iATPSnFR2: A high-dynamic-range fluorescent sensor for monitoring intracellular ATP.

We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted superfolder green fluorescent protein (GFP) inserted between the ATP-binding helices of the ε-subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a ~fivefold to sixfold improvement in the dynamic range compared to the previous-generation sensor, with excellent discrimination against other analytes, and affinity variants varying from 4 µM to 500 µM. A chimeric version of this sensor fused to either the HaloTag protein or a suitable spectrally separated fluorescent protein provides an optional ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting the sensor to nerve terminals reveals previously uncharacterized single-synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.

Rational Design of Bioavailable Photosensitizers for Manipulation and Imaging of Biological Systems.

Light-mediated chemical reactions are powerful methods for manipulating and interrogating biological systems. Photosensitizers, compounds that generate reactive oxygen species upon excitation with light, can be utilized for numerous biological experiments, but the repertoire of bioavailable photosensitizers is limited. Here, we describe the synthesis, characterization, and utility of two photosensitizers based upon the widely used rhodamine scaffold and demonstrate their efficacy for chromophore-assisted light inactivation, cell ablation in culture and in vivo, and photopolymerization of diaminobenzidine for electron microscopy. These chemical tools will facilitate a broad range of applications spanning from targeted destruction of proteins to high-resolution imaging.

A general method to optimize and functionalize red-shifted rhodamine dyes.

Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted 'Janelia Fluor' (JF) dyes useful for biological imaging experiments in cells and in vivo.

Bright photoactivatable fluorophores for single-molecule imaging.

Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.

High-density three-dimensional localization microscopy across large volumes.

Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high-localization precision, ultrahigh-labeling density, multicolor localization microscopy in samples up to 20 µm thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes.

Web-based screening and brief intervention for student marijuana use in a university health center: pilot study to examine the implementation of eCHECKUP TO GO in different contexts.

This pilot study sought to test the feasibility of procedures to screen students for marijuana use in Student Health Services (SHS) and test the efficacy of a web-based intervention designed to reduce marijuana use and consequences. Students were asked to participate in voluntary screening of health behaviors upon arrival at SHS. One hundred and twenty-three students who used marijuana at least monthly completed assessments and were randomized to one of four intervention conditions in a 2 (intervention: Marijuana eCHECKUP TO GO vs. control)×2 (site of intervention: on-site vs. off-site) between-groups design. Follow-up assessments were conducted online at 3 and 6 months. Latent growth modeling was used to provide effect size estimates for the influence of intervention on outcomes. One thousand and eighty undergraduate students completed screening. The intervention did not influence marijuana use frequency. However, there was evidence of a small overall intervention effect on marijuana-related consequences and a medium effect in stratified analyses in the on-site condition. Analyses of psychological variables showed that the intervention significantly reduced perceived norms regarding peer marijuana use. These findings demonstrate that it is feasible to identify marijuana users in SHS and deliver an automated web-based intervention to these students in different contexts. Effect size estimates suggest that the intervention has some promise as a means of correcting misperceptions of marijuana use norms and reducing marijuana-related consequences. Future work should test the efficacy of this intervention in a full scale randomized controlled trial.

Depressive symptoms and the incidence of adult-onset asthma in African American women.

BACKGROUND: Some evidence suggests that depression may increase the risk of adult-onset asthma. No data are available for African American women, in whom the prevalence of depression and asthma is high. OBJECTIVE: To conduct prospective analyses of the relation of depressive symptoms to asthma incidence in the Black Women's Health Study, a prospective cohort of US black women followed since 1995 with mailed biennial questionnaires. METHODS: Of 31,848 participants followed from 1999 to 2011, 771 reported incident asthma. Depressive symptoms were ascertained on 1999 and 2005 follow-up questionnaires with the Center for Epidemiological Studies-Depression Scale (CES-D). Participants rated the frequency of 20 symptoms. A score was calculated by summing the responses to all questions. Cox regression models were used to derive incidence rate ratios and 95% confidence intervals for 4 categories of the CES-D score in relation to incident asthma, adjusted for body mass index, smoking, and other covariates. RESULTS: The multivariable incidence rate ratio in the highest category of CES-D score (≥ 33) compared with the lowest (<16) was 2.08 (95% confidence interval 1.58-2.74), with a significant trend (P < .0001). The incidence rate ratio was higher in women who took antidepressants, were current or former smokers, were not obese, and were at least 40 years old, although there were no statistically significant interactions. CONCLUSION: A positive association was observed between CES-D score and the incidence of adult-onset asthma. If the hypothesis is confirmed, depression could contribute substantially to the burden of asthma in adults.

Differences between daily smokers, chippers, and nonsmokers with co-occurring anxiety and alcohol-use disorders.

Tobacco use is disproportionately represented among both alcohol-use disorders (AUDs) and anxiety disorders (ANX) compared to the general population [Kalman, D. A., Morissette, S. B., & George, T. P. (2005). Co-morbidity of nicotine and tobacco use in psychiatric and substance use disorders. The American Journal on Addictions, 14, 1-18]. Despite this common overlap, little is known about how smokers with co-occurring AUD-ANX differ from their nonsmoking counterparts. Seventy-two patients participated in a larger clinical trial evaluating the efficacy of venlafaxine and cognitive-behavioral therapy for AUD-ANX. Differences between daily smokers (n=23), chippers (n=12) and nonsmokers (n=37) with AUD-ANX were examined with respect to intensity and frequency of alcohol use, anxiety symptoms, depressed mood, and stress. Point prevalence of current daily smoking was 31.9%, which is considerably lower than traditionally reported in AUD studies. Consistent with predictions, daily smokers reported higher levels of alcohol dependence, average drinks per drinking occasion, and peak blood concentration levels in a day than nonsmokers during the 90 days prior to assessment. Chippers were nonsignificantly different from either smokers or nonsmokers. Smokers and nonsmokers did not differ with respect to percent heavy drinking days or emotional symptoms.

Genesis and wanderings: origins and migrations in asymmetrically replicating mitochondrial DNA.

Mammalian mitochondria maintain a small circular genome that encodes RNA and polypeptides that are essential for the generation of ATP through oxidative phosphorylation. The mechanism of replication of mammalian mitochondrial DNA (mtDNA) has recently been a topic of controversy. New evidence has led to a modified strand-displacement model that reconciles much of the current data. This revision stems from a new appreciation for alternative light-strand origins. We consider here some of the potential mechanisms for light-strand origin initiation. We also consider further the susceptibility of branch migration within replicating mtDNA molecules. The existence of alternative light-strand origins and a propensity for branch migration in replicating mtDNA molecules exposes a new array of possible configurations of mtDNA. The assortment and assignment of these forms is relevant to the interpretation of experimental data and may also yield insight into the molecular basis of replication errors.

Differences between smokers and nonsmokers with anxiety disorders.

Epidemiological data suggest that early smoking increases the risk for emergence of certain anxiety disorders (e.g., panic disorder, generalized anxiety disorder (GAD)), and that presence of certain anxiety disorders (e.g., social anxiety) increases the risk for later development of nicotine dependence. Although some studies report a high prevalence of smoking among anxiety disorders, the extent to which smokers with anxiety disorders differ from their nonsmoking counterparts remains uncertain. Differences between smokers and nonsmokers with anxiety disorders (N=527) were examined with respect to multiple measures of theoretical and clinical interest. Compared to nonsmokers, smokers with anxiety disorders reported greater anxiety sensitivity, anxiety symptoms, agoraphobic avoidance, depressed mood, negative affect, stress and life interference; however, these differences were largely accounted for by panic disorder. No differences were found between smokers and nonsmokers regarding social anxiety, worry, obsessive-compulsive symptoms or positive affect. Differential patterns were observed when evaluating constructs within anxiety disorder diagnoses.

Differences in nuclear gene expression between cells containing monomer and dimer mitochondrial genomes.

It is known that point mutations and rearrangements (deletions and duplications) of mammalian mitochondrial DNA (mtDNA) can result in mitochondrial dysfunction and human disease. Very little attention has been paid to mtDNA circular dimers (a complex form consisting of two genomes joined head-to-tail) despite their close association with human neoplasia. MtDNA dimers are frequently found in human leukemia, but the clinical relevance of their presence remains unknown. To begin to investigate the role of circular dimer mtDNA in the tumorigenic phenotype, we have created isogenic cell lines containing monomer and dimer mitochondrial genomes and compared the respective nuclear mRNA expression using Affymetrix gene array analysis. Surprisingly, a large number of nuclear gene changes were observed, with one of the largest category of genes being associated with remodeling of the cell surface and extracellular matrix. Since cell growth, migration, apoptosis, and many other cellular processes are influenced by signals initiating from the cell surface, the changes associated with the presence of mtDNA dimers could lead to significant alterations in tumorigenic potential and/or progression.