Domain-Specific Common Data Elements for Rare Disease Registration: Conceptual Approach of a European Joint Initiative Toward Semantic Interoperability in Rare Disease Research.

BACKGROUND: With hundreds of registries across Europe, rare diseases (RDs) suffer from fragmented knowledge, expertise, and research. A joint initiative of the European Commission Joint Research Center and its European Platform on Rare Disease Registration (EU RD Platform), the European Reference Networks (ERNs), and the European Joint Programme on Rare Diseases (EJP RD) was launched in 2020. The purpose was to extend the set of common data elements (CDEs) for RD registration by defining domain-specific CDEs (DCDEs). OBJECTIVE: This study aims to introduce and assess the feasibility of the concept of a joint initiative that unites the efforts of the European Platform on Rare Disease Registration Platform, ERNs, and European Joint Programme on Rare Diseases toward extending RD CDEs, aiming to improve the semantic interoperability of RD registries and enhance the quality of RD research. METHODS: A joint conference was conducted in December 2020. All 24 ERNs were invited. Before the conference, a survey was communicated to all ERNs, proposing 18 medical domains and requesting them to identify highly relevant choices. After the conference, a 3-phase plan for defining and modeling DCDEs was drafted. Expected outcomes included harmonized lists of DCDEs. RESULTS: All ERNs attended the conference. The survey results indicated that genetic, congenital, pediatric, and cancer were the most overlapping domains. Accordingly, the proposed list was reorganized into 10 domain groups and recommunicated to all ERNs, aiming at a smaller number of domains. CONCLUSIONS: The approach described for defining DCDEs appears to be feasible. However, it remains dynamic and should be repeated regularly based on arising research needs.

Tissue-specific targeting of DNA nanodevices in a multicellular living organism.

Nucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are localized in specific organs, and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell types in vivo. Here, we show that by exploiting either endogenous or synthetic receptor-ligand interactions and leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in Caenorhabditis elegans, with subcellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology.

The Occurrence of Skeletons of Silicoflagellata and Other Siliceous Bioparticles in Floral Honeys.

Siliceous marine microfossils were unexpectedly discovered during the analysis of flower honey samples from Poland and Tunisia. The microfossils were represented by protist with siliceous skeletons: silicoflagellates, diatoms, and endoskeletal dinoflagellates. This is the first record of such microfossils in honeys. Based on the high percent of anemophilous pollen grains and spores in the sample, it was hypothesized that silicoflagellates were deposited from the air onto the nectariferous flowers, then bees harvested them with the nectar. Based on the comparison of pollen content of honeys and flowering calendar of Tunisia, the harvest time of honey was identified as a period between 1 April and 31 May 2011. Trajectory analysis of air masses in this period confirmed that siliceous microfossils could be aerosolized by wind from the rocks of the so-called Tripoli Formation of Messinian age (6-7 Ma). Similar to the Tunisian case, the Polish trajectory simulation also supports the hypothesis of atmospheric transport of silicoflagellates from outcrops of Oligocene age in the Polish Outer Carpathians. In the case of diatom content of honey, however, the source can be both natural (wind) and artificial (diatomaceous earth filters). For a correct determination, natural sources of siliceous bioparticles, such as wind transport from nearby outcrops should be also considered. Silicoflagellates could be used as complementary indicators of the geographical origin of honeys collected in areas characterized by diatomite outcrops, supporting the results obtained with other methods; thus, such indicators merit further studies within the area of honey authenticity.

A sustainable solution for the activities of the European network for surveillance of congenital anomalies: EUROCAT as part of the EU Platform on Rare Diseases Registration.

The European Commission through its Directorates-General Joint Research Centre (DG JRC) and Health and Food Safety (DG SANTE) is developing the European Platform on Rare Diseases Registration (EU RD Platform) with the objective to set European-level standards for data collection and data sharing. In the field of rare diseases the EU RD Platform will be a source of information on available rare disease patient data with large transnational European coverage. One main function of the EU RD Platform is to enable interoperability for the >600 existing RD registries in Europe. The second function is to offer a sustainable solution for two large European surveillance networks: European Surveillance of Congenital Anomalies (EUROCAT) and Surveillance of Cerebral Palsy in Europe (SCPE). EUROCAT is European network of population-based registries for the epidemiological surveillance of congenital anomalies. It covers about one third of the European birth population. The Central Database contains about 800,000 cases with congenital anomalies among livebirths, stillbirths and terminations of pregnancy, reported using the same standardised classification and coding. These high quality data enables epidemiological surveillance of congenital anomalies, which includes estimating prevalence, prenatal diagnosis and perinatal mortality rates and the detection of teratogenic exposures among others. The network also develops recommendations for primary prevention in the Rare Diseases National Plans for medicinal drugs, food/nutrition, lifestyle, health services, and environmental pollution. The network has received the European Commission's support since its inception. In order to offer a sustainable solution for the continuation of EUROCAT activities, it was agreed that EUROCAT would become part of the EU RD Platform. In 2015, the European level-coordination activities and the Central Database were transferred to the DG JRC, where the JRC-EUROCAT Central Registry is now located. This paper describes the functioning of EUROCAT in the new setting, and gives an overview of the activities and the organisation of the JRC-EUROCAT Central Registry.

Micromachining of Polyurethane Membranes for Tissue Engineering Applications.

Engineered tissue barrier models offer in vitro alternatives in toxicology and disease research. To mimic barrier-tissue microenvironment, a porous membrane that can approach the stiffness of physiological basement membranes is required. While several biocompatible membranes with micrometer range thickness (10 µm) and a stiffness less than polystyrene (3 GPa) or polyethylene (PET, 2 GPa), have been developed, there has been little effort to optimize the process to enable rapid and reproducible pore production in these membranes. Here, we investigate the use of laser irradiation with femtosecond (fs) pulses because the combination of high-precision and cold-ablation causes minimal damage to polymeric membranes. This process enables automated, high-throughput and reproducible fabrication of thin, microporous membranes that can be utilized to culture cells at air-liquid interface (ALI), a unique culture technique that simulates the tissue-barrier microenvironment. We show the optimization of laser parameters on a thin polyurethane membrane and patterned pores with an average diameter of 5 µm. Tissue was cultured at ALI for 28 days to demonstrate the membrane's utility in constructing a tissue barrier model. These results confirm the utilization of fs laser machining as a viable method for creating a porous barrier substrate in tissue engineering platforms.

Evidence for marine microfossils from amber.

Amber usually contains inclusions of terrestrial and rarely limnetic organisms that were embedded in the places were they lived in the amber forests. Therefore, it has been supposed that amber could not have preserved marine organisms. Here, we report the discovery amber-preserved marine microfossils. Diverse marine diatoms as well as radiolarians, sponge spicules, a foraminifer, and a spine of a larval echinoderm were found in Late Albian and Early Cenomanian amber samples of southwestern France. The highly fossiliferous resin samples solidified approximately 100 million years ago on the floor of coastal mixed forests dominated by conifers. The amber forests of southwestern France grew directly along the coast of the Atlantic Ocean and were influenced by the nearby sea: shells and remnants of marine organisms were probably introduced by wind, spray, or high tide from the beach or the sea onto the resin flows.

Rituximab long-term maintenance therapy after autologous stem cell transplantation in patients with B-cell non-Hodgkin's lymphoma.

Treatment of B-cell non-Hodgkin's lymphomas (NHL) with either Rituximab alone or in combination with cytotoxic chemotherapy has been effective without major side effects. Thus, Rituximab maintenance therapy after autologous peripheral blood stem cell transplantation (PBSCT) might represent an improvement in NHL therapy. We therefore retrospectively analyzed the efficacy and side effects of monthly long-term Rituximab maintenance therapy after PBSCT in 27 patients with NHL. In median 10 infusions of Rituximab were given after PBSCT in time intervals of 1 month. Molecular monitoring of t(14;18) was performed using nested as well as quantitative real time polymerase chain reaction (RT-PCR) based on the LightCycler technology. Side effects according to common toxicity criteria (CTC) > II did mainly affect the hematopoietic system. In total, 10 patients (37%) suffered form grade III-IV hematotoxicity. Except for two patients with cutaneous Varicella-Zoster infection no serious infectious complications (CTC grade III/IV) occurred. No patient died because of treatment-related causes. This adverse event data compared favorably to the published data. Three patients had t(14;18) nested RT-PCR positive results before Rituximab therapy and converted to negativity after Rituximab therapy. We conclude that a prolonged Rituximab maintenance therapy after PBSCT with monthly administration is reliable and safe.

LightCycler-based quantitative real-time PCR monitoring of patients with follicular lymphoma receiving rituximab in combination with conventional or high-dose cytotoxic chemotherapy.

OBJECTIVES: Quantitative real-time polymerase chain reaction (qPCR) is a suitable method to measure residual disease in hematological malignancies. Our objective was to assess a LightCycler-based qPCR for t(14;18)(q32;q21)(IgH/bcl-2)-positive cells quantification in the context of clinical and morphopathological characteristics of patients with follicular lymphoma treated with rituximab (R) in combination with conventional or high-dose chemotherapy. METHODS: A total of 270 bone marrow (BM) and peripheral blood (PB) samples collected from 52 patients with follicular lymphoma at diagnosis or at relapse before or sequentially during therapy were examined by qPCR and nested-PCR. RESULTS: A greater amount of t(14;18)-positive cells was observed in BM in comparison with PB in 76% of paired samples. The presence and number of t(14;18)-positive cells in BM and PB correlated with lymphoma activity. Significantly higher numbers of lymphoma cells were found in patients under non-remission compared with patients in clinical remission. During non-remission, 10-fold higher numbers were measured at relapse than at diagnosis. During remission, significantly higher levels were found in partial compared with complete remission. During first-line therapy, R/cyclophosphamide/adriamycin/vincristine/prednisone (CHOP) had higher in vivo purging ability than R/fludarabine/mitoxantrone (FM). After R/high-dose cytosine-arabinoside and mitoxantrone (HAM) or R/carmustine/etoposide/cytarabine/melphalan (BEAM), the level of t(14;18)-positive cells dropped below the detection limit in 80% of patients. CONCLUSIONS: LightCycler qPCR is a reliable method for quantitative molecular monitoring of t(14;18)-positive cells in BM and PB of patients with follicular lymphoma. It reflects the clinical characteristics of patients and allows assessment of response to different treatment regimens on a molecular level.

Cell surface sialylation and ecto-sialyltransferase activity of human CD34 progenitors from peripheral blood and bone marrow.

Surface expressed negatively charged sialoglycans contribute to the regulation of adhesive cellular interactions and are thus involved in the growth and differentiation of hematopoietic progenitor cells. In particular, the cell surface sialylation state may govern the liberation of CD34+ hematopoietic precursors from bone marrow stroma cells and extracellular matrix. In order to assess the overall surface sialylation of live human CD34+ hematopoietic precursor cells, we applied a previously described flow cytometric enzyme assay. Cells with and without sialidase pretreatment were incubated in the presence of fluorescent CMP-sialic acid and exogenous ST6GalI. Thus sialylation of surface-expressed lactosamine residues was analysed. We demonstrated that surface lactosamines of CD34+ precursors derived from bone marrow and peripheral blood are over 95% sialylated, predominantly in alpha2-6 linkage. These results are in accordance with flow cytometric analysis of surface lectin staining. Sialic acid specific lectins MAA and SNA were strongly bound whereas SBA, VVA, and PNA became reactive only after sialidase pretreatment. CD34+ leukemia cell lines TF1 and KG1a also showed a high degree of surface sialylation, whereas cell line KG1 expressed to the largest extent free lactosamines. In these cell lines, alpha2-6 and alpha2-3 sialylated residues were present in equal amounts. In a variation of the flow cytometric enzyme assay, live cells were incubated without exogenous STGal I to measure the activity of endogenous ecto-sialyltransferase. Ecto sialyltransferase activity was observed in all CD34+ cells which was able to resialylate major surface glycoproteins such as HLA Class I, CD45, CD43, and CD34. The ecto-sialyltransferase may serve to maintain or increase surface sialylation rapidly without de novo synthesis.

Molecular biology of hematopoietic stem cells.

Human CD34+ hematopoietic stem and progenitor cells are capable of maintaining a life-long supply of the entire spectrum of blood cells dependent on systemic needs. Recent studies suggest that hematopoietic stem cells are, beyond their hematopoietic potential, able to differentiate into nonhematopoietic cell types, which could open novel avenues in the field of cellular therapy. Here, we concentrate on the molecular biology underlying basic features of hematopoietic stem cells. Immunofluorescence analyses, culture assays, and transplantation models permit an extensive immunological as well as functional characterization of human hematopoietic stem and progenitor cells. New methods such as cDNA array technology have demonstrated that distinct gene expression patterns of transcription factors and cell cycle genes molecularly control self-renewal, differentiation, and proliferation. Furthermore, several adhesion molecules have been shown to play an important role in the regulation of hematopoiesis and stem cell trafficking. Progress has also been made in elucidating molecular mechanisms of stem cell aging that limit replicative potential. Finally, more recent data provide the first molecular basis for a better understanding of transdifferentiation and developmental plasticity of hematopoietic stem cells. These findings could be helpful for non-hematopoietic cell therapeutic approaches.