Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images. Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3]. Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons. One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, and maintaining general cell health. Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons. A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde. Our approach to imaging GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events. The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available

The Impact of Socioeconomic Status on the Neural Substrates Associated with Pleasure

Low socio-economic status (SES) is associated with increased morbidity and premature mortality. Because tonic adversity relates to a diminished ability to experience pleasure, we hypothesized that subjects living in poverty would show diminished neural responsivity to positive stimuli in regions associated with positive experience and reward. Visual images were presented to twenty-two subjects in the context of a EPI-BOLD fMRI paradigm. Significant differences in neural responses between SES groups to poverty vs. neutral images were assessed, examining group, condition, and interaction effects. The data suggest that persons living in low-SES have neural experiences consistent with diminished interest in things generally enjoyed and point toward a possible explanation for the relationship between socioeconomic inequalities and mood disorders, such as depression, by SES

Potential implications of coronary artery calcium testing for guiding aspirin use among asymptomatic individuals with diabetes.

ObjectiveIt is unclear whether coronary artery calcium (CAC) is effective for risk stratifying patients with diabetes in whom treatment decisions are uncertain.Research design and methodsOf 44,052 asymptomatic individuals referred for CAC testing, we studied 2,384 individuals with diabetes. Subjects were followed for a mean of 5.6 ± 2.6 years for the end point of all-cause mortality.ResultsThere were 162 deaths (6.8%) in the population. CAC was a strong predictor of mortality across age-groups (age <50, 50-59, ≥60), sex, and risk factor burden (0 vs. ≥1 additional risk factor). In individuals without a clear indication for aspirin per current guidelines, CAC stratified risk, identifying patients above and below the 10% risk threshold of presumed aspirin benefit.ConclusionsCAC can help risk stratify individuals with diabetes and may aid in selection of patients who may benefit from therapies such as low-dose aspirin for primary prevention

Intracellular Amyloid β Oligomers Impair Organelle Transport and Induce Dendritic Spine Loss in Primary Neurons

Introduction Synaptic dysfunction and intracellular transport defects are early events in Alzheimer’s disease (AD). Extracellular amyloid β (Aβ) oligomers cause spine alterations and impede the transport of proteins and organelles such as brain-derived neurotrophic factor (BDNF) and mitochondria that are required for synaptic function. Meanwhile, intraneuronal accumulation of Aβ precedes its extracellular deposition and is also associated with synaptic dysfunction in AD. However, the links between intracellular Aβ, spine alteration, and mechanisms that support synaptic maintenance such as organelle trafficking are poorly understood. Results We compared the effects of wild-type and Osaka (E693Δ)-mutant amyloid precursor proteins: the former secretes Aβ into extracellular space and the latter accumulates Aβ oligomers within cells. First we investigated the effects of intracellular Aβ oligomers on dendritic spines in primary neurons and their tau-dependency using tau knockout neurons. We found that intracellular Aβ oligomers caused a reduction in mushroom, or mature spines, independently of tau. We also found that intracellular Aβ oligomers significantly impaired the intracellular transport of BDNF, mitochondria, and recycling endosomes: cargoes essential for synaptic maintenance. A reduction in BDNF transport by intracellular Aβ oligomers was also observed in tau knockout neurons. Conclusions Our findings indicate that intracellular Aβ oligomers likely contribute to early synaptic pathology in AD and argue against the consensus that Aβ-induced spine loss and transport defects require tau

Tight Glycemic Control After Pediatric Cardiac Surgery in High-Risk Patient Populations

Background—Our previous randomized, clinical trial showed that postoperative tight glycemic control (TGC) for children undergoing cardiac surgery did not reduce the rate of health care–associated infections compared with standard care (STD). Heterogeneity of treatment effect may exist within this population. Methods and Results—We performed a post hoc exploratory analysis of 980 children from birth to 36 months of age at the time of cardiac surgery who were randomized to postoperative TGC or STD in the intensive care unit. Significant interactions were observed between treatment group and both neonate (age ≤30 days; P=0.03) and intraoperative glucocorticoid exposure (P=0.03) on the risk of infection. The rate and incidence of infections in subjects ≤60 days old were significantly increased in the TGC compared with the STD group (rate: 13.5 versus 3.7 infections per 1000 cardiac intensive care unit days, P=0.01; incidence: 13% versus 4%, P=0.02), whereas infections among those \u3e60 days of age were significantly reduced in the TGC compared with the STD group (rate: 5.0 versus 14.1 infections per 1000 cardiac intensive care unit days, P=0.02; incidence: 2% versus 5%, P=0.03); the interaction of treatment group by age subgroup was highly significant (P=0.001). Multivariable logistic regression controlling for the main effects revealed that previous cardiac surgery, chromosomal anomaly, and delayed sternal closure were independently associated with increased risk of infection. Conclusions—This exploratory analysis demonstrated that TGC may lower the risk of infection in children \u3e60 days of age at the time of cardiac surgery compared with children receiving STD. Meta-analyses of past and ongoing clinical trials are necessary to confirm these findings before clinical practice is altered

Exploring Individual and Structural Factors Associated with Employment Among Young Transgender Women of Color Using a No-Cost Transgender Legal Resource Center

Purpose: The purpose of this study was to explore individual and structural factors associated with employment among young transgender women (TW) of color. Methods: Sixty-five trans women of color were recruited from the Transgender Legal Defense and Education Fund to complete a 30-min interviewer-assisted survey assessing sociodemographics, housing, workplace discrimination, job-seeking self-efficacy, self-esteem, perceived public passability, and transactional sex work. Results: Logistic regression models revealed that stable housing (structural factor) and job-seeking self-efficacy (individual factor) were significantly associated with currently being employed. Conclusion: Our findings underscore the need for multilevel approaches to assist TW of color gain employment

Epidemiology, genetics, and subtyping of preserved ratio impaired spirometry (PRISm) in COPDGene.

BackgroundPreserved Ratio Impaired Spirometry (PRISm), defined as a reduced FEV1 in the setting of a preserved FEV1/FVC ratio, is highly prevalent and is associated with increased respiratory symptoms, systemic inflammation, and mortality. Studies investigating quantitative chest tomographic features, genetic associations, and subtypes in PRISm subjects have not been reported.MethodsData from current and former smokers enrolled in COPDGene (n = 10,192), an observational, cross-sectional study which recruited subjects aged 45-80 with ≥10 pack years of smoking, were analyzed. To identify epidemiological and radiographic predictors of PRISm, we performed univariate and multivariate analyses comparing PRISm subjects both to control subjects with normal spirometry and to subjects with COPD. To investigate common genetic predictors of PRISm, we performed a genome-wide association study (GWAS). To explore potential subgroups within PRISm, we performed unsupervised k-means clustering.ResultsThe prevalence of PRISm in COPDGene is 12.3%. Increased dyspnea, reduced 6-minute walk distance, increased percent emphysema and decreased total lung capacity, as well as increased segmental bronchial wall area percentage were significant predictors (p-value <0.05) of PRISm status when compared to control subjects in multivariate models. Although no common genetic variants were identified on GWAS testing, a significant association with Klinefelter's syndrome (47XXY) was observed (p-value < 0.001). Subgroups identified through k-means clustering include a putative "COPD-subtype", "Restrictive-subtype", and a highly symptomatic "Metabolic-subtype".ConclusionsPRISm subjects are clinically and genetically heterogeneous. Future investigations into the pathophysiological mechanisms behind and potential treatment options for subgroups within PRISm are warranted.Trial registrationClinicaltrials.gov Identifier: NCT000608764

Athena\u27s Prism - A Diplomatic Strategy Role Playing Simulation for Generating Ideas and Exploring Alternatives

Intelligence analysts must clear at least three hurdles to get good product out the door: cognitive biases, social biases and self-imposed organizational impediments. Others (e.g., Gilovich, et al., Heuer, and Kahneman and Tversky), explain the cognitive processes that can help or trip us. A less well mapped set of dangers arises in the social dynamics of communicating tasking, working with other analysts, editing and customer interaction. Finally, the mere fact of a unit\u27s published record creates analytic inertia - an argument at rest tends to stay at rest and one in motion (i.e., ambiguous or uncertain) tends to stay in motion. (A variation of this includes groupthink.

Licensing an assured isolation facility for low-level radioactive waste. Volume 2: Recommendations on the content and review of an application

This report provides a detailed set of proposed criteria and guidance for the preparation of a license application for an assured isolation facility (AIF). The report is intended to provide a detailed planning basis upon which a prospective applicant may begin pre-licensing discussions with the Nuclear Regulatory Commission and initiate development of a license application. The report may also be useful to the NRC or to state regulatory agencies that may be asked to review such an application. Volume 1 of this report provides background information, and describes the licensing approach and methodology. Volume 2 identifies specific information that is recommended for inclusion in a license application

Висновок експерта в криміналістичних технологіях

Досліджується висновок експерта в аспекті технологій експертних та технологій слідчих. Встановлюються умови технологічності цього процесуального документу. Конкретизуються критерії та дії експерта з забезпечення процесуальних вимог при складанні висновку за результатами проведених досліджень, та операції з оцінки висновку експерта слідчим.Исследуется заключение эксперта в аспекте технологий экспертных и технологий следственных. Устанавливаются условия технологичности этого процессуального документа. Конкретизируются критерии и действия эксперта по обеспечению процессуальных требований при составлении заключения по результатам проведенных исследований, и операции по оценке заключения эксперта следователем.The expert's conclusion is investigated in the aspect of expertise technologies and investigation technologies . The conditions for manufacturability of this procedural document are established. The criteria and expert's actions providing the procedural requirements while preparation of the report on the research results, and the operation on the assessment of the expert conclusion by investigator are concretized