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Purpose: This study aimed to investigate if a deep learning model trained with a single institution's data has comparable accuracy to that trained with multi-institutional data for segmenting kidney and cyst regions in magnetic resonance (MR) images of patients affected by autosomal dominant polycystic kidney disease (ADPKD)., Methods: We used TensorFlow with a Keras custom UNet on 2D slices of 756 MRI images of kidneys with ADPKD obtained from four institutions in the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease (CRISP) study. The ground truth was determined via a manual plus global thresholding method. Five models were trained with 80 % of all institutional data (n = 604) and each institutional data (n = 232, 172, 148, or 52), respectively, and validated with 10 % and tested on an unseen 10 % of the data. The model's performance was evaluated using the Dice Similarity Coefficient (DSC)., Results: The DSCs by the model trained with all institutional data ranged from 0.92 to 0.95 for kidney image segmentation, only 1-2 % higher than those by the models trained with single institutional data (0.90-0.93).In cyst segmentation, however, the DSCs by the model trained with all institutional data ranged from 0.83 to 0.89, which were 2-20 % higher than those by the models trained with single institutional data (0.66-0.86)., Conclusion: The UNet performance, when trained with a single institutional dataset, exhibited similar accuracy to the model trained on a multi-institutional dataset. Segmentation accuracy increases with models trained on larger sample sizes, especially in more complex cyst segmentation., Competing Interests: Declaration of competing interest M. M. reports grants and consulting fees outside the submitted work from Otsuka Pharmaceuticals, Sanofi, Palladio Biosciences, Reata, Natera, Chinook Therapeutics, Goldilocks Therapeutics, and Carraway Therapeutics., (Copyright © 2023. Published by Elsevier Inc.)

Duplex sequencing (DS) is an error-corrected next-generation sequencing method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors in consensus sequences. The resulting background of less than one artifactual mutation per 10 7 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DS-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues⁠, a considerable advancement compared to currently used in vivo gene mutation assays., Competing Interests: Declaration of Competing Interest G.A.P., F.Y.L, J.E.H., E.K.S., L.N.W., D.N., C.C.V., and J.J.S. are employees (or former employees) and equity holders of TwinStrand Biosciences, Inc. F.Y.L, E.K.S., L.N.W., C.C.V., and J.J.S. are authors on one or more duplex sequencing-related patents., (Published by Elsevier B.V.)

Error-corrected duplex sequencing (DS) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DS to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, N-ethyl-N-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25-200 µM) and DNA was sequenced 48, 72, and 96 h post-exposure. A DS mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A significant increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (r = 0.97). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The consistent results across the three time points suggest that 48 h may be sufficient for mutation analysis post-exposure. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DS for characterizing chemical mutagenicity in both research and regulatory evaluation., Competing Interests: Declaration of Competing Interest EC, CDS, MR, AW, CLY, FM, LR, KLW, and SLSR declare that they have no conflict of interest. EKS, JY, THS, PV, FYL, CCV, and JJS are employees and equity holders at TwinStrand Biosciences, Inc. and are authors on one or more duplex sequencing-related patents., (Crown Copyright © 2023. Published by Elsevier B.V. All rights reserved.)

Background: Individuals with intellectual and developmental disabilities demonstrate disparities in sexual and reproductive health (SRH) compared to individuals without disabilities (e.g., lack of sexual education and knowledge, increased rates of abuse, unplanned pregnancies and sexually transmitted infections). Therefore, the purpose of this study was to identify topics healthcare providers address and perceived barriers and supports to SRH education., Methods: We conducted semi-structured interviews with healthcare providers (N = 12)., Results: Providers address relationships, safety, protection and appropriate sexual behaviours with clients with intellectual and developmental disabilities. Parent education and client-centred care were identified as supports, while the patient's level of understanding, the provider's lack of knowledge or access to resources and to appropriate referrals were identified as barriers to SRH education., Conclusion: Future studies are needed to link providers to resources they can use to provide comprehensive, accessible SRH education for clients with intellectual and developmental disabilities., (© 2021 John Wiley & Sons Ltd.)

Importance: Occupational therapy practitioners are expected to translate promising discoveries from empirical research into routine practice with their clients. However, complex barriers can influence practitioners' knowledge translation (KT) efforts, leading the American Occupational Therapy Association's Evidence-Based Practice (EBP) group to develop the KT Toolkit tailored to the perceived needs of occupational therapists and occupational therapy assistants., Objective: To identify common barriers to implementing EBPs and potential strategies to support EBP uptake., Design: Cross-sectional survey., Setting: United States., Participants: Occupational therapy practitioners., Outcomes and Measures: Data underwent descriptive and directed content analysis, the latter of which was guided by the Consolidated Framework for Implementation Research., Results: Occupational therapy survey respondents (N = 818) identified common EBP implementation barriers (e.g., lack of time and resources, difficulty understanding research findings). Initial KT Toolkit content was developed to address these barriers and included resources for searching for, analyzing, and applying evidence in practice., Conclusions and Relevance: Survey findings have informed the development of the KT Toolkit, which includes resources designed to support occupational therapy practitioners' EBP implementation efforts. This KT Toolkit is available at AOTA.org and will be continuously revised and updated on an ongoing basis. What This Article Adds: Several barriers limit the extent to which occupational therapy practitioners can implement evidence with their client populations. The KT Toolkit is directly informed by practitioner input and provides resources to support practitioners in their efforts to translate knowledge into real-world practice., (Copyright © 2022 by the American Occupational Therapy Association, Inc.)

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational 'hot-spots' or 'cold-spots'. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: People with disabilities and chronic health conditions rely on a range of services and supports to complete daily tasks, maintain health, and participate in the community. Preliminary research suggests the COVID-19 pandemic greatly disrupted these services and this population may be particularly susceptible to unemployment., Objective: Describe employment and service disruptions for individuals with disabilities and chronic health conditions during the onset of community-based spread of COVID-19 in the United States., Methods: Adults with disabilities and chronic health conditions completed online surveys to report employment and service changes via multiple choice and open-ended questions. Multiple choice questions were analyzed using descriptive statistics; open-ended responses were coded using content analysis., Results: Participants (n = 109): 79.8% female, 88.1% white, 77.121% completed a 4-year college degree or greater, 61.4% had annual income ≥$45,000. Only 14.9% of survey respondents reported disruptions in employment. On average, 54.0% of service changes were due to discontinuation, including loss of physical therapy, job coaching, community organizations, transportation, and peer supports. Other changes included a shift to virtual service delivery and family members taking the role of service providers., Conclusions: Individuals with chronic health conditions and disabilities experienced service disruptions, even in a sample with considerably more economic, social, and educational privilege than the general population of people with chronic health conditions and disabilities in the United States., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The organotypic human air-liquid-interface (ALI) airway tissue model has been used as an in vitro cell culture system for evaluating the toxicity of inhaled substances. ALI airway cultures are highly differentiated, which has made it challenging to evaluate genetic toxicology endpoints. In the current study, we assayed DNA damage with the high-throughput CometChip assay and quantified mutagenesis with Duplex Sequencing, an error-corrected next-generation sequencing method capable of detecting a single mutation per 10 7 base pairs. Fully differentiated human ALI airway cultures were treated from the basolateral side with 6.25 to 100 μg/mL ethyl methanesulfonate (EMS) over a period of 28 days. CometChip assays were conducted after 3 and 28 days of treatment, and Duplex Sequencing after 28 days of treatment. Treating the airway cultures with EMS resulted in time- and concentration-dependent increases in DNA damage and a concentration-dependent increase in mutant frequency. The mutations observed in the EMS-treated cultures were predominantly C → T transitions and exhibited a unique trinucleotide signature relative to the negative control. Measurement of physiological endpoints indicated that the EMS treatments had no effect on anti-p63-positive basal cell frequency, but produced concentration-responsive increases in cytotoxicity and perturbations in cell morphology, along with concentration-responsive decreases in culture viability, goblet cell and anti-Ki67-positive proliferating cell frequency, cilia beating frequency, and mucin secretion. The results indicate that a unified 28-day study can be used to measure several important safety endpoints in physiologically relevant human in vitro ALI airway cultures, including DNA damage, mutagenicity, and tissue-specific general toxicity., (© 2021 The Authors. Emergency Medicine Australasia published by John Wiley & Sons Australia, Ltd on behalf of Australasian College for Emergency Medicine. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Importance: People with intellectual and developmental disabilities (IDD) express a clear interest in intimate relationships but face many barriers to receiving sex education (SE) that would support their engagement in these relationships., Objective: To understand barriers to, the context of, and recommendations for SE for people with IDD., Design: Qualitative study design with interviews and focus groups with four key stakeholder groups. Data were analyzed using a constant comparative approach., Participants: Participants were 8 youths with IDD, 9 parents, 12 health care providers, and 8 educators., Results: Four barriers to SE were identified: (1) values and cultural issues, (2) parental attitudes toward their child's sexuality, (3) a lack of organizational policies and standards, and (4) limited professional education or societal biases. These barriers contribute to a SE context primarily initiated by people with IDD or provided reactively. The participants recommended proactive, formal SE provided by multiple stakeholders throughout adulthood., Conclusions and Relevance: Stakeholders should advocate for policies, standards, and additional training for parents, educators, and health care providers to support SE for people with IDD throughout adulthood. What This Article Adds: Barriers to SE contribute to the current context in which SE is shared with people with IDD. Stakeholders can advocate for policies, standards, and training to overcome these barriers and support recommendations for proactive, formal SE provided by multiple stakeholders through adulthood., (Copyright © 2021 by the American Occupational Therapy Association, Inc.)

Activities of daily living (ADLs) are activities that people engage in on a routine basis, such as brushing their teeth, preparing a meal, and caring for their child. Independence with ADLs is associated with better outcomes in independent living, education, employment, relationships, and mental health. Therefore, this perspective piece includes a review of the literature and assessment databases to identify and summarize ADL assessments for Autistic adults. These assessments were compared and analyzed using the neurodiversity paradigm. Specifically, we compared assessments using predetermined priorities the authors identified: (1) assessment type, (2) inclusivity, and (3) performance factors. We identified five unique norm-referenced measures, four performance-based measures, and a variety of checklists, surveys, questionnaires, and/or interviews used to assess ADL performance among Autistic adults. The authors present their perspectives regarding the challenges with the current assessments, including the high-frequency use of norm-referenced assessments, lack of inclusivity, and failure to consider performance factors (e.g., sensory, motor, and emotional), and the paucity of assessments designed specifically for Autistic adults older than 30 years. In response to these challenges, we recommend researchers partner with Autistic adults to develop a new assessment tool. If researchers or clinicians are using existing measures, we recommend that they utilize self-report over proxy-report and include methods to improve the accessibility of the assessment. We also recommend that clinicians and researchers offer breaks, comfort objects, or sensory modifications during the assessment to decrease anxiety; and ask follow-up questions to understand whether environment or emotional health are impacting one's ADL performance., Lay Summary: Why is this topic important?: Activity of daily living (ADL) assessments are used to determine what Autistics can and cannot do in their day-to-day life, what services they may be eligible for, and to monitor gains. However, we struggled to find an assessment that was useful and relevant from an Autistic point-of-view. What is the purpose of this article?: The purpose of this article was to review and evaluate current ADL assessments usefulness for Autistics and provide recommendations for improving the ADL assessment process. What is the perspective of the authors?: The first author is an Autistic social worker and the second author is an occupational therapist and postdoctoral fellow. The authors' perspective is based in the neurodiversity paradigm and social model of disability, which centers on respecting and acknowledging differences in the brain and their effects on Autistics' lives. We believe in strengths-based approaches versus deficit-based models. What did you find about this topic?: We found 17 measurement tools, some that compared Autistic ADL performance with neurotypical performance, a few that were observation-based meaning the researcher or clinician watched the Autistic person complete the ADLs, and many forms or guides that asked questions about ADL performance. Only six measures allowed Autistic people to respond to the questions themselves, whereas the rest of the measures had someone else respond for them. These measures did not include questions about how sensory differences (e.g., feeling upset by the feeling of jeans or the taste of minty toothpaste) or feeling sad or nervous may also impact ADL performance. What do the authors recommend?: We recommend that researchers partner with Autistic adults to make new ADL assessments. If researchers or clinicians are using previously made ADL assessments, we recommend that they use self-report and adapt the materials to make it easier to understand (e.g., using pictures). We also recommend that researchers and clinicians ask Autistics what they need or want to make the assessment easier and more comfortable for them. Finally, researchers and clinicians should ask follow-up questions about sensory differences and whether someone is feeling sad or nervous to know how this impacts their ability to do their ADL tasks. How will these recommendations help autistic adults now or in the future?: These recommendations will help Autistic adults be more involved in the evaluation process, which will make the assessments more trustworthy and relevant to Autistics. This also could help more Autistic people get services and supports that are useful to them. Finally, this may help researchers when monitoring if these supports or services actually work., Competing Interests: No competing financial interests exist., (Copyright 2021, Mary Ann Liebert, Inc., publishers.)

Sexual and reproductive health is an important aspect of human development, but discussions with adolescents and young adults on this topic are often challenging for health-care providers. As a result, many adolescents and young adults do not receive appropriate, comprehensive sexual education, despite recognition from WHO and the UN that access to this education is a human right. Adolescents and young adults with mild to moderate intellectual or developmental disability, or both, are just as likely to be sexually active as are their peers without disability; however, these individuals are less likely to receive comprehensive sexual education. To ensure adequate comprehensive sexual education for adolescents and young adults with intellectual and developmental disabilities, sexual health educators should facilitate conversations about sexual and reproductive health that are non-judgmental and sexually inclusive. Such initiatives should use an educational framework grounded in universal design for learning, including use of multiple media types with clear, concise language and images., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Hereditary gelsolin (AGel) amyloidosis is an autosomal dominantly inherited systemic amyloidosis that manifests with the characteristic triad of progressive ophthalmological, neurological and dermatological signs and symptoms. The National Finnish Gelsolin Amyloidosis Registry (FIN-GAR) was founded in 2013 to collect clinical data on patients with AGel amyloidosis, including altogether approximately one third of the Finnish patients. We aim to deepen knowledge on the disease burden and life span of the patients using data from the updated FIN-GAR registry. We sent an updated questionnaire concerning the symptoms and signs, symptomatic treatments and subjective perception on disease progression to 240 members of the Finnish Amyloidosis Association (SAMY). We analyzed the lifespan of 478 patients using the relative survival (RS) framework., Results: The updated FIN-GAR registry includes 261 patients. Symptoms and signs corresponding to the classical triad of ophthalmological (dry eyes in 93%; corneal lattice amyloidosis in 89%), neurological (numbness, tingling and other paresthesias in 75%; facial paresis in 67%), and dermatological (drooping eyelids in 86%; cutis laxa in 84%) manifestations were highly prevalent. Cardiac arrhythmias were reported by 15% of the patients and 5% had a cardiac pacemaker installed. Proteinuria was reported by 13% and renal failure by 5% of the patients. A total of 65% of the patients had undergone a skin or soft tissue surgery, 26% carpal tunnel surgery and 24% at least unilateral cataract surgery. As regards life span, relative survival estimates exceeded 1 for males and females until the age group of 70-74 years, for which it was 0.96., Conclusions: AGel amyloidosis causes a wide variety of ophthalmological, neurological, cutaneous, and oral symptoms that together with repeated surgeries cause a clinically significant disease burden. Severe renal and cardiac manifestations are rare as compared to other systemic amyloidoses, explaining in part the finding that AGel amyloidosis does not shorten the life span of the patients at least for the first 75 years.

Self-advocacy is a client's ability to represent one's interests when managing disease or disability. Self-advocacy may increase one's ability to seek, evaluate, and use information to promote health, yet little is known about the role of occupational therapy in promoting self-advocacy. This scoping review aims to identify interventions within occupational therapists' scope of practice to improve self-advocacy. A literature search was conducted through Academic Search Complete to identify interventions within the scope of occupational therapy that promote self-advocacy. All levels of evidence were included, and articles were reviewed for inclusion by two authors. Included articles were charted for level of evidence, objectives, participants, and results. Fourteen articles met the criteria. Interventions included interactive multimedia interventions, peer-led educational groups, writing interventions, job counseling and advocacy, and disease-specific advocacy programs. Occupational therapists are well-equipped to intervene and promote self-advocacy through workplace modification, utilizing assistive technologies, and facilitating peer-led educational groups.

Objectives: Previous clinical studies have shown frequent cardiac symptoms in patients with hereditary gelsolin (AGel) amyloidosis, possibly related to amyloid deposition in the heart and other internal organs. Previous studies on internal organ amyloid deposition in AGel amyloidosis have been based on small patient series. Methods: Paraffin-embedded tissue sections from 25 autopsied individuals (age at death 44.4-88.6 years) with AGel amyloidosis were stained with HE, Congo red and Herovici stains and immunohistochemistry against the low molecular weight gelsolin fraction was performed. The amount of amyloid was estimated semi-quantitatively. Results: AGel-based amyloid deposits were found in the myocardium and cardiac blood vessels in every patient. The deposits were mainly small and co-localized with regions with excess fibrosis in the myocardium. The lungs were positive for amyloid in 79%, renal parenchyma in 54% and renal blood vessels in 71% of the cases. The amount of myocardial, renal and hepatic amyloid correlated with age at death of the patients. Conclusions: We show the constant presence of AGel amyloid in the hearts of patients with AGel amyloidosis. Although the deposits were mainly small, the co-localization of amyloid with fibrosis may amplify the effect of pure amyloid deposition, possibly leading to clinical signs and symptoms.

Somatic mutations in the mitochondrial genome (mtDNA) have been linked to multiple disease conditions and to ageing itself. In Drosophila, knock-in of a proofreading deficient mtDNA polymerase (POLG) generates high levels of somatic point mutations and also small indels, but surprisingly limited impact on organismal longevity or fitness. Here we describe a new mtDNA mutator model based on a mitochondrially-targeted cytidine deaminase, APOBEC1. mito-APOBEC1 acts as a potent mutagen which exclusively induces C:G>T:A transitions with no indels or mtDNA depletion. In these flies, the presence of multiple non-synonymous substitutions, even at modest heteroplasmy, disrupts mitochondrial function and dramatically impacts organismal fitness. A detailed analysis of the mutation profile in the POLG and mito-APOBEC1 models reveals that mutation type (quality) rather than quantity is a critical factor in impacting organismal fitness. The specificity for transition mutations and the severe phenotypes make mito-APOBEC1 an excellent mtDNA mutator model for ageing research.

Aim: Individuals with intellectual and developmental disabilities (I/DD) may have an increased risk of sexually transmitted infections (STIs) due to limited sexual health education and higher rates of sexual abuse, yet little is known about the prevalence of STIs and STI testing in this population. Methods: This study compared national samples of privately insured individuals with (n = 25,193) and without I/DD (n = 25,193) on the prevalence of STIs and STI testing. Results: In multivariable models, individuals with I/DD were significantly less likely to have an STI diagnosis and no difference was found between groups on the odds of STI testing overall. Conclusion: Findings may, in part, be explained by fewer sexual experiences, increased supervision in social settings and delayed onset of sexual activity among those with I/DD.

Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of TP53 and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic TP53 mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment., (© 2018 Nachmanson et al.; Published by Cold Spring Harbor Laboratory Press.)

Finnish gelsolin amyloidosis (AGel amyloidosis) is an autosomal dominantly inherited systemic disorder with ophthalmologic, neurologic and dermatologic symptoms. Only the gelsolin (GSN) c.640G>A variant has been found in the Finnish patients thus far. The purpose of this study was to examine whether the Finnish patients have a common ancestor or whether multiple mutation events have occurred at c.640G, which is a known mutational hot spot. A total of 79 Finnish AGel amyloidosis families including 707 patients were first discovered by means of patient interviews, genealogic studies and civil and parish registers. From each family 1-2 index patients were chosen. Blood samples were available from 71 index patients representing 64 families. After quality control, SNP array genotype data were available from 68 patients from 62 nuclear families. All the index patients had the same c.640G>A variant (rs121909715). Genotyping was performed using the Illumina CoreExome SNP array. The homozygosity haplotype method was used to analyse shared haplotypes. Haplotype analysis identified a shared haplotype, common to all studied patients. This shared haplotype included 17 markers and was 361 kb in length (GRCh37 coordinates 9:124003326-124364349) and this level of haplotype sharing was found to occur highly unlikely by chance. This GSN haplotype ranked as the largest shared haplotype in the 68 patients in a genome-wide analysis of haplotype block lengths. These results provide strong evidence that although there is a known mutational hot spot at GSN c.640G, all of the studied 62 Finnish AGel amyloidosis families are genetically linked to a common ancestor.

Background: Finnish type of hereditary gelsolin amyloidosis (AGel amyloidosis) is an autosomal dominant disorder. Until recently, there has only been little knowledge of fatal complications of the disease and its possible impact on the patients' life span., Methods: We identified 272 deceased patients based on patient interviews and genealogical data. After collecting their death certificates, we recorded the patients' underlying and immediate causes of death (CoD) and life span and compared them to the general Finnish population. We then calculated proportional mortality ratios (PMR), standardised for age and sex, for the CoDs., Results: The underlying CoD in 20% of the patients was AGel amyloidosis (PMR = 114.2; 95% CI: 85.6-149.4). The frequency of fatal cancers (10%) was significantly diminished (PMR = 0.47; 95% CI: 0.31-0.69). Renal complications were overrepresented as the immediate CoD in female patients (PMR = 2.82 95% CI: 1.13-5.81). The mean life span for male patients was 73.9 years (95% CI: 72.0-75.6) and 78.0 years for female patients (95% CI: 76.4-79.5) compared to 72.1 and 80.1 years for the general population., Conclusions: Our results suggest that the disease increases the risk of fatal renal complications but does not substantially shorten the life span, possibly due to the significantly lower frequency of fatal cancers. Key Messages AGel amyloidosis may increase the risk of renal complications, especially among female patients. The frequency of fatal cancers is significantly lower. The patients' life span is comparable to that of the general population.

Purpose: To investigate gender differences in Finnish gelsolin amyloidosis (AGel amyloidosis)., Patients and Methods: AGel amyloidosis patients, who were members of Finnish Amyloidosis Association (SAMY), filled in a questionnaire compiling known and suspected aspects of their disease. Telephone interviews and hospital medical records, when available, complemented the questionnaire. The data were entered to the database in order to create a national AGel amyloidosis patient registry (FIN-GAR)., Results: A total of 227 patients, 156 women and 71 men, participated in the study. The women in our registry noticed their first symptoms at the median age of 39 years versus 43 years for men (p = 0.01). At the age in which the diagnosis was made there was a trend to be observed between men and women (women: 39 years versus men: 43 years, p = 0.053). Corneal lattice dystrophy was diagnosed in significantly younger women than men (median ages 41 versus 49 years, respectively, p = 0.01). Of other ophthalmological manifestations, corneal ulcer, impaired vision and glaucoma were all diagnosed at least 5 years earlier in women, although differences were not statistically significant. Ophthalmological manifestations, such as dry eyes and corneal ulcer; dermatological signs, such as blepharochalasis, and also neurological symptoms, such as myokymia and carpal tunnel syndrome, were more prevalent among women., Conclusions: In the largest so far available study on AGel amyloidosis we show that women developed symptoms and signs of AGel amyloidosis at younger age. Especially eye-related problems occurred earlier and together with nerve and skin manifestations, the characteristic clinical triad in AGel amyloidosis, were more common in women. However, a clear limitation of our study was a selection bias caused by a significant underrepresentation of men in the study population.

Background: Finnish type of hereditary gelsolin amyloidosis (FGA) is one of the most common diseases of Finnish disease heritage. Existing FGA knowledge is based only on smaller patient series, so our aim was to elucidate the natural course of the disease in a comprehensive sample of patients and to build up a national FGA patient registry., Methods: An inquiry about the known and suspected signs of FGA, sent to the members of Finnish Amyloidosis Association, telephone contacts, and hospital records were utilized to create the registry., Results: A total of 227 patients were entered to the database. The first symptom was ophthalmological for 167 patients (73.6%) at the mean age of 39 years. Corneal lattice dystrophy (CLD) was reported at the mean age of 43 years. Impaired vision, polyneuropathy, facial nerve paresis, and cutis laxa appeared on average between 52 and 57 years. Carpal tunnel syndrome (CTS) was reported by 86 patients (37.9%). Nine patients (4.0%) had a pacemaker, and 12 (6.1%) had cardiomyopathy., Conclusions: The first symptom was ophthalmological in most cases. Except for CLD no prominent difference in the age of appearance was found between the major symptoms. CTS, cardiac pacemakers, and cardiomyopathy were remarkably more common compared to the general population.

This paper discusses the development of a mixed methods approach to analyse research funding. Research policy has taken on an increasingly prominent role in the broader political scene, where research is seen as a critical factor in maintaining and improving growth, welfare and international competitiveness. This has motivated growing emphasis on the impacts of science funding, and how funding can best be designed to promote socio-economic progress. Meeting these demands for impact assessment involves a number of complex issues that are difficult to fully address in a single study or in the design of a single methodology. However, they point to some general principles that can be explored in methodological design. We draw on a recent evaluation of the impacts of research grant funding, discussing both key issues in developing a methodology for the analysis and subsequent results. The case of research grant funding, involving a complex mix of direct and intermediate effects that contribute to the overall impact of funding on research performance, illustrates the value of a mixed methods approach to provide a more robust and complete analysis of policy impacts. Reflections on the strengths and weaknesses of the methodology are used to examine refinements for future work., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation that exhibits specific mechanisms to control the immune response. Here we show that in response to polyriboinosinic:polyribocytidylic acid (pI:C), DCs mount a specific integrated stress response during which the transcription factor ATF4 and the growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), a phosphatase 1 (PP1) cofactor, are expressed. In agreement with increased GADD34 levels, an extensive dephosphorylation of the translation initiation factor eIF2α was observed during DC activation. Unexpectedly, although DCs display an unusual resistance to protein synthesis inhibition induced in response to cytosolic dsRNA, GADD34 expression did not have a major impact on protein synthesis. GADD34, however, was shown to be required for normal cytokine production both in vitro and in vivo. These observations have important implications in linking further pathogen detection with the integrated stress response pathways.

Nucleic acid sensing by cells is a key feature of antiviral responses, which generally result in type-I Interferon production and tissue protection. However, detection of double-stranded RNAs in virus-infected cells promotes two concomitant and apparently conflicting events. The dsRNA-dependent protein kinase (PKR) phosphorylates translation initiation factor 2-alpha (eIF2α) and inhibits protein synthesis, whereas cytosolic DExD/H box RNA helicases induce expression of type I-IFN and other cytokines. We demonstrate that the phosphatase-1 cofactor, growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), an important component of the unfolded protein response (UPR), is absolutely required for type I-IFN and IL-6 production by mouse embryonic fibroblasts (MEFs) in response to dsRNA. GADD34 expression in MEFs is dependent on PKR activation, linking cytosolic microbial sensing with the ATF4 branch of the UPR. The importance of this link for anti-viral immunity is underlined by the extreme susceptibility of GADD34-deficient fibroblasts and neonate mice to Chikungunya virus infection.

In this study, the principles of surface sensing of translation (SUnSET) were used to develop a nonradioactive method for ex vivo and in vivo measurements of protein synthesis (PS). Compared with controls, we first demonstrate excellent agreement between SUnSET and a [(3)H]phenylalanine method when detecting synergist ablation-induced increases in skeletal muscle PS ex vivo. We then show that SUnSET can detect the same synergist ablation-induced increase in PS when used in vivo (IV-SUnSET). In addition, IV-SUnSET detected food deprivation-induced decreases in PS in the heart, kidney, and skeletal muscles, with similar changes being visualized with an immunohistochemical version of IV-SUnSET (IV-IHC-SUnSET). By combining IV-IHC-SUnSET with in vivo transfection, we demonstrate that constitutively active PKB induces a robust increase in skeletal muscle PS. Furthermore, transfection with Ras homolog enriched in brain (Rheb) revealed that a PKB-independent activation of mammalian target of rapamycin is also sufficient to induce an increase in skeletal muscle PS. Finally, IV-IHC-SUnSET exposed the existence of fiber type-dependent differences in skeletal muscle PS, with PS in type 2B and 2X fibers being significantly lower than that in type 2A fibers within the same muscle. Thus, our nonradioactive method allowed us to accurately visualize and quantify PS under various ex vivo and in vivo conditions and revealed novel insights into the regulation of PS in skeletal muscle.

Considerable evidence indicates that the general blockade of protein synthesis prevents both the initial consolidation and the postretrieval reconsolidation of long-term memories. These findings come largely from studies of drugs that block ribosomal function, so as to globally interfere with both cap-dependent and -independent forms of translation. Here we show that intra-amygdala microinfusions of 4EGI-1, a small molecule inhibitor of cap-dependent translation that selectively disrupts the interaction between eukaryotic initiation factors (eIF) 4E and 4G, attenuates fear memory consolidation but not reconsolidation. Using a combination of behavioral and biochemical techniques, we provide both in vitro and in vivo evidence that the eIF4E-eIF4G complex is more stringently required for plasticity induced by initial learning than for that triggered by reactivation of an existing memory.

Background: Dendritic cells (DCs) are the sentinels of the mammalian immune system, characterized by a complex maturation process driven by pathogen detection. Although multiple studies have described the analysis of activated DCs by transcriptional profiling, recent findings indicate that mRNAs are also regulated at the translational level. A systematic analysis of the mRNAs being translationally regulated at various stages of DC activation was performed using translational profiling, which combines sucrose gradient fractionation of polysomal-bound mRNAs with DNA microarray analysis., Results: Total and polysomal-bound mRNA populations purified from immature, 4 h and 16 h LPS-stimulated human monocyte-derived DCs were analyzed on Affymetrix microarrays U133 2.0. A group of 375 transcripts was identified as translationally regulated during DC-activation. In addition to several biochemical pathways related to immunity, the most statistically relevant biological function identified among the translationally regulated mRNAs was protein biosynthesis itself. We singled-out a cluster of 11 large ribosome proteins mRNAs, which are disengaged from polysomes at late time of maturation, suggesting the existence of a negative feedback loop regulating translation in DCs and linking ribosomal proteins to immuno-modulatory function., Conclusion: Our observations highlight the importance of translation regulation during the immune response, and may favor the identification of novel protein networks relevant for immunity. Our study also provides information on the potential absence of correlation between gene expression and protein production for specific mRNA molecules present in DCs.

We developed a nonradioactive fluorescence-activated cell sorting-based assay, called surface sensing of translation (SUnSET), which allows the monitoring and quantification of global protein synthesis in individual mammalian cells and in heterogeneous cell populations. We demonstrate here, using mouse dendritic and T cells as a model, that SUnSET offers a technical alternative to classical radioactive labeling methods for the study of mRNA translation and cellular activation.

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control antigen processing and presentation. Here, we show that in response to lipopolysaccharides, protein synthesis is rapidly enhanced in DCs. This enhancement occurs via a PI3K-dependent signaling pathway and is key for DC activation. In addition, we show that later on, in a manner similar to viral or apoptotic stress, DC activation leads to the phosphorylation and proteolysis of important translation initiation factors, thus inhibiting cap-dependent translation. This inhibition correlates with major changes in the origin of the peptides presented by MHC class I and the ability of mature DCs to prevent cell death. Our observations have important implications in linking translation regulation with DC function and survival during the immune response.

Further insight into the behavior of suppositional diradicals in a caldera is sought in the thermal rearrangements among the four "Delta2-thujenes", two 1-isopropyl-4-methylbicyclo[3.1.0]hex-2-enes [(-)-cis-1 and (+)-trans-2] and two isomers, exo- and endo-3-isopropyl-6-methylbicyclo[3.1.0]hex-2-ene [(+)-exo-3 and (-)-endo-4]. Optically pure trans-3-isopropyl-5-vinylcyclopentene (5) is the final, strongly thermochemically favored product, the result of an intramolecular homodienyl shift of a methyl hydrogen atom in (-)-endo-4. The set of twelve specific rate constants, four sets of three each, that define the interrelations among the four isomers has been extracted from data acquired starting from each isomer. An attractive mechanistic hypothesis involving an intermediate diradical of iso conformation, common, for example, to both (-)-cis-1 and (+)-exo-3 (as educts), that proceeds to an anticonformer common to both (+)-trans-2 and (-)-endo-4 does not lead to a satisfactory rationalization of the product distribution. Addition of a second mechanistic conceptual scheme, that of a diradical-in-transit behaving as if there were a measure of continuous bonding (for example, (+)-trans-2 proceeding directly to (-)-cis-1), improves agreement with experiment. Over a 30 degrees C range of temperature, there is no credible change in product distribution.

Background: Erythropoietin is a multifunctional cytokine which regulates the number of erythrocytes circulating in mammalian blood. This is crucial in order to maintain an appropriate oxygen supply throughout the body. Stimulation of primary human erythroid progenitors (PEPs) with erythropoietin (Epo) leads to the activation of the mitogenic kinases (MEKs and Erks). How this is accomplished mechanistically remained unclear., Results: Biochemical studies with human cord blood-derived PEPs now show that Ras and the class Ib enzyme of the phosphatidylinositol-3 kinase (PI3K) family, PI3K gamma, are activated in response to minimal Epo concentrations. Surprisingly, three structurally different PI3K inhibitors block Ras, MEK and Erk activation in PEPs by Epo. Furthermore, Erk activation in PEPs is insensitive to the inhibition of Raf kinases but suppressed upon PKC inhibition. In contrast, Erk activation induced by stem cell factor, which activates c-Kit in the same cells, is sensitive to Raf inhibition and insensitive to PI3K and PKC inhibitors., Conclusions: These unexpected findings contrast with previous results in human primary cells using Epo at supraphysiological concentrations and open new doors to eventually understanding how low Epo concentrations mediate the moderate proliferation of erythroid progenitors under homeostatic blood oxygen levels. They indicate that the basal activation of MEKs and Erks in PEPs by minimal concentrations of Epo does not occur through the classical cascade Shc/Grb2/Sos/Ras/Raf/MEK/Erk. Instead, MEKs and Erks are signal mediators of PI3K, probably the recently described PI3K gamma, through a Raf-independent signaling pathway which requires PKC activity. It is likely that higher concentrations of Epo that are induced by hypoxia, for example, following blood loss, lead to additional mitogenic signals which greatly accelerate erythroid progenitor proliferation.

Protoporphyrin IX is the last common intermediate of tetrapyrrole biosynthesis. The chelation of a Mg2+ ion by magnesium chelatase and of a ferrous ion by ferrochelatase directs protoporphyrin IX towards the formation of chlorophyll and heme, respectively. A full length cDNA clone encoding a ferrochelatase was identified from a Nicotiana tabacum cDNA library. The encoded protein consists of 497 amino acid residues with a molecular weight of 55.4 kDa. In vitro import of the protein into chloroplasts and its location in stroma and thylakoids confirm its close relationship to the previously described Arabidopsis thaliana plastid-located ferrochelatase (FeChII). A 1700-bp tobacco FeCh cDNA sequence was expressed in Nicotiana tabacum cv. Samsun NN under the control of the CaMV 35S promoter in antisense orientation allowing investigation into the consequences of selective reduction of the plastidic ferrochelatase activity for protoporphyrin IX channeling in chloroplasts and for interactions between plastidic and mitochondrial heme synthesis. Leaves of several transformants showed a reduced chlorophyll content and, during development, a light intensity-dependent formation of necrotic leaf lesions. In comparison with wild-type plants the total ferrochelatase activity was decreased in transgenic lines leading to an accumulation of photosensitizing protoporphyrin IX. Ferrochelatase activity was reduced only in plastids but not in mitochondria of transgenic plants. By means of the specifically diminished ferrochelatase activity consequences of the selective inhibition of protoheme formation for the intracellular supply of heme can be investigated in the future.

Chronic myelogenous leukemia (CML) is commonly characterized by the presence of the p210(Bcr-Abl) oncoprotein. Many downstream effectors of Bcr-Abl have been described, including activation of the Grb2-SoS-Ras-MAP kinase (Erk) pathway. The precise contributions of these signal-transduction proteins in CML blast cells in human patients are not yet well defined. To gain further insight into the importance of Grb2 for CML, peptides that disrupt Grb2-SoS complexes were tested. These high-affinity Grb2-binding peptides (HAGBPs) can autonomously shuttle into cells and function by binding to the N-terminal SH3 domain of Grb2. The HAGBPs were analyzed for their effects on Bcr-Abl-expressing cell lines and freshly isolated CML blast cells from patients. They induced a dramatic decrease in the proliferation of CML cell lines. This was not observed with point-mutated control peptides with abolished Grb2SH3(N) binding. As expected, Grb2-SoS complexes were greatly diminished in the HAGBP-treated cells, and MAP kinase activity was significantly reduced as determined by an activation-specific phospho-MAPK antibody. Furthermore, cell fractions that are enriched for blast cells from CML patients with active disease were also incubated with the Grb2 blocker peptides. The HAGBPs led to a significant proliferation reduction of these cells in the majority of the isolates, but not in all patients' cells. These results show that, in addition to the direct targeting of Bcr-Abl, selective inhibition of Grb2 protein complexes may be a therapeutic option for a significant number of CML patients.

The adapter Grb2 is an important mediator of normal cell proliferation and oncogenic signal transduction events. It consists of a central SH2 domain flanked by two SH3 domains. While the binding specificities of the Grb2 SH2 and N-terminal SH3 domain [Grb2 SH3(N)] have been studied in detail, binding properties of the Grb2 SH3(C) domain remained poorly defined. Gab1, a receptor tyrosine kinase substrate which associates with Grb2 and the c-Met receptor, was previously shown to bind Grb2 via a region which lacks a Grb2 SH3(N)-typical motif (P-x-x-P-x-R). Precipitation experiments with the domains of Grb2 show now that Gab1 can bind stably to the Grb2 SH3(C) domain. For further analyses, Gab1 mutants were generated by PCR to test in vivo residues thought to be crucial for Grb2 SH3(C) binding. The Grb2 SH3(C) binding region of Gab1 has significant homology to a region of the adapter protein SLP-76. Peptides corresponding to epitopes SLP-76, Gab1, SoS and other proteins with related sequences, as well as mutant peptides were synthesized and analysed by tryptophan-fluorescence spectrometry and by in vitro competition experiments. These experiments define a 13 amino acid sequence with the unusual consensus motif P-x-x-x-R-x-x-K-P as required for a stable binding to the SH3(C) domain of Grb2. Additional analyses point to a distinct binding specificity of the Grb2-homologous adapter protein Mona (Gads), indicating that the proteins of the Grb2 adapter family may have partially overlapping, yet distinct protein binding properties.

A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS). Phosphorylation-specific marker ions (m/z 79, PO(3)(-), and m/z 97, H(2)PO(4)(-)) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO(3)(-)) and m/z 97 (H(2)PO(4)(-)) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C(2)F(3)O(-) fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic beta-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by (mu)LC/ESI-sCID-MS and (mu)LC/ESI-MS analysis., (Copyright 2001 John Wiley & Sons, Ltd.)

Our strategy to provide the structural basis for the build-up of functional tethered membranes focuses on three approaches: the first one is based on the pre-organization of a monomolecular layer of a lipopolymer at the water/air interface which is then transferred to a solid support. Prior to deposition, the substrate is coated with a layer of benzophenone-derivatized silane molecules that allow for a stable covalent attachment by photo-cross-linking of some of the monomer units of the lipopolymer to the support. An alternative concept realizes a layer-by-layer deposition of the various structural elements: (1) the attachment layer with the reactive sites for the chemical stabilization; (2) a polymer 'cushion' prepared by adsorption and simultaneous or subsequent partial covalent binding to the reactive sites; and (3) a lipid monolayer transferred from the water/air interface, that contains a certain amount of lipids with reactive headgroups which, upon binding to the polymer tether, act as anchor lipids stabilizing the whole monolayer/cushion-composite. And finally, we build peptide-supported monolayers by first (self-) assembling amino acid sequences of various lengths via a SH-group near their N-terminus onto Au substances and use then their COO(-)-terminus to chemically attach phosphatidyl-ethanolamine lipids to form a stable monolayer of lipid-peptide conjugates. All the individual preparation steps and the various resulting (multi-) layers are characterized by surface plasmon spectroscopy, X-ray and neutron-reflectometry, contact angle measurements, IR spectroscopy, fluorescence microscopy, scanning probe microscopies, as well as, electrochemical techniques. For all tethering systems, the final membranes' architecture is obtained by fusing lipid vesicles onto the lipid monolayer. Proteins can be incorporated by either fusing vesicles that are loaded with the respective receptors, pores, or ion pumps via a reconstitution procedure, or via a transfer directly from a micellar solution to the pre-formed lipid bilayer at the solid support by a dialysis step. Two structural/dynamical features of tethered membranes which are considered to be of particular functional relevance, i.e. the degree of water uptake and, hence, the degree of swelling of the polymer support, as well as the lateral mobility of the lipid molecules in the membrane, are tested by surface plasmon optics and by measurements of the fluorescence recovery after photobleaching (FRAP), respectively. The results confirm that the presented preparation protocols yield fluid bilayers that mimic certain relevant properties of biological membranes. The functional characterization of tethered membranes, which is briefly summarized, is based on various electrochemical techniques, in particular, impedance spectroscopy, cyclic voltammetry, and chronoamperometric studies. The results obtained for reconstituted H(+)-ATPase from chloroplasts and E. coli and for cytochrome oxidase (with and without cytochrome c) confirm the incorporation of the proteins in an active form, thus, opening opportunities for novel sensor formats or offering a completely new model membrane system.

Background and Objectives: To determine the sentinel node detection rate and the accuracy with which the sentinel node histology reflects that of the axilla in a series of patients with palpable invasive breast cancer., Methods: Forty-four patients with clinically node-negative palpable invasive T1 or T2 breast tumors underwent sentinel node biopsy using isosulfan blue dye, followed immediately by either local excision of the primary lesion with standard axillary lymph node dissection or modified radical mastectomy. All surgeries were performed at Northwest Hospital, Seattle, Washington, between January 1996 and October 1997., Results: The sentinel node was successfully identified in 73% of the patients (32/44). The frequency of sentinel node detection was greater for tumors in the outer quadrants than the inner quadrants (z-test, P < 0.001). Of the 32 patients in whom a sentinel node was identified, 10 (31%) had histologically positive sentinel nodes: 5 (16%) by frozen section, 2 additional patients (6%) after permanent hematoxalin-eosin (H&E) stained sections, and the remaining 3 (9%) after immunohistochemical stains for cytokeratins when the FS and permanent H&E-stained sections were benign. Twenty patients had benign axilla. The sentinel node was falsely negative in 2 patients, yielding an accuracy of 93.8%, sensitivity of 83.3%, and negative predictive value of 91%., Conclusions: Lymphatic mapping is technically feasible for patients with small (T1 or T2) palpable invasive breast tumors. The sentinel node can be reliably identified in the majority of these patients, and its histology reflects that of the axilla with a high degree of accuracy. Immunohistochemical stains and permanent H&E-stained sections of the sentinel node increased the test's ability to correctly identify axillary metastases. Improving this sensitivity remains a primary goal, however, if benign sentinel node histology is to be used as a criterion to preclude axillary dissection., (Copyright 1999 Wiley-Liss, Inc.)

The dimer species (M(r) 580,000) of the nicotinic acetylcholine receptor, isolated from the electric organ of Torpedo californica, was incorporated into a thiopeptide supported lipid bilayer. The incorporation was achieved by fusion of liposomes with reconstituted receptor onto a gold-supported thiopeptide lipid monolayer. Surface plasmon resonance spectroscopy (SPS) was used to monitor in real time the fusion process as well as the specific binding of the antagonist alpha-bungarotoxin. A recently developed extension of SPS offering enhanced sensitivity and specificity, surface plasmon fluorescence spectroscopy (SPFS), was then used to monitor subsequent binding of the monoclonal WF6 and polyclonal antibody, respectively. The latter was fluorescence labeled with Cy5. The different binding assays indicate the successful incorporation of the receptor in the lipid bilayer.

Objective: This was a randomised, open, three-way crossover study in 12 healthy male volunteers to determine the effect of a single oral dose of cimetidine on the pharmacokinetics of a single oral dose of the angiotensin II receptor antagonist valsartan and vice versa. The volunteers received either valsartan alone (160 mg), or cimetidine alone (800 mg), or valsartan 1 h after cimetidine. The study was designed primarily to detect a possible influence of cimetidine on the rate and extent of absorption of valsartan., Methods: Plasma concentrations of valsartan and cimetidine, measured by means of high-performance liquid chromatography, were used to calculate pharmacokinetic parameters. The rate of absorption of valsartan and the fraction of the dose absorbed and systemically available after oral administration were calculated using data from an i.v. study with valsartan in healthy young volunteers., Results: The pharmacokinetics of cimetidine area under curve (AUC0-48 h), maximum concentration (Cmax), time to reach Cmax(tmax) and apparent terminal plasma half-life (t1/2) was not changed by co-administration of valsartan. For valsartan, the AUC0-48 h increased by 7% and the Cmax by 51% (ratio of geometric means) with co-administration of cimetidine. The higher value for Cmax was attributed to the initial increase in the rate of absorption of valsartan: ka was increased 2.7-fold and another indicator for the rate of absorption, Cmax/tmax, 2.2-fold. This effect was ascribed to inhibition of acid secretion by cimetidine, which leads to a higher gastric pH, thereby increasing the solubility of valsartan; the t1/2 of valsartan was not changed. After valsartan alone, 19% of the dose was absorbed, 23% with co-administration of cimetidine. It was estimated that only 2.2% of the possible change in AUC might be missed by giving a single high dose of cimetidine instead of multiple doses, with the aim to optimally inhibit formation of the inactive metabolite of valsartan. Cimetidine-related changes in the rate of elimination of valsartan were not anticipated, since the clearance from plasma occurs mainly by biliary excretion of unchanged valsartan; metabolism and renal excretion are only minor contributors. Therefore, even in the clinically relevant situation with multiple doses of valsartan and cimetidine, notable changes in the pharmacokinetics of valsartan, except for an increase in Cmax, are not to be expected. This increase in Cmax appears to be of no clinical significance. Valsartan alone and in combination with cimetidine was well tolerated by healthy subjects.

One of the best strategies for understanding an animal's behavior is to study the function of the brain by experimentally modifying brain chemistry temporarily or on a long-term basis. This can be achieved by direct manipulation of neurochemistry of a targeted brain area with various drugs whose in vitro specificity and sensitivity are known. We assume that an animal's behavior is primarily controlled by the integrated performance of neural networks, rather than the action of a "superstar" single neuron which has narrowly tuned selectivity, in a specified brain region. Therefore, the former must be regulated by a large number of combinations of various transmitter/modulator receptors, hormones, growth factors, and other biochemically identifiable and yet unidentified substances. Under certain conditions, the activation of receptor-bound second messenger systems is thought to cause the enhanced expression of particular genes. Given the wide possibilities in manipulating brain chemistry, which may otherwise result in a variety of consequences, it is crucial to have a dependable means of sustaining the steady-state action of a drug for a sufficiently long time period at a targeted area in the brain of behaving animals. In most cases the continuous application of a drug is necessary to counteract its secondary mitigating effect, which is set in action through negative feedback loops and which in effect reduces the primary action of the drug in use. We have developed a technique to answer this need, using the Alzet osmotic minipump as the source of the continuous infusion force. A drug solution is continuously and directly infused, guided through a chronically implanted cannula, into a targeted area in the brain of behaving animals. The consequences of such an infusion are assessed, during as well as after the infusion, using various types of measurements in behavior, biochemistry, neurophysiology, pharmacology and morphology. The method has been successfully applied, for example, to the study of developmentally regulated neural plasticity in cat visual cortex. A few preconditions should be satisfied for the method to be properly applied to the brains of live animals. Those are: (1) manufacturing a suitable guide system, i.e., cannula-minipump assembly, for the infusion solution; (2) stereotaxic implantation of a cannula-minipump assembly into a selected brain region; and (3) estimating the concentration gradient of the continuously infused solution. This is crucial to assess the specificity and sensitivity of a drug for its assumed effects in vivo.

CGP 28,014 is a specific inhibitor of catechol-O-methyltransferase (COMT) in vivo. In humans, the inhibition was assessed by measuring urinary excretion of isoquinolines and with the levodopa test. Following administration of CGP 28,014, urinary excretion of isoquinolines was significantly increased. In rats, CGP 28,014 reduced plasma and striatal concentrations of 3-O-methyldopa (30MD) in a dose-dependent manner. Acute and subchronic administration of CGP 28,014 alone or in combination with the peripherally acting decarboxylase inhibitor benserazide decreased plasma 30MD as an index of COMT inhibition by about 50%. There seems to be a close relationship between the time-course of plasma concentrations of CGP 28,014 and the extent of COMT inhibition assessed by the 30MD/DOPA ratio in plasma.

A sensitive assay for prenylamine and dideuteroprenylamine (racemic or pseudo-racemate) has been developed and used in human pharmacokinetic studies. Plasma levels of prenylamine could be measured up to 50 h after a single oral therapeutic dose. The extracted drug was derivatized with pentafluoropropionic anhydride in acetonitrile. The dried samples were reconstituted in decane; an aliquot was injected into a fused-silica capillary in a cooled on-column injector. The base peaks in the electron impact mass spectra of the compounds--derived by loss of a benzyl radical--at m/z 384, 386 and 390 were measured for prenylamine, (D2)-prenylamine and the internal standard hexahydroprenylamine, respectively. The sensitivity of this assay--limit of detection 0.2 ng ml-1 plasma with a signal-to-noise ratio of 5:1--allowed measurement of the kinetics of the racemate and of both stereoisomers for the first time. In man, the (+)-isomer was eliminated considerably faster than the (-)-prenylamine; the area under the plasma concentration time curve (AUC) of the (+)-isomer was only about 1/4 of the AUC of (-)-prenylamine.

Experiments in animals have indicated that CGS 5,649 B [6-(2-isopropylaminopropyl)-3-pyridinol fumarate] might enhance memory and learning processes and might be a valuable drug for treatment of impairment of vigilance and mental performance in the elderly. CGS 5,649 (I) is extensively metabolized. Therefore, we investigated the excretion of the drug and its conjugates (I-SULF,I-GLUC) into urine after oral administration of 1200 mg of the fumarate salt of I to one healthy male volunteer. Three HPLC methods were developed to analyze the three compounds in urine: a. Solid-phase extraction and UV-measurement at 280 nm; b. Dansylation followed by fluorometric detection (lambda ex 340, lambda em 525 nm); c. Direct injection of diluted urine, gradient system with ion-pair reagent and UV-detection at 280 nm. The conjugates were measured after enzymatic hydrolysis with glucuronidase/sulfatase and sulfatase. I-GLUC was also measured directly since a synthetic sample had become available. Results from the different analytical methods showed agreement: about 100% of the administered dose was excreted within 24 h; the relative amounts of I, I-SULF and I-GLUC were about 1:2:2; after oral administration, CGS 5,649 B is rapidly and completely absorbed; it is eliminated from plasma by conjugation and direct excretion into urine.

Pharmacokinetics of racemic prenylamine were investigated in 6 healthy volunteers. Plasma levels were determined by gas chromatography/mass spectrometry. Concentration-time profiles were analyzed both by compartment-dependent and compartment-independent pharmacokinetic models. Terminal elimination half-life was 14.1 h (SD: 6.9 h). The apparent total clearance was 5.8 l/min. Mean residence time of racemic prenylamine was found to be 14.7 h (SD: 3.8 h). The relative bioavailability of prenylamine (Segontin 100) was 82.2% (SD: 9.9%) determined in six healthy volunteers. The volunteers received simultaneously the film tablet and 100 mg racemic dideuteroprenylamine as an aqueous solution of the lactate. This procedure is known to exclude intraindividual changes in absorption, first-pass metabolism or volume of distribution that might occur on sequential administration. The absolute bioavailability was estimated to be in the order of 15%. In a pilot study the pharmacokinetics of the enantiomers were investigated in 2 healthy volunteers. S-(+)-prenylamine was eliminated considerably faster from plasma than R-(-)-prenylamine suggesting a stereoselective metabolism. The AUC of the (+)-enantiomer was 20% of that of the R-(-)-prenylamine.

A rapid high-performance liquid chromatographic (HPLC) method for the determination of the novel benzodiazepine receptor antagonist 2-phenylpyrazolo[4,3-c]quinolin-3(5H)-one (CGS 8216) and its hydroxy metabolite is described. The method involves a solid-phase extraction with C18 disposable columns and quantification by HPLC with UV-detection. In plasma 20 ng/ml of the antagonist and 10 ng/ml of metabolite can be measured with a coefficient of variation of about 10%. The high sensitivity and selectivity of the assay makes it suitable for use in pharmacokinetic studies.

The diagnosis of Beckwith-Wiedemann syndrome was made in a 580-g, stillborn infant with an omphalocele. This case reemphasizes the importance of complete postmortem examinations. Appropriate counseling is dependent on an accurate diagnosis.

The formation of the two major metabolites of the antiarrhythmic and oxytocic drug sparteine (2- and 5-dehydrosparteine) exhibits a genetic polymorphism. Two phenotypes, extensive (EM) and poor metabolizers (PM) are observed in the population. The frequency of the PM phenotype in various populations (Caucasian and Japanese) ranges from 2.3 to 9%. The metabolism of sparteine is determined by two allelic genes at a single gene locus. PM subjects are homozygous for an autosomal recessive gene. The metabolism of sparteine is predominantly under genetic control as treatment with drugs such as antipyrine and rifampicin known to induce oxidative drug metabolism elicited only marginal changes in sparteine metabolism. The formation of 2-dehydrosparteine in human liver microsomes from EM and PM subjects showed a more than 40-fold difference in Km between EM and PM subjects. However, Vmax-values were almost identical in both groups. These data indicate that the basis of the differences in oxidative capacity between EM and PM subjects is more likely to be due to a variant isozyme with defective catalytic properties than to a decreased amount of the isozyme.