Direct dorsal root ganglia (DRG) injection in mice for analysis of adeno-associated viral (AAV) gene transfer to peripheral somatosensory neurons.

BACKGROUND: Delivering optogenetic genes to the peripheral sensory nervous system provides an efficient approach to study and treat neurological disorders and offers the potential to reintroduce sensory feedback to prostheses users and those who have incurred other neuropathies. Adeno-associated viral (AAV) vectors are a common method of gene delivery due to efficiency of gene transfer and minimal toxicity. AAVs are capable of being designed to target specific tissues, with transduction efficacy determined through the combination of serotype and genetic promoter selection, as well as location of vector administration. The dorsal root ganglia (DRGs) are collections of cell bodies of sensory neurons which project from the periphery to the central nervous system (CNS). The anatomical make-up of DRGs make them an ideal injection location to target the somatosensory neurons in the peripheral nervous system (PNS). COMPARISON TO EXISTING METHODS: Previous studies have detailed methods of direct DRG injection in rats and dorsal horn injection in mice, however, due to the size and anatomical differences between rats and strains of mice, there is only one other published method for AAV injection into murine DRGs for transduction of peripheral sensory neurons using a different methodology. NEW METHOD/RESULTS: Here, we detail the necessary materials and methods required to inject AAVs into the L3 and L4 DRGs of mice, as well as how to harvest the sciatic nerve and L3/L4 DRGs for analysis. This methodology results in optogenetic expression in both the L3/L4 DRGs and sciatic nerve and can be adapted to inject any DRG.

iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells.

To address the limitation associated with degron based systems, we have developed iTAG, a synthetic tag based on IMiDs/CELMoDs mechanism of action that improves and addresses the limitations of both PROTAC and previous IMiDs/CeLMoDs based tags. Using structural and sequence analysis, we systematically explored native and chimeric degron containing domains (DCDs) and evaluated their ability to induce degradation. We identified the optimal chimeric iTAG(DCD23 60aa) that elicits robust degradation of targets across cell types and subcellular localizations without exhibiting the well documented "hook effect" of PROTAC-based systems. We showed that iTAG can also induce target degradation by murine CRBN and enabled the exploration of natural neo-substrates that can be degraded by murine CRBN. Hence, the iTAG system constitutes a versatile tool to degrade targets across the human and murine proteome.

Short sleep duration is associated with a wide variety of medical conditions among United States military service members.

OBJECTIVES: This cross-sectional study investigated self-reported sleep duration and its association with a comprehensive range of clinically-diagnosed medical condition categories (CDMCs), as well as the relationship between short sleep duration (≤6 h) and demographic/lifestyle factors, among United States military service members (SMs). METHODS: A stratified random sample of SMs (n = 20,819) completed an online questionnaire on usual daily hours of sleep and demographic/lifestyle characteristics. CDMCs for a six-month period prior to questionnaire completion were obtained from a comprehensive military electronic medical surveillance system and grouped into 33 CDMCs covering both broad and specific medical conditions. Prevalence of CDMCs was compared among three sleep duration categories (≤4, 5-6 and ≥7 h). RESULTS: SMs reported a mean ± standard deviation of 6.3 ± 1.4 h of sleep per day. After adjustment for demographic/lifestyle characteristics, shorter sleep duration was associated with higher odds of a medical condition in 25 of 33 CDMCs, with most (n = 20) demonstrating a dose-response relationship. The five CDMCs with the largest differences between ≤4 vs ≥ 7 h sleep were: diseases of the nervous system (odds ratio [OR] = 2.9, 95% confidence interval [95%CI] = 2.4-3.4), mental/behavioral diseases (OR = 2.7, 95%CI = 2.3-3.2), diseases of the musculoskeletal system (OR = 1.9, 95%CI = 1.6-2.1), diseases of the circulatory system (OR = 1.7, 95%CI = 1.3-2.2), and diseases of the digestive system (OR = 1.6, 95%CI = 1.2-2.0). Six hours of sleep or less was independently associated with older age, less formal education, race, Hispanic ethnicity, higher body mass index, smoking, and military service branch. CONCLUSIONS: In this young, physically active population, reporting shorter sleep duration was associated with a higher risk of multiple CDMCs.

SimPLIT: Simplified Sample Preparation for Large-Scale Isobaric Tagging Proteomics.

Large scale proteomic profiling of cell lines can reveal molecular signatures attributed to variable genotypes or induced perturbations, enabling proteogenomic associations and elucidation of pharmacological mechanisms of action. Although isobaric labeling has increased the throughput of proteomic analysis, the commonly used sample preparation workflows often require time-consuming steps and costly consumables, limiting their suitability for large scale studies. Here, we present a simplified and cost-effective one-pot reaction workflow in a 96-well plate format (SimPLIT) that minimizes processing steps and demonstrates improved reproducibility compared to alternative approaches. The workflow is based on a sodium deoxycholate lysis buffer and a single detergent cleanup step after peptide labeling, followed by quick off-line fractionation and MS2 analysis. We showcase the applicability of the workflow in a panel of colorectal cancer cell lines and by performing target discovery for a set of molecular glue degraders in different cell lines, in a 96-sample assay. Using this workflow, we report frequently dysregulated proteins in colorectal cancer cells and uncover cell-dependent protein degradation profiles of seven cereblon E3 ligase modulators (CRL4CRBN). Overall, SimPLIT is a robust method that can be easily implemented in any proteomics laboratory for medium-to-large scale TMT-based studies for deep profiling of cell lines.

Optical vagus nerve modulation of heart and respiration via heart-injected retrograde AAV.

Vagus nerve stimulation has shown many benefits for disease therapies but current approaches involve imprecise electrical stimulation that gives rise to off-target effects, while the functionally relevant pathways remain poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate targeted neural communication with light-sensitive actuators (opsins) and can be targeted to organs of interest based on the location of viral delivery. Here, we tested whether retrograde adeno-associated virus (rAAV2-retro) injected in the heart can be used to selectively express opsins in vagus nerve fibers controlling cardiac function. Furthermore, we investigated whether perturbations in cardiac function could be achieved with photostimulation at the cervical vagus nerve. Viral injection in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Photostimulation using both one-photon stimulation and two-photon holography with a GRIN-lens incorporated nerve cuff, was tested on the pilot-cohort of injected mice. Changes in heart rate, surface electrocardiogram, and respiratory responses were observed in response to both one- and two-photon photostimulation. The results demonstrate feasibility of retrograde labeling for organ targeted optical neuromodulation.

Cyp1 Inhibition Prevents Doxorubicin-Induced Cardiomyopathy in a Zebrafish Heart-Failure Model.

Doxorubicin is a highly effective chemotherapy agent used to treat many common malignancies. However, its use is limited by cardiotoxicity, and cumulative doses exponentially increase the risk of heart failure. To identify novel heart failure treatment targets, a zebrafish model of doxorubicin-induced cardiomyopathy was previously established for small-molecule screening. Using this model, several small molecules that prevent doxorubicin-induced cardiotoxicity both in zebrafish and in mouse models have previously been identified. In this study, exploration of doxorubicin cardiotoxicity is expanded by screening 2271 small molecules from a proprietary, target-annotated tool compound collection. It is found that 120 small molecules can prevent doxorubicin-induced cardiotoxicity, including 7 highly effective compounds. Of these, all seven exhibited inhibitory activity towards cytochrome P450 family 1 (CYP1). These results are consistent with previous findings, in which visnagin, a CYP1 inhibitor, also prevents doxorubicin-induced cardiotoxicity. Importantly, genetic mutation of cyp1a protected zebrafish against doxorubicin-induced cardiotoxicity phenotypes. Together, these results provide strong evidence that CYP1 is an important contributor to doxorubicin-induced cardiotoxicity and highlight the CYP1 pathway as a candidate therapeutic target for clinical cardioprotection.

Intravascular injections of adenoassociated viral vector serotypes rh10 and PHP.B transduce murine sciatic nerve axons.

Adenoassociated viral vectors provide a safe and robust method for expression of transgenes in nondividing cells such as neurons. Intravenous injections of these vectors provide a means of transducing motoneurons of peripheral nerves. Previous research has demonstrated that serotypes 1, rh10 and PHP.B can transduce motor neuron cell bodies in the spinal cord, but has not quantified expression in the peripheral nerve axon. Axonal labeling is crucial for optogenetic stimulation and detection of action potentials in peripheral nerve. Therefore, in this study, serotypes 1, PHP.B, and rh10 were tested for their ability to label axons of the murine sciatic and tibial nerve following intravenous injection. Serotype rh10 elicits expression in 10% of acetylcholine transferase positive axons of the sciatic nerve in immunohistochemically-stained sections. Serotype rh10 transduces a variety of axon diameters from <1-12 µm, while PHP.B transduces larger axons of diameter (4-16 µm). Expression was not seen with serotype 1. These results show the potential of serotypes PHP.B and rh10 delivery of transgenic products to axons of the peripheral nerve.

An automated method for the quantification of transgene expression in motor axons of the peripheral nerve.

BACKGROUND: Determination of transgene expression in motor axons of peripheral nerves is important in evaluating the effectiveness of viral transduction. Currently only manual and semi-automatic methods of quantification have been employed for quantification in immunolabeled nerve sections, but automatic methods exist for axon counting only in brightfield sections. Manual and semi-automatic methods can suffer from inter- and intraobserver bias, sampling bias and can be time consuming to implement. NEW METHOD: A fully automated method using ImageJ and the Nucleus Counter plugin was developed to quantify the fraction of green fluorescent protein (GFP) labeled acetylcholine transferase positive axons in triple immunolabeled peripheral nerve sections. This method utilizes the Nucleus Counter to generate axonal regions of interest which are quantified for colocalization with GFP expression and nonoverlap with Laminin. Thresholding using histograms generated from control animals is used to remove noise. RESULTS: The automated method is able to successfully distinguish transgenic GFP expressing mice from wild type. Using computer generated peripheral nerve sections, the automated method has less than 5% error at signal-to-noise ratios greater than 10% of baseline. COMPARISONS WITH EXISTING METHODS: This method has comparable performance in false positive rates (<1%) and a 95% predictive interval that closely matches existing fully automated methods for quantification in brightfield sections. It outperforms the intra- and interobserver differences of manual and semi-automated methods for quantification. CONCLUSIONS: This automated quantification method provides a fast and robust means of determining the fraction of labeled axons in peripheral nerve sections.

Imaging of electrical activity in small diameter fibers of the murine peripheral nerve with virally-delivered GCaMP6f.

Current neural interfaces are hampered by lack of specificity and selectivity for neural interrogation. A method that might improve these interfaces is an optical peripheral nerve interface which communicates with individual axons via optogenetic reporters. To determine the feasibility of such an interface, we delivered the genetically encoded calcium indicator GCaMP6f to the mouse peripheral nerve by intramuscular injection of adenoassociated viral vector (AAV1) under the control of the CAG (chicken beta actin- cytomegalovirus hybrid promoter). Small diameter axons in the common peroneal nerve were transduced and demonstrated electrically inducible calcium transients ex vivo. Responses to single electrical stimuli were resolvable, and increasing the number of stimuli resulted in a monotonic increase in maximum fluorescence and a prolongation of calcium transient kinetics. This work demonstrates the viability of using a virally-delivered, genetically-encoded calcium indicator to read-out from peripheral nerve axons.

Optical read-out and modulation of peripheral nerve activity.

Numerous clinical and research applications necessitate the ability to interface with peripheral nerve fibers to read and control relevant neural pathways. Visceral organ modulation and rehabilitative prosthesis are two areas which could benefit greatly from improved neural interfacing approaches. Therapeutic neural interfacing, or 'bioelectronic medicine', has potential to affect a broad range of disorders given that all the major organs of the viscera are neurally innervated. However, a better understanding of the neural pathways that underlie function and a means to precisely interface with these fibers are required. Existing peripheral nerve interfaces, consisting primarily of electrode-based designs, are unsuited for highly specific (individual axon) communication and/or are invasive to the tissue. Our laboratory has explored an optogenetic approach by which optically sensitive reporters and actuators are targeted to specific cell (axon) types. The nature of such an approach is laid out in this short perspective, along with associated technologies and challenges.

Chemical approaches to targeted protein degradation through modulation of the ubiquitin-proteasome pathway.

Manipulation of the ubiquitin-proteasome system to achieve targeted degradation of proteins within cells using chemical tools and drugs has the potential to transform pharmacological and therapeutic approaches in cancer and other diseases. An increased understanding of the molecular mechanism of thalidomide and its analogues following their clinical use has unlocked small-molecule modulation of the substrate specificity of the E3 ligase cereblon (CRBN), which in turn has resulted in the advancement of new immunomodulatory drugs (IMiDs) into the clinic. The degradation of multiple context-specific proteins by these pleiotropic small molecules provides a means to uncover new cell biology and to generate future drug molecules against currently undruggable targets. In parallel, the development of larger bifunctional molecules that bring together highly specific protein targets in complexes with CRBN, von Hippel-Lindau, or other E3 ligases to promote ubiquitin-dependent degradation has progressed to generate selective chemical compounds with potent effects in cells and in vivo models, providing valuable tools for biological target validation and with future potential for therapeutic use. In this review, we survey recent breakthroughs achieved in these two complementary methods and the discovery of new modes of direct and indirect engagement of target proteins with the proteasome. We discuss the experimental characterisation that validates the use of molecules that promote protein degradation as chemical tools, the preclinical and clinical examples disclosed to date, and the future prospects for this exciting area of chemical biology.

2D-LC as an on-line desalting tool allowing peptide identification directly from MS unfriendly HPLC methods.

The increasing interest in peptides and proteins in pharmaceutical research and development has led to many challenges for the researchers tasked with characterizing and analyzing these larger molecules. Due to the more complicated impurity profile of peptides and proteins, multiple liquid chromatography techniques are often needed to achieve comprehensive analysis. However, many of these separation conditions require buffers, salts or additives that render them incompatible with mass spectrometry (MS) detection. Previous researchers have demonstrated proof of concept for the use of two dimensional liquid chromatography (2D-LC) to provide convenient second dimension online desalting of components purified in the first chromatographic dimension. In this paper, we evaluated the Agilent heart-cutting 2D-LC system connected with an Agilent Q-TOF mass spectrometer to address this frequently encountered analytical challenge. On this 2D-LC/MS system, fractions containing the compounds of interest are separated by the first dimension using an MS incompatible mobile phase, then sent to a second dimension HPLC method where fast desalting using an MS compatible mobile phase is performed prior to MS analysis. The system allows for fast and direct collection of MS information for chromatographic peaks eluted in MS incompatible mobile phases, without requiring difficult, time consuming and error-prone translation of chromatographic methods from MS incompatible to MS compatible eluents, or off-line fraction collection and preparation. Several examples showing the application of the approach to complex mixtures containing peptides with impurities and positional isomers are presented.

A review of caffeine's effects on cognitive, physical and occupational performance.

Caffeine is consumed by over 80% of U.S. adults. This review examines the effects caffeine has on cognitive and physical function, since most real-world activities require complex decision making, motor processing and movement. Caffeine exerts its effects by blocking adenosine receptors. Following low (∼40mg or ∼0.5mgkg-1) to moderate (∼300mg or 4mgkg-1) caffeine doses, alertness, vigilance, attention, reaction time and attention improve, but less consistent effects are observed on memory and higher-order executive function, such as judgment and decision making. Effects on physical performance on a vast array of physical performance metrics such as time-to-exhaustion, time-trial, muscle strength and endurance, and high-intensity sprints typical of team sports are evident following doses that exceed about 200mg (∼3mgkg-1). Many occupations, including military, first responders, transport workers and factory shift workers, require optimal physical and cognitive function to ensure success, workplace safety and productivity. In these circumstances, that may include restricted sleep, repeated administration of caffeine is an effective strategy to maintain physical and cognitive capabilities.

Synthesis and Evaluation of a 2,11-Cembranoid-Inspired Library.

The 2,11-cembranoid family of natural products has been used as inspiration for the synthesis of a structurally simplified, functionally diverse library of octahydroisobenzofuran-based compounds designed to augment a typical medicinal chemistry library screen. Ring-closing metathesis, lactonisation and SmI2 -mediated methods were exemplified and applied to the installation of a third ring to mimic the nine-membered ring of the 2,11-cembranoids. The library was assessed for aqueous solubility and permeability, with a chemical-space analysis performed for comparison to the family of cembranoid natural products and a sample set of a screening library. Preliminary investigations in cancer cells showed that the simpler scaffolds could recapitulate the reported anti-migratory activity of the natural products.

Rho kinase inhibitors block melanoma cell migration and inhibit metastasis.

There is an urgent need to identify new therapeutic opportunities for metastatic melanoma. Fragment-based screening has led to the discovery of orally available, ATP-competitive AKT kinase inhibitors, AT13148 and CCT129254. These compounds also inhibit the Rho-kinases ROCK 1 and ROCK 2 and we show they potently inhibit ROCK activity in melanoma cells in culture and in vivo. Treatment of melanoma cells with CCT129254 or AT13148 dramatically reduces cell invasion, impairing both "amoeboid-like" and mesenchymal-like modes of invasion in culture. Intravital imaging shows that CCT129254 or AT13148 treatment reduces the motility of melanoma cells in vivo. CCT129254 inhibits melanoma metastasis when administered 2 days after orthotopic intradermal injection of the cells, or when treatment starts after metastases have arisen. Mechanistically, our data suggest that inhibition of ROCK reduces the ability of melanoma cells to efficiently colonize the lungs. These results suggest that these novel inhibitors of ROCK may be beneficial in the treatment of metastasis.

Seasonal and reproductive effects on wound healing in the flight membranes of captive big brown bats.

The flight membranes of bats serve a number of physiological functions important for survival. Although flight membrane injuries are commonly observed in wild-caught bats, in most cases the damage heals completely. Previous studies examining wound healing in the flight membranes of bats have not taken into consideration energy constraints that could influence healing times. Wound healing results in increased energy demands, therefore we hypothesized that wound healing times would be slower during periods of energy conservation and/or energy output. In this study we used an 8 mm diameter circular punch tool to biopsy the wing membranes of healthy adult female big brown bats (Eptesicus fuscus) from a captive research colony to test the hypothesis that healing times will vary with seasonal temperature changes between the summer and winter seasons, and with reproductive condition between lactating and non-reproductive females. As expected, membrane biopsies took significantly longer to heal during the winter when bats were hibernating compared to the summer when bats were active. Surprisingly, no difference in healing time was observed between lactating and non-reproductive females. The wings of most bats fully healed, although some individuals showed wound expansion demonstrating that impaired healing is occasionally observed in otherwise healthy subjects.

Synthesis and evaluation of heteroaryl substituted diazaspirocycles as scaffolds to probe the ATP-binding site of protein kinases.

With the success of protein kinase inhibitors as drugs to target cancer, there is a continued need for new kinase inhibitor scaffolds. We have investigated the synthesis and kinase inhibition of new heteroaryl-substituted diazaspirocyclic compounds that mimic ATP. Versatile syntheses of substituted diazaspirocycles through ring-closing metathesis were demonstrated. Diazaspirocycles directly linked to heteroaromatic hinge binder groups provided ligand efficient inhibitors of multiple kinases, suitable as starting points for further optimization. The binding modes of representative diazaspirocyclic motifs were confirmed by protein crystallography. Selectivity profiles were influenced by the hinge binder group and the interactions of basic nitrogen atoms in the scaffold with acidic side-chains of residues in the ATP pocket. The introduction of more complex substitution to the diazaspirocycles increased potency and varied the selectivity profiles of these initial hits through engagement of the P-loop and changes to the spirocycle conformation, demonstrating the potential of these core scaffolds for future application to kinase inhibitor discovery.

Overexpression of GATA1 confers resistance to chemotherapy in acute megakaryocytic Leukemia.

It has been previously shown that acute myeloid leukemia (AML) patients with higher levels of GATA1 expression have poorer outcomes. Furthermore, pediatric Down syndrome (DS) patients with acute megakaryocytic leukemia (AMKL), whose blast cells almost universally harbor somatic mutations in exon 2 of the transcription factor gene GATA1, demonstrate increased overall survival relative to non-DS pediatric patients, suggesting a potential role for GATA1 in chemotherapy response. In this study, we confirmed that amongst non-DS patients, GATA1 transcripts were significantly higher in AMKL blasts compared to blasts from other AML subgroups. Further, GATA1 transcript levels significantly correlated with transcript levels for the anti-apoptotic protein Bcl-xL in our patient cohort. ShRNA knockdown of GATA1 in the megakaryocytic cell line Meg-01 resulted in significantly increased cytarabine (ara-C) and daunorubicin anti-proliferative sensitivities and decreased Bcl-xL transcript and protein levels. Chromatin immunoprecipitation (ChIP) and reporter gene assays demonstrated that the Bcl-x gene (which transcribes the Bcl-xL transcripts) is a bona fide GATA1 target gene in AMKL cells. Treatment of the Meg-01 cells with the histone deacetylase inhibitor valproic acid resulted in down-regulation of both GATA1 and Bcl-xL and significantly enhanced ara-C sensitivity. Furthermore, additional GATA1 target genes were identified by oligonucleotide microarray and ChIP-on-Chip analyses. Our findings demonstrate a role for GATA1 in chemotherapy resistance in non-DS AMKL cells, and identified additional GATA1 target genes for future studies.

The development and implementation of an in-service exam for medical genetics residency programs.

PURPOSE: In-service exams are a commonly used educational tool in postgraduate medical education. Although most specialties utilize such an exam, medical genetics did not. It was decided in the spring of 2009 at the inaugural Medical Genetics Residency Program Directors (PDs) Group meeting to develop and implement such a test. METHODS: Using questions sent in from PDs, a 125-question exam was created, with 125 multiple-choice questions according to the format of the National Board of Medical Examiners. The exam covered genetics in the following areas: basic/molecular (~45 questions), cancer and adult (20), prenatal (20), biochemical (20), pediatric/dysmorphology (20). The exam was administered for the first time in February 2010, and again with modifications in 2011. RESULTS: In total, 174 trainees from 35 programs completed the exam in 2010; in 2011 the number increased to 214, representing 39 US programs, and 4 Canadian programs. For both years, most participants were medical genetics residents (106 in 2010; 127 in 2011), but a substantial number of clinical laboratory fellows also participated (68 in 2010; 85 in 2011). CONCLUSION: The development and implementation of this test were an overall success, in that in two years we were able to secure almost 100% participation from medical genetics residency programs, and that we created an infrastructure to develop and implement this exam on a yearly basis. There is need for improvement, notably in the relatively low mean score and relatively narrow spread of scores. However, we believe that, with efforts under way to improve the quality of the questions, the in-service exam will become a fundamental tool in medical genetics residency education.

High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction.

BACKGROUND: Therapeutic irreversible electroporation (IRE) is an emerging technology for the non-thermal ablation of tumors. The technique involves delivering a series of unipolar electric pulses to permanently destabilize the plasma membrane of cancer cells through an increase in transmembrane potential, which leads to the development of a tissue lesion. Clinically, IRE requires the administration of paralytic agents to prevent muscle contractions during treatment that are associated with the delivery of electric pulses. This study shows that by applying high-frequency, bipolar bursts, muscle contractions can be eliminated during IRE without compromising the non-thermal mechanism of cell death. METHODS: A combination of analytical, numerical, and experimental techniques were performed to investigate high-frequency irreversible electroporation (H-FIRE). A theoretical model for determining transmembrane potential in response to arbitrary electric fields was used to identify optimal burst frequencies and amplitudes for in vivo treatments. A finite element model for predicting thermal damage based on the electric field distribution was used to design non-thermal protocols for in vivo experiments. H-FIRE was applied to the brain of rats, and muscle contractions were quantified via accelerometers placed at the cervicothoracic junction. MRI and histological evaluation was performed post-operatively to assess ablation. RESULTS: No visual or tactile evidence of muscle contraction was seen during H-FIRE at 250 kHz or 500 kHz, while all IRE protocols resulted in detectable muscle contractions at the cervicothoracic junction. H-FIRE produced ablative lesions in brain tissue that were characteristic in cellular morphology of non-thermal IRE treatments. Specifically, there was complete uniformity of tissue death within targeted areas, and a sharp transition zone was present between lesioned and normal brain. CONCLUSIONS: H-FIRE is a feasible technique for non-thermal tissue ablation that eliminates muscle contractions seen in IRE treatments performed with unipolar electric pulses. Therefore, it has the potential to be performed clinically without the administration of paralytic agents.