Fieldwork in Upstate New York: Methods

production and perception experiments for the NCS and Low Back Merger in Ogdensburg, NY

[Renal transplantation - new developments]

Renal transplantation has become an established option for renal replacement therapy in many patients with end stage renal disease. Living donation is a possibility for timely transplantation, hampered in 20 % of all possible donors and recipients byincompatible blood groups. AB0-incompatible renal transplantation overcomes this hurdle with acceptable allograft survival compared to conventional living-donor renal transplantation. During the last 10 years, the number of patients awaiting renal transplantation older than 65 years has nearly doubled. The decision to transplant those patients and their medical treatment is a growing challenge in transplantation. On the other hand donor age is increasing with potential negative consequences for long-term outcome of organ function. Antibody-mediated humoral rejection have been identified lately as an important cause for allograft failure during long-term follow up of renal transplant patients. New immunological methods to detect donor-specific antibodies, like solid-phase assays (Luminex®), have increased the knowledge and understanding of humoral rejection processes. This will lead hopefully to modified immunosuppressive strategies to minimize organ failure due to chronic rejection.

Highlights in Analytical Chemistry in Switzerland: OMA & OPA - A Software Tool for Mass Spectrometric Sequencing of Nucleic Acids

Oligonucleotides comprising unnatural building blocks, which interfere with the translation machinery, have gained increased attention for the treatment of gene-related diseases (e.g. antisense, RNAi). Due to structural modifications, synthetic oligonucleotides exhibit increased biostability and bioavailability upon administration. Consequently, classical enzyme-based sequencing methods are not applicable to their sequence elucidation and verification. Tandem mass spectrometry is the method of choice for performing such tasks, since gas-phase dissociation is not restricted to natural nucleic acids. However, tandem mass spectrometric analysis can generate product ion spectra of tremendous complexity, as the number of possible fragments grows rapidly with increasing sequence length. The fact that structural modifications affect the dissociation pathways greatly increases the variety of analytically valuable fragment ions. The gas-phase dissociation of oligonucleotides is characterized by the cleavage of one of the four bonds along the phosphodiester chain, by the accompanying loss of nucleases, and by the generation of internal fragments due to secondary backbone cleavage. For example, an 18-mer oligonucleotide yields a total number of 272’920 theoretical fragment ions. In contrast to the processing of peptide product ion spectra, which nowadays is highly automated, there is a lack of tools assisting the interpretation of oligonucleotide data. The existing web-based and stand-alone software applications are primarily designed for the sequence analysis of natural nucleic acids, but do not account for chemical modifications and adducts. Consequently, we developed a software to support the interpretation of mass spectrometric data of natural and modified nucleic acids and their adducts with chemotherapeutic agents.

cDNA Library Generation for the Analysis of Small RNAs by High-Throughput Sequencing

The RNome of a cell is highly diverse and consists besides messenger RNAs (mRNAs), transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs) also of other small and long transcript entities without apparent coding potential. This class of molecules, commonly referred to as non-protein-coding RNAs (ncRNAs), is involved in regulating numerous biological processes and thought to contribute to cellular complexity. Therefore, much effort is put into their identification and further functional characterization. Here we provide a cost-effective and reliable method for cDNA library construction of small RNAs in the size range of 20-500 residues. The effectiveness of the described method is demonstrated by the analysis of ribosome-associated small RNAs in the eukaryotic model organism Trypanosoma brucei.

Development and Evaluation of a New Tablet Computer Application for the Therapy of Brain-Injured Patients with Aphasia

Introduction Language is the most important mean of communication and plays a central role in our everyday life. Brain damage (e.g. stroke) can lead to acquired disorders of lan- guage affecting the four linguistic modalities (i.e. reading, writing, speech production and comprehension) in different combinations and levels of severity. Every year, more than 5000 people (Aphasie Suisse) are affected by aphasia in Switzerland alone. Since aphasia is highly individual, the level of difficulty and the content of tasks have to be adapted continuously by the speech therapists. Computer-based assignments allow patients to train independently at home and thus increasing the frequency of ther- apy. Recent developments in tablet computers have opened new opportunities to use these devices for rehabilitation purposes. Especially older people, who have no prior experience with computers, can benefit from the new technologies. Methods The aim of this project was to develop an application that enables patients to train language related tasks autonomously and, on the other hand, allows speech therapists to assign exercises to the patients and to track their results online. Seven categories with various types of assignments were implemented. The application has two parts which are separated by a user management system into a patient interface and a therapist interface. Both interfaces were evaluated using the SUS (Subject Usability Scale). The patient interface was tested by 15 healthy controls and 5 patients. For the patients, we also collected tracking data for further analysis. The therapist interface was evaluated by 5 speech therapists. Results The SUS score are xpatients = 98 and xhealthy = 92.7 (median = 95, SD = 7, 95% CI [88.8, 96.6]) in case of the patient interface and xtherapists = 68 in case of the therapist interface. Conclusion Both, the patients and the healthy subjects, attested high SUS scores to the patient interface. These scores are considered as "best imaginable". The therapist interface got a lower SUS score compared to the patient interface, but is still considered as "good" and "usable". The user tracking system and the interviews revealed that there is room for improvements and inspired new ideas for future versions.

Essential hypertension - which genes can be held responsible for it?

Hypertension is a common heritable cardiovascular risk factor. Some rare monogenic forms of hypertension have been described, but the majority of patients suffer from essential hypertension, for whom the underlying genetic mechanisms are not clear. Essential hypertension is a complex trait, involving multiple genes and environmental factors. Recently, progress in the identification of common genetic variants associated with essential hypertension has been made due to large-scale international collaborative projects. In this article we review the new research methods used as well as selected recent findings in this field.

A brief update on lung stereology

Lung stereology has a long and successful tradition. From mice to men, the application of new stereological methods at several levels (alveoli, parenchymal cells, organelles, proteins) has led to new insights into normal lung architecture, parenchymal remodelling in emphysema-like pathology, alveolar type II cell hyperplasia and hypertrophy and intracellular surfactant alterations as well as distribution of surfactant proteins. The Euler number of the network of alveolar openings, estimated using physical disectors at the light microscopic level, is an unbiased and direct estimate of alveolar number. Surfactant-producing alveolar type II cells can be counted and sampled for local size estimation with physical disectors at a high magnification light microscopic level. The number of their surfactant storage organelles, lamellar bodies, can be estimated using physical disectors at the EM level. By immunoelectron microscopy, surfactant protein distribution can be analysed with the relative labelling index. Together with the well-established classical stereological methods, these design-based methods now allow for a complete quantitative phenotype analysis in lung development and disease, including the structural characterization of gene-manipulated mice, at the light and electron microscopic level.

Internalization of sst2, sst3, and sst5 receptors: effects of somatostatin agonists and antagonists

The uptake of radiolabeled somatostatin analogs by tumor cells through receptor-mediated internalization is a critical process for the in vivo targeting of tumoral somatostatin receptors. In the present study, the somatostatin receptor internalization induced by a variety of somatostatin analogs was measured with new immunocytochemical methods that allow characterization of trafficking of the somatostatin receptor subtype 2 (sst2), somatostatin receptor subtype 3 (sst3), and somatostatin receptor subtype 5 (sst5) in vitro at the protein level. METHODS: Human embryonic kidney 293 (HEK293) cells expressing the sst2, sst3, or the sst5 were used in a morphologic immunocytochemical internalization assay using specific sst2, sst3 and sst5 antibodies to qualitatively and quantitatively determine the capability of somatostatin agonists or antagonists to induce somatostatin receptor internalization. In addition, the internalization properties of a selection of these agonists have been compared and quantified in sst2-expressing CHO-K1 cells using an ELISA. RESULTS: Agonists with a high sst2-binding affinity were able to induce sst2 internalization in the HEK293 and CHO-K1 cell lines. New sst2 agonists, such as Y-DOTA-TATE, Y-DOTA-NOC, Lu-DOTA-BOC-ATE (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; TATE is [Tyr3, Thr8]-octreotide; NOC is [1-NaI3]-octreotide; and BOC-ATE is [BzThi3, Thr8]-octreotide), iodinated sugar-containing octreotide analogs, or BIM-23244 were considerably more potent in internalizing sst2 than was DTPA-octreotide (where DTPA is diethylenetriaminepentaacetic acid). Similarly, compounds with high sst3 affinity such as KE108 were able to induce sst3 internalization. In sst2- or sst3-expressing cell lines, agonist-induced receptor internalization was efficiently abolished by sst2- or sst3-selective antagonists, respectively. Antagonists alone had no effect on sst2 or sst3 internalization. We also showed that somatostatin-28 and somatostatin-14 can induce sst5 internalization. Unexpectedly, however, potent sst5 agonists such as KE108, BIM-23244, and L-817,818 were not able to induce sst5 internalization under the same conditions. CONCLUSION: Using sensitive and reproducible immunocytochemical methods, the ability of various somatostatin analogs to induce sst2, sst3, and sst5 internalization has been qualitatively and quantitatively determined. Whereas all agonists triggered sst2 and sst3 internalization, sst5 internalization was induced by natural somatostatin peptides but not by synthetic high-affinity sst5 agonists. Such assays will be of considerable help for the future characterization of ligands foreseen for nuclear medicine applications.

Unexpected metastatic pheochromocytoma - an unusual presentation

The classic triad of pheochromocytoma consists of episodic headache, sweating, and tachycardia. General clinicians should be aware, however, that this rare entity might present with a wide spectrum of clinical symptoms. We recently observed a noteworthy case of malignant pheochromocytoma where there was a lack of specific symptoms despite an advanced tumor stage. Malignancy is an important cause of mortality. Reliable diagnosis of malignancy depends upon evidence of local invasion, distant metastases, or recurrence. As in our case, new scintigraphic methods, such as 111-In-pentetreotide scintigraphy (Octreoscan), may occasionally reveal 123-I-metaiodobenzylguanidine-negative distant metastases and help to establish an early diagnosis of malignancy. Tumor size, and perhaps even biochemical profile, may be factors increasing the likelihood of a malignant process and may contribute to early identification of patients at risk.

Management of acute bronchiolitis: can evidence based guidelines alter clinical practice?

BACKGROUND: Acute bronchiolitis is the most common lower respiratory tract infection in infants and there is no evidence that drug treatment alters its natural course. Despite this, most Swiss paediatricians reported in 2001 prescribing bronchodilators and inhaled corticosteroids (ICS). This situation led to the creation of national guidelines followed by a tailored implementation programme. The aim of this study was to examine if treatment practices changed after the implementation of the new guidelines. METHODS: A questionnaire on treatment of bronchiolitis was sent to all Swiss paediatricians before (2001) and after (2006) creation and implementation of national guidelines (2003-2005). Guidelines were created in collaboration with all paediatric pulmonologists and implemented carefully using a multifaceted approach. RESULTS: Questionnaires were returned by 541 paediatricians (58%) in 2001 and by 639 (54%) in 2006. While both surveys showed a wide variation in the treatment of bronchiolitis between physicians, reported drug prescription decreased significantly between the two surveys. For outpatients, general use (for all patients) of bronchodilators dropped from 60% to 23%, and general use of ICS from 34% to 6%. For inpatients, general use of bronchodilators and ICS dropped from 55% to 18% and from 26% to 6%, respectively (all p<0.001). The decrease was evident in all regions, among hospital and primary care physicians, and among general paediatricians and paediatric pulmonologists. CONCLUSIONS: National guidelines together with a tailored implementation programme can have a major impact on medical management practices in a country.

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice

BACKGROUND: Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. METHODS: Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. RESULTS: 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as "potentially pathogenic". Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. CONCLUSIONS: Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered "new syndromes" were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype-phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

Challenges and strategies in the repair of ruptured annulus fibrosus

Lumbar discectomy is the surgical procedure most frequently performed for patients suffering from low back pain and sciatica. Disc herniation as a consequence of degenerative or traumatic processes is commonly encountered as the underlying cause for the painful condition. While discectomy provides favourable outcome in a majority of cases, there are conditions where unmet requirements exist in terms of treatment, such as large disc protrusions with minimal disc degeneration; in these cases, the high rate of recurrent disc herniation after discectomy is a prevalent problem. An effective biological annular repair could improve the surgical outcome in patients with contained disc herniations but otherwise minor degenerative changes. An attractive approach is a tissue-engineered implant that will enable/stimulate the repair of the ruptured annulus. The strategy is to develop three-dimensional scaffolds and activate them by seeding cells or by incorporating molecular signals that enable new matrix synthesis at the defect site, while the biomaterial provides immediate closure of the defect and maintains the mechanical properties of the disc. This review is structured into (1) introduction, (2) clinical problems, current treatment options and needs, (3) biomechanical demands, (4) cellular and extracellular components, (5) biomaterials for delivery, scaffolding and support, (6) pre-clinical models for evaluation of newly developed cell- and material-based therapies, and (7) conclusions. This article highlights that an interdisciplinary approach is necessary for successful development of new clinical methods for annulus fibrosus repair. This will benefit from a close collaboration between research groups with expertise in all areas addressed in this review.

Who is likely to develop persistent low back pain? A longitudinal analysis of prognostic occupational factors

OBJECTIVE: Occupational low back pain (LBP) is considered to be the most expensive form of work disability, with the socioeconomic costs of persistent LBP exceeding the costs of acute and subacute LBP by far. This makes the early identification of patients at risk of developing persistent LBP essential, especially in working populations. The aim of the study was to evaluate both risk factors (for the development of persistent LBP) and protective factors (preventing the development of persistent LBP) in the same cohort. PARTICIPANTS: An inception cohort of 315 patients with acute to subacute or with recurrent LBP was recruited from 14 health practitioners (twelve general practitioners and two physiotherapists) across New Zealand. METHODS: Patients with persistent LBP at six-month follow-up were compared to patients with non-persistent LBP looking at occupational, psychological, biomedical and demographic/lifestyle predictors at baseline using multiple logistic regression analyses. All significant variables from the different domains were combined into a one predictor model. RESULTS: A final two-predictor model with an overall predictive value of 78% included social support at work (OR 0.67; 95%CI 0.45 to 0.99) and somatization (OR 1.08; 95%CI 1.01 to 1.15). CONCLUSIONS: Social support at work should be considered as a resource preventing the development of persistent LBP whereas somatization should be considered as a risk factor for the development of persistent LBP. Further studies are needed to determine if addressing these factors in workplace interventions for patients suffering from acute, subacute or recurrent LBP prevents subsequent development of persistent LBP.

DNA-based diagnosis of rare diseases in veterinary medicine: a 4.4 kb deletion of ITGB4 is associated with epidermolysis bullosa in Charolais cattle.

BACKGROUND Rare diseases in livestock animals are traditionally poorly diagnosed. Other than clinical description and pathological examination, the underlying causes have, for the most part, remained unknown. A single case of congenital skin fragility in cattle was observed, necropsy, histological and ultrastructural examinations were carried out and whole genome sequencing was utilized to identify the causative mutation. RESULTS A single purebred female Charolais calf with severe skin lesions was delivered full-term and died spontaneously after birth. The clinical and pathological findings exactly matched the gross description given by previous reports on epitheliogenesis imperfecta and epidermolysis bullosa (EB) in cattle. Histological and ultrastructural changes were consistent with EB junctionalis (EBJ). Genetic analysis revealed a previously unpublished ITGB4 loss-of-function mutation; the affected calf was homozygous for a 4.4 kb deletion involving exons 17 to 22, and the dam carried a single copy of the deletion indicating recessive inheritance. The homozygous mutant genotype did not occur in healthy controls of various breeds but some heterozygous carriers were found among Charolais cattle belonging to the affected herd. The mutant allele was absent in a representative sample of unrelated sires of the German Charolais population. CONCLUSION This is the first time in which a recessively inherited ITGB4 associated EBJ has been reported in cattle. The identification of heterozygous carriers is of importance in avoiding the transmission of this defect in future. Current DNA sequencing methods offer a powerful tool for understanding the genetic background of rare diseases in domestic animals having a reference genome sequence available.

Elucidation of the gas-phase structure of a sugar-modified DNA analogue

Antisense oligonucleotides are medical agents for the treatment of genetic diseases that are designed to interact specifically with mRNA. This interaction either induces enzymatic degradation of the targeted RNA or modifies processing pathways, e.g. by inducing alternative splicing of the pre-mRNA. The latter mechanism applies to the treatment of Duchenne muscular dystrophy with a sugar-modified DNA analogue called tricyclo-DNA (tcDNA). In tcDNA the ribose sugar-moiety is extended to a three-membered ring system, which augments the binding affinity and the selectivity of the antisense oligonucleotide for its target. The advent of chemically modified nucleic acids for antisense therapy presents a challenge to diagnostic tools, which must be able to cope with a variety of structural analogues. Mass spectrometry meets this demand for non-enzyme based sequencing methods ideally, because the technique is largely unaffected by structural modifications of the analyte. Sequence coverage of a fully modified tcDNA 15mer can be obtained in a single tandem mass spectrometric experiment. Beyond sequencing experiments, tandem mass spectrometry was applied to elucidate the gas-phase structure and stability of tcDNA:DNA and tcDNA:RNA hybrid duplexes. Most remarkable is the formation of truncated duplexes upon collision-induced dissociation of these structures. Our data suggest that the cleavage site within the duplex is directed by the modified sugar-moiety. Moreover, the formation of truncated duplexes manifests the exceptional stability of the hybrid duplexes in the gas-phase. This stability arises from the modified sugar-moiety, which locks the tcDNA single strand into a conformation that is similar to RNA in A-form duplexes. The conformational particularity of tcDNA in the gas-phase was confirmed by ion mobility-mass spectrometry experiments on tcDNA, DNA, and RNA.