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Background: Identifying and addressing heavy drinking represents a major public health priority worldwide. Whilst the majority of alcohol screening and brief intervention (ASBI) research has been conducted in western, high-income countries, evidence is growing that ASBI can also impact positively on heavy drinkers in low- and middle-income country populations. This mixed methods study aims to assess the feasibility of conducting a fully randomised controlled trial of the effectiveness of ASBI in primary care in Kazakhstan and explore the feasibility and acceptability of implementing ASBI in this setting from patients' and physicians' perspectives., Methods: Six primary health care units in the region of Pavlodar will be cluster randomised to either an intervention (WHO manualised 5 min alcohol brief intervention plus alcohol leaflet) or control group (simple feedback plus alcohol leaflet). Primary feasibility measures will be rates of participation at baseline and retention of eligible patients at the 3-month follow-up point. Patient/physician questionnaires and physician focus groups will assess additional dimensions of feasibility, as well as acceptability, according to the RE-AIM framework: Reach (rates of eligible patients screened/received advice); Effectiveness (change in AUDIT-C score); Adoption (rate/representativeness of participating physicians); Implementation (quality of ASBI/barriers and facilitators to delivery); and Maintenance (potential sustainability of intervention)., Discussion: This is the first trial of the feasibility and acceptability of ASBI in Kazakhstan. As the planning and assessment of implementation determinants is based on the RE-AIM framework, the project outcomes will be relevant for the future development, tailoring and implementation of ASBI in Kazakhstan., Trial Registration: DRKS, DRKS00015882, Registered 17 December 2018., Competing Interests: Competing interestsThe authors declare that they have no competing interests, (© The Author(s). 2020.)

Cognitive deficits, core features of mental illness, largely result from dysfunction of prefrontal networks. This dysfunction emerges during early development, before a detectable behavioral readout, yet the cellular elements controlling the abnormal maturation are still unknown. Here, we address this open question by combining in vivo electrophysiology, optogenetics, neuroanatomy, and behavioral assays during development in mice mimicking the dual genetic-environmental etiology of psychiatric disorders. We report that pyramidal neurons in superficial layers of the prefrontal cortex are key elements causing disorganized oscillatory entrainment of local circuits in beta-gamma frequencies. Their abnormal firing rate and timing relate to sparser dendritic arborization and lower spine density. Administration of minocycline during the first postnatal week, potentially acting via microglial cells, rescues the neuronal deficits and restores pre-juvenile cognitive abilities. Elucidation of the cellular substrate of developmental miswiring causing later cognitive deficits opens new perspectives for identification of neurobiological targets amenable to therapies., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: According to the German guidelines, people with severe alcohol use disorders (AUDs) should receive withdrawal treatment. Compared to somatic withdrawal treatment (SWT), extended duration and psychosocial elements of so-called "qualified withdrawal treatment" (QWT) aim to reduce relapse rates. Despite promising results of prospective studies on QWT, only few German inpatients seeking withdrawal treatment receive QWT. We estimated the potential effects on mortality and morbidity for higher proportions of treatment-seeking patients receiving QWT rather than SWT in the German city of Bremen., Methods: In 2016 and 2017, 2051 inpatients were admitted to two specialised hospitals for withdrawal treatment. The potential beneficial effects of QWT over SWT were estimated by simulating treatment outcomes taken from two prospective studies. Outcomes comprised number and length of all-cause hospitalisations within 5 years, as well as abstinence and all-cause mortality rates within 28 months. Outcomes were estimated for actual and increased rates of QWT (25, 50%) among inpatients seeking alcohol treatment., Results: In the selected hospitals, 170 patients (8%) received QWT. If 25% of AUD inpatients were to receive QWT, benefits in abstinence rates (+ 18%), the total number of hospitalisations (- 9%) and hospital days (- 10%) could be expected. If 50% of AUD inpatients were to receive QWT, benefits in abstinence rates (+ 45%), the total number of hospitalisations (- 23%) and hospital days (- 26%) were more pronounced, in addition to reductions in mortality (- 20%)., Conclusion: Increasing the proportion of people with severe AUD enrolled in extended withdrawal treatment programs (such as QWT) may contribute to reduce overall alcohol-attributable burden of disease. Randomised controlled trials or other prospective studies controlling for confounding factors are needed to determine the potential at the population level.

Background: Early detection of risky alcohol use and severe alcohol use disorders (AUDs) is crucial to avoid adverse health consequences. The German "Guidelines on Screening, Diagnosis and Treatment of Alcohol Use Disorders" recommend to routinely screen patients for hazardous alcohol use and to subsequently conduct brief interventions, for example in primary healthcare. For severe AUDs, provision of withdrawal treatment is recommended in inpatient settings if complications are anticipated., Objectives: To estimate the proportion of people with hazardous alcohol use or severe AUDs receiving healthcare as stipulated by the guidelines., Materials and Methods: The prevalence of hazardous use (female ≥12 g; male ≥24 g) and severe AUDs (female ≥60 g; male ≥90 g) was estimated using per capita consumption of pure alcohol. Treatment rates were estimated using survey data (for hazardous use) and inpatient admissions (for severe AUDs). All estimates refer to the adult population (15 years or older) of the federal state of Bremen for 2016., Results: Physicians screened 2.9% of all people with hazardous alcohol use and conducted brief interventions with 1.4%. Among people with severe AUDs, 7.1% received inpatient treatment. Among people with severe AUDs who required inpatient treatment, 14.1% received withdrawal treatment in inpatient settings. Treatment rates below average were registered among 21- to 39-year-olds., Conclusions: In Bremen, provision of guideline-conform healthcare for hazardous alcohol use and severe AUDs is insufficient, especially among 21- to 39-year-olds.

With climate warming, a widespread expectation is that events in spring, such as flowering, bird migrations, and insect bursts, will occur earlier because of increasing temperature. At high latitudes, increased ocean temperature is suggested to advance the spring phytoplankton bloom due to earlier stabilization of the water column. However, climate warming is also expected to cause browning in lakes and rivers due to increases in terrestrial greening, ultimately reducing water clarity in coastal areas where freshwater drain. In shallow areas, decreased retention of sediments on the seabed will add to this effect. Both browning and resuspension of sediments imply a reduction of the euphotic zone and Sverdrup's critical depth leading to a delay in the spring bloom, counteracting the effect of increasing temperature. Here, we provide evidence that such a transparency reduction has already taken place in both the deep and shallow areas of the North Sea during the 20th century. A sensitivity analysis using a water column model suggests that the reduced transparency might have caused up to 3 weeks delay in the spring bloom over the last century. This delay stands in contrast to the earlier bloom onset expected from global warming, thus highlighting the importance of including changing water transparency in analyses of phytoplankton phenology and primary production. This appears to be of particular relevance for coastal waters, where increased concentrations of absorbing and scattering substances (sediments, dissolved organic matter) have been suggested to lead to coastal darkening., (© 2019 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

Background: The aim of the study was to investigate psychological distress and health-related quality of life (HrQoL) in patients with bone marrow edema syndrome (BMES) of the hip or knee joint., Methods: This retrospective study included patients with the diagnosis BMES treated in the period 2016-2017. As well as analyzing the epidemiological data (age, sex, vascular disease, hypertension, etc.), we used the Hospital Anxiety and Depression Scale (HADS) to document anxiety and depression and the five-level version of the EuroQol Group's EQ-5D instrument (EQ-5D-5L) to assess HrQoL and compared it to historical controls of the healthy population., Results: The study group comprised 56 patients (26 females, 30 males) with a mean age of 55.8 (range, 15-84) years. HADS: there was no difference between the study and control cohorts in the rates of anxiety (P=0.595) or depression (P=0.241). EQ-5D-5L: the HrQoL was significantly lower in the patients with BMES than in the healthy controls both for parameters of the EQ-5D-5L index and in the various age groups. No difference in HrQoL was seen between BMES of the hip and the knee or among the different radiological stages of BMES., Conclusions: The patients with BMES displayed a clear reduction in HrQoL, but this was not associated with psychological distress with regard to significant anxiety and depression. Patients with BMES and a high score for anxiety and depression are at great risk of chronic pain, and we recommend they should receive psychological counseling., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare., (2019 Annals of Translational Medicine. All rights reserved.)

Lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria provide the first line of defense against antibiotics and other harmful compounds. LPS biosynthesis critically depends on LpxC catalyzing the first committed enzyme in this process. In Escherichia coli , the cellular concentration of LpxC is adjusted in a growth rate-dependent manner by the FtsH protease making sure that LPS biosynthesis is coordinated with the cellular demand. As a result, LpxC is stable in fast-growing cells and prone to degradation in slow-growing cells. One of the factors involved in this process is the alarmone guanosine tetraphosphate (ppGpp) but previous studies suggested the involvement of yet unknown factors in LpxC degradation. We established a quantitative proteomics approach aiming at the identification of proteins that are associated with LpxC and/or FtsH at high or low growth rates. The identification of known LpxC and FtsH interactors validated our approach. A number of proteins involved in fatty acid biosynthesis and degradation, including the central regulator FadR, were found in the LpxC and/or FtsH interactomes. Another protein associated with LpxC and FtsH was WaaH, a LPS-modifying enzyme. When overproduced, several members of the LpxC/FtsH interactomes were able to modulate LpxC proteolysis. Our results go beyond the previously established link between LPS and phospholipid biosynthesis and uncover a far-reaching network that controls LPS production by involving multiple enzymes in fatty acid metabolism, phospholipid biosynthesis and LPS modification.

Background: Despite a decreasing population of children and adolescents, the cumulative total amount of dispensed methylphenidate (MPH), the first-choice treatment of attention-deficit/hyperactivity disorder (ADHD) in this age group, has increased dramatically in Germany. We investigated potential reasons for this increase such as changes in the ADHD prevalence over time and other potential explanations including the cumulative amount of dispensed MPH per person., Methods: Based on German claims data, we calculated standardized annual ADHD prevalence rates, proportions of ADHD cases treated with MPH and/or psychotherapy, and mean cumulative defined daily doses of ADHD drugs for 3- to 17-year-old children and adolescents from 2004 to 2013., Results: The ADHD prevalence increased continuously from 2004 to 2011 and remained stable thereafter. In ADHD cases, there was little variation in the proportion of individuals treated with drugs and in the frequency of psychotherapeutic treatment during the whole study period. The annual cumulative mean amount of MPH defined daily doses increased by approximately 30% from 2004 to 2008., Conclusions: Our analyses suggest that the increase in MPH use in Germany was mainly influenced by an increasing ADHD prevalence and increasing amounts of dispensed MPH per person.

Controlling the cellular abundance and proper function of proteins by proteolysis is a universal process in all living organisms. In Escherichia coli, the ATP-dependent Lon protease is crucial for protein quality control and regulatory processes. To understand how diverse substrates are selected and degraded, unbiased global approaches are needed. We employed a quantitative Super-SILAC (stable isotope labeling with amino acids in cell culture) mass spectrometry approach and compared the proteomes of a lon mutant and a strain producing the protease to discover Lon-dependent physiological functions. To identify Lon substrates, we took advantage of a Lon trapping variant, which is able to translocate substrates but unable to degrade them. Lon-associated proteins were identified by label-free LC-MS/MS. The combination of both approaches revealed a total of 14 novel Lon substrates. Besides the identification of known pathways affected by Lon, for example, the superoxide stress response, our cumulative data suggests previously unrecognized fundamental functions of Lon in sulfur assimilation, nucleotide biosynthesis, amino acid and central energy metabolism., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nutrient acquisition is a critical determinant for the competitive advantage for auto- and osmohetero- trophs alike. Nutrient limited growth is commonly described on a whole cell basis through reference to a maximum growth rate (Gmax) and a half-saturation constant (KG). This empirical application of a Michaelis-Menten like description ignores the multiple underlying feedbacks between physiology contributing to growth, cell size, elemental stoichiometry and cell motion. Here we explore these relationships with reference to the kinetics of the nutrient transporter protein, the transporter rate density at the cell surface (TRD; potential transport rate per unit plasma-membrane area), and diffusion gradients. While the half saturation value for the limiting nutrient increases rapidly with cell size, significant mitigation is afforded by cell motion (swimming or sedimentation), and by decreasing the cellular carbon density. There is thus potential for high vacuolation and high sedimentation rates in diatoms to significantly decrease KG and increase species competitive advantage. Our results also suggest that Gmax for larger non-diatom protists may be constrained by rates of nutrient transport. For a given carbon density, cell size and TRD, the value of Gmax/KG remains constant. This implies that species or strains with a lower Gmax might coincidentally have a competitive advantage under nutrient limited conditions as they also express lower values of KG. The ability of cells to modulate the TRD according to their nutritional status, and hence change the instantaneous maximum transport rate, has a very marked effect upon transport and growth kinetics. Analyses and dynamic models that do not consider such modulation will inevitably fail to properly reflect competitive advantage in nutrient acquisition. This has important implications for the accurate representation and predictive capabilities of model applications, in particular in a changing environment.

AAA + proteases (ATPases associated with various cellular activities) shape the cellular protein pool in response to environmental conditions. A prerequisite for understanding the underlying recognition and degradation principles is the identification of as many protease substrates as possible. Most previous studies made use of inactive protease variants to trap substrates, which were identified by 2D-gel based proteomics. Since this method is known for limitations in the identification of low-abundant proteins or proteins with many transmembrane domains, we established a trapping approach that overcomes these limitations. We used a proteolytically inactive FtsH variant (FtsH trap ) of Escherichia coli (E. coli) that is still able to bind and translocate substrates into the proteolytic chamber but no longer able to degrade proteins. Proteins associated with FtsH trap or FtsH wt (proteolytically active FtsH) were purified, concentrated by an 1D-short gel, and identified by LC-coupled mass spectrometry (LC-MS) followed by label-free quantification. The identification of four known FtsH substrates validated this approach and suggests that it is generally applicable to AAA + proteases.

Turbulence and coherent circulation structures, such as submesoscale and mesoscale eddies, convective plumes and Langmuir cells, play a critical role in shaping phytoplankton spatial distribution and population dynamics. We use a framework of advection-reaction-diffusion equations to investigate the effects of turbulent transport on the phytoplankton population growth and its spatial structure in a vertical two-dimensional vortex flow field. In particular, we focus on how turbulent flow velocities and sinking influence phytoplankton growth and biomass aggregation. Our results indicate that conditions in mixing and growth of phytoplankton can drive different vertical spatial structures in the mixed layer, with the depth of the mixed layer being a critical factor to allow coexistence of populations with different sinking speed. With increasing mixed layer depth, positive growth for sinking phytoplankton can be maintained with increasing turbulent flow velocities, allowing the apparently counter-intuitive persistence of fast sinking phytoplankton populations in highly turbulent and deep mixed layers. These dynamics demonstrate the role of considering advective transport within a turbulent vortex and can help to explain observed phytoplankton biomass during winter in the North Atlantic, where the overturn of deep convection has been suggested to play a critical role in phytoplankton survival., (© 2017 The Author(s).)

Hsp90 is a molecular chaperone involved in the regulation and maturation of kinases and transcription factors. In Caenorhabditis elegans, it contributes to the development of fertility, maintenance of muscle structure, the regulation of heat-shock response and dauer state. To understand the consequences of Hsp90-depletion, we studied Hsp90 RNAi-treated nematodes by DNA microarrays and mass spectrometry. We find that upon development of phenotypes the levels of chaperones and Hsp90 cofactors are increased, while specific proteins related to the innate immune response are depleted. In microarrays, we further find many differentially expressed genes related to gonad and larval development. These genes form an expression cluster that is regulated independently from the immune response implying separate pathways of Hsp90-involvement. Using fluorescent reporter strains for the differentially expressed immune response genes skr-5, dod-24 and clec-60 we observe that their activity in intestinal tissues is influenced by Hsp90-depletion. Instead, effects on the development are evident in both gonad arms. After Hsp90-depletion, changes can be observed in early embryos and adults containing fluorescence-tagged versions of SEPA-1, CAV-1 or PUD-1, all of which are downregulated after Hsp90-depletion. Our observations identify molecular events for Hsp90-RNAi induced phenotypes during development and immune responses, which may help to separately investigate independent Hsp90-influenced processes that are relevant during the nematode's life and development.

Background: Attention deficit/hyperactivity disorder (ADHD) is a common neuropsychiatric disorder in children and adolescents worldwide, and children with ADHD have elevated risk of injuries. Our aim was to assess the risk of hospitalizations with injury diagnoses and their various subtypes in children and adolescents with newly diagnosed ADHD compared to those without ADHD, as well as to study sex effects on this risk in the setting of the German health care system., Methods: The German Pharmacoepidemiological Research Database, in which 20 million insured from four statutory health insurances in Germany are included, was used to set up a matched cohort study of 3- to 17-year-old children and adolescents with and without ADHD. We calculated age-specific incidence rates and used Cox regression to obtain hazard ratios (HRs) for hospitalizations with injury diagnoses. We used the injury mortality diagnosis matrix for classification of injuries., Results: The matched cohort comprised a total of 75,300 children. The age-specific incidence rates for hospitalization with injury diagnosis for males with ADHD displayed a u-shaped form with highest incidences in the in the age groups 3-6 years [26.2 per 1,000 person-years; 95% confidence interval (CI) 20.5-33.0] and 18-21 years (28.6; 22.4-36.0). Girls with ADHD were less affected in younger age-groups, but the incidence rate for 18-21 year olds was similar to boys with ADHD (26.4; 17.4-38.4). The adjusted HR for children with ADHD was 1.40 (95% CI 1.30-1.49) compared to non-affected children. With respect to nature of injury, ADHD was associated with hospitalization with injury diagnoses of the internal organs, open wounds, and contusions but not with other injuries. With respect to body regions, children with ADHD were more prone to hospitalizations with injuries of the abdomen, traumatic brain injuries, other head injuries, and system-wide injuries such as poisoning and intoxication. No significant associations were seen for the other body regions. Differences between sexes were only seen for system-wide injuries., Conclusion: Children and adolescents with ADHD are at an increased risk for hospitalizations with diagnoses of injuries compared to non-affected children. Despite differences in health-care systems, the risk increase is at a similar level in Germany as in other countries.

One fifth to one quarter of the human population is asymptomatically, naturally and persistently colonised by Staphylococcus aureus. Observational human studies indicate that although the whole population is intermittently exposed, some individuals lose S. aureus rapidly. Others become persistent carriers, as assessed by nasal cultures, with many individuals colonised for decades. Current animal models of S. aureus colonisation are expensive and normally require antibiotics. Importantly, these animal models have not yet contributed to our poor understanding of the dichotomy in human colonisation status. Here, we identify a single strain of S. aureus found to be persistently colonising the gastrointestinal tract of BALB/c mice. Phylogenetic analyses suggest it diverged from a human ST15 lineage in the recent past. We show that murine carriage of this organism occurs in the bowel and nares, is acquired early in life, and can persist for months. Importantly, we observe the development of persistent and non-persistent gastrointestinal carriage states in genetically identical mice. We developed a needle- and antibiotic-free model in which we readily induced S. aureus colonisation of the gastrointestinal tract experimentally by environmental exposure. Using our experimental model, impact of adaptive immunity on S. aureus colonisation could be assessed. Vaccine efficacy to eliminate colonisation could also be investigated using this model.

Quantitative mass spectrometry approaches are used for absolute and relative quantification in global proteome studies. To date, relative and absolute quantification techniques are available that differ in quantification accuracy, proteome coverage, complexity and robustness. This review focuses on most common relative or absolute quantification strategies exemplified by three experimental studies. A label-free relative quantification approach was performed for the investigation of the membrane proteome of sensory cilia to the depth of olfactory receptors in Mus musculus. A SILAC-based relative quantification approach was successfully applied for the identification of core components and transient interactors of the peroxisomal importomer in Saccharomyces cerevisiae. Furthermore, AQUA using stable isotopes was exemplified to unraveling the prenylome influenced by novel prenyltransferase inhibitors. Characteristic enrichment and fragmentation strategies for a robust quantification of the prenylome are also summarized.

Cognitive deficits represent a major burden of neuropsychiatric disorders and result in part from abnormal communication within hippocampal-prefrontal circuits. While it has been hypothesized that this network dysfunction arises during development, long before the first clinical symptoms, experimental evidence is still missing. Here, we show that pre-juvenile mice mimicking genetic and environmental risk factors of disease (dual-hit GE mice) have poorer recognition memory that correlates with augmented coupling by synchrony and stronger directed interactions between prefrontal cortex and hippocampus. The network dysfunction emerges already during neonatal development, yet it initially consists in a diminished hippocampal theta drive and consequently, a weaker and disorganized entrainment of local prefrontal circuits in discontinuous oscillatory activity in dual-hit GE mice when compared with controls. Thus, impaired maturation of functional communication within hippocampal-prefrontal networks switching from hypo- to hyper-coupling may represent a mechanism underlying the pathophysiology of cognitive deficits in neuropsychiatric disorders., (© The Author 2016. Published by Oxford University Press.)

Environmental water samples from the Lower Rhine area in Germany were investigated via immunofluorescence assays (IFAs), nested polymerase chain reaction (nested PCR) and loop-mediated isothermal amplification (LAMP) to detect the presence of Giardia spp. (n=185) and Cryptosporidium spp. (n=227). The samples were concentrated through filtration or flocculation, and oocysts were purified via centrifugation through a sucrose density gradient. For all samples, IFA was performed first, followed by DNA extraction for the nested PCR and LAMP assays. Giardia cysts were detected in 105 samples (56.8%) by IFA, 62 samples (33.5%) by nested PCR and 79 samples (42.7%) by LAMP. Cryptosporidium spp. were detected in 69 samples (30.4%) by IFA, 95 samples (41.9%) by nested PCR and 99 samples (43.6%) by LAMP. According to these results, the three detection methods are complementary for monitoring Giardia and Cryptosporidium in environmental waters., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is a major bacterial pathogen, which causes severe blood and tissue infections that frequently emerge by autoinfection with asymptomatically carried nose and skin populations. However, recent studies report that bloodstream isolates differ systematically from those found in the nose and skin, exhibiting reduced toxicity toward leukocytes. In two patients, an attenuated toxicity bloodstream infection evolved from an asymptomatically carried high-toxicity nasal strain by loss-of-function mutations in the gene encoding the transcription factor repressor of surface proteins (rsp). Here, we report that rsp knockout mutants lead to global transcriptional and proteomic reprofiling, and they exhibit the greatest signal in a genome-wide screen for genes influencing S. aureus survival in human cells. This effect is likely to be mediated in part via SSR42, a long-noncoding RNA. We show that rsp controls SSR42 expression, is induced by hydrogen peroxide, and is required for normal cytotoxicity and hemolytic activity. Rsp inactivation in laboratory- and bacteremia-derived mutants attenuates toxin production, but up-regulates other immune subversion proteins and reduces lethality during experimental infection. Crucially, inactivation of rsp preserves bacterial dissemination, because it affects neither formation of deep abscesses in mice nor survival in human blood. Thus, we have identified a spontaneously evolving, attenuated-cytotoxicity, nonhemolytic S. aureus phenotype, controlled by a pleiotropic transcriptional regulator/noncoding RNA virulence regulatory system, capable of causing S. aureus bloodstream infections. Such a phenotype could promote deep infection with limited early clinical manifestations, raising concerns that bacterial evolution within the human body may contribute to severe infection.

Purpose: To develop and validate a sensitive and specific method of abscess enumeration and quantification in a preclinical model of Staphylococcus aureus infection., Methods: S. aureus infected murine kidneys were fixed in paraformaldehyde, impregnated with gadolinium, and embedded in agar blocks, which were subjected to 3D magnetic resonance microscopy on a 9.4T MRI scanner. Image analysis techniques were developed, which could identify and quantify abscesses. The result of this imaging was compared with histological examination. The impact of a S. aureus Sortase A vaccination regime was assessed using the technique., Results: Up to 32 murine kidneys could be imaged in a single MRI run, yielding images with voxels of about 25 μm3. S. aureus abscesses could be readily identified in blinded analyses of the kidneys after 3 days of infection, with low inter-observer variability. Comparison with histological sections shows a striking correlation between the two techniques: all presumptive abscesses identified by MRI were confirmed histologically, and histology identified no abscesses not evident on MRI. In view of this, simulations were performed assuming that both MRI reconstruction, and histology examining all sections of the tissue, were fully sensitive and specific at abscess detection. This simulation showed that MRI provided more sensitive and precise estimates of abscess numbers and volume than histology, unless at least 5 histological sections are taken through the long axis of the kidney. We used the MRI technique described to investigate the impact of a S. aureus Sortase A vaccine., Conclusion: Post mortem MRI scanning of large batches of fixed organs has application in the preclinical assessment of S. aureus vaccines.

Background: The attention deficit hyperactivity disorder (ADHD) is associated with substantial impairment and psychiatric comorbidities. Thus, an optimized treatment is essential. In 2011, a new multidisciplinary treatment strategy (so-called Versorgungsvertrag) was contracted for the model region of Bremerhaven, Germany. This manuscript describes the results of the feasibility testing, focusing on the effects of the Versorgungsvertrag on patients' ADHD symptoms and on the treatment satisfaction of patients' kins., Material and Methods: Patients with ADHD (4-17 years) were assessed at baseline and at 9 months follow-up. Kins documented the current symptomatology using the FBB-ADHS questionnaire on both occasions, and rated their satisfaction with the Versorgungsvertrag at follow-up. The FBB-ADHS gives information on the severity of the ADHD core symptoms (0=normal, 3=very noticeable)., Results: 69 patients (77 kins) were included. At follow-up, data from 59 patients (67 kins) were available. FBB-ADHS data of both occasions was available for 44 patients. Improvements regarding the ADHD total score (1.27 at follow-up vs. 1.59 at baseline, p=0.003) and the subdomains inattention (1.42 vs. 1.81, p=0.001) and hyperactivity (0.96 vs. 1.22, p=0.032) were documented. In the subgroup of boys (n=34), inattention (p=0.001), impulsivity (p=0.019) and the ADHD total score (p=0.002) improved, while no changes were observed in the subgroup of girls (n=10). The majority of kins (52.4 to 68.4%) rated the treatment as helpful., Discussion: Our study shows improvements for the ADHD core symptoms after 9 months and a high satisfaction of kins with the treatment strategy. Due to the lack of a control group from routine care, no certain statement about the additional benefit of the treatment strategy can be made. The null effect in the subgroup of girls might be explained by the underrepresentation of girls, but the gender distribution observed in our study is commonly observed in patients with ADHD., Conclusion: The positive effects during the observation period should be confirmed in further studies including a control group from routine care., (© Georg Thieme Verlag KG Stuttgart · New York.)

Spindle oscillations have been described during early brain development and in the adult brain. Besides similarities in temporal patterns and involved brain areas, neonatal spindle bursts (NSBs) and adult sleep spindles (ASSs) show differences in their occurrence, spatial distribution, and underlying mechanisms. While NSBs have been proposed to coordinate the refinement of the maturating neuronal network, ASSs are associated with the implementation of acquired information within existing networks. Along with these functional differences, separate synaptic plasticity mechanisms seem to be recruited. Here, we review the generation of spindle oscillations in the developing and adult brain and discuss possible implications of their differences for synaptic plasticity. The first part of the review is dedicated to the generation and function of ASSs with a particular focus on their role in healthy and impaired neuronal networks. The second part overviews the present knowledge of spindle activity during development and the ability of NSBs to organize immature circuits. Studies linking abnormal maturation of brain wiring with neurological and neuropsychiatric disorders highlight the importance to better elucidate neonatal plasticity rules in future research.

Objective: The aim of this study was to identify individual therapy goals (ITGs) of children and adolescents with ADHD and their primary caregivers., Methods: Within the evaluation of the selective contract for children and adolescents with ADHD in Bremerhaven, Germany, ITGs of 42 study participants (aged 8-17) and their primary caregivers were collected with the psychotherapy basis documentation for children and adolescents (Psy-BaDo-KJ). ITGs were analysed following the classification of categories for individual therapy goals (KITZ) and their modification for children and adolescents. Analysis focused on the most frequently named ITGs and the agreement of patients and primary caregivers ITGs on the individual level., Results: 235 ITGs were named. The greatest proportion of ADHD patients and their caregivers (47%) focused on interactional, psychosocial conflicts. In 19% of the cases (n=8) patients and their caregivers had the same main goal. 38% of patients and of caregivers (n=16) named the other ones main goal in one of his/her ITGs as well., Conclusions: ADHD patients and their primary caregivers both pursue ITGs related to ADHD symptoms. Few ITGs address medication related aspects. In case of differences in the ITGs of a patient and his/her primary caregivers, therapists should check whether differing ITGs address the same problem from different perspectives., (© Georg Thieme Verlag KG Stuttgart · New York.)

The size of an individual organism is a key trait to characterize its physiology and feeding ecology. Size-based scaling laws may have a limited size range of validity or undergo a transition from one scaling exponent to another at some characteristic size. We collate and review data on size-based scaling laws for resource acquisition, mobility, sensory range, and progeny size for all pelagic marine life, from bacteria to whales. Further, we review and develop simple theoretical arguments for observed scaling laws and the characteristic sizes of a change or breakdown of power laws. We divide life in the ocean into seven major realms based on trophic strategy, physiology, and life history strategy. Such a categorization represents a move away from a taxonomically oriented description toward a trait-based description of life in the oceans. Finally, we discuss life forms that transgress the simple size-based rules and identify unanswered questions.

Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes. Using crosslinking experiments coupled with mass spectrometric analysis and structural modelling we identify sites, which link the middle/C-terminal domain interface of C. elegans Hsp90 to the phosphatase domain of the corresponding kinase. Studying the relevance of the domains of Hsp90 for turnover of native substrates we find that ternary complexes with the glucocorticoid receptor (GR) are cooperatively formed by full-length Hsp90 and PPH-5. Our data suggest that the direct stimulation of the phosphatase activity by C-terminal Hsp90 fragments leads to increased dephosphorylation rates. These are further modulated by the binding of clients to the N-terminal and middle domain of Hsp90 and their presentation to the phosphatase within the phosphatase-Hsp90 complex.

Survival in aquatic environments requires organisms to have effective means of collecting information from their surroundings through various sensing strategies. In this study, we explore how sensing mode and range depend on body size. We find a hierarchy of sensing modes determined by body size. With increasing body size, a larger battery of modes becomes available (chemosensing, mechanosensing, vision, hearing and echolocation, in that order) while the sensing range also increases. This size-dependent hierarchy and the transitions between primary sensory modes are explained on the grounds of limiting factors set by physiology and the physical laws governing signal generation, transmission and reception. We theoretically predict the body size limits for various sensory modes, which align well with size ranges found in literature. The treatise of all ocean life, from unicellular organisms to whales, demonstrates how body size determines available sensing modes, and thereby acts as a major structuring factor of aquatic life., (© 2015 The Author(s).)

Second mitochondria-derived activator of caspase (Smac) mimetics are considered as promising anticancer therapeutics that are currently under investigation in early clinical trials. They induce apoptosis by antagonizing inhibitor of apoptosis proteins, which are frequently overexpressed in cancer. We previously reported that Smac mimetics, such as BV6, additionally exert non-apoptotic functions in glioblastoma (GBM) cells by stimulating migration and invasion in a nuclear factor kappa B (NF-κB)-dependent manner. Because NF-κB target genes mediating these effects are largely unknown, we performed whole-genome expression analyses. Here, we identify chemokine (C-C motif) ligand 2 (CCL2) as the top-listed NF-κB-regulated gene being upregulated upon BV6 treatment in GBM cells. BV6-induced upregulation and secretion of CCL2 are required for migration and invasion of GBM cells because knockdown of CCL2 in GBM cells abolishes these effects. Co-culture experiments of GBM cells with non-malignant astroglial cells reveal that BV6-stimulated secretion of CCL2 by GBM cells into the supernatant triggers migration of astroglial cells toward GBM cells because CCL2 knockdown in BV6-treated GBM cells impedes BV6-stimulated migration of astroglial cells. In conclusion, we identify CCL2 as a BV6-induced NF-κB target gene that triggers migration and invasion of GBM cells and exerts paracrine effects on the GBM's microenvironment by stimulating migration of astroglial cells. These findings provide novel insights into the biological functions of Smac mimetics with important implications for the development of Smac mimetics as cancer therapeutics., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Importance: Children and adolescents with attention-deficit/hyperactivity disorder (ADHD) have an increased risk of injuries. Attention-deficit/hyperactivity disorder is often treated with medication, but the evidence regarding prevention of injuries is inconclusive., Objective: To determine via a case-only design whether the use of methylphenidate hydrochloride or atomoxetine hydrochloride reduces the risk of injuries among children and adolescents with ADHD., Design, Setting, and Participants: We used the German Pharmacoepidemiological Research Database, which includes records from about 17 million insurees (approximately 20% of the population) from 4 statutory health insurance providers in Germany to identify children aged 3 to 17 years with new diagnoses of ADHD in 2005 and 2006. We identified 37,650 children with ADHD based on inpatient and outpatientdiagnostic codes (F90.0, F90.1, and F90.9) from the German modification of the International Statistical Classification of Diseases and Related Health Problems, 10th Revision. Among them, we identified those with an inpatient injury diagnosis during follow-up until 2009. A total of 2128 children with any injury diagnosis at hospitalization, 821 of whom had a brain injury diagnosis, were included in the analysis. We applied the self-controlled case series design to control for time-invariant characteristics of the patients and time trends in the exposure., Exposures: Treatment with methylphenidate or atomoxetine based on prescription data., Main Outcomes and Measures: Hospitalization because of any injury or brain injury according to the injury mortality diagnosis matrix., Results: Incidence rate ratios for the periods with medication compared with nonmedicated periods were 0.87 (95% CI, 0.74-1.02) for hospitalization with any injuries and 0.66 (95% CI, 0.48-0.91) for brain injuries only in the full sample. These estimates remained stable in sensitivity analyses restricting the sample to a narrower age range or to patients with a single hospitalization. There was no indication that medication prescriptions are increased after hospitalizations., Conclusions and Relevance: No significant risk reduction for hospitalizations with injury diagnoses was observed during periods of ADHD medication, but there was a preventive effect on the risk of brain injuries (34% risk reduction). The effects were controlled for time-invariant characteristics of the patients by the study design.

Sixteen indolizidine-based alkaloids (IBAs) that were isolated as poison constituents of the skin of frogs were synthesized in a highly flexible and stereoselective manner. As a key step, a three-component, organocatalytic, highly enantio- and diastereoselective vinylogous Mukaiyama-Mannich reaction was employed furnishing optically highly enriched butyrolactams as central intermediates on a multigram scale. The attached six-membered ring was constructed through cyclization of the pendant enoate moiety onto the pyrrolidine ring. The absolute configuration of the bridgehead chiral center and the adjacent 8-position was established in the initial vinylogous Mannich reaction, whereas the 3- and 5-substituents were introduced through organometallic addition at a late stage of the synthesis with full stereochemical control from the substrate. With this strategy, simple as well as even more complex alkaloids were accessible in good overall yields as single stereoisomers. These syntheses also served to establish the absolute and relative configuration of those IBAs that had never been synthesized before., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lesions of the rat nigrostriatal dopamine system by injection of 6-hydroxydopamine (6-OHDA) lead to abnormal neuronal activity in the basal ganglia (BG) motor loop similar to that found in Parkinson's disease (PD). In the BG motor loop the subthalamic nucleus (STN) represents an important structure, which, however, also comprises areas of the BG associative and limbic loops. We were interested whether neuronal activity would differ between the STN medial associative-limbic and lateral motor part, and whether selective 6-OHDA-induced lesions of the dorsolateral striatum, the entrance region of the BG motor loop, would differently affect these subregions. In male Sprague-Dawley rats 6-OHDA (n = 12) or vehicle (n = 10) was bilaterally injected in the dorsolateral striatum. Four weeks later extracellular single-unit activity and local field potentials were recorded in medial and lateral STN neurons of urethane-anesthetized rats. In sham-lesioned rats the discharge rate and burst activity were higher in the lateral compared to the medial STN. Similar differences were found for other neuronal activity measures (coefficient of variation of interspike interval, skewness, kurtosis, approximate entropy). After 6-OHDA injection neuronal burst activity was enhanced, while the discharge rate was not affected. In addition, in 6-OHDA-lesioned rats β-band oscillatory activity was enhanced, with no difference between STN subregions. We found important differences of neuronal activity between STN subregions, indicating functional segregation. However, selective 6-OHDA lesions of the dorsolateral striatum also had a pronounced effect on the medial STN subregion, indicating interaction between BG loops., (© 2013 Wiley Periodicals, Inc.)

Peroxynitrite is a highly reactive chemical species with antibacterial properties that are synthesized in immune cells. In a proteomic approach, we identified specific target proteins of peroxynitrite-induced modifications in Escherichia coli. Although peroxynitrite caused a fairly indiscriminate nitration of tyrosine residues, reversible modifications of protein thiols were highly specific. We used a quantitative redox proteomic method based on isotope-coded affinity tag chemistry and identified four proteins consistently thiol-modified in cells treated with peroxynitrite as follows: AsnB, FrmA, MaeB, and RidA. All four were required for peroxynitrite stress tolerance in vivo. Three of the identified proteins were modified at highly conserved cysteines, and MaeB and FrmA are known to be directly involved in the oxidative and nitrosative stress response in E. coli. In in vitro studies, we could show that the activity of RidA, a recently discovered enamine/imine deaminase, is regulated in a specific manner by the modification of its single conserved cysteine. Mutation of this cysteine 107 to serine generated a constitutively active protein that was not susceptible to peroxynitrite.

Background: Determinants of regional variation in caesarean sections can contribute explanations for the observed overall increasing trend of caesarean sections. We assessed which mechanism explains the higher rate of caesarean sections in the former West than East Germany: a more liberal use of caesarean sections in the case of relative indications or more common caesarean sections without indications., Methods: We used a health insurance database from all regions of Germany with approximately 14 million insured individuals (about 17% of the total population in Germany). We selected women who gave birth in the years 2004 to 2006 and identified indications for caesarean section on the basis of hospital diagnoses in 30 days around birth. We classified pregnancies into three groups: those with strong indications for caesarean section (based on classification of absolute indications recommended by the Unmet Obstetrics Need network), those with moderate indications (other indications increasing the probability of caesarean section) and those with no indications. We investigated the percentage of caesarean sections among all births, presence of strong or moderate indications in all pregnancies, the probability of caesarean sections in the presence of indications and the fraction of caesarean sections attributable to strong, moderate and no indications., Results: In total, 294,841 births from 2004-2006 were included in the analysis. In the former West Germany, 30% births occurred by caesarean section, while in the former East Germany the caesarean section rate was 22%. Proportions of pregnancies with strong and moderate indications for caesarean section were similar in both regions. For strong indications the probability of caesarean section was similar in East and West Germany, but the probability of caesarean section among women with moderate indications was substantially higher in the former West Germany. Caesarean sections were also more common among women with no indications in the former West (8%) than in the former East (4-5%). The higher probability of caesarean section in the case of strong or moderate indications in the former West than in the East explained 87% of the difference between section rates in these two regions, while caesarean sections without indications contributed to only 13% of the difference observed., Conclusions: The observed difference between caesarean section rates in the former East and West Germany was most likely due to different medical practice in handling relative indications.

Human toxoplasmosis is potentially contracted due to consumption of contaminated drinking water and represents an increasing public health risk worldwide. Toxoplasma gondii oocysts can be resistant to standard disinfection processes, including UV radiation. Increased awareness of the risk of waterborne toxoplasmosis outbreaks has led to an increase in research interest in the detection of oocysts in environmental water systems. Ninety-five environmental water samples from the Lower Rhine area in Germany have been included in the study and examined for the presence of Toxoplasma. Water samples were filtered or flocculated by aluminum sulfate and purified by sucrose density gradient. DNA was then extracted, and the DNA samples were then examined by LAMP analysis. T. gondii DNA was detected in eight out of 83 (9.6%) influent and effluent samples obtained from wastewater treatment plants. All samples (n=12) from the surface, ground, raw and tap waters tested negative. The purpose of this work was to investigate the occurrence and distribution of Toxoplasma oocysts on the Lower Rhine in Germany. Our study provides evidence that the assay is a sensitive, specific, rapid and cost effective method for the detection of T. gondii and is useful for both the investigations of cases of waterborne outbreaks and for identifying the source of contamination., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Methylphenidate (MPH) is the most common drug treatment of attention deficit / hyperactivity disorder (ADHD) in children. Treatment with MPH is contraindicated in the presence of certain psychiatric, cerebro- and cardiovascular conditions. We assessed MPH treatment prevalence and incidence and the frequency of comorbid conditions related to these contraindications in new MPH users compared to a control group without ADHD and ADHD medication., Methods: We used health care data for the years 2004 to 2006 from the German Pharmacoepidemiological Research Database (GePaRD) which includes about 18% of the German population. MPH treatment prevalence and incidence was assessed based on at least one MPH prescription in the given year. In MPH users, the prevalence of psychiatric and other comorbidities was assessed in the quarter of the first MPH prescription and the three preceding quarters, whereas in controls it was assessed in the earliest four quarters of continuous insurance time starting at 01.01.2004 or the start of insurance if this was later. Differences in the presence of comorbid diagnoses between MPH users and controls were tested by logistic regression., Results: In 2005, 1.5% of all children and adolescents aged 3 to 17 years (2.3% of males and 0.6% of females) received MPH in Germany. The proportion of children with a record of a psychiatric comorbidity in any of the nine ICD categories of diagnoses was substantially higher in new MPH users (83%) compared to controls (20%). Cerebro- and cardiovascular comorbidities were rare in general. Still, among new MPH users, 2% of males and females had a diagnosis of a pre-existing cardiovascular disorder but only 1.2% of controls., Conclusions: Besides MPH treatment prevalence we first publish age-specific incidence rates for Germany. A high proportion of children who were started on MPH had a record of a psychiatric comorbidity preceding the first prescription. Cerebro- and cardiovascular conditions were rare in the studied age range, but still higher among children who received MPH than in the control group. Results show that in a substantial subgroup of patients, comorbidities require a thorough weighting of possible risks of MPH medication against the risks of untreated ADHD.

Samples from different water sources (n = 396) were collected during 2009 and 2011. Wastewater (2-5 l) was purified by aluminium sulphate flocculation. Surface, ground and drinking waters (400-6400 l) were collected by filtration. Cryptosporidium oocysts and Giardia cysts were further concentrated by sucrose centrifugation. (Oo)cysts were identified by IFT (immunofluorescence test), DAPI (4',6-diamidino-2-phenylindole) staining and DICM (difference interference contrast microscopy). Out of 206 wastewater samples, 134 (65·0%) were found to be positive for Giardia cysts and 64 (31·1%) for Cryptosporidium oocysts. Parasite numbers ranged from 0 to 2436 cysts/l and 0 to 1745 oocysts/l. Eight (4·2%) surface and drinking water samples (n = 190) were found to be positive for Giardia cysts (0-56000/100 l), and 18 (9·5%) for Cryptosporidium oocysts (2400/100 l). The purpose of this study was to establish the prevalence and concentrations of Giardia lamblia and Cryptosporidium spp. by detecting (oo)cysts from water samples. This study provides substantial evidence that G. lamblia cysts and Cryptosporidium spp. oocysts are able to enter and circulate in the aquatic environment with negative implications for public health.

Four indolizidine based alkaloids (IBAs) have been synthesized in a highly enantioselective, straightforward, and flexible manner. As a key step our previously developed Brønsted acid catalyzed vinylogous Mannich reaction was employed which easily afforded gram amounts of an optically pure central intermediate which can be converted into a wide range of diversely substituted IBAs.

Objective: The purpose of this study was to estimate the prevalence and incidence of new diagnoses of attention-deficit/hyperactivity disorder (ADHD) and assess drug treatment of ADHD in the 3-17 year age group in Germany., Method: We analysed data from the German Pharmacoepidemiological Research Database (GePaRD) for the years 2004-2006. The GePaRD includes claim records of 14,000,000 members of four statutory health insurances, representing 17% of the German population. The assessment of ADHD diagnoses was based on International Classification of Diseases version 10 (ICD-10) codes in outpatient and hospital data., Results: In 2005, the age-standardized prevalence and incidence of new diagnoses were 2.5% and 9/1000 person-years, respectively. Both measures were 3-4 times higher for males than for females. Incidence of new ADHD diagnoses increased linearly up to the age of 8 years for boys and 9 years for girls and decreased abruptly thereafter. In the calendar quarter of the initial ADHD diagnosis, 9.4% (95% confidence interval [CI] 8.9-9.8%) received methylphenidate or atomoxetine and 36.8% (95% CI 36.1-37.6%) received at least one prescription of either drug within the first year. Initiation of drug treatment and choice of drug were similar for both sexes., Conclusions: ADHD is a common condition among children and youth in Germany. There are substantial differences by sex in the prevalence and incidence of new ADHD diagnoses, but only a small difference in drug treatment among those diagnosed with ADHD. A relatively low percentage of children receives drug treatment in the first year after the initial diagnosis of ADHD.

Introduction: In patients, a transient decrease in peripheral blood lymphocyte counts was observed following intraperitoneal administration of the trifunctional monoclonal antibody catumaxomab (anti-human EpCAM x anti-human CD3). The aim of this study was to clarify the observed effect in a preclinical mouse model and to analyse the related mechanism of action in vitro., Materials and Methods: A related antibody, BiLu (antihuman EpCAM x anti-mouse CD3), was administered to mice and blood leukocytes were analysed. In vitro studies measured activation and cytokine secretion from human peripheral blood mononuclear cells (PBMC). For the analysis of T cell adhesion, PBMC were preincubated with catumaxomab and then co-cultured with human endothelial cells (HUVEC); T cell adhesion was assessed in the presence or absence of endothelial cell preactivation by TNFα. Adherent T cells were determined by flow cytometry., Results: Treatment of mice with BiLu resulted in a dosedependent transient decrease in CD3+ T cells (both CD4+ and CD8+) that returned to the normal range within 48 h. Catumaxomab physiologically activated T cells in vitro (increased CD69 expression) and induced cytokine release (TNFα, IFNγ). TNFα increased expression of adhesion molecules CD54 and CD62E on endothelial cells. Furthermore, catumaxomab dose-dependently enhanced adhesion of T cells to endothelial cells. Adhesion was further increased when endothelial cells were preactivated with TNFα., Conclusions: Catumaxomab increases adhesion of T cells to endothelial cells due to antibody-mediated activation of T cells and production of T cell cytokines that up-regulate endothelial cell adhesion molecules. These results provide a mechanistic rationale for the transient, reversible decrease in lymphocyte counts observed following catumaxomab administration in patients, which is likely to be due to redistribution of lymphocytes.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is effectively used to treat motor symptoms in Parkinson's disease (PD). Recently more attention has been paid to behavioral disturbances caused by PD itself and by STN DBS. In the 6-hydroxydopamine (6-OHDA) PD rat model we investigated the effect of STN DBS on deficient prepulse inhibition (PPI) induced by the dopamine (DA) receptor agonist apomorphine, which is an operative measure for disturbed sensorimotor gating seen in certain neuropsychiatric disturbances. Male Sprague Dawley rats with bilateral lesions of the nigrostriatal DA system (striatal injection of 6-OHDA or vehicle for sham-lesion) were bilaterally implanted with electrodes for DBS into the STN. After determination of individual thresholds rats were stimulated (130Hz, 80μs pulse width) or sham-stimulated for epochs of six days. On the sixth day of each epoch rats were tested for PPI of the acoustic startle response after apomorphine or vehicle injection in a within randomized cross-over design. Stimulation of the STN improved PPI in vehicle-treated (control) rats, but deteriorated PPI after apomorphine treatment. This effect was more pronounced in sham-lesioned rats. Furthermore, in lesioned rats the startle reaction was marginally enhanced without effect of stimulation or apomorphine treatment. These data suggest that STN DBS interacts with dopaminergic action. With respect to functional neurosurgery, STN DBS alone may improve certain aspects of psychiatric disturbances, but may have a different impact when combined with dopaminergic medication., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Because of its versatile chemical properties, the amino acid cysteine plays a variety of vital roles in proteins. It can form structure-stabilizing elements (e.g., disulfide bonds), coordinate metal cofactors and is part of the catalytic center of many enzymes. Recently, a new role has been discovered for cysteine: so-called redox-sensitive proteins use the thiol group of cysteine as a specific sensor for Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS). The oxidation of such a redox-active cysteine, e.g., under conditions of elevated cellular ROS or RNS levels (oxidative or nitrosative stress), often results in a reversible thiol modification. This, in turn, might lead to structural changes and altered protein activity. When the oxidative stress subsides, cellular antioxidant systems, including thioredoxin and glutathione can reduce the redox-active cysteine and restore the original structure and activity of the redox-sensitive protein. This makes oxidative thiol modifications an attractive mechanism for cellular redox sensing and signaling.To study the target cysteines of oxidative and nitrosative stress and to quantify the extent of the thiol modifications generated under these conditions, we have recently developed a thiol trapping technique using isotope coded affinity tag (ICAT) chemistry (1). With this method, reduced cysteines are selectively labeled with the isotopically light form of ICAT and oxidized cysteines with the isotopically heavy form of ICAT. Thus we could globally quantify the ratio of reduced and oxidized cysteines in cellular proteins based on the modified peptide masses. Here, we present an expansion of this method, which we term NOxICAT, because it uses ICAT chemistry to detect changes in thiol modifications of proteins upon Nitrosative and Oxidative stress. The NOxICAT-method is a highly specific and quantitative method to study the global changes in the thiol redox state of cellular proteins under a variety of physiological and pathological stress conditions.

BACKGROUND Trifunctional antibodies, such as catumaxomab (anti-EpCAM×anti-CD3) and ertumaxomab (anti- HER-2/neu×anti-CD3), transiently link immune effector cells to tumour cells, which results in cellular cytotoxicity towards the tumour cells. A functional immune system is therefore essential for effective anti-tumour activity. However, the commonly observed haematotoxicity of chemotherapeutics and radiation therapy may be associated with some degree of immunosuppression. Combining chemotherapy and trifunctional antibodies in cancer treatment requires understanding of the impact of chemotherapeutics on immune cell function and, thus, on the activity of trifunctional antibodies. METHODS The effect of chemotherapeutic treatment on trifunctional antibody-mediated anti-tumour activity was assessed in vitro. Blood samples were collected from 12 head and neck squamous cell carcinoma patients after chemotherapy (5-fluorouracil, cisplatin) and radiotherapy, and from one healthy control donor. The immune cell status was analysed and mononuclear cells (MNC) were isolated. The potency of catumaxomab and ertumaxomab was assessed in a cytotoxicity assay using MNC isolated from each patient sample in co-culture with a tumour target cell line. The release of infl ammatory cytokines was also monitored in the cell culture supernatant. RESULTS Most patients included in this study had decreased immune cell counts during the course of chemotherapy. Nonetheless, an effective and concentration-dependent anti- tumour activity mediated by trifunctional antibodies was demonstrated using these patient immune effector cells. The immune response activity of the patient immune cells was not impaired one week after cisplatin administration or even three days after the last 5-fluorouracil treatment. CONCLUSION This study shows for the first time that immune effector cells from cancer patients undergoing standard chemotherapy and radiotherapy can be activated by trifunctional antibodies for efficient killing of tumour cells.

We have developed a synthesis of phosphoarginine containing peptides using a bis(2,2,2-trichloroethyl) protected phosphoarginine derivative as building block. Binding studies and computer modelling demonstrate the ability of the SH2 domain from Src kinase to recognize a phosphoarginine-containing peptide in a phosphoryl group-dependent manner., (This journal is © The Royal Society of Chemistry 2011)

The suppressor of cytokine signaling 3 (SOCS3) gene is one of eight structurally related genes of the SOCS family and has been suggested to function as a tumor suppressor by inhibition of the JAK/STAT signaling pathway. We investigated 60 human gliomas of different histological types for SOCS3 alterations and found frequent SOCS3 promoter hypermethylation and transcriptional downregulation. However, SOCS3 promoter hypermethylation was virtually absent in primary glioblastomas, which are characterized by frequent epidermal growth factor receptor (EGFR) amplification and overexpression. Assessment of the relationship between SOCS3 and EGFR aberrations revealed that SOCS3 promoter hypermethylation was inversely related to both the EGFR gene dosage as well as the EGFR protein expression, thus suggesting SOCS3 inactivation as a mechanism substituting for EGFR activation in a subset of gliomas. In support of this hypothesis, stable shRNA-mediated SOCS3 knock-down in U251 glioblastoma cells resulted in an activation of EGFR-related signaling pathways, i.e. an increase in the activation levels of STAT3, FAK and to a lesser extent MAPK, while the AKT phosphorylation levels remained unaffected. Functionally, SOCS3-depletion caused strongly increased tumor cell invasion with no obvious effect on tumor cell proliferation. In summary, our findings suggest that SOCS3 inactivation by promoter hypermethylation is mutually exclusive to EGFR activation in gliomas and preferentially promotes glioma cell invasion through STAT3 and FAK activation.

Purpose: Administrative healthcare databases are increasingly being used to investigate potential drug risks in pregnancy. Our study aimed to develop an algorithm for linkage of mother-baby pairs (MBPs) in the German Pharmacoepidemiological Research Database (GePaRD) as a prerequisite for such studies., Methods: GePaRD contains sociodemographic data, drug dispensations, ambulatory, and hospital information on more than 14 million insurants from four German statutory health insurances (SHIs) covering all regions in Germany. Linkage was based on co-insurance information of the newborn with the potential mother (direct linkage) or of both potential mother and newborn with the potential father (indirect linkage). Linkage is not possible if the baby is co-insured with the father and the mother is self-insured. Further information on birth or childbed was used to validate the potential mother as true mother in MBP. Descriptive comparisons between linked and unlinked mothers were conducted., Results: Of 323,993 newborns identified between 2004 and 2006, 250,355 (77.3%) could be linked in MBP. Of those, 189,702 (75.8%) MBP were based on direct linkage. Mean age was similar in linked (31.1 years, standard deviation (SD = 5.4) and unlinked (31.8 years, SD = 5.5) mothers as was the proportion of caesarean sections in both groups (28.9% vs. 29.3%)., Conclusions: The developed algorithm permits linkage of a great number of newborns with their mothers and creates a potential data source for investigation of drug risks in pregnancy. Further validation studies are needed also including information on pregnancies not resulting in live births., (Copyright © 2010 John Wiley & Sons, Ltd.)

Outbred rats are widely used in biomedical research studies. The genotypic variation, different housing and handling conditions during development, and gender of used rats can be expected to cause variations in behavior and epileptogenesis. We tested the hypothesis that adult female Wistar rats from different breeders vary in anxiety-like behavior, seizure susceptibility, and epileptogenesis in the kindling model of temporal lobe epilepsy. In female Wistar rats from three different commercial breeders ([HsdCpb:WU], [Crl:WI(Han)], and [RjHan:WI]), anxiety-like behavior was monitored in the open field and the elevated plus maze. Wistar rats from Charles River showed lower locomotor activity and higher anxiety-like behavior compared to Wistar rats from Harlan-Winkelmann and Janvier. Female Wistar rats from Harlan-Winkelmann showed the lowest anxiety-like behavior and the highest exploratory behavior. Subsequently, rats were kindled by daily electrical stimulation of the right amygdala as a model of temporal lobe epilepsy. Wistar rats from Charles River exhibited significantly increased cumulative motor seizure duration and cumulative afterdischarge duration until seizure generalization compared to Harlan-Winkelmann and Janvier rats. In other words, Wistar rats from Charles River showed a longer time of focal seizures until generalization, reflecting a slower epileptogenesis in these rats. The initial afterdischarge threshold did not differ significantly between Wistar rats from the three breeders, indicating that baseline seizure susceptibility is not different between the three groups. In conclusion, female Wistar outbred rats from different breeders showed variations in anxiety-like behavior and epileptogenesis. Decisions about appropriate commercial colonies used for biomedical research should be taken with caution., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Many Parkinson's disease (PD) patients suffer from anxiety disorders, which often precede the onset of classical motor symptoms. So far, there is no evidence from randomized, placebo-controlled trials for successful treatment of anxiety in patients with PD. Grafts of fetal nigral neurons are currently explored as a restorative cell therapy for PD. In PD animal models, intrastriatal transplantations of embryonic dopaminergic neurons have been shown to ameliorate behavioral defects. In our previous study we showed that expanded and differentiated neural progenitors improved drug-induced rotation behavior and posture balance as a more complex motor task. However, it is not clear whether grafting of these cells affected spontaneous locomotor activity and anxiety-like behavior in 6-OHDA lesioned rats. Therefore, we analyzed behavior of control, lesioned, sham-transplanted, and transplanted rats using open field (OF) and elevated plus maze (EPM). After unilateral 6-OHDA lesion of the medial forebrain bundle, we observed reduced locomotor activity in the EPM (wall-rearing, entries in closed arms) in lesioned and sham-transplanted rats, which correlated with the loss of dopaminergic neurons and apomorphine-induced rotation behavior. Furthermore, anxiety-like behavior in the EPM (entries and time in open arms) was increased in lesioned and sham-transplanted rats. Although exogenous cell replacement improved apomorphine-induced rotation behavior, locomotor activity and anxiety-like behavior was not reconstituted in transplanted rats. However, we provided evidence for an interaction of locomotor activity/anxiety-like behavior with graft localization in the host striatum. These results emphasize the crucial role of graft localization for benefit of restorative cell therapy for PD., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Despite a lower prevalence of opioid dependence among females, drug-related problems and risk factors such as prostitution have a negative effect for women in treatment. This study was conducted with the purpose of analyzing gender differences in the German trial on heroin-assisted treatment (HAT), which compared HAT with methadone maintenance treatment (MMT). Significant baseline gender differences were found, with females showing a greater extent of mental distress. Differences in retention and outcome were significant for male patients, but no differences between treatment options were found for female patients. Ongoing prostitution was found to influence drug use outcomes. Other outcome criteria may need to be stressed when assessing the effect of HAT for women.

The aim of the present study was to assess the feasibility of a 3D tumor cell culture model, that is, multicellular tumor spheroids (MCTSs) as an adequate model for micrometastases and therefore as a pharmacological model for efficacy testing of trifunctional therapeutic antibodies. Unlike conventional monolayer cultures, spheroids allow researchers to study parameters, such as 3D cell shape, 3D cell arrangement and microenvironment, and penetration efficiency of defense cells that may largely influence the efficacy of antibody treatment in vivo. The authors established a long-term coculture of human MCTSs with peripheral blood mononuclear cells (PBMCs) to test the anticancer effect of the trifunctional, bispecific antibody catumaxomab (anti-EpCAM x anti-CD3) or similar therapeutic molecules. The test system is accessible to various analytical methods and thus allows for characterizing multiple parameters, which can help elucidate the mode of action of immunotherapeutic anticancer treatment. For example, the novel approach enables precise, reproducible volume growth analysis of MCTSs under immunotherapeutic treatments. For evaluation of changes within individual spheroids, cryosections can be stained (e.g., for proliferating or apoptotic cells as well as infiltrating PBMCs). Molecular PCR-based assays or flow cytometric analyses allow for discrimination between different cell types, particularly leukocyte subtypes. Furthermore, MCTSs can be disaggregated to form standard monolayers for cell viability or plating efficiency experiments. For these reasons, the MCTS model is a powerful tool to analyze drug efficacy with various endpoints under highly reproducible, standardized conditions.