Optical inhibition of motor nerve and muscle activity

Introduction: There is no therapeutic approach that provides precise and rapidly reversible inhibition of motor nerve and muscle activity for treatment of spastic hypertonia. Methods: We used optogenetics to demonstrate precise and rapidly reversible light-mediated inhibition of motor nerve and muscle activity in vivo in transgenic Thy1::eNpHR2.0 mice. Results: We found optical inhibition of motor nerve and muscle activity to be effective at all muscle force amplitudes and determined that muscle activity can be modulated by changing light pulse duration and light power density. Conclusions: This demonstration of optical inhibition of motor nerves is an important advancement toward novel optogenetics-based therapies for spastic hypertonia.Stanford University (Stanford Bio-X Interdisciplinary Initiatives Award)Stanford University (National Institutes of Health Graduate Training Program in Biotechnology grant)W. M. Keck Foundation (grant)National Institutes of Health (U.S.) (NIH grant R01NS080954

Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery

BACKGROUND: Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. RESULTS: Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependent manner. More than 90% of transduced cells were small and medium sized neurons (< 700 microm 2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (approximately 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types. CONCLUSION: We have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain

New Methods for ALK Status Diagnosis in Non–Small-Cell Lung Cancer: An Improved ALK Immunohistochemical Assay and a New, Brightfield, Dual ALK IHC–In Situ Hybridization Assay

Introduction:The demonstration of anaplastic lymphoma kinase (ALK) positivity in non–small-cell lung cancer (NSCLC) has been hindered by the technical complexity and interpretative challenges of fluorescence in situ hybridization methods for detection of ALK gene rearrangement and by the inadequate sensitivity of existing immunohistochemistry (IHC) methods for ALK protein detection. In this study, we sought to increase the sensitivity of ALK IHC detection and to develop a brightfield assay for concurrent detection of ALK protein expression and ALK gene rearrangement.Methods:We developed a horseradish peroxidase–based IHC detection system using the novel, nonendogenous hapten 3-hydroxy-2-quinoxaline (HQ) and tyramide. We also developed a dual gene protein ALK assay combining a brightfield break-apart in situ hybridization ALK assay with another sensitive IHC method using the novel, nonendogenous hapten 5-nitro-3-pyrazole. We examined the sensitivity and accuracy of these methods using surgically resected NSCLC cases examined with ALK fluorescence in situ hybridization.Results:The new HQ-tyramide IHC detection system offered readily interpretable staining with substantially greater sensitivity than conventional ALK IHC, and produced heterogeneous and homogeneous patterns of ALK protein staining among ALK-positive NSCLC surgical cases. The new 5-nitro-3-pyrazole–based IHC detection system was similar in ALK detection sensitivity to the HQ-tyramide IHC system and was compatible with the brightfield in situ hybridization assay.Conclusion:The new HQ-tyramide IHC reagent system allows more sensitive assessment of ALK protein status in NSCLC cases. The new ALK gene-protein assay allows the concurrent visualization of ALK gene and ALK protein status in single cells, allowing more accurate ALK status determination even in heterogeneous specimens

Migration of breast cancer cells: understanding the roles of volume exclusion and cell-to-cell adhesion

We study MCF-7 breast cancer cell movement in a transwell apparatus. Various experimental conditions lead to a variety of monotone and nonmonotone responses which are difficult to interpret. We anticipate that the experimental results could be caused by cell-to-cell adhesion or volume exclusion. Without any modeling, it is impossible to understand the relative roles played by these two mechanisms. A lattice-based exclusion process random-walk model incorporating agent-to-agent adhesion is applied to the experimental system. Our combined experimental and modeling approach shows that a low value of cell-to-cell adhesion strength provides the best explanation of the experimental data suggesting that volume exclusion plays a more important role than cell-to-cell adhesion. This combined experimental and modeling study gives insight into the cell-level details and design of transwell assays

Practice and Procedure Before the Growth Planning Hearings Boards

In 1990, the Washington State Legislature took the first significant step toward growth management when it enacted the Washington Growth Management Act (GMA). The GMA directs cities and counties to protect natural features and to begin planning to accommodate anticipated population increases. The legislature examined the recommendation of the Growth Strategies Commission\u27 to create an independent dispute resolution system to resolve conflicts under the GMA. The Commission recommended the use of a panel of independent arbitrators with mediation and binding arbitration. Appeals would be limited to the Washington State Court of Appeals only on constitutional and procedural issues. The legislature concluded, however, that the dispute resolution mechanism should instead be administered by an independent state agency, and, in its 1991 amendments to the GMA, directed the establishment of three Growth Planning Hearings Boards. Our goal in the following commentary is to provide how-to assistance that clarifies the Boards\u27 Rules of Practice and Procedure and that illustrates the appeals process. This Article reflects the opinions of the authors, who are Board members, and does not represent an official position of the Boards

Insulin-Like Growth Factor-II Bound to Vitronectin Enhances MCF-7 Breast Cancer Cell Migration

We have previously reported that IGF-II binds the extracellular matrix protein vitronectin (VN) with an affinity similar to that for the type-1 IGF receptor (IGF-1R). In view of this finding, and given the cited role of VN in cell motility and adhesion, we aimed to elucidate the functional consequences of this interaction on cellular processes relevant to breast carcinoma. We demonstrate that this complex slightly inhibits cell attachment and has little effect on protein synthesis in MCF-7 breast cancer cells. However, prebinding IGF-II to immobilized VN was found to significantly enhance breast cancer cell migration through Transwells. Interestingly, IGF-II bound to VN, and not IGF-II in solution in the presence of VN, seems to be responsible for the effects on cell migration. Furthermore, studies using analogs of IGF-II with reduced affinity for the IGF-1R or IGF binding proteins indicate that this response involves the IGF-1R but is independent of IGF binding proteins. This is the first study demonstrating that IGF-II:VN complexes enhance migration of cells. This may prove to be especially relevant, given that overexpression of IGF-II and VN are features of many tumors