial sources of S100B, especially from adipose tissue, may confound its interpretation in the clinical setting. The objective of this study was to characterize the tissue specificity of S100B and assess how extracranial sources of S100B affect serum levels. The extracranial sources of S100B were determined by analyzing nine different types of human tissues by ELISA and Western blot. In addition, brain and adipose tissue were further analyzed by mass spectrometry. A study of 200 subjects was undertaken to determine the relationship between body mass index (BMI) and S100B serum levels. We also measured the levels of S100B homo- and heterodimers in serum quantitatively after blood-brain barrier disruption. Analysis of human tissues by ELISA and Western blot revealed variable levels of S100B expression. By ELISA, brain tissue expressed the highest S100B levels. Similarly, Western blot measurements revealed that brain tissue expressed high levels of S100B but comparable levels were found in skeletal muscle. Mass spectrometry of brain and adipose tissue confirmed the presence of S100B but also revealed the presence of S100A1. The analysis of 200 subjects revealed no statistically significant relationship between BMI and S100B levels. The main species of S100B released from the brain was the B-B homodimer. Our results show that extracranial sources of S100B do not affect serum levels. Thus, the diagnostic value of S100B and its negative predictive value in neurological diseases in intact subjects (without traumatic brain or bodily injury from accident or surgery) are not compromised in the clinical setting

Modulation of coxsackie and adenovirus receptor expression for gene transfer to normal and dystrophic skeletal muscle

Background: Efficient adenovirus (AdV)-mediated gene transfer is possible only in immature muscle or regenerating muscle, suggesting that a developmentally regulated event plays a major role in limiting AdV uptake in mature skeletal muscle. Previously, we showed that the expression of the primary coxsackie and adenovirus receptor (CAR) is severely down-regulated during muscle maturation and that, in muscle-specific CAR transgenic mice, there is significant enhancement of AdV-mediated gene transfer to mature skeletal muscle. Methods: To evaluate whether increasing CAR expression can also augment gene transfer to dystrophic muscle that has many regenerating fibers, we crossed CAR transgenics with dystrophin-deficient mice (mdx/CAR). We also tested a two-step protocol in which CAR levels were increased in the target muscle, prior to administration of AdV, through the use of recombinant adeno-associated virus (AAV2) expressing CAR. Lastly, we assessed the effect of histone deacetylase inhibitors on CAR and AdV transduction efficiency in myoblasts and mdx muscle. Results: Although somewhat higher rates of transduction can be achieved in adult mdx mice than in normal mice as a result of ongoing muscle regeneration in these animals, CAR expression in the mdx background (mdx/CAR transgenics) still markedly improved the susceptibility of mature muscle to AdV-mediated gene transfer of dystrophin. Prior administration of AAV2-CAR to normal muscle led to significantly increased transduction by subsequent injection of AdV. The histone deacetylase inhibitor valproate increased CAR transcript and protein levels in myoblasts and mdx muscle, and also increased AdV-mediated gene transfer. Conclusions: We have developed a method of increasing CAR levels in both normal and regenerating muscle.Contexte : un transfert efficace de g\ue8nes par ad\ue9novirus est possible uniquement dans le muscle immature ou en r\ue9g\ue9n\ue9ration, ce qui suppose qu\u2019un \ue9v\ue9nement r\ue9gul\ue9 par le d\ue9veloppement joue un r\uf4le important dans la limitation de l\u2019absorption ad\ue9novirale dans le muscle squelettique mature. Nous avons d\ue9j\ue0 montr\ue9 que l\u2019expression du r\ue9cepteur primaire CAR [coxsackie and adenovirus receptor] est r\ue9gul\ue9e fortement \ue0 la baisse au cours de la maturation musculaire et que chez la souris transg\ue9nique pour le r\ue9cepteur CAR sp\ue9cifique du muscle, on observe une am\ue9lioration importante du transfert de g\ue8nes par ad\ue9novirus au muscle squelettique mature. M\ue9thodes : pour \ue9valuer si l\u2019augmentation de l\u2019expression du r\ue9cepteur CAR peut \ue9galement augmenter le transfert de g\ue8nes au muscle dystrophique qui pr\ue9sente de nombreuses fibres en r\ue9g\ue9n\ue9ration, nous avons crois\ue9 des souris transg\ue9niques pour le r\ue9cepteur CAR avec des souris d\ue9ficientes en dystrophine (mdx/CAR). Nous avons \ue9galement essay\ue9 un protocole en deux \ue9tapes qui consiste \ue0 augmenter le taux de r\ue9cepteurs CAR dans le muscle cible au moyen du virus recombinant ad\ue9no-associ\ue9 (AAV2) exprimant le r\ue9cepteur CAR, avant d\u2019administrer l\u2019ad\ue9novirus. Enfin, nous avons \ue9valu\ue9 l\u2019effet des inhibiteurs de l\u2019histone d\ue9sac\ue9tylase sur l\u2019efficacit\ue9 de la transduction du r\ue9cepteur CAR et de l\u2019ad\ue9novirus dans des myoblastes et des muscles de souris mdx. R\ue9sultats : bien qu\u2019il soit possible d\u2019obtenir des taux de transduction un peu plus \ue9lev\ue9s chez la souris mdx adulte que chez la souris normale en raison de la r\ue9g\ue9n\ue9ration musculaire continue chez ces animaux, l\u2019expression du r\ue9cepteur CAR coupl\ue9e \ue0 l\u2019absence de dystrophine (croisement mdx/CAR) a encore am\ue9lior\ue9 nettement la sensibilit\ue9 du muscle mature au transfert de g\ue8nes par ad\ue9novirus de la dystrophine. L\u2019administration de l\u2019AAV2 CAR au muscle normal avant l\u2019injection de l\u2019ad\ue9novirus a men\ue9 \ue0 une augmentation importante de la transduction. Un inhibiteur de l\u2019histone d\ue9sac\ue9tylase, le valproate, a augment\ue9 les taux de transcrits et de prot\ue9ines du r\ue9cepteur CAR dans les myoblastes et le muscle de souris mdx, et a \ue9galement augment\ue9 le transfert de g\ue8nes par ad\ue9novirus. Conclusion : nous avons mis au point une m\ue9thode qui permet d\u2019augmenter le taux des r\ue9cepteurs CAR dans le muscle normal et le muscle en r\ue9g\ue9n\ue9ration.Peer reviewed: YesNRC publication: Ye

Viral clostridial light chain gene-based control of penicillin-induced neocortical seizures.

International audienceRestraining excitatory neurotransmission within a seizure focus provides a nondestructive treatment strategy for intractable neocortical epilepsy. Clostridial toxin light chain (LC) inhibits synaptic transmission by digesting a critical vesicle-docking protein, synaptobrevin, without directly altering neuronal health. This study tests the treatment efficacy of adenoviral vector delivered LC (AdLC) on a model of seizures in rats induced by motor cortex penicillin (PCN) injection. LC expression significantly reduced electroencephalogram (EEG) frequency, amplitude, duration, and latency compared to control groups injected with either an adenoviral vector bearing green fluorescent protein (AdGFP) or phosphate buffered solution (PBS). Correspondingly, LC gene expression improved behavioral manifestations including seizure severity and latency. There was no statistical difference in motor function before and after vector administration between treatment and control groups. Histological analysis revealed spatially discrete LC expression with corresponding synaptobrevin depletion in the cortex surrounding the injection site. Thus, vector-mediated LC gene delivery is capable of improving both the EEG and behavioral manifestations of PCN-induced focal neocortical seizures through synaptobrevin depletion

Authors: Motor Neuron Inhibition-Based Gene Therapy for Spasticity REVIEW &amp; ANALYSIS MECHANISM OF SPASTICITY

ABSTRACT Spasticity is a condition resulting from excess motor neuron excitation, leading to involuntary muscle contraction in response to increased velocity of movement, for which there is currently no cure. Existing symptomatic therapies face a variety of limitations. The extent of relief that can be delivered by ablative techniques such as rhizotomy is limited by the potential for sensory denervation. Pharmacological approaches, including intrathecal baclofen, can be undermined by tolerance. One potential new approach to the treatment of spasticity is the control of neuromuscular overactivity through the delivery of genes capable of inducing synaptic inhibition. A variety of experiments in cell culture and animal models have demonstrated the ability of neural gene transfer to inhibit neuronal activity and suppress transmission. Similarly, enthusiasm for the application of gene therapy to neurodegenerative diseases of motor neurons has led to the development of a variety of strategies for motor neuron gene delivery. In this review, we discuss the limitations of existing spasticity therapies, the feasibility of motor neuron inhibition as a gene-based treatment for spasticity, potential inhibitory transgene candidates, strategies for control of transgene expression, and applicable motor neuron gene targeting strategies. Finally, we discuss future directions and the potential for gene-based motor neuron inhibition in therapeutic clinical trials to serve as an effective treatment modality for spasticity, either in conjunction with or as a replacement for presently available therapies. Key Words: Spasticity, Transgene Expression, Motor Neuron, Gene Targeting Spasti city is a condition resulting from excess motor neuron excitation caused by lesions in the upper motor neuron pathway that lead to the absence of inhibition of alpha and/or gamma motor neurons. This loss of inhibition results in involuntary muscle contraction, causing stiffness interfering with movement, speech, and locomotion. 1,2 Affecting more than 12 million people worldwide, spasticity is commonly caused by stroke, multiple sclerosis, cerebral palsy, cerebral infection (encephalitis/meningitis), and/or cerebrospinal trauma. REVIEW &amp; ANALYSIS Spasticity MECHANISM OF SPASTICITY The exact mechanism of spasticity in humans is incompletely understood, primarily because it is multifactorial in nature. It is generally understood that spasticity is caused by pathology involving the stretch reflex, which normally causes a muscle to contract to resist the force that is stretching it. For normal movement to occur, the brain must be able to selectively turn this reflex off, usually via inhibitory signals relayed to the spinal cord via the corticospinal tract. However, damage to this circuit results in disinhibition of the stretch reflex; over time, this reduces the triggering threshold until excessive and complete muscle contraction can occur even at rest, making the limb virtually impossible to move. Specific causes proposed include (a) alpha motor neuron hyperexcitability resulting from an imbalance in excitatory vs. inhibitory alpha motor neurons, and (b) gamma motor neuron hyperactivity manifesting as increased sensitivity of muscle spindle to stretch (fusimotor hyperactivity). 9 Additional causes involve damage to descending tracts that control interneurons responsible for (a) mediating presynaptic inhibition of the Ia terminals on the alpha motor neuron, (b) mediating type II afferents, and (c) reciprocal Ia inhibition. Such damage results in greater afferent stimulus to the alpha motor neuron as a result of stretch, decreased inhibition from type II afferents, and loss of normal inhibition of antagonist muscle during muscle stretch. Finally, a mechanism of decreased recurrent inhibition from Renshaw cells as a consequence of supraspinal damage has also been proposed. Whereas each individual cause contributes to the clinical picture observed in spasticity, it is unlikely that any single hypothesis is sufficient to explain the exact mechanism of spasticity. EXISTING THERAPIES AND LIMITATIONS Medical Therapies Although there are a number of oral medications available to treat spasticity, almost none are effective without significant side effects. The most common medications are diazepam, baclofen, and progabide, all of which are designed to increase presynaptic inhibition of alpha motor neurons by activation of ␥-aminobutyric acid (GABA) receptors. However, these treatments are associated with a high incidence of adverse effects, including sedation, weakness, fever without infection, and elevated liver enzymes. 10,11 Agents affecting ion flux in skeletal muscle, such as dantrolene, lamotrigine, and riluzole, share side effects of muscle weakness, sedation, and idiosyncratic hepatitis. 11,13 These significant toxicities limit the doses of medication that can be employed, thereby limiting efficacy. Furthermore, the issue of tolerance significantly hinders the longterm efficacy of any pharmacologic therapy, particularly for baclofen and diazepam. An alternative medical therapy involves the use of clostridial toxin (i.e., botulinum toxin), which acts by decreasing acetylcholine release at the neuromuscular junction, resulting in a neuromuscular blocking effect. However, the results from this therapy are often transient, with redosing complicated by tachyphylaxis, and increasing dosage complicated by severe muscle weakness. 14 -16 The issue of cost is another consideration. The cost of clostridial toxin treatment might hinder its extensive clinical applications because conventional oral therapies are much less expensive. 17 Surgical Therapies Surgery for spasticity is reserved for cases refractory to medical management or for those that cannot be medically managed because of intolerable side effects. The most common surgical options are generally orthopedic (consisting primarily of tendon-release operations) and neurosurgical. 18,19 Neurosurgical procedures fall into two categories: nonablative and ablative. The most frequently used nonablative procedure is intrathecal baclofen (ITB), which is generally offered for refractory patients with chronic spasticity (Ͼ12 mos). To be considered a candidate, a patient must demonstrate a positive response to ITB at a test dose of less than 100 g, compared with no response to placebo. 20 Although highly effective in improving muscle tone and reducing postoperative spasticity, ITB is fraught with catheter-and wound-related morbidity, both at the time of implantation and throughout the life of the implanted device. 20,21 There are limited data characterizing the problem of tolerance to ITB. [22][23][24][25] In many cases, ITB tolerance is ascribed to progression of the underlying disease (in amyotrophic lateral sclerosis and multiple sclerosis) or to dynamic catheter obstruction (kinking), which is difficult to demonstrate on standard pump contrast injections (pumpograms) or nuclear medicine studies. 23,26 In our practice, we use inpatient externalized catheter ITB trials to address the question of baclofen tolerance. In this context, it is easy to assess the patency of the catheter and document the threshold for response to an intrathecal drug. Outpatient trials of bolus intrathecal injection can be attempted, but these are often misleading because of the inherent differences in the pharmacokinetics of bolus and pump injection. However, the majority of our patients require gradually escalated doses of ITB to maintain adequate control of spasticity. May 2007 Gene Therapy for Spasticity 413 Another nonablative option is spinal cord stimulation, which has been shown to facilitate spasticity control, spasm inhibition, and gait improvement in spastic patients, 27-29 likely by selective modification of segmental spinal reflexes. 33 The most common ablative procedure is selective dorsal rhizotomy, which uses intraoperative electromyography and stimulation to identify the rootlets most responsible for causing severe spasticity. 42-45 POSSIBLE FUTURE THERAPIES Despite the wide range of medical and surgical treatments for spasticity, there is currently no treatment modality that is hardware free, reversible, adjustable, nondestructive, and not subject to tolerance. 2,3,14 -16,46 A potential modality for satisfying these criteria is the use of viral vectors to elicit effects on muscle contraction via gene transfer, because selective control of certain genes has been shown to modulate neuronal activity in multiple applications. POTENTIAL INHIBITORY TRANSGENES One of the most widely studied inhibitory transgenes is the gene encoding glutamate decarboxylase (GAD), the rate-limiting enzyme required to produce the inhibitory neurotransmitter GABA. In vitro and in vivo studies using retroviral vectors and adeno-associated virus (AAV) vectors have suggested that it is feasible to achieve long-term GAD expression in the CNS. 57 GAD was expressed, and the expression of GAD induced production of GABA in these neurons. These findings suggest that AAV-mediated GAD gene transfer might provide a treatment option for overactive diseases such as Parkinson disease. 57,58 These results have led to an ongoing Phase I trial of GAD gene transfer to the human subthalamic nucleus for medically refractory Parkinson disease patients. Tetanus Toxin Light Chain Our laboratory has focused on the gene for the light chain (LC) fragment of clostridial neurotoxin. The expression of this gene in neurons provides inhibition of synaptic function in transgenic mice via reversible suppression of glutamatergic neurotransmission. 59,60 Clostridial intoxication in neurons involves the production of inactive single-chain clostridial neurotoxin polypeptides, which are released after bacterial lysis. This release converts the polypeptide from an inactive single-chain molecule to an active di-chain molecule composed of a heavy chain (HC) and an LC fragment linked by a single disulfide bond. HC binds axon terminals and triggers internalization of the toxin. Once inside the neuron, reduction of the disulfide bond releases the active LC fragment. The activated LC cleaves the soluble N-ethylmaleimide-sensitive factor attachment receptor proteins responsible for synaptic vesicle membrane fusion. 59 Such specific, reversible, effective neuronal inhibition makes LC a viable transgene candidate for exploring gene-based treatment of spasticity. Inwardly Rectifying Potassium Channel Kir2.1 Another gene of interest as a feasible modulator of motor neuron activity is Kir2.1, which encodes inwardly rectifying potassium (Kir) channels in the heart and brain. For inwardly rectifying potassium channels, the inward flow of potassium ions at subthreshold is greater than the outward flow of potassium ions for the opposite driving force. This inward rectification results when intracellular magnesium ions and polyamines enter the ion channel pore from the cytoplasmic side but are unable to pass through it to the extracellular solution. The block is more intense at decreased membrane potentials as the larger depolarization facilitates the movement of magnesium ions and polyamines into the pore. In contrast, as membrane potentials approach the resting membrane potential, the decreasing depolarization hinders magnesium ion movement into the pore. When the membrane potential exceeds the resting membrane potential (hyperpolarization), magnesium ions become prevented from entering the channel. Inwardly rectifying potassium channels prevent the membrane potential from depolarizing by increasing the membrane potassium conductance. This increase in potassium permeability counterbalances the excitatory synaptic potentials that drive the initial membrane depolarization, hence inhibiting the formation of the action potential. In this way, the Kir2.1 contributes to stabilizing the resting potential at a sufficiently negative level to prevent enough sodium channel availability for action potential in the CNS and heart. Kir2.1 has been demonstrated to inhibit both evoked and spontaneous activity of neurons in vitro. STRATEGIES FOR CONTROL OF TRANSGENE EXPRESSION Inducible Gene Expression System Gene therapy will only prove beneficial as a treatment modality for spasticity if it provides advantages over existing pharmacologic and lesionbased modalities. For this to be accomplished, transgene expression must be both adjustable and reversible. One way to control viral vector-mediated transgene expression is to use inducible promoter elements. Several inducible promoter systems have been developed for this purpose, such as tetracycline, RU-486, rapamycin responsive systems, and the chimeric drosophila/bombyx ecdysone receptor system

The Overexpression of Peanut (<i>Arachis hypogaea</i> L.) <i>AhALDH2B6</i> in Soybean Enhances Cold Resistance

Soybeans are the main source of oils and protein for humans and animals; however, cold stress jeopardizes their growth and limits the soybean planting area. Aldehyde dehydrogenases (ALDH) are conserved enzymes that catalyze aldehyde oxidation for detoxification in response to stress. Additionally, transgenic breeding is an efficient method for producing stress-resistant germplasms. In this study, the peanut ALDH gene AhALDH2B6 was heterologously expressed in soybean, and its function was tested. We performed RNA-seq using transgenic and wild-type soybeans with and without cold treatment to investigate the potential mechanism. Transgenic soybeans developed stronger cold tolerance, with longer roots and taller stems than P3 soybeans. Biochemically, the transgenic soybeans exhibited a decrease in malondialdehyde activity and an increase in peroxidase and catalase content, both of which are indicative of stress alleviation. They also possessed higher levels of ALDH enzyme activity. Two phenylpropanoid-related pathways were specifically enriched in up-regulated differentially expressed genes (DEGs), including the phenylpropanoid metabolic process and phenylpropanoid biosynthetic process. Our findings suggest that AhALDH2B6 specifically up-regulates genes involved in oxidoreductase-related functions such as peroxidase, oxidoreductase, monooxygenase, and antioxidant activity, which is partially consistent with our biochemical data. These findings established the function of AhALDH2B6, especially its role in cold stress processes, and provided a foundation for molecular plant breeding, especially plant-stress-resistance breeding