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DNA-binding-domain fusions enhance the targeting range and precision of Cas9

Wed, 12/23/2015 - 9:30am

The CRISPR-Cas9 system is commonly used in biomedical research; however, the precision of Cas9 is suboptimal for applications that involve editing a large population of cells (for example, gene therapy). Variations on the standard Cas9 system have yielded improvements in the precision of targeted DNA cleavage, but they often restrict the range of targetable sequences. It remains unclear whether these variants can limit lesions to a single site in the human genome over a large cohort of treated cells. Here we show that by fusing a programmable DNA-binding domain (pDBD) to Cas9 and attenuating Cas9's inherent DNA-binding affinity, we were able to produce a Cas9-pDBD chimera with dramatically improved precision and an increased targeting range. Because the specificity and affinity of this framework can be easily tuned, Cas9-pDBDs provide a flexible system that can be tailored to achieve extremely precise genome editing at nearly any genomic locus.

Create, activate, destroy, repeat: Cdk1 controls proliferation by limiting transcription factor activity

Wed, 12/23/2015 - 9:30am

Progression through the cell cycle is controlled by a network of transcription factors that coordinate gene expression with cell-cycle events. One transcriptional activator in this network in budding yeast is the forkhead protein Hcm1, which controls the expression of genes that are transcribed during S-phase. Hcm1 activity is coordinated with the cell cycle via its regulation by cyclin-dependent kinase (Cdk1), which both activates Hcm1 and targets it for degradation, through phosphorylation of distinct sites. The mechanisms controlling the differential phosphorylation timing of the activating and destabilizing phosphosites are not clear. However, a recent study shows that the phosphatase calcineurin specifically removes activating phosphates from Hcm1 when cells are exposed to environmental stress, thus extinguishing its activity and slowing proliferation under unfavorable growth conditions. This regulatory mechanism, whereby a phosphatase actively alters the distribution of phosphosites on a cell cycle-regulatory transcription factor to elicit a change in cellular proliferation, adds an additional layer of complexity to the regulatory network controlling the cell cycle. Furthermore, this regulatory paradigm is likely to be a conserved mode of phosphoregulation that controls the cell cycle in diverse systems.

Ral GTPase and the exocyst regulate autophagy in a tissue-specific manner

Wed, 12/23/2015 - 9:30am

Autophagy traffics cellular components to the lysosome for degradation. Ral GTPase and the exocyst have been implicated in the regulation of stress-induced autophagy, but it is unclear whether they are global regulators of this process. Here, we investigate Ral function in different cellular contexts in Drosophila and find that it is required for autophagy during developmentally regulated cell death in salivary glands, but does not affect starvation-induced autophagy in the fat body. Furthermore, knockdown of exocyst subunits has a similar effect, preventing autophagy in dying cells but not in cells of starved animals. Notch activity is elevated in dying salivary glands, this change in Notch signaling is influenced by Ral, and decreased Notch function influences autophagy. These data indicate that Ral and the exocyst regulate autophagy in a context-dependent manner, and that in dying salivary glands, Ral mediates autophagy, at least in part, by regulation of Notch.

Improving the Outcomes of Transition Age Youth with Psychiatric Disabilities through the Adoption and Use of Best Practice Transition Planning

Tue, 12/22/2015 - 11:44am

The goal of the newly funded Translating Evidence to Support Transitions (TEST) grant is to increase the use and adoption of research-based best practices in transition planning services for high school students with emotional and behavioral disturbances (EBD) receiving special education services.

A conserved flagella-associated protein in Chlamydomonas, FAP234, is essential for axonemal localization of tubulin polyglutamylase TTLL9

Tue, 12/22/2015 - 11:33am

Tubulin undergoes various posttranslational modifications, including polyglutamylation, which is catalyzed by enzymes belonging to the tubulin tyrosine ligase-like protein (TTLL) family. A previously isolated Chlamydomonas reinhardtii mutant, tpg1, carries a mutation in a gene encoding a homologue of mammalian TTLL9 and displays lowered motility because of decreased polyglutamylation of axonemal tubulin. Here we identify a novel tpg1-like mutant, tpg2, which carries a mutation in the gene encoding FAP234, a flagella-associated protein of unknown function. Immunoprecipitation and sucrose density gradient centrifugation experiments show that FAP234 and TTLL9 form a complex. The mutant tpg1 retains FAP234 in the cell body and flagellar matrix but lacks it in the axoneme. In contrast, tpg2 lacks both TTLL9 and FAP234 in all fractions. In fla10, a temperature-sensitive mutant deficient in intraflagellar transport (IFT), both TTLL9 and FAP234 are lost from the flagellum at nonpermissive temperatures. These and other results suggest that FAP234 functions in stabilization and IFT-dependent transport of TTLL9. Both TTLL9 and FAP234 are conserved in most ciliated organisms. We propose that they constitute a polyglutamylation complex specialized for regulation of ciliary motility.

The impact of equilibrium assumptions on tests of selection

Tue, 12/22/2015 - 11:33am

With the increasing availability and quality of whole genome population data, various methodologies of population genetic inference are being utilized in order to identify and quantify recent population-level selective events. Though there has been a great proliferation of such methodology, the type-I and type-II error rates of many proposed statistics have not been well-described. Moreover, the performance of these statistics is often not evaluated for different biologically relevant scenarios (e.g., population size change, population structure), nor for the effect of differing data sizes (i.e., genomic vs. sub-genomic). The absence of the above information makes it difficult to evaluate newly available statistics relative to one another, and thus, difficult to choose the proper toolset for a given empirical analysis. Thus, we here describe and compare the performance of four widely used tests of selection: SweepFinder, SweeD, OmegaPlus, and iHS. In order to consider the above questions, we utilize simulated data spanning a variety of selection coefficients and beneficial mutation rates. We demonstrate that the LD-based OmegaPlus performs best in terms of power to reject the neutral model under both equilibrium and non-equilibrium conditions-an important result regarding the relative effectiveness of linkage disequilibrium relative to site frequency spectrum based statics. The results presented here ought to serve as a useful guide for future empirical studies, and provides a guide for statistical choice depending on the history of the population under consideration. Moreover, the parameter space investigated and the Type-I and Type-II error rates calculated, represent a natural benchmark by which future statistics may be assessed.

Intracranial hypotension with multiple complications: an unusual case report

Tue, 12/22/2015 - 11:33am

Background. Undiagnosed intracranial hypotension can result in several complications including subdural hematoma (SDH), subarachnoid hemorrhage (SAH), dural venous sinuses thrombosis (CVT), cranial nerve palsies, and stupor resulting from sagging of the brain. It is rare to see all the complications in one patient. Furthermore, imaging of the brain vasculature may reveal incidental asymptomatic small aneurysms. Given the combination of these imaging findings and a severe headache, the patients are often confused to have a primary subarachnoid hemorrhage.

Case Report. We present a patient with spontaneous intracranial hypotension (SIH) who had an incidental ophthalmic artery aneurysm on MR imaging, and this presentation led to coiling of the aneurysm. The key aspect in the history "postural headaches" was missed, and this led to life threatening complications and unnecessary interventions. Revisiting the history and significant improvement in symptoms following an epidural blood patch resulted in the diagnosis of SIH.

Conclusion. We strongly emphasize that appropriate history taking is the key in the diagnosis of SIH and providing timely treatment with an epidural blood patch could prevent potentially life threatening complications.

Birth of a new therapeutic platform: 47 years of adeno-associated virus biology from virus discovery to licensed gene therapy

Tue, 12/22/2015 - 11:33am

Within American biomedical research, as supported primarily by the National Institutes of Health (NIH) since the post–World War II era, it has been considered axiomatic that investments in discovery research will ultimately lead to the development of novel approaches to diagnostics and therapeutics that will improve the health and well-being of citizens of the United States and the broader world. I reflect here in a historical context on how one such series of investments in discovery has resulted in the emergence of the first licensed human gene therapy product in Europe, namely Glybera. Glybera (alipogene tiparvovec) is a recombinant adeno-associated virus (rAAV) vector with AAV2 inverted terminal repeats (ITRs) encapsidated into AAV1 capsids (so-called rAAV2/1, or, for this purpose, rAAV1), which, when administered intramuscularly, can result in expression of sufficient levels of lipoprotein lipase (LPL) to be considered a safe and effective treatment of LPL deficiency. It was licensed in the European Union in November 2012, 47 years after the near simultaneous discovery of AAV in laboratories at the NIH Bethesda campus and at the University of Pittsburgh. I trace key milestones in the progress from the discovery of the virus, to its use as a platform for an approved therapeutic product.

The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor

Tue, 12/22/2015 - 11:33am

In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex.

Influenza infection control guidance provided to staff at Veterans Affairs facilities for veterans with spinal cord injury during a pandemic(dagger)

Tue, 12/22/2015 - 11:33am

CONTEXT/OBJECTIVE: To assess guidance provided to staff at Veterans Affairs (VA) healthcare facilities on H1N1 influenza infection control for veterans with spinal cord injuries and disorders (SCI/D).

STUDY DESIGN: Cross-sectional qualitative semi-structured interviews.

SETTING: Thirty-three VA healthcare facilities from throughout the United States that provide care to veterans with SCI/D.

PARTICIPANTS: Thirty-three infection control key informants, each representing a VA healthcare facility.

INTERVENTIONS: None.

OUTCOME MEASURES: Infection control practices, including vaccination practices, hospital preparedness, and recommendations for future pandemics, both in general and specifically to SCI/D.

RESULTS: Most (n = 26, 78.8%) infection control key informants believed veterans with SCI/D were at increased risk for influenza and complications, but only 17 (51.5%) said veterans with SCI/D were treated as a priority group for vaccination at their facilities. There was little special guidance provided for treating veterans with SCI/D, and most (n = 28, 84.8%) informants said that infection control procedures and recommendations were applied universally. Yet, 10 key informants discussed 'unique challenges' to infection control in the SCI/D population. Informants discussed the potential for infectious agents to be spread through shared and common use equipment and the necessity of including caregivers in any vaccination or educational campaigns.

CONCLUSION: Greater input by experts knowledgeable about SCI/D is recommended to adequately address pandemic influenza within healthcare facilities where individuals with SCI/D receive care.

Overcoming current limitations in humanized mouse research

Tue, 12/22/2015 - 11:33am

Immunodeficient mice engrafted with human cells and tissues have provided an exciting alternative to in vitro studies with human tissues and nonhuman primates for the study of human immunobiology. A major breakthrough in the early 2000s was the introduction of a targeted mutation in the interleukin 2 (IL-2) receptor common gamma chain (IL2rg(null)) into mice that were already deficient in T and B cells. Among other immune defects, natural killer (NK) cells are disrupted in these mice, permitting efficient engraftment with human hematopoietic cells that generate a functional human immune system. These humanized mouse models are becoming increasingly important for preclinical studies of human immunity, hematopoiesis, tissue regeneration, cancer, and infectious diseases. In particular, humanized mice have enabled studies of the pathogenesis of human-specific pathogens, including human immunodeficiency virus type 1, Epstein Barr virus, and Salmonella typhi. However, there are a number of limitations in the currently available humanized mouse models. Investigators are continuing to identify molecular mechanisms underlying the remaining defects in the engrafted human immune system and are generating "next generation" models to overcome these final deficiencies. This article provides an overview of some of the emerging models of humanized mice, their use in the study of infectious diseases, and some of the remaining limitations that are currently being addressed.

Intimate host attachment: enteropathogenic and enterohaemorrhagic Escherichia coli

Tue, 12/22/2015 - 11:33am

Enteropathogenic and enterohaemorrhagic Escherichia coli use a novel infection strategy to colonize the gut epithelium, involving translocation of their own receptor, Tir, via a type III secretion system and subsequent formation of attaching and effecting (A/E) lesions. Following integration into the host cell plasma membrane of cultured cells, and clustering by the outer membrane adhesin intimin, Tir triggers multiple actin polymerization pathways involving host and bacterial adaptor proteins that converge on the host Arp2/3 actin nucleator. Although initially thought to be involved in A/E lesion formation, recent data have shown that the known Tir-induced actin polymerization pathways are dispensable for this activity, but can play other major roles in colonization efficiency, in vivo fitness and systemic disease. In this review we summarize the roadmap leading from the discovery of Tir, through the different actin polymerization pathways it triggers, to our current understanding of their physiological functions.

Prefrontal cortical dysfunction after overexpression of histone deacetylase 1

Tue, 12/22/2015 - 11:33am

BACKGROUND: Postmortem brain studies have shown that HDAC1-a lysine deacetylase with broad activity against histones and nonhistone proteins-is frequently expressed at increased levels in prefrontal cortex (PFC) of subjects diagnosed with schizophrenia and related disease. However, it remains unclear whether upregulated expression of Hdac1 in the PFC could affect cognition and behavior.

METHODS: Using adeno-associated virus, an Hdac1 transgene was expressed in young adult mouse PFC, followed by behavioral assays for working and long-term memory, repetitive activity, and response to novelty. Prefrontal cortex transcriptomes were profiled by microarray. Antipsychotic drug effects were explored in mice treated for 21 days with haloperidol or clozapine.

RESULTS: Hdac1 overexpression in PFC neurons and astrocytes resulted in robust impairments in working memory, increased repetitive behaviors, and abnormal locomotor response profiles in novel environments. Long-term memory remained intact. Over 300 transcripts showed subtle but significant changes in Hdac1-overexpressing PFC. Major histocompatibility complex class II (MHC II)-related transcripts, including HLA-DQA1/H2-Aa, HLA-DQB1/H2-Ab1, and HLA-DRB1/H2-Eb1, located in the chromosome 6p21.3-22.1 schizophrenia and bipolar disorder risk locus, were among the subset of genes with a more robust ( > 1.5-fold) downregulation in expression. Hdac1 levels declined during the course of normal PFC development. Antipsychotic drug treatment, including the atypical clozapine, did not affect Hdac1 levels in PFC but induced expression of multiple MHC II transcripts.

CONCLUSIONS: Excessive HDAC1 activity, due to developmental defects or other factors, is associated with behavioral alterations and dysregulated expression of MHC II and other gene transcripts in the PFC.

Reduction of coil mass artifacts in high-resolution flat detector conebeam CT of cerebral stent-assisted coiling

Tue, 12/22/2015 - 11:33am

BACKGROUND AND PURPOSE: Developments in flat panel angiographic C-arm systems have enabled visualization of both the neurovascular stents and host arteries in great detail, providing complementary spatial information in addition to conventional DSA. However, the visibility of these structures may be impeded by artifacts generated by adjacent radio-attenuating objects. We report on the use of a metal artifact reduction algorithm for high-resolution contrast-enhanced conebeam CT for follow-up imaging of stent-assisted coil embolization.

MATERIALS AND METHODS: Contrast-enhanced conebeam CT data were acquired in 25 patients who underwent stent-assisted coiling. Reconstructions were generated with and without metal artifact reduction and were reviewed by 3 experienced neuroradiologists by use of a 3-point scale.

RESULTS: With metal artifact reduction, the observers agreed that the visibility had improved by at least 1 point on the scoring scale in > 40% of the cases (kappa = 0.6) and that the streak artifact was not obscuring surrounding structures in 64% of all cases (kappa = 0.6). Metal artifact reduction improved the image quality, which allowed for visibility sufficient for evaluation in 65% of the cases, and was preferred over no metal artifact reduction in 92% (kappa = 0.9). Significantly higher scores were given with metal artifact reduction (P < .0001).

CONCLUSIONS: Although metal artifact reduction is not capable of fully removing artifacts caused by implants with high x-ray absorption, we have shown that the image quality of contrast-enhanced conebeam CT data are improved drastically. The impact of the artifacts on the visibility varied between cases, and yet the overall visibility of the contrast-enhanced conebeam CT with metal artifact reduction improved in most the cases.

Advancing the university mission through partnerships with state Medicaid programs

Tue, 12/22/2015 - 11:33am

State Medicaid programs are playing an increasingly important role in the U.S. health care system and represent a major expenditure as well as a major source of revenue for state budgets. The size and complexity of these programs will only increase with the implementation of the Patient Protection and Affordable Care Act. Yet, many state Medicaid programs lack the resources and breadth of expertise to maximize the value of their programs not only for their beneficiaries but also for all those served by the health care system.Universities, especially those with medical schools and other health science programs, can serve as valuable partners in helping state Medicaid programs achieve higher levels of performance, including designing and implementing new approaches for monitoring the effectiveness and outcomes of health services and developing and sharing knowledge about program outcomes. In turn, universities can expand their role in public policy decision making while taking advantage of opportunities for additional research, training, and funding. As of 2013, approximately a dozen universities have developed formal agreements to provide faculty and care delivery resources to support their state Medicaid programs. These examples offer a road map for how others might approach developing similar, mutually beneficial partnerships.

Enhancement of the assessment of physician-patient communication skills in the United States Medical Licensing Examination

Tue, 12/22/2015 - 11:33am

The National Board of Medical Examiners (NBME) reviewed all components of the United States Medical Licensing Examination as part of a strategic planning activity. One recommendation generated from the review called for enhancements of the communication skills component of the Step 2 Clinical Skills (Step 2 CS) examination. To address this recommendation, the NBME created a multidisciplinary team that comprised experts in communication content, communication measurement, and implementation of standardized patient (SP)-based examinations. From 2007 through 2012, the team reviewed literature in physician-patient communication, examined performance characteristics of the Step 2 CS exam, observed case development and quality assurance processes, interviewed SPs and their trainers, and reviewed video recordings of examinee-SP interactions. The authors describe perspectives gained by their team from the review process and outline the resulting enhancements to the Step 2 CS exam, some of which were rolled out in June 2012.

Working as a team to improve patient care in the intensive care unit

Tue, 12/22/2015 - 11:33am

Doctors and nurses share the same goals for patient care. We are at our best when we function as a part of a multidisciplinary team, with individual team members contributing the perspectives and expertise afforded by their professions. Here, we share our perspectives on what a doctor is and what a nurse is, based on our respective experiences as a critical care physician and an acute care nurse practitioner.

Dopamine transporter endocytic trafficking in striatal dopaminergic neurons: differential dependence on dynamin and the actin cytoskeleton

Tue, 12/22/2015 - 11:33am

Dopaminergic signaling profoundly impacts rewarding behaviors, movement, and executive function. The presynaptic dopamine (DA) transporter (DAT) recaptures released DA, thereby limiting synaptic DA availability and maintaining dopaminergic tone. DAT constitutively internalizes and PKC activation rapidly accelerates DAT endocytosis, resulting in DAT surface loss. Longstanding evidence supports PKC-stimulated DAT trafficking in heterologous expression studies. However, PKC-stimulated DAT internalization is not readily observed in cultured dopaminergic neurons. Moreover, conflicting reports implicate both classic and nonclassic endocytic mechanisms mediating DAT trafficking. Prior DAT trafficking studies relied primarily upon chronic gene disruption and dominant-negative protein expression, or were performed in cell lines and cultured neurons, yielding results difficult to translate to adult dopaminergic neurons. Here, we use newly described dynamin inhibitors to test whether constitutive and PKC-stimulated DAT internalization are dynamin-dependent in adult dopaminergic neurons. Ex vivo biotinylation studies in mouse striatal slices demonstrate that acute PKC activation drives native DAT surface loss, and that surface DAT surprisingly partitions between endocytic-willing and endocytic-resistant populations. Acute dynamin inhibition reveals that constitutive DAT internalization is dynamin-independent, whereas PKC-stimulated DAT internalization is dynamin-dependent. Moreover, total internal reflection fluorescence microscopy experiments demonstrate that constitutive DAT internalization occurs equivalently from lipid raft and nonraft microdomains, whereas PKC-stimulated DAT internalization arises exclusively from lipid rafts. Finally, DAT endocytic recycling relies on a dynamin-dependent mechanism that acts in concert with the actin cytoskeleton. These studies are the first comprehensive investigation of native DAT trafficking in ex vivo adult neurons, and reveal that DAT surface dynamics are governed by complex multimodal mechanisms.

Lymphoma occurring during pregnancy: antenatal therapy, complications, and maternal survival in a multicenter analysis

Tue, 12/22/2015 - 11:33am

PURPOSE: Lymphoma is the fourth most frequent cancer in pregnancy; however, current clinical practice is based largely on small series and case reports.

PATIENTS AND METHODS: In a multicenter retrospective analysis, we examined treatment, complications, and outcomes for Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) occurring during pregnancy.

RESULTS: Among 90 patients (NHL, n = 50; HL, n = 40), median age was 30 years (range, 18 to 44 years) and median diagnosis occurred at 24 weeks gestation. Of patients with NHL, 52% had advanced-stage versus 25% of patients with HL (P = .01). Pregnancy was terminated in six patients. Among the other 84 patients, 28 (33%) had therapy deferred to postpartum; these patients were diagnosed at a median 30 weeks gestation. This compared with 56 patients (67%) who received antenatal therapy with median lymphoma diagnosis at 21 weeks (P < .001); 89% of these patients received combination chemotherapy. The most common preterm complication was induction of labor (33%). Gestation went to full term in 56% of patients with delivery occurring at a median of 37 weeks. There were no differences in maternal complications, perinatal events, or median infant birth weight based on deferred versus antenatal therapy. At 41 months, 3-year progression-free survival (PFS) and overall survival (OS) for NHL were 53% and 82%, respectively, and 85% and 97%, respectively, for HL. On univariate analysis for NHL, radiotherapy predicted inferior PFS, and increased lactate dehydrogenase and poor Eastern Cooperative Oncology Group performance status (ECOG PS) portended worse OS. For HL patients, nulliparous status and "B" symptoms predicted inferior PFS.

CONCLUSION: Standard (non-antimetabolite) combination chemotherapy administered past the first trimester, as early as 13 weeks gestation, was associated with few complications and expected maternal survival with lymphoma occurring during pregnancy.

Cell biology in neuroscience: Architects in neural circuit design: glia control neuron numbers and connectivity

Tue, 12/22/2015 - 11:32am

Glia serve many important functions in the mature nervous system. In addition, these diverse cells have emerged as essential participants in nearly all aspects of neural development. Improved techniques to study neurons in the absence of glia, and to visualize and manipulate glia in vivo, have greatly expanded our knowledge of glial biology and neuron-glia interactions during development. Exciting studies in the last decade have begun to identify the cellular and molecular mechanisms by which glia exert control over neuronal circuit formation. Recent findings illustrate the importance of glial cells in shaping the nervous system by controlling the number and connectivity of neurons.