Microbial rRNA sequencing analysis of evaporative cooler indoor environments located in the Great Basin Desert region of the United States.

Recent studies conducted in the Great Basin Desert region of the United States have shown that skin test reactivity to fungal and dust mite allergens are increased in children with asthma or allergy living in homes with evaporative coolers (EC). The objective of this study was to determine if the increased humidity previously reported in EC homes leads to varying microbial populations compared to homes with air conditioners (AC). Children with physician-diagnosed allergic rhinitis living in EC or AC environments were recruited into the study. Air samples were collected from the child's bedroom for genomic DNA extraction and metagenomic analysis of bacteria and fungi using the Illumina MiSeq sequencing platform. The analysis of bacterial populations revealed no major differences between EC and AC sampling environments. The fungal populations observed in EC homes differed from AC homes. The most prevalent species discovered in AC environments belonged to the genera Cryptococcus (20%) and Aspergillus (20%). In contrast, the most common fungi identified in EC homes belonged to the order Pleosporales and included Alternaria alternata (32%) and Phoma spp. (22%). The variations in fungal populations provide preliminary evidence of the microbial burden children may be exposed to within EC environments in this region.

Oxandrolone for the treatment of bone marrow failure in Fanconi anemia.

BACKGROUND: A majority of Fanconi anemia (FA) patients will experience bone marrow failure (BMF) and androgen therapy (most often oxymetholone) may be utilized as a treatment to improve BMF-related cytopenias. However, oxymetholone is associated with toxicities making identification of other agents of interest. In this study we aimed to evaluate the toxicity profile and hematologic response in patients with FA who are treated with low-dose oxandrolone, a synthetic non-fluorinated anabolic steroid, similar to oxymetholone, with known dosing thresholds for virilization. PROCEDURE: A single arm, Phase I/II study was designed to treat patients on low-dose oxandrolone. If no toxicity or hematologic response was noted at 16 weeks, a single dose escalation was offered. Subjects were regularly assessed for toxicity, including determinations of virilization, behavioral changes, and liver and kidney function. At 32 weeks, those who demonstrated hematologic response were allowed to continue study treatment, and those without improvement were deemed non-responsive. RESULTS: Nine subjects completed the study and were followed for a median of 99 weeks (46-136 weeks). Three (33.3%) subjects developed mild sub-clinical virilization and continued treatment with a dose reduction. None (0%) had adverse behavioral changes. Two (22.2%) developed elevated liver function tests at 42 and 105 weeks. Seven (77.8%) subjects had a hematologic response. CONCLUSION: Oxandrolone appears to be well-tolerated, has limited toxicities at the administered doses in FA with patients, and may be an alternative androgen for the treatment of BMF in FA.

Interdomain interactions support interdomain communication in human Pin1.

Pin1 is an essential mitotic regulator consisting of a peptidyl-prolyl isomerase (PPIase) domain flexibly tethered to a smaller Trp-Trp (WW) binding domain. Communication between these domains is important for Pin1 in vivo activity; however, the atomic basis for this communication has remained elusive. Our previous nuclear magnetic resonance (NMR) studies of Pin1 functional dynamics suggested that weak interdomain contacts within Pin1 enable allosteric communication between the domain interface and the distal active site of the PPIase domain.1,2 A necessary condition for this hypothesis is that the intrinsic properties of the PPIase domain should be sensitive to interdomain contact. Here, we test this sensitivity by generating a Pin1 mutant, I28A, which weakens the wild-type interdomain contact while maintaining the overall folds of the two domains. Using NMR, we show that I28A leads to altered substrate binding affinity and isomerase activity. Moreover, I28A causes long-range perturbations to conformational flexibility in both domains, for both the apo and substrate-complexed states of the protein. These results show that the distribution of conformations sampled by the PPIase domain is sensitive to interdomain contact and strengthen the hypothesis that such contact supports interdomain allosteric communication in Pin1. Other modular systems may exploit interdomain interactions in a similar manner.

Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification.

Recent studies have shown that zinc finger nucleases (ZFNs) are powerful reagents for making site-specific genomic modifications. The generic structure of these enzymes includes a ZF DNA-binding domain and nuclease domain (Fn) are separated by an amino acid "linker" and cut genomic DNA at sites that have a generic structure (site1)-(spacer)-(site2) where the "spacer" separates the two binding sites. In this work, we compare the activity of ZFNs with different linkers on target sites with different spacer lengths. We found those nucleases with linkers' lengths of 2 or 4 amino acid (aa) efficiently cut at target sites with 5 or 6 base pair (bp) spacers, and that those ZFNs with a 5-aa linker length efficiently cut target sites with 6 or 7 bp spacers. In addition, we demonstrate that the Oligomerized Pool ENgineering (OPEN) platform used for making three-fingered ZF proteins (ZFPs) can be modified to incorporate modular assembly fingers (including those recognizing ANNs, CNNs, and TNNs) and we were able to generate nucleases that efficiently cut cognate target sites. The ability to use module fingers in the OPEN platform at target sites of 5-7 bp spacer lengths increases the probability of finding a ZFN target site to 1 in 4 bp. These findings significantly expand the range of sites that can be potentially targeted by these custom-engineered proteins.Molecular Therapy - Nucleic Acids (2013) 2, e88; doi:10.1038/mtna.2013.13; published online 30 April 2013.

Design and Development of Artificial Zinc Finger Transcription Factors and Zinc Finger Nucleases to the hTERT Locus.

The ability to direct human telomerase reverse transcriptase (hTERT) expression through either genetic control or tunable regulatory factors would advance not only our understanding of the transcriptional regulation of this gene, but also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and ZF nucleases (ZFNs) to target sequences within the hTERT promoter and exon-1. We were able to identify several active ZFTFs that demonstrate a broadly tunable response when screened by a cell-based transcriptional reporter assay. Using the same DNA-binding domains, we generated ZFNs that were screened in combinatorial pairs in cell-based extrachromosomal single-strand annealing (SSA) assays and in gene-targeting assays using stably integrated constructs. Selected ZFN pairs were tested for the ability to induce sequence changes in a Cel1 assay and we observed frequencies of genomic modification up to 18.7% at the endogenous hTERT locus. These screening strategies have pinpointed several ZFN pairs that may be useful in gene editing of the hTERT locus. Our work provides a foundation for using engineered ZF proteins (ZFPs) for modulation of the hTERT locus.Molecular Therapy - Nucleic Acids (2013) 2, e87; doi:10.1038/mtna.2013.12; published online 23 April 2013.

Attentional biases for threat in at-risk daughters and mothers with lifetime panic disorder.

Children of parents with panic disorder (PD) have high risk for developing anxiety disorders. However, the mechanisms involved in transmission of risk are uncertain. Cognitive models of anxiety propose that information-processing biases underlie anxiety vulnerability; in particular, attentional biases for threat. Consequently, this study examined attentional biases in mothers with lifetime PD and their daughters (aged 9-14 years). Sixty mother-daughter dyads (n = 120) were recruited to the study; half the mothers had lifetime PD (i.e., either a current or past history of PD), and half had no psychiatric history. Attentional biases were assessed using a visual-probe task with pictorial and word stimuli related to physical-health threat. Stimulus duration was varied to examine the time-course of attentional biases (initial orienting and maintained attention). Results showed an attentional bias for threat in daughters of mothers with lifetime PD, compared with daughters of mothers with no PD history. Specifically, at-risk daughters had an attentional bias for physical-health threat cues (words and pictures) at the longer stimulus duration of 1250 ms (but not at 500 ms). In addition, attentional bias for threat in girls was associated with increased physical-health threat worries. Mothers with lifetime PD did not significantly differ from mothers with no PD history on the indices of attentional bias. The findings are discussed in terms of an attentional threat-monitoring strategy in at-risk girls and argue against the view that there is simple transmission of an anxiety-related attentional processing style across generations.

Stereospecific gating of functional motions in Pin1.

Pin1 is a modular enzyme that accelerates the cis-trans isomerization of phosphorylated-Ser/Thr-Pro (pS/T-P) motifs found in numerous signaling proteins regulating cell growth and neuronal survival. We have used NMR to investigate the interaction of Pin1 with three related ligands that include a pS-P substrate peptide, and two pS-P substrate analogue inhibitors locked in the cis and trans conformations. Specifically, we compared the ligand binding modes and binding-induced changes in Pin1 side-chain flexibility. The cis and trans binding modes differ, and produce different mobility in Pin1. The cis-locked inhibitor and substrate produced a loss of side-chain flexibility along an internal conduit of conserved hydrophobic residues, connecting the domain interface with the isomerase active site. The trans-locked inhibitor produces a weaker conduit response. Thus, the conduit response is stereoselective. We further show interactions between the peptidyl-prolyl isomerase and Trp-Trp (WW) domains amplify the conduit response, and alter binding properties at the remote peptidyl-prolyl isomerase active site. These results suggest that specific input conformations can gate dynamic changes that support intraprotein communication. Such gating may help control the propagation of chemical signals by Pin1, and other modular signaling proteins.

Everolimus for subependymal giant-cell astrocytomas in tuberous sclerosis.

BACKGROUND: Neurosurgical resection is the standard treatment for subependymal giant-cell astrocytomas in patients with the tuberous sclerosis complex. An alternative may be the use of everolimus, which inhibits the mammalian target of rapamycin, a protein regulated by gene products involved in the tuberous sclerosis complex. METHODS: Patients 3 years of age or older with serial growth of subependymal giant-cell astrocytomas were eligible for this open-label study. The primary efficacy end point was the change in volume of subependymal giant-cell astrocytomas between baseline and 6 months. We gave everolimus orally, at a dose of 3.0 mg per square meter of body-surface area, to achieve a trough concentration of 5 to 15 ng per milliliter. RESULTS: We enrolled 28 patients. Everolimus therapy was associated with a clinically meaningful reduction in volume of the primary subependymal giant-cell astrocytoma, as assessed on independent central review (P<0.001 for baseline vs. 6 months), with a reduction of at least 30% in 21 patients (75%) and at least 50% in 9 patients (32%). Marked reductions were seen within 3 months and were sustained. There were no new lesions, worsening hydrocephalus, evidence of increased intracranial pressure, or necessity for surgical resection or other therapy for subependymal giant-cell astrocytoma. Of the 16 patients for whom 24-hour video electroencephalography data were available, seizure frequency for the 6-month study period (vs. the previous 6-month period) decreased in 9, did not change in 6, and increased in 1 (median change, -1 seizure; P=0.02). The mean (±SD) score on the validated Quality-of-Life in Childhood Epilepsy questionnaire (on which scores can range from 0 to 100, with higher scores indicating a better quality of life) was improved at 3 months (63.4±12.4) and 6 months (62.1±14.2) over the baseline score (57.8±14.0). Single cases of grade 3 treatment-related sinusitis, pneumonia, viral bronchitis, tooth infection, stomatitis, and leukopenia were reported. CONCLUSIONS: Everolimus therapy was associated with marked reduction in the volume of subependymal giant-cell astrocytomas and seizure frequency and may be a potential alternative to neurosurgical resection in some cases, though long-term studies are needed. (Funded by Novartis; ClinicalTrials.gov number, NCT00411619.).

Toward flexibility-activity relationships by NMR spectroscopy: dynamics of Pin1 ligands.

Drug design involves iterative ligand modifications. For flexible ligands, these modifications often entail restricting conformational flexibility. However, defining optimal restriction strategies can be challenging if the relationship between ligand flexibility and biological activity is unclear. Here, we describe an approach for ligand flexibility-activity studies using Nuclear Magnetic Resonance (NMR) spin relaxation. Specifically, we use (13)C relaxation dispersion measurements to compare site-specific changes in ligand flexibility for a series of related ligands that bind a common macromolecular receptor. The flexibility changes reflect conformational reorganization resulting from formation of the receptor-ligand complex. We demonstrate this approach on three structurally similar but flexibly differentiated ligands of human Pin1, a peptidyl-prolyl isomerase. The approach is able to map the ligand dynamics relevant for activity and expose changes in those dynamics caused by conformational locking. Thus, NMR flexibility-activity studies can provide information to guide strategic ligand rigidification. As such, they help establish an experimental basis for developing flexibility-activity relationships (FAR) to complement traditional structure-activity relationships (SAR) in molecular design.

Patient motivation for participating in clinical trials for depression: validation of the motivation for clinical trials inventory-depression.

The motivation for clinical trials inventory-depression (MCTI-D) was developed and evaluated for assessing motivations to participate in clinical trials on depression. Sixty-four participants completed the MCTI-D: 40 individuals expressing interest in participating in a randomized clinical trial (RCT); and 24 clinic patients receiving traditional care for depression. Content validity was supported by feedback derived from a panel of experts in depression research and respondents completing the measure. The motivation most frequently endorsed for participating in an RCT was the desire to help others and/or to further science. The potential stigma associated with seeing a psychiatrist was reported to have the least influence. Patients expressed a greater likelihood to participate in RCTs that involved psychotherapy than in experimental medication or placebo-controlled trials. Data from the MCTI-D may provide useful information for depression researchers to consider as possible influences on patients' decisions about whether or not they will participate.

Mindfulness and rumination: does mindfulness training lead to reductions in the ruminative thinking associated with depression?

The purpose of this study was to investigate the impact of mindfulness-based stress reduction (MBSR; Kabat-Zinn, 1982, 1990) training on a self-selected adult community sample in the areas of mindfulness, rumination, depressive symptomatology and overall well-being. Targeting rumination was considered particularly important because a tendency toward rumination in nondepressed populations has been found to be predictive of subsequent onset of depression. As hypothesized, completers of the MBSR class showed increases in mindfulness and overall wellbeing, and decreases in rumination and symptoms of depression. Limitations of the study are discussed, as are the implications of these findings.

Transcription of ENOD8 in Medicago truncatula nodules directs ENOD8 esterase to developing and mature symbiosomes.

In Medicago truncatula nodules, the soil bacterium Sinorhizobium meliloti reduces atmospheric dinitrogen into nitrogenous compounds that the legume uses for its own growth. In nitrogen-fixing nodules, each infected cell contains symbiosomes, which include the rhizobial cell, the symbiosome membrane surrounding it, and the matrix between the bacterium and the symbiosome membrane, termed the symbiosome space. Here, we describe the localization of ENOD8, a nodule-specific esterase. The onset of ENOD8 expression occurs at 4 to 5 days postinoculation, before the genes that support the nitrogen fixation capabilities of the nodule. Expression of an ENOD8 promoter-gusA fusion in nodulated hairy roots of composite transformed M. truncatula plants indicated that ENOD8 is expressed from the proximal end of interzone II to III to the proximal end of the nodules. Confocal immunomicroscopy using an ENOD8-specific antibody showed that the ENOD8 protein was detected in the same zones. ENOD8 protein was localized in the symbiosome membrane or symbiosome space around the bacteroids in the infected nodule cells. Immunoblot analysis of fractionated symbiosomes strongly suggested that ENOD8 protein was found in the symbiosome membrane and symbiosome space, but not in the bacteroid. Determining the localization of ENOD8 protein in the symbiosome is a first step in understanding its role in symbiosome membrane and space during nodule formation and function.

The developmental psychopathology of social anxiety in adolescents.

To evaluate a developmental psychopathology approach for understanding adolescent social anxiety, parent-reported predictors of social anxiety were examined in a nonclinical sample of adolescents. Structured diagnostic interviews were obtained from biological parents of 770 participants. Potential risk factors assessed included child characteristics: negative affect, shyness, separation anxiety disorder, and childhood chronic illness, as well as parent characteristics: major depression, panic disorder, and agoraphobia. Adolescent social anxiety was measured multiple times during high school. Findings indicate stability in social anxiety symptoms across time. Parent-reported, childhood negative affect, shyness, and chronic illness as well as parental panic disorder or agoraphobia were associated with adolescent social anxiety. Interactions were observed between parent-reported childhood shyness and gender and between parent-reported childhood shyness and parent-reported childhood chronic illness in the prediction of social anxiety. Parent-reported childhood shyness was a stronger predictor of adolescent social anxiety in females compared to males. The combined effect of subjects being positive for both parent-reported childhood shyness and parent-reported childhood chronic illness was greater than would be expected based on additive effects. This study provides support for a multifactorial and developmentally informed understanding of adolescent social anxiety.

Puberty and gender interact to predict social anxiety symptoms in early adolescence.

This study examines direct and interactive effects of puberty and gender on social anxiety symptoms in early adolescence. One hundred-six participants were assessed at ages 9.5 and 11 years. Results suggest that gender and puberty interact to predict social anxiety symptoms. Advanced puberty was associated with increased symptoms for girls only.

Preferential loss of central (trunk) adiposity in adolescents and young adults after laparoscopic gastric bypass.

BACKGROUND: An increasing number of young people are developing severe obesity with adult-like co-morbidities and undergoing bariatric surgery. Although a number of studies have described major weight loss after bariatric surgery, none have examined the proportions of lean body and fat mass lost or the potentially more important issue of changes in regional fat mass distribution after laparoscopic gastric bypass surgery. METHODS: Five morbidly obese females (mean age 18) were evaluated by standard anthropometric measures and dual energy x-ray absorptiometry at baseline and 1 year after bariatric surgery. The mean and SD values for the anthropometric and dual energy x-ray absorptiometry body composition variables were calculated, and the differences were evaluated using paired t tests. RESULTS: Significant body mass index and weight loss were seen in all subjects at 1 year, with the percentage of excess weight loss at 63.4%. Overall fat mass loss exceeded lean mass loss by threefold in this cohort (P <.01), demonstrating the relative sparing of lean mass. Their waist circumference also decreased significantly. Using dual energy x-ray absorptiometry analysis, the vast majority (83%) of central mass loss consisted of adipose tissue. Central fat loss significantly exceeded peripheral fat loss by 1.6-fold (P = .03). CONCLUSION: These results have demonstrated the preferential loss of central adiposity in morbidly obese young women after 1 year of surgical weight loss and may be more informative than anthropometric measurements alone. Given the association between central adiposity and the risk of subsequent cardiovascular disease, these results are suggestive of reduced cardiac risk.

Anxiety sensitivity: a missing piece to the agoraphobia-without-panic puzzle.

This article reviews the controversy surrounding the diagnosis of agoraphobia without panic attacks and proposes a key role for anxiety sensitivity in explaining agoraphobic avoidance among those who have never experienced panic. Although rare in clinical samples, agoraphobia without panic is commonly observed in population-based surveys, including more recent studies in which misclassification bias is addressed. Differential treatment seeking may partially explain these discrepant findings; however, it remains unclear why agoraphobic avoidance develops in the absence of panic. Because anxiety sensitivity is a dispositional analogue of panic, it is proposed that high anxiety sensitivity is a risk factor for agoraphobic avoidance in the absence of frank panic attacks. Preliminary evidence to support this contention is reviewed.

Lifetime prevalence and pseudocomorbidity in psychiatric research.

CONTEXT: Comorbidity is the rule rather than the exception with psychiatric disorders and is consequently of great interest to both researchers and clinicians. However, many studies of psychiatric comorbidity have been based on lifetime prevalence with mixed-age samples, a practice that (1) biases the assessment of epidemiologic comorbidity and (2) creates the appearance of comorbidity even when disorders are randomly associated. This bias is what we refer to as pseudocomorbidity. OBJECTIVES: To clarify the source of the problem and to discuss strategies that might be adopted to deal hereafter with lifetime prevalence data. METHODS: A simulated example is presented to show that even when there is only random association between disorders, there will appear to be nonrandom comorbidity when lifetime prevalence is used with mixed-age samples. An actual example relating psychosis to phobia is presented to show the bias that can result and to illustrate one way of dealing with lifetime prevalence data. CONCLUSIONS: Use of lifetime prevalence with mixed-age samples, used almost exclusively in psychiatric research, generates problematic results, especially when used for assessment of comorbidity, and should be viewed with some skepticism. Hereafter, we recommend that any future use of lifetime prevalence should require determination of the age of onset, even if only by retrospective report. Comorbidity then should be reported by age.

Unique contributions of anxiety sensitivity to avoidance: a prospective study in adolescents.

Examination of the prospective relation between anxiety sensitivity (AS) and behavioral avoidance is largely absent from the literature. In a longitudinal study of a community sample of 2246 adolescents, participants completed the Anxiety Sensitivity Index (ASI; Reiss, Peterson, Gursky, & McNally (1986). Behaviour Research & Therapy, 24, 1-8), State-Trait Anxiety Inventory (STAI; Spielberger (1983). STAI: Manual for the Stait-Trait Anxiety Inventory. Palo Alto: Consulting Psychologists Press), and the Fear Questionnaire (Marks & Matthews (1979). Behaviour Research & Therapy, 17, 263-267) on an annual basis. To stringently test AS's ability to prospectively predict behavioral avoidance, linear regression was used to test whether AS factors predicted variance in follow-up behavioral avoidance scores after controlling for gender, trait anxiety, panic attacks, and baseline avoidance. Results indicted that the mental and physical subscales of the ASI predicted change in behavioral avoidance. The findings of the study are consistent with the view that AS may serve as a precursor to avoidant behavior and that, regardless of whether or not acute panic has been experienced, those who fear autonomic arousal may be more likely to avoid situations in which those sensations may be present.

Cognitive therapy for obsessive-compulsive disorder.

A multiple baseline across subjects design was used to test the efficacy of cognitive therapy for obsessive-compulsive disorder (OCD). Six people with OCD received 10-18 sessions of weekly, outpatient cognitive therapy. Assessment included both structured interviews and diary data. For three to four of the six patients, stable baseline periods were followed by reductions of symptoms during intervention. Two clients met stringent criteria for Jacobson and Truax's (J. Consulting Clin. Psychol. 59 (1991) 12) recovered status at posttest according to the Yale-Brown Obsessive-Compulsive Scale (Arch. Gen. Psychiatry 46 (1989) 1006). For the group, large pretest-posttest effect sizes were found.

A prospective evaluation of agoraphobia and depression symptoms following panic attacks in a community sample of adolescents.

In a community sample of high schoolers who experienced their first panic attack, we examined the prospective relationships among pre-panic vulnerabilities, panic attack severity, and post-panic agoraphobia and depression symptoms. Students were evaluated yearly over 4 years to test the following four hypotheses: (1) pre-panic anxiety sensitivity, negative affect, and childhood behavioral inhibition will serve as vulnerabilities that predict agoraphobia and depression symptoms following a panic attack; (2) these vulnerabilities will lead to more severe panic attacks; (3) severe and spontaneous panic attacks will predict subsequent agoraphobia and depressive symptoms; and (4) the interaction between panic severity and vulnerabilities will be associated with worse outcomes following a panic attack. Results supported the first three hypotheses, but no evidence emerged for an interactive effect. Findings are discussed in light of recent modernized classical conditioning models that address factors contributing to development of more severe panic related psychopathology after panic attacks.