<Emphasis Type="Italic">FACT</Emphasis> – a framework for the functional interpretation of high-throughput experiments

Background  

Interpreting the results of high-throughput experiments, such as those obtained from DNA-microarrays, is an often time-consuming task due to the high number of data-points that need to be analyzed in parallel. It is usually a matter of extensive testing and unknown beforehand, which of the possible approaches for the functional analysis will be the most informative     

Reliability of computerized image analysis for the evaluation of serial synovial biopsies in randomized controlled trials in rheumatoid arthritis

Analysis of biomarkers in synovial tissue is increasingly used in the evaluation of new targeted therapies for patients with rheumatoid arthritis (RA). This study determined the intrarater and inter-rater reliability of digital image analysis (DIA) of synovial biopsies from RA patients participating in clinical trials. Arthroscopic synovial biopsies were obtained before and after treatment from 19 RA patients participating in a randomized controlled trial with prednisolone. Immunohistochemistry was used to detect CD3⁺ T cells, CD38⁺ plasma cells and CD68⁺ macrophages. The mean change in positive cells per square millimetre for each marker was determined by different operators and at different times using DIA. Nonparametric tests were used to determine differences between observers and assessments, and to determine changes after treatment. The intraclass correlations (ICCs) were calculated to determine the intrarater and inter-rater reliability. Intrarater ICCs showed good reliability for measuring changes in T lymphocytes (R = 0.87), plasma cells (R = 0.62) and macrophages (R = 0.73). Analysis by Bland–Altman plots showed no systemic differences between measurements. The smallest detectable changes were calculated and their discriminatory power revealed good response in the prednisolone group compared with the placebo group. Similarly, inter-rater ICCs also revealed good reliability for measuring T lymphocytes (R = 0.68), plasma cells (R = 0.69) and macrophages (R = 0.72). All measurements identified the same cell types as changing significantly in the treated patients compared with the placebo group. The measurement of change in total positive cell numbers in synovial tissue can be determined reproducibly for various cell types by DIA in RA clinical trials.     

Valid and reliable instruments for arm-hand assessment at ICF activity level in persons with hemiplegia: a systematic review

Background

Loss of arm-hand performance due to a hemiparesis as a result of stroke or cerebral palsy (CP), leads to large problems in daily life of these patients. Assessment of arm-hand performance is important in both clinical practice and research. To gain more insight in e.g. effectiveness of common therapies for different patient populations with similar clinical characteristics, consensus regarding the choice and use of outcome measures is paramount. To guide this choice, an overview of available instruments is necessary. The aim of this systematic review is to identify, evaluate and categorize instruments, reported to be valid and reliable, assessing arm-hand performance at the ICF activity level in patients with stroke or cerebral palsy.

Methods

A systematic literature search was performed to identify articles containing instruments assessing arm-hand skilled performance in patients with stroke or cerebral palsy. Instruments were identified and divided into the categories capacity, perceived performance and actual performance. A second search was performed to obtain information on their content and psychometrics.

Results

Regarding capacity, perceived performance and actual performance, 18, 9 and 3 instruments were included respectively. Only 3 of all included instruments were used and tested in both patient populations. The content of the instruments differed widely regarding the ICF levels measured, assessment of the amount of use versus the quality of use, the inclusion of unimanual and/or bimanual tasks and the inclusion of basic and/or extended tasks.

Conclusions

Although many instruments assess capacity and perceived performance, a dearth exists of instruments assessing actual performance. In addition, instruments appropriate for more than one patient population are sparse. For actual performance, new instruments have to be developed, with specific focus on the usability in different patient populations and the assessment of quality of use as well as amount of use. Also, consensus about the choice and use of instruments within and across populations is needed.     

Elevated levels of fibrinogen-derived endogenous citrullinated peptides in synovial fluid of rheumatoid arthritis patients

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and the presence of autoantibodies directed against proteins containing the non-standard arginine-derived amino acid citrulline. The protein fibrinogen, which has an essential role in blood clotting, is one of the most prominent citrullinated autoantigens in RA, particularly because it can be found in the inflamed tissue of affected joints. Here, we set out to analyze the presence of citrullinated endogenous peptides in the synovial fluid of RA and arthritic control patients.

Methods

Endogenous peptides were isolated from the synovial fluid of RA patients and controls by filtration and solid phase extraction. The peptides were identified and quantified using high-resolution liquid chromatography-mass spectrometry.

Results

Our data reveal that the synovial fluid of RA patients contains soluble endogenous peptides, derived from fibrinogen, containing significant amounts of citrulline residues and, in some cases, also phosphorylated serine. Several citrullinated peptides are found to be more abundantly present in the synovial fluid of RA patients compared to patients suffering from other inflammatory diseases affecting the joints.

Conclusions

The increased presence of citrullinated peptides in RA patients points toward a possible specific role of these peptides in the immune response at the basis of the recognition of citrullinated peptides and proteins by RA patient autoantibodies.     

Nos2 inactivation promotes the development of medulloblastoma in Ptch1(+/-) mice by deregulation of Gap43-dependent granule cell precursor migration

Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1(+/-) mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1(+/-) mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1(+/-) Nos2(-/-) mice compared to Ptch1(+/-) Nos2(+/+) mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1(+/+) Nos2(-/-) mice but not from Ptch1(+/-) Nos2(-/-) mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1(+/+) Nos2(-/-) mice but increased in Ptch1(+/-) Nos2(-/) (-) mice relative to Ptch1(+/-) Nos2(+/+) mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1(+/-) mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during morphogenesis is crucial for their malignant progression.     

Attributes of short linear motifs

Traditionally, protein-protein interactions were thought to be mediated by large, structured domains. However, it has become clear that the interactome comprises a wide range of binding interfaces with varying degrees of flexibility, ranging from rigid globular domains to disordered regions that natively lack structure. Enrichment for disorder in highly connected hub proteins and its correlation with organism complexity hint at the functional importance of disordered regions. Nevertheless, they have not yet been extensively characterised. Shifting the attention from globular domains to disordered regions of the proteome might bring us closer to elucidating the dense and complex connectivity of the interactome. An important class of disordered interfaces are the compact mono-partite, short linear motifs (SLiMs, or eukaryotic linear motifs (ELMs)). They are evolutionarily plastic and interact with relatively low affinity due to the limited number of residues that make direct contact with the binding partner. These features confer to SLiMs the ability to evolve convergently and mediate transient interactions, which is imperative to network evolution and to maintain robust cell signalling, respectively. The ability to discriminate biologically relevant SLiMs by means of different attributes will improve our understanding of the complexity of the interactome and aid development of bioinformatics tools for motif discovery. In this paper, the curated instances currently available in the Eukaryotic Linear Motif (ELM) database are analysed to provide a clear overview of the defining attributes of SLiMs. These analyses suggest that functional SLiMs have higher levels of conservation than their surrounding residues, frequently evolve convergently, preferentially occur in disordered regions and often form a secondary structure when bound to their interaction partner. These results advocate searching for small groupings of residues in disordered regions with higher relative conservation and a propensity to form the secondary structure. Finally, the most interesting conclusions are examined in regard to their functional consequences.     

Cytomegalovirus seropositivity is associated with glucose regulation in the oldest old. Results from the Leiden 85-plus Study

ABSTRACT: BACKGROUND: Cytomegalovirus (CMV) infection has been reported to contribute to the pathogenesis of type 1 diabetes and post-transplantation diabetes. However, CMV infection has not been evaluated as a possible risk factor for type 2 diabetes. Our aim was to investigate potential associations between CMV seropositivity, CMV IgG antibody level and glucose regulation in the oldest old. RESULTS: CMV seropositive subjects were more likely to have type 2 diabetes (17.2% vs 7.9%, p = 0.016), had a higher level of HbA1c (p = 0.014) and higher non-fasting glucose (p = 0.024) in the oldest olds. These associations remained significant after adjustment for possible confounders. CMV IgG antibody level was not significantly associated with glucose regulation (all p > 0.05). CONCLUSIONS: In the oldest old, CMV seropositivity is significantly associated with various indicators of glucose regulation. This finding suggests that CMV infection might be a risk factor for the development of type 2 diabetes in the elderly.     

A new brace treatment similar for adolescent scoliosis and kyphosis based on restoration of thoracolumbar lordosis. Radiological and subjective clinical results after at least one year of treatment

Study design

A prospective treatment study with a new brace was conducted Objective. To evaluate radiological and subjective clinical results after one year conservative brace treatment with pressure onto lordosis at the thoracolumbar joint in children with scoliosis and kyphosis.

Summary of background data

Conservative brace treatment of adolescent scoliosis is not proven to be effective in terms of lasting correction. Conservative treatment in kyphotic deformities may lead to satisfactory correction. None of the brace or casting techniques is based on sagittal forces only applied at the thoracolumbar spine (TLI= thoracolumbar lordotic intervention). Previously we showed in patients with scoliosis after forced lordosis at the thoracolumbar spine a radiological instantaneous reduction in both coronal curves of double major scoliosis.

Methods

A consecutive series of 91 children with adolescent scoliosis and kyphosis were treated with a modified symmetric 30 degrees Boston brace to ensure only forced lordosis at the thoracolumbar spine. Scoliosis was defined with a Cobb angle of at least one of the curves [greater than or equal to] 25 degrees and kyphosis with or without a curve <25 degrees in the coronal plane. Standing radiographs were made i) at start, ii) in brace at beginning and iii) after one year treatment without brace.

Results

Before treatment start ??in brace?? radiographs showed a strong reduction of the Cobb angles in different curves in kyphosis and scoliosis groups (sagittal n = 5 all p < 0.001, pelvic obliquity p < 0.001). After one year of brace treatment in scoliosis and kyphosis group the measurements on radiographs made without brace revealed an improvement in 3 Cobb angles each.

Conclusion

Conservative treatment using thoracolumbar lordotic intervention in scoliotic and kyphotic deformities in adolescence demonstrates a marked improvement after one year also in clinical and postural criteria. An effect not obtained with current brace techniques.     

Architectural design influences the diversity and structure of the built environment microbiome

Buildings are complex ecosystems that house trillions of microorganisms interacting with each other, with humans and with their environment. Understanding the ecological and evolutionary processes that determine the diversity and composition of the built environment microbiome—the community of microorganisms that live indoors—is important for understanding the relationship between building design, biodiversity and human health. In this study, we used high-throughput sequencing of the bacterial 16S rRNA gene to quantify relationships between building attributes and airborne bacterial communities at a health-care facility. We quantified airborne bacterial community structure and environmental conditions in patient rooms exposed to mechanical or window ventilation and in outdoor air. The phylogenetic diversity of airborne bacterial communities was lower indoors than outdoors, and mechanically ventilated rooms contained less diverse microbial communities than did window-ventilated rooms. Bacterial communities in indoor environments contained many taxa that are absent or rare outdoors, including taxa closely related to potential human pathogens. Building attributes, specifically the source of ventilation air, airflow rates, relative humidity and temperature, were correlated with the diversity and composition of indoor bacterial communities. The relative abundance of bacteria closely related to human pathogens was higher indoors than outdoors, and higher in rooms with lower airflow rates and lower relative humidity. The observed relationship between building design and airborne bacterial diversity suggests that we can manage indoor environments, altering through building design and operation the community of microbial species that potentially colonize the human microbiome during our time indoors.     

Use of the EM algorithm to detect QTL affecting multiple-traits in an across half-sib family analysis

QTL detection experiments in livestock species commonly use the half-sib design. Each male is mated to a number of females, each female producing a limited number of progeny. Analysis consists of attempting to detect associations between phenotype and genotype measured on the progeny. When family sizes are limiting experimenters may wish to incorporate as much information as possible into a single analysis. However, combining information across sires is problematic because of incomplete linkage disequilibrium between the markers and the QTL in the population. This study describes formulæ for obtaining MLEs via the expectation maximization (EM) algorithm for use in a multiple-trait, multiple-family analysis. A model specifying a QTL with only two alleles, and a common within sire error variance is assumed. Compared to single-family analyses, power can be improved up to fourfold with multi-family analyses. The accuracy and precision of QTL location estimates are also substantially improved. With small family sizes, the multi-family, multi-trait analyses reduce substantially, but not totally remove, biases in QTL effect estimates. In situations where multiple QTL alleles are segregating the multi-family analysis will average out the effects of the different QTL alleles.