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Mechanisms Regulating Early Mesendodermal Differentiation of Human Embryonic Stem Cells: A Dissertation

Wed, 11/30/2016 - 12:16pm

Key regulatory events take place at very early stages of human embryonic stem cell (hESC) differentiation to accommodate their ability to differentiate into different lineages; this work examines two separate regulatory events.

To investigate precise mechanisms that link alterations in the cell cycle and early differentiation, we examined the initial stages of mesendodermal lineage commitment and observed a cell cycle pause that occurred concurrently with an increase in genes that regulate the G2/M transition, including WEE1. Inhibition of WEE1 prevented the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. These findings reveal a novel G2 cell cycle pause required for endodermal differentiation.

A role for phenotypic transcription factors in very early differentiation is unknown. From a screen of candidate factors during early mesendodermal differentiation, we found that RUNX1 is selectively and transiently up-regulated. Transcriptome and functional analyses upon RUNX1 depletion established a role for RUNX1 in promoting cell motility. In parallel, we discovered a loss of repression for several epithelial genes, indicating that RUNX1 knockdown impaired an epithelial to mesenchymal transition during differentiation. Cell biological and biochemical approaches revealed that RUNX1 depletion compromised TGFβ2 signaling. Both the decrease in motility and deregulated epithelial marker expression upon RUNX1 depletion were rescued by reintroduction of TGFβ2, but not TGFβ1. These findings identify novel roles for RUNX1-TGFβ2 signaling in mesendodermal lineage commitment.

Evaluating Acceptability, Feasibility and Efficacy of a Diabetes Care Support Program Facilitated by Cellular-Enabled Glucose Meters: A Dissertation

Wed, 11/30/2016 - 12:16pm

Background. Diabetes requires significant disease management, patient-provider communication, and interaction between patients, family members, caregivers, and care teams. Emerging patient-facing technologies, such as cellular-enabled glucose meters, can facilitate additional care support and improve diabetes self-management. This study evaluated patient acceptability, feasibility, and efficacy of a diabetes care support program facilitated by cellular-enabled glucose meters.

Methods. A two-phase study approach was taken. Get In Touch – Phase 1 (GIT-1) was a 1-month pilot involving patients with type 1 and type 2 diabetes. Get In Touch – Phase 2 (GIT-2) was a 12-month randomized controlled crossover trial involving patients with poorly-controlled type 2 diabetes. Results from GIT-1 and preliminary results from GIT-2 are presented.

Results. GIT-1 participants with type 1 (n=6) and type 2 (n=10) diabetes reported the intervention and cellular-enabled glucose meter were easy to use and useful while identifying potential areas of improvement. GIT-2 participants in both the intervention (n=60) and control (n=60) groups saw significant improvements in treatment satisfaction and A1c change, with intervention participants experiencing slightly greater improvements in each after 6 months (p=0.09 and p=0.16, respectively) compared to control participants.

Conclusions. Patients reported favorable acceptability of the intervention. Preliminary results from a randomized trial demonstrated potential of intervention to improve patient-reported and physiological health outcomes. Future studies should evaluate feasibility and efficacy over a longer period of time, with a greater number of participants, and targeting different populations of patients with diabetes. Provider perspectives and changes in provider behavior, clinical work flow, and caregiver burden should also be assessed.

The Role of MDM2 Phosphorylation in P53 Responses to DNA Damage and Tumor Suppression: A Dissertation

Wed, 11/30/2016 - 12:16pm

The p53 tumor suppressor protein is upregulated in response to DNA damage and other stress signals. The upregulation of p53 involves freeing it from negative regulation imposed by Mdm2 and MdmX (Mdm4). Accumulating evidence indicates that phosphorylation of Mdm proteins by different stress-activated kinases such as ATM or c-Abl significantly impacts p53 functions. We have previously shown that ATM phosphorylation of Mdm2 Ser394 is required for robust p53 stabilization and activation following DNA damage.

This dissertation describes in vivo examination of the mechanism by which Mdm2 Ser394 phosphorylation impacts p53 activities and its contribution to suppression of oncogene and DNA damage-induced tumors. We determine that phosphorylation of Mdm2 Ser394 regulates p53 activity by modulating Mdm2 stability and paradoxically delays Myc-driven lymphomagenesis while increasing lymphomagenesis in sub-lethally irradiated mice. c-Abl phosphorylates the residue neighboring Mdm2 Ser394, Mdm2 Tyr393.

This dissertation describes the generation of a novel Mdm2Y393F mutant mouse to determine if c-Abl phosphorylation of Mdm2 regulates p53-mediated DNA damage responses or tumor suppression in vivo. Mdm2Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2Y393F/S394A mice and Mdm2S394A mice display similar phenotypes.

The studies presented herein further our understanding of the mechanisms by which DNA damage-associated kinases stabilize and activate p53, and influence p53-dependent responses and tumor suppression. A better understanding of the in vivo effects of Mdm2 phosphorylation may facilitate the development of novel therapeutics capable of stimulating p53 anti-tumor activity or alleviating p53- dependent toxicities in non-malignant tissues.

Investigation of RNA Binding Protein Pumilio as a Genetic Modifier of Mutant CHMP2B in Frontotemporal Dementia (FTD): A Masters Thesis

Wed, 11/30/2016 - 12:16pm

Frontotemporal dementia (FTD) is the second most common early-onset dementia. A rare mutation in CHMP2B gene was found to be associated with FTD linked to chromosome 3. Previous studies have shown that mutant CHMP2B could lead to impaired autophagy pathway and altered RNA metabolism. However, it is still unknown what genes mediate the crosstalk between different pathways affected by mutant CHMP2B. Genetic screens designed to identify genes interacting with mutant CHMP2B represents a key approach in solving the puzzle. Expression of mutant CHMP2B (CHMP2Bintron5) in Drosophila eyes leads to a neurodegenerative phenotype including melanin deposition and disrupted internal structure of ommatidia. The phenotype is easily quantified by estimating the percentage of black dots on the surface of the eyes. Using this established Drosophila model, I searched for genes encoding RNA binding proteins that genetically modify CHMP2Bintron5 toxicity. I found that partial loss of Pumilio, a translation repressor, mitigates CHMP2Bintron5 induced toxicity in the fly eyes. Western blot analysis showed that down regulation of Pumilio does not significantly decrease CHMP2Bintron5 protein level, indicating indirect regulation involved in suppression of the phenotype. The molecular targets regulated by Pumilio and the mechanism underlying CHMP2Bintron5 toxicity suppression by Pumilio down-regulation requires further investigation.

mTORC2 Promotes Lipid Storage and Suppresses Thermogenesis in Brown Adipose Tissue in Part Through AKT-Independent Regulation of FoxO1: A Dissertation

Wed, 11/30/2016 - 12:16pm

Recent studies suggest adipose tissue plays a critical role in regulating whole body energy homeostasis in both animals and humans. In particular, activating brown adipose tissue (BAT) activity is now appreciated as a potential therapeutic strategy against obesity and metabolic disease. However, the signaling circuits that coordinate nutrient uptake and BAT function are poorly understood. Here, I investigated the role of the nutrient-sensing mTOR signaling pathway in BAT by conditionally deleting Rictor, which encodes an essential component of mTOR Complex 2 (mTORC2) either in brown adipocyte precursors or mature brown adipocytes. In general, inhibiting BAT mTORC2 reduces glucose uptake and de novo lipogenesis pathways while increases lipid uptake and oxidation pathways indicating a switch in fuel utilization. Moreover, several key thermogenic factors (Ucp1, Pgc1α, and Irf4) are elevated in Rictor-deficient BAT, resulting in enhanced thermogenesis. Accordingly, mice with mTORC2 loss in BAT are protected from HFD-induced obesity and metabolic disease at thermoneutrality. In vitro culture experiments further suggest that mTORC2 cell-autonomously regulates the BAT thermogenic program, especially Ucp1 expression, which depends on FoxO1 activity. Mechanistically, mTORC2 appears to inhibit FoxO1 by facilitating its lysine-acetylation but not through the canonical AKT-mediated phosphorylation pathway. Finally, I also provide evidence that β-adrenergic signaling which normally triggers thermogenesis also induces FoxO1 deacetylation in BAT. Based on these data, I propose a model in which mTORC2 functions in BAT as a critical signaling hub for coordinating nutrient uptake, fuel utilization, and thermogenic gene expression. These data provide a foundation for future studies into the mTORC2-FoxO1 signaling axis in different metabolic tissues and physiological conditions.

Structural Mechanisms of the Sliding Clamp and Sliding Clamp Loader: Insights into Disease and Function: A Dissertation

Wed, 11/30/2016 - 12:16pm

Chromosomal replication is an essential process in all life. This dissertation highlights regulatory roles for two critical protein complexes at the heart of the replication fork: 1) the sliding clamp, the major polymerase processivity factor, and 2) the sliding clamp loader, a spiral-shaped AAA+ ATPase, which loads the clamp onto DNA.

The clamp is a promiscuous binding protein that interacts with at least 100 binding partners to orchestrate many processes on DNA, but spatiotemporal regulation of these binding interactions is unknown. Remarkably, a recent disease-causing mutant of the sliding clamp showed specific defects in DNA repair pathways. We aimed to use this mutant as a tool to understand the binding specificity of clamp interactions, and investigate the disease further. We solved three structures of the mutant, and biochemically showed perturbation of partnerbinding for some, but not all, ligands. Using a fission yeast model, we showed that mutant cells are sensitive to select DNA damaging agents. These data revealed significant flexibility within the binding site, which likely regulates partner binding.

Before the clamp can act on DNA, the sliding clamp loader places the clamp onto DNA at primer/template (p/t) junctions. The clamp loader reaction couples p/t binding and subsequent ATP hydrolysis to clamp closure. Here we show that composition (RNA vs. DNA) of the primer strand affects clamp loader binding, and that the order of ATP hydrolysis around the spiral is likely sequential. These studies highlight additional details into the clamp loader mechanism, which further elucidate general mechanisms of AAA+ machinery.

A Walk on the Fine Line Between Reward and Risk: AAV-IFNβ Gene Therapy for Glioblastoma: A Dissertation

Wed, 11/30/2016 - 12:16pm

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The current standard-of-care treatment including surgery, radiation and temozolomide (TMZ) chemotherapy does not prolong the survival satisfactorily. Here we have tested the feasibility, efficacy and safety of a potential gene therapy approach using AAV as gene delivery vehicle for treatment of GBM.

Interferon-beta (IFNβ) is a cytokine molecule also having pleiotropic anticancerous properties. Previously it has been shown by our group that AAV mediated local (intracranial) gene delivery of human IFNβ (hIFNβ) could be an effective treatment for non-invasive human glioblastoma (U87) in orthotopic xenograft mouse model.But as one of the major challenges to treat GBM effectively in clinics is its highly invasive property, in the current study we first sought to test the efficacy of our therapeutic model in a highly invasive human GBM (GBM8) xenograft mouse model.

One major limitation of using the xenograft mouse model is that these mice are immune-compromised. Moreover, as IFNβ does not interact with cross-species receptors, the influence of immune systems on GBM remains largely untested. Therefore to test the therapeutic approach in an immune-competent mouse model, we next treated a syngeneic mouse GBM model (GL261) in an immune-competent mouse (C57B6) with the gene encoding the species-matched IFNβ (mIFNβ). We also tested if combination of this IFNβ gene therapy with the current standard chemotherapeutic drug (TMZ) is more effective than any one of the therapeutic modes alone. Finally, we tested the long term safety of the AAV-mIFNβ local gene therapy in healthy C57B6 mice.

Next, we hypothesized that global genetic engineering of brain cells expressing secretory therapeutic protein like hIFNβ could be more beneficial for treatment of invasive, migratory and distal multifocal GBM. We tested this hypothesis using systemic delivery of AAV9 vectors encoding hIFNβ gene for treatment of GBM8 tumor in nude mice.

Using in vivo bioluminescence imaging of tumor associated firefly luciferase activity, long term survival assay and histological analysis of the brains we have shown that local treatment of AAV-hIFNβ for highly invasive human GBM8 is therapeutically beneficial at an early growth phase of tumor. However, systemic delivery route treatment is far superior for treating multifocal distal GBM8 tumors. Nonetheless, for both delivery routes, treatment efficacy is significantly reduced when treated at a later growth phase of the tumor.

In syngeneic GL261 tumor model study, we show that local AAV-mIFNβ gene therapy alone or in combination with TMZ treatment can provide significant survival benefit over control or only TMZ treatment, respectively. However, the animals eventually succumb to the tumor. Safety study in the healthy animals shows significant body weight loss in some treatment groups, whereas one group shows long term survival without any weight loss or any noticeable changes in the external appearances. However, histological analysis indicates marked demyelinating neurotoxic effects upon long term exposures to mIFNβ over-expressions in brain. Overall, we conclude from this study that AAV-IFNβ gene therapy has great therapeutic potential for GBM treatment in future, but the therapeutic window is small and long term continuous expression could have severe deleterious effects on health.

Roles of the Mother Centriole Appendage Protein Cenexin in Microtubule Organization during Cell Migration and Cell Division: A Dissertation

Tue, 11/29/2016 - 12:01pm

Epithelial cells are necessary building blocks of the organs they line. Their apicalbasolateral polarity, characterized by an asymmetric distribution of cell components along their apical-basal axis, is a requirement for normal organ function. Although the centrosome, also known as the microtubule organizing center, is important in establishing cell polarity the mechanisms through which it achieves this remain unclear. It has been suggested that the centrosome influences cell polarity through microtubule cytoskeleton organization and endosome trafficking. In the first chapter of this thesis, I summarize the current understanding of the mechanisms regulating cell polarity and review evidence for the role of centrosomes in this process.

In the second chapter, I examine the roles of the mother centriole appendages in cell polarity during cell migration and cell division. Interestingly, the subdistal appendages, but not the distal appendages, are essential in both processes, a role they achieve through organizing centrosomal microtubules. Depletion of subdistal appendages disrupts microtubule organization at the centrosome and hence, affects microtubule stability. These microtubule defects affect centrosome reorientation and spindle orientation during cell migration and division, respectively. In addition, depletion of subdistal appendages affects the localization and dynamics of apical polarity proteins in relation to microtubule stability and endosome recycling. Taken together, our results suggest the mother centriole subdistal appendages play an essential role in regulating cell polarity. A discussion of the significance of these results is included in chapter three.

Viral Proteases as Drug Targets and the Mechanisms of Drug Resistance: A Dissertation

Tue, 11/29/2016 - 12:01pm

Viral proteases have been shown to be effective targets of anti-viral therapies for human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, under the pressure of therapy including protease inhibitors, the virus evolves to select drug resistance mutations both in the protease and substrates. In my thesis study, I aimed to understand the mechanisms of how this protease−substrate co-evolution contributes to drug resistance. Currently, there are no approved drugs against dengue virus (DENV); I investigated substrate recognition by DENV protease and designed cyclic peptides as inhibitors targeting the prime site of dengue protease.

First, I used X-ray crystallography and subsequent structural analysis to investigate the molecular basis of HIV-1 protease and p1-p6 substrate coevolution. I found that co-evolved p1-p6 substrates rescue the HIV-1 I50V protease’s binding activity by forming more van der Waals contacts and hydrogen bonds, and that co-evolution restores the dynamics at the active site for all three mutant substrates.

Next, I used aprotinin as a platform to investigate DENV protease–substrate recognizing pattern, which revealed that the prime side residues significantly modulate substrate affinity to protease and the optimal interactions at each residue position. Based on these results, I designed cyclic peptide inhibitors that target the prime site pocket of DENV protease. Through optimizing the length and sequence, the best inhibitor achieved a 2.9 micromolar Ki value against DENV3 protease. Since dengue protease does not share substrate sequence with human serine proteases, these cyclic peptides can be used as scaffolds for inhibitor design with higher specificity.

Understanding Drug Resistance and Antibody Neutralization Escape in Antivirals: A Dissertation

Tue, 11/29/2016 - 12:01pm

Antiviral drug resistance is a major problem in the treatment of viral infections, including influenza and hepatitis C virus (HCV). Influenza neuraminidase (NA) is a viral sialidase on the surface of the influenza virion and a primary antiviral target in influenza. Two subtypes of NA predominate in humans, N1 and N2, but different patterns of drug resistance have emerged in each subtype. To provide a framework for understanding the structural basis of subtype specific drug resistance mutations in NA, we used molecular dynamics simulations to define dynamic substrate envelopes for NA to determine how different patterns of drug resistance have emerged in N1 and N2 NA. Furthermore, we used the substrate envelope to analyze HCV NS3/4A protease inhibitors in clinical development. In addition, influenza hemagglutinin (HA) is a primary target of neutralizing antibodies against influenza. Novel broadly neutralizing antibodies (BnAbs) against the stem region of HA have been described and inhibit several influenza viral subtypes, but antibody neutralization escape mutations have emerged. We identified potential escape mutations in broadly neutralizing antibody F10 that may impact protein dynamics in HA that are critical for function. We also solved crystal structures of antibody fragments that are important for understanding the structural basis of antibody binding for influenza BnAbs. These studies can inform the design of improved therapeutic strategies against viruses by incorporating an understanding of structural elements that are critical for function, such as substrate processing and protein dynamics, into the development of novel therapeutics that are robust against resistance.

The Drosophila Homolog of the Intellectual Disability Gene ACSL4 Acts in Glia to Regulate Morphology and Neuronal Activity: A Dissertation

Tue, 11/29/2016 - 12:01pm

Recent developments in neurobiology make it clear that glia play fundamental and active roles, in the adult and in development. Many hereditary cognitive disorders have been linked to developmental defects, and in at least two cases, Rett Syndrome and Fragile X Mental Retardation, glia are important in pathogenesis. However, most studies of developmental disorders, in particular intellectual disability, focus on neuronal defects. An example is intellectual disability caused by mutations in ACSL4, a metabolic enzyme that conjugates long-chain fatty acids to Coenzyme A (CoA). Depleting ACSL4 in neurons is associated with defects in dendritic spines, a finding replicated in patient tissue, but the etiology of this disorder remains unclear. In a genetic screen to discover genes necessary for visual function, I identified the Drosophila homolog of ACSL4, Acsl, as a gene important for the magnitude of neuronal transmission, and found that it is required in glia. I determined that Acsl is required in a specific subtype of glia in the Drosophila optic lobe, and that depletion of Acsl from this population causes morphological defects. I demonstrated that Acsl is required in development, and that the phenotype can be rescued by human ACSL4. Finally, I discovered that ACSL4 is expressed in astrocytes in the mouse hippocampus. This study is highly significant for understanding glial biology and neurodevelopment. It provides information on the role of glia in development, substantiates a novel role for Acsl in glia, and advances our understanding of the potential role that glia play in the pathogenesis of intellectual disability.

The Mechanistic Role and Therapeutic Potential of microRNA-122 in Alcoholic Liver Disease: A Dissertation

Tue, 11/29/2016 - 12:01pm

Chronic alcohol use results in accelerated liver injury, leading to alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. However, due to the complex nature of this disease process, a central, druggable mechanism has remained elusive. microRNAs are potent post-transcriptional regulators of gene expression. A single miRNA has the ability to regulate hundreds of pathways simultaneously, defining cellular fate and function. microRNA-122 (miR-122), the most abundant miRNA in hepatocytes, has a demonstrated role as an tumor suppressor, regulator of hepatocyte metabolism, and hepatic differentiation.

In this dissertation I demonstrate the role of miR-122 on alcoholic liver disease (ALD) pathogenesis over four parts. In chapter II, I will demonstrate chronic alcoholic patients, free of neoplastic changes, have a reduction of miR-122 and that this miRNA regulates HIF-1α, a determinant of ALD pathogenesis. In chapter III, using hepatocytetropic adeno-associated virus 8 (AAV8) vector, I demonstrate that miR-122 inhibition mimics ALD pathogenesis, and furthermore, using hepatocyte-specific HIF-1α-null (HIF1hepKO) mice that this phenomenon is HIF-1α dependent. Given this finding, in chapter IV, I demonstrate that ectopic expression of miR-122 in vivo can reverse alcoholinduced liver damage, steatosis, and inflammation by directly targeting HIF-1α. Finally, in chapter V, I present evidence that alcohol-induced dysregulation of grainyhead-like proteins 1 and 2 (GRHL2), mediate the inhibition of miR-122 at the transcriptional level. These findings dissect a novel mechanistic regulatory axis of miR-122 and indicate a potential opportunity for restoration of miR-122 as a therapy in early ALD.

A Role for TNMD in Adipocyte Differentiation and Adipose Tissue Function: A Dissertation

Tue, 11/29/2016 - 12:01pm

Adipose tissue is one of the most dynamic tissues in the body and is vital for metabolic homeostasis. In the case of excess nutrient uptake, adipose tissue expands to store excess energy in the form of lipids, and in the case of reduced nutrient intake, adipose tissue can shrink and release this energy. Adipocytes are most functional when the balance between these two processes is intact. To understand the molecular mechanisms that drive insulin resistance or conversely preserve the metabolically healthy state in obese individuals, our laboratory performed a screen for differentially regulated adipocyte genes in insulin resistant versus insulin sensitive subjects who had been matched for BMI. From this screen, we identified the type II transmembrane protein tenomodulin (TNMD), which had been previously implicated in glucose tolerance in gene association studies. TNMD was upregulated in omental fat samples isolated from the insulin resistant patient group compared to insulin sensitive individuals. TNMD was predominantly expressed in primary adipocytes compared to the stromal vascular fraction from this adipose tissue. Furthermore, TNMD expression was greatly increased in human preadipocytes by differentiation, and silencing TNMD blocked adipogenic gene induction and adipogenesis, suggesting its role in adipose tissue expansion.

Upon high fat diet feeding, transgenic mice overexpressing Tnmd specifically in adipose tissue developed increased epididymal adipose tissue (eWAT) mass without a difference in mean cell size, consistent with elevated in vitro adipogenesis. Moreover, preadipocytes isolated from transgenic epididymal adipose tissue demonstrated higher BrdU incorporation than control littermates, suggesting elevated preadipocyte proliferation. In TNMD overexpressing mice, lipogenic genes PPARG, FASN, SREBP1c and ACLY were upregulated in eWAT as was UCP-1 in brown fat, while liver triglyceride content was reduced. Transgenic animals displayed improved systemic insulin sensitivity, as demonstrated by decreased inflammation and collagen accumulation and increased Akt phosphorylation in eWAT. Thus, the data we present here suggest that TNMD plays a protective role during visceral adipose tissue expansion by promoting adipogenesis and inhibiting inflammation and tissue fibrosis.

Plague and the Defeat of Mammalian Innate Immunity: Systematic Genetic Analysis of Yersinia pestis Virulence Factors: A Dissertation

Tue, 11/29/2016 - 12:00pm

Yersinia pestis, the causative agent of plague, specializes in causing dense bacteremia following intradermal deposition of a small number of bacteria by the bite of an infected flea. This robust invasiveness requires the ability to evade containment by the innate immune system. Of the various mechanisms employed by Y. pestis to subvert the innate immune response and to proliferate rapidly in mammalian tissue, only a few are well-characterized. Here, I present two complementary genetic analyses of Y. pestis adaptations to the mammalian environment. In the first, genome-wide fitness profiling for Y. pestis by Tn-seq demonstrates that the bacterium has adapted to overcome limitation of diverse nutrients during mammalian infection. In the second, a series of combinatorial targeted mutations disentangles apparent functional redundancy among the effectors of the Y. pestis type III secretion system, and we report that YpkA, YopT, and YopJ contribute to virulence in mice. We have also begun to investigate a novel relationship between Y. pestis and mammalian platelets, a highly abundant cell type in plasma. I present evidence that Y. pestis has evolved specific mechanisms to interfere with platelet activation, likely in order to evade immune responses and promote maintenance of bacteremia by undermining platelet thrombotic and innate immune functions. The principles guiding this work – systematic genetic analysis of complex systems, coupled with rational modification of in vitro assays to more closely mimic the in vivo environment – are a generalizable approach for increasing the efficiency of discovering new virulence determinants in bacterial pathogens.

Neuromodulators signal through astrocytes to alter neural circuit activity and behaviour

Mon, 11/28/2016 - 11:32am

Astrocytes associate with synapses throughout the brain and express receptors for neurotransmitters that can increase intracellular calcium (Ca2+). Astrocytic Ca2+ signalling has been proposed to modulate neural circuit activity, but the pathways that regulate these events are poorly defined and in vivo evidence linking changes in astrocyte Ca2+ levels to alterations in neurotransmission or behaviour is limited. Here we show that Drosophila astrocytes exhibit activity-regulated Ca2+ signalling in vivo. Tyramine and octopamine released from neurons expressing tyrosine decarboxylase 2 (Tdc2) signal directly to astrocytes to stimulate Ca2+ increases through the octopamine/tyramine receptor (Oct-TyrR) and the transient receptor potential (TRP) channel Water witch (Wtrw), and astrocytes in turn modulate downstream dopaminergic neurons. Application of tyramine or octopamine to live preparations silenced dopaminergic neurons and this inhibition required astrocytic Oct-TyrR and Wtrw. Increasing astrocyte Ca2+ signalling was sufficient to silence dopaminergic neuron activity, which was mediated by astrocyte endocytic function and adenosine receptors. Selective disruption of Oct-TyrR or Wtrw expression in astrocytes blocked astrocytic Ca2+ signalling and profoundly altered olfactory-driven chemotaxis and touch-induced startle responses. Our work identifies Oct-TyrR and Wtrw as key components of the astrocytic Ca2+ signalling machinery, provides direct evidence that octopamine- and tyramine-based neuromodulation can be mediated by astrocytes, and demonstrates that astrocytes are essential for multiple sensory-driven behaviours in Drosophila.

Circadian rhythms identified in Caenorhabditis elegans by in vivo long-term monitoring of a bioluminescent reporter

Mon, 11/28/2016 - 11:32am

Circadian rhythms are based on endogenous clocks that allow organisms to adjust their physiology and behavior by entrainment to the solar day and, in turn, to select the optimal times for most biological variables. Diverse model systems-including mice, flies, fungi, plants, and bacteria-have provided important insights into the mechanisms of circadian rhythmicity. However, the general principles that govern the circadian clock of Caenorhabditis elegans have remained largely elusive. Here we report robust molecular circadian rhythms in C elegans recorded with a bioluminescence assay in vivo and demonstrate the main features of the circadian system of the nematode. By constructing a luciferase-based reporter coupled to the promoter of the suppressor of activated let-60 Ras (sur-5) gene, we show in both population and single-nematode assays that C elegans expresses approximately 24-h rhythms that can be entrained by light/dark and temperature cycles. We provide evidence that these rhythms are temperature-compensated and can be re-entrained after phase changes of the synchronizing agents. In addition, we demonstrate that light and temperature sensing requires the photoreceptors LITE and GUR-3, and the cyclic nucleotide-gated channel subunit TAX-2. Our results shed light on C elegans circadian biology and demonstrate evolutionarily conserved features in the circadian system of the nematode.

Obesity, health-care utilization, and health-related quality of life after fracture in postmenopausal women: Global Longitudinal Study of Osteoporosis in Women (GLOW)

Tue, 11/22/2016 - 2:21pm

Fractures may be associated with higher morbidity in obese postmenopausal women than in nonobese women. We compared health-care utilization, functional status, and health-related quality of life (HRQL) in obese, nonobese, and underweight women with fractures. Information from the GLOW study, started in 2006, was collected at baseline and at 1, 2, and 3 years. In this subanalysis, self-reported incident clinical fractures, health-care utilization, HRQL, and functional status were recorded and examined. Women in GLOW (n = 60,393) were aged ≥55 years, from 723 physician practices at 17 sites in 10 countries. Complete data for fracture and body mass index were available for 90 underweight, 3,270 nonobese, and 941 obese women with one or more incident clinical fractures during the 3-year follow-up. The median hospital length of stay, adjusted for age, comorbidities, and fracture type, was significantly greater in obese than nonobese women (6 vs. 5 days, p = 0.017). Physical function and vitality score were significantly worse in obese than in nonobese women, both before and after fracture; but changes after fracture were similar across groups. Use of antiosteoporosis medication was significantly lower in obese than in nonobese or underweight women. In conclusion, obese women with fracture undergo a longer period of hospitalization for treatment and have poorer functional status and HRQL than nonobese women. Whether these differences translate into higher economic costs and adverse effects on longer-term outcomes remains to be established.

Biomedical Research Data Management Open Online Education: Challenges & Lessons Learned

Sun, 11/20/2016 - 7:52pm

The Best Practices for Biomedical Big Data project is a two year collaboration between Harvard Medical School and University of Massachusetts Medical School, funded by the NIH Big Data to Knowledge (BD2K) Initiative for Resource Development. The Best Practices for Biomedical Research Data Management Massive Open Online Course (MOOC) provides training to librarians, biomedical researchers, undergraduate and graduate biomedical students, and other interested individuals on recommended practices facilitating the discoverability, access, integrity, reuse value, privacy, security, and long term preservation of biomedical research data. This poster highlights lessons learned from the first year of this project.

Built upon the New England Collaborative Data Management Curriculum, the development team sought to use existing curricular materials to create a fully online course. The course is designed with an open course platform, WordPress Learning Management System (WPLMS), in order to facilitate broad access. Each of the MOOC’s nine modules is dedicated to a specific component of data management best practices and includes video lectures, presentation slides, research teaching cases, readings, activities, and interactive quizzes.

The project team overcame multiple challenges related to creating an open online course: curriculum, audience and software.

Working towards overcoming these, the Best Practices for Biomedical Research Data Management MOOC development team has moved slowly and deliberately, created additional content, and added content experts to provide guidance. These lessons learned will assist course development beyond this project, adding to best practices for creating massive open online courseware. Lessons learned include: teaching method influences the curriculum and content should not be developed in isolation from the teaching method; content is dependent on audience and supplementary content can be used to bridge audience gaps; and implementing new or unfamiliar technologies is challenging so allow more time in the timeline for project team to work with open source platform.

Gallbladder opacification on gadoxetate disodium-enhanced CT scan

Fri, 11/18/2016 - 8:23am

This study aimed to evaluate the radiologist's ability to identify excreted gadoxetate disodium within the gallbladder on CT scan. Thirty three healthy adults underwent imaging of the liver during work-up for potential liver donation. Three patients had undergone prior cholecystectomy and therefore were excluded. Imaging consisted of gadoxetate disodium-enhanced magnetic resonance cholangiography (MRC) and multiphase contrast-enhanced CT scan of the abdomen and pelvis. Two fellowship-trained abdominal imaging radiologists, who were blinded to the MRC images and the contrast agent used during MRC, independently reviewed the CT scans of the 30 patients that were included. The scans were evaluated for the presence or absence of abnormal hyperdensity within the gallbladder. Three patients did not receive intravenous gadoxetate disodium, 4 patients had their MRC after the CT scan, and 1 patient had the CT scans 5 days following the MRC. Twenty two patients had the CT scan within 24 h following the gadoxetate disodium-enhanced MRC. Of the 22 patients expected to have gadolinium in the gallbladder, both reviewers identified hyperdensity in the same 20 patients (90%). Both reviewers reported no abnormal hyperdensity within the gallbladder in the remaining 10 patients. CT scan can reveal excreted gadoxetate disodium within the gallbladder lumen and therefore gadoxetate disodium-enhanced CT scan can potentially play a role in the evaluation of cystic duct patency and work-up of acute cholecystitis.

Transfusion practices in evolution, not revolution*

Thu, 11/17/2016 - 12:31pm

Comment on: Survey on stated transfusion practices in PICUs*. [Pediatr Crit Care Med. 2014]