Recombinant Viral Vectors as Neuroscience Tools.

Recombinant viruses are highly efficient vehicles for in vivo gene delivery. Viral vectors expand the neurobiology toolbox to include direct and rapid anterograde, retrograde, and trans-synaptic delivery of tracers, sensors, and actuators to the mammalian brain. Each viral type offers unique advantages and limitations. To establish strategies for selecting a suitable viral type, this article aims to provide readers with an overview of viral recombinant technology, viral structure, tropism, and differences between serotypes and pseudotypes for three of the most commonly used vectors in neurobiology research: adeno-associated viruses, retro/lentiviruses, and glycoprotein-deleted rabies viruses. © 2019 by John Wiley & Sons, Inc.

Production of Viral Constructs for Neuroanatomy, Calcium Imaging, and Optogenetics.

Advances in design and use of light-sensitive and light-emitting sensors have facilitated observation, measurement, and control of neuronal activities. Viruses are effective vectors for delivery of these valuable research tools to mammalian brains. Recombinant viruses are optimized to mediate regulatable, long-term, and cell-specific gene expression. Here, we describe production methods for three of the most commonly used types of recombinant viruses in neurobiology research: adeno-associated virus (AAV), retrovirus/lentivirus, and glycoprotein-deleted rabies virus. These viral constructs are frequently used for calcium imaging or to deliver neural tracers and optogenetic tools. Popular constructs are readily obtained commercially; however, customized virus production through commercial sources is time consuming and costly. This article aims to provide readers with detailed technical information for rapid production and validation of high-quality viral particles in a laboratory setting while highlighting advantages and limitations of each viral type. © 2019 by John Wiley & Sons, Inc.

Effect of Nasal Valve Shape on Downstream Volume, Airflow, and Pressure Drop: Importance of the Nasal Valve Revisited.

OBJECTIVES: The relative importance of the nasal valve relative to the remainder of the nasal airway remains unknown. The goal of this article was to objectively measure the shape of the nasal inlet and its effect on downstream airflow and nasal cavity volume using a physical model and a physiologic flow model. METHODS: A patient who had isolated nasal valve surgery and had pre- and postoperative computed tomography scans available for analysis was studied. Nasal inlet shape measurements, computational fluid dynamics, and nasal volume analysis were performed using the computed tomography data. In addition, a physical model was used to determine the effect of nasal obstruction on downstream soft tissue. RESULTS: The postoperative shape of the nasal inlet was improved in terms of length and degree of tortuosity. Whereas the operated-on region at the nasal inlet showed an only 25% increase in cross-sectional area postoperatively, downstream nonoperated sites in the nasal cavity revealed increases in area ranging from 33% to 51%. Computational fluid dynamics analysis showed that airway resistance decreased by 42%, and pressure drop was reduced by 43%. Intraluminal mucosal expansion was found with nasal obstruction in the physical model. CONCLUSION: By decreasing the degree of bending and length at the nasal valve, inspiratory downstream nonoperated sites of the nasal cavity showed improvement in volume and airflow, suggesting that the nasal valve could play an oversized role in modulating the aerodynamics of the airway. This was confirmed with the physical model of nasal obstruction on downstream mucosa.

In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation.

Development of efficacious in vivo delivery platforms for CRISPR-Cas9-based epigenome engineering will be critical to enable the ability to target human diseases without permanent modification of the genome. Toward this, we utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner.

Discovery of Novel Small-Molecule Inhibitors of LIM Domain Kinase for Inhibiting HIV-1.

A dynamic actin cytoskeleton is necessary for viral entry, intracellular migration, and virion release. For HIV-1 infection, during entry, the virus triggers early actin activity by hijacking chemokine coreceptor signaling, which activates a host dependency factor, cofilin, and its kinase, the LIM domain kinase (LIMK). Although knockdown of human LIM domain kinase 1 (LIMK1) with short hairpin RNA (shRNA) inhibits HIV infection, no specific small-molecule inhibitor of LIMK has been available. Here, we describe the design and discovery of novel classes of small-molecule inhibitors of LIMK for inhibiting HIV infection. We identified R10015 as a lead compound that blocks LIMK activity by binding to the ATP-binding pocket. R10015 specifically blocks viral DNA synthesis, nuclear migration, and virion release. In addition, R10015 inhibits multiple viruses, including Zaire ebolavirus (EBOV), Rift Valley fever virus (RVFV), Venezuelan equine encephalitis virus (VEEV), and herpes simplex virus 1 (HSV-1), suggesting that LIMK inhibitors could be developed as a new class of broad-spectrum antiviral drugs.IMPORTANCE The actin cytoskeleton is a structure that gives the cell shape and the ability to migrate. Viruses frequently rely on actin dynamics for entry and intracellular migration. In cells, actin dynamics are regulated by kinases, such as the LIM domain kinase (LIMK), which regulates actin activity through phosphorylation of cofilin, an actin-depolymerizing factor. Recent studies have found that LIMK/cofilin are targeted by viruses such as HIV-1 for propelling viral intracellular migration. Although inhibiting LIMK1 expression blocks HIV-1 infection, no highly specific LIMK inhibitor is available. This study describes the design, medicinal synthesis, and discovery of small-molecule LIMK inhibitors for blocking HIV-1 and several other viruses and emphasizes the feasibility of developing LIMK inhibitors as broad-spectrum antiviral drugs.

Differential protein stability of EGFR mutants determines responsiveness to tyrosine kinase inhibitors.

Non-small cell lung cancer (NSCLC) patients carrying specific EGFR kinase activating mutations (L858R, delE746-A750) respond well to tyrosine kinase inhibitors (TKIs). However, drug resistance develops within a year. In about 50% of such patients, acquired drug resistance is attributed to the enrichment of a constitutively active point mutation within the EGFR kinase domain (T790M). To date, differential drug-binding and altered ATP affinities by EGFR mutants have been shown to be responsible for differential TKI response. As it has been reported that EGFR stability plays a role in the survival of EGFR driven cancers, we hypothesized that differential TKI-induced receptor degradation between the sensitive L858R and delE746-A750 and the resistant T790M may also play a role in drug responsiveness. To explore this, we have utilized an EGFR-null CHO overexpression system as well as NSCLC cell lines expressing various EGFR mutants and determined the effects of erlotinib treatment. We found that erlotinib inhibits EGFR phosphorylation in both TKI sensitive and resistant cells, but the protein half-lives of L858R and delE746-A750 were significantly shorter than L858R/T790M. Third generation EGFR kinase inhibitor (AZD9291) inhibits the growth of L858R/T790M-EGFR driven cells and also induces EGFR degradation. Erlotinib treatment induced polyubiquitination and proteasomal degradation, primarily in a c-CBL-independent manner, in TKI sensitive L858R and delE746-A750 mutants when compared to the L858R/T790M mutant, which correlated with drug sensitivity. These data suggest an additional mechanism of TKI resistance, and we postulate that agents that degrade L858R/T790M-EGFR protein may overcome TKI resistance.

Discovery of novel inhibitors of HIV-1 reverse transcriptase through virtual screening of experimental and theoretical ensembles.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are potent anti-HIV chemotherapeutics. Although there are FDA-approved NNRTIs, challenges such as the development of resistance have limited their utility. Here, we describe the identification of novel NNRTIs through a combination of computational and experimental approaches. Based on the known plasticity of the NNRTI binding pocket (NNIBP), we adopted an ensemble-based virtual screening strategy: coupling receptor conformations from 10 X-ray crystal structures with 120 snapshots from a total of 480 ns of molecular dynamics (MD) trajectories. A screening library of 2864 National Cancer Institute (NCI) compounds was built and docked against the ensembles in a hierarchical fashion. Sixteen diverse compounds were tested for their ability to block HIV infection in human tissue cultures using a luciferase-based reporter assay. Three promising compounds were further characterized, using a HIV-1 RT-based polymerase assay, to determine the specific mechanism of inhibition. We found that 2 of the three compounds inhibited the polymerase activity of RT (with potency similar to the positive control, the FDA-approved drug nevirapine). Through a computational approach, we were able to discover two compounds which inhibit HIV replication and block the activity of RT, thus offering the potential for optimization into mature inhibitors.

Enveloped viruses disable innate immune responses in dendritic cells by direct activation of TAM receptors.

Upon activation by the ligands Gas6 and Protein S, Tyro3/Axl/Mer (TAM) receptor tyrosine kinases promote phagocytic clearance of apoptotic cells and downregulate immune responses initiated by Toll-like receptors and type I interferons (IFNs). Many enveloped viruses display the phospholipid phosphatidylserine on their membranes, through which they bind Gas6 and Protein S and engage TAM receptors. We find that ligand-coated viruses activate TAM receptors on dendritic cells (DCs), dampen type I IFN signaling, and thereby evade host immunity and promote infection. Upon virus challenge, TAM-deficient DCs display type I IFN responses that are elevated in comparison to wild-type cells. As a consequence, TAM-deficient DCs are relatively resistant to infection by flaviviruses and pseudotyped retroviruses, but infection can be restored with neutralizing type I IFN antibodies. Correspondingly, a TAM kinase inhibitor antagonizes the infection of wild-type DCs. Thus, TAM receptors are engaged by viruses in order to attenuate type I IFN signaling and represent potential therapeutic targets.

Identification of a 3-aminoimidazo[1,2-a]pyridine inhibitor of HIV-1 reverse transcriptase.

BACKGROUND: Despite the effectiveness of highly active antiretroviral therapy (HAART), there remains an urgent need to develop new human immunodeficiency virus type 1 (HIV-1) inhibitors with better pharmacokinetic properties that are well tolerated, and that block common drug resistant virus strains. METHODS: Here we screened an in-house small molecule library for novel inhibitors of HIV-1 replication. RESULTS: An active compound containing a 3-aminoimidazo[1,2-a]pyridine scaffold was identified and quantitatively characterized as a non-nucleoside reverse transcriptase inhibitor (NNRTI). CONCLUSIONS: The potency of this compound coupled with its inexpensive chemical synthesis and tractability for downstream SAR analysis make this inhibitor a suitable lead candidate for further development as an antiviral drug.

Delayed toxicity associated with soluble anthrax toxin receptor decoy-Ig fusion protein treatment.

Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig) fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA) toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream.

Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion.

A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.

Poststroke rehabilitation: outcomes and reimbursement of inpatient rehabilitation facilities and subacute rehabilitation programs.

BACKGROUND AND PURPOSE: To assess whether poststroke rehabilitation outcomes and reimbursement for Medicare beneficiaries differ across inpatient rehabilitation facilities (IRFs) and skilled nursing facility (SNF) subacute rehabilitation programs. METHODS: Clinical data were linked with Medicare claims for 58,724 Medicare beneficiaries with a recent stroke who completed treatment in 1996 or 1997 in IRFs and subacute rehabilitation SNFs that subscribed to the Uniform Data System for Medical Rehabilitation. Outcome measures were discharge destination, discharge FIM ratings and Medicare Part A reimbursement during the institutional stay. RESULTS: IRF patients that were more likely to have a community-based discharge, compared with rehabilitation SNF patients, were patients with mild motor disabilities and FIM cognitive ratings of 23 or greater (adjusted odds ratio [AOR]=2.19; 95% CI: 1.52 to 3.14), patients with moderate motor disabilities (AOR=1.98; 95% CI: 1.49 to 2.61), patients with significant motor disabilities (AOR=1.26; 95% CI: 1.01 to 1.57) and patients younger than 82 with severe motor disabilities (AOR=1.43; 95% CI: 1.25 to 1.64). IRF patients with significant and severe motor disabilities achieved greater motor function of 2 or more FIM units compared with rehabilitation SNF patients. Medicare Part A payments for IRFs were higher than rehabilitation SNF payments across all subgroups. CONCLUSIONS: For most patients, poststroke rehabilitation in the more costly and intensive IRFs resulted in higher functional outcomes compared with care in a SNF-based rehabilitation program. IRF and SNF outcomes were similar for patients with minimal motor disabilities and patients with mild motor disabilities and significant cognitive disabilities. Cost-effectiveness analyses require considering the costs of the full episode of care.

Outcomes and reimbursement of inpatient rehabilitation facilities and subacute rehabilitation programs for Medicare beneficiaries with hip fracture.

OBJECTIVE: We sought to assess whether outcomes and reimbursement differ for Medicare beneficiaries with hip fracture when treated in an inpatient rehabilitation facility (IRF) compared with a skilled nursing facility (SNF) subacute rehabilitation program. PARTICIPANTS: Clinical data were linked with Medicare claims for 29,793 Medicare fee-for-service beneficiaries with a recent hip fracture who completed treatment in 1996 or 1997 in rehabilitation facilities that subscribed to the Uniform Data System for Medical Rehabilitation. OUTCOME MEASURES: We measured discharge destination, change in motor FIM rating, and Medicare Part A reimbursement. RESULTS: For patients with moderate-to-severe and severe disabilities, case mix groups (CMGs) 704 and 705, the percentage of patients discharged to the community from IRFs was lower than for patients treated in subacute rehabilitation SNFs, after controlling for covariates. Adjusted odds ratios were 0.71 (95% confidence interval 0.55-0.92) for CMG 704 and 0.72 (95% confidence interval 0.63-0.83) for CMG 705. For patients in the 3 other CMGs, no significant differences were detected. Improvement in motor functional status was roughly equivalent for patients treated in IRFs and those treated in the subacute rehabilitation programs across all 5 CMGs, after controlling for covariates. Medicare Part A payments for IRFs were significantly higher than SNF payments across all CMGs. CONCLUSION: SNF-based subacute rehabilitation was less costly and outcomes were in most, but not all, instances similar or better than IRF-based rehabilitation for Medicare fee-for-service beneficiaries who had a recent hip fracture.

Myoangiogenesis after cell patch cardiomyoplasty and omentopexy in a patient with ischemic cardiomyopathy.

We describe a procedure to promote angiogenesis and impregnation of skeletal myoblast into infarcted myocardium. At the completion of coronary artery bypass surgery, the midline sternotomy incision was extended to open the abdomen, and the greater omentum was tailored to reach the myocardium. Four pieces of autologous rectus muscle were applied to the infarcted left ventricle. This implantation was reinforced by the greater omentum. Incisions were closed in the usual manner. Postoperatively, the patient showed significant improvements in left ventricular ejection fraction (from 0.15 to 0.40) and in exercise tolerance (from 3 METs to 6 METs, or 100%). Computed tomographic angiography and positron emission tomography demonstrated improved myocardial viability and vascularity in the ischemic segments of the left ventricle. Omentopexy and cell patch cardiomyoplasty in conjunction with coronary artery bypass surgery may stimulate myogenesis and angiogenesis in avascular, dyskinetic scar tissue of left ventricle; in this preliminary study, this procedure appeared to improve the functional capacity of the left ventricle.