Multicenter Preparedness Exercise Enables Rapid Development of Cluster-Specific PCR-Based Screening Assays from Bacterial Genomic Data.

PCR-based screening assays targeting strain-specific genetic markers allow the timely detection and specific differentiation of bacterial strains. Especially in situations where an infection cluster occurs, fast assay development is crucial for supporting targeted control measures. However, the turnaround times (TATs) for assay setup may be high due to insufficient knowledge about screening assay methods, workflows, and software tools. Here, two blind-coded and quality-controlled ring trials were performed in which five German laboratories established PCR-based screening assays from genomic data that specifically target selected bacterial clusters within two bacterial monospecies sample panels. While the first ring trial was conducted without a time limit to train the participants and assess assay feasibility, in the second ring trial, a challenging time limit of 2 weeks was set to force fast assay development as soon as genomic data were available. During both ring trials, we detected high interlaboratory variability regarding the screening assay methods and targets, the TATs for assay setup, and the number of screening assays. The participants designed between one and four assays per cluster that targeted cluster-specific unique genetic sequences, genes, or single nucleotide variants using conventional PCRs, high-resolution melting assays, or TaqMan PCRs. Assays were established within the 2-week time limit, with TATs ranging from 4 to 13 days. TaqMan probe delivery times strongly influenced TATs. In summary, we demonstrate that a specific exercise improved the preparedness to develop functional cluster-specific PCR-based screening assays from bacterial genomic data. Furthermore, the parallel development of several assays enhances assay availability.

[Assessment of available and currently applied typing methods including genome-based methods for zoonotic pathogens with a focus on Salmonella enterica]. / Bestandsaufnahme der verfügbaren und aktuell eingesetzten Typisierungsmethoden einschließlich genombasierter Verfahren von Zoonoseerregern am Beispiel von Salmonella enterica.

BACKGROUND: In recent years, whole genome sequencing (WGS) in combination with bioinformatic analyses has become state of the art in evaluating the pathogenicity/resistance potential and relatedness of bacteria. WGS analysis thus represents a central tool in the investigation of the resistance and virulence potential of pathogens, as well as their dissemination via outbreak clusters and transmission chains within the framework of molecular epidemiology. In order to gain an overview of the available genotypic and phenotypic methods used for pathogen typing of Salmonella and Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany at state and federal level, along with the availability of WGS-based typing and corresponding analytical methods, a survey of laboratories was conducted. METHODS: An electronic survey of laboratories working for public health protection and consumer health protection was conducted from February to June 2020. RESULTS AND CONCLUSION: The results of the survey showed that many of the participating laboratories provide a wide range of phenotypic and molecular methods. Molecular typing is most commonly used for species identification of Salmonella. In many cases, WGS-based methods have already been established at federal and state institutions or are in the process of being established. The Illumina sequencing technology is the most widely used technology. The survey confirms the importance of molecular biology and whole genome typing technologies for laboratories in the diagnosis of bacterial zoonotic pathogens.

[Assessment of available and currently applied typing methods of zoonotic pathogens using the example of Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC)]. / Bestandsaufnahme der verfügbaren und aktuell eingesetzten Typisierungsmethoden von zoonotischen Erregern am Beispiel von Shiga Toxin-bildenden bzw. enterohämorrhagischen Escherichia coli (STEC/EHEC).

INTRODUCTION: In order to improve patient care and to increase food safety within the framework of One Health, the project "Integrated Genomic Surveillance of Zoonotic Agents (IGS-Zoo)" aims to develop concepts for a genomic surveillance of Shiga toxin(Stx)-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany. METHODS: An online survey was conducted to assess the currently available and applied STEC/EHEC typing methods in the federal laboratories of veterinary regulation, food control, and public health service. RESULTS: Twenty-six questionnaires from 33 participants were evaluated with regard to STEC/EHEC. The number of STEC/EHEC-suspected samples that the laboratories process per year ranges between 10 and 3500, and out of these they obtain between 3 and 1000 pathogenic isolates. Currently the most frequently used typing method is the determination of Stx- and intimin-coding genes using polymerase chain reaction (PCR). Whole genome sequencing (WGS) is currently used by eight federal state laboratories, and nine are planning to implement it in the future. The most common obstacle for further typing of STEC/EHEC is that isolation from sample material is often unsuccessful despite apparent PCR detection of the stx genes. DISCUSSION: The results of the survey should facilitate the integration of the analysis methods developed in the project and emphasize the target groups' individual needs for corresponding training concepts.

2D pelvic floor ultrasound imaging in identifying levator ani muscle trauma agrees highly with 4D ultrasound imaging.

INTRODUCTION AND HYPOTHESIS: The objective was to evaluate the agreement between 2D and 4D translabial ultrasound (TLUS) technique in showing levator ani muscle (LAM) states after vaginal birth. METHODS: In a prospective observational cohort study between March 2017 and April 2019 we evaluated LAM states (intact, hematoma, partial, complete avulsion) of primiparous women having given birth vaginally with singletons in vertex presentation ≥ 36+0 gestational weeks by using 2D and 4D TLUS within 1-4 days postpartum (assessment A1) and again 6-10 weeks postpartum (assessment A2). Cohen's Kappa analysis was performed for each side separately to evaluate the test agreement between the two ultrasound techniques at every assessment period. RESULTS: A total of 224 women participated at A1 and 213 at A2. The agreement between the two ultrasound techniques was good to very good at A1 (Cohen`s kappa right-sided 0.78, left-sided 0.82) and very good at A2 (Cohen`s kappa both sides 0.88). The agreement was best when assessing an intact LAM or a complete avulsion (Cohen`s kappa between 0.78-0.92 for complete avulsions). CONCLUSIONS: The comparison between 2D and 4D TLUS showed a good to very good agreement in LAM trauma immediately after birth as well as 6-10 weeks postpartum. Therefore, 2D ultrasound could also be a valuable method for demonstrating a LAM abnormality and could be used in settings where 3D/4D ultrasound equipment is not available.

The evolution of levator ani muscle trauma over the first 9 months after vaginal birth.

INTRODUCTION AND HYPOTHESIS: The objective was to investigate the evolution of levator ani muscle (LAM) trauma over the first 9 months after birth and to evaluate their agreement between different assessment periods. METHODS: From March 2017 to April 2019 we prospectively evaluated LAM states (intact, hematoma, partial or complete avulsion) of primiparous women after vaginal birth by using 4D translabial ultrasound (TLUS) at three different assessment periods. All women were examined 1-4 days (A1) and 6-10 weeks (A2) postpartum, and women with a trauma additionally 6-9 months postpartum (A3). Cohen's Kappa analysis was performed to evaluate the test agreement between the assessment periods. RESULTS: Thirty-two percent of the women at A1 had a LAM trauma and 24% at A2. The higher number of LAM injuries at A1 can be explained by hematomas (14%), of which 51% spontaneously resolved at A2, 35% revealed themselves as partial, and 12% as complete avulsions. At A3, we observed anatomical improvement from complete to partial avulsions (23%) and few partial avulsions changed into an intact LAM (3%); none of the complete avulsions changed into an intact LAM. The agreement of 4D TLUS between A1 and A2 was moderate to good (0.64 for the right-sided LAM/0.60 for the left-sided LAM) and between A2 and A3 good to very good (0.76 right-sided/0.84 left-sided). CONCLUSIONS: Levator ani muscle trauma can reliably be diagnosed during all assessment periods. However, the agreement between A1 and A2 was only moderate to good. This can be explained by hematomas inside the LAM that were only observed early postpartum. We observed some anatomical improvement at A3, but no complete avulsion improved to an intact LAM.

Chemoradiotherapy by intensity-modulated radiation therapy with simultaneous integrated boost in locally advanced or oligometastatic non-small-cell lung cancer-a two center experience.

PURPOSE: Integrating moderate hypofractionation to the macroscopic tumor with elective nodal irradiation while sparing the organs at risk (OAR) in chemoradiotherapy of locally advanced non-small-cell lung cancer. METHODS: From 2010-2018, treatment, patient and tumor characteristics of 138 patients from two radiation therapy centers were assessed. Chemoradiotherapy by intensity-modulated radiation therapy (IMRT) with a simultaneous integrated boost (SIB) to the primary tumor and macroscopic lymph node metastases was used. RESULTS: A total of 124 (90%) patients received concurrent chemotherapy. 106 (76%) patients had UICC (Union for International Cancer Control) stage ≥IIIB and 21 (15%) patients had an oligometastatic disease (UICC stage IV). Median SIB and elective total dose was 61.6 and 50.4 Gy in 28 fractions, respectively. Furthermore, 64 patients (46%) had an additional sequential boost to the primary tumor after the SIB-IMRT main series: median 6.6 Gy in median 3 fractions. The median cumulative mean lung dose was 15.6 Gy (range 6.2-29.5 Gy). Median follow-up and radiological follow-up for all patients was 18.0 months (range 0.6-86.9) and 16.0 months (range 0.2-86.9), respectively. Actuarial local control rates at 1, 2 and 3 years were 80.4, 68.4 and 57.8%. Median overall survival and progression-free survival was 30.0 months (95% confidence interval [CI] 23.5-36.4) and 12.1 months (95% CI 8.2-16.0), respectively. Treatment-related toxicity was moderate. Radiation-induced pneumonitis grade 2 and grade 3 occurred in 13 (9.8%) and 3 (2.3%) patients. CONCLUSIONS: Chemoradiotherapy using SIB-IMRT showed promising local tumor control rates and acceptable toxicity in patients with locally advanced and in part oligometastatic lung cancer. The SIB concept, resulting in a relatively low mean lung dose, was associated with low numbers of clinically relevant pneumonitis. The overall survival appears promising in the presence of a majority of patients with UICC stage ≥IIIB disease.

Comparison of treatment plans for hypofractionated high-dose prostate cancer radiotherapy using the Varian Halcyon and the Elekta Synergy platforms.

PURPOSE: To compare radiotherapy plans between an O-ring and a conventional C-arm linac for hypofractionated high-dose prostate radiotherapy in terms of plan quality, dose distribution, and quality assurance in a multi-vendor environment. METHODS: Twenty prostate cancer treatment plans were irradiated on the O-ring Varian Halcyon linac and were re-optimized for the C-arm Elekta Synergy Agility linac. Dose-volume histogram metrics for target coverage and organ at risk dose, quality assurance, and monitor units were retrospectively compared. Patient-specific quality assurance with ion chamber measurements, gamma index analysis, and portal dosimetry was performed using the Varian Portal Dosimetry system and the ArcCHECK® phantom (Sun Nuclear Corporation). Prostate-only radiotherapy was delivered with simultaneous integrated boost (SIB) volumetric modulated arc therapy (VMAT) in 20 fractions of 2.5/3.0 Gy each. RESULTS: For both linacs, target coverage was excellent and plan quality comparable. Homogeneity in PTVBoost was high for Synergy as well as Halcyon with a mean homogeneity index of 0.07 ± 0.01 and 0.05 ± 0.01, respectively. Mean dose for the organs at risk rectum and bladder differed not significantly between the linacs but were higher for the femoral heads and penile bulb for Halcyon. Quality assurance showed no significant differences in terms of ArcCHECK gamma pass rates. Median pass rate for 3%/2 mm was 99.3% (96.7 to 99.8%) for Synergy and 99.8% (95.6 to 100%) for Halcyon. Agreement between calculated and measured dose was high with a median deviation of -0.6% (-1.7 to 0.8%) for Synergy and 0.2% (-0.6 to 2.3%) for Halcyon. Monitor units were higher for the Halcyon by approximately 20% (p < 0.001). CONCLUSION: Hypofractionated high-dose prostate cancer SIB VMAT on the Halcyon system is feasible with comparable plan quality in reference to a standard C-arm Elekta Synergy linac.

Non-rigid image registration of 4D-MRI data for improved delineation of moving tumors.

BACKGROUND: To increase the image quality of end-expiratory and end-inspiratory phases of retrospective respiratory self-gated 4D MRI data sets using non-rigid image registration for improved target delineation of moving tumors. METHODS: End-expiratory and end-inspiratory phases of volunteer and patient 4D MRI data sets are used as targets for non-rigid image registration of all other phases using two different registration schemes: In the first, all phases are registered directly (dir-Reg) while next neighbors are successively registered until the target is reached in the second (nn-Reg). Resulting data sets are quantitatively compared using diaphragm and tumor sharpness and the coefficient of variation of regions of interest in the lung, liver, and heart. Qualitative assessment of the patient data regarding noise level, tumor delineation, and overall image quality was performed by blinded reading based on a 4 point Likert scale. RESULTS: The median coefficient of variation was lower for both registration schemes compared to the target. Median dir-Reg coefficient of variation of all ROIs was 5.6% lower for expiration and 7.0% lower for inspiration compared with nn-Reg. Statistical significant differences between the two schemes were found in all comparisons. Median sharpness in inspiration is lower compared to expiration sharpness in all cases. Registered data sets were rated better compared to the targets in all categories. Over all categories, mean expiration scores were 2.92 ± 0.18 for the target, 3.19 ± 0.22 for nn-Reg and 3.56 ± 0.14 for dir-Reg and mean inspiration scores 2.25 ± 0.12 for the target, 2.72 ± 215 0.04 for nn-Reg and 3.78 ± 0.04 for dir-Reg. CONCLUSIONS: In this work, end-expiratory and inspiratory phases of a 4D MRI data sets are used as targets for non-rigid image registration of all other phases. It is qualitatively and quantitatively shown that image quality of the targets can be significantly enhanced leading to improved target delineation of moving tumors.

Mould-reactive T cells for the diagnosis of invasive mould infection-A prospective study.

Invasive mould infections (IMI) in immunocompromised patients are difficult to diagnose. Early and targeted treatment is paramount, but minimally invasive tests reliably identifying pathogens are lacking. We previously showed that monitoring pathogen-specific CD4+T cells in peripheral blood using upregulation of induced CD154 positive lymphocytes can be used to diagnose acute IMI. Here, we validate our findings in an independent patient cohort. We stimulated peripheral blood cells from at-risk patients with Aspergillus spp. and Mucorales lysates and quantitated mould-reactive CD4/CD69/CD154 positive lymphocytes via flow cytometry. Mould-reactive lymphocytes were quantitated in 115 at-risk patients. In 38 (33%) patients, the test was not evaluable, mainly due to low T cell counts or non-reactive positive control. Test results were evaluable in 77 (67%) patients. Of these, four patients (5%) had proven IMI and elevated mould-reactive T cell signals. Of 73 (95%) patients without proven IMI, 59 (81%) had mould-reactive T cell signals within normal range. Fourteen (19%) patients without confirmed IMI showed elevated T cell signals and 11 of those received antifungal treatment. The mould-reactive lymphocyte assay identified presence of IMI with a sensitivity of 100% and specificity of 81%. The mould-reactive lymphocyte assay correctly identified all patients with proven IMI. Assay applicability is limited by low T cell counts during bone marrow suppression. The assay has the potential to support diagnosis of invasive mould infection to facilitate tailored treatment even when biopsies are contraindicated or cultures remain negative.

Building implementation capacity (BIC): a longitudinal mixed methods evaluation of a team intervention.

BACKGROUND: Managers and professionals in health and social care are required to implement evidence-based methods. Despite this, they generally lack training in implementation. In clinical settings, implementation is often a team effort, so it calls for team training. The aim of this study was to evaluate the effects of the Building Implementation Capacity (BIC) intervention that targets teams of professionals, including their managers. METHODS: A non-randomized design was used, with two intervention cases (each consisting of two groups). The longitudinal, mixed-methods evaluation included pre-post and workshop-evaluation questionnaires, and interviews following Kirkpatrick's four-level evaluation framework. The intervention was delivered in five workshops, using a systematic implementation method with exercises and practical working materials. To improve transfer of training, the teams' managers were included. Practical experiences were combined with theoretical knowledge, social interactions, reflections, and peer support. RESULTS: Overall, the participants were satisfied with the intervention (first level), and all groups increased their self-rated implementation knowledge (second level). The qualitative results indicated that most participants applied what they had learned by enacting new implementation behaviors (third level). However, they only partially applied the implementation method, as they did not use the planned systematic approach. A few changes in organizational results occurred (fourth level). CONCLUSIONS: The intervention had positive effects with regard to the first two levels of the evaluation model; that is, the participants were satisfied with the intervention and improved their knowledge and skills. Some positive changes also occurred on the third level (behaviors) and fourth level (organizational results), but these were not as clear as the results for the first two levels. This highlights the fact that further optimization is needed to improve transfer of training when building teams' implementation capacity. In addition to considering the design of such interventions, the organizational context and the participants' characteristics may also need to be considered to maximize the chances that the learned skills will be successfully transferred to behaviors.

Birth Lacerations in Different Genital Compartments and their Effect on Maternal Subjective Outcome: A Prospective Observational Study.

INTRODUCTION: Lacerations are common in vaginal births, but little is known about tears other than perineal tears and their association with maternal impairment. This study aimed to evaluate the frequency and distribution of birth lacerations and their association with maternal discomfort. METHODS: From 2/2015 to 12/2016, we conducted a prospective observational study on 140 women with singletons in vertex presentation at term, who gave birth vaginally in our center and were affected by a laceration. The lacerations were assigned objectively and subjectively to eight genital tract compartments. The presence and effect of lacerations on maternal health were assessed by questionnaires for the time before birth (T1), 1-4 days (T2), and 6-8 weeks postpartum (T3). RESULTS: The number of affected compartments was 1.33 objectively and 2.99 at T2 and 1.27 at T3 subjectively. The most affected compartment was the right perineum (73%) followed by the right inner posterior (21%) and the right outer anterior (14%) compartment. Subjective and objective assessment concurred in 83% at T2 and 69% of cases at T3. Overall, impairment of women was low, reversible, and not directly associated with the location of lacerations, although women were psychologically affected. CONCLUSION: Birth lacerations predominantly appear at the right perineum. Physical impairment from these lacerations is generally low, reversible, and not directly associated with the location of lacerations, although psychological impairment is not negligible.

Hampered motility promotes the evolution of wrinkly phenotype in Bacillus subtilis.

BACKGROUND: Selection for a certain trait in microbes depends on the genetic background of the strain and the selection pressure of the environmental conditions acting on the cells. In contrast to the sessile state in the biofilm, various bacterial cells employ flagellum-dependent motility under planktonic conditions suggesting that the two phenotypes are mutually exclusive. However, flagellum dependent motility facilitates the prompt establishment of floating biofilms on the air-medium interface, called pellicles. Previously, pellicles of B. subtilis were shown to be preferably established by motile cells, causing a reduced fitness of non-motile derivatives in the presence of the wild type strain. RESULTS: Here, we show that lack of active flagella promotes the evolution of matrix overproducers that can be distinguished by the characteristic wrinkled colony morphotype. The wrinkly phenotype is associated with amino acid substitutions in the master repressor of biofilm-related genes, SinR. By analyzing one of the mutations, we show that it alters the tetramerization and DNA binding properties of SinR, allowing an increased expression of the operon responsible for exopolysaccharide production. Finally, we demonstrate that the wrinkly phenotype is advantageous when cells lack flagella, but not in the wild type background. CONCLUSIONS: Our experiments suggest that loss of function phenotypes could expose rapid evolutionary adaptation in bacterial biofilms that is otherwise not evident in the wild type strains.

Candida-reactive T cells for the diagnosis of invasive Candida infection of the lumbar vertebral spine.

Invasive Candida infection is the fourth most common bloodstream infection. Blood cultures are the current gold standard diagnostic method, however, false negatives remain a clinical challenge. We developed a new technique measuring Candida-reactive T cells as diagnostic read-out for invasive Candida infection. In a pilot study, we followed the treatment course of a patient with an invasive Candida infection of the lumbar vertebral spine. We present the case of a 56-year-old patient with HIV-associated Burkitt lymphoma who developed septic shock during chemotherapy-induced neutropenia. For the first time, we provide flow cytometry-based diagnostics with Candida-reactive T cells for invasive candidiasis with comprehensive MRI imaging. The Candida-reactive T cell assay has potential to complement current diagnostic assays for invasive Candida infection and thus to support targeted treatment.

The development and validation of a scale to explore staff experience of governance of economic efficiency and quality (GOV-EQ) of health care.

BACKGROUND: In publicly funded health care systems, governance models are developed to push public service providers to use tax payers' money more efficiently and maintain a high quality of service. Although this implies change in staff behaviors, evaluation studies commonly focus on organizational outputs. Unintended consequences for staff have been observed in case studies, but theoretical and methodological development is necessary to enable studies of staff experience in larger populations across various settings. The aim of the study is to develop a self-assessment scale of staff experience of the governance of economic efficiency and quality of health care and to assess its psychometric properties. METHODS: Factors relevant to staff members' experience of economic efficiency and quality requirements of health care were identified in the literature and through interviews with practitioners, and then compared to a theoretical model of behavior change. Relevant experiences were developed into sub-factors and items. The scale was tested in collaboration with the Department of Rehabilitation Medicine at a university hospital. 93 staff members participated. The scale's psychometric properties were assessed using exploratory factor analysis, analysis of internal consistency and criterion-related validity. RESULTS: The analysis revealed an eight factor structure (including sub-factors knowledge and awareness, opportunity to influence, motivation, impact on professional autonomy and organizational alignment), and items showed strong factor loadings and high internal consistency within sub-factors. Sub-factors were interrelated and contributed to the prediction of impact on clinical behavior (criterion). CONCLUSIONS: The scale clearly distinguishes between various experiences regarding economic efficiency and quality requirements among health care staff, and shows satisfactory psychometric quality. The scale has broad applications for research and practice, as it serves as a tool for capturing staff members' perspectives when evaluating and improving health care governance. The scale could also be useful for understanding the underlying processes of changes in provider performance and for adapting management strategies to engage staff in driving change that contributes to increased economic efficiency and quality, for the benefit of health care systems, patients and staff.

Analysis of the functional WT1-specific T-cell repertoire in healthy donors reveals a discrepancy between CD4(+) and CD8(+) memory formation.

The Wilms' tumour-1 (WT1) protein is considered a prime target for cancer immunotherapy based on its presumptive immunogenicity and widespread expression across a variety of malignancies. However, little is known about the naturally occurring WT1-specific T-cell repertoire because self-derived antigens typically elicit low frequency responses that challenge the sensitivity limits of current detection techniques. In this study, we used highly efficient cell enrichment procedures based on CD137, CD154, and pHLA class I tetramer staining to conduct a detailed analysis of WT1-specific T cells from the peripheral blood. Remarkably, we detected WT1-specific CD4(+) and CD8(+) T-cell populations in the vast majority of healthy individuals. Memory responses specific for WT1 were commonly present in the CD4(+) T-cell compartment, whereas WT1-specific CD8(+) T cells almost universally displayed a naive phenotype. Moreover, memory CD4(+) and naive CD8(+) T cells with specificity for WT1 were found to coexist in some individuals. Collectively, these findings suggest a natural discrepancy between the CD4(+) and CD8(+) T-cell lineages with respect to memory formation in response to a self-derived antigen. Nonetheless, WT1-specific T cells from both lineages were readily activated ex vivo and expanded in vitro, supporting the use of strategies designed to exploit this expansive reservoir of self-reactive T cells for immunotherapeutic purposes.

BMP4 promotes EMT and mesodermal commitment in human embryonic stem cells via SLUG and MSX2.

Bone morphogenetic proteins (BMPs) initiate differentiation in human embryonic stem cells (hESCs) but the exact mechanisms have not been fully elucidated. We demonstrate here that SLUG and MSX2, transcription factors involved in epithelial-mesenchymal transitions, essential features of gastrulation in development and tumor progression, are important mediators of BMP4-induced differentiation in hESCs. Phosphorylated Smad1/5/8 colocalized with the SLUG protein at the edges of hESC colonies where differentiation takes place. The upregulation of the BMP target SLUG was direct as shown by the binding of phosphorylated Smad1/5/8 to its promoter, which interrupted the formation of adhesion proteins, resulting in migration. Knockdown of SLUG by short hairpin RNA blocked these changes, confirming an important role for SLUG in BMP-mediated mesodermal differentiation. Furthermore, BMP4-induced MSX2 expression leads to mesoderm formation and then preferential differentiation toward the cardiovascular lineage.

Disulfide Bonds of Proteins Displayed on Spores of Bacillus subtilis Can Occur Spontaneously.

Surface display using spores of Bacillus subtilis is widely used to anchor antigens and enzymes of different sources. One open question is whether anchored proteins are able to form disulfide bonds. To answer this important question, we anchored the Escherichia coli alkaline phosphatase PhoA on the spore surface using two different surface proteins, CotB and CotZ. This enzyme needs two disulfide bonds to become active. Subsequently, we purified the spores and assayed for alkaline phosphatase activity. In both cases, we were able to recover enzymatic activity. Next, we asked whether formation of disulfide bonds occurs spontaneous or is catalyzed by thiol-disulfide oxidoreductases upon lysis of the cells. The experiment was repeated in a double-knockout mutant ΔbdbC and ΔbdbD. Since the disulfide bonds are also present on spores prepared from the double knockout, we conclude that oxidative environment after cell lysis is sufficient for disulfide formation of alkaline phosphatase.

Work-family conflict, emotional exhaustion and performance-based self-esteem: reciprocal relationships.

OBJECTIVES: The three constructs of work-family conflict, emotional exhaustion and performance-based self-esteem are all related to tremendous negative consequences for the individual, the organization as well as for society. Even though there are studies that connect two of those constructs, the prospective relations between all three of them have not been studied yet. METHODS: We explored the prospective relations between the three constructs in a large Swedish data set representative of the Swedish workforce. Gender differences in the relations were investigated. Longitudinal data with a 2-year time lag were gathered from 3,387 working men and women who responded to the 2006 and 2008 waves of the Swedish Longitudinal Occupational Survey of Health. Four different cross-lagged models were analysed. RESULTS: In the best fitting model, higher levels of work-family conflict at time 1 were associated with an increased level of performance-based self-esteem at time 2, but not with emotional exhaustion, after controlling for having children, gender, education and age. Also, relationships between emotional exhaustion at time 1 and work-family conflict and performance-based self-esteem at time 2 could be established. Furthermore, relationships between performance-based self-esteem time 1 and work-family conflict and emotional exhaustion time 2 were found. Multiple-group analysis did not show any differences in the relations of the tested constructs over time for either men or women. CONCLUSIONS: We conclude that the three constructs are interrelated and best understood through a reciprocal model. No differences were found between men and women.

Neural activation during processing of aversive faces predicts treatment outcome in alcoholism.

Neuropsychological studies reported decoding deficits of emotional facial expressions in alcohol-dependent patients, and imaging studies revealed reduced prefrontal and limbic activation during emotional face processing. However, it remains unclear whether this reduced neural activation is mediated by alcohol-associated volume reductions and whether it interacts with treatment outcome. We combined analyses of neural activation during an aversive face-cue-comparison task and local gray matter volumes (GM) using Biological Parametric Mapping in 33 detoxified alcohol-dependent patients and 33 matched healthy controls. Alcoholics displayed reduced activation toward aversive faces-neutral shapes in bilateral fusiform gyrus [FG; Brodmann areas (BA) 18/19], right middle frontal gyrus (BA46/47), right inferior parietal gyrus (BA7) and left cerebellum compared with controls, which were explained by GM differences (except for cerebellum). Enhanced functional activation in patients versus controls was found in left rostral anterior cingulate cortex (ACC) and medial frontal gyrus (BA10/11), even after GM reduction control. Increased ACC activation correlated significantly with less (previous) lifetime alcohol intake [Lifetime Drinking History (LDH)], longer abstinence and less subsequent binge drinking in patients. High LDH appear to impair treatment outcome via its neurotoxicity on ACC integrity. Thus, high activation of the rostral ACC elicited by affective faces appears to be a resilience factor predicting better treatment outcome. Although no group differences were found, increased FG activation correlated with patients' higher LDH. Because high LDH correlated with worse task performance for facial stimuli in patients, elevated activation in the fusiform 'face' area may reflect inefficient compensatory activation. Therapeutic interventions (e.g. emotion evaluation training) may enable patients to cope with social stress and to decrease relapses after detoxification.

Nanoscale artificial antigen presenting cells for T cell immunotherapy.

Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for both adoptive and active immunotherapy. Thus far, aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to micron-sized beads. Nanoscale platforms have different trafficking and biophysical interaction properties and may allow development of new immunotherapeutic strategies. We therefore manufactured aAPC based on two types of nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles (50-100 nm in diameter) and avidin-coated quantum dot nanocrystals (~30 nm). Nanoscale aAPC induced antigen-specific T cell proliferation from mouse splenocytes and human peripheral blood T cells. When injected in vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T cell proliferation in vitro and lead to effective T cell stimulation and inhibition of tumor growth in vivo. FROM THE CLINICAL EDITOR: Artifical antigen presenting cells could revolutionize the field of cancer-directed immunotherapy. This team of investigators have manufactured two types of nanoscale particle platform-based aAPCs and demonstrates that both iron-dextran particles and quantum dot nanocrystals enhance tumor rejection in a melanoma model, providing the first description of nanoscale aAPCs that lead to effective T cell stimulation and inhibition of tumor growth.