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The Role of Signal 3 Cytokine Timing in CD8 T Cell Activation: A Dissertation

Tue, 10/27/2015 - 3:18pm

During an acute virus infection, antigen-specific CD8 T cells undergo clonal expansion and differentiation into effector cells in order to control the infection. Efficient clonal expansion and differentiation of CD8 T cells are required to develop protective memory CD8 T cells. Antigen specific cells require 3 distinct signals for their activation: TCR engagement of peptide-MHC (signal 1), costimulation between B7 and CD28 (signal 2), and inflammatory cytokines including IL-12 or type 1 IFN (signal 3). CD8 T cells that encounter antigen and costimulation undergo programmed cell division, but these two signals alone are not sufficient for full effector cell differentiation and survival into memory. CD8 T cells need a third signal for efficient clonal expansion, differentiation into various effector populations, acquisition of cytolytic effector functions, and memory formation. The requirements for signal 3 cytokines in CD8 T cell activation have only been recently described; however, the timing of exposure to these signals has yet to be investigated. During the course of an immune response not all T cells will see antigen, costimulation, and inflammatory cytokines at the same time or in the same order. I sought to examine how the timing of signal 3 cytokines affected CD8 T cell activation. I questioned how the order of these signals effected CD8 T cell priming and subsequent activation, expansion and differentiation.

In order to study the in vivo effects of out-of-sequence signaling on CD8 T cell activation, I utilized poly(I:C), a dsRNA analogue, which is known to induce a strong type 1 IFN response. Through the use of various congenic transgenic and polyclonal CD8 T cell populations, in conjunction with adoptive transfer models, specific T cells which had been exposed to poly(I:C) induced environments could be identified and tracked over time. I wanted to characterize how out-of-sequence signaling affected T cell activation immediately after cognate antigen stimulation (4-5hours), and after prolonged exposure to cognate antigen (days-weeks).

Considering type 1 IFN can have both inhibitory and stimulatory effects on CD8 T cell proliferation, and when type 1 IFN provides signal 3 cytokine activity, it has positive effects on CD8 T cell expansion, I wanted to investigate the role of type 1 IFN as an out-of-sequence signal during CD8 T cell activation. We identified a transient defect in the phosphorylation of downstream STAT molecules after IFNβ signaling within poly(I:C) pretreated CD8 T cells. The inability of poly(I:C) pretreated CD8 T cells to respond to IFNβ signaling makes these cells behave in a manner more similar to T cells that only received 2 signals, rather than ones that received all 3 signals in the appropriate order. Consequently, poly(I:C) pretreated, or out-of-sequence, CD8 T cells were found to have defects in clonal expansion, effector differentiation and function as well as memory generation resulting in reduced efficacy of viral clearance.

Out-of-sequence CD8 T cells showed suppression of CD8 T cell responses after prolonged exposure to cognate antigen, but naïve CD8 T cells pre-exposed to poly(I:C) exhibited immediate effector function within hours of cognate antigen stimulation, prior to cell division. Poly(I:C) pretreated naïve CD8 T cells acquired an early activated phenotype associated with alterations of transcription factors and surface markers. Changes in naïve CD8 T cell phenotype are thought to be mediated by poly(I:C)-induced upregulation of self-MHC and costimulatory molecules on APCs through direct type 1 IFN signaling. Inoculating with poly(I:C) enabled naive CD8 T cells to produce effector functions immediately upon stimulation with high density cognate antigen, reduced affinity altered peptide ligands (APLs), and in response to reduced concentrations of cognate antigen. Unlike conventional naïve CD8 T cells, poly(I:C) pretreated naïve CD8 T cells acquired the ability to specifically lyse target cells. These studies identified how the timing of activation signals can dramatically affect the acquisition of CD8 T cell effector function.

This thesis describes how CD8 T cell exposure to activation signals in an unconventional order may result in altered response to antigen stimulation. Exposure of naïve CD8 T cells to type 1 IFN and costimulatory molecules in the presence of self-peptides enabled them to respond immediately upon antigen stimulation. Primed naïve CD8 T cells produced multiple cytokines in response to low-affinity, and low-density antigens, and gained ability to specifically lyse target cells. However, immediate effector function may come at the expense of clonal expansion and effector cell differentiation in response to prolonged antigen exposure as out-of-sequence CD8 T cells showed reduced proliferation, effector function and memory formation. The findings presented here may seem contradictory because out-of-sequence signaling can prime T cells to produce immediate effector functions and yet cause defects in T cell expansion and effector differentiation. However, these two models ascertained T cell function at different points after antigen exposure; one where functions were evaluated within hours after seeing cognate antigen, and the other showing T cell responses after days of antigen stimulation.

Studies described in this thesis highlight the growing complexity of CD8 T cell activation. Not only do the presence or absence of signals 1-3 contribute to T cell activation, but the timing of these signals also proves to be of great importance. These studies may describe how both latecomer and third party antigen specific T cells behave when and if they encounter cognate antigen in the midst of an ongoing infection. Out-of-sequence exposure to IFN initially stimulates effector function but at the expense of efficient clonal expansion and subsequent memory formation. The immediate effector function that naïve T cells gain during out-of-sequence priming may explain how some individuals are more resistant to superinfections, whereas the impairment in proliferation describes a universal mechanism of virus-induced immune suppression, explaining how other individuals can be more susceptible to secondary infections. Ultimately, results identified here can be applied to developing better and more effective vaccines.

Attention Deficit/Hyperactivity Disorder, Screen Time, Physical Activity, and Diet Quality: A Dissertation

Tue, 10/27/2015 - 3:18pm

Background. Emerging evidence suggests that youth with attention deficit/hyperactivity disorder (ADHD) may engage in sub-optimal health behaviors including high levels of screen time, low physical activity participation, and consumption of poor diets. These are independent risk factors for adverse health outcomes, and health-related behavior patterns established in childhood can track into adulthood. Thus, identifying and addressing dietary and physical activity habits in sub-populations of youth have important implications for health over the lifespan. The specific aims of this dissertation were to: (1) compare screen time between youth with and without ADHD and to assess its relationship to ADHD symptomatology; (2) compare participation in physical activity (PA) between adolescents with and without ADHD and to assess the relationship of PA participation to ADHD symptomatology; and (3) evaluate the association of diet quality and dietary patterns to ADHD symptomatology among youth ages 8-15 years.

Methods. The aforementioned outcomes of interest were analyzed using data from the continuous National Health and Nutrition Examination Survey (NHANES) 2001-2004. These waves of NHANES included a structured DSM-IV-based interview administered to parents that identified youth with ADHD and also yielded symptom counts for hyperactivity/impulsivity and inattention. Screen time and physical activity data were obtained from questionnaires that queried the amount of time spent watching television, playing videos, or using the computer outside of school time, and also surveyed the types, frequency, and duration of PA in which youth participated. Diet quality and dietary patterns, which included consumption of sugar-sweetened beverages (SSBs), total calorie intake, and eating frequency, were obtained by a 24-hour dietary recall using the Automated Multiple Pass Method of interviewing. Linear and logistic regression models adjusted for sociodemographic factors and anxiety/depression were employed to address the specific aims.

Results. The findings suggest that youth with ADHD are at the same, if not higher, risk for engaging in suboptimal health behaviors. Overall, youth participating in NHANES engaged in excessive amounts of screen time, failed to acquire sufficient physical activity, and consumed diets of poor quality. However, our findings suggest that ADHD symptomatology places youth at higher risk for sedentary behavior and poor diet quality. Relative to screen time, youth with ADHD showed a trend toward increased screen time, as did youth who took medication. ADHD symptoms were also associated with over two hours of daily TV viewing and overall increased screen time, and this was particularly true for children ages 8-11 years. Relative to physical activity, the outcomes did not differ between youth with and without ADHD, but the majority of youth did not meet the recommended guidelines of 60 minutes or more of moderate-to-vigorous PA each day. Diet quality was poor across the population of youth who participated in NHANES, and hyperactive/impulsive symptoms were associated with an even greater decrease in diet quality in both children and adolescents. In males, the presence of hyperactive/impulsive symptoms was associated with a decrease in diet quality, whereas in females, inattentive symptoms accounted for a decrease in diet quality. No differences in the other dietary patterns (i.e., SSB consumption, total energy intake, and eating frequency) were observed.

Conclusions. The diagnosis of ADHD and/or its symptoms are associated with less-than-recommended levels of screen time and poor diet quality, though youth in general were found to be engaging in suboptimal sedentary, physical activity, and dietary behaviors. The mechanisms for why youth with ADHD may have increased vulnerability to poorer health behaviors are not yet well understood. The findings from this dissertation support the need for ongoing efforts to address lifestyle factors among the nation’s youth generally, but may also stimulate new hypotheses about the needs of youth with ADHD from both public health and clinical perspectives, and encourage research on the implications of ADHD symptomatology on health-related behaviors and lifestyle factors.

TAK1-Mediated Post-Translational Modifications Modulate Immune Response: A Dissertation

Tue, 10/27/2015 - 3:18pm

Innate immunity is the first line of defense against invading pathogens. It provides immediate protection by initiating both cellular and humoral immune reactions in response to a wide range of infections. It is also important to the development of long-lasting and pathogen-specific adaptive immunity. Thus, studying of the innate immunity, especially the pathogen recognition and signaling modulation, is crucial for understanding the intrinsic mechanisms underlying the host defense, as well as contributing the development of the fight against infectious diseases. Drosophila is an ideal model organism for study of innate immunity. Comparing to mammals, Drosophila immunity is relative conserved and less redundant. A variety of molecular and genetic tools available add further convenience to the research in this system. My work is focused on the signaling modulation by post-translational modification after activation. In these studies I demonstrated in the center of Imd pathway, the Imd protein undergoes proteolytic cleavage, K63-polyubiquitination, phosphorylation, K63-deubiquitination and K48-polyubiquitination/degradation in a stimulation-dependent manner. These modifications of Imd play a crucial role in regulating signaling in response to infection. The characterization of ubiquitin-editing event provides a new insight into the molecular mechanisms underlying the activation and termination of insect immune signaling pathway.

Function and Regulation of the Tip60-p400 Complex in Embryonic Stem Cells: A Dissertation

Tue, 10/27/2015 - 3:18pm

The following work examines the mechanisms by which Tip60-p400 chromatin remodeling complex regulates gene expression in embryonic stem cells (ESCs). Tip60-p400 complex has distinct functions in undifferentiated and differentiated cells. While Tip60-p400 is often associated with gene activation in differentiated cells, its most prominent function in ESCs is to repress differentiation-related genes. I show that Tip60-p400 interacts with Hdac6 and other proteins to form a unique form of the complex in ESCs. Tip60-Hdac6 interaction is stem cell specific and is necessary for Tip60-p400 mediated gene regulation, indicating that Tip60- p400 function is controlled in part through the regulation of Hdac6 during development. Furthermore, I find that Hdac6 is required for the binding of Tip60- p400 to many of its target genes, indicating Hdac6 is necessary for the unique function of Tip60-p400 in ESCs. In addition to accessory proteins like Hdac6, Tip60-p400 also interacts with thousands of coding and noncoding RNAs in ESCs. I show that R-loops, DNA-RNA hybrids formed during transcription of many genes, are important for regulation of chromatin binding by at least two chromatin regulators (Tip60-p400 and PRC2). This finding suggests that transcripts produced by many genes in ESC may serve as a signal to modulate binding of chromatin regulators. However, R-loops might also function to regulate chromatin architecture in differentiated cells as well. Future studies based on this work will be necessary to understand the full repertoire of cell types and chromatin regulators regulated by these structures.

Potent monoclonal antibodies against Clostridium difficile toxin A elicited by DNA immunization

Sun, 10/25/2015 - 7:56pm

Recent studies have demonstrated that DNA immunization is effective in eliciting antigen-specific antibody responses against a wide range of infectious disease targets. The polyclonal antibodies elicited by DNA vaccination exhibit high sensitivity to conformational epitopes and high avidity. However, there have been limited reports in literature on the production of monoclonal antibodies (mAb) by DNA immunization. Here, by using Clostridium difficile (C. diff) toxin A as a model antigen, we demonstrated that DNA immunization was effective in producing a panel of mAb that are protective against toxin A challenge and can also be used as sensitive reagents to detect toxin A from various testing samples. The immunoglobulin (Ig) gene usage for such mAb was also investigated. Further studies should be conducted to fully establish DNA immunization as a unique platform to produce mAb in various hosts.

Design of the Vitesse Intracranial Stent Study for Ischemic Therapy (VISSIT) trial in symptomatic intracranial stenosis

Sun, 10/25/2015 - 7:56pm

BACKGROUND: Patients with high-grade symptomatic intracranial stenosis ( > /= 70%) have an increased risk of recurrent stroke despite medical treatment with antiplatelet or anticoagulant therapy. Intracranial stenting has been proposed as a viable treatment option for this high-risk patient population; however, evaluation of this therapy in randomized multicenter trials is needed. In this article, we present the design and methods of the Vitesse Intracranial Stent Study for Ischemic Therapy (VISSIT) trial for symptomatic intracranial stenosis.

METHODS: The VISSIT trial is a randomized control study designed to evaluate the safety, probable benefit, and effectiveness of the PHAROS Vitesse neurovascular balloon-expandable stent system plus medical therapy versus medical therapy alone in patients with cerebral or retinal ischemia due to neurovascular stenosis ( > /= 70%) for preventing the primary composite end point: stroke in the same territory (distal to the target lesion) as the presenting event within 12 months of randomization or hard transient ischemic attack in the same territory (distal to the target lesion) as the presenting event from day 2 through month 12 postrandomization.

RESULTS: Enrollment began in February 2009 and was halted in January 2012 with 112 subjects enrolled into the study. Clinical follow-up will continue for the planned period of 12 months postrandomization.

CONCLUSIONS: The VISSIT trial may provide valuable insight into the use of balloon-expandable intracranial stent as a treatment option for high-risk patients. Lessons learned from this trial may better guide future clinical trial design on best patient selection, stenting techniques, and periprocedural management.

Progressive bilateral lipoma arborescens of the knee complicated by juvenile spondyloarthropathy: a case report and review of the literature

Sun, 10/25/2015 - 7:56pm

OBJECTIVES: To report an unusual case of lipoma aborescens (LA) presented in a patient with treatment-responsive juvenile spondyloarthropathy (JSPA) and to summarize the clinical manifestations, therapy and prognosis of LA by literature review.

METHODS: We report an atypical case of a 17-year-old patient with an initial presentation of juvenile spondyloarthropathy, whose inflammatory condition was improved successfully by traditional anti-rheumatic drugs and an anti-TNF alpha agent but developed progressive swelling of bilateral knees. Lipoma arborescens were diagnosed in each knee by synovial biopsy obtained by arthroscopic surgery. Fifty-one cases of LA have been reported and are reviewed in detail.

RESULTS: Clinically, LA could present as monoarthritis or oligoarthritis. The lateral compartment of the knee is the most common site of involvement. Several cases were reported as a comorbidity of inflammatory diseases, but were not improved by anti-inflammatory therapy. Most patients were diagnosed by classic MRI and biopsy findings. The lesions can be managed by open or arthroscopic surgery, but a minority of the cases may have reoccurrence in the same or opposite joint.

CONCLUSIONS: LA is a very rare lesion of the synovial and bursal tissue with an unknown etiology. It is considered to be a benign proliferation of the synovial fat associated with trauma, degenerative or inflammatory conditions. LA should be considered as a secondary or comorbid condition in inflammatory arthropathies if other joints respond well to intensive therapy and one or more do not.

Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1)

Sun, 10/25/2015 - 7:56pm

In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8(+) T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses.

Oncogenic RAS directs silencing of tumor suppressor genes through ordered recruitment of transcriptional repressors

Sun, 10/25/2015 - 7:56pm

We previously identified 28 cofactors through which a RAS oncoprotein directs transcriptional silencing of Fas and other tumor suppressor genes (TSGs). Here we performed RNAi-based epistasis experiments and found that RAS-directed silencing occurs through a highly ordered pathway that is initiated by binding of ZFP354B, a sequence-specific DNA-binding protein, and culminates in recruitment of the DNA methyltransferase DNMT1. RNAi and pharmacological inhibition experiments reveal that silencing requires continuous function of RAS and its cofactors and can be rapidly reversed, which may have therapeutic implications for reactivation of silenced TSGs in RAS-positive cancers.

Relation of N-terminal pro-B-type natriuretic peptide with diastolic function in hypertensive heart disease

Sun, 10/25/2015 - 7:56pm

BACKGROUND: Elevated natriuretic peptide levels in asymptomatic individuals without heart failure are associated with increased risk of adverse cardiovascular outcomes and may reflect subclinical cardiac dysfunction.

METHODS: In a sample of 313 asymptomatic individuals (51% women, mean age 61 years) with hypertension and diastolic dysfunction, we examined the association of plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) with both conventional and advanced echocardiographic measures of systolic and diastolic function, including myocardial strain, using speckle-tracking-based analyses.

RESULTS: In univariate analyses, higher NT-proBNP was associated with greater left ventricular mass index (P = 0.003), left atrial volume index (P = 0.007), lateral E' velocity (P < 0.0001), E/E' ratio (P < 0.0001), peak global longitudinal systolic strain (P = 0.015), systolic strain rate (P = 0.021), and early diastolic strain rate (P < 0.0001). In multivariable analyses, NT-proBNP remained associated with measures of diastolic dysfunction, including lateral E' velocity (P = 0.013) and the E/E' ratio (P = 0.008). However, early diastolic strain rate was the echocardiographic parameter most strongly associated with NT-proBNP (P = 0.003).

CONCLUSIONS: In the setting of asymptomatic hypertensive heart disease and preserved ejection fraction, elevation in natriuretic peptide levels is predominantly associated with subclinical diastolic dysfunction.

Antigenic peptide trimming by ER aminopeptidases--insights from structural studies

Sun, 10/25/2015 - 7:56pm

Generation and destruction of antigenic peptides by ER resident aminopeptidases ERAP1 and ERAP2 have been shown in the last few years to be important for the correct functioning and regulation of the adaptive immune response. These two highly homologous aminopeptidases appear to have evolved complex mechanisms well suited for their biological role in antigen presentation. Furthermore, polymorphic variability in these enzymes appears to affect their function and predispose individuals to disease. This review discusses our current understanding of the molecular mechanisms behind ERAP1/2 function as suggested by several recently determined crystallographic structures of these enzymes.

RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

Sun, 10/25/2015 - 7:56pm

Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE-DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo.

Alternative spliceosome assembly pathways revealed by single-molecule fluorescence microscopy

Sun, 10/25/2015 - 7:56pm

Removal of introns from nascent transcripts (pre-mRNAs) by the spliceosome is an essential step in eukaryotic gene expression. Previous studies have suggested that the earliest steps in spliceosome assembly in yeast are highly ordered and the stable recruitment of U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site necessarily precedes recruitment of U2 snRNP to the branch site to form the "prespliceosome." Here, using colocalization single-molecule spectroscopy to follow initial spliceosome assembly on eight different S. cerevisiae pre-mRNAs, we demonstrate that active yeast spliceosomes can form by both U1-first and U2-first pathways. Both assembly pathways yield prespliceosomes functionally equivalent for subsequent U5.U4/U6 tri-snRNP recruitment and for intron excision. Although fractional flux through the two pathways varies on different introns, both are operational on all introns studied. Thus, multiple pathways exist for assembling functional spliceosomes. These observations provide insight into the mechanisms of cross-intron coordination of initial spliceosome assembly.

Teaching health care students about disability within a cultural competency context

Sun, 10/25/2015 - 7:56pm

The training of health care providers has been identified as key to resolving the health disparities experienced by persons with disabilities. We contend that: 1) cultural competency provides a useful conceptual framework for teaching disability-related content to health professions students; 2) educational experiences can be structured to reflect the socio-cultural complexity of the 'disability culture;' 3) desired competencies associated with culture can be defined with regard to professionals' approach to patients with disabilities; 4) exposure to persons who have disabilities in their homes allows the student to make connections between the nuances of daily life with a disability and one's health care needs; 5) the framework allows the disability culture to be integrated with other cultural contexts, including race and ethnicity; and 6) the framework acknowledges the potential impact of providers' conscious or unconscious recognition of their potential membership in the disability culture on their approach to patients with disabilities.

NKp30 enables NK cells to act naturally with fungi

Sun, 10/25/2015 - 7:56pm

NK cells have direct activity against fungal pathogens. Using an unbiased systematic approach, Li et al. (2013) find that NKp30 is a major NK cell receptor responsible for fungal recognition. Moreover, diminished NKp30 expression is associated with reduced antifungal activity in NK cells isolated from HIV-infected persons.

Differentiating reversible cerebral vasoconstriction syndrome with subarachnoid hemorrhage from other causes of subarachnoid hemorrhage

Sun, 10/25/2015 - 7:56pm

IMPORTANCE: Reversible cerebral vasoconstriction syndrome (RCVS) is a clinical-angiographic syndrome characterized by recurrent thunderclap headaches and reversible segmental multifocal cerebral artery narrowing. More than 30% of patients with RCVS develop subarachnoid hemorrhage (SAH). Patients with RCVS with SAH (RCVS-SAH) are often misdiagnosed as having potentially ominous conditions such as aneurysmal SAH (aSAH) or cryptogenic "angiogram-negative" SAH (cSAH) owing to overlapping clinical and imaging features.

OBJECTIVE: To identify predictors that can distinguish RCVS-SAH from aSAH and cSAH at the time of clinical presentation.

DESIGN: Retrospective analysis of 3 patient cohorts: patients with RCVS (1998-2009), patients with aSAH (1995-2003), and patients with cSAH (1995-2003).

SETTING: Academic hospital and tertiary referral center.

PARTICIPANTS: Consecutive patients with RCVS-SAH (n = 38), aSAH (n = 515), or cSAH (n = 93) whose conditions were diagnosed using standard criteria.

MAIN OUTCOMES AND MEASURES: Multivariate logistic regression analysis was used to identify predictors that differentiate RCVS-SAH from aSAH and cSAH.

RESULTS: Predictors differentiating RCVS-SAH from aSAH were younger age, chronic headache disorder, prior depression, prior chronic obstructive pulmonary disease, lower Hunt-Hess grade, lower Fisher SAH group, higher number of affected arteries, and the presence of bilateral arterial narrowing. Predictors differentiating RCVS-SAH from cSAH were younger age, female sex, prior hypertension, chronic headache disorder, lower Hunt-Hess grade, lower Fisher SAH group, and the presence of bilateral arterial narrowing.

CONCLUSIONS AND RELEVANCE: We identified important clinical and imaging differences between RCVS-SAH, aSAH, and cSAH that may be useful for improving diagnostic accuracy, clinical management, and resource utilization.

Syntaxin 13, a genetic modifier of mutant CHMP2B in frontotemporal dementia, is required for autophagosome maturation

Sun, 10/25/2015 - 7:56pm

Phagophore maturation is a key step in the macroautophagy pathway, which is critical in many important physiological and pathological processes. Here we identified Drosophila N-ethylmaleimide-sensitive fusion protein 2 (dNSF2) and soluble NSF attachment protein (Snap) as strong genetic modifiers of mutant CHMP2B, an ESCRT-III component that causes frontotemporal dementia and autophagosome accumulation. Among several SNAP receptor (SNARE) genes, Drosophila syntaxin 13 (syx13) exhibited a strong genetic interaction with mutant CHMP2B. Knockdown of syntaxin 13 (STX13) or its binding partner Vti1a in mammalian cells caused LC3-positive puncta to accumulate and blocks autophagic flux. STX13 was present on LC3-positive phagophores induced by rapamycin and was highly enriched on multilamellar structures induced by dysfunctional ESCRT-III. Loss of STX13 also caused the accumulation of Atg5-positive puncta and the formation of multilamellar structures. These results suggest that STX13 is a genetic modifier of ESCRT-III dysfunction and participates in the maturation of phagophores into closed autophagosomes.

Analysis of in vitro insulin-resistance models and their physiological relevance to in vivo diet-induced adipose insulin resistance

Sun, 10/25/2015 - 7:56pm

Diet-induced obesity (DIO) predisposes individuals to insulin resistance, and adipose tissue has a major role in the disease. Insulin resistance can be induced in cultured adipocytes by a variety of treatments, but what aspects of the in vivo responses are captured by these models remains unknown. We use global RNA sequencing to investigate changes induced by TNF-alpha, hypoxia, dexamethasone, high insulin, and a combination of TNF-alpha and hypoxia, comparing the results to the changes in white adipose tissue from DIO mice. We found that different in vitro models capture distinct features of DIO adipose insulin resistance, and a combined treatment of TNF-alpha and hypoxia is most able to mimic the in vivo changes. Using genome-wide DNase I hypersensitivity followed by sequencing, we further examined the transcriptional regulation of TNF-alpha-induced insulin resistance, and we found that C/EPBbeta is a potential key regulator of adipose insulin resistance.

Extremes of lineage plasticity in the Drosophila brain

Sun, 10/25/2015 - 7:56pm

An often-overlooked aspect of neural plasticity is the plasticity of neuronal composition, in which the numbers of neurons of particular classes are altered in response to environment and experience. The Drosophila brain features several well-characterized lineages in which a single neuroblast gives rise to multiple neuronal classes in a stereotyped sequence during development. We find that in the intrinsic mushroom body neuron lineage, the numbers for each class are highly plastic, depending on the timing of temporal fate transitions and the rate of neuroblast proliferation. For example, mushroom body neuroblast cycling can continue under starvation conditions, uncoupled from temporal fate transitions that depend on extrinsic cues reflecting organismal growth and development. In contrast, the proliferation rates of antennal lobe lineages are closely associated with organismal development, and their temporal fate changes appear to be cell cycle-dependent, such that the same numbers and types of uniglomerular projection neurons innervate the antennal lobe following various perturbations. We propose that this surprising difference in plasticity for these brain lineages is adaptive, given their respective roles as parallel processors versus discrete carriers of olfactory information.

Impact of mirabegron extended-release on the treatment of overactive bladder with urge urinary incontinence, urgency, and frequency

Sun, 10/25/2015 - 7:56pm

Overactive bladder is a highly prevalent disorder with a significant impact on quality of life. Antimuscarinic agents are commonly used, but persistence is limited due to unsatisfactory efficacy and/or tolerability. Mirabegron is the first beta-3 adrenoceptor agonist approved for the treatment of overactive bladder syndrome. This paper reviews the pharmacology, mechanism of action, efficacy, and safety of mirabegron. A PubMed search of all English articles pertaining to mirabegron was performed. An alternative to antimuscarinics, mirabegron has a unique mechanism, improves overactive bladder symptoms and quality of life, and has limited adverse effects and few contraindications.