Mutagenesis techniques for evolutionary engineering of microbes - exploiting CRISPR-Cas, oligonucleotides, recombinases, and polymerases.

The natural process of evolutionary adaptation is often exploited as a powerful tool to obtain microbes with desirable traits. For industrial microbes, evolutionary engineering is often used to generate variants that show increased yields or resistance to stressful industrial environments, thus obtaining superior microbial cell factories. However, even in large populations, the natural supply of beneficial mutations is typically low, which implies that obtaining improved microbes is often time-consuming and inefficient. To overcome this limitation, different techniques have been developed that boost mutation rates. While some of these methods simply increase the overall mutation rate across a genome, others use recent developments in DNA synthesis, synthetic biology, and CRISPR-Cas techniques to control the type and location of mutations. This review summarizes the most important recent developments and methods in the field of evolutionary engineering in model microorganisms. It discusses how both in vitro and in vivo approaches can increase the genetic diversity of the host, with a special emphasis on in vivo techniques for the optimization of metabolic pathways for precision fermentation.

Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast.

Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed Gene Expression Modification by LoxPsym-Cre Recombination (GEMbLeR). GEMbLeR exploits orthogonal LoxPsym sites to independently shuffle promoter and terminator modules at distinct genomic loci. This approach facilitates creation of large strain libraries, in which expression of every pathway gene ranges over 120-fold and each strain harbors a unique expression profile. When applied to the biosynthetic pathway of astaxanthin, an industrially relevant antioxidant, a single round of GEMbLeR improved pathway flux and doubled production titers. Together, this shows that GEMbLeR allows rapid and efficient gene expression optimization in heterologous biosynthetic pathways, offering possibilities for enhancing the performance of microbial cell factories.

Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts.

Site-specific recombinases such as the Cre-LoxP system are routinely used for genome engineering in both prokaryotes and eukaryotes. Importantly, recombinases complement the CRISPR-Cas toolbox and provide the additional benefit of high-efficiency DNA editing without generating toxic DNA double-strand breaks, allowing multiple recombination events at the same time. However, only a handful of independent, orthogonal recombination systems are available, limiting their use in more complex applications that require multiple specific recombination events, such as metabolic engineering and genetic circuits. To address this shortcoming, we develop 63 symmetrical LoxP variants and test 1192 pairwise combinations to determine their cross-reactivity and specificity upon Cre activation. Ultimately, we establish a set of 16 orthogonal LoxPsym variants and demonstrate their use for multiplexed genome engineering in both prokaryotes (E. coli) and eukaryotes (S. cerevisiae and Z. mays). Together, this work yields a significant expansion of the Cre-LoxP toolbox for genome editing, metabolic engineering and other controlled recombination events, and provides insights into the Cre-LoxP recombination process.

Polyhexanide based contact lens storage fluids frequently exhibit insufficient antifungal activity against Fusarium species.

PURPOSE: Fusarium keratitis is a severe infection of the anterior eye, frequently leading to keratoplasty or surgical removal of the affected eye. A major risk factor for infection is the use of contact lenses. Inadequate hygiene precautions and mold-growth permissive storage fluids are important risk factors for fungal keratitis. The aim of this study was to comparatively analyze contact lens storage fluids disinfection efficacy against Fusarium species. METHODS: Eleven commercially available storage fluids were tested. The storage fluids were classified according to their active ingredients myristamidopropyldimethylamine (Aldox), polyhexanide and hydrogen peroxide. Efficacy was tested against isolates belonging to the Fusarium solani and Fusarium oxysporum species complexes as the most common agents of mould keratitis. Tests were carried out based on DIN EN ISO 14729. RESULTS: All Aldox and hydrogen peroxide (H2O2) based fluids were effective against Fusarium spp., while the majority of polyhexanide based storage fluids showed only limited or no antifungal effects. Efficacy of polyhexanide could be restored by the addition of the pH-regulating agent tromethamine - an additive component in one commercially available product. CONCLUSIONS: In summary, the use of Aldox- or hydrogen peroxide-based storage fluids may reduce the risk of Fusarium keratitis, while polyhexanide-based agents largely lack efficacy against Fusarium.

Neoadjuvant Intratumoral Immunotherapy with Cowpea Mosaic Virus Induces Local and Systemic Antitumor Efficacy in Canine Mammary Cancer Patients.

The lack of optimal models to evaluate novel agents is delaying the development of effective immunotherapies against human breast cancer (BC). In this prospective open label study, we applied neoadjuvant intratumoral immunotherapy with empty cowpea mosaic virus-like particles (eCPMV) to 11 companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that two neoadjuvant intratumoral eCPMV injections resulted in tumor reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of injected dogs. Tumor reduction was independent of clinical stage, tumor size, histopathologic grade, and tumor molecular subtype. RNA-seq-based analysis of injected tumors indicated a decrease in DNA replication activity and an increase in activated dendritic cell infiltration in the tumor microenvironment. Immunohistochemistry analysis demonstrated significant intratumoral increases in neutrophils, T and B lymphocytes, and plasma cells. eCPMV intratumoral immunotherapy demonstrated antitumor efficacy without any adverse effects. This novel immunotherapy has the potential for improving outcomes for human BC patients.

A Cas3-base editing tool for targetable in vivo mutagenesis.

The generation of genetic diversity via mutagenesis is routinely used for protein engineering and pathway optimization. Current technologies for random mutagenesis often target either the whole genome or relatively narrow windows. To bridge this gap, we developed CoMuTER (Confined Mutagenesis using a Type I-E CRISPR-Cas system), a tool that allows inducible and targetable, in vivo mutagenesis of genomic loci of up to 55 kilobases. CoMuTER employs the targetable helicase Cas3, signature enzyme of the class 1 type I-E CRISPR-Cas system, fused to a cytidine deaminase to unwind and mutate large stretches of DNA at once, including complete metabolic pathways. The tool increases the number of mutations in the target region 350-fold compared to the rest of the genome, with an average of 0.3 mutations per kilobase. We demonstrate the suitability of CoMuTER for pathway optimization by doubling the production of lycopene in Saccharomyces cerevisiae after a single round of mutagenesis.

Hand Hygiene Evaluation Using Two Different Evaluation Tools and Hand Contamination of Veterinary Healthcare Workers in a Swiss Companion Animal Clinic.

Hand hygiene (HH) is the most important measure to prevent nosocomial infections. HH compliance in companion animal clinics has been reported to be poor. The present study compared an online application with the WHO evaluation form to assess the WHO five moments of HH in a Swiss companion animal clinic. In 202 hand swabs from 87 staff members, total viable count (TVC) before and after patient contact was evaluated and the swabs were tested for selected antimicrobial resistant microorganisms of public health importance. HH compliance (95% confidence interval) was 36.6% (33.8-39.5%) and was similar when assessed with the two evaluation tools. HH differed between hospital areas (p = 0.0035) and HH indications (p < 0.0001). Gloves were worn in 22.0% (18.0-26.6%) of HH observations and were indicated in 37.2% (27.3-48.3%) of these observations. Mean TVC before patient contact was lower (0.52 log CFU/cm2) than after patient contact (1.02 log CFU/cm2) but was similar before patient contact on gloved and ungloved hands. Three hand swabs (1.5% (0.4-4.3%)) were positive for methicillin-resistant Staphylococcus aureus. Gloving should not be regarded as a substitute for HH. Overall, HH in companion animal medicine should urgently be fostered.

Eye Infections Caused by Filamentous Fungi: Spectrum and Antifungal Susceptibility of the Prevailing Agents in Germany.

Fungal eye infections can lead to loss of vision and blindness. The disease is most prevalent in the tropics, although case numbers in moderate climates are increasing as well. This study aimed to determine the dominating filamentous fungi causing eye infections in Germany and their antifungal susceptibility profiles in order to improve treatment, including cases with unidentified pathogenic fungi. As such, we studied all filamentous fungi isolated from the eye or associated materials that were sent to the NRZMyk between 2014 and 2020. All strains were molecularly identified and antifungal susceptibility testing according to the EUCAST protocol was performed for common species. In total, 242 strains of 66 species were received. Fusarium was the dominating genus, followed by Aspergillus, Purpureocillium, Alternaria, and Scedosporium. The most prevalent species in eye samples were Fusarium petroliphilum, F. keratoplasticum, and F. solani of the Fusarium solani species complex. The spectrum of species comprises less susceptible taxa for amphotericin B, natamycin, and azoles, including voriconazole. Natamycin is effective for most species but not for Aspergillus flavus or Purpureocillium spp. Some strains of F. solani show MICs higher than 16 mg/L. Our data underline the importance of species identification for correct treatment.

Acute mental stress drives vascular inflammation and promotes plaque destabilization in mouse atherosclerosis.

AIMS: Mental stress substantially contributes to the initiation and progression of human disease, including cardiovascular conditions. We aim to investigate the underlying mechanisms of these contributions since they remain largely unclear. METHODS AND RESULTS: Here, we show in humans and mice that leucocytes deplete rapidly from the blood after a single episode of acute mental stress. Using cell-tracking experiments in animal models of acute mental stress, we found that stress exposure leads to prompt uptake of inflammatory leucocytes from the blood to distinct tissues including heart, lung, skin, and, if present, atherosclerotic plaques. Mechanistically, we found that acute stress enhances leucocyte influx into mouse atherosclerotic plaques by modulating endothelial cells. Specifically, acute stress increases adhesion molecule expression and chemokine release through locally derived norepinephrine. Either chemical or surgical disruption of norepinephrine signalling diminished stress-induced leucocyte migration into mouse atherosclerotic plaques. CONCLUSION: Our data show that acute mental stress rapidly amplifies inflammatory leucocyte expansion inside mouse atherosclerotic lesions and promotes plaque vulnerability.

ZmOrphan94 Transcription Factor Downregulates ZmPEPC1 Gene Expression in Maize Bundle Sheath Cells.

Spatial separation of the photosynthetic reactions is a key feature of C4 metabolism. In most C4 plants, this separation requires compartmentation of photosynthetic enzymes between mesophyll (M) and bundle sheath (BS) cells. The upstream region of the gene encoding the maize PHOSPHOENOLPYRUVATE CARBOXYLASE 1 (ZmPEPC1) has been shown sufficient to drive M-specific ZmPEPC1 gene expression. Although this region has been well characterized, to date, only few trans-factors involved in the ZmPEPC1 gene regulation were identified. Here, using a yeast one-hybrid approach, we have identified three novel maize transcription factors ZmHB87, ZmCPP8, and ZmOrphan94 as binding to the ZmPEPC1 upstream region. Bimolecular fluorescence complementation assays in maize M protoplasts unveiled that ZmOrphan94 forms homodimers and interacts with ZmCPP8 and with two other ZmPEPC1 regulators previously reported, ZmbHLH80 and ZmbHLH90. Trans-activation assays in maize M protoplasts unveiled that ZmHB87 does not have a clear transcriptional activity, whereas ZmCPP8 and ZmOrphan94 act as activator and repressor, respectively. Moreover, we observed that ZmOrphan94 reduces the trans-activation activity of both activators ZmCPP8 and ZmbHLH90. Using the electromobility shift assay, we showed that ZmOrphan94 binds to several cis-elements present in the ZmPEPC1 upstream region and one of these cis-elements overlaps with the ZmbHLH90 binding site. Gene expression analysis revealed that ZmOrphan94 is preferentially expressed in the BS cells, suggesting that ZmOrphan94 is part of a transcriptional regulatory network downregulating ZmPEPC1 transcript level in the BS cells. Based on both this and our previous work, we propose a model underpinning the importance of a regulatory mechanism within BS cells that contributes to the M-specific ZmPEPC1 gene expression.

ZmbHLH80 and ZmbHLH90 transcription factors act antagonistically and contribute to regulate PEPC1 cell-specific gene expression in maize.

Compartmentation of photosynthetic reactions between mesophyll and bundle sheath cells is a key feature of C4 photosynthesis and depends on the cell-specific accumulation of major C4 enzymes, such as phosphoenolpyruvate carboxylase 1. The ZmPEPC1 upstream region, which drives light-inducible and mesophyll-specific gene expression in maize, has been shown to keep the same properties when introduced into rice (C3 plant), indicating that rice has the transcription factors (TFs) needed to confer C4 -like gene expression. Using a yeast one-hybrid approach, we identified OsbHLH112, a rice basic Helix-Loop-Helix (bHLH) TF that interacts with the maize ZmPEPC1 upstream region. Moreover, we found that maize OsbHLH112 homologues, ZmbHLH80, and ZmbHLH90, also interact with the ZmPEPC1 upstream region, suggesting that these C4 regulators were co-opted from C3 plants. A transactivation assay in maize mesophyll protoplasts revealed that ZmbHLH80 represses, whereas ZmbHLH90 activates, ZmPEPC1 expression. In addition, ZmbHLH80 was shown to impair the ZmPEPC1 promoter activation caused by ZmbHLH90. We showed that ZmbHLH80 and ZmbHLH90 bind to the same cis-element within the ZmPEPC1 upstream region either as homodimers or heterodimers. The formation of homo- and heterodimers with higher oligomeric forms promoted by ZmbHLH80 may explain its negative effect on gene transcription. Gene expression analysis revealed that ZmbHLH80 is preferentially expressed in bundle sheath cells, whereas ZmbHLH90 does not show a clear cell-specific expression pattern. Altogether, our results led us to propose a model in which ZmbHLH80 contributes to mesophyll-specific ZmPEPC1 gene expression by impairing ZmbHLH90-mediated ZmPEPC1 activation in the bundle sheath cells.

Outpatient Psychotherapy Improves Symptoms and Reduces Health Care Costs in Regularly and Prematurely Terminated Therapies.

Background: In view of a shortage of health care costs, monetary aspects of psychotherapy become increasingly relevant. The present study examined the pre-post reduction of impairment and direct health care costs depending on therapy termination (regularly terminated, dropout with an unproblematic reason, and dropout with a quality-relevant reason) and the association of symptom and cost reduction. Methods: In a naturalistic longitudinal study, we examined a disorder heterogeneous sample of N = 584 outpatients who were either treated with cognitive-behavioral, psychodynamic, or psychoanalytic therapy. Depression, anxiety, stress, and somatization were assessed with the Patient Health Questionnaire (PHQ). Annual amounts of inpatient costs, outpatient costs, medication costs, days of hospitalization, work disability days, utilization of psychotherapy, and utilization of pharmacotherapy 1 year before therapy and 1 year after therapy were provided by health care insurances. Symptom and cost reduction were analyzed using t-tests. Associations between symptom and cost reduction were examined using partial correlations and hierarchical linear models. Results: Patients who terminated therapy regularly showed the largest symptom reduction (d = 0.981-1.22). Patients who dropped out due to an unproblematic reason and patients who terminated early due to a quality-relevant reason showed significant but small effects of symptom reductions (e.g., depression: d = 0.429 vs. d = 0.366). For patients with a regular end and those dropping out due to a quality-relevant reason, we observed a significant reduction of work disability (diff in % of pre-test value = 56.3 vs. 42.9%) and hospitalization days (52.8 vs. 35.0%). Annual inpatient costs decreased in the group with a regular therapy end (31.5%). Furthermore, reduction of symptoms on the one side and reduction of work disability days and psychotherapy utilization on the other side were significant correlated (r = 0.091-0.135). Conclusion: Health care costs and symptoms were reduced in each of the three groups. The average symptom and cost reduction of patients with a quality-relevant dropout suggested that not each dropout might be seen as therapy failure.

Treatment outcomes by histology in REVEL: A randomized phase III trial of Ramucirumab plus docetaxel for advanced non-small cell lung cancer.

OBJECTIVES: Ramucirumab, a recombinant human immunoglobulin G1 monoclonal antibody inhibiting vascular endothelial growth factor receptor-2, increased overall survival (OS) combined with docetaxel versus docetaxel alone in non-small cell lung cancer (NSCLC) in the REVEL trial. Pre-specified exploratory analysis examined efficacy and safety by histology. MATERIALS AND METHODS: 1253 patients with NSCLC were randomized to receive ramucirumab (10mg/kg; n=628) plus docetaxel (75mg/m2) or placebo plus docetaxel (n=625) after disease progression on or after platinum-based therapy, with or without bevacizumab or maintenance therapy. OS was analyzed using Kaplan-Meier method. Hazard ratios (HRs) and 95% confidence intervals (CIs) were obtained using an unstratified Cox proportional hazards model. Primary quality-of-life analysis was time to deterioration (TtD) of the Lung Cancer Symptom Scale (LCSS) scores using the Kaplan-Meier method and Cox regression. RESULTS: Median OS for adenocarcinoma was 11.2 months for ramucirumab-docetaxel (n = 377) and 9.8 months for placebo-docetaxel (n=348); HR=0.83 (95% CI: 0.69-0.99). In squamous disease, median OS was 9.5 months for ramucirumab-docetaxel (n=157) versus 8.2 months for placebo-docetaxel (n=171); HR 0.88 (95% CI: 0.69-1.13). Median OS for other nonsquamous was 10.8 months for ramucirumab-docetaxel (n=74) and 9.3 months for placebo-docetaxel (n=78); HR=0.86 (95% CI: 0.59-1.26). Treatment-emergent adverse events were comparable between treatment arms across histologic subgroups. TtD for LCSS scores was similar between treatment arms in the nonsquamous and squamous subgroups. CONCLUSION: REVEL demonstrated similar favorable efficacy and manageable safety for ramucirumab-docetaxel across histologic subgroups of NSCLC.

Impact of Cleaning Procedures on Adhesion of Living Cells to Three Abutment Materials.

PURPOSE: To test the adhesion properties of live gingival fibroblasts to three different implant abutment materials after five different cleaning procedures. MATERIALS AND METHODS: Highly polished discs of lithium disilicate (LS), zirconium dioxide (Zr), and titanium alloy (Ti) were fabricated. The specimens were cleaned by one of five different methods: steam (S), argon plasma (AP), ultrasound and disinfection (UD), ultrasound and sterilization in an autoclave (UA), or photofunctionalization with high-intensity ultraviolet light (PF). Cell detachment force (adhesion) was measured by single-cell force spectroscopy, which is a method to quantify cell adhesion at the single cell level. Data were statistically analyzed using parametric tests (analysis of variance [ANOVA], t tests). RESULTS: Cell detachment forces in the low nN regime were recorded in all experiments. Significant differences in cell adhesion on the different materials were found as a function of the cleaning method (P ≤ .0001). For LS abutments, no significant differences between the cleaning methods could be found (P > .05). For Zr specimens, the AP method showed the highest cell detachment forces, followed by UD, PF, S, and UA (S/UD, S/UA, S/PF, AP/UD, and UD/PF were not significantly different from each other). For Ti abutments, UD showed the highest cell detachment forces, followed by S, AP, and UA/PF (S/UD, S/UA, S/PF, AP/U, and UA/PF were not significantly different from each other). CONCLUSION: All cleaning methods provided comparable cell detachment forces for LS abutments. AP/PF or ultrasonic cleaning were the most suitable methods for strong cell adhesion on Zr. UD provided the best cell adhesion for Ti.

Prediction of human sensory irritation due to ethyl acrylate: the appropriateness of time-weighted average concentration × time models for varying concentrations.

Human data about the potency of ethyl acrylate to evoke sensory irritation is currently not available. Therefore, we conducted an experimental exposure study and the magnitude of chemosensory effects in healthy human volunteers was mathematically modeled by combining the factors current concentration (c) and duration/time (t). In a repeated-measures design, 19 subjects were exposed for 4 h to constant and varying concentrations (including peaks of 5 and 10 ppm) of ethyl acrylate with either a 2.5 or 5 ppm time-weighted average (TWA) concentration. Clean air served as control condition. Nasal lavage fluid, eye blinking frequencies, and rhinomanometry were used as physiological measures of sensory irritation. Several subjective ratings assessed olfactory and trigeminal perceptions. The blinking frequency was significantly increased during the varying 5 ppm condition. Regardless of the TWA concentration, varying exposures caused stronger effects than constant exposures. Our mathematical modeling showed that olfactory perceptions generally decreased over time while ratings of eye irritation increased over time even under the constant 5 ppm condition. Including the current concentration in the mathematical modeling always increased the goodness of fit substantially. The results showed that the intensity of sensory irritation could be predicted best with a complex c × t model. During the 2.5 ppm conditions, only the current concentration predicted the ratings and time-dependent processes could not be observed. However, in both 5 ppm TWA conditions strong eye irritations and increased blinking frequency, only at the end of the 4-h exposures a dose-dependency of these adverse effects was clearly shown.

Mastite bovina por Prototheca zopfii na região oeste do Paraná: Relato de casos / Bovine mastitis by Prototheca zopfii in western Paraná: Case reports / Mastitis bovina por Prototheca zopfii en la región oeste de Paraná: Relato de casos

Relatam-se três casos de mastite clínica em vacas da raça Jersey de uma propriedade localizada na região Oeste do estado do Paraná, refratários ao tratamento com antimicrobianos. Após realização de cultura das amostras de leite, foi identificada Prototheca zopfii, uma alga aclorofilada, unicelular, presente em matéria orgânica e resistente a antimicrobianos. Foi indicado o descarte dos animais positivos e, como medida de profilaxia, a higienização de caixas de abastecimento de água e bebedouros, uma vez que o manejo da ordenha e higienização da ordenhadeira era realizada de maneira adequada na propriedade. De acordo com a literatura consultada, este é o primeiro relato de mastite por Prototheca zopfii na região Oeste do Paraná, salientando a importância da realização da cultura de amostras de leite de vacas portadoras de mastite, especialmente em casos refratários ao tratamento.(AU)

Three cases of clinical mastitis were reported in Jersey cows at a farm located in western Parana, with a history of refractory treatment with antimicrobials. Milk sample cultures presented Prototheca zopfii, an aclorophylated, unicellular algae, present in organic matter and resistant to antimicrobial treatment. The disposal of positive animals was indicated, and the prophylactic measure of cleaning the water storage and drinking containers were encouraged, since the milking and cleaning of the milking machine were properly performed at the farm. According to the literature, this is the first report of mastitis by Prototheca zopfii in western Parana, thus emphasizing the importance of performing cultures to the milk samples of cows presenting mastitis, especially in cases that are refractory to treatment.(AU)

Tres casos de mastitis clínica se ha relatado en vacas de la raza Jersey, de una propiedad ubicada en la región Oeste del Estado de Paraná, resistentes al tratamiento con antimicrobianos. Después de la realización de cultura de las muestras de leche, se identificó Prototheca zopfii, un alga aclorofilada, unicelular, presente en materia orgánica y resistente a antimicrobianos. Se aconsejó el descarte de los animales positivos y, como medida de profilaxis, la higienización de las cajas de abastecimiento de agua y bebederos, una vez que el manejo de ordeña e higienización de la ordeñadera era realizada de forma adecuada en la propiedad. De acuerdo con la literatura consultada, este es el primer relato de mastitis por Prototheca zopfii en la región Oeste de Paraná, resaltando la importancia de realización de cultura de las muestras de leche de vacas portadoras de mastitis, especialmente en casos resistentes al tratamiento.(AU)

Outpatient Psychotherapy Reduces Health-Care Costs: A Study of 22,294 Insurants over 5 Years.

BACKGROUND: The project "Quality Assurance in Ambulatory Psychotherapy in Bavaria" (QS-PSY-BAY) focuses on the quality assurance of outpatient psychotherapy (OPT) in Germany in terms of symptom reduction and cost reduction under naturalistic conditions. In this study, we examined the effectiveness of psychotherapy in terms of pre-post cost reduction. METHOD: The health-care costs of N = 22,294 insurants over a 5-year period were examined in a naturalistic longitudinal design. Six participating health insurance funds provided data on costs related to inpatient treatment, outpatient treatment, drugs, and hospitalization and work disability days. RESULTS: We found that the average annual total costs for inpatient and outpatient treatments as well as drug costs and work disability days increased from the second to the first year before OPT. Besides a large and significant reduction of work disability days (41.8%), hospitalization days (27.4%), and inpatient costs (21.5%) from the first year before versus the first year following OPT, we found evidence for long-term effects: the number of work disability days in the second year after OPT was lower (23.8%), and drug costs were higher than in the second year before OPT (41.5%). CONCLUSION: We conclude that OPT as a part of the health insurance system is an investment which can pay off in the future especially in terms of lower inpatient costs and work disability.

Benefits and challenges in practice-oriented psychotherapy research in Germany: the TK and the QS-PSY-BAY projects of quality assurance in outpatient psychotherapy.

OBJECTIVE: Two patient-focused long-term research projects performed in the German outpatient psychotherapy system are focused on in this article. The TK (Techniker Krankenkasse) project is the first study to evaluate a quality assurance and feedback system with regard to its practical feasibility in German routine care. The other study ("Quality Assurance in Outpatient Psychotherapy in Bavaria"; QS-PSY-BAY) was designed to test a new approach for quality assurance in outpatient psychotherapy using electronic documentation of patient characteristics and outcome parameters. In addition this project provides the opportunity to analyze data on health-related costs for the patients undergoing outpatient psychotherapy. METHOD: Both projects and their results indicating high effect sizes are briefly described. RESULTS: From the perspectives of the research teams, advisory boards and other stakeholders, the experiences with these projects are discussed focusing on obstacles, challenges, difficulties, and benefits in developing and implementing the studies. The triangle collaboration of therapists, researchers, and health insurance companies/health service institutions turned out to be fruitful in both studies. CONCLUSIONS: Despite some controversies between the partners the experiences indicate the importance of practiced-research collaborations to provide relevant information about the delivery of outpatient psychotherapy in the health system.

[Non-completion of insurance-funded outpatient psychotherapy: frequency, risk factors, outcome]. / Abbrüche antragspflichtiger ambulanter Psychotherapien: Häufigkeit, Risikofaktoren, Outcome.

OBJECTIVE: Because premature discontinuation of psychotherapy limits the effectiveness of the interventions, in a naturalistic design we examined the prevalence, predictors, and outcome of premature discontinuation. METHODS: The sample included N = 584 patients with various mental disorders. Risk factors were identified using regression analysis. As outcome Patient Health Questionnaire scales were considered. We compared pre and post averages as well as post averages of premature discontinuation versus regular termination. RESULTS: Risk factors were: female and/or unemployed patient, low patient and/or therapist ratings regarding therapy success, and extraordinarily high therapist ratings of the therapeutic alliance. Despite premature discontinuation we found significant reductions of depression, anxiety, somatic symptoms, and stress (ES = 0.30, …, 0.44). Compared to regular termination though, patients with premature discontinuation were more impaired at last measurement (ES = 0.17, …, 0.37). CONCLUSION: Not each premature discontinuation is a psychotherapeutic failure. Warning signals for looming premature discontinuation are low ratings of therapy success while psychotherapeutic treatment.

Hepatocyte expression of TRAIL pathway regulators correlates with histopathological and clinical parameters in chronic HCV infection.

BACKGROUND AND AIMS: Treatment with pegylated interferon-alpha (PEG-IFN) and ribavirin is the backbone of standard therapy of HCV by mechanisms that are not completely understood. Besides a direct antiviral effect, different immunomodulatory and apoptotic effects have been discussed. Tumor necrosis factor-related apoptosis inducing-ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family with immunomodulatory as well as pro- and antiapoptotic effects and is putatively involved in control of HCV infection. Thus, we analyzed the expression of the TRAIL/TRAIL-receptor system, caspase-8 and cFLIP and examined their prognostic and predictive value for HCV infection and antiviral therapy, respectively. METHODS: We immunohistochemically analyzed liver biopsies of 116 therapy-naive HCV patients before treatment with PEG-IFNα and ribavirin in comparison to healthy liver tissue. Expression levels of TRAIL, TRAIL-R1 to TRAIL-R4, caspase-8 and cFLIP were correlated with sustained virologic response (SVR), genotype and staging of chronic hepatitis. RESULTS: Caspase-8, cFLIP, TRAIL-R2 and TRAIL-R4 were strongly upregulated in HCV patients, whereas TRAIL-R3 was downregulated. SVR correlated with high expression of TRAIL and pro-apoptotic TRAIL-R2 on HCV infected hepatocytes. CONCLUSIONS: Our results suggest a pathophysiological role of TRAIL in both, HCV infection and therapy. Further studies need to elaborate possible TRAIL-related targets for clinical applications.