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Most N-Nitrosamine compounds are found to be genotoxic in several animal species. Some are classified as probable or possible human carcinogens and very low acceptable daily intake has been established such as 96 ng/day for N-nitrosodimethylamine (NDMA) and 26.5 ng/N-nitrosodiethylamine (NDEA). The pharmaceutical industry has considered all processing areas for potential formation or contamination of N-nitrosamine. One risk is the potential contamination of nitrosamine during drug product blister packaging using lidding foils containing nitrocellulose, and different approaches have been used by pharmaceutical companies to evaluate and mitigate this risk. Herein we share a perspective from IQ Consortium N-nitrosamine Working Group on some of the approaches and corresponding results. From these assessments, it was concluded that the risk of nitrosamine contamination during blister packaging is negligible. The approaches shared in this perspective can be incorporated into risk assessment for nitrosamine contamination during drug product packaging at other pharmaceutical companies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The potential presence of N-nitrosamines in medicinal products has become a matter of concern for health authorities and pharmaceutical companies. However, very little information is available in published literature on N-nitrosamine formation within pharmaceutical drug products. In response, experiments were undertaken to test if secondary and tertiary amines present in solid drug products could undergo nitrosation due to the presence of nitrite in the excipients used in the manufacture of the drug product. This work focused on solid dosage forms exploring several model amines of varying chemical structure, solubility and pKa which were formulated using common excipients with and without added nitrite. Monitoring the formation of the N-nitrosamines after processing and upon stressed stability conditions showed that N-nitrosamine formation can occur in solid drug product formulations. The results show that the rate and extent of N-nitrosamine formation depend upon the solubility of the amine, level of nitrite, expected local acidity in water layers within the drug product and mode of processing. Our findings agree with the rank order of dosage form risk from the published EFPIA workflows for quality risk management of N-nitrosamine risks in medicines (EFPIA, 2022): amorphous > wet granulation > direct compression > dry blends. In all cases the level of N-nitrosamine formation in solid dosage forms plateaued at a level that was significantly lower than the maximum theoretical yield based on the level of nitrite present. Trace secondary amine impurities were shown to be a significantly lower risk relative to cases containing a secondary amine present at drug substance levels. A comparison of secondary and simple tertiary alkylamine reactivity showed the tertiary amine to be significantly less reactive with nitrite., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Breast cancer diagnosis and treatment can lead to longer term cognitive and emotional vulnerability, making the ability to efficiently adapt to setbacks critical. Whilst cancer-related cognitive impairments (CRCI) are often reported amongst breast cancer survivors, investigation into the capacity to efficiently process errors is limited. The present study investigated the neurocognitive correlates of cognitive-control related performance monitoring, an important function influencing behavioural adjustment to mistakes. 62 participants (30 Breast Cancer Survivors, 32 Non-Cancer) completed a modified flanker task designed to challenge response inhibition as we measured neurocognitive indices of performance monitoring (ERN, the error-related negativity; CRN, the correct-response negativity; Pe, the error positivity). Findings indicated a blunted CRN and larger ∆ERN in the breast cancer survivors compared to the non-cancer group, in the absence of performance effects. This was followed by a larger Pe in the breast cancer survivors' group, indicating an exaggerated performance monitoring response. For women affected by breast cancer, findings suggest an early disrupted neural response to monitoring cognitive performance, followed by the requirement for more effortful processing in the conscious response to errors, indicating deficits in neurocognitive efficiency. These findings have important implications for developing cognitive rehabilitation programmes for breast cancer survivors affected by cognitive dysfunction to assist in the monitoring and adjustment of performance required to meet established goals in the face of adversity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The concept of fetal consciousness is a widely discussed topic. In this study, we applied a hierarchical rule learning paradigm to investigate the possibility of fetal conscious processing during the last trimester of pregnancy. We used fetal magnetoencephalography, to assess fetal brain activity in 56 healthy fetuses between gestational week 25 and 40, during an auditory oddball paradigm containing first- and second-order regularities. The comparison of fetal brain responses towards standard and deviant tones revealed that the investigated fetuses show signs of hierarchical rule learning, and thus the formation of a memory trace for the second-order regularity. This ability develops over the course of the last trimester of gestation, in accordance with processes in physiological brain development and was only reliably present in fetuses older than week 35 of gestation. Analysis of fetal autonomic nervous system activity replicates findings in newborns, showing importance of activity state for cognitive processes. On the whole, our results support the assumption that fetuses in the last weeks of gestation are capable of consciously processing stimuli that reach them from outside the womb., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Estimating the extent to which newborn humans process input from their environment, especially regarding the depth of processing, is a challenging question. To approach this problem, we measured brain responses in 20 newborns with magnetoencephalography (MEG) in a "local-global" auditory oddball paradigm in which two-levels of hierarchical regularities are presented. Results suggest that infants in the first weeks of life are able to learn hierarchical rules, yet a certain level of vigilance seems to be necessary. Newborns detected violations of the first-order regularity and displayed a mismatch response between 200-400 ms. Violations of the second-order regularity only evoked a late response in newborns in an active state, which was expressed by a high heart rate variability. These findings are in line with those obtained in human adults and older infants suggesting a continuity in the functional architecture from term-birth on, despite the immaturity of the human brain at this age., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Endothelial dysfunction leading to vascular permeability and plasma leakage are characteristic features of severe dengue and sepsis. However, the mechanisms underlying these immune-pathologies remain unclear. The risk of severe dengue and sepsis development depend on patient-related and pathogen-related factors. Additionally, comorbidities increase the risk of severe disease and their incidence hampers correct diagnosis and treatments. To date, there is no efficient therapy to combat severe dengue and sepsis. Here, we discuss the differences and similarities between the pathogenesis of severe dengue and that of bacterial sepsis. We identify gaps in knowledge that need to be better understood in order to move towards the rational development and/or usage of therapeutic strategies to ameliorate severe dengue disease., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objective: This systematic review highlights the influence of developmental changes of the central nervous system on habituation assessment during child development. Therefore, studies on age dependant changes in event-related brain responses as well as studies on behavioural and neurophysiological habituation during child development are compiled and discussed., Methods: Two PubMed searches with terms "(development evoked brain response (fetus OR neonate OR children) (electroencephalography OR magnetoencephalography))" and with terms "(psychology habituation (fetal OR neonate OR children) (human brain))" were performed to identify studies on developmental changes in event-related brain responses as well as habituation studies during child development., Results: Both search results showed a wide diversity of subjects' ages, stimulation protocols and examined behaviour or components of event-related brain responses as well as a demand for more longitudinal study designs., Conclusions: A conclusive statement about clear developmental trends in event-related brain responses or in neurophysiological habituation studies is difficult to draw. Future studies should implement longitudinal designs, combination of behavioural and neurophysiological habituation measurement and more complex habituation paradigms to assess several habituation criteria., Significance: This review emphasizes that event-related brain responses underlie certain changes during child development which should be more considered in the context of neurophysiological habituation studies., (Copyright © 2019 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.)

The error-related negativity (ERN) is a neurophysiologic response to errors that associates with anxiety. Despite the potential relevance of the ERN for understanding mechanisms of early anxiety problems in the developing brain, the relation between ERN and anxious symptoms in young children remains poorly understood. Emerging evidence suggests that ERN-anxiety associations could vary by developmental stage, but this work requires replication and consideration of gender effects, given earlier maturation of the ERN and higher rates of anxiety problems in girls relative to boys. To address this gap, the ERN was collected in 49 preschool- to school-aged children (ages 4-9; 26 girls) sampled across a wide range of anxiety severity. Regression analyses revealed that ERN - anxiety associations depended on age and gender. Specifically, larger (more negative) ERN associated with more anxiety in older girls, whereas smaller ERN associated with more anxiety symptoms in younger girls. No ERN-anxiety association was found in boys. These findings suggest that age and gender moderate the direction of the relation between ERN and anxiety in early childhood and could have important implications for the development of ERN-based risk identification and targeted treatment strategies tailored to individual children., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The role of continuing anti-HER2 therapy beyond progression on front-line therapy in patients with metastatic HER2 positive gastro-oesophageal cancer (GEC) is unclear. Continued chemotherapy plus trastuzumab (CT) has never been compared with the current standard second-line treatment, chemotherapy plus ramucirumab (CR)., Methods: The Flatiron Health electronic health record derived database, a nationwide database comprising patient-level structured and unstructured data, curated via technology-enabled abstraction, was reviewed for patients with metastatic HER2 positive GEC who received first-line CT, followed by second-line CT or CR. Survival from second-line therapy (SST) and time to next therapy or death (TTNTD) were compared using Kaplan-Meier curves and logrank analysis., Results: 133 patients with metastatic HER2 positive GEC who received first-line CT were identified. 32 received second-line CR and 101 received CT. Median SST for patients treated with CT versus CR was 10.2 months (IQR 5.1-20.8) and 6.8 months (IQR 2.4-20.2), respectively (p=0.29). Median TTNTD for second-line CT versus CR was 4.9 months (IQR 2.8-9.8) and 5.1 months (IQR 2.3-7.5), respectively (p=0.65). Patients who received second-line CT were more likely to receive a multiagent chemotherapy backbone (76% vs 3%, p≤0.001)., Conclusions: This analysis showed no significant difference in SST for patients treated with second-line CT versus CR. Further studies are needed to clarify the role of trastuzumab in the second line, especially in patients with confirmed retention of HER2 positivity following progression., Competing Interests: Competing interests: None declared.

The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions. It highlights the need to develop standardized protocols when conducting toxicity-related predictions. This contribution articulates the information needed for protocols to support in silico predictions for major toxicological endpoints of concern (e.g., genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity) across several industries and regulatory bodies. Such novel in silico toxicology (IST) protocols, when fully developed and implemented, will ensure in silico toxicological assessments are performed and evaluated in a consistent, reproducible, and well-documented manner across industries and regulatory bodies to support wider uptake and acceptance of the approaches. The development of IST protocols is an initiative developed through a collaboration among an international consortium to reflect the state-of-the-art in in silico toxicology for hazard identification and characterization. A general outline for describing the development of such protocols is included and it is based on in silico predictions and/or available experimental data for a defined series of relevant toxicological effects or mechanisms. The publication presents a novel approach for determining the reliability of in silico predictions alongside experimental data. In addition, we discuss how to determine the level of confidence in the assessment based on the relevance and reliability of the information., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Arthroscopic sub-acromial decompression (decompressing the sub-acromial space by removing bone spurs and soft tissue arthroscopically) is a common surgery for subacromial shoulder pain, but its effectiveness is uncertain. We did a study to assess its effectiveness and to investigate the mechanism for surgical decompression., Methods: We did a multicentre, randomised, pragmatic, parallel group, placebo-controlled, three-group trial at 32 hospitals in the UK with 51 surgeons. Participants were patients who had subacromial pain for at least 3 months with intact rotator cuff tendons, were eligible for arthroscopic surgery, and had previously completed a non-operative management programme that included exercise therapy and at least one steroid injection. Exclusion criteria included a full-thickness torn rotator cuff. We randomly assigned participants (1:1:1) to arthroscopic subacromial decompression, investigational arthroscopy only, or no treatment (attendance of one reassessment appointment with a specialist shoulder clinician 3 months after study entry, but no intervention). Arthroscopy only was a placebo as the essential surgical element (bone and soft tissue removal) was omitted. We did the randomisation with a computer-generated minimisation system. In the surgical intervention groups, patients were not told which type of surgery they were receiving (to ensure masking). Patients were followed up at 6 months and 1 year after randomisation; surgeons coordinated their waiting lists to schedule surgeries as close as possible to randomisation. The primary outcome was the Oxford Shoulder Score (0 [worst] to 48 [best]) at 6 months, analysed by intention to treat. The sample size calculation was based upon a target difference of 4·5 points (SD 9·0). This trial has been registered at ClinicalTrials.gov, number NCT01623011., Findings: Between Sept 14, 2012, and June 16, 2015, we randomly assigned 313 patients to treatment groups (106 to decompression surgery, 103 to arthroscopy only, and 104 to no treatment). 24 [23%], 43 [42%], and 12 [12%] of the decompression, arthroscopy only, and no treatment groups, respectively, did not receive their assigned treatment by 6 months. At 6 months, data for the Oxford Shoulder Score were available for 90 patients assigned to decompression, 94 to arthroscopy, and 90 to no treatment. Mean Oxford Shoulder Score did not differ between the two surgical groups at 6 months (decompression mean 32·7 points [SD 11·6] vs arthroscopy mean 34·2 points [9·2]; mean difference -1·3 points (95% CI -3·9 to 1·3, p=0·3141). Both surgical groups showed a small benefit over no treatment (mean 29·4 points [SD 11·9], mean difference vs decompression 2·8 points [95% CI 0·5-5·2], p=0·0186; mean difference vs arthroscopy 4·2 [1·8-6·6], p=0·0014) but these differences were not clinically important. There were six study-related complications that were all frozen shoulders (in two patients in each group)., Interpretation: Surgical groups had better outcomes for shoulder pain and function compared with no treatment but this difference was not clinically important. Additionally, surgical decompression appeared to offer no extra benefit over arthroscopy only. The difference between the surgical groups and no treatment might be the result of, for instance, a placebo effect or postoperative physiotherapy. The findings question the value of this operation for these indications, and this should be communicated to patients during the shared treatment decision-making process., Funding: Arthritis Research UK, the National Institute for Health Research Biomedical Research Centre, and the Royal College of Surgeons (England)., (Copyright © 2018 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Gastric sonography is emerging as a valuable clinical point-of-care tool to assess aspiration risk. A recent study proposed that a single cut-off cross-sectional area (CSA) in the supine position could diagnose an empty stomach in the parturient. This study establishes the sensitivity and specificity of a single CSA cut-off measurement in both supine and right lateral decubitus (RLD) positions in the diagnosis of an empty antrum in paediatric patients., Methods: Following induction of anaesthesia, antral sonography was performed in supine and RLD positions in 100 fasted paediatric patients prior to upper endoscopic evaluation. Following upper endoscopy, any residual stomach content was suctioned under direct visualization and antral sonography was immediately performed. Antral CSA values were compared using Wilcoxon signed rank test. Receiver operator characteristic (ROC) curves were plotted to estimate the discriminating power of antral sonography position in the diagnosis of an empty antrum., Results: Significant differences were found between pre-suctioned and post-suctioned CSA values in the RLD position. The cut-off CSAs of the empty antrum in the supine and RLD positions were 2.19 cm2 (sensitivity 75%, specificity 36%) and 3.07 cm2 (sensitivity 76%, specificity 67%), respectively., Conclusions: The RLD position produces the most sensitive and specific CSA cut-off value where an antral CSA of ≤ 3.07 cm2 in the RLD position presents with acceptable performance in the ability to discriminate an empty antrum in paediatric patients over 1 yr of age. As age increases, the sensitivity and specificity of this test increases in the RLD position., (© The Author 2017. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Several endoparasites of dogs cannot only be detrimental to their primary host but might also represent a threat to human health because of their zoonotic potential. Due to their high dog population densities, metropolitan areas can be highly endemic for such parasites. We aimed to estimate the prevalence of endoparasites in dogs in the Austrian capital of Vienna by examining a representative number of canine faecal samples and to compare the prevalences with two neighbouring peri-urban and rural regions. In addition we analysed whether the density of dog populations and cleanliness of dog zones correlated with parasite occurrence. We collected 1001 anonymous faecal samples from 55 dog zones from all 23 districts of the federal state of Vienna, as well as 480 faecal samples from the Mödling district and Wolkersdorf with a peri-urban and rural character, respectively. Faeces were examined by flotation and by Baermann technique. Additionally we evaluated 292 Viennese, 102 peri-urban and 50 rural samples for Giardia and Cryptosporidium by GiardiaFASTest ® and CryptoFASTest ® . Samples from "clean" dog zones were compared to samples from "dirty" zones. The infection rate of Toxocara was surprisingly low, ranging from 0.6% to 1.9%. Trichuris was the most frequent helminth (1.8-7.5%) and Giardia the most frequent protozoan (4.0-10.8%). Ancylostomatidae, Crenosoma, Capillaria, Taeniidae, Cystoisospora and Sarcocystis were found in 1.8-2.2%, 0-0.9%, 0-0.9%, 0-0.6%, 0.3-3.1% and 0-0.6% of the samples, respectively. Samples from "dirty" dog zones in Vienna showed a significantly higher rate of parasites overall (p=0.003) and of Trichuris (p=0.048) compared to samples from "clean" dog zones. There were no statistically significant differences in densely vs. less densely populated areas of Vienna. Samples from the rural region of Wolkersdorf had significantly higher overall parasite, Trichuris and Cystoisospora prevalences than the peri-urban Mödling district and Vienna (p=0.000-0.039), while samples from the Mödling district had a significantly higher Giardia, Crenosoma and Capillaria prevalence than those from Vienna (p=0.002-0.047). Parasite excretion is dynamic and representative sampling and monitoring are necessary for parasite surveillance. Dog owners should be informed about the zoonotic risk and encouraged to remove dog faeces and dispose of them properly to reduce the infection risk for both other dogs and humans., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A feeding trial was conducted with laying hens where either 10% or 20% regular-fat distiller's dried grains with solubles (R-DDGS) or low-fat DDGS (L-DDGS) were incorporated into the feed. Production parameters and the effect of DDGS on egg nutritional quality, focusing on yolk lipids, were evaluated. Neither R-DDGS nor L-DDGS at up to 20% of laying hen feeds had a statistically significant impact on hen weight gain, egg production, feed intake, feed efficiency, egg mass, or egg weight. Specific gravity was slightly lower for eggs from hens fed 10% R-DDGS or 20% L-DDGS. Eggs from layers fed DDGS had enhanced levels of tocopherols, tocotrienols, and xanthophylls in the yolk, as well as also increased yolk yellow and red color. Eggs from L-DDGS diet had higher tocopherol content, but eggs from R-DDGS diets had higher xanthophylls. Fatty acid composition in eggs was slightly altered by DDGS, but the ratio of saturated to unsaturated fatty acids was very similar. Feeding DDGS to layer hens had no effect on lecithin or cholesterol content of the eggs. Thus, inclusion of DDGS in the diet of laying hens resulted in increases of several beneficial lipophilic nutrients in egg yolks with no apparent detrimental effects., (© 2016 Poultry Science Association Inc.)

Background: The dynamics of PD-L1 expression may limit its use as a tissue-based predictive biomarker. We sought to expand our understanding of the dynamics of PD-L1 expression and tumor-infiltrating lymphocytes (TILs) in patients with lung cancer-related brain metastases., Experimental Design: Paired primary lung cancers and brain metastases were identified and assessed for PD-L1 and CD3 expression by immunohistochemistry. Lesions with 5% or greater PD-L1 expression were considered positive. Agreement statistics and the χ(2) or Fisher's exact test were used for analysis., Results: We analyzed 146 paired lesions from 73 cases. There was disagreement of tumor cell PD-L1 expression in 10 cases (14%, κ = 0.71), and disagreement of TIL PD-L1 expression in 19 cases (26%, κ = 0.38). Most paired lesions with discordant tumor cell expression of PD-L1 were obtained 6 or more months apart. When specimens were categorized using a proposed tumor microenvironment categorization scheme based on PD-L1 expression and TILs, there were significant changes in the classifications because many of the brain metastases lacked either PD-L1 expression, tumor lymphocyte infiltration or both even when they were present in the primary lung cancer specimens (P = 0.009)., Conclusions: We identified that there are significant differences between the tumor microenvironment of paired primary lung cancers and brain metastases. When physicians decide to treat patients with lung cancer with a PD-1 or PD-L1 inhibitor, they must do so in the context of the spatial and temporal heterogeneity of the tumor microenvironment., (© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology.)

Objective: To describe the design and ongoing conduct of the Million Veteran Program (MVP), as an observational cohort study and mega-biobank in the Department of Veterans Affairs (VA) health care system., Study Design and Setting: Data are being collected from participants using questionnaires, the VA electronic health record, and a blood sample for genomic and other testing. Several ongoing projects are linked to MVP, both as peer-reviewed research studies and as activities to help develop an infrastructure for future, broad-based research uses., Results: Formal planning for MVP commenced in 2009; the protocol was approved in 2010, and enrollment began in 2011. As of August 3, 2015, and with a steady state of ≈50 recruiting sites nationwide, N = 397,104 veterans have been enrolled. Among N = 199,348 with currently available genotyping data, most participants (as expected) are male (92.0%) between the ages of 50 and 69 years (55.0%). On the basis of self-reported race, white (77.2%) and African American (13.5%) populations are well represented., Conclusions: By helping to promote the future integration of genetic testing in health care delivery, including clinical decision making, the MVP is designed to contribute to the development of precision medicine., (Published by Elsevier Inc.)

Glutamyl-tRNA reductase (GluTR) is the first enzyme committed to tetrapyrrole biosynthesis by the C(5)-pathway. This enzyme transforms glutamyl-tRNA into glutamate-1-semi-aldehyde, which is then transformed into 5-amino levulinic acid by the glutamate-1-semi-aldehyde 2,1-aminomutase. Binding of heme to GluTR seems to be relevant to regulate the enzyme function. Recombinant GluTR from Acidithiobacillus ferrooxidans an acidophilic bacterium that participates in bioleaching of minerals was expressed in Escherichia coli and purified as a soluble protein containing type b heme. Upon control of the cellular content of heme in E. coli, GluTR with different levels of bound heme was obtained. An inverse correlation between the activity of the enzyme and the level of bound heme to GluTR suggested a control of the enzyme activity by heme. Heme bound preferentially to dimeric GluTR. An intact dimerization domain was essential for the enzyme to be fully active. We propose that the cellular levels of heme might regulate the activity of GluTR and ultimately its own biosynthesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Previous studies have shown that sera of patients with severe atopic dermatitis (AD) contain IgE specific for self-proteins, supporting the hypothesis of autoreactivity as a pathogenic factor in AD. In this study, we screened a large panel of AD patients (n=192) by western blotting (WB) for IgE reactivity not only against the human epithelial cell line A431 but also against primary keratinocytes (KCs). To investigate autoantigenic cell structures in detail, normal human skin and primary KCs were incubated with sera from both WB-reactive patients and, for control purposes, healthy individuals, and analyzed by immunohistology, confocal laser microscopy, and flow cytometry. Our analysis revealed that 28% of AD patients, but not healthy individuals, display serum IgE autoreactivity by WB analysis. The individual IgE reaction patterns of the sera pointed to the existence of unique as well as common specificities against epidermal or A431-derived proteins. Immunostainings identified cytoplasmic and, occasionally, also cell membrane-associated moieties as targets for autoreactive IgE antibodies. Interestingly, in certain autoreactive patients, the surface-staining pattern was accentuated at cellular contact sites. We conclude that IgE autoreactivity is common, particularly among severe AD patients, and that non-transformed primary cells are needed for characterization of the entire spectrum of IgE-defined autoantigens.

Glutamate-1-semialdehyde 2,1-aminomutase (GSAM) is the second enzyme in the C(5) pathway of tetrapyrrole biosynthesis found in most bacteria, in archaea and in plants. It catalyzes the transamination of glutamate-1-semialdehyde to 5-aminolevulinic acid (ALA) in a pyridoxal 5'-phosphate (PLP)-dependent manner. We present the crystal structure of GSAM from the thermophilic cyanobacterium Thermosynechococcus elongatus (GSAM(Tel)) in its PLP-bound form at 2.85A resolution. GSAM(Tel) is a symmetric homodimer, whereas GSAM from Synechococcus (GSAM(Syn)) has been described as asymmetric. The symmetry of GSAM(Tel) thus challenges the previously proposed negative cooperativity between monomers of this enzyme. Furthermore, GSAM(Tel) reveals an extensive flexible region at the interface of the proposed complex of GSAM with glutamyl-tRNA reductase (GluTR), the preceding enzyme in tetrapyrrole biosynthesis. Compared to GSAM(Syn), the monomers of GSAM(Tel) are rotated away from each other along the dimerization interface by 10 degrees . The associated flexibility of GSAM may be essential for complex formation with GluTR to occur. Unexpectedly, we find that GSAM is structurally related to 5-aminolevulinate synthase (ALAS), the ALA-producing enzyme in the Shemin pathway of alpha-proteobacteria and non-plant eukaryotes. This structural relationship applies also to the corresponding subfamilies of PLP-dependent enzymes. We thus propose that the CoA-subfamily (including ALAS) and the aminotransferase subfamily II (including GSAM) are evolutionarily closely related and that ALAS may thus have evolved from GSAM.

Previous studies point to the XPC-hHR23B complex as the principal initiator of global genome nucleotide excision repair (NER) pathway, responsible for the repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) in human cells. However, the UV-damaged DNA binding protein (UV-DDB) has also been proposed as a damage recognition factor involved in repair of UV-photoproducts, especially CPD. Here, we show in human XP-E cells (UV-DDB deficient) that the incision complex formation at UV-induced lesions was severely diminished in locally damaged nuclear spots. Repair kinetics of CPD and 6-4PP in locally and globally UV-irradiated normal human and XP-E cells demonstrate that UV-DDB can mediate efficient targeting of XPC-hHR23B and other NER factors to 6-4PP. The data is consistent with a mechanism in which UV-DDB forms a stable complex when bound to a 6-4PP, allowing subsequent repair proteins--starting with XPC-hHR23B--to accumulate, and verify the lesion, resulting in efficient 6-4PP repair. These findings suggest that (i) UV-DDB accelerates repair of 6-4PP, and at later time points also CPD, (ii) the fraction of 6-4PP that can be bound by UV-DDB is limited due to its low cellular quantity and fast UV dependent degradation, and (iii) in the absence of UV-DDB a slow XPC-hHR23B dependent pathway is capable to repair 6-4PP, and to some extent also CPD.

This study examined the psychometric properties of the Teasing Questionnaire-Revised (TQ-R) and the relationships among recalled childhood teasing and current psychosocial distress in 414 undergraduate students. Participants were administered the TQ-R, Beck Depression Inventory-II, State-Trait Anxiety Inventory-Trait Version, Brief Fear of Negative Evaluation Scale, and UCLA Loneliness Scale. Confirmatory factor analysis supported a five-factor model assessing teasing related to performance, academic issues, social behavior, family background, and appearance. Internal consistency of the TQ-R and its factors was acceptable, and intercorrelations among subscales were moderate, suggesting that the factors measure related but conceptually distinct teasing experiences. Defining Pearson product-moment correlations with a magnitude of greater than.25 as conceptually meaningful, we found that the TQ-R Total score was meaningfully related to depressive symptoms, anxiety, fear of negative evaluation, and loneliness. Being teased in the Performance and Social domains as a child was moderately related to current psychopathology. Implications of these findings for clinical practice and future research are discussed.

Cognitive-behavioral therapy (CBT) and pharmacotherapy demonstrate efficacy across the anxiety disorders, but recognition of their limitations has sparked interest in combining modalities to maximize benefit. This article reviews the empirical literature to examine whether combining treatments influences efficacy of either monotherapy. We conducted a comprehensive literature search of published randomized trials that compared combined treatment with pharmacologic or CBT monotherapies. Ten studies that met our inclusion criteria were reviewed in detail, and within-subjects effect sizes were calculated to compare treatment conditions within and across studies. At posttreatment and follow-up, effect size and percentage responder data failed to clearly demonstrate an advantage or disadvantage of combined treatment over CBT alone for obsessive-compulsive disorder, social phobia, and generalized anxiety disorder. Some advantage of combined treatment over pharmacotherapy alone emerged from the few studies that allowed for such a direct comparison. In contrast, combined treatment for panic disorder seems to provide an advantage over CBT alone at posttreatment, but is associated with greater relapse after treatment discontinuation. The advantage of combined treatment may vary across the anxiety disorders. The potential differences in usefulness of combined treatment are discussed, directions for future research are suggested, and implications for clinical practice are considered.

The chromosome-type exchange aberrations induced by ionizing radiation during the G(0)/G(1) phase of the cell cycle are believed to be the result of illegitimate rejoining of chromosome breaks. From numerous studies using chromosome painting, it has emerged that even after a moderate dose of radiation, a substantial fraction of these exchanges is complex. Most of them are derived from the free interaction between the ends of three or more breaks. Other studies have demonstrated that chromosomes occupy distinct territories in the interphase nucleus. Since breaks that are in close proximity have an enhanced interaction probability, it seems likely that after ionizing radiation many of the interacting breaks will be present within one chromosome or chromosome arm. Unfortunately, the majority of these intrachanges remain undetected, even when sophisticated molecular cytogenetic detection methods (i.e. mFISH) are applied to paint all chromosome pairs in distinct colors. In the present paper, we evaluate the limitations of full-color painting for the detection of complex exchanges and the correct interpretations of break interactions.

We provide the first demonstration for a Na+/Ca2+ exchanger, NCX-1, in the osteoclast. We speculate that by using Na+ exchange, NCX-1 couples H+ extrusion with Ca2+ fluxes during bone resorption. Microspectrofluorimetry of fura-2-loaded osteoclasts revealed a rapid and sustained, but reversible, cytosolic Ca2+ elevation upon Na+ withdrawal. This elevation was abolished by the cytosolic introduction (by gentle permeabilization) of a highly specific Na+/Ca2+ exchange inhibitor peptide, XIP, but not its inactive analogue, sXIP. Confocal microscopy revealed intense plasma membrane immunofluorescence with an isoform-specific monoclonal anti-NCX-1 antibody applied to gently permeabilized osteoclasts. Electrophysiological studies using excised outside-in membrane patches showed a low-conductance, Na+-selective, dichlorobenzamil-sensitive, amiloride-insensitive channel that we tentatively assigned as being an NCX. Finally, to examine for physiological relevance, an osteoclast resorption (pit) assay was performed. There was a dramatic reduction of bone resorption following NCX-1 inhibition by dichlorobenzamil and XIP (but not with S-XIP). Together, the results suggest that a functional NCX, likely NCX-1, is involved in the regulation of osteoclast cytosolic Ca2+ and bone resorption., (Copyright 2001 Academic Press.)

The X-ray crystal structure of the phosphatidylinositol-specific phospholipase C (PI-PLC) from the human pathogen Listeria monocytogenes has been determined both in free form at 2.0 A resolution, and in complex with the competitive inhibitor myo-inositol at 2.6 A resolution. The structure was solved by a combination of molecular replacement using the structure of Bacillus cereus PI-PLC and single isomorphous replacement. The enzyme consists of a single (beta alpha)8-barrel domain with the active site located at the C-terminal side of the beta-barrel. Unlike other (beta alpha)8-barrels, the barrel in PI-PLC is open because it lacks hydrogen bonding interactions between beta-strands V and VI. myo-Inositol binds to the active site pocket by making specific hydrogen bonding interactions with a number of charged amino acid side-chains as well as a coplanar stacking interaction with a tyrosine residue. Despite a relatively low sequence identity of approximately 24%, the structure is highly homologous to that of B.cereus PI-PLC with an r.m.s. deviation for 228 common C alpha positions of 1.46 A. Larger differences are found for loop regions that accommodate most of the numerous amino acid insertions and deletions. The active site pocket is also well conserved with only two amino acid replacements directly implicated in inositol binding.

Myocardial cells from newborn rats were held in a spinner type culture for 2 days and then explanted into culture flasks. Three main cell types were observed: single multipolar cells of embryonic type, cell aggregates containing 10 to 50 connected cells, and bipolar cells retaining some adult characteristics. Except for the latter, up to 95% of intact cells settled and were beating 6 hours after explantation. The percentage of fibroblast-like cells was drastically reduced when compared with conventional cultures. Cell debris could be removed 2 hours after explantation by changing the culture medium, or more effectively by a density step centrifugation using Lymphoprep or Lymphoprep-Ficoll mixtures.

Eyeglasses with protective filters are recommended for users of visible light-curing units to decrease the intensity of blue light reaching the eyes. The transmission curves of 20 commercially available filter glasses are obtained. The irradiance from eight curing units through these glasses is measured. The reduction in irradiance through the filter glasses is related to the transmission curves.