The intrinsically „not-so-unfolded“ protein Osteopontin

Osteopontin (OPN) ist ein Mitglied aus der Familie der small integrin binding ligand N- linked glycoproteins (SIBLINGs). Seine molekulare Struktur beinhaltet Motive, die für Zell-Zell und Zell-Matrix Signaling via der αvβ-Integrin- oder CD44 Rezeptoren in normalen sowie pathologischen Prozessen notwendig sind. v-myc and v-mil(raf) co-transformierte Hühner Fibroblasten besitzen eine erhöhte Konzentration des Transkriptionsfaktors AP-1, welcher zu einer erhöhten Expression des für OPN kodierenden Gens OPN führt. Eine erhöhte OPN Expression ist häufig in Tumorzellen zu beobachten, wo das Protein an einer Reihe von Prozessen wie Remodeling, Inflammation, Knochen Resorption und Tumorwachstum. OPN ist ein sogenanntes intrinsically unfolded protein (IUP) mit keiner definierten Tertiärstruktur. IUPs stellen das gängige Struktur-Funktions Paradigma der Biologie in Frage. Trotz des Fehlens einer definierten Struktur sind sie an wichtigen Prozessen wie Transkriptions, Translations und Signaltransduktionsprozessen beteiligt. Seine konformationelle Flexibilität macht es jedoch auch zum Ziel von Misfolding was häufig zu Krankheiten führt. NMR eignet sich hervorragend dazu, die Dynamik und Struktur solcher ungefaltenen Proteine genauer unter die Lupe zu nehmen. Eine Kombination aus 15N- Relaxationsmessungen, pulsed-field gradient (PFG) diffusion measurements, residual dipolar couplings (RDCs) und paramagnetic relaxation enhancement (PRE) Experimenten wurde verwendet, um den dynamischen Charakter von OPN genauer zu untersuchen. Diese Arbeit versucht, die molekulare Struktur von OPN mit seinen vielschichtigen physiologischen Aufgaben in Verbindung zu bringen.Osteopontin (OPN) is a member of the small integrin binding ligand N-linked glycoprotein (SIBLING) family of proteins. The molecular structure of OPN contains unique structural motifs, which mediate cell-matrix and cell–cell signaling through the αvβ integrin family and CD44 receptors in a variety of normal and pathologic processes. Avian fibroblasts simultaneously transformed by v-myc and v-mil(raf) display significantly elevated levels of the transcription factor complex AP-1 which subsequently leads to upregulation of the osteopontin (OPN) encoding gene (OPN). Elevated OPN expression is commonly observed in tumor cells where it plays crucial roles in remodeling processes such as inflammation, bone resorption and tumor progression. OPN belongs to the class of intrinsically unfolded proteins (IUP) with no apparent ordered tertiary structure detectable. Intrinsically unfolded proteins have challenged the structure–function paradigm in current biology, since they are now recognized to play essential roles in transcription, translation and signal transduction pathways. The absence of defined structure allows disordered regions of proteins to acquire a high degree of plasticity but also represents an easy target for misfolding, often leading to disease. NMR is uniquely suited to elucidate the structural and dynamic features of these unfolded states. A combination of 15N-relaxation time measurements, pulsed-field gradient (PFG) diffusion measurements, residual dipolar couplings and paramagnetic relaxation enhancement experiments were applied to characterize the dynamic properties of OPN. This work will address the connection between the molecular structure of Osteopontin and its versatile physiological functions

KCNJ3 is a new independent prognostic marker for estrogen receptor positive breast cancer patients

Numerous studies showed abnormal expression of ion channels in different cancer types. Amongst these, the potassium channel gene KCNJ3 (encoding for GIRK1 proteins) has been reported to be upregulated in tumors of patients with breast cancer and to correlate with positive lymph node status. We aimed to study KCNJ3 levels in different breast cancer subtypes using gene expression data from the TCGA, to validate our findings using RNA in situ hybridization in a validation cohort (GEO ID GSE17705), and to study the prognostic value of KCNJ3 using survival analysis. In a total of > 1000 breast cancer patients of two independent data sets we showed a) that KCNJ3 expression is upregulated in tumor tissue compared to corresponding normal tissue (p < 0.001), b) that KCNJ3 expression is associated with estrogen receptor (ER) positive tumors (p < 0.001), but that KCNJ3 expression is variable within this group, and c) that ER positive patients with high KCNJ3 levels have worse overall (p < 0.05) and disease free survival probabilities (p < 0.01), whereby KCNJ3 is an independent prognostic factor (p <0.05). In conclusion, our data suggest that patients with ER positive breast cancer might be stratified into high risk and low risk groups based on the KCNJ3 levels in the tumor

C/EBP beta-LIP induces cancer-type metabolic reprogramming by regulating the let-7/LIN28B circuit in mice

The transcription factors LAP1, LAP2 and LIP are derived from the Cebpb-mRNA through the use of alternative start codons. High LIP expression has been associated with human cancer and increased cancer incidence in mice. However, how LIP contributes to cellular transformation is poorly understood. Here we present that LIP induces aerobic glycolysis and mitochondrial respiration reminiscent of cancer metabolism. We show that LIP-induced metabolic programming is dependent on the RNA-binding protein LIN28B, a translational regulator of glycolytic and mitochondrial enzymes with known oncogenic function. LIP activates LIN28B through repression of the let-7 microRNA family that targets the Lin28b-mRNA. Transgenic mice overexpressing LIP have reduced levels of let-7 and increased LIN28B expression, which is associated with metabolic reprogramming as shown in primary bone marrow cells, and with hyperplasia in the skin. This study establishes LIP as an inducer of cancer-type metabolic reprogramming and as a regulator of the let-7/LIN28B regulatory circuit

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8-13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05-6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50-75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Familial hypercholesterolaemia in children and adolescents from 48 countries : a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11848 individuals. Median age at registry entry was 9·6 years (IQR 5·8–13·2). 10 099 (89·9%) of 11235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05–6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50–75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life.peer-reviewe

Osteopontin and the molecular principles that orchestrate its rheomorphic ensemble

Proteine sind an einer Vielzahl von Prozessen in lebenden Organsimen beteiligt. Diese reichen von der Umsetzung von Metaboliten, Transformation, Replikation und Reparatur von DNS, über zellulären Transport bis hin zu Signaltransduktionsprozessen. Obwohl viele Proteine auf eine wohldefinierte dreidimensionale Struktur angewiesen sind um ihre physiologische Rolle auszuüben, finden sich in höheren Lebewesen viele Proteine die teilweise unstrukturierte Bereiche enthalten bis hin zu komplett unstrukturierten Proteinen. Diese sogenannten Intrinsisch Ungefaltenen Proteine (IUPs) haben in den letzten Jahren viel Interesse geweckt, besonders angesichts ihrer Beteiligung an einer großen Anzahl an physiologischen und pathophysiologischen Prozessen. In dieser Arbeit untersuchen wir die dynamischen und strukturellen Eigenschaften des IUPs Osteopontin (OPN). OPN ist ein hoch negativ geladenes Polypeptid und Ziel einer Vielzahl von post-translationalen Modifikationen, die ausschlaggebend für seine in vivo Funktion sind. OPN wird von vielen verschiedenen Zellen exprimiert und ist, je nach zellulärer Lokalisation an unterschiedlichen Prozessen beteiligt, unter anderem Biomineralisierung, Immunantwort, Abbau von Komponenten der extrazellulären Matrix und Signaltransduktionsprozessen. Zusätzlich wird OPN in vielen Tumoren überexprimiert wo es durch Interaktion mit Zellrezeptoren wie Integrinen und CD44 in tumorfördernden Prozessen wechselwirkt. Da Röntgendiffraktometrie, die klassische Methode zur Strukturbestimmung von Proteinen im Falle von IUPs nicht anwendbar ist, verwenden wir in unserer Arbeit eine Kombination von NMR, EPR, SAXS und CD-Spektroskopie um die hoch flexible Beschaffenheit von OPN zu untersuchen. Unsere Resultate zeigen dass OPN sich durch ein vielschichtiges Strukturensemble in Lösung auszeichnet das ausgedehnte sowie kooperativ gefaltete Konformationen enthält. Schlussendlich untersuchen wir die Interaktion von OPN mit seinem natürlichen Ligand, Heparin.Proteins perform a vast array of functions within a living organism ranging from the catalysis of metabolic reactions and DNA synthesis to cellular transport, signaling and many others. While many proteins rely on a well-defined tertiary structure to fulfill their physiological roles, a large number of proteins in higher organisms are found to contain unstructured regions or in extreme cases display a total lack of any tertiary structure in solution. These so-called intrinsically disordered proteins (IDPs) have recently attracted a lot of attention in the scientific community given their interesting biophysical properties and their involvement in a multitude of physiological and pathological processes. In this work we investigate the dynamic and structural properties of the IDP Osteopontin (OPN). OPN is a highly negatively charged polypeptide and is a target of several post-translational modifications (PTMs) that govern its function in vivo. It is expressed in a variety of cell-types and depending on its localization, is involved in variety of cellular events including biomineralization, immune response, extracellular matrix (ECM)-degradation and cell signaling. Additionally, the protein is overexpressed in malignant tissues where it exerts its tumorigenic function by interacting with cell-surface receptors like Integrins and CD44, therfore rendering it a highly interesting research target. Since X-ray crystallography, as the classical approach for protein-structure determination is not applicable in the case of IDPs, we use a methodologically integrated approach of NMR, EPR, SAXS and CD-spectroscopy to gain insights into the highly flexible nature of OPN. Our findings show that OPN displays a highly diverse conformational ensemble comprising cooperative folding and unfolding events between extended and compact structures in solution. Finally, we address the implications of disorder on the interaction with Osteopontin’s naturally occurring ligand heparin