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Adeno-associated virus (AAV) is a DNA virus with a small ( approximately 4.7 kb) single-stranded genome. It is a naturally replication-defective parvovirus of the dependovirus group. Recombinant AAV (rAAV), for use as a gene transfer vector, is created by replacing the viral rep and cap genes with the transgene of interest along with promoter and polyadenylation sequences. Only the viral inverted terminal repeats (ITRs) are required in cis for replication and packaging during production. The ITRs are also necessary and sufficient for vector genome processing and persistence during transduction. The tissue tropism of the rAAV vector is determined by the AAV capsid. In this unit we will discuss several methods to deliver rAAV in order to transduce cardiac and/or skeletal muscle, including intravenous delivery, intramuscular delivery, isolated limb infusion, intrapericardial injection in neonatal mice, and left ventricular wall injection in adult rats.

Ocular gene therapy is a fast-growing area of research. The eye is an ideal organ for gene therapy since it is immune privileged and easily accessible, and direct viral delivery results primarily in local infection. Because the eye is not a vital organ, mutations in eye-specific genes tend to be more common. To date, over 40 eye-specific genes have been identified harboring mutations that lead to blindness. Gene therapy with recombinant adeno-associated virus (rAAV) holds the promise to treat patients with such mutations. However, proof-of-concept and safety evaluation for gene therapy remains to be established for most of these diseases. This unit describes the in vivo delivery of genes to the mouse eye by rAAV-mediated gene transfer and plasmid DNA electroporation. Advantages and limitations of these methods are discussed, and detailed protocols for gene delivery, required materials, and subsequent tissue processing methods are described.

The rough endoplasmic reticulum is a major site of protein biosynthesis in all eukaryotic cells, serving as the entry point for the secretory pathway and as the initial integration site for the majority of cellular integral membrane proteins. The core components of the protein translocation machinery have been identified, and high-resolution structures of the targeting components and the transport channel have been obtained. Research in this area is now focused on obtaining a better understanding of the molecular mechanism of protein translocation and membrane protein integration.

Described in this unit are basic protocols frequently used in the research of human immunodeficiency viruses (HIVs). Provided are methods for propagating and quantifying HIV, as well as recommendations for long-term storage. Background information about these methods is also provided and includes their advantages, disadvantages, and troubleshooting.

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.

INTRODUCTION: The largest randomized controlled trial that compared the efficacy of carotid endarterectomy (CEA) with carotid artery stenting (CAS) showed equivalent outcomes for the composite end point of postoperative stroke, myocardial infarction (MI), or death. However, CAS had a higher risk of postoperative stroke, and CEA had a higher risk of MI. We hypothesize that there is a differential association of postoperative stroke, compared with that of postoperative MI, with reduced long-term survival after carotid revascularization when compared with neither complication.

METHODS: The Vascular Study Group of New England database was used to identify all CEA and CAS procedures performed between 2003 and 2011. Patients were stratified according to whether they experienced an in-hospital postoperative stroke (minor or major), MI (troponin elevation, electrocardiographic changes, or clinical symptoms), or neither. Primary study end point was survival during the first year and the first 5 years postoperatively. Multivariable Cox proportional hazards models compared the magnitude of association of stroke and MI on survival.

RESULTS: Of 8315 patients, 81 (0.97%) experienced postoperative MI, and 63 (0.76%) experienced stroke. During the first year after operation, survival significantly differed among the three groups: neither, 96%; MI, 84%; stroke, 77% (log-rank P < .0001). After adjusting for confounders, survival after postoperative stroke (hazard ratio [HR], 6.6; 95% confidence interval [CI], 3.7-12; P < .0001) was nearly twofold less than that after postoperative MI (HR, 3.6; 95% CI, 2-6.8; P < .0001). During the first 5 years postoperatively, multivariable modeling showed postoperative stroke and postoperative MI remained independent predictors of decreased survival, but the magnitude of association was similar (HR, 2.7; 95% CI, 1.7-4.3 [P < .0001] vs HR, 2.8; 95% CI, 1.8-4.3 [P < .0001]).

CONCLUSIONS: During the first year after operation, postoperative stroke conferred a twofold lower survival than that after postoperative MI. By 5 years after operation, these survival curves converged, and the survival disadvantage associated with stroke became similar to that of MI. These data suggest that different postoperative complications after carotid revascularization have different implications for patients, with decreased short-term survival in patients experiencing a postoperative stroke. These findings help to inform our interpretation of studies that have used a composite end point in order to evaluate the comparative effectiveness of revascularization strategies.

Dendrite and synapse development are critical for establishing appropriate neuronal circuits, and disrupted timing of these events can alter neural connectivity. Using microarrays, we have identified a nuclear factor I (NFI)-regulated temporal switch program linked to dendrite formation in developing mouse cerebellar granule neurons (CGNs). NFI function was required for upregulation of many synapse-related genes as well as downregulation of genes expressed in immature CGNs. Chromatin immunoprecipitation analysis revealed that a central feature of this program was temporally regulated NFI occupancy of late-expressed gene promoters. Developing CGNs undergo a hyperpolarizing shift in membrane potential, and depolarization inhibits their dendritic and synaptic maturation via activation of calcineurin (CaN) (Okazawa et al., 2009). Maintaining immature CGNs in a depolarized state blocked NFI temporal occupancy of late-expressed genes and the NFI switch program via activation of the CaN/nuclear factor of activated T-cells, cytoplasmic (NFATc) pathway and promotion of late-gene occupancy by NFATc4, and these mechanisms inhibited dendritogenesis. Conversely, inhibition of the CaN/NFATc pathway in CGNs maturing under physiological nondepolarizing conditions upregulated the NFI switch program, NFI temporal occupancy, and dendrite formation. NFATc4 occupied the promoters of late-expressed NFI program genes in immature mouse cerebellum, and its binding was temporally downregulated with development. Further, NFI temporal binding and switch gene expression were upregulated in the developing cerebellum of Nfatc4 (-/-) mice. These findings define a novel NFI switch and temporal occupancy program that forms a critical link between membrane potential/CaN and dendritic maturation in CGNs. CaN inhibits the program and NFI occupancy in immature CGNs by promoting NFATc4 binding to late-expressed genes. As maturing CGNs become more hyperpolarized, NFATc4 binding declines leading to onset of NFI temporal binding and the NFI switch program.

Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne Syndrome (PAPA syndrome) is an autoinflammatory disease caused by aberrant production of the proinflammatory cytokine interleukin-1. Mutations in the gene encoding proline serine threonine phosphatase-interacting protein-1 (PSTPIP1) have been linked to PAPA syndrome. PSTPIP1 is an adaptor protein that interacts with PYRIN, the protein encoded by the Mediterranean Fever (MEFV) gene whose mutations cause Familial Mediterranean Fever (FMF). However, the pathophysiological function of PSTPIP1 remains to be elucidated. We have generated mouse strains that either are PSTPIP1 deficient or ectopically express mutant PSTPIP1. Results from analyzing these mice suggested that PSTPIP1 is not an essential regulator of the Nlrp3, Aim2, or Nlrc4 inflammasomes. Although common features of human PAPA syndrome such as pyogenic arthritis and skin inflammation were not recapitulated in the mouse model, ectopic expression of the mutant but not the wild type PSTPIP1 in mice lead to partial embryonic lethality, growth retardation, and elevated level of circulating proinflammatory cytokines.

OBJECTIVE: In the majority of literature concerning age in TBI, specifically in subdural hematomas (SDH), the mean age of patients considered elderly is 55-65. Limited data in SDH patients>75 years suggest an increased mortality rate. The impact of medical decision making on these data is not well-documented.

PATIENTS/METHODS: We use the Nationwide Inpatient Sample (NIS) database to compare outcomes between SDH patients 60-79 and >/=80. As administrative databases have some shortcomings, i.e. in-hospital data only, acute and chronic SDHs listed together, we examined institutional data to evaluate the impact of these factors on medical decision making which may falsely elevate mortality rates.

RESULTS: In-hospital mortality was increased in NIS patients>80 treated both surgically and non-surgically (P80 with SDHs as a group. However, the SDH patients>80 who underwent surgery at our institution had much lower mortality rates. We found that patients>/=80 made up 87% of all patients with "surgical lesions" that were not operated on. Type of subdural, admission GCS, and baseline cognitive status appeared to have a significant impact on surgical decision making.

CONCLUSION: This study examines mortality rates in patients>80 with SDHs who are managed surgically and non-surgically using a large administrative database and institutional data. It provides preliminary insight into medical decision making which make affect mortality rates of the very elderly.

The septum initiation network (SIN) regulates multiple functions during late mitosis to ensure successful completion of cytokinesis in Schizosaccharomyces pombe. One mechanism by which the SIN promotes cytokinesis is by inhibiting a competing polarity pathway called the MOR, which is required for initiation of polarized growth following completion of cytokinesis. Mutual antagonism between the two NDR kinase pathways, SIN and MOR, is required to coordinate cytoskeletal rearrangements during the mitosis-interphase transition. To determine how the SIN regulates the MOR pathway, we developed a proteomics approach that allowed us to identify multiple substrates of the SIN effector kinase Sid2, including the MOR pathway components Nak1 kinase and an associated protein, Sog2. We show that Sid2 phosphorylation of Nak1 causes removal of Nak1 from the spindle pole bodies, which may both relieve Nak1 inhibition of the SIN and block MOR signaling by preventing interaction of Nak1 with the scaffold protein Mor2. Because the SIN and MOR are conserved in mammalian cells (Hippo and Ndr1/2 pathways, respectively), this work may provide important insight into how the activities of these essential pathways are coordinated.

PURPOSE: Proliferative vitreoretinopathy (PVR) is a complication of retinal detachment that can lead to surgical failure and vision loss. Previous studies suggest that a variety of retinal cells, including RPE and Muller glia, may be responsible. Platelet-derived growth factor receptor alpha (PDGFRalpha) has been strongly implicated in the pathogenesis, and found to be intrinsic to the development of PVR in rabbit models. We examine whether SU9518, a tyrosine kinase inhibitor with PDGFRalpha specificity, can inhibit the development of PVR in fibroblast and Muller cell rabbit models of PVR.

METHODS: SU9518 was injected in rabbit eyes along with fibroblasts, Muller cells (MIO-M1), or Muller cells transfected to increase their expression of PDGFRalpha (MIO-M1alpha). Indirect ophthalmoscopy and histopathology were used to assess efficacy and toxicity.

RESULTS: SU9518 was an effective inhibitor of PVR in both fibroblast and Muller cell models of PVR. No toxic effects were identified by indirect ophthalmoscopy or histopathology.

CONCLUSIONS: SU9518 is an effective and safe inhibitor of PVR in rabbit models, and could potentially be used in humans for the treatment of this and other proliferative diseases of the retina involving fibrosis and gliosis. Further animal studies need to be performed to examine retinal toxicity and sustained delivery mechanisms.

Synaptic transmission depends on the matching and alignment of presynaptically released transmitters and postsynaptic neurotransmitter receptors. Neuroligin (NL) and Neurexin (Nrxn) proteins are trans-synaptic adhesion molecules that are important in validation and maturation of specific synapses. NL isoforms NL1 and NL2 have specific functional roles in excitatory and inhibitory synapses, respectively, but the molecular basis behind this distinction is still unclear. We show here that the extracellular domain of NL2 confers its unique ability to enhance inhibitory synaptic function when overexpressed in rat hippocampal pyramidal neurons, whereas NL1 normally only promotes excitatory synapses. This specificity is conferred by presynaptic Nrxn isoforms, as NL1 can also induce functional inhibitory synapse connections when the presynaptic interneurons ectopically express an Nrxn isoform that binds to NL1. Our results indicate that trans-synaptic interaction with differentially expressed presynaptic Nrxns underlies the distinct functions of NL1 and NL2, and is sufficient to induce functional inhibitory synapse formation.

In this issue of Molecular Cell, van Bemmel and colleagues (2013) report the genome-wide mapping of 42 novel chromatin factors, systematically identifying new components of the various chromatin domains present in fly cells.

BACKGROUND: Necrotizing soft-tissue infections (NSTI) are rare, potentially fatal, operative emergencies. We studied a national cohort of patients to determine recent trends in incidence, treatment, and outcomes for NSTI.

METHODS: We queried the Nationwide Inpatient Sample (1998-2010) for patients with a primary diagnosis of NSTI. Temporal trends in patient characteristics, treatment (debridement, amputation, hyperbaric oxygen therapy [HBOT]), and outcomes were determined with Cochran-Armitage trend tests and linear regression. To account for trends in case mix (age, sex, race, insurance, Elixhauser index) or receipt of HBOT on outcomes, multivariable analyses were conducted to determine the independent effect of year of treatment on mortality, any major complication, and hospital length of stay (LOS) for NSTI.

RESULTS: We identified 56,527 weighted NSTI admissions, with an incidence ranging from approximately 3,800-5,800 cases annually. The number of cases peaked in 2004 and then decreased between 1998 and 2010 (P < .0001). The percentage of female patients decreased slightly over time (38.6-34.1%, P < .0001). Patients were increasingly in the 18- to 34-year-old (8.8-14.6%, P < .0001) and 50- to 64-year-old age groups (33.2-43.5, P < .0001), Hispanic (6.8-10.5%, P < .0001), obese (8.9-24.6%, P < .0001), and admitted with >3 comorbidities (14.5-39.7%, P < .0001). The percentage of patients requiring only one operative debridement increased somewhat (43.2-46.2%, P < .0001), whereas the use of HBOT was rare and decreasing (1.6-0.8%, P < .0001). The percentage of patients requiring operative wound closure decreased somewhat (23.5-20.8%, P < .0001). Although major complication rates increased (30.9-48.2%, P < .0001), hospital LOS remained stable (18-19 days) and mortality decreased (9.0-4.9%, P < .0001) on univariate analyses. On multivariable analyses each 1-year incremental increase in year was associated with a 5% increased odds of complication (odds ratio 1.05), 0.4 times decrease in hospital LOS (coefficient -0.41), and 11% decreased odds of mortality (odds ratio 0.89).

CONCLUSION: There were potentially important national trends in patient characteristics and treatment patterns for NSTI between 1998 and 2010. Importantly, though patient acuity worsened and complication rates increased, but LOS remained relatively stable and mortality decreased. Improvements in early diagnosis, wound care, and critical care delivery may be the cause.

OBJECTIVE: Emergency airway situations are relatively rare events in pediatrics with most graduating residents having little exposure to intubate. Newer video technology offers the promise of reducing complications associated with intubation. This study proposes that video laryngoscopy (VL) should aid less skilled residents to intubate an infant mannequin with greater success and speed as compared with traditional direct laryngoscopy (DL).

METHODS: Pediatric (PED) and emergency medicine (EM) residents were randomized in a prospective controlled study. A standard respiratory failure scenario was conducted using SimBaby with an uncomplicated airway. Residents who inadvertently performed esophageal intubation were made aware as part of the scenario and allowed to reattempt until successful.

RESULTS: Sixty-nine residents voluntarily participated, 49% EM and 51% PED. Seven subjects in the DL group required multiple attempts (21%), compared with 6 subjects in the VL group (17%) (P = 0.718). Median time to intubation was 30 seconds (95% confidence interval [CI], 19-41 seconds) for DL and 39 seconds (95% CI, 36-42 seconds) for VL (P = 0.111). Comparison of programs revealed a 77% PED success rate versus 85% EM success rate (P = 0.578) and median time to intubation of 38 seconds (95% CI, 31-45 seconds) for PED compared with 32 seconds (95% CI, 23-41 seconds) for EM residents (P = 0.316). In a subanalysis, subjects successful at first attempt revealed a 13-second median difference (DL, 23 seconds [95% CI, 18-28 seconds] vs. VL, 36 seconds [95% CI, 29-43 seconds; P = 0.01).

CONCLUSIONS: In a simulated respiratory failure scenario involving residents, VL provided no additional success over DL with slightly longer time to intubation.

Around a century ago, the midbody (MB) was described as a structural assembly within the intercellular bridge during cytokinesis that served to connect the two future daughter cells. The MB has become the focus of intense investigation through the identification of a growing number of diverse cellular and molecular pathways that localize to the MB and contribute to its cytokinetic functions, ranging from selective vesicle trafficking and regulated microtubule (MT), actin, and endosomal sorting complex required for transport (ESCRT) filament assembly and disassembly to post-translational modification, such as ubiquitination. More recent studies have revealed new and unexpected functions of MBs in post-mitotic cells. In this review, we provide a historical perspective, discuss exciting new roles for MBs beyond their cytokinetic function, and speculate on their potential contributions to pluripotency.

RadioGraphics continues to publish radiologic-pathologic case material selected from the American Institute for Radiologic Pathology (AIRP) “best case” presentations. The AIRP conducts a 4-week Radiologic Pathology Correlation Course, which is offered five times per year. On the penultimate day of the course, the best case presentation is held at the American Film Institute Silver Theater and Cultural Center in Silver Spring, Md. The AIRP faculty identifies the best cases, from each organ system, brought by the resident attendees. One or more of the best cases from each of the five courses are then solicited for publication in RadioGraphics. These cases emphasize the importance of radiologic-pathologic correlation in the imaging evaluation and diagnosis of diseases encountered at the institute and its predecessor, the Armed Forces Institute of Pathology (AFIP).

Studies on chronic myeloid leukemia (CML) have served as a paradigm for cancer research and therapy. These studies involve the identification of the first cancer-associated chromosomal abnormality and the subsequent development of tyrosine kinase inhibitors (TKIs) that inhibit BCR-ABL kinase activity in CML. It becomes clear that leukemia stem cells (LSCs) in CML which are resistant to TKIs, and eradication of LSCs appears to be extremely difficult. Therefore, one of the major issues in current CML biology is to understand the biology of LSCs and to investigate why LSCs are insensitive to TKI monotherapy for developing curative therapeutic strategies. Studies from our group and others have revealed that CML LSCs form a hierarchy similar to that seen in normal hematopoiesis, in which a rare stem cell population with limitless self-renewal potential gives rise to progenies that lack such potential. LSCs also possess biological features that are different from those of normal hematopoietic stem cells (HSCs) and are critical for their malignant characteristics. In this review, we summarize the latest progress in CML field, and attempt to understand the molecular mechanisms of survival regulation of LSCs.

The genome of influenza A virus consists of eight separate RNA segments, which are selectively packaged into virions prior to virus budding. The microscopic mechanism of highly selective packaging involves molecular interactions between packaging signals in the genome segments and remains poorly understood. We propose that the condition of proper packaging can be formulated as a large gap between RNA-RNA interaction energies in the viable virion with eight unique segments and in improperly packed assemblages lacking the complete genome. We then demonstrate that selective packaging of eight unique segments into an infective influenza virion can be achieved by self-repulsion of identical segments at the virion assembly stage, rather than by previously hypothesized intricate molecular recognition of particular segments. Using Monte Carlo simulations to maximize the energy gap, without any other assumptions, we generated model eight-segment virions, which all display specific packaging, strong self-repulsion of the segments, and reassortment patterns similar to natural influenza. The model provides a biophysical foundation of influenza genome packaging and reassortment and serves as an important step towards robust sequence-driven prediction of reassortment patterns of the influenza virus.

The nuclear envelope forms a cocoon that surrounds the cellular genome keeping it out of harm's way and can be utilized by the cell as a means of functionally regulating chromatin structure and gene expression. At the same time, this double-layered membrane system constitutes a formidable obstacle to the unimpeded flow of genetic information between the genome and the rest of the cell. The nuclear pore has been long considered the sole passageway between nucleus and cytoplasm. A new report challenges this view and proposes a novel mechanism by which RNA transcripts destined for localized translation in highly polarized cell types, cross both inner and outer nuclear envelope membranes and reach the cytoplasm without utilizing the nuclear pore route.