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OBJECTIVES: The aim of this study was to define a smoking cessation ‘cascade’ among USA women with and without HIV and examine differences by sociodemographic characteristics. DESIGN: An observational cohort study using data from smokers participating in the Women’s Interagency HIV Study between 2014 and 2019. METHODS: We followed 1165 women smokers with and without HIV from their first study visit in 2014 or 2015 until an attempt to quit smoking within approximately 3 years of follow-up, initial cessation (i.e. no restarting smoking within approximately 6 months of a quit attempt), and sustained cessation (i.e. no restarting smoking within approximately 12 months of a quit attempt). Using the Aalen-Johansen estimator, we estimated the cumulative probability of achieving each step, accounting for the competing risk of death. RESULTS: Forty-five percent of smokers attempted to quit, 27% achieved initial cessation, and 14% achieved sustained cessation with no differences by HIV status. Women with some post-high school education were more likely to achieve each step than those with less education. Outcomes did not differ by race. Thirty-six percent [95% confidence interval (95% CI): 31–42] of uninsured women attempted to quit compared with 47% (95% CI: 44–50) with Medicaid and 49% (95% CI: 41–59) with private insurance. CONCLUSION: To decrease smoking among USA women with and without HIV, targeted, multistage interventions, and increased insurance coverage are needed to address shortfalls along this cascade.

Inverse probability weights are increasingly used in epidemiological analysis, and estimation and application of weights to address a single bias are well discussed in the literature. Weights to address multiple biases simultaneously (i.e. a combination of weights) have almost exclusively been discussed related to marginal structural models in longitudinal settings where treatment weights (estimated first) are combined with censoring weights (estimated second). In this work, we examine two examples of combined weights for confounding and missingness in a time-fixed setting in which outcome or confounder data are missing, and the estimand is the marginal expectation of the outcome under a time-fixed treatment. We discuss the identification conditions, construction of combined weights and how assumptions of the missing data mechanisms affect this construction. We use a simulation to illustrate the estimation and application of the weights in the two examples. Notably, when only outcome data are missing, construction of combined weights is straightforward; however, when confounder data are missing, we show that in general we must follow a specific estimation procedure which entails first estimating missingness weights and then estimating treatment probabilities from data with missingness weights applied. However, if treatment and missingness are conditionally independent, then treatment probabilities can be estimated among the complete cases.

Background Understanding advances in the care and treatment of adults with HIV as well as remaining gaps requires comparing differences in mortality between persons entering care for HIV and the general population. Objective To assess the extent to which mortality among persons entering HIV care in the United States is elevated over mortality among matched persons in the general U.S. population and trends in this difference over time. Design Observational cohort study. Setting Thirteen sites from the U.S. North American AIDS Cohort Collaboration on Research and Design. Participants 82 766 adults entering HIV clinical care between 1999 and 2017 and a subset of the U.S. population matched on calendar time, age, sex, race/ethnicity, and county using U.S. mortality and population data compiled by the National Center for Health Statistics. Measurements Five-year all-cause mortality, estimated using the Kaplan-Meier estimator of the survival function. Results Overall 5-year mortality among persons entering HIV care was 10.6%, and mortality among the matched U.S. population was 2.9%, for a difference of 7.7 (95% CI, 7.4 to 7.9) percentage points. This difference decreased over time, from 11.1 percentage points among those entering care between 1999 and 2004 to 2.7 percentage points among those entering care between 2011 and 2017. Limitation Matching on available covariates may have failed to account for differences in mortality that were due to sociodemographic factors rather than consequences of HIV infection and other modifiable factors. Conclusion Mortality among persons entering HIV care decreased dramatically between 1999 and 2017, although those entering care remained at modestly higher risk for death in the years after starting care than comparable persons in the general U.S. population. Primary funding source National Institutes of Health.

BackgroundMortality among adults with human immunodeficiency virus (HIV) remains elevated over those in the US general population, even in the years after entry into HIV care. We explore whether the elevation in 5-year mortality would have persisted if all adults with HIV had initiated antiretroviral therapy within 3 months of entering care.MethodsAmong 82 766 adults entering HIV care at North American AIDS Cohort Collaboration clinical sites in the United States, we computed mortality over 5 years since entry into HIV care under observed treatment patterns. We then used inverse probability weights to estimate mortality under universal early treatment. To compare mortality with those for similar individuals in the general population, we used National Center for Health Statistics data to construct a cohort representing the subset of the US population matched to study participants on key characteristics.ResultsFor the entire study period (1999–2017), the 5-year mortality among adults with HIV was 7.9% (95% confidence interval [CI]: 7.6%–8.2%) higher than expected based on the US general population. Under universal early treatment, the elevation in mortality for people with HIV would have been 7.2% (95% CI: 5.8%–8.6%). In the most recent calendar period examined (2011–2017), the elevation in mortality for people with HIV was 2.6% (95% CI: 2.0%–3.3%) under observed treatment patterns and 2.1% (.0%–4.2%) under universal early treatment.ConclusionsExpanding early treatment may modestly reduce, but not eliminate, the elevation in mortality for people with HIV.

Segmentation of brain tissue types from diffusion MRI (dMRI) is an important task, required for quantification of brain microstructure and for improving tractography. Current dMRI segmentation is mostly based on anatomical MRI (e.g., T1- and T2-weighted) segmentation that is registered to the dMRI space. However, such inter-modality registration is challenging due to more image distortions and lower image resolution in dMRI as compared with anatomical MRI. In this study, we present a deep learning method for diffusion MRI segmentation, which we refer to as DDSeg. Our proposed method learns tissue segmentation from high-quality imaging data from the Human Connectome Project (HCP), where registration of anatomical MRI to dMRI is more precise. The method is then able to predict a tissue segmentation directly from new dMRI data, including data collected with different acquisition protocols, without requiring anatomical data and inter-modality registration. We train a convolutional neural network (CNN) to learn a tissue segmentation model using a novel augmented target loss function designed to improve accuracy in regions of tissue boundary. To further improve accuracy, our method adds diffusion kurtosis imaging (DKI) parameters that characterize non-Gaussian water molecule diffusion to the conventional diffusion tensor imaging parameters. The DKI parameters are calculated from the recently proposed mean-kurtosis-curve method that corrects implausible DKI parameter values and provides additional features that discriminate between tissue types. We demonstrate high tissue segmentation accuracy on HCP data, and also when applying the HCP-trained model on dMRI data from other acquisitions with lower resolution and fewer gradient directions.

OBJECTIVES Within the past several years, innovative communication tools have been established as viable quality improvement mechanisms in health care. Meanwhile, the economic and societal burdens of hospital-acquired conditions (HACs) continue to rise. Various interventions have been attempted to reduce HACs. This study evaluated the effectiveness of a communication tool in reducing risk factors for HACs. METHODS A communication tool aimed at reducing HAC risk factors was developed by an interdisciplinary team of physicians and nurses and tested in a simple before-after quality improvement study. It included 8 components: ambulation/fall risk, blood glucose greater than 200 mg/dL, central venous catheters, deep venous thrombosis prophylaxis, erosions of the skin/dermal ulcers, Foley/urinary catheters, got communication, and heart monitor/telemetry. This communication tool facilitated multidisciplinary communication. The nurses completed it nightly, and the physicians reviewed the communication tool each morning and, when appropriate, addressed components of care that were out of compliance with best practices. RESULTS The use of the ABCs of Hospitalized Patients Communication Tool led to daily improvements with reduction in the percentage of patients with blood glucose greater than 200 mg/dL from 43.3% to 35.1%, reduction in the use of central venous catheters from 8.2% to 1.0% of patients, increase in the use of chemical deep venous thrombosis prophylaxis from 45.4% of patients to 56.7%, reduction in the use of urinary catheters from 27.6% to 13.2%, and decrease in use of telemetry from 67.5% to 55.1%. All of the results have P < 0.05. These improvements were sustained over time. CONCLUSIONS Implementation of a multidisciplinary communication tool serves as a simple, resource-conscious, and customizable instrument to reduce the risk factors for developing HACs. This communication tool can be easily disseminated and used by other institutions.

Illustrations of the g-computation algorithm to evaluate population average treatment and intervention effects have been predominantly implemented in settings with complete exposure information. Thus, worked examples of approaches to handle missing data in this causal framework are needed to facilitate wider use of these estimators. We illustrate two-stage g-computation estimators that leverage partially observed information on the full study sample and complete exposure information on a subset to estimate causal effects. In a hypothetical cohort of 1,623 human immunodeficiency virus (HIV)-positive women with 30% complete opioid prescription information, we illustrate a two-stage extrapolation g-computation estimator for the average treatment effect of shorter or longer duration opioid prescriptions; we further illustrate two-stage inverse probability weighting and imputation g-computation estimators for the average intervention effect of shortening the duration of prescriptions relative to the status quo. Two-stage g-computation estimators approximated the true risk differences for the population average treatment and intervention effects while g-computation fit to the subset of complete cases was biased. In 10,000 Monte Carlo simulations, two-stage approaches considerably reduced bias and mean squared error and improved the coverage of 95% confidence limits. Although missing data threaten validity and precision, two-stage g-computation designs offer principled approaches to handling missing information.

The first step of SARS-CoV-2 infection is binding of the spike protein's receptor binding domain to the host cell's ACE2 receptor on the plasma membrane. Here, we have generated a versatile imaging probe using recombinant Spike receptor binding domain conjugated to fluorescent quantum dots (QDs). This probe is capable of engaging in energy transfer quenching with ACE2-conjugated gold nanoparticles to enable monitoring of the binding event in solution. Neutralizing antibodies and recombinant human ACE2 blocked quenching, demonstrating a specific binding interaction. In cells transfected with ACE2-GFP, we observed immediate binding of the probe on the cell surface followed by endocytosis. Neutralizing antibodies and ACE2-Fc fully prevented binding and endocytosis with low nanomolar potency. Importantly, we will be able to use this QD nanoparticle probe to identify and validate inhibitors of the SARS-CoV-2 Spike and ACE2 receptor binding in human cells. This work enables facile, rapid, and high-throughput cell-based screening of inhibitors for coronavirus Spike-mediated cell recognition and entry.

International audience; In recent years, substantial evidence has emerged to suggest that spreading of pathological proteins contributes to disease pathology in numerous neurodegenerative disorders. Work from our laboratory and others have shown that, despite its strictly genetic nature, Huntington's disease (HD) may be another condition in which this mechanism contributes to pathology. In this study, we set out to determine if the mutant huntingtin protein (mHTT) present in post-mortem brain tissue derived from HD patients can induce pathology in mice and/or non-human primates. For this, we performed three distinct sets of experiments where homogenates were injected into the brains of adult a) Wild-type (WT) and b) BACHD mice or c) non-human primates. Neuropathological assessments revealed that, while changes in the endogenous huntingtin were not apparent, mHTT could spread between cellular elements and brain structures. Furthermore, behavioural differences only occurred in the animal model of HD which already overexpressed mHTT. Taken together, our results indicate that mHTT derived from human brains has only a limited capacity to propagate between cells and does not depict prion-like characteristics. This contrasts with recent work demonstrating that other forms of mHTT - such as fibrils of a pathological polyQ length or fibroblasts and induced pluripotent stem cells derived from HD cases - can indeed disseminate disease throughout the brain in a prion-like fashion.

Background There are few methodologic examples of how multiple causes of death may be summarized in cause-specific mortality analyses to address limitations of attributing death to a single underlying cause. We propose a cause-of-death weighting approach to estimate the set of risk functions of specific causes of mortality using both underlying and contributing cause-of-death information. Methods We constructed weights according to a user-specified function. Using data from four southern US human immunodeficiency virus (HIV) clinics, we constructed a cause of death-weighted Aalen-Johansen estimator of the cumulative incidence function to estimate risks of five specific causes of mortality in the full sample and by injection drug use history. Results Among 7740 HIV-positive patients initiating antiretroviral therapy between 1999 and 2014, the 8-year risk of all-cause mortality was 17.5% [95% confidence interval (CI) = 16.5, 18.4]. The cause of death-weighted risk of HIV-related mortality was 6.7% (95% CI = 6.0, 7.3) and accounted for 39% (95% CI = 35, 42) of total mortality risk. This compared with 10.2% (95% CI = 9.2, 11.2) using only the underlying cause, in which case HIV-related deaths accounted for nearly 60% of total mortality risk. The proportion attributable to cardiovascular disease among those whose HIV risk factor was injection drug use was twice as high using cause-of-death weights compared with only the underlying cause (8%; 95% CI = 5, 11 vs 4%; 95% CI = 1, 6). Conclusion Using cause of death-weighted estimators to incorporate multiple causes of death may yield different conclusions regarding the importance of certain causes of mortality. See video abstract: http://links.lww.com/EDE/B706.

International audience; In order to model various aspects of Huntington's disease (HD) pathology, in particular protein spread, we administered adeno-associated virus (AAV) expressing green fluorescent protein (GFP) or GFP coupled to HTT-Exon1 (19Q or 103Q) to the central nervous system of adult wild-type (WT) mice and non-human primates. All animals underwent behavioral testing and post-mortem analyses to determine the long-term consequences of AAV injection. Both mice and non-human primates demonstrated behavioral changes at 2-3 weeks post-surgery. In mice, these changes were absent after 3 months while in non-human primates, they persisted in the majority of tested animals. Post-mortem analysis revealed that spreading of the aggregates was limited, although the virus did spread between synaptically-connected brain regions. Despite the small degree of spreading, the presence of mHTT generated changes in endogenous huntingtin (HTT) levels in both models. Together, these results suggest that viral expression of mHTTExon1 can induce spreading and seeding of HTT in both mice and non-human primates.

We demonstrate the viability of using ultra-thin sheets of microbially grown nanocellulose to build functional medical sensors. Microbially grown nanocellulose is an interesting alternative to plastics, as it is hydrophilic, biocompatible, porous, and hydrogen bonding, thereby allowing the potential development of new application routes. Exploiting the distinguishing properties of this material enables us to develop solution-based processes to create nanocellulose printed circuit boards, allowing a variety of electronics to be mounted onto our nanocellulose. As proofs of concept, we have demonstrated applications in medical sensing such as heart rate monitoring and temperature sensing&mdash, potential applications fitting the wide-ranging paradigm of a future where the Internet of Things is dominant.

Diabetic macular edema (DME) and retina vein occlusion (RVO) are macular diseases in which central photoreceptors are affected due to pathological accumulation of fluid. Optical coherence tomography allows to visually assess and evaluate photoreceptor integrity, whose alteration has been observed as an important biomarker of both diseases. However, the manual quantification of this layered structure is challenging, tedious and time-consuming. In this paper we introduce a deep learning approach for automatically segmenting and characterising photoreceptor alteration. The photoreceptor layer is segmented using an ensemble of four different convolutional neural networks. En-face representations of the layer thickness are produced to characterize the photoreceptors. The pixel-wise standard deviation of the score maps produced by the individual models is also taken to indicate areas of photoreceptor abnormality or ambiguous results. Experimental results showed that our ensemble is able to produce results in pair with a human expert, outperforming each of its constitutive models. No statistically significant differences were observed between mean thickness estimates obtained from automated and manually generated annotations. Therefore, our model is able to reliable quantify photoreceptors, which can be used to improve prognosis and managment of macular diseases.

The chemical identity of RNA molecules beyond the four standard ribonucleosides has fascinated scientists since pseudouridine was characterized as the "fifth" ribonucleotide in 1951. Since then, the ever-increasing number and complexity of modified ribonucleosides have been found in viruses and throughout all three domains of life. Such modifications can be as simple as methylations, hydroxylations, or thiolations, complex as ring closures, glycosylations, acylations, or aminoacylations, or unusual as the incorporation of selenium. While initially found in transfer and ribosomal RNAs, modifications also exist in messenger RNAs and noncoding RNAs. Modifications have profound cellular outcomes at various levels, such as altering RNA structure or being essential for cell survival or organism viability. The aberrant presence or absence of RNA modifications can lead to human disease, ranging from cancer to various metabolic and developmental illnesses such as Hoyeraal-Hreidarsson syndrome, Bowen-Conradi syndrome, or Williams-Beuren syndrome. In this review article, we summarize the characterization of all 143 currently known modified ribonucleosides by describing their taxonomic distributions, the enzymes that generate the modifications, and any implications in cellular processes, RNA structure, and disease. We also highlight areas of active research, such as specific RNAs that contain a particular type of modification as well as methodologies used to identify novel RNA modifications. This article is categorized under: RNA Processing > RNA Editing and Modification.

Although a fundamental understanding of the pathogenicity of most biothreat agents has been elucidated and available treatments have increased substantially over the past decades, they still represent a significant public health threat in this age of (bio)terrorism, indiscriminate warfare, pollution, climate change, unchecked population growth, and globalization. The key step to almost all prevention, protection, prophylaxis, post-exposure treatment, and mitigation of any bioagent is early detection. Here, we review available methods for detecting bioagents including pathogenic bacteria and viruses along with their toxins. An introduction placing this subject in the historical context of previous naturally occurring outbreaks and efforts to weaponize selected agents is first provided along with definitions and relevant considerations. An overview of the detection technologies that find use in this endeavor along with how they provide data or transduce signal within a sensing configuration follows. Current "gold" standards for biothreat detection/diagnostics along with a listing of relevant FDA approved in vitro diagnostic devices is then discussed to provide an overview of the current state of the art. Given the 2014 outbreak of Ebola virus in Western Africa and the recent 2016 spread of Zika virus in the Americas, discussion of what constitutes a public health emergency and how new in vitro diagnostic devices are authorized for emergency use in the U.S. are also included. The majority of the Review is then subdivided around the sensing of bacterial, viral, and toxin biothreats with each including an overview of the major agents in that class, a detailed cross-section of different sensing methods in development based on assay format or analytical technique, and some discussion of related microfluidic lab-on-a-chip/point-of-care devices. Finally, an outlook is given on how this field will develop from the perspective of the biosensing technology itself and the new emerging threats they may face.

PurposeThe purpose of the present study is to develop fast automated quantification of retinal fluid in optical coherence tomography (OCT) image sets.MethodsWe developed an image analysis pipeline tailored towards OCT images that consists of five steps for binary retinal fluid segmentation. The method is based on feature extraction, pre-segmention, dimension reduction procedures, and supervised learning tools.ResultsFluid identification using our pipeline was tested on two separate patient groups: one associated to neovascular age-related macular degeneration, the other showing diabetic macular edema. For training and evaluation purposes, retinal fluid was annotated manually in each cross-section by human expert graders of the Vienna Reading Center. Compared with the manual annotations, our pipeline yields good quantification, visually and in numbers.ConclusionsBy demonstrating good automated retinal fluid quantification, our pipeline appears useful to expert graders within their current grading processes. Owing to dimension reduction, the actual learning part is fast and requires only few training samples. Hence, it is well-suited for integration into actual manufacturer's devices, further improving segmentation by its use in daily clinical life.

Inpatient psychotherapy might trigger adverse effects among others due to short but intensive treatment. Thus, in this pilot study, certain adverse effects of the multidisciplinary inpatient Cognitive Behavioral Analysis System of Psychotherapy (CBASP) for treatment-resistant chronically depressed patients as well as their relationship to treatment outcome (response-, remission-, and relapse-rates) are examined.50 patients with treatment-resistant and chronic depression completed the structured 12-weeks inpatient treatment program. Adverse effects were assessed by 1) deterioration of depressive symptoms (measured by the Hamilton Depression Rating Scale, HDRS) at discharge and 2) a self-report questionnaire measuring Adverse Effects of Inpatient Psychotherapy (ADEFIP), which were filled out 6 to 12 months after discharge by the patients.After 12 weeks of treatment, 84% could be classified as responder, of whom 44% fulfilled the remission criterion. 16% were Non-Responder. According to HDRS, none of the patients showed objective deterioration of the depressive symptoms. Six months after discharge, 40% of the responders suffered from relapse. Concerning the ADEFIP, 66% of the patients reported transient deterioration of symptoms. These patients were less likely to achieve remission. Over 50% reported interpersonal conflicts with treatment team members or other patients without any relation to outcome. Finally, more than half of the patients reported significant changes in social relationships after discharge. These patients were less likely to relapse. Overall, 94% of the patients reported at least one of the in this study assessed adverse effects.Despite some limitations, this pilot study suggests that the CBASP inpatient program could indeed cause adverse effects. However, only subjective transient deterioration appeared to have a negative impact on the individual treatment outcome in the short-term. Results encourage further research concerning adverse treatment effects in the context of short- and long-term treatment outcome investigating how relevant adverse effects are.Stationäre Psychotherapien können u. a. aufgrund der kurzen, aber intensiven Behandlung Nebenwirkungen auslösen. In der vorliegenden Pilotstudie wurden daher bestimmte Nebenwirkungen eines multidisziplinären, stationären CBASP-Programms (Cognitive Behavioral Analysis System of Psychotherapy) für Patienten mit therapieresistenter chronischer Depression sowie deren Zusammenhang mit dem Therapieerfolg (Response-, Remissions- und Rückfallraten) untersucht.50 Patienten mit therapieresistenter chronischer Depression nahmen an einem strukturierten 12-wöchigen, stationären CBASP-Programm teil. Nebenwirkungen wurden erfasst durch 1) eine Verschlechterung der depressiven Symptomatik im Fremdbericht bei Entlassung (Wert auf der Hamilton Depression Rating Scale, HDRS24) und 2) einem Selbstbeurteilungsfragebogen zur Erfassung von Nebenwirkungen in der stationären Psychotherapie (NESTAP), welcher von Patienten 6–12 Monate nach Entlassung ausgefüllt wurde.Nach der 12-wöchigen Behandlung konnten 84% der Patienten als Responder klassifiziert werden, von denen 44% das Remissionskriterium erfüllten. 16% waren Non-Responder, wobei kein Patient gemäß HDRS eine fremdbeurteilte Verschlechterung der depressiven Symptomatik zeigte. Sechs Monate nach Entlassung erlitten 40% der Responder einen Rückfall. Im NESTAP gaben 66% der Patienten an, dass sich während der Behandlung ihre Symptomatik vorübergehend verschlechtert habe. Bei diesen Patienten zeigte sich eine geringere Remissionsrate. Über 50% berichteten von interpersonellen Konflikten mit Teammitgliedern oder anderen Patienten, wobei kein Zusammenhang mit dem Therapieerfolg nachweisbar war. Schließlich berichteten über 50% der Patienten, nach Entlassung Veränderungen in sozialen Beziehungen erlebt zu haben. Diese Patienten hatten seltener einen Rückfall. Insgesamt gaben 94% aller Patienten an, mindestens eine der in dieser Studie erhobenen Nebenwirkungen erlebt zu haben.Diese Pilotstudie gibt trotz einiger Limitationen erste Hinweise, dass das stationäre CBASP-Programm Nebenwirkungen verursacht, wobei nur eine subjektiv wahrgenommene vorübergehende Verschlechterung den individuellen Therapieerfolg kurzfristig negativ zu beeinflussen scheint. Die Ergebnisse sprechen dafür, Nebenwirkungen im Kontext des kurz- und langfristigen Therapieerfolges zu untersuchen, um herauszufinden, wie relevant Nebenwirkungen sind.

Parkinson's disease (PD) is a progressive neurological disorder characterised by aberrant accumulation of insoluble proteins, including alpha-synuclein, and a loss of dopaminergic neurons in the substantia nigra. The extended neurodegeneration leads to a drop of striatal dopamine levels responsible for disabling motor and non-motor impairments. Although the causes of the disease remain unclear, it is well accepted among the scientific community that the disorder may also have a genetic component. For that reason, the number of genetically engineered animal models has greatly increased over the past two decades, ranging from invertebrates to more complex organisms such as mice and rats. This trend is growing as new genetic variants associated with the disease are discovered. The EU Joint Programme - Neurodegenerative Disease Research (JPND) has promoted the creation of an online database aiming at summarising the different features of experimental models of Parkinson's disease. This review discusses available genetic models of PD and the extent to which they adequately mirror the human pathology and reflects on future development and uses of genetically engineered experimental models for the study of PD.

Importance Depression commonly affects adults with HIV and complicates the management of HIV. Depression among individuals with HIV tends to be chronic and cyclical, but the association of this chronicity with HIV outcomes (and the related potential for screening and intervention to shorten depressive episodes) has received little attention. Objective To examine the association between increased chronicity of depression and multiple HIV care continuum indicators (HIV appointment attendance, treatment failure, and mortality). Design, Setting, and Participants The study comprised an observational clinical cohort of 5927 patients with 2 or more assessments of depressive severity who were receiving HIV primary care at 6 geographically dispersed US academic medical centers from September 22, 2005, to August 6, 2015. Main Outcomes and Measures Missing a scheduled HIV primary care visit, detectable HIV RNA viral load (≥75 copies/mL), and all-cause mortality. Consecutive depressive severity measures were converted into a time-updated measure: percentage of days with depression (PDD), following established methods for determining depression-free days. Results During 10 767 person-years of follow-up, the 5927 participants (5000 men, 926 women, and 1 intersex individual; median age, 44 years [range, 35-50 years]) had a median PDD of 14% (interquartile range, 0%-48%). During follow-up, 10 361 of 55 040 scheduled visits (18.8%) were missed, 6191 of 28 455 viral loads (21.8%) were detectable, and the mortality rate was 1.5 deaths per 100 person-years. Percentage of days with depression showed a dose-response relationship with each outcome. Each 25% increase in PDD led to an 8% increase in the risk of missing a scheduled appointment (risk ratio, 1.08; 95% CI, 1.05-1.11), a 5% increase in the risk of a detectable viral load (risk ratio, 1.05; 95% CI, 1.01-1.09), and a 19% increase in the mortality hazard (hazard ratio, 1.19; 95% CI, 1.05-1.36). These estimates imply that, compared with patients who spent no follow-up time with depression (PDD, 0%), those who spent the entire follow-up time with depression (PDD, 100%) faced a 37% increased risk of missing appointments (risk ratio, 1.37; 95% CI, 1.22-1.53), a 23% increased risk of a detectable viral load (risk ratio, 1.23; 95% CI, 1.06-1.43), and a doubled mortality rate (hazard ratio, 2.02; 95% CI, 1.20-3.42). Conclusions and Relevance Greater chronicity of depression increased the likelihood of failure at multiple points along the HIV care continuum. Even modest increases in the proportion of time spent with depression led to clinically meaningful increases in negative outcomes. Clinic-level trials of protocols to promptly identify and appropriately treat depression among adults living with HIV should be conducted to understand the effect of such protocols on shortening the course and preventing the recurrence of depressive illness and improving clinical outcomes.

Background Research linking depression to mortality among people living with human immunodeficiency virus (PLWH) has largely focused on binary "always vs never" characterizations of depression. However, depression is chronic and is likely to have cumulative effects on mortality over time. Quantifying depression as a cumulative exposure may provide a better indication of the clinical benefit of enhanced depression treatment protocols delivered in HIV care settings. Methods Women living with HIV (WLWH), naive to antiretroviral therapy, from the Women's Interagency HIV Study were followed from their first visit in or after 1998 for up to 10 semiannual visits (5 years). Depressive symptoms were assessed using the Center for Epidemiologic Studies Depression (CES-D) scale. An area-under-the-curve approach was used to translate CES-D scores into a time-updated measure of cumulative days with depression (CDWD). We estimated the effect of CDWD on all-cause mortality using marginal structural Cox proportional hazards models. Results Overall, 818 women contributed 3292 woman-years over a median of 4.8 years of follow-up, during which the median (interquartile range) CDWD was 366 (97-853). Ninety-four women died during follow-up (2.9 deaths/100 woman-years). A dose-response relationship was observed between CDWD and mortality. Each additional 365 days spent with depression increased mortality risk by 72% (hazard ratio, 1.72; 95% confidence interval, 1.34-2.20). Conclusions In this sample of WLWH, increased CDWD elevated mortality rates in a dose-response fashion. More frequent monitoring and enhanced depression treatment protocols designed to reduce CDWD may interrupt the accumulation of mortality risk among WLWH.

Although a causative role of α-synuclein (α-syn) is well established in Parkinson’s disease pathogenesis, available animal models of synucleinopathy do not replicate the full range of cellular and behavioral changes characteristic of the human disease. This study was designed to generate a more faithful model of Parkinson’s disease by injecting human α-syn fibril seeds into the rat substantia nigra (SN), in combination with adenoassociated virus (AAV)-mediated overexpression of human α-syn, at levels that, by themselves, are unable to induce acute dopamine (DA) neurodegeneration. We show that the ability of human α-syn fibrils to trigger Lewy-like α-synuclein pathology in the affected DA neurons is dramatically enhanced in the presence of elevated levels of human α-syn. This synucleinopathy was fully developed already 10 days after fibril injection, accompanied by progressive degeneration of dopaminergic neurons in SN, neuritic swelling, reduced striatal DA release, and impaired motor behavior. Moreover, a prominent inflammatory response involving both activation of resident microglia and infiltration of CD4+ and CD8+ T lymphocytes was observed. Hypertrophic microglia were found to enclose or engulf cells and processes containing Lewy-like α-syn aggregates. α-Syn aggregates were also observed inside these cells, suggesting transfer of phosphorylated α-syn from the affected nigral neurons. The nigral pathology triggered by fibrils in combination with AAV-mediated overexpression of α-syn reproduced many of the cardinal features of the human disease. The short time span and the distinct sequence of pathological and degenerative changes make this combined approach attractive as an experimental model for the assessment of neuroprotective and disease-modifying strategies.

Poor retention in care is common among HIV-positive adults in sub-Saharan Africa settings and remains a key barrier to HIV management. We quantify the associations of disclosure of HIV status and referral to disclosure counseling with successful retention in care using data from three Cameroon clinics participating in the Phase 1 International epidemiologic Databases to Evaluate AIDS Central Africa cohort. Of 1646 patients newly initiating antiretroviral therapy between January 2008 and January 2011, 43% were retained in care following treatment initiation. Self-disclosure of HIV status to at least one person prior to treatment initiation was associated with a minimal increase in the likelihood of being retained in care (risk ratio [RR] = 1.14; 95% confidence interval (CI): 0.94, 1.38). However, referral to disclosure counseling was associated with a moderate increase in retention (RR = 1.37; 95% CI: 1.21, 1.55) and was not significantly modified by prior disclosure status (p = .3). Our results suggest that while self-disclosure may not significantly improve retention among patients receiving care at these Cameroon sites, counseling services may play an important role regardless of prior disclosure status.

One of the well-recognized problems of long-term l-3,4-dihydroxyphenylalanine (l-DOPA) therapy in the treatment of Parkinson’s disease is the development of l-DOPA induced dyskinesia. These abnormal movements cause significant disability and narrow the therapeutic window of l-DOPA. Cell transplantation is one of the most promising upcoming therapies for the treatment of Parkinson’s disease, and may help alleviate or avoid l-DOPA-induced dyskinesia. However, the more recently acknowledged phenomenon of graft-induced dyskinesia is posing a major obstacle to the success of this treatment. This motor side-effect closely resembles abnormal movements induced by chronic l-DOPA treatment, yet they remain after withdrawal of the medication indicating their origins lie in the transplant. In this review, we compare these two therapy-induced adverse effects, from the way they manifest in patients to the possible mechanisms underlying their development.

People can get oral cancers from UV (290-400 nm) exposures. Besides high outdoor UV exposures, high indoor UV exposures to oral tissues can occur when consumers use UV-emitting tanning devices to either tan or whiten their teeth. We compared the carcinogenic risks of skin to oral tissue cells after UVB (290-320 nm) exposures using commercially available 3D-engineered models for human skin (EpiDerm™), gingival (EpiGing™) and oral (EpiOral™) tissues. To compare the relative carcinogenic risks, we investigated the release of cytokines, initial DNA damage in the form of cyclobutane pyrimidine dimers (CPDs), repair of CPDs and apoptotic cell numbers. We measured cytokine release using cytometric beads with flow cytometry and previously developed a fluorescent immunohistochemical assay to quantify simultaneously CPD repair rates and apoptotic cell numbers. We found that interleukin-8 (IL-8) release and the initial CPDs are significantly higher, whereas the CPD repair rates and apoptotic cell numbers are significantly lower for oral compared with skin tissue cells. Thus, the increased release of the inflammatory cytokine IL-8 along with inefficient CPD repair and decreased death rates for oral compared with skin tissue cells suggests that mutations are accumulating in the surviving population of oral cells increasing people's risks for getting oral cancers.

With continued advancements in nanoparticle (NP) synthesis and in the interfacing of NPs with biological systems has come the exponential growth in the use of NPs for therapeutic drug delivery and imaging applications. In recent years, the advent of NP multifunctionality-the ability to perform multiple, disparate functions on a single NP platform-has garnered much excitement for the potential realization of highly functional NP-mediated drug delivery for use in the clinical setting. This Overview will survey the current state of the art (reports published within the last 5 years) of multifunctional NPs for therapeutic drug delivery, imaging or a combination thereof. We provide extensive examples of both soft (micelles, liposomes, polymeric NPs) and hard (noble metals, quantum dots, metal oxides) NP formulations that have been used for multimodal drug delivery and imaging. The criteria for inclusion, herein, is that there must be at least two therapeutic drug cargos or imaging agents or a combination of the two. We next offer an assessment of the cytotoxicity of therapeutic NP constructs in biological systems. We then conclude with a forward-looking perspective on how we expect this field to develop in the coming years. WIREs Nanomed Nanobiotechnol 2017, 9:e1466. doi: 10.1002/wnan.1466 For further resources related to this article, please visit the WIREs website.

A primary envisioned use for nanoparticles (NPs) in a cellular context is for controlled drug delivery where the full benefit of NP attributes (small size, large drug cargo loading capacity) can improve the pharmacokinetics of the drug cargo. This requires the ability to controllably manipulate the release of the drug cargo from the NP vehicle or ‘controlled actuation’. In this review, we highlight new developments in this field from 2013 to 2015. The number and breadth of reports are a testament to the significant advancements made in this field over this time period. We conclude with a perspective of how we envision this field to continue to develop in the years to come.

Fusariosis is an emerging infectious complication of immune deficiency, but models to study this infection are lacking. The use of the soil nematode Caenorhabditis elegans as a model host to study the pathogenesis of Fusarium spp. was investigated. We observed that Fusarium conidia consumed by C. elegans can cause a lethal infection and result in more than 90% killing of the host within 120 hours, and the nematode had a significantly longer survival when challenged with Fusarium proliferatum compared to other species. Interestingly, mycelium production appears to be a major contributor in nematode killing in this model system, and C. elegans mutant strains with the immune response genes, tir-1 (encoding a protein containing a TIR domain that functions upstream of PMK-1) and pmk-1 (the homolog of the mammalian p38 MAPK) lived significantly shorter when challenged with Fusarium compared to the wild type strain. Furthermore, we used the C. elegans model to assess the efficacy and toxicity of various compounds against Fusarium. We demonstrated that amphotericin B, voriconazole, mancozeb, and phenyl mercury acetate significantly prolonged the survival of Fusarium-infected C. elegans, although mancozeb was toxic at higher concentrations. In conclusion, we describe a new model system for the study of Fusarium pathogenesis and evolutionarily preserved host responses to this important fungal pathogen.

Freud's creation of psychoanalysis is the starting place for a great many forms of contemporary psychodynamic psychotherapy, despite the fact that his work is very much a mixture of brilliant insights and misconceptions. Here, I will attempt to describe both the valuable contributions and the wrong turns, and to show how both can be understood in terms of the traumas he suffered as a child, as well as his particular adaptations to them. A review of his treatment of a number of patients will illustrate this discussion.

Study Design Systematic review. Introduction Contrast baths are used as an intervention in hand therapy, yet it is unclear which patients, if any, benefit from this intervention. Purpose of the Study To examine the nature and quality of the evidence regarding the use of contrast baths using a systematic review process. Methods Of a total of 28 clinical research articles on contrast baths, from 1938 forward, ten met the inclusion criteria set by the authors. Results These studies addressed the physiological changes of hot and cold on blood flow, intramuscular temperature, subcutaneous temperature, and the influence of room temperature and age. The subjects included normal/healthy volunteers and patients with a diagnosis of rheumatoid arthritis, diabetes, or foot/ankle injuries. The diversity of conditions, protocols, and outcomes limited the ability to make definitive conclusions on efficacy. Conclusions The contrast bath procedure may increase superficial blood flow and skin temperature, though the evidence on the impact on edema is conflicting. No relationship between physiologic effects and functional outcomes has been established. Level of Evidence: 2A

OBJECTIVE The purpose of this study was to accumulate behavioral validity evidence for physical activity Stage of Change (SOC). DESIGN Nine studies used a common physical activity SOC measure and examined self-report, objective, and performance physical activity indicators to accumulate behavioral validity evidence for SOC. Type of measure, the strength of the expected relationship between the measure and SOC, and the predicted SOC differences were examined. Validity evidence for the SOC was also examined by population and sampling method. MAIN OUTCOME MEASURES Validity evidence for physical activity SOC was classified with respect to the type of measurement instrument and the hypothesized magnitude of the relationship between the measure and the SOC. RESULTS Physical activity SOC was found to be behaviorally valid as evidenced by self-reported physical activity, self-reported exercise, self-reported sedentary behaviors, pedometers, and physical functioning. Physical activity SOC does not appear to be related to physical fitness or weight indicators. CONCLUSIONS This study highlights a successful multi-site collaboration. Physical activity data from nine large-scale, health trials was combined and accumulated behavioral validation evidence for the physical activity SOC.

PHLAME's (Promoting Healthy Lifestyles: Alternative Models' Effects) objective was to assess and compare two means to promote healthy lifestyles.Prospective trial among 599 firefighters randomized by station to 1) team-centered curriculum, 2) one-on-one motivational interviewing (MI), and 3) controls. Assessment included dietary behavior, physical activity, weight, and general well-being at baseline and 12 months. Program effects were determined using an analysis of covariance (ANCOVA) based approach, and models for relationships were evaluated with path analysis.Both interventions were acceptable and delivered with high fidelity. The team and MI programs increased fruit and vegetable consumption (P0.01 and 0.05, respectively) and general well-being (P0.01). Significantly less weight gain occurred in both (P0.05). A cross-sectional model was consistent with mediation differing between interventions.Both a team-centered and individual-oriented intervention promoted healthy behaviors. The scripted team curriculum is innovative, exportable, and may enlist influences not accessed with individual formats.

Gene therapy represents a promising approach for the treatment of monogenic and multifactorial neurological disorders. It can be used to replace a missing gene and mutated gene or downregulate a causal gene. Despite the versatility of gene therapy, one of the main limitations lies in the irreversibility of the process: once delivered to target cells, the gene of interest is constitutively expressed and cannot be removed. Therefore, efficient, safe and long-term gene modification requires a system allowing fine control of transgene expression.Different systems have been developed over the past decades to regulate transgene expression after in vivo delivery, either at transcriptional or post-translational levels. The purpose of this chapter is to give an overview on current regulatory system used in the context of gene therapy for neurological disorders. Systems using external regulation of transgenes using antibiotics are commonly used to control either gene expression using tetracycline-controlled transcription or protein levels using destabilizing domain technology. Alternatively, specific promoters of genes that are regulated by disease mechanisms, increasing expression as the disease progresses or decreasing expression as disease regresses, are also examined. Overall, this chapter discusses advantages and drawbacks of current molecular methods for regulated gene therapy in the central nervous system.

To describe a technique for extemporaneously drawing up bevacizumab for intravitreal injection (IVT) and report the rate of post-injection endophthtalmitis.Retrospective monocentric analysis (January 2010-December 2014) of all IVT of bevacizumab drawn up with the following technique: in the operating room (class ISO 7) through a mini-spike with an integrated bacteria retentive air filter. The surgeon was wearing sterile gloves and a mask. The assisting nurse wore a mask. The bevacizumab vial was discarded at the end of each session.Six thousand two hundred and thirty-six bevacizumab injections were performed. One case of endophthalmitis was noted (0.016%). During the same period, 4 cases of endophthalmitis were found after IVT of other drugs (4/32,992; 0.012%. P=0.8).Intravitreal injection of bevacizumab after extemporaneous withdrawal through a mini-spike filter is a simple and safe technique. The risk of postoperative endophthalmitis is very low. This simple technique facilitates access to compounded bevacizumab.

There is an enormous literature devoted to Freud and psychoanalysis, including full biographies by Jones (1953, 1955, 1957), Gay (1988), and myself (Breger, 2000, 2009); wide ranging critical studies: Roazen (1975), Sulloway (1979), Holt (1989), Rudnytsky (2002), and Makari (2008); a large number of Freud’s letters to friends and colleagues—including the revealing letters to his close colleague Wilhelm Fliess (Masson, 1985); evaluations of his published cases, and much more. These works range from the loyalty of orthodox Freudians to the most severe criticisms, with all shades in between. We can ask where Adam Phillips (2014) fits in all this, what does he offer that has not been discussed already, and what is the evidence for his version of Freud and psychoanalysis?

To evaluate prospectively the safety and effectiveness of aortoiliac magnetic resonance (MR) angiography enhanced with MS-325 (gadofosveset trisodium) at a dose of 0.03 mmol/kg; effectiveness was defined as accuracy relative to the reference standard, conventional angiography.Study was approved by institutional review boards of participating institutions, and required national approvals were obtained. Study protocol conformed to Good Clinical Practice guidelines, and informed patient consent was obtained. Patients with known or suspected peripheral vascular disease received 0.03 mmol/kg MS-325 for aortoiliac MR angiography. They were also examined with conventional angiography. MS-325-enhanced MR was evaluated for safety and effectiveness. Along with unenhanced two-dimensional time-of-flight MR angiography, it was compared with conventional angiography for presence of vascular stenosis. Student t tests were used to identify significant improvement in diagnostic sensitivity, specificity, and accuracy, as well as quantitative characterization of stenoses by three blinded readers. Correlations between readers of conventional angiograms were calculated and compared with MR results.In 174 patients, MS-325-enhanced MR angiography showed significant improvement (Por = .001) in sensitivity, specificity, and accuracy for diagnosis of clinically significant (or =50%) stenosis, compared with unenhanced MR. For all readers, areas under the receiver operating characteristic curve for both quantitative and qualitative measures of significant disease increased (P.001) for MS-325-enhanced MR compared with time-of-flight MR. All readers also expressed higher confidence in diagnosis (P.001) and found fewer images uninterpretable with MS-325 enhancement. All measures of interpretation accuracy approached corresponding measures of correlation between readers of conventional angiograms. Incidence of severe and serious adverse events with MS-325 was low. No patients were withdrawn from study due to adverse events or abnormalities in laboratory results. There were no clinically important trends in findings at hematology, blood chemistry, urinalysis, electrocardiography, or physical examination.MR angiography with MS-325 provides significant improvement in effectiveness over unenhanced MR (and minimal and transient side effects) at a dose of 0.03 mmol/kg and was safe and effective for MR evaluation of patients with aortoiliac occlusive disease.

Certain chromosome rearrangements, found in cancer cells or in cells exposed to ionizing radiation, exhibit a chromosome-wide delay in replication timing (DRT) that is associated with a delay in mitotic chromosome condensation (DMC). We have developed a chromosome engineering strategy that allows the generation of chromosomes with this DRT/DMC phenotype. We found that approximately 10% of inter-chromosomal translocations induced by two distinct mechanisms, site-specific recombination mediated by Cre or non-homologous end joining of DNA double-strand breaks induced by I-Sce1, result in DRT/DMC. Furthermore, on certain balanced translocations only one of the derivative chromosomes displays the phenotype. Finally, we show that the engineered DRT/DMC chromosomes acquire gross chromosomal rearrangements at an increased rate when compared with non-DRT/DMC chromosomes. These results indicate that the DRT/DMC phenotype is not the result of a stochastic process that could occur at any translocation breakpoint or as an epigenetic response to chromosome damage. Instead, our data indicate that the replication timing of certain derivative chromosomes is regulated by a cis-acting mechanism that delays both initiation and completion of DNA synthesis along the entire length of the chromosome. Because chromosomes with DRT/DMC are common in tumor cells and in cells exposed to ionizing radiation, we propose that DRT/DMC represents a common mechanism responsible for the genomic instability found in cancer cells and for the persistent chromosomal instability associated with cells exposed to ionizing radiation.

Treatment fidelity plays an important role in the research team's ability to ensure that a treatment has been implemented as intended and that the treatment has been accurately tested. Developing, implementing, and evaluating a treatment fidelity plan can be challenging. The treatment fidelity workgroup within the Behavior Change Consortium (BCC) developed guidelines to comprehensively evaluate treatment fidelity in behavior change research. The guidelines include evaluation of treatment fidelity with regard to study design, training of interventionists, delivery and receipt of the intervention, and enactment of the intervention in real-life settings. This article describes these guidelines and provides examples from four BCC studies as to how these recommended guidelines for fidelity were considered. Future work needs to focus not only on implementing treatment fidelity plans but also on quantifying the evaluations performed, developing specific criteria for interpretation of the findings, and establishing best practices of treatment fidelity.

Certain chromosome rearrangements display a significant delay in replication timing that is associated with a delay in mitotic chromosome condensation. Chromosomes with delay in replication timing/delay in mitotic chromosome condensation participate in frequent secondary rearrangements, indicating that cells with delay in replication timing/delay in mitotic chromosome condensation display chromosomal instability. In this report, we show that exposing cell lines or primary blood lymphocytes to ionizing radiation results in chromosomes with the delay in replication timing/delay in mitotic chromosome condensation phenotype, and that the delay in replication timing/delay in mitotic chromosome condensation phenotype occurs predominantly on chromosome translocations. In addition, exposing mice to ionizing radiation also induces cells with delay in replication timing/delay in mitotic chromosome condensation chromosomes that persist for as long as 2 years. Cells containing delay in replication timing/delay in mitotic chromosome condensation chromosomes frequently display hyperdiploid karyotypes, indicating that delay in replication timing/delay in mitotic chromosome condensation is associated with aneuploidy. Finally, using a chromosome engineering strategy, we show that only a subset of chromosome translocations displays delay in replication timing/delay in mitotic chromosome condensation. Our results indicate that specific chromosome rearrangements result in the generation of the delay in replication timing/delay in mitotic chromosome condensation phenotype and that this phenotype occurs frequently in cells exposed to ionizing radiation both in vitro and in vivo.

Introduction : In planning for the introduction of vaginal microbicides and other new antiretroviral (ARV)-based prevention products for women, an in-depth understanding of potential end-users will be critically important to inform strategies to optimize uptake and long-term adherence. User-centred private sector companies have contributed to the successful launch of many different types of products, employing methods drawn from behavioural and social sciences to shape product designs, marketing messages and communication channels. Examples of how the private sector has adapted and applied these techniques to make decisions around product messaging and targeting may be instructive for adaptation to microbicide introduction. Discussion : In preparing to introduce a product, user-centred private sector companies employ diverse methods to understand the target population and their lifestyles, values and motivations. ReD Associates’ observational research on user behaviours in the packaged food and diabetes fields illustrates how ‘tag along’ or ‘shadowing’ techniques can identify sources of non-adherence. Another open-ended method is self-documentation, and IDEO’s mammography research utilized this to uncover user motivations that extended beyond health. Mapping the user journey is a quantitative approach for outlining critical decision-making stages, and Monitor Inclusive Markets applied this framework to identify toilet design opportunities for the rural poor. Through an iterative process, these various techniques can generate hypotheses on user drop-off points, quantify where drop-off is highest and prioritize areas of further research to uncover usage barriers. Although research constraints exist, these types of user-centred techniques have helped create effective messaging, product positioning and packaging of health products as well as family planning information. These methods can be applied to microbicide acceptability testing outside of clinical trials to design microbicide marketing that enhances product usage. Conclusions : The introduction of microbicide products presents an ideal opportunity to draw on the insights from user-centred private sector companies’ approaches, which can complement other methods that have been more commonly utilized in microbicide research to date. As microbicides move from clinical trials to real-world implementation, there will be more opportunities to combine a variety of approaches to understand end-users, which can lead to a more effective product launch and ultimately greater impact on preventing HIV infections. Keywords: HIV; prevention; microbicides; end-user; user research; women; adherence; introduction planning. (Published: 8 September 2014) Citation: Lin AH et al. Journal of the International AIDS Society 2014, 17 (Suppl 2):19162 http://www.jiasociety.org/index.php/jias/article/view/19162 | http://dx.doi.org/10.7448/IAS.17.3.19162

There is a comprehensive early defibrillation program in Bochum (Germany); since 2003 a total of 175 automated external defibrillators (AEDs) have been installed in urban areas by the city of Bochum and private companies. These were preferably installed in places with high foot traffic, e.g., public buildings, companies, and event/shopping centers. Approximately 15,000 laypeople who work in the vicinity of the AED locations were trained in the use of defibrillators and in basic resuscitation. In addition, rescue workers on fire trucks and medically trained personnel in physicians' medical practices were equipped as "first responders" with AEDs.After an initiation phase, all available information after each AED use since August 2004 has been collected by the project coordinator. During the period of data collection (August 2004 to August 2013), an AED was used in a total of 17 patients who had suffered sudden cardiac death (SCD) under the project in Bochum. Eleven patients had primary ventricular fibrillation (VF). Six of these survived without neurological deficit. In another 6 patients, a nondefibrillatable rhythm disorder was diagnosed. The AEDs are reliable and showed impeccable rhythm analysis before the instructions to provide any necessary shock.Compared to the number of existing units and an estimated number of 37-100 SCD/100,000, the use of the AEDs only 17 times appears relatively small. To improve the effectiveness of the AED program in Bochum, an analysis of the emergency service responses, which were necessary because of sudden circulatory collapse, is currently being performed. This will allow areas with an increased incidence of SCD to be identified and a plan for the strategic placement of AED and emergency services can be made.

Contamination and adulterants in both naturally derived and synthetic drugs pose a serious threat to the worldwide medical community. Developing rapid and sensitive sensors/devices to detect these hazards is thus a continuing need. We describe a hydrophilic semiconductor quantum dot (QD)-peptide Förster resonance energy transfer (FRET) nanosensor for monitoring the activity of kallikrein, a key proteolytic enzyme functioning at the initiation of the blood clotting cascade. Kallikrein is also activated by the presence of an oversulfated contaminant recently found in preparations of the drug heparin. Quantitatively monitoring the activity of this enzyme within a nanosensor format has proven challenging because of inherent steric and kinetic considerations. Our sensor is designed around a central QD donor platform which displays controlled ratios of a modular peptidyl substrate. This peptide, in turn, sequentially expresses a terminal oligohistidine motif that mediates the rapid self-assembly of peptides to the QD surface, a linker-spacer sequence to extend the peptide away from the QD surface, a kallikrein recognized-cleavage site, and terminates in an acceptor dye-labeling site. Hydrophilic QDs prepared with compact, zwitterionic surface coatings were first evaluated for their ability to self-assemble the dye-labeled peptide substrates. An optimized two-step protocol was then utilized where high concentrations of peptide were initially digested with purified human kallikrein and samples collected at distinct time points were subsequently diluted into QD-containing solutions for assaying. This sensor provided a quantitative FRET-based readout for monitoring kallikrein activity and comparison to a calibration curve allowed estimation of the relevant Michaelis-Menten kinetic descriptors. The results further suggest that almost any protease should be amenable to a QD-based FRET assay format with appropriate design considerations.

The biological applications of luminescent semiconductor quantum dots (QDs) continue to grow at a nearly unabated pace. This growth is driven, in part, by their unique photophysical and physicochemical properties which have allowed them to be used in many different roles in cellular biology including: as superior fluorophores for a wide variety of cellular labeling applications; as active platforms for assembly of nanoscale sensors; and, more recently, as a powerful tool to understand the mechanisms of nanoparticle mediated drug delivery. Given that controlled cellular delivery is at the intersection of all these applications, the latest progress in delivering QDs to cells is examined here. A brief discussion of relevant considerations including the importance of materials preparation and bioconjugation along with the continuing issue of endosomal sequestration is initially provided for context. Methods for the cellular delivery of QDs are then highlighted including those based on passive exposure, facilitated strategies that utilize peptides or polymers and fully active modalities such as electroporation and other mechanically based methods. Following on this, the exciting advent of QD cellular delivery using multiple or combined mechanisms is then previewed. Several recent methods reporting endosomal escape of QD materials in cells are also examined in detail with a focus on the mechanisms by which access to the cytosol is achieved. The ongoing debate over QD cytotoxicity is also discussed along with a perspective on how this field will continue to evolve in the future.

The generation of functional mRNA in eukaryotes requires the accurate removal of noncoding regions (introns) from pre-mRNA by a process termed pre-mRNA splicing (14, 25). Splicing takes place in the spliceosome, a dynamic RNA-protein complex that assembles in a stepwise manner on the pre-mRNA (10, 14). The spliceosome is composed of small nuclear ribonucleoprotein particles (snRNPs) and extrinsic (non-snRNP) factors. The recognition of exon/intron boundaries, the splice sites, by the splicing apparatus is a critical step in processing of both constitutively and alternatively spliced pre-mRNAs. U1 snRNP defines the 5′ splice site, and U2 snRNP defines the branchpoint sequence (3, 10, 14, 17). Since in most cases the first AG dinucleotide downstream of the branchpoint is used as the 3′ splice site, by defining the branchpoint, U2 snRNP defines the 3′ splice site (18, 27). Targeting of U2 snRNP to the branch site requires the extrinsic splicing factor U2 snRNP auxiliary factor (U2AF). U2AF binds site specifically to the intron pyrimidine tract between the branchpoint sequence and 3′ splice site at an early step in spliceosome assembly and recruits U2 snRNP to the branch site (22, 29, 42). Regulation of 3′ splice site choice, both positive and negative, can be realized by influencing the pyrimidine tract binding of U2AF (33, 35, 45). Because U2AF is a major determinant in 3′ splice site selection, it has been the subject of extensive biochemical and genetic investigation. Human U2AF is a heterodimer composed of a 65-kDa large subunit (hU2AF65) and a 35-kDa small subunit (hU2AF35) (41). Both subunits are conserved in other organisms (40), and U2AF homologs have been identified in Drosophila melanogaster (9, 21), Schizosaccharomyces pombe (16, 36), and Caenorhabditis elegans (3a, 39). The Drosophila U2AF large (dU2AF50)- and small (dU2AF38)-subunit homologs are 50 and 38 kDa, respectively (9, 21). The U2AF large subunit contains three RNA recognition motifs (RRMs) and an amino-terminal arginine-serine-rich (RS) domain (42). The small subunit contains a highly degenerate RRM (pseudo-RRM) (2), two Zn2+ binding motifs (37), and a carboxyl-terminal RS domain and glycine-rich region (43). Both U2AF subunits are involved in recognition of the intron pyrimidine tract. The large subunit (hU2AF65 and dU2AF50) is required for site-specific pyrimidine tract binding (9, 42). The small subunit acts as a cofactor to stabilize the large subunit on the pyrimidine tract, apparently through protein-protein interactions with constitutive and alternative splicing factors (38, 45). While it has been firmly established that all three RRMs on hU2AF65 are necessary for high-affinity RNA binding, a role for the large-subunit RS domain in RNA binding remains unresolved (11, 42). In one study removal of the hU2AF65 RS domain had a modest effect on RNA binding (42). In a second study, the RS domain was found to be absolutely required for RNA binding (11). In vitro splicing assays using U2AF-depleted extracts prepared by two independent methods have identified independent and essential roles for the two U2AF RS domains: the large-subunit RS domain is required to target U2 snRNP to the branch site (34, 42), and in the immunodepleted extracts under certain conditions, the small-subunit RS domain is apparently necessary for protein-protein interactions with constitutive and alternative splicing factors to stabilize hU2AF65 on the pyrimidine tract (38, 45). In contrast to the essential roles assigned to the two U2AF RS domains in vitro, molecular genetic analysis of the Drosophila U2AF RS domains indicates that either one of the RS domains is dispensable in vivo (19). Importantly, at least one RS domain on U2AF is essential for viability (19). The observation that the dU2AF38 RS domain is not essential in vivo (19) refocused our attention on domains present in the U2AF small subunit that are phylogenetically conserved. In an exhaustive database search for proteins containing RRMs, some of the signature sequences of this motif were identified in hU2AF35 (2). These sequences are also present in the Drosophila and S. pombe small-subunit homologs (21, 36). Although some of the most conserved residues in the RNA recognition motif are present in the U2AF small subunits, the RNP-1 octamer is highly degenerate and the RNP-2 hexamer is absent. Since these defining submotifs and other conserved residues are not present in the U2AF small-subunit RRM, it was termed a degenerate RRM or pseudo-RRM (2). Degenerate RRMs have been identified in a collection of RNA binding proteins, including several of the SR proteins, the pyrimidine tract binding protein, and the large subunit of U2AF (10). The degenerate RRMs in SRp30a (ASF/SF2) (4, 46), pyrimidine tract-binding protein (15), and hU2AF65 (42) were all found to be required for high-affinity RNA binding. Two putative Zn2+ binding domains, one on either side of the pseudo-RRM, were recently identified in hU2AF35 in a database search (37). These Cys3His Zn2+ binding motifs are conserved in all three small subunit homologs. Though sequence-specific RNA binding has not been described for proteins that contain this type of Zn2+ binding motif, several proteins that have this domain are involved in RNA metabolism (37). The evolutionary conservation of the pseudo-RRM and the two Zn2+ binding motifs in all three U2AF small subunit homologs suggested to us that these domains are important for function. The lack of requirement for the dU2AF38 RS domain in vivo prompted us to search for novel biochemical activities associated with the small subunit. The phylogenetically conserved, degenerate RRM (2) and two Zn2+ binding motifs (37) in the small subunit suggested that it might participate in RNA binding. While we detected weak RNA binding activity for the Drosophila small subunit on its own, we found that when complexed with the large subunit, dU2AF pyrimidine tract binding affinity increased 20-fold. This increase in RNA binding activity was not specific to Drosophila U2AF; the human U2AF heterodimer bound RNA with 15-fold-higher affinity than hU2AF65. Surprisingly, removal of the dU2AF38 RS domain abolished the increase in binding activity of the dU2AF heterodimer, indicating that the RS domain is necessary for high-affinity binding. Deletion of the dU2AF50 RS domain (dU2AF50ΔRS) dramatically reduced RNA binding activity of the large-subunit monomer. High-affinity binding was restored when the dU2AF38 RS domain was supplied in trans to dU2AF50ΔRS. These data suggest that high-affinity RNA binding activity requires at least one RS domain on U2AF, which is consistent with the requirement for at least one RS domain in vivo.

The pre-mRNA splicing factor U2AF (U2 snRNP auxiliary factor) has an essential role in 3' splice site selection. U2AF binds the intron pyrimidine tract between the branchpoint and the 3' splice site and recruits U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. Both subunits contain a domain enriched in arginine-serine dipeptide repeats termed an RS domain. The two U2AF RS domains have been assigned essential and independent roles in spliceosome assembly in vitro-the hU2AF65 RS domain is required to target U2 snRNP to the branch site and the hU2AF35 RS domain is necessary for protein-protein interactions with constitutive and alternative splicing factors. We have investigated the functional requirements for the RS domains on the Drosophila U2AF homolog in vivo. In sharp contrast to its essential role in U2 snRNP recruitment in vitro, the RS domain on the Drosophila large subunit homolog (dU2AF50) was completely dispensable in vivo. Prompted by this unexpected result, we analyzed the RS domain on the Drosophila small subunit homolog (dU2AF38). Despite its requirement for enhancer-dependent splicing activity in vitro, the dU2AF38 RS domain was also inessential in vivo. Finally, we have tested whether the Drosophila U2AF heterodimer requires any RS domain. Flies mutant for both the small and large subunits could not be rescued by dU2AF50deltaRS and dU2AF38deltaRS transgenes. Therefore, in contrast to the separate roles assigned to the U2AF RS domains in vitro, our genetic data suggest that they may have redundant functions in vivo.

Inflammatory reactions to biomaterials may include macrophage-mediated generation of nitric oxide (NO), which may harm patient tissue or potentially interfere with proper function of an implanted device. RAW 264.7 cells were grown in culture and treated at various times with lipopolysaccharide (LPS, endotoxin), murine recombinant γ-interferon (mrIFN-γ), and different preparations of hyaluronic acid (HA). Increase in fluorescence of 2,3-diaminonaphthalene (DAN) allowed for detection of initial (24 h or less) NO inflammatory responses of RAW 264.7 to LPS from E. coli O26:B6. By looking at early time points, mrIFN-γ augmentation of the LPS effect was observed, simulating a complex immune reaction. Activation through nuclear factor-κB (NF-κB), was confirmed in this system by parthenolide inhibition of LPS stimulation. Stimulation of RAW 264.7 by different HA preparations resulted in NO responses that correlated with the amount of LPS present. In the presence of mrIFN-γ, a significant inflammatory reaction to HA was observed when the concentration of contaminating LPS was as low as 0.15 EU/mL. NO production in the presence of mrIFN-γ by RAW 264.7 may serve as a convenient in vitro system to routinely screen biomaterials for potentially harmful macrophage-mediated inflammation whereby the safety of implanted medical devices might be compromised. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009

Motivational Interviewing (MI) has been established as an effective psychotherapeutic treatment for problem drinking in clinical settings. Consequently, there is a growing interest in applying MI to facilitate change across other health behaviors, such as tobacco use, eating habits, and physical activity in a variety of community-based research settings. These extended applications pose new challenges regarding implementation and evaluation. For instance, investigators must consider how best to train intervention counselors; implement strategies for preserving the MI spirit, despite limited client contact time; incorporate adjunctive strategies that support brief MI sessions; and develop a plan for monitoring and evaluating MI treatment fidelity. This article highlights specific examples of how several behavior change research projects applied MI across a variety of settings and populations, provides lessons learned from our experience as a collaborative workgroup, and offers strategies for consideration in future community-based research.