Zooming in on size distribution patterns underlying species coexistence in Baltic Sea phytoplankton.

Scale is a key to determining which processes drive community structure. We analyse size distributions of phytoplankton to determine time scales at which we can observe either fixed environmental characteristics underlying communities structure or competition-driven size distributions. Using multiple statistical tests, we characterise size distributions of phytoplankton from 20-year time series in two sites of the Baltic Sea. At large temporal scales (5-20 years), size distributions are unimodal, indicating that fundamental barriers to existence are here subtler than in other systems. Frequency distributions of the average size of the species weighted by biovolume are multimodal over large time scales, although this is the product of often unimodal short-term (<1 year) patterns. Our study represents a much-needed structured, high-resolution analysis of phytoplankton size distributions, revealing that short-term analyses are necessary to determine if, and how, competition shapes them. Our results provide a stepping-stone on which to further investigate the intricacies of competition and coexistence.

The rising incidence of anal cancer in England 1990-2010: a population-based study.

AIM: Although anal cancer is rare, its incidence has been reported to be rising in several countries. This study aimed to determine whether there have been any changes in incidence over time in England. METHOD: In the cancer registry component of the English National Cancer Data Repository, 13 940 patients were identified with a primary diagnosis of anal cancer made between 1990 and 2010. Tumours were grouped according to the ICD-O morphology codes into squamous cell carcinoma, basaloid and cloacogenic carcinoma, adenocarcinoma and other cancer types. The incidence over this period was investigated in relation to type of tumour, age and sex. RESULTS: In men there was a 69% increase in squamous cell anal carcinoma from 0.43 per 100 000 population in 1990-94 to 0.73 in 2006-10. For women these rates were 0.50 in 1990-94 and 1.13 in 2006-10, a rise of 126%. CONCLUSION: The study showed that between 1990 and 2010 there was a substantial rise in the incidence of anal cancer in England. This effect was more marked in women than men.

Early mortality from colorectal cancer in England: a retrospective observational study of the factors associated with death in the first year after diagnosis.

BACKGROUND: The United Kingdom performs poorly in international comparisons of colorectal cancer survival with much of the deficit owing to high numbers of deaths close to the time of diagnosis. This retrospective cohort study investigates the patient, tumour and treatment characteristics of those who die in the first year after diagnosis of their disease. METHODS: Patients diagnosed with colon (n=65,733) or rectal (n=26,123) cancer in England between 2006 and 2008 were identified in the National Cancer Data Repository. Multivariable logistic regression was used to investigate the odds of death within 1 month, 1-3 months and 3-12 months after diagnosis. RESULTS: In all, 11.5% of colon and 5.4% of rectal cancer patients died within a month of diagnosis: this proportion decreased significantly over the study period. For both cancer sites, older age, stage at diagnosis, deprivation and emergency presentation were associated with early death. Individuals who died shortly after diagnosis were also more likely to have missing data about important prognostic factors such as disease stage and treatment. CONCLUSION: Using routinely collected data, at no inconvenience to patients, we have identified some important areas relating to early deaths from colorectal cancer, which merit further research.

Plasma and rectal mucosal oxylipin levels during aspirin and eicosapentaenoic acid treatment in the seAFOod polyp prevention trial.

BACKGROUND: Aspirin and eicosapentaenoic acid (EPA) have colorectal polyp prevention activity, alone and in combination. This study measured levels of plasma and rectal mucosal oxylipins in participants of the seAFOod 2 × 2 factorial, randomised, placebo-controlled trial, who received aspirin 300 mg daily and EPA 2000 mg free fatty acid, alone and in combination, for 12 months. METHODS: Resolvin (Rv) E1, 15-epi-lipoxin (LX) A4 and respective precursors 18-HEPE and 15-HETE (with chiral separation) were measured by ultra-high performance liquid chromatography-tandem mass spectrometry in plasma taken at baseline, 6 months and 12 months, as well as rectal mucosa obtained at trial exit colonoscopy at 12 months, in 401 trial participants. RESULTS: Despite detection of S- and R- enantiomers of 18-HEPE and 15-HETE in ng/ml concentrations, RvE1 or 15­epi-LXA4 were not detected above a limit of detection of 20 pg/ml in plasma or rectal mucosa, even in individuals randomised to both aspirin and EPA. We have confirmed in a large clinical trial cohort that prolonged (12 months) treatment with EPA is associated with increased plasma 18-HEPE concentrations (median [inter-quartile range] total 18-HEPE 0.51 [0.21-1.95] ng/ml at baseline versus 0.95 [0.46-4.06] ng/ml at 6 months [P<0.0001] in those randomised to EPA alone), which correlate strongly with respective rectal mucosal 18-HEPE levels (r = 0.82; P<0.001), but which do not predict polyp prevention efficacy by EPA or aspirin. CONCLUSION: Analysis of seAFOod trial plasma and rectal mucosal samples has not provided evidence of synthesis of the EPA-derived specialised pro-resolving mediator RvE1 or aspirin-trigged lipoxin 15­epi-LXA4. We cannot rule out degradation of individual oxylipins during sample collection and storage but readily measurable precursor oxylipins argues against widespread degradation.

Are lower rates of surgery amongst older women with breast cancer in the UK explained by co-morbidity?

BACKGROUND: Around 60% of women ≥ 80 years old, in the UK do not have surgery for their breast cancer (vs<10% of younger age groups). The extent to which this difference can be accounted for by co-morbidity has not been established. METHODS: A Cancer Registry/Hospital Episode Statistics-linked data set identified women aged ≥ 65 years diagnosed with invasive breast cancer (between 1 April 1997 and 31 March 2005) in two regions of the UK (n=23038). Receipt of surgery by age was investigated using logistic regression, adjusting for co-morbidity and other patient, tumour and treatment factors. RESULTS: Overall, 72% of older women received surgery, varying from 86% of 65-69-year olds to 34% of women aged ≥ 85 years. The proportion receiving surgery fell with increasing co-morbidity (Charlson score 0=73%, score 1=66%, score 2+=49%). However, after adjustment for co-morbidity, older age still predicts lack of surgery. Compared with 65-69-year olds, the odds of surgery decreased from 0.74 (95% CI: 0.66-0.83) for 70-74-year olds to 0.13 (95% CI: 0.11-0.14) for women aged ≥ 85 years. CONCLUSION: Although co-morbidity is associated with a reduced likelihood of surgery, it does not explain the shortfall in surgery amongst older women in the UK. Routine data on co-morbidity enables fairer comparison of treatment across population groups but needs to be more complete.

Pharmacogenetic analysis of the mGlu2/3 agonist LY2140023 monohydrate in the treatment of schizophrenia.

The goal of this study was to identify genetic markers associated with LY2140023 monohydrate response in patients with schizophrenia. Variants in eight candidate genes related to the mechanism of action of LY2140023 or olanzapine were investigated in a genetic cohort collected from two clinical trials. Results from this genetic analysis indicate that 23 single nucleotide polymorphisms (SNPs) were associated with a change in Positive and Negative Syndrome Scale total score in response to LY2140023 at 28 days (P<0.01; false discovery rate <0.2). Sixteen of these SNPs were located in the serotonin 2A receptor (HTR2A). Bioinformatic analyses identified a putative antisense nested gene in intron 2 of HTR2A in the region of the genetic markers associated with LY2140023 response. These data suggest a genetic association exists between SNPs in several genes, such as HTR2A, and response to LY2140023 treatment. Additional clinical trials are needed to establish replication of these results.

Time to Surgery Following Short-Course Radiotherapy in Rectal Cancer and its Impact on Postoperative Outcomes. A Population-Based Study Across the English National Health Service, 2009-2014.

AIMS: Preoperative short-course radiotherapy (SCRT) is an important treatment option for rectal cancer. The length of time between completing SCRT and surgery may influence postoperative outcomes, but the evidence available to determine the optimal interval is limited and often conflicting. MATERIALS AND METHODS: Information was extracted from a colorectal cancer data repository (CORECT-R) on all surgically treated rectal cancer patients who received SCRT in the English National Health Service between April 2009 and December 2014. The time from radiotherapy to surgery was described across the population. Thirty-day postoperative mortality, returns to theatre, length of stay and 1-year survival were investigated in relation to the interval between radiotherapy and surgery. RESULTS: Within the cohort of 3469 patients, the time to surgery was 0-7 days for 76% of patients, 8-14 days for 19% of patients and 15-27 days for 5% of patients. There was a clear variation in relation to different patient characteristics. There was, however, no evidence of differences in postoperative outcomes in relation to interval length. CONCLUSIONS: This study suggests that the time interval between SCRT and surgery does not influence postoperative outcomes up to a year after surgery. The study provides population-level, real-world evidence to complement that from clinical trials.

Genomewide association for schizophrenia in the CATIE study: results of stage 1.

Little is known for certain about the genetics of schizophrenia. The advent of genomewide association has been widely anticipated as a promising means to identify reproducible DNA sequence variation associated with this important and debilitating disorder. A total of 738 cases with DSM-IV schizophrenia (all participants in the CATIE study) and 733 group-matched controls were genotyped for 492,900 single-nucleotide polymorphisms (SNPs) using the Affymetrix 500K two-chip genotyping platform plus a custom 164K fill-in chip. Following multiple quality control steps for both subjects and SNPs, logistic regression analyses were used to assess the evidence for association of all SNPs with schizophrenia. We identified a number of promising SNPs for follow-up studies, although no SNP or multimarker combination of SNPs achieved genomewide statistical significance. Although a few signals coincided with genomic regions previously implicated in schizophrenia, chance could not be excluded. These data do not provide evidence for the involvement of any genomic region with schizophrenia detectable with moderate sample size. However, a planned genomewide association study for response phenotypes and inclusion of individual phenotype and genotype data from this study in meta-analyses hold promise for eventual identification of susceptibility and protective variants.

Designing Trials of Disease Modifying Agents for Early and Preclinical Alzheimer's Disease Intervention: What Evidence is Meaningful to Patients, Providers, and Payers?

BACKGROUND: Drug development for disease modifying agents in Alzheimer's disease (AD) is focused increasingly on targeting underlying pathology in very early stages of AD or in cognitively normal patients at elevated risk of developing dementia due to Alzheimer's. Very early interventional studies of this type have many uncertainties, including whether they can provide the clinical results that payers, providers, and patients will wish to see for decisions. This paper describes an initiative to create greater transparency for researchers to anticipate these decision needs. OBJECTIVE: To create multi-stakeholder-vetted recommendations for the design of studies in later phases of drug development to evaluate the ability of disease modifying agents to delay or prevent the onset of dementia due to Alzheimer's disease (AD). DESIGN: A multi-stakeholder expert workgroup and overseeing steering group were convened to discuss current advances in early interventional clinical trial design and the evidence needs of patients, providers, and payers. Eight teleconferences and one in-person all-day meeting were held. Meetings were recorded and summary notes prepared between sessions. Final conclusions were consolidated by the project team with the workgroup Chair based on these discussions and were reviewed by group members. SETTING: The in-person meeting was held in Baltimore, MD. PARTICIPANTS: In total, 36 stakeholders representing life sciences industry, payers or health technology assessors, patient advocates and research advocacy organizations, regulators, clinical experts and academic or NIH researchers. INTERVENTION: N/A. MEASUREMENTS: N/A. RESULTS: Certain aspects of clinical trial design were deemed important to address stakeholder decision needs for future Alzheimer's prevention drugs even as the field rapidly progresses. These include the need for more robust behavioral and psychological outcome data in early symptomatic disease and the need to update activities of daily living measures to include "digital independence." CONCLUSIONS: Amyloid, tau, and biomarkers of neurodegeneration should be included in trials and studied in relation to other early measures of change meaningful to individuals with AD, their families, and health plans. These measures include early sensitive changes in behavioral and psychological measures and ability to navigate the contemporary digital landscape. Additional work is needed to generate more robust behavioral and psychological outcome data in early symptomatic disease, and to generate multi-stakeholder consensus on early measures of change and magnitudes of change that will be meaningful to patients, providers, and payers.

Three-dimensional solution structure of the pleckstrin homology domain from dynamin.

BACKGROUND: The pleckstrin homology (PH) domain is a region of approximately 100 amino acids, defined by sequence similarity, that has been found in about 60 proteins, many of which are involved in signal transduction downstream of cell surface receptors; the function of PH domains is unknown. The only clue to the function of PH domains is the circumstantial evidence that they may link beta gamma subunits of G proteins to second messenger systems. Knowledge of the three-dimensional structures of PH domains should help to elucidate the roles they play in the proteins that contain them. RESULTS: Using homonuclear and heteronuclear magnetic resonance spectroscopy, we have determined the solution structure of the PH domain of the GTPase dynamin, one of a number of proteins that have PH domains and interact with GTP. The fold of the dynamin PH domain is composed of two antiparallel beta-sheets, which pack face-to-face at an angle of approximately 60 degrees. The first beta-sheet comprises four strands (residues 13-58) from the amino-terminal half of the protein sequence; the second beta-sheet contains three strands (residues 63-99). A single alpha-helix (residues 102-116) flanks one edge of the interface between the two sheets, parallel in orientation to the second sheet, in an alpha/beta roll motif similar to that of the B oligomer of verotoxin-1 from Escherichia coli. CONCLUSIONS: The structure of the dynamin PH domain is very similar to the recently reported structures of the pleckstrin and spectrin PH domains. This shows that, despite the low level of sequence similarity between different PH domains, they do have a characteristic polypeptide fold. On the basis of our structure, the suggestion that PH domains engage in coiled-coil interactions with G protein beta gamma subunits seems unlikely and should be re-evaluated.

Development Review of the BACE1 Inhibitor Lanabecestat (AZD3293/LY3314814).

Several ongoing clinical development programs are investigating potential disease-modifying treatments for Alzheimer's disease (AD), including lanabecestat (AZD3293/LY3314814). Lanabecestat is a brain-permeable oral inhibitor of human beta-site amyloid (Aß) precursor protein-cleaving enzyme 1 (BACE1) that reduces Aß production. As a potent BACE1 inhibitor, lanabecestat significantly reduced soluble Aß species and soluble amyloid precursor proteins (sAPPß) in mouse, guinea pig, and dog in a time- and dose-dependent manner. Significant reductions in plasma and cerebrospinal fluid (CSF) Aß1-40 and Aß1-42 were observed in Phase 1 studies of healthy subjects and AD patients treated with lanabecestat. Three lanabecestat trials are ongoing and intended to support registration in Early AD: (1) Phase 2/3 study in patients with mild cognitive impairment (MCI) due to AD and mild AD dementia (AMARANTH, NCT02245737); (2) Delayed-start extension study (AMARANTH-EXTENSION, NCT02972658) for patients who have completed treatment in the AMARANTH Study; and (3) Phase 3 study in mild AD dementia (DAYBREAK-ALZ, NCT02783573). This review will discuss the development of lanabecestat, results from the completed nonclinical and clinical studies, as well as describe the ongoing Phase 3 clinical trials.

Amino acid requirements for formation of the TGF-beta-latent TGF-beta binding protein complexes.

Transforming growth factor beta (TGF-beta) is secreted primarily as a latent complex consisting of the TGF-beta homodimer, the TGF-beta propeptides (called the latency-associated protein or LAP) and the latent TGF-beta binding protein (LTBP). Mature TGF-beta remains associated with LAP by non-covalent interactions that block TGF-beta from binding to its receptor. Complex formation between LAP and LTBP is mediated by an intramolecular disulfide exchange between the third 8-cysteine (8-Cys3) domain of LTBP with a pair of cysteine residues in LAP. Only the third 8-Cys domains of LTBP-1, -3, and -4 bind LAP. From comparison of the 8-Cys3(LTBP-1) structure with that of the non-TGF-beta-binding 8-Cys6(fibrillin-1), we observed that a two-residue insertion in 8-Cys3(LTBP-1) increased the potential for disulfide exchange of the 2-6 disulfide bond. We further proposed that five negatively charged amino acid residues surrounding this bond mediate initial protein-protein association. To validate this hypothesis, we monitored binding by fluorescence resonance energy transfer (FRET) analysis and co-expression assays with TGF-beta1 LAP (LAP-1) and wild-type and mutant 8-Cys3 domains. FRET experiments demonstrated ionic interactions between LAP-1 and 8-Cys3. Mutation of the five amino acid residues revealed that efficient complex formation is most dependent on two of these residues. Although 8-Cys3(LTBP-1) binds proTGF-betas effectively, the domain from LTBP-4 does so poorly. We speculated that this difference was due to the substitution of three acidic residues by alanine, serine, and arginine in the LTBP-4 sequence. Additional experiments with 8-Cys3(LTBP-4) indicated that enhanced binding of LAP to 8-Cys3(LTBP-4) is achieved if the residues A, S, and R are changed to those in 8-Cys3(LTBP1) (D, D, and E) and the QQ dipeptide insertion of LTBP-4 is changed to the FP in 8-Cys3(LTBP-1). These studies identify surface residues that contribute to the interactions of 8-Cys3 and LAP-1 and may yield information germane to the interaction of 8-Cys domains and additional TGF-beta superfamily propeptides, an emerging paradigm for growth factor regulation.

The solution structure and backbone dynamics of the fibronectin type I and epidermal growth factor-like pair of modules of tissue-type plasminogen activator.

BACKGROUND: The thrombolytic serine protease tissue-type plasminogen activator (t-PA) is a classical modular protein consisting of three types of domain in addition to the serine protease domain: F1 (homologous to fibronectin type I); G (epidermal growth factor-like) and kringle. Biochemical data suggest that the F1 and G modules play a major role in the binding of t-PA to fibrin and to receptors on hepatocytes. RESULTS: We have derived the solution structure of the F1 and G pair of modules from t-PA by two- and three-dimensional NMR techniques, in combination with dynamical simulated annealing calculations. We have also obtained information about the molecule's backbone dynamics through measurement of amide 15N relaxation parameters. CONCLUSIONS: Although the F1 and G modules each adopt their expected tertiary structure, the modules interact intimately to bury a hydrophobic core, and the inter-module linker makes up the third strand of the G module's major beta-sheet. The new structural results allow the interpretation of earlier mutational data relevant to fibrin-binding and hepatocyte-receptor binding.

Solution structure of the LDL receptor EGF-AB pair: a paradigm for the assembly of tandem calcium binding EGF domains.

BACKGROUND: From the observed structure and sequence of a pair of calcium binding (cb) epidermal growth factor-like (EGF) domains from human fibrillin-1, we proposed that many tandem cbEGF domains adopt a conserved relative conformation. The low-density lipoprotein receptor (LDLR), which is functionally unrelated to fibrillin-1, contains a single pair of EGF domains that was chosen for study in the validation of this hypothesis. The LDLR is the protein that is defective in familial hypercholesterolaemia, a common genetic disorder that predisposes individuals to cardiovascular complications and premature death. RESULTS: Here, we present the solution structure of the first two EGF domains from the LDL receptor, determined using conventional NMR restraints and residual dipolar couplings. The cbEGF domains have an elongated, rod-like arrangement, as predicted. The new structure allows a detailed assessment of the consequences of mutations associated with familial hypercholesterolaemia to be made. CONCLUSIONS: The validation of the conserved arrangement of EGF domains in functionally distinct proteins has important implications for structural genomics, since multiple tandem cbEGF pairs have been identified in many essential proteins that are implicated in human disease. Our results provide the means to use homology modeling to probe structure-function relationships in this diverse family of proteins and may hold the potential for the design of novel diagnostics and therapies in the future.

Structural and functional properties of the human notch-1 ligand binding region.

We present NMR structural and dynamics analysis of the putative ligand binding region of human Notch-1, comprising EGF-like domains 11-13. Functional integrity of an unglycosylated, recombinant fragment was confirmed by calcium-dependent binding of tetrameric complexes to ligand-expressing cells. EGF modules 11 and 12 adopt a well-defined, rod-like orientation rigidified by calcium. The interdomain tilt is similar to that found in previously studied calcium binding EGF pairs, but the angle of twist is significantly different. This leads to an extended double-stranded beta sheet structure, spanning the two EGF modules. Based on the conservation of residues involved in interdomain hydrophobic packing, we propose this arrangement to be prototypical of a distinct class of EGF linkages. On this premise, we have constructed a model of the 36 EGF modules of the Notch extracellular domain that enables predictions to be made about the general role of calcium binding to this region.

A study of childhood attendance at emergency departments in the West Midlands region.

INTRODUCTION: Research into childhood attendance at EDs in the UK has focused mainly on injury rather than medical conditions and studies have been relatively small. This study looks at all types of ED attendance by children across a large population. DATA AND METHODS: Routine data on all new attendances by children under 16 years were available for 12 EDs in the West Midlands (period: 1 April 2002 to 31 March 2004, 365 695 records). The data were split into four age groups (<1, 1-4, 5-9, and 10-15 years). RESULTS: Injury related conditions increased with age (with the exception of head injury). Respiratory and gastrointestinal were the most common medical conditions decreased with age. 11.5% of children were admitted to hospital and this varied from 8.2% (10-15 years) to 24.2% (<1 year). CONCLUSIONS: This study has shown substantial variations in ED attendance by age and has given an insight into the variation among hospitals. This is the largest study of childhood ED attendance undertaken in the UK, and it is hoped that the questions raised will prompt more research in this field.

Bridging the Gap between Research and Clinical Practice in Asymptomatic Alzheimer's Disease.

Due to the growing global health impact of Alzheimer's disease (AD), there is a greater need for interventions that prevent or delay the onset of clinical symptoms of this debilitating disease. Clinical trials for disease-modifying compounds in AD have shifted towards earlier stages in the spectrum of illness, including the stage prior to cognitive symptoms. A population of specific interest for clinical research includes individuals with evidence of Alzheimer's disease pathology who are asymptomatic (ADPa). The challenges and barriers regarding medical treatment of ADPa must be identified and addressed prior to the completion of a positive clinical trial in order to accelerate the translation of research findings to clinical practice. This report applies an existing public health impact model from Spencer and colleagues (2013) to evaluate the readiness of the clinical practice environment to treat ADPa individuals if a disease-modifying agent achieves approval. We contrast the current clinical practice environment with a potential future state through investigating the effectiveness, reach, feasibility, sustainability, and transferability of the practice of treating ADPa individuals.

Interaction of normal and unusually modified microbial DNA with cultured mammalian cells. Breakdown and reincorporation vs. uptake of polymerized DNA.

The uptake of radioactively labeled bacterial and phage DNA and the incorporation of acid-soluble DNAase I digests of these DNAs by cultures of human foreskin and 3T3 cells were studied. The presence of large amounts of unusually modified pyrimidine residues in donor phage DNAs allowed radioactive donor DNA in the nuclei of DNA-treated cells to be distinguished from host DNA labeled with breakdown products derived from donor DNA. This distinction could be made because it was found that radioactively labeled 5-methylcytosine residues in predigested XP-12 DNA and glucosylated 5-hydroxymethylcytosine residues in predigested T4 DNA could not be incorporated in an unaltered form into animal cell DNA. The results obtained from the study of uptake of these DNAs suggest that approx. 4--40 ng of phage DNA per 10(6) cells was transported to the nuclei of DEAE-dextran-pretreated cells during 3 days of incubation in medium after treatment with the DNA. However, interpretation of the results is complicated by the finding of considerable amounts of donor DNA binding to and persisting at the cell surface, which might attach to nuclei during subcellular fractionation.

The effects of variable glycosylation on the functional activities of ribonuclease, plasminogen and tissue plasminogen activator.

The relatively large size and dynamics of oligosaccharides can result in substantial shielding of functionally important areas of proteins to which they are attached, modulate the interactions of glycoconjugates with other molecules and affect the rate of processes which involve conformational changes. This review focuses on the occupancy of N-linked glycosylation sites on three enzymes, ribonuclease, plasminogen and tissue plasminogen activator. Each of these proteins occurs naturally as two populations of molecules, distinguished from each other only by the presence or absence of an oligosaccharide at one glycosylation site. The presence of an oligomannose sugar on ribonuclease (at Asn-34) alters its overall dynamics, increases its stability towards proteinases and decreases its functional activity towards double-stranded RNA. The N-linked sugar on plasminogen (at Asn-288) within kringle 3 reduces the rate of the beta- to alpha-conformational change, modulates the transport of plasminogen into the extravascular compartment, decreases plasminogen binding to U937 cells and downregulates the activation of plasminogen by both urokinase and tissue plasminogen activator. Additionally, in fibrinolysis, within a ternary complex of fibrin, plasminogen and tissue plasminogen activator, the N-linked sugar of plasminogen hinders the initial interaction with tissue plasminogen activator (i.e., it alters Km). The presence of an N-linked glycan (at Asn-184) in the kringle 2 domain of tissue plasminogen activator hinders the rearrangement of this ternary complex, decreasing the turnover rate (Kcat).

Calcium binding properties of an epidermal growth factor-like domain pair from human fibrillin-1.

Ca2+ binding epidermal growth factor-like (EGF-like) domains are found in a large number of extracellular proteins with diverse functions, including those involved in blood coagulation, determination of cell fate, cell adhesion and connective tissue architecture. Their importance is emphasised by the identification of mutations in these domains in patients with haemophilia B (defective in coagulation factor IX) and the Marfan syndrome (defective in the connective tissue protein fibrillin-1). The X-ray crystal structure of a single Ca2+ binding EGF-like domain from human coagulation factor IX has recently been solved. It shows that the Ca2+ ligands form a pentagonal bipyramid, where one ligand is provided by an adjacent (N-terminal) EGF-like domain in the crystal. The N and C termini of the neighbouring domains are only approximately 4 angstrum apart, hence the crystal packing has been proposed as a model for the association of contiguous EGF-like domains in proteins. Since the adjacent EGF-like domain in the crystal, although close, is not covalently linked to its neighbour, this model requires verification. In this study we have expressed and purified a Ca2+ binding EGF-like domain pair from human fibrillin-1 and used an in vitro refolding system to obtain protein with the correct EGF fold. The Ca2+ binding properties of the protein have been investigated by two-dimensional NMR. The affinity of the C-terminal domain for Ca2+ is approximately 25-fold higher than that of the N-terminal domain, consistent with the two Ca2+ binding sites having different local environments. In addition, these data provide the first direct experimental evidence that Ca2+ plays a major role in defining the interdomain linkage in multiple repeats of Ca2+ binding EGF-like domains.