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Large RNAs are central to cellular functions, but characterizing such RNAs remains challenging by solution NMR. We present two labeling technologies based on [2- 19 F, 2- 13 C]-adenosine, which allow the incorporation of aromatic 19 F- 13 C spin pairs. The labels when coupled with the transverse relaxation optimized spectroscopy (TROSY) enable us to probe RNAs comprising up to 124 nucleotides. With our new [2- 19 F, 2- 13 C]-adenosine-phosphoramidite, all resonances of the human hepatitis B virus epsilon RNA could be readily assigned. With [2- 19 F, 2- 13 C]-adenosine triphosphate, the 124 nt pre-miR-17-NPSL1-RNA was produced via in vitro transcription and the TROSY spectrum of this 40 kDa [2- 19 F, 2- 13 C]-A-labeled RNA featured sharper resonances than the [2- 1 H, 2- 13 C]-A sample. The mutual cancelation of the chemical-shift-anisotropy and the dipole-dipole-components of TROSY-resonances leads to narrow linewidths over a wide range of molecular weights. With the synthesis of a non-hydrolysable [2- 19 F, 2- 13 C]-adenosine-triphosphate, we facilitate the probing of co-factor binding in kinase complexes and NMR-based inhibitor binding studies in such systems. Our labels allow a straightforward assignment for larger RNAs via a divide-and-conquer/mutational approach. The new [2- 19 F, 2- 13 C]-adenosine precursors are a valuable addition to the RNA NMR toolbox and will allow the study of large RNAs/RNA protein complexes in vitro and in cells., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Heat shock protein 70 (Hsp70) isoforms are key players in the regulation of protein homeostasis and cell death pathways and are therefore attractive targets in cancer research. Developing nucleotide-competitive inhibitors or allosteric modulators, however, has turned out to be very challenging for this protein family, and no Hsp70-directed therapeutics have so far become available. As the field could profit from alternative starting points for inhibitor development, we present the results of a fragment-based screening approach on a two-domain Hsp70 construct using in-solution NMR methods, together with X-ray-crystallographic investigations and mixed-solvent molecular dynamics simulations. The screening protocol resulted in hits on both domains. In particular, fragment binding in a deeply buried pocket at the substrate-binding domain could be detected. The corresponding site is known to be important for communication between the nucleotide-binding and substrate-binding domains of Hsp70 proteins. The main fragment identified at this position also offers an interesting starting point for the development of a dual Hsp70/Hsp90 inhibitor.

Due to the unprecedented situation caused by a global pandemic, the traditional way of teaching that is reliant on face-to-face interaction between teachers and students has been dismantled. This article looks into university teachers' experiences of teaching under lockdown, with an intention to understand what the change meant in terms of social practice. The research follows a qualitative design, in which ten university teachers were interviewed using a semi-structured interview guide. Three themes interwoven with a common thread were identified through teachers' reflections, including displacement, routine, and role. The common thread was identified as the interaction between teachers and students, and analysing the quality of this interaction led to understanding the social kernel of teaching as embedded in social practice, suggesting that physical dislocation demands teachers to recreate meaning in the new situation. This change has been seen as difficult, yet unpacking teachers' perceptions provided valuable lessons for the future., Competing Interests: Conflict of interestThe authors declare no competing interests., (© The Author(s) 2022.)

The increasing demand for rare earth elements (REEs) for modern industry has led to a surge in mining activities and consequently has released these metals into the environment. Intensifying REEs in a habitat has impacts on its ecosystem, but on the other side, it also provides the opportunity to recover REEs from low-grade minerals. Phytomining has emerged as an ecologically sound technique to extract these valuable elements from contaminated soils where traditional mining is not competitive. This paper presents and reviews the concept of REE phytomining from three scientific areas. The accumulation of rare earth metals in plants is the first stage, referred to as the phytoextraction process. This is followed by elevating REE concentrations into bio-ores via the enrichment phase. Ultimately, extraction is the final step to complete the phytomining pathway for reclaiming REEs in brownfield land., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Incisional hernia (IH) is a postoperative defect of the abdominal wall through which the contents of the peritoneal cavity are externalized beneath the skin in a peritoneal sac. IH differs in anatomic complexity, but also in its associated comorbidities and surgical history. As IH enlarges, complications occur and these become part of its natural history. The goal of the study is to review the impact of loss of domain upon abdominal wall before and after abdominal wall reconstruction. The absence of anatomical and functional linea alba leads to a combination of muscular disturbances, chronic respiratory and gastrointestinal conditions, and psychosocial issues. The pathophysiological changes are also due to the decrease of the intra-abdominal pressure (IAP). During repair, the sudden reintroduction of the viscera into an unprepared cavity leads to a sudden increase in cavity volume and an increase in IAP with fatal pathophysiological implications. For an optimal repair, preoperatively, the abdominal wall must be augmented by achieving the following objectives: reducing the volume of the sac contents, optimizing compliance, enlargement of the container. At the same time, for the optimal repair, the following must be taken into account: increased knowledge about this condition to manage systemic and local changes, CT scan evaluation, monitoring IAP, plateau pressure (PP), and Positive End Expiratory Pressure (PEEP). In conclusion, the goals can be achieved by systemic optimization with a multidisciplinary team, using Preoperative Progressive Pneumoperitoneum (PPP) and/or Botox (BTX), and abdominal wall reconstruction through a mesh with augmented component separation technique., (Celsius.)

Phytomining of noble metals (NMs) offers a promising possibility of metal extraction at sites where traditional mining activities or recovering NMs from low-grade minerals are not competitive. In addition to conventional mining, producing NMs from secondary resources strengthening the circular economy has been paid worldwide attention. The review presented in this paper links three scientific areas as the essential elements to form the phytomining chain of NMs. The accumulation of NMs in plants is the first step, referred as the phytoextraction process. This is followed by heightening the concentration of NMs via the enrichment stage. Eventually, although less well understood, extraction methods of NMs from biomass solid remains as well as from diverse secondary sources particularly incineration ashes are discussed that assist to visualize the potential pathways in phytomining., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The 10-23 DNAzyme is one of the most prominent catalytically active DNA sequences 1,2 . Its ability to cleave a wide range of RNA targets with high selectivity entails a substantial therapeutic and biotechnological potential 2 . However, the high expectations have not yet been met, a fact that coincides with the lack of high-resolution and time-resolved information about its mode of action 3 . Here we provide high-resolution NMR characterization of all apparent states of the prototypic 10-23 DNAzyme and present a comprehensive survey of the kinetics and dynamics of its catalytic function. The determined structure and identified metal-ion-binding sites of the precatalytic DNAzyme-RNA complex reveal that the basis of the DNA-mediated catalysis is an interplay among three factors: an unexpected, yet exciting molecular architecture; distinct conformational plasticity; and dynamic modulation by metal ions. We further identify previously hidden rate-limiting transient intermediate states in the DNA-mediated catalytic process via real-time NMR measurements. Using a rationally selected single-atom replacement, we could considerably enhance the performance of the DNAzyme, demonstrating that the acquired knowledge of the molecular structure, its plasticity and the occurrence of long-lived intermediate states constitutes a valuable starting point for the rational design of next-generation DNAzymes., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: In the case of patients admitted with acute abdomen at the emergency department, interstitial pulmonary pathology (Covid-19 infections) represents a significant operative risk for the patients. The rate of postoperative complications is high with increased morbidity and mortality, a real challenge for the medical staff and surgical/intensive care unit teams. In emergency settings, patients were examined with targeted clinical and paraclinical parameters that assure a fast diagnosis to optimize a rapid medical and surgical treatment. Methods: We conducted a retrospective comparative study that included patients enrolled and diagnosed with an acute surgical abdomen in Surgical Clinic 1 Tg. Mures Emergency County Hospital. Patients were examined and analyzed at the emergency department UPU-SMURD. We included patients admitted over the two years (2019 and 2020) and divided them into two groups. Results: The total number of patients admitted in the UPU-Smurd emergency department Surgical Clinic I over the two years was 1033. There was a significant reduction in total cases diagnosed with the acute surgical abdomen in the pandemic period (p=0.033). The average time from the admission to the surgical procedure was significantly higher in the pandemic period 380Ã+-2 min in comparison with 222+-3 min (p=0.001) and also with an increased average operative time 223+-3 min versus 145+-2 min (p=0.002). Average hospitalization time was higher in the pandemic period 10+-1 (p=0.031) with no significant difference between the groups regarding Intensive Care Unit (ICU) admission (p=0.122). Overall mortality has more than doubled, with 31 cases (19%) in the pandemic and 28 (9%) in the non-pandemic. (p=0.001). Conclusions: The COVID-19 pandemic has played an essential role in treating acute surgical abdomen cases. The high solicitation rate of the emergency department delayed the diagnosis and treatment of severe surgical cases. As the scale of this pandemic is unprecedented, standard protocols with minor changes do not provide adequate results., (Celsius.)

In this paper we examine the impacts of the global pandemic in 2020 on different levels of education system, particularly looking at the changes in teaching practice. The health emergency caused closure of schools, and online distance education became a temporary solution, creating discomfort for many teachers for whom this was the first time engaged with online education. In our research we investigated two important dimensions, namely, how technology was used and what the newfound distance meant in terms of the teacher-student relationship. The article offers insights into experiences of teaching from lockdown reported by 41 teachers at primary, vocational and higher education level in the region of Vaud, Switzerland. This comparative qualitative research has provided an opportunity for an in-depth analysis of the main similarities and differences at three distinctly different educational levels and a possibility to learn more about common coping practices in teaching. The study gives a contribution to a lack of comparative studies of teacher experiences at different educational levels. Results show two dimensions in handling the lockdown crisis: mastering the digital tools and the importance of student-teacher interaction. Whilst the interviewed teachers largely overcame the challenges of mastering digital tools, optimizing the quality interaction and ensuring the transactional presence online remained a problem. This indicates the importance of the social aspect in education at all levels, and implies that teacher support needs to expand beyond technical pedagogical knowledge of online distance education., (© The Author(s) 2021.)

Fluorine NMR has recently gained high popularity in drug discovery as it allows efficient and sensitive screening of large numbers of ligands. However, the positive hits found in screening must subsequently be ranked according to their affinity in order to prioritize them for follow-up chemistry. Unfortunately, the primary read-out from the screening experiments, namely the increased relaxation rate upon binding, is not proportional to the affinity of the ligand, as it is polluted by effects such as exchange broadening. Here we present the method CSAR (Chemical Shift-anisotropy-based Affinity Ranking) for reliable ranking of fluorinated ligands by NMR, without the need of isotope labeled protein, titrations or setting up a reporter format. Our strategy is to produce relaxation data that is directly proportional to the binding affinity. This is achieved by removing all other contributions to relaxation as follows: (i) exchange effects are efficiently suppressed by using high power spin lock pulses, (ii) dipolar relaxation effects are approximately subtracted by measuring at two different magnetic fields and (iii) differences in chemical shift anisotropy are normalized using calculated values. A similar ranking can be obtained with the simplified approach FastCSAR that relies on a measurement of a single relaxation experiment at high field (preferably > 600 MHz). An affinity ranking obtained in this simple way will enable prioritizing ligands and thus improve the efficiency of fragment-based drug design.

Atomic-level information about the structure and dynamics of biomolecules is critical for an understanding of their function. Nuclear magnetic resonance (NMR) spectroscopy provides unique insights into the dynamic nature of biomolecules and their interactions, capturing transient conformers and their features. However, relaxation-induced line broadening and signal overlap make it challenging to apply NMR spectroscopy to large biological systems. Here we took advantage of the high sensitivity and broad chemical shift range of 19 F nuclei and leveraged the remarkable relaxation properties of the aromatic 19 F- 13 C spin pair to disperse 19 F resonances in a two-dimensional transverse relaxation-optimized spectroscopy spectrum. We demonstrate the application of 19 F- 13 C transverse relaxation-optimized spectroscopy to investigate proteins and nucleic acids. This experiment expands the scope of 19 F NMR in the study of the structure, dynamics, and function of large and complex biological systems and provides a powerful background-free NMR probe.

Reducing or preventing damage caused by environmental pollution is a significant goal nowadays. Phytoextraction, as remediation technique is widely used, but during the process, the heavy metal content of the biomass grown on these sites special treatment and disposal techniques are required, for example liquid extraction, direct disposal, composting, and combustion. These processes are discussed in this review in economical and environmental aspects. The following main properties are analyzed: form and harmful element content of remains, utilization of the main and byproducts, affect to the environment during the treatment and disposal. The thermal treatment (combustion, gasification) of contaminated biomass provides a promising alternative disposal option, because the energy production affects the rate of return, and the harmful elements are riched in a small amount of solid remains depending on the ash content of the plant (1-2%). The biomass combustion technology is a wildely used energy production process in residential and industrial scale, but the ordinary biomass firing systems are not suited to burn this type of fuel without environmental risk., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Recording NMR signals of several nuclear species simultaneously by using parallel receivers provides more information from a single measurement and at the same time increases the measurement sensitivity per unit time. Here we present a comprehensive series of the most frequently used NMR experiments modified for simultaneous direct detection of two of the most sensitive NMR nuclei - (1) H and (19) F. We hope that the presented material will stimulate interest in and further development of this technique., (Copyright © 2016 John Wiley & Sons, Ltd.)

In RNA secondary structure determination, it is essential to determine whether a nucleotide is base-paired and not. Base-pairing of nucleotides is mediated by hydrogen bonds. The NMR characterization of hydrogen bonds relies on experiments correlating the NMR resonances of exchangeable protons and can be best performed for structured parts of the RNA, where labile hydrogen atoms are protected from solvent exchange. Functionally important regions in RNA, however, frequently reveal increased dynamic disorder which often leads to NMR signals of exchangeable protons that are broadened beyond (1)H detection. Here, we develop (13)C direct detected experiments to observe all nucleotides in RNA irrespective of whether they are involved in hydrogen bonds or not. Exploiting the self-decoupling of scalar couplings due to the exchange process, the hydrogen bonding behavior of the hydrogen bond donor of each individual nucleotide can be determined. Furthermore, the adaption of HNN-COSY experiments for (13)C direct detection allows correlations of donor-acceptor pairs and the localization of hydrogen-bond acceptor nucleotides. The proposed (13)C direct detected experiments therefore provide information about molecular sites not amenable by conventional proton-detected methods. Such information makes the RNA secondary structure determination by NMR more accurate and helps to validate secondary structure predictions based on bioinformatics.

We propose several significant improvements to the PANSY (Parallel NMR SpectroscopY) experiments-PANSY COSY and PANSY-TOCSY. The improved versions of these experiments provide sufficient spectral information for structure elucidation of small organic molecules from just two 2D experiments. The PANSY-TOCSY-Q experiment has been modified to allow for simultaneous acquisition of three different types of NMR spectra-1D C-13 of non-protonated carbon sites, 2D TOCSY and multiplicity edited 2D HETCOR. In addition the J-filtered 2D PANSY-gCOSY experiment records a 2D HH gCOSY spectrum in parallel with a (1) J-filtered HC long-range HETCOR spectrum as well as offers a simplified data processing. In addition to parallel acquisition, further time savings are feasible because of significantly smaller F1 spectral windows as compared to the indirect detection experiments. Use of cryoprobes and multiple receivers can significantly alleviate the sensitivity issues that are usually associated with the so called direct detection experiments. In cases where experiments are sampling limited rather than sensitivity limited further reduction of experiment time is achieved by using Hadamard encoding. In favorable cases the total recording time for the two PANSY experiments can be reduced to just 40 s. The proposed PANSY experiments provide sufficient information to allow the CMCse software package (Bruker) to solve structures of small organic molecules., (Copyright © 2015 John Wiley & Sons, Ltd.)

In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of (13)C-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly (13)C-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-(13)C)-sucrose, 342 Da] and one compound of medium molecular weight ((13)C-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, ~10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The (13)C resonances are traced using (13)C-(13)C correlations from homonuclear experiments, such as (H)CC-CT-COSY, (H)CC-NOESY, CC-CT-TOCSY and/or virtually decoupled (H)CC-TOCSY. Based on the assignment of the (13)C resonances, the (1)H chemical shifts are derived in a straightforward manner using one-bond (1)H-(13)C correlations from heteronuclear experiments (HC-CT-HSQC). In order to avoid the (1) J CC splitting of the (13)C resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either (13)C or (1)H detected experiments, namely CC-CT-COSY, band-selective (H)CC-TOCSY, HC-CT-HSQC-NOESY or long-range HC-CT-HSQC. However, due to the short T2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the (1)H-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the (13)C-enriched polysaccharide were assigned by using HC-H2BC spectra. The assignment of the N-acetyl groups with (15)N at natural abundance was completed by using HN-SOFAST-HMQC, HNCA, HNCO and (13)C-detected (H)CACO spectra.

Three improved ¹³C-spinlock experiments for side chain assignments of isotope labelled proteins in liquid state are presented. These are based on wide bandwidth spinlock techniques that have become possible with contemporary cryogenic probes. The first application, the H(C(ali)C(aro))H-TOCSY, is an HCCH-TOCSY in which all CHn moieties of a protein are detected in a single experiment, including the aromatic ones. This enables unambiguous assignment of aromatic and aliphatic amino acids in a single, highly sensitive experiment. In the second application, the ¹³C-detected C(all)-TOCSY, magnetization transfer comprises all carbons--aliphatic, aromatic as well as the carbonyl carbons--making the complete carbon assignment possible using one spectrum only. Thirdly, the frequently used HC(CCO)NH experiment was redesigned by replacing the long C-carbonyl refocused INEPT transfer step by direct ¹³C-¹³C-TOCSY magnetization transfer from side chain carbons to the backbone carbonyls. The resulting HC(CCO)NH experiment minimizes relaxation losses because it is shorter and represents a more sensitive alternative particularly for larger proteins. The performance of the experiments is demonstrated on isotope labeled proteins up to the size of 43 kDa.

Biomass, as a renewable energy source, is an excellent alternative for the partial replacement of fossil fuels in thermal and electric energy production. A new fuel type as biomass for energy utilisation includes ligneous plants with considerable heavy metal content. The combustion process must be controlled during the firing of significant quantities of contaminated biomass grown on brownfield lands. By implementing these measures, air pollution and further soil contamination caused by the disposal of the solid burning residue, the ash, can be prevented. For the test samples from ligneous plants grown on heavy metal-contaminated fields, an ore mine (already closed for 25 years) was chosen. With our focus on the determination of the heavy metal content, we have examined the composition of the soil, the biomass and the combustion by-products (ash, fly ash). Our results confirm that ash resulting from the combustion must be treated as toxic waste and its deposition must take place on hazardous waste disposal sites. Biomass of these characteristics can be burnt in special combustion facility that was equipped with means for the disposal of solid burning residues as well as air pollutants.

Using the case of the catalytic domain of MMP-12 in complex with the known inhibitor CGS27023A, a recently assembled 3D (15)N-edited/(14)N,(12)C-filtered ROESY experiment is used to monitor and distinguish protein amide protons in fast exchange with bulk water from amide protons close to water molecules with longer residence times, the latter possibly reflecting water molecules of structural or functional importance. The (15)N-edited/(14)N,(12)C-filtered ROESY spectra were compared to the original (15)N-edited/(14)N,(12)C-filtered NOESY and the conventional amide-water exchange experiment, CLEANEX. Three protein backbone amide protons experiencing direct dipolar cross relaxation with water in the (15)N-edited/(14)N,(12)C-filtered ROESY spectrum were assigned. In an ensemble of six crystal structures, two conserved water molecules within 3 Å of the three amide protons were identified. These two water molecules are buried into cavities in the protein surface and thus sufficiently slowed down by the protein topology to account for the observed dipolar interaction. Structural analysis of an ensemble of six crystal structures ruled out any exchange-relayed contributions for the amide-water interactions of interest.

Objectives: Currently there are no reliable biomarkers in the synovial fluid available to differentiate between septic and non-septic arthritis or to predict the prognosis of osteoarthritis, respectively. Nuclear magnetic resonance (NMR) spectroscopy is an analytical technique that allows a rapid, high throughput metabolic profiling of biological fluids or tissues., Methods: Proton (1H)-nuclear magnetic resonance (NMR) spectroscopy was performed in synovial fluid samples from patients with septic arthritis, crystal arthropathy, different forms of inflammatory arthritis or osteoarthritis (OA). The metabolic environment based on the low molecular weight components was compared in disease subsets and principal component analysis (PCA) was performed., Results: Fifty-nine samples from patients with OA, gout, calcium pyrophosphate disease, spondylarthritis, septic arthritis and rheumatoid arthritis (RA) were analysed. NMR yielded stable and reproducible metabolites over time. Thirty-five different metabolites as well as paracetamol and ibuprofen were identified in synovial fluid. The metabolic profile of septic arthritis assessed by PCA was distinguishable from the other samples whereas no differences were seen in OA compared to crystal-associated arthritis, RA or spondylarthritis., Conclusions: 1H-NMR is a fast analytic tool with possible implications in synovial fluid diagnostics. A distinctive metabolism is observed in septic arthritis whereas metabolites in OA are similar to those in inflammatory arthritis.

We present here a set of (13)C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose (13)C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4' nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1',H1' ribose signals. The experiments were applied to two RNA hairpin structures. The current set of (13)C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, (13)C-direct detected NMR methods constitute useful complements to the conventional (1)H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs.

The N7 of purine nucleotides presents one of the most dominant metal ion binding sites in nucleic acids. However, the interactions between kinetically labile metal ions like Mg(2+) and these nitrogen atoms are inherently difficult to observe in large RNAs. Rather than using the insensitive direct (15)N detection, here we have used (2)J-[(1)H,(15)N]-HSQC (Heteronuclear Single Quantum Coherence) NMR experiments as a fast and efficient method to specifically observe and characterize such interactions within larger RNA constructs. Using the 27 nucleotides long branch domain of the yeast-mitochondrial group II intron ribozyme Sc.ai5gamma as an example, we show that direct N7 coordination of a Mg(2+) ion takes place in a tetraloop nucleotide. A second Mg(2+) ion, located in the major groove at the catalytic branch site, coordinates mainly in an outer-sphere fashion to the highly conserved flanking GU wobble pairs but not to N7 of the sandwiched branch adenosine.

A human matrix metalloproteinase (MMP) hydroxamic acid inhibitor (CGS27023A) was cross-docked into 15 MMP-12, MMP-13, MMP-9, and MMP-1 cocrystal structures. The aim was to validate a fast protocol for ligand binding conformation elucidation and to probe the feasibility of using inhibitor-protein NMR contacts to dock an inhibitor into related MMP crystal structures. Such an approach avoids full NMR structure elucidation, saving both spectrometer- and analysis time. We report here that for the studied MMPs, one can obtain docking results well within 1 A compared to the corresponding reference X-ray structure, using backbone amide contacts only. From the perspective of the pharmaceutical industry, these results are relevant for the binding studies of inhibitor series to a common target and have the potential advantage of obtaining information on protein-inhibitor complexes that are difficult to crystallize.

Background: The epsilon4 allele of the gene encoding apolipoprotein E (apoE) is associated with elevated serum levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), as well as an increased risk of coronary heart disease (CHD), greater disease severity, and higher CHD mortality. ApoE epsilon4 allele carriers have also shown reduced lipid and lipoprotein responses to lipid-modifying pharmacotherapy and lifestyle modifications., Objective: To provide preliminary descriptive data on the effects of apoE genotype on lipid and lipoprotein responses to the cholesterol absorption inhibitor ezetimibe (Ezetrol/Zetia) in Hungarian subjects not at cholesterol goals at baseline., Methods: This prospective open-label study compared the effects of the cholesterol absorption inhibitor ezetimibe 10 mg/day for up to 6 weeks added to existing therapies on the lipid profiles of 14 epsilon4 allele carriers and 14 age- and gender-matched APOE3/APOE3 homozygotes., Results: Treatment with ezetimibe reduced TC, LDL-, and triglycerides, and increased high-density lipoprotein cholesterol (HDL-) significantly from baseline, and to similar extents, in both groups, lowering TC from baseline by 13.7% in APOE3/APOE3 homozygotes compared with 12.1% in epsilon4 allele carriers (p = 0.139); LDL-C by 22.8% (vs. 19.6%; p = 0.081); and triglycerides by 9.2% (vs. 9.1%; p = 0.120). Ezetimibe also increased HDL-C by 8.0% in subjects with the epsilon3/epsilon3 genotype compared with 8.9% in epsilon4 allele carriers (p = 0.263)., Conclusions: Ezetimibe significantly improved lipid and lipoprotein profiles from baseline irrespective of apoE epsilon3/epsilon3 or epsilon4 genotype in Hungarian subjects not at cholesterol goals. Limitations of our study include its open-label nature and small sample population, as well as the facts that patients with the epsilon4 allele were not included and data were not collected on initial cholesterol levels, initial statin doses, cholesterol responses to statins, and the safety and tolerability of ezetimibe. Further randomized controlled studies in larger numbers of people followed for longer intervals are warranted to confirm these findings.

Flaxseed's oil and lignan, secoisolariciresinol diglucoside (SDG), are implicated in attainment of health and treatment of renal injury and osteoporosis. To test for these benefits, weanling Han:SPRD-cy rats (n=171) with or without kidney disease were randomized to diets made with either corn oil or flaxseed oil and with or without SDG for 12 weeks. In females, weight was lower with the SDG diet. In males fed flaxseed oil, lean mass was higher and fat % was lower. In both sexes, fat % was lower in diseased rats. Bone mineral content (BMC) and density were higher in rats fed flaxseed oil and lower in diseased rats, additionally; BMC was lower in SDG-supplemented females. The benefit of flaxseed oil on body composition is sex specific but the effect on bone mass is not. Lastly, reduced weight due to early rat kidney disease is not due to loss of lean body mass.

Recent advances in instrumentation and isotope labeling methodology allow proteins up to 100 kDa in size to be studied in detail using NMR spectroscopy. Using 2H/13C/15N enrichment and selective methyl protonation, we show that newly developed 13C direct detection methods can be used to rapidly yield proton and carbon resonance assignments for the methyl groups of Val, Leu, and Ile residues. We present a highly sensitive 13C-detected CH3-TOCSY experiment that, in combination with standard 1H-detected backbone experiments, allows the full assignment of side chain resonances in methyl-protonated residues. Selective methyl protonation, originally developed by Kay and co-workers (Rosen, M. K.; Gardner, K. H.; Willis, R. C.; Parris, W. E.; Pawson, T.; Kay, L. E. J. Mol. Biol. 1996, 263, 627-636; Gardner, K. G.; Kay, L. E. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 357-406; Goto, N. K.; Kay, L. E. Curr. Opin. Struct. Biol. 2000, 10, 585-592), improves the nuclear relaxation behavior of larger proteins compared to their fully protonated counterparts, allows significant simplification of spectra, and facilitates NOE assignments. Here, we demonstrate the usefulness of the 13C-detected CH3-TOCSY experiment through studies of (i) a medium-sized protein (CbpA-R1; 14 kDa) with a repetitive primary sequence that yields highly degenerate NMR spectra, and (ii) a larger, bimolecular protein complex (p21-KID/Cdk2; 45 kDa) at low concentration in a high ionic strength solution. Through the analysis of NOEs involving amide and Ile, Leu, and Val methyl protons, we determined the global fold of CbpA-R1, a bacterial protein that mediates the pathogenic effects of Streptococcus pneumoniae, demonstrating that this approach can significantly reduce the time required to determine protein structures by NMR.

Long-chain polyunsaturated fatty acids (LC PUFA) are associated with bone mass in animals and human adults, yet no data exist for human infants. Thus, the objective of this study was to establish that LC PUFA status is associated with bone mass in healthy infants. Thirty mother-infant pairs were studied for LC PUFA status by measuring maternal and cord blood red blood cells (RBC) for arachidonic acid (AA), eicosapentaenoic acid (EPA), and DHA. Infant anthropometry and lumbar spine 1-4, femur and whole-body bone mineral content (BMC) were measured within 15 d of delivery. Maternal and infant LC PUFA were tested for their relationship to BMC using Pearson correlation and backward step-wise regression analyses. At birth, the average gestational age was 39.3+/-1.1 wk and body weight was 3433+/-430 g. Cord RBC AA was positively correlated with whole-body BMC, AA:EPA positively correlated with lumbar spine 1-4 BMC and femur BMC. Maternal RBC AA was positively correlated with whole-body BMC. After accounting for infant weight using regression, whole-body BMC was positively predicted by cord RBC AA but none of the maternal LC PUFA; lumbar spine 1-4 BMC was positively predicted by cord RBCAA:EPA ratio but negatively by maternal DHA; and femur BMC was not predicted by cord LC PUFA but was negatively predicted by maternal DHA. Imbalances among the n-6 and n-3 LC PUFA by term gestation are associated with lower bone mass, suggesting that the maternal diet should be balanced in n-6 and n-3 LC PUFA.

Echistatin is a potent antagonist of the integrins alpha(v)beta3, alpha5beta1 and alpha(IIb)beta3. Its full inhibitory activity depends on an RGD (Arg-Gly-Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The full-length C-terminal polypeptide is visible as a beta-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30 degrees . Internal angular motions in the 100-300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length echistatin-alpha(v)beta3 docking model suggests that echistatin's C-terminal amino acids may contact alpha(v)-subunit residues and provides new insights to delineate structure-function correlations.

Background: Vitamin D is required for normal bone growth and mineralization. We sought to determine whether vitamin D deficiency at birth is associated with bone mineral content (BMC) of Canadian infants., Methods: We measured plasma 25-hydroxyvitamin D [25(OH)D] as an indicator of vitamin D status in 50 healthy mothers and their newborn term infants. In the infants, anthropometry and lumbar, femur and whole-body BMC were measured within 15 days of delivery. Mothers completed a 24-hour recall and 3-day food and supplement record. We categorized the vitamin D status of mothers and infants as deficient or adequate and then compared infant bone mass in these groups using nonpaired t tests. Maternal and infant variables known to be related to bone mass were tested for their relation to BMC using backward stepwise regression analysis., Results: Twenty-three (46%) of the mothers and 18 (36%) of the infants had a plasma 25(OH)D concentration consistent with deficiency. Infants who were vitamin D deficient were larger at birth and follow-up. Absolute lumbar spine, femur and whole-body BMC were not different between infants with adequate vitamin D and those who were deficient, despite larger body size in the latter group. In the regression analysis, higher whole-body BMC was associated with greater gestational age and weight at birth as well as higher infant plasma 25(OH)D., Conclusion: A high rate of vitamin D deficiency was observed among women and their newborn infants. Among infants, vitamin D deficiency was associated with greater weight and length but lower bone mass relative to body weight. Whether a return to normal vitamin D status, achieved through supplements or fortified infant formula, can reset the trajectory for acquisition of BMC requires investigation.

Significant resolution improvement in 13C,13C-TOCSY spectra of uniformly deuterated and 13C, 15N-labeled protein and 13C,15N-labeled RNA samples is achieved by introduction of multiple-band-selective 13C-homodecoupling applied simultaneously with 1H- or 2H- and 15N-decoupling at all stages of multidimensional experiments including signal acquisition period. The application of single, double or triple band-selective 13C-decoupling in 2D-[13C,13C]-TOCSY experiments during acquisition strongly simplifies the homonuclear splitting pattern. The technical aspects of complex multiple-band homonuclear decoupling and hardware requirements are discussed. The use of this technique (i) facilitates the resonance assignment process as it reduces signal overlap in homonuclear 13C-spectra and (ii) possibly improves the signal-to-noise ratio through multiplet collapse. It can be applied in any 13C-detected experiment.

(13)C-detected experiments are still limited by their inherently lower sensitivity, as compared to the equivalent (1)H-detected experiments. Improving the sensitivity of (13)C detection methods remains a significant area of NMR research that may provide better means for studying large macromolecular systems by NMR. In this communication, we show that (13)C-detected experiments are less sensitive to the salt concentration of the sample solution than (1)H-detected experiments. In addition, acquisition can be started with anti-phase coherence, resulting in higher sensitivity due to the elimination of the final INEPT transfer step.

13C-only spectroscopy was used to measure multiple residual (13)C-(13)C dipolar couplings (RDCs) in uniformly deuterated and (13)C-labeled proteins. We demonstrate that (13)C-start and (13)C-observe spectra can be routinely used to measure an extensive set of the side-chain residual (13)C-(13)C dipolar couplings upon partial alignment of human ubiquitin in the presence of bacteriophages Pf1. We establish that, among different broadband polarization transfer schemes, the FLOPSY family can be used to exchange magnetization between a J coupled network of spins while largely decoupling dipolar interactions between these spins. An excellent correlation between measured RDCs and the 3D structure of the protein was observed, indicating a potential use of the (13)C-(13)C RDCs in the structure determination of perdeuterated proteins.

Obtustatin is a potent and selective inhibitor of the alpha1beta1 integrin in vitro and of angiogenesis in vivo. It possesses an integrin recognition loop that harbors, in a lateral position, the inhibitory 21KTS23 motif. We report an analysis of the dynamics of the backbone and side-chain atoms of obtustatin by homonuclear NMR methods. Angular mobility has been calculated for 90 assigned cross-peaks from 22 off-resonance rotating frame nuclear Overhauser effect spectroscopy spectra recorded at three magnetic fields. Our results suggest that the integrin binding loop and the C-terminal tail display concerted motions, which can be interpreted by hinge effects. Among the integrin-binding motif, threonine 22 and serine 23 exhibit the lowest and the highest side-chain flexibility, respectively. It is noteworthy that the side chain of threonine 22 is not solvent-exposed, although based on synthetic peptides it appears to be the most critical residue for the inhibitory activity of obtustatin on the binding of integrin alpha1beta1 to collagen IV. Instead, the side chain of threonine 22 is oriented toward the loop center and hydrogen-bonded to residues Thr25 and Ser26. This network of interactions explains the restrained mobility of threonine 22 and suggests that its functional importance lies in maintaining the active conformation of the alpha1beta1 inhibitory loop.

The assignment of the aliphatic (13)C resonances of trimeric Bacillus Subtilis chorismate mutase, a protein with a molecular mass of 44 kDa, consisting of three 127-residue monomers is presented by use of two-dimensional (2D) (13)C-start and (13)C-observe NMR experiments. These experiments start with (13)C excitation and end with (13)C observation while relying on the long transverse relaxation times of (13)C spins in uniformly deuterated and (13)C,(15)N-labeled large proteins. Gains in sensitivity are achieved by the use of a paramagnetic relaxation enhancement agent to reduce (13)C T(1) relaxation times with little effect on (13)C T(2) relaxation times. Such 2D (13)C-only NMR experiments circumvent problems associated with the application of conventional experiments for side-chain assignment to proteins of larger sizes, for instance, the absence or low concentration of the side-chain (1)H spins, the transfer of the side-chain spin polarization to the (1)H(N) spins for signal acquisition, or the necessity of a quantitative reprotonation of the methyl moieties in the otherwise fully deuterated side-chains. We demonstrate that having obtained a nearly complete assignment of the side-chain aliphatic (13)C resonances, the side-chain (1)H chemical shifts can be assigned in a semiautomatic fashion using 3D (15)N-resolved and (13)C-resolved NOESY experiments measured with a randomly partially protonated protein sample. We also discuss perspectives for structure determination of larger proteins by using novel strategies which are based on the (1)H,(1)H NOEs in combination with multiple residual dipolar couplings between adjacent (13)C spins determined with 2D (13)C-only experiments.

Surfactant protein D (SP-D) is one of four known protein components of the pulmonary surfactant lining the lung alveoli. It is involved in immune and allergic responses. SP-D occurs as a tetramer of trimers. Trimerization is thought to be initiated by a coiled coil domain. We have determined the solution structure of a 64-residue peptide encompassing the coiled coil domain of human SP-D. As predicted, the domain forms a triple-helical parallel coiled coil. As with all symmetric oligomers, the structure calculation was complicated by the symmetry degeneracy in the NMR spectra. We used the symmetry-ADR (ambiguous distance restraint) structure calculation method to solve the structure. The results demonstrate that the leucine zipper region of SP-D is an autonomously folded domain. The structure is very similar to the independently determined X-ray crystal structure, differing mainly at a single residue, Tyr248. This residue is completely symmetric in the solution structure, and markedly asymmetric in the crystalline phase. This difference may be functionally important, as it affects the orientation of the antigenic surface presented by SP-D.

Hyperparathyroidism, secondary to renal disease, is thought to cause high bone turnover via prostaglandin E2 (PGE2). Diets high in n-3 fatty acids reduced PGE2. Thus the objective was to compare the effect of diets high in n-6 and n-3 fatty acids on hyperparathyroidism, bone turnover, and PGE2 in Han:SPRD- cy rats that develop polycystic kidney disease (PKD). Weanling male rats ( n=58) were randomized to diets made with either corn or flaxseed oil (5%) for 8 weeks, followed by measurement of plasma parathyroid hormone (PTH), osteocalcin, urinary N-telopeptide (NTX), and ex vivo release of PGE2from femur. Plasma PTH was elevated ( P<0.01) as a result of PKD. Mean values for plasma osteocalcin and urinary NTX were elevated ( P<0.01) by PKD but not altered by diet. In contrast, values for PGE2 were lowest in the PKD rats fed flaxseed oil compared with PKD rats fed corn oil and compared with non-affected rats fed either oil. Rats with PKD have high-turnover bone disease, likely due to hyperparathyroidism, that is unaffected by feeding corn or flaxseed oils. Since PGE2 release is lower in the presence of high bone turnover, the high bone turnover in evolving rat uremia is not likely to be mediated by PGE2.

The role of leptin in neonatal growth and bone metabolism has been investigated, but not simultaneously. The objectives of this study were to determine if leptin relates to bone mass during rapid growth; if consumption of maternal milk is related to elevated circulating concentrations of leptin resulting in higher fat mass; and if glucocorticoids result in higher fat mass and reduced bone mass due to elevated leptin. Thirty-two piglets were randomized to either a suckling or milk substitute plus either dexamethasone (DEX) or placebo injection for 15 days beginning at 5 days of age. Milk and blood samples were obtained at baseline, and after 15 days, blood was sampled again for measurement of leptin and bone biochemistry. Weight at baseline plus weight and length after 15 days were recorded, followed by measurement of whole body bone mineral content, bone area, and fat mass using dual energy x-ray absorptiometry. At baseline, plasma leptin was elevated in suckled piglets. Piglets that suckled had elevated fat mass as did those who received DEX. However, DEX resulted in suppressed weight and length, bone mass, and bone metabolism. Leptin was similar among groups after the 15 days. After accounting for body size and treatment effects, piglet plasma leptin was predictive of bone and fat mass. Leptin circulating early postnatally is linked to body composition, specifically fat and bone mass. Elevations in fat mass and reductions in bone mass observed after 15 days of DEX treatment are not related to leptin metabolism. Both human and porcine neonates share similar characteristics with respect to relationships of leptin with fat and bone mass.

Molecular dynamics simulations were conducted to estimate the free energy barrier of unfolding surfactant-associated polypeptide C (SP-C) from an alpha-helical conformation. Experimental studies indicate that while the helical fold of SP-C is thermodynamically stable in phospholipid micelles, it is metastable in a mixed organic solvent of CHCl3/CH3OH/0.1 M HCl at 32:64:5 (v/v/v), in which it undergoes an irreversible transformation to an insoluble aggregate that contains beta-sheet. On the basis of experimental observations, the free energy barrier was estimated to be approximately 100 kJ/mole by applying Eyring's transition state theory to the experimental rate of unfolding [Protein Sci 1998;7:2533-2540]. These studies prompted us to carry out simulations to investigate the unwinding process of two helical turns encompassing residues 25-32 in water and in methanol. The results give an upper bound estimation for the free energy barrier of unfolding of SP-C of approximately 20 kJ/mole. The results suggest a need to reconsider the applicability of a single-mode activated process theory to protein unfolding., (Copyright 2001 Wiley-Liss, Inc.)

SpoIIAA participates in a four-component mechanism for phosphorylation-dependent transcription control at the outset of sporulation. We report the refinement of the solution structure of SpoIIAA by using the automated iterative NOE assignment method ARIA. To complement the structural data, the protein dynamics were determined by measuring the T1, T2 and NOE of the backbone 15N-nuclei. The refined structure permits a discussion of the structural features that are important for the function of SpoIIAA in the regulation of the sporulation sigma factor sigmaF, and for homologous regulatory pathways present in B. subtilis and in other bacilli.

Calreticulin (CRT) is located predominantly in the endoplasmic reticulum (ER) of cells, where it functions as a quality control controller of protein folding. However, CRT is also a prevalent autoantigen in patients with systemic lupus erythematosus (SLE), where its release from the cell may arise as a results of dysfunctional apoptosis and inefficient removal of ER vesicles, which are an abundant source of CRT and other autoantigens. Indicative of this is the presence of autoantibodies against CRT in the sera of 40-60% of all SLE patients. Once released into the circulation, CRT might bind directly to C1q and we have suggested that this association may result in a defect in C1q-mediated clearance of antigen-antibody complexes. It has been previously shown that CRT under physiological salt conditions binds to the globular head of C1q. It is known that the globular head region of C1q binds to the CH2 domain in the Fc portion of immunoglobulin gamma (IgG). The N-terminal half of CRT contains a number of short regions of 7-10 amino acids that show sequence similarity to the putative C1q binding region in the CH2 domain of IgG. By use of a series of 92 overlapping CRT synthetic peptides, a number of C1q binding sites on the CRT molecule have been identified, including several containing a CH2-like motif similar to the ExKxKx C1q binding motif found in the CH2 domain of IgG. A number of these peptides were shown to inhibit binding of C1q to IgG and reduce binding of native CRT to C1q. Moreover, several of the peptides were capable of inhibiting the classical pathway of complement activation. These studies have identified specific binding sites on the CRT molecule for C1q and lend support to the hypothesis that interaction of CRT with C1q may interfere with the ability of C1q to associate with immune complexes in autoimmune-related disorders.

We present the results of studies of an aqueous sample of a highly [15N,2H] enriched protein, the SH3 domain from Fyn. Measurements of 1H relaxation and interactions between H2O solvent and exchangeable protons are given, as well as a method for increasing the effective longitudinal relaxation of solvent exchangeable proton resonances. The long-range isotope shifts are measured, for 1H and 15N, which arise due to perdeuteration. Simulations, which employed a 7 or 8 spin relaxation matrix analysis, were compared to the experimental data from a time series of 2D NOESY datasets for some resonances. The agreement between experiment and simulation suggest that, with this 1H dilute sample, relatively long mixing times (up to 1.2 s) can be used to detect specific dipolar interactions between amide protons up to about 7A apart. A set of 155 inter-amide NOEs and 7 side chain NOEs were thus identified in a series of 3D HSQC-NOESY-HSQC experiments. These data, alone and in combination with previously collected restraints, were used to calculate sets of structures using X-PLOR. These results are compared to the available X-ray and NMR structures of the Fyn SH3 domain.

The establishment of differential gene expression in sporulating Bacillus subtilis involves four protein components, one of which, SpoIIAA, undergoes phosphorylation and dephosphorylation. We have used NMR spectroscopy to determine the solution structure of the nonphosphorylated form of SpoIIAA. The structure shows a fold consisting of a four-stranded beta-sheet and four alpha-helices. Knowledge of the structure helps to account for the phenotype of several strains of B. subtilis that carry known spoIIAA mutations and should facilitate investigations of the conformational consequences of phosphorylation.

The impact of an extensive, uniform and hydrophobic protein surface on the behavior of the surrounding solvent is investigated. In particular, focus is placed on the possible enhancement of the structure of water at the interface, one model for the hydrophobic effect. Solvent residence times and radial distribution functions are analyzed around three types of atomic sites (methyl, polar, and positively charged sites) in 1 ns molecular dynamics simulations of the alpha-helical polypeptide SP-C in water, in methanol and in chloroform. For comparison, water residence times at positively and negatively charged sites are obtained from a simulation of a highly charged alpha-helical polypeptide from the protein titin in water. In the simulations the structure of water is not enhanced at the hydrophobic protein surface, but instead is disrupted and devoid of positional correlation beyond the first solvation sphere. Comparing solvents of different polarity, no clear trend toward the most polar solvent being more ordered is found. In addition, comparison of the water residence times at nonpolar, polar, positively charged, or negatively charged sites on the surface of SP-C or titin does not reveal pronounced or definite differences. It is shown, however, that the local environment may considerably affect solvent residence times. The implications of this work for the interpretation of the hydrophobic effect are discussed.

Structural, dynamic and energetic properties of proteins in solution can be studied in atomic detail by molecular dynamics computer simulation. Protein unfolding can be caused by a variety of driving forces induced in different ways: increased temperature or pressure, change of solvent composition, or protein amino acid mutation. The stability and unfolding of four different proteins (bovine pancreatic trypsin inhibitor, hen egg white lysozyme, the surfactant protein C and the DNA-binding domain of the 434 repressor) have been studied by applying the afore-mentioned driving forces and also to some artificial forces. The results give a picture of protein (in)stability and possible unfolding pathways, and are compared to experimental data where possible.

A series of three molecular dynamics simulations at 300 K in explicit solvent environments of chloroform, methanol and water has been performed on the pulmonary surfactant lipoprotein, SP-C, comprising several consecutive valine residues in order to investigate the stability of the alpha-helical conformation. Two additional simulations were performed on truncated SP-C with a five-residue N-terminal deletion at 300 K and 500 K in water, the high temperature run in order to increase the rate of peptide denaturation. Indications of destabilization appear in chloroform during 1 ns while the SP-C alpha-helix is remarkably stable during 1 ns in methanol and water. In particular the polyvalyl part comprising residues Val15 to Val21 remains intact even at elevated temperature, and the valines do not disrupt the alpha-helical conformation. The valyl-rotamer sampling is partly restricted. Unfolding takes place successively along the primary sequence starting from the C-terminal end. Factors affecting polypeptide stability in molecular dynamics simulations are addressed. The intrinsic helix-forming tendency of valine residues and its dependence on the sequence context, and the role of the solvent environment in stabilizing or destabilizing an alpha-helical fold, are discussed.

The sensitivity of the radiofibrinogen test was investigated to determine the accuracy of the test in diagnosis of venographically proven venous thrombosis in 70 heparinized patients. 125J-fibrinogen was administered between 2 h and 4 days (mean: 1.5 days) after initiating heparin therapy. There were two criteria for a positive test: if the difference between the counts for adjacent points on the same leg or equivalent sites on opposite legs was at least 15%; and/or if there were three adjacent points each with counts greater than 5% that of the same three points on the other leg. The fibrinogen uptake test was positive at the first examination in 67 of 70 patients, giving a sensitivity of 96%. The respective frequencies of truly abnormal results for the 3 X 5% and the 1 X 15% criteria were 94% and 71%. As far as localization and extension of thrombosis were concerned, the fibrinogen uptake test agreed with phlebography in 73% of the cases, when the counts in the groin and the upper third of the thigh were ignored. The 125J-fibrinogen uptake test is an accurate method for detecting established deep leg vein thrombosis even in anticoagulated patients.

Thirteen European libraries were visited and are described with special attention given to the Turkish and Hungarian library systems. Information and personal impressions give insight into European ideas of librarianship.