Participation and outcome in manualized self-help for bulimia nervosa and binge eating disorder — A systematic review and metaregression analysis

There is a growing body of research on manualized self-help interventions for bulimia nervosa (BN) and binge eating disorder (BED). Study and treatment dropout and adherence represent particular challenges in these studies. However, systematic investigations of the relationship between study, intervention and patient characteristics, participation, and intervention outcomes are lacking. We conducted a systematic literature review using electronic databases and hand searches of relevant journals. In metaregression analyses, we analyzed study dropout as well as more specific measures of treatment participation in manualized self-help interventions, their association with intervention characteristics (e.g. duration, guidance, intervention type [bibliotherapy, CD-ROM or Internet based intervention]) and their association with treatment outcomes. Seventy-three publications reporting on 50 different trials of manualized self-help interventions for binge eating and bulimia nervosa published through July 9th 2012 were identified. Across studies, dropout rates ranged from 1% to 88%. Study dropout rates were highest in CD-ROM interventions and lowest in Internet-based interventions. They were higher in samples of BN patients, samples of patients with higher degrees of dietary restraint at baseline, lower age, and lower body mass index. Between 6% and 88% of patients completed the intervention to which they had been assigned. None of the patient, study and intervention characteristics predicted intervention completion rates. Intervention outcomes were moderated by the provision of personal guidance by a health professional, the number of guidance sessions as well as participants' age, BMI, and eating disorder related attitudes (Restraint, Eating, Weight and Shape Concerns) at baseline (after adjusting for study dropout and intervention completion rates). Guidance particularly improved adherence and outcomes in samples of patients with bulimia nervosa; specialist guidance led to higher intervention completion rates and larger intervention effects on some outcomes than non-specialist guidance. Self-help interventions have a place in the treatment of BN and BED, especially if the features of their delivery and indications are considered carefully. To better determine who benefits most from what kind and “dosage” of self-help interventions, we recommend the use of consistent terminology as well as uniform standards for reporting adherence and participation in future self-help trials.     

OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines

The application of designer nucleases allows the induction of DNA double-strand breaks (DSBs) at user-defined genomic loci. Due to imperfect DNA repair mechanisms, DSBs can lead to alterations in the genomic architecture, such as the disruption of the reading frame of a critical exon. This can be exploited to generate somatic knockout cell lines. While high genome editing activities can be achieved in various cellular systems, obtaining cell clones that contain all-allelic frameshift mutations at the target locus of interest remains a laborious task. To this end, we have developed an easy-to-follow deep sequencing workflow and the evaluation tool OutKnocker (www.OutKnocker.org), which allows convenient, reliable, and cost-effective identification of knockout cell lines.Advances in targeted genome editing technologies have opened new avenues for addressing challenging questions in the field of life sciences. The recent introduction of designer nucleases such as ZFNs (Carroll 2011), TALENs (Miller et al. 2011), or CRISPR/Cas systems (Jinek et al. 2012; Cong et al. 2013; Mali et al. 2013) allows for highly efficient, flexible, and specific induction of DNA double-strand breaks (DSB) in eukaryotic genomes. DSBs trigger two distinct repair pathways that can be exploited to specifically modify gene architecture (Carroll 2011). While the process of homologous recombination (HR) accurately repairs DSBs using the sister chromatid as a template, nonhomologous end-joining (NHEJ) repair is an error-prone end-joining mismatch repair pathway that frequently leads to genetic alterations (Lieber 2010; Chiruvella et al. 2013). Providing a donor construct with appropriate homology arms as a template, the pathway of DSB-triggered HR can be used to site-specifically introduce heterologous genetic material into cells (Carroll 2011). For example, it is possible to generate gene knockouts in somatic cell lines by introducing marker cassettes with premature stop codons. However, this strategy is time consuming and laborious and therefore not optimal for high-throughput approaches. The DSB-induced NHEJ repair pathway, on the other hand, leads to insertions or deletions (indels) (Lieber 2010) that can result in frameshift mutations and thus loss-of-function phenotypes if located within early coding exons.While in HR-based genome editing approaches marker genes can be introduced to select for the desired genotype starting from a polyclonal cell culture, frameshift mutations induced by NHEJ are difficult to select for unless the editing event provides a survival benefit. To this end, single-cell cloning and subsequent sequencing of the genetic locus is required to obtain cells with the desired gene disruption. Sanger sequencing is most commonly used to identify modified alleles. However, in addition to being costly, this method requires a locus-specific PCR to be subcloned in order to sequence single alleles, and thus is not practical for large-scale projects. Moreover, the ploidy of the genome may vary between cell lines and even between loci, which may require the sequencing of a considerable number of PCR subclones to reliably identify cell clones with all-allelic frameshift mutations. Small benchtop deep sequencing machines can achieve a far greater throughput. Theoretically, even low sequencing capacities are sufficient to analyze hundreds of clones in parallel, without the need to subclone PCR products. However, analysis of deep sequencing data remains challenging and no streamlined workflow has been described that would allow full exploitation of deep sequencing capacities in gene disruption projects.Here we describe OutKnocker, a web-based application that facilitates the analysis of deep sequencing data to identify knockout cells obtained from designer nuclease-mediated genome editing. We aimed at developing an evaluation tool to genotype single-cell clones at a confined genomic region for indel mutations, as they are typically induced by designer nuclease targeting. As such, we established an algorithm that focuses on identifying a single indel event per sequencing read around a predefined target site, while ignoring SNPs or point mutations originated during sequencing. Optionally, our software also allows the detection of specific point mutations introduced by targeted mutagenesis. To fully exploit sequencing capacities, OutKnocker was designed to analyze data of sequencing runs that have been multiplexed to evaluate the same or different genomic target regions in parallel, while only requiring a limited number of unidirectional sequencing reads. OutKnocker is operated from a web browser making it conveniently accessible to any user.     

The phosphoproteome and its physiological dynamics in Staphylococcus aureus

Phosphorylation events on proteins during growth and stress/starvation can represent crucial regulation processes inside the bacterial cell. Therefore, serine, threonine and tyrosine phosphorylation patterns were analyzed by two powerful complementary proteomic methods for the human pathogen Staphylococcus aureus. Using 2D-gel analysis with a phosphosensitive stain (Pro-Q Diamond) and gel-free titanium dioxide based phosphopeptide enrichment, 103 putative phosphorylated proteins with successfully mapped 68 different phosphorylation sites were found in the soluble proteome of S. aureus. Additionally, in a proof of concept study, 8 proteins phosphorylated on arginine residues have been identified. Most important for functional analyses of S. aureus, proteins related to pathogenicity and virulence were found to be phosphorylated: the virulence regulator SarA, the potential antimicrobial target FbaA and the elastin-binding protein EbpS. Besides newly identified phosphorylation sites we compared our dataset with existing data from literature and subsequent experiments revealed additional phosphorylation events on highly conserved localizations in FbaA. Differential analysis of phosphorylation signals on the 2D-gels showed significant changes in phosphorylation under different physiological conditions for 10 proteins. Among these, we were able to detect newly appearing signals for phosphorylated isoforms of FdaB and HchA under nitrosative stress conditions.     

Concentration-dependent Effect of Sodium Hypochlorite on Stem Cells of Apical Papilla Survival and Differentiation

Introduction

Intracanal disinfection is a crucial step in regenerative endodontic procedures. Most published cases suggest the use of sodium hypochlorite (NaOCl) as the primary irrigant. However, the effect of clinically used concentrations of NaOCl on the survival and differentiation of stem cells is largely unknown. In this study, we tested the effect of various concentrations of NaOCl on the stem cells of the apical papilla (SCAPs) survival and dentin sialophosphoprotein (DSPP) expression.

Methods

Standardized root canals were created in extracted human teeth and irrigated with NaOCl (0.5%, 1.5%, 3%, or 6%) followed by 17% EDTA or sterile saline. SCAPs in a hyaluronic acid–based scaffold were seeded into the canals and cultured for 7 days. Next, viable cells were quantified using a luminescence assay, and DSPP expression was evaluated using quantitative real-time polymerase chain reaction.

Results

There was a significant reduction in survival and DSPP expression in the group treated with 6% NaOCl compared with the untreated control group. Comparable survival was observed in the groups treated with the lower concentrations of NaOCl, but greater DSPP expression was observed in the 1.5% NaOCl group. In addition, 17% EDTA resulted in increased survival and DSPP expression partially reversing the deleterious effects of NaOCl.

Conclusions

Collectively, the results suggest that dentin conditioning with high concentrations of NaOCl has a profound negative effect on the survival and differentiation of SCAPs. However, this effect can be prevented with the use of 1.5% NaOCl followed by 17% EDTA. The inclusion of this irrigation regimen might be beneficial in regenerative endodontic procedures.     

Cover Image

Prehensile tails are defined as having the ability to grasp objects and are commonly used as a fifth appendage during arboreal locomotion. Despite the independent evolution of tail prehensility in numerous mammalian genera, data relating muscle structure, physiology, and function of prehensile tails are largely incomplete. Didelphid marsupials make an excellent model to relate myosin heavy chain (MHC) isoform fiber type with structure/function of caudal muscles, as all opossums have a prehensile tail and tail use varies between arboreal and terrestrial forms. Expanding on our previous work in the Virginia opossum, this study tests the hypothesis that arboreal and terrestrial opossums differentially express faster versus slower MHC isoforms, respectively. MHC isoform expression and percent fiber type distribution were determined in the flexor caudae longus (FCL) muscle of Caluromys derbianus (arboreal) and Monodelphis domestica (terrestrial), using a combination of gel electrophoresis and immunohistochemistry analyses. C. derbianus expresses three MHC isoforms (1, 2A, 2X) that are distributed (mean percentage) as 8.2% MHC‐1, 2.6% 1/2A, and 89.2% 2A/X hybrid fibers. M. domestica also expresses MHC‐1, 2A, and 2X, in addition to the 2B isoform, distributed as 17.0% MHC‐1, 1.3% 1/2A, 9.0% 2A, 75.2% 2A/X, and 0.3% 2X/B hybrid fibers. The distribution of similar isoform fiber types differed significantly between species (P < 0.001). Although not statistically significant, C. derbianus was observed to have larger cross‐sectional area (CSA) for each corresponding fiber type along with a greater amount of extra‐cellular matrix. An overall faster fiber type composition (and larger fibers) in the tail of an arboreal specialist supports our hypothesis, and correlates with higher muscle force required for tail hanging and arboreal maneuvering on terminal substrates. Conversely, a broader distribution of highly oxidative fibers in the caudal musculature is well suited for tail nest building/remodeling behaviors of terrestrial opossums. Anat Rec, 297:1364–1376, 2014. © 2014 Wiley Periodicals, Inc.     

Diagnostic accuracy and clinical utility of biopsy in musculoskeletal lesions: A comparison of fine‐needle aspiration,core, and open biopsy techniques

Selection of biopsy technique for musculoskeletal lesions is complex. Fine‐needle aspiration (FNA) is uncommonly used due to concerns regarding accuracy. We compared diagnostic accuracy of FNA, core, and open biopsy in a series of musculoskeletal lesions. Records of the University of Utah were searched for biopsy and resection specimens of musculoskeletal lesions. Results of corresponding imaging studies were obtained. Biopsy and FNA diagnoses were correlated with resection diagnoses. For each technique, diagnostic accuracy, utility, and frequency of subsequent biopsy were calculated. Open biopsy had the highest diagnostic accuracy (89%) followed by FNA (82%) and core biopsy (78%). Clinically significant errors occurred with all methods. The likelihood of an open biopsy being performed was affected by prior performance of an FNA or core biopsy and by diagnostic imaging and FNA results. Diagn. Cytopathol. 2014;42:476–486. © 2014 Wiley Periodicals, Inc.     

Significance of atypia in pancreatic and bile duct brushings: Follow‐Up analysis of the categories atypical and suspicious for malignancy

Brushing cytology is frequently utilized for the investigation of pancreatic and biliary strictures but is associated with low diagnostic sensitivity. The Papanicolaou Society of Cytopathology has presented a system for diagnostic classification which includes the categories benign, atypical, suspicious for malignancy and malignant. We studied a series of 216 pancreatic and biliary brushings with either histologic follow‐up or a minimum of 6 months clinical follow‐up to determine outcomes for the diagnostic categories (“benign,” “atypical, favor reactive,” “atypical, not otherwise specified,” “atypical, suspicious” and “malignant”). Eighty‐six of the 216 (39.8%) were designated “atypical” with 10 of these designated as “atypical favor reactive.” Forty‐five were called “atypical not otherwise specified” and 31 were interpreted as “atypical suspicious for malignancy.” On follow‐up, 2 of 10 (20%) “atypical favor reactive” were eventually associated with a malignant diagnosis and 23 of 31 (74.2%) “atypical, suspicious for malignancy” demonstrated a malignant outcome. The remaining 45 brushings in the “atypical” category were “atypical not otherwise specified,” and 62% of these were associated with malignancy on follow‐up. Stratification of the “atypical” category into “atypical favor reactive,” “atypical, not otherwise specified” and “atypical, suspicious for malignancy” improves diagnostic accuracy. The “atypical suspicious for malignancy” category has a follow‐up similar to the “malignant” category while the “atypical favor reactive” category is associated with a clinical outcome similar to that of the “benign” category. Diagn. Cytopathol. 2014;42:285–291. © 2013 Wiley Periodicals, Inc.     

Efficacy of various side-to-side toothbrushes for noncontact biofilm removal

Objectives

The aim of this study was to evaluate the efficacy of four different powered toothbrushes with side-to-side action for noncontact biofilm removal in vitro.

Materials and methods

A three-species biofilm was formed in vitro on protein-coated titanium disks using a flow chamber combined with a static biofilm growth model. Subsequently, the biofilm-coated substrates were exposed to four different side-to-side toothbrushes (A, B, C, and D) with various brushing times (2, 4, and 6 s) and brushing (bristle-to-disk) distances (0, 2, and 4 mm). The biofilm volumes were measured using volumetric analyses with confocal laser scanning microscope images and Imaris version 7.5.2 software.

Results

The median percentages of biofilm reduction by the analyzed toothbrushes ranged from 9 % to 80 %. The abilities of the tested toothbrushes to remove the in vitro biofilm differed significantly (p?<?0.05). Two of the tested toothbrushes (C and D) were capable of significant biofilm reduction by noncontact brushing.

Conclusions

It was possible to reduce a three-species in vitro biofilm by noncontact brushing with two out of four side-to-side toothbrushes.

Clinical relevance

Toothbrushes C and D show in vitro a high efficacy in biofilm removal without bristle contact.     

Tracking of Tumor Cell–Derived Extracellular Vesicles In Vivo Reveals a Specific Distribution Pattern with Consecutive Biological Effects on Target Sites of Metastasis

Purpose

Extracellular vesicles, small vesicles carrying inter alia proteins, miRNA and RNA, are important mediators of intercellular communication. The purpose of this study was to assess the distribution of extracellular vesicles from highly malignant breast cancer and their subsequent effect on the immune cell infiltrate in target organs of metastasis.

Procedures

Extracellular vesicles were isolated from the tissue culture supernatant of highly malignant 4T1 breast cancer cells or the serum of healthy BALB/c mice. The purity of the isolate was verified by electron microscopy and western blotting. Extracellular vesicles were additionally subjected to proteome analysis. After labeling with the fluorescent dye DiR, extracellular vesicles were injected into healthy BALB/c mice and their in vivo distribution was assessed using fluorescence reflectance imaging (FRI). Following ex vivo imaging of the organs, lung tissue samples were analyzed for extracellular vesicle-mediated changes of myeloid cells and T cell numbers, using flow cytometry. Proteome analysis revealed major differences in the cargo of tumor cell–derived versus extracellular vesicles from healthy serum.

Results

In contrast to control extracellular vesicles, DiR-labeled extracellular vesicles from tumor cells preferentially accumulated in lung, liver, and spine. Subsequent flow cytometry of the immune cell composition of lung tissue samples revealed an increase of cytotoxic CD8+ T cells and a decrease of CD4+ T-helper cells as well as an increase in mature macrophages in response to tumor cell EV.

Conclusions

In conclusion, distribution of tumor cell–derived extracellular vesicles follows a specific pattern and can be monitored, using dedicated imaging. Extracellular vesicles alter the immune cell composition in target organs of metastasis, using a specific proteome cargo.