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Biological or biomedical microelectromechanical systems (BioMEMS) are poised to have a significant impact on clinical and biomedical applications. These devices-also termed lab-on-chip or point-of-care (POC) sensors-rep- resent a significant opportunity in various patient-centric settings, including at home, at the doctor's office, in ambulances on the way to the hospital, in emergency rooms (ERs), at the hospital bedside, in rural and global health settings, and in clinical or commercial diagnostic laboratories. The potential impact of these technologies on the early diagnosis and management of disease can be very high for sensing and reporting on parameters ranging from physiological to biomolecular. As health-care delivery and management be- come increasingly personalized and individualized and as genomic, proteomic, and metabolic technologies unravel the human genetic and epigenetic dispositions to disease, detection of multiple markers (at any of the Omics scale) at an individualized level to assess the state of health and disease will become even more important.

On 7 November 2011, the permanent deferral from blood donation of men who have sex with men (MSM) will change in England, Scotland and Wales, to a 12-month deferral since last relevant sexual contact. This follows an evidence-based policy review by the Advisory Committee on the Safety of Blood, Tissues and Organs (SaBTO) which concluded that the available evidence supported the introduction of a 12-month fixed period deferral (Donor Selection Criteria Review, 2011). The review found that the risks associated with a 12-month deferral of MSM were equivalent to a permanent deferral and the existing policy was inappropriate. In many countries, MSM are permanently deferred from donating blood. These policies were introduced as a response to the spread of acquired immunodeficiency syndrome (AIDS) in the early 1980s. Since that time our understanding of the causative agent, human immunodeficiency virus (HIV), and our ability to detect it have improved significantly. It is accepted that policies relating to blood donation should be equitable and based on the latest available scientific evidence. In order to achieve this, regular reviews of blood donor selection policies are required. The UK policy on blood donation by MSM was last reviewed in 2006, but at that time data on compliance with the permanent deferral were not available. Recently, the findings of a study on the level of compliance with the permanent deferral and attitudes towards alternative policies have become available (Grenfell et al., 2011). This, together with new data for the residual risk of an infectious donation being released for transfusion under a number of deferral periods, prompted a further policy review. The findings of this review conducted by a Steering Group of SaBTO, have recently been published (SaBTO, 2011). The Steering Group was comprised of leading experts in the field, joined by patient groups and key stakeholders. Its remit was to ‘Review the evidence base for donor

Objective Despite growing awareness that hospital design can impact meaningful outcomes, the same-handed medical-surgical inpatient unit configuration has not been empirically investigated. This study measured differences in patient and nurse outcomes between the same-handed and mirrored unit configurations. Background It has been hypothesized that the same-handed unit configuration may contribute to operational efficiencies, fewer adverse events, and reduced noise levels. Methods A natural experiment of 8 medical-surgical inpatient units used 2 questionnaires developed for the study. The first questionnaire was available to registered nurses. The second was available to the nurses' patients. Results Compared with participants on the mirrored unit configuration, participants on the same-handed unit configuration reported lower noise levels, better sleep quality, more frequent approaches to patients' right side, and improved satisfaction with organization of the workspace at patients' bedsides. The increased right-side approach was related to fewer instances of patients catching themselves from falling. Conclusion The same-handed unit configuration benefits patient experience, patient safety, and operational outcomes. Before renovation or new construction of units, nurse executives should consider the advantages of a same-handed unit configuration.

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

We have developed a microfabricated biochip that enumerates CD4+ T lymphocytes from leukocyte populations obtained from human blood samples using electrical impedance sensing and immunoaffinity chromatography. T cell counts are found by obtaining the difference between the number of leukocytes before and after depleting CD4+ T cells with immobilized antibodies in a capture chamber. This differential counting technique has been validated to analyze physiological concentrations of leukocytes with an accuracy of ∼9 cells per µL by passivating the capture chamber with bovine serum albumin. In addition, the counter provided T cell counts which correlated closely with an optical control (R(2) = 0.997) for CD4 cell concentrations ranging from approximately 100 to 700 cells per µL. We believe that this approach can be a promising method for bringing quantitative HIV/AIDS diagnostics to resource-poor regions in the world.

Box C/D snoRNP catalysed methylation is aided by additional pre-rRNA base-pairing Box C/D small nucleolar RNPs catalyse 2′-O-methylation of eukaryotic ribosomal RNA. A large-scale analysis of yeast box C/D snoRNAs reveals conserved ‘extra base-pairing' between snoRNAs and regions adjacent to their rRNA methylation site and points to a role for the non-catalytic protein subunits Nop56 and Nop58 in rRNA binding., 2′-O-methylation of eukaryotic ribosomal RNA (r)RNA, essential for ribosome function, is catalysed by box C/D small nucleolar (sno)RNPs. The RNA components of these complexes (snoRNAs) contain one or two guide sequences, which, through base-pairing, select the rRNA modification site. Adjacent to the guide sequences are protein-binding sites (the C/D or C′/D′ motifs). Analysis of >2000 yeast box C/D snoRNAs identified additional conserved sequences in many snoRNAs that are complementary to regions adjacent to the rRNA methylation site. This ‘extra base-pairing' was also found in many human box C/D snoRNAs and can stimulate methylation by up to five-fold. Sequence analysis, combined with RNA–protein crosslinking in Saccharomyces cerevisiae, identified highly divergent box C′/D′ motifs that are bound by snoRNP proteins. In vivo rRNA methylation assays showed these to be active. Our data suggest roles for non-catalytic subunits (Nop56 and Nop58) in rRNA binding and support an asymmetric model for box C/D snoRNP organization. The study provides novel insights into the extent of the snoRNA–rRNA interactions required for efficient methylation and the structural organization of the snoRNPs.

Background: It is widely accepted that atherosclerosis and inflammation are intimately linked. Monocytes play a key role in both of these processes and we hypothesized that activation of inflammatory pathways in monocytes would lead to, among others, proatherogenic changes in the monocyte transcriptome. Such differentially expressed genes in circulating monocytes would be strong candidates for further investigation in disease association studies, Methods: Endotoxin, lipopolysaccharide (LPS), or saline control was infused in healthy volunteers. Monocyte RNA was isolated, processed and hybridized to Hver 2.1.1 spotted cDNA microarrays. Differential expression of key genes was confirmed by RT-PCR and results were compared to in vitro data obtained by our group to identify candidate genes, Results: All subjects who received LPS experienced the anticipated clinical response indicating successful stimulation. One hour after LPS infusion, 11 genes were identified as being differentially expressed; 1 down regulated and 10 up regulated. Four hours after LPS infusion, 28 genes were identified as being differentially expressed; 3 being down regulated and 25 up regulated. No genes were significantly differentially expressed following saline infusion. Comparison with results obtained in in vitro experiments lead to the identification of 6 strong candidate genes (BATF, BID, C3aR1, IL1RN, SEC61B and SLC43A3), Conclusion: In vivo endotoxin exposure of healthy individuals resulted in the identification of several candidate genes through which systemic inflammation links to atherosclerosis., peer-reviewed

We investigate single charged particle dynamics in the Earth's magnetotail using a simple, scale free magnetic field model which is explicitly time dependent, with a corresponding induction electric field. The time-dependent Hamiltonian of particle motion in the simple model describes a system of two coupled oscillators which is driven. When the (time-dependent) ratio of the oscillation frequencies is different from unity, the motion is regular, but if it approaches unity at some point on the trajectory, the motion becomes chaotic. A parameter α is found which characterizes the adiabaticity of the system, and the transition time for behavior in the system is given by at αt → 1. The condition at αt ≈ 1 is equivalent to κ ≈ 1 in the static parabolic field model discussed by previous authors (Buchner and Zelenyi, 1989). The explicit time dependence produces two possible classes of motion, ordered by α. If the reversal is thick and is folding slowly, so that α ≪ 1, the motion is a transition in behavior, from regular μ conserving to chaotic “cucumberlike” trajectories, when t ≈ 1/α. If on the other hand, the reversal is thin and folds quickly, so that α ≫ 1, the particles execute regular “ring type” trajectories once t > 1/α. Simple estimates of presubstorm magnetotail parameters indicate that electrons in a slowly thinning (15-min time scale), thick (1 RE) sheet have α ≪ 1, whereas protons in a thin (several hundreds of kilometers) sheet which thins on a 5-min time scale have α ≫ 1. Hence the behavior of the particles, and by implication, the field reversal which they support, will depend upon the adiabaticity of the system α as well as the “chaotization” parameter αt; this is shown only to be the case in a model which is explicitly time dependent and which includes the induction electric field.

The healthcare design community is adopting evidence-based design (EBD) at a startling rate. However, the role of research within an architectural practice is unclear. Reasons for the lack of clarity include multiple connotations of EBD, the tension between a research-driven market and market-driven research, and the competing expectations and standards of design practitioners and researchers. Research as part of EBD should be integral with the design process so that research directly contributes to building projects. Characteristics of a comprehensive programming methodology to close the gap between design and EBD are suggested.

Archaeal and eukaryotic box C/D RNPs catalyze the 2′-O-methylation of ribosomal RNA, a modification that is essential for the correct folding and function of the ribosome. Each archaeal RNP contains three core proteins—L7Ae, Nop5, and fibrillarin (methyltransferase)—and a box C/D sRNA. Base-pairing between the sRNA guide region and the rRNA directs target site selection with the C/D and related C′/D′ motifs functioning as protein binding sites. Recent structural analysis of in vitro assembled archaeal complexes has produced two divergent models of box C/D sRNP structure. In one model, the complex is proposed to be monomeric, while the other suggests a dimeric sRNP. The position of the RNA in the RNP is significantly different in each model. We have used UV-cross-linking to characterize protein–RNA contacts in the in vitro assembled Pyrococcus furiosus box C/D sRNP. The P. furiosus sRNP components assemble into complexes that are the expected size of di-sRNPs. Analysis of UV-induced protein–RNA cross-links revealed a novel interaction between the ALFR motif, in the Nop domain of Nop5, and the guide/spacer regions of the sRNA. We show that the ALFR motif and the spacer sequence adjacent to box C or C′ are important for box C/D sRNP assembly in vitro. These data therefore reveal new RNA–protein contacts in the box C/D sRNP and suggest a role for Nop5 in substrate binding and/or release.

Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 [leucine-rich repeat (in FLII) interacting protein 1]. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.

The number and volume of cells in the blood affect a wide range of disorders including cancer and cardiovascular, metabolic, infectious and immune conditions. We consider here the genetic variation in eight clinically relevant hematological parameters, including hemoglobin levels, red and white blood cell counts and platelet counts and volume. We describe common variants within 22 genetic loci reproducibly associated with these hematological parameters in 13,943 samples from six European population-based studies, including 6 associated with red blood cell parameters, 15 associated with platelet parameters and 1 associated with total white blood cell count. We further identified a long-range haplotype at 12q24 associated with coronary artery disease and myocardial infarction in 9,479 cases and 10,527 controls. We show that this haplotype demonstrates extensive disease pleiotropy, as it contains known risk loci for type 1 diabetes, hypertension and celiac disease and has been spread by a selective sweep specific to European and geographically nearby populations.

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis. ispartof: BLOOD vol:113 issue:19 pages:4754-4762 ispartof: location:United States status: published

Eukaryotic 18S rRNA processing is mediated by the small subunit (SSU) processome, a machine comprised of the U3 small nucleolar RNP (U3 snoRNP), tUTP, bUTP, MPP10, and BMS1/RCL1 subcomplexes. We report that the human SSU processome is a dynamic structure with the recruitment and release of subcomplexes occurring during the early stages of ribosome biogenesis. A novel 50S U3 snoRNP accumulated when either pre-rRNA transcription was blocked or the tUTP proteins were depleted. This complex did not contain the tUTP, bUTP, MPP10, and BMS1/RCL1 subcomplexes but was associated with the RNA-binding proteins nucleolin and RRP5 and the RNA helicase DBP4. Our data suggest that the 50S U3 snoRNP is an SSU assembly intermediate that is likely recruited to the pre-rRNA through the RNA-binding proteins nucleolin and RRP5. We predict that nucleolin is only transiently associated with the SSU processome and likely leaves the complex not long after 50S U3 snoRNP recruitment. The nucleolin-binding site potentially overlaps that of several other key factors, and we propose that this protein must leave the SSU processome for pre-rRNA processing to occur.

Mean platelet volume (MPV) and platelet count (PLT) are highly heritable and tightly regulated traits. We performed a genome-wide association study for MPV and identified one SNP, rs342293, as having highly significant and reproducible association with MPV (per-G allele effect 0.016 ± 0.001 log fL; P < 1.08 × 10−24) and PLT (per-G effect −4.55 ± 0.80 109/L; P < 7.19 × 10−8) in 8586 healthy subjects. Whole-genome expression analysis in the 1-MB region showed a significant association with platelet transcript levels for PIK3CG (n = 35; P = .047). The G allele at rs342293 was also associated with decreased binding of annexin V to platelets activated with collagen-related peptide (n = 84; P = .003). The region 7q22.3 identifies the first QTL influencing platelet volume, counts, and function in healthy subjects. Notably, the association signal maps to a chromosome region implicated in myeloid malignancies, indicating this site as an important regulatory site for hematopoiesis. The identification of loci regulating MPV by this and other studies will increase our insight in the processes of megakaryopoiesis and proplatelet formation, and it may aid the identification of genes that are somatically mutated in essential thrombocytosis.

Mean platelet volume (MPV) is increased in myocardial and cerebral infarction and is an independent and strong predictor for postevent morbidity and mortality. We conducted a genome-wide association study (GWAS), the KORA (Kooperative Gesundheitsforschung in der Region Augsburg) F3 500K study, and found MPV to be strongly associated with three common single-nucleotide polymorphisms (SNPs): rs7961894 located within intron 3 of WDR66 on chromosome 12q24.31, rs12485738 upstream of the ARHGEF3 on chromosome 3p13-p21, and rs2138852 located upstream of TAOK1 on chromosome 17q11.2. We replicated all three SNPs in another GWAS from the UK and in two population-based samples from Germany. In a combined analysis including 10,048 subjects, the SNPs had p values of 7.24 x 10(-48) for rs7961894, 3.81 x 10(-27) for rs12485738, and 7.19 x 10(-28) for rs2138852. These three quantitative trait loci together accounted for 4%-5% of the variance in MPV. In-depth sequence analysis of WDR66 in 382 samples from the extremes revealed 20 new variants and a haplotype with three coding SNPs and one SNP at the transcription start site associated with MPV (p = 6.8 x 10(-5)). In addition, expression analysis indicated a direct correlation of WDR66 transcripts and MPV. These findings may not only enhance our understanding of platelet activation and function, but may also provide a focus for several novel research avenues.

The accurate estimation of scaling exponents is central in the observational study of scale-invariant phenomena. Natural systems unavoidably provide observations over restricted intervals; consequently a stationary stochastic process (time series) can yield anomalous time variation in the scaling exponents, suggestive of non-stationarity. The variance in the estimates of scaling exponents computed from an interval of N observations is known for finite variance processes to vary as ~1/N as N goes to infinity for certain statistical estimators; however, the convergence to this behaviour will depend on the details of the process, and may be slow. We study the variation in the scaling of second order moments of the time series increments with N, for a variety of synthetic and `real world' time series; and find that in particular for heavy tailed processes, for realizable N, one is far from this 1/N limiting behaviour. We propose a semi-empirical estimate for the minimum N needed to make a meaningful estimate of the scaling exponents for model stochastic processes and compare these with some `real world' time series., 12 pages, 5 figures, accepted Physical Review E

Levy flights and fractional Brownian motion (fBm) have become exemplars of the heavy tailed jumps and long-ranged memory widely seen in physics. Natural time series frequently combine both effects, and linear fractional stable motion (lfsm) is a model process of this type, combining alpha-stable jumps with a memory kernel. In contrast complex physical spatiotemporal diffusion processes where both the above effects compete have for many years been modelled using the fully fractional (FF) kinetic equation for the continuous time random walk (CTRW), with power laws in the pdfs of both jump size and waiting time. We derive the analogous kinetic equation for lfsm and show that it has a diffusion coefficient with a power law in time rather than having a fractional time derivative like the CTRW. We discuss some preliminary results on the scaling of burst "sizes" and "durations" in lfsm time series, with applications to modelling existing observations in space physics and elsewhere., Comment: 9 pages including 11 figures. Shortened from 0803.2833 [math-ph] in which kinetic equation was first obtained. Latest revised version now improves pointers to relevant maths literature and expands simulation section

Platelet Glycoprotein VI (GPVI) is the activatory collagen signalling receptor that transmits an outside-in signal via the FcR gamma-chain. In Caucasians two GP6 haplotypes have been identified which encode GPVI isoforms that differ by five amino-acids. The minor haplotype is associated with a modest but statistically significant reduction in GPVI abundance and reduced downstream signalling events. As GPVI is also expressed on megakaryocytes, different GPVI isoforms may imprint on the platelet transcriptome. We investigated the association of GP6 haplotype with transcription by comparing the transcriptomes of platelets from individuals homozygous for the major ('a') and minor ('b') haplotypes to identify differentially expressed (DE) transcripts. Platelet RNA was isolated from apheresis concentrates from 16 'aa' donors and eight 'bb' donors. mRNA was amplified using a template-switching PCR based protocol and fluorescently labelled. Samples were randomly paired both within and between haplotypes and compared on a cDNA microarray. No consistently DE transcripts were identified within the 'aa' haplotype but 52 significantly DE transcripts were observed between haplotypes. Generally the fold differences were low (two to four-fold) but were confirmed by qRT-PCR for selected transcripts (TUBB1, P = 0.0004; VWF, P = 0.0126). The results of this study indicate that there are subtle differences between the platelet transcriptomes of individuals who differ by GP6 haplotype. The identification of DE genes may identify critical pathways and nodes not previously known to be involved in platelet development and function.

1. We compared the velocity waveforms in the superficial femoral artery measured by multichannel Doppler ultrasound in 45 subjects: 21 patients with untreated essential hypertension and 24 normal subjects of similar age and sex. 2. The pattern of arterial flow was abnormal in hypertensive patients, with the acceleration time, the duration of reverse flow and the time to maximum flow reversal being abbreviated. The internal arterial diameter, calculated from the velocity profile, was reduced despite raised pressure, suggesting altered arterial wall mechanics in essential hypertension. 3. These abnormalities will influence the wall shear stress, a major determinant of arterial function. The abnormal arterial wall mechanics and abnormal blood flow pattern may contribute to the increased risk of arterial disease in patients with untreated hypertension.

We report results of a nonsynonymous SNP scan for ulcerative colitis and identify a previously unknown susceptibility locus at ECM1. We also show that several risk loci are common to ulcerative colitis and Crohn's disease (IL23R, IL12B, HLA, NKX2-3 and MST1), whereas autophagy genes ATG16L1 and IRGM, along with NOD2 (also known as CARD15), are specific for Crohn's disease. These data provide the first detailed illustration of the genetic relationship between these common inflammatory bowel diseases.

The S. typhimurium genome encodes proteins, designated EngA and YhbZ, which have a high sequence identity with the GTPases EngA/Der and ObgE/CgtAE of Escherichia coli. The wild-type activity of the E. coli proteins is essential for normal ribosome maturation and cell viability. In order to characterize the potential involvement of the Salmonella typhimurium EngA and YhbZ proteins in ribosome biology, we used high stringency affinity chromatography experiments to identify strongly binding ribosomal partner proteins. A combination of biochemical and microcalorimetric analysis was then used to characterize these protein:protein interactions and quantify nucleotide binding affinities. These experiments show that YhbZ specifically interacts with the pseudouridine synthase RluD (KD=2 microM and 1:1 stoichiometry), and we show for the first time that EngA can interact with the ribosomal structural protein S7. Thermodynamic analysis shows both EngA and YhbZ bind GDP with a higher affinity than GTP (20-fold difference for EngA and 3.8-fold for YhbZ), and that the two nucleotide binding sites in EngA show a 5.3-fold difference in affinity for GDP. We report a fluorescence assay for nucleotide binding to EngA and YhbZ, which is suitable for identifying inhibitors specific for this ligand-binding site, which would potentially inhibit their biological functions. The interactions of YhbZ with ribosome structural proteins that we identify may demonstrate a previously unreported additional function for this class of GTPase: that of ensuring delivery of rRNA modifying enzymes to the appropriate region of the ribosome.

Box C/D snoRNPs, factors essential for ribosome biogenesis, are proposed to be assembled in the nucleoplasm before localizing to the nucleolus. However, recent work demonstrated the involvement of nuclear export factors in this process, suggesting that export may take place. Here we show that there are distinct distributions of U8 pre-snoRNAs and pre-snoRNP complexes in HeLa cell nuclear and cytoplasmic extracts. We observed differential association of nuclear export (PHAX, CRM1, and Ran) factors with complexes in the two extracts, consistent with nucleocytoplasmic transport. Furthermore, we show that the U8 pre-snoRNA in one of the cytoplasmic complexes contains an m3G cap and is associated with the nuclear import factor Snurportin1. Using RNA interference, we show that loss of either PHAX or Snurportin1 results in the incorrect localization of the U8 snoRNA. Our data therefore show that nuclear export and import factors are directly involved in U8 box C/D snoRNP biogenesis. The distinct distribution of U8 pre-snoRNP complexes between the two cellular compartments together with the association of both nuclear import and export factors with the precursor complex suggests that the mammalian U8 snoRNP is exported during biogenesis. In the eukaryotic nucleolus, small nucleolar RNAs (snoRNAs) are essential for the production of functional ribosomes (2, 29, 47). The U3 and U8 box C/D snoRNAs are essential for rRNA processing and are believed to function as chaperones directing the folding and cleavage of the pre-rRNA (47). These two snoRNAs are stably associated with larger, pre-rRNA processing complexes in mammalian cell extracts (48). A subset of mammalian box C/D snoRNAs, which include U3, U8, and U13, are independently transcribed by RNA polymerase II, and the mature transcripts possess an m3G cap (24). The initial transcripts contain an m 7 G cap and a short 3 extension. The precursors are matured by removal of the 3

[1] The solar wind exhibits scaling typical of intermittent turbulence in the statistics of in situ fluctuations in both the magnetic and velocity fields. Intriguingly, quantities not directly accessed by theories of ideal, incompressible, MHD turbulence, such as magnetic energy density, B2, nevertheless show evidence of simple fractal (self-affine) statistical scaling. We apply a novel statistical technique which is a sensitive discriminator of fractality to the B2 timeseries from WIND and ACE. We show that robust fractal behaviour occurs at solar maximum and determine the scaling exponents. The probability density function (PDF) of fluctuations at solar maximum and minimum are distinct. Power law tails are seen at maximum, and the PDF is reminiscent of a Levy flight.

Polygenic autoimmune diseases, such as systemic lupus erythematosus (SLE), are a significant cause of morbidity and mortality worldwide. In recent years, functionally important genetic polymorphisms conferring susceptibility to SLE have been identified, but the evolutionary pressures driving their retention in the gene pool remain elusive. A defunctioning, SLE-associated polymorphism of the inhibitory receptor FcγRIIb is found at an increased frequency in African and Asian populations, broadly corresponding to areas where malaria is endemic. Here, we show that FcγRIIb-deficient mice have increased clearance of malarial parasites (Plasmodium chabaudi chabaudi) and develop less severe disease.In vitro, the human lupus associated FcγRIIb polymorphism enhances phagocytosis ofPlasmodium falciparum-infected erythrocytes. These results demonstrate that FcγRIIb is important in controlling the immune response to malarial parasites and suggests that the higher frequency of human FcγRIIb polymorphisms predisposing to SLE in Asians and Africans may be maintained because these variants reduce susceptibility to malaria.

We present a detailed analysis of the spatial structure of the ionospheric plasma velocity in the nightside F-region ionosphere, poleward of the open-closed magnetic field line boundary (OCB), i.e. in regions magnetically connected to the turbulent solar wind. We make use of spatially distributed measurements of the ionospheric plasma velocity made with the Halley Super Dual Auroral Radar Network (SuperDARN) radar between 1996 and 2003. We analyze the spatial structure of the plasma velocity using structure functions and P(0) scaling (where P(0) is the value of the probability density function at 0), which provide simple methods for deriving information about the scaling, intermittency and multi-fractal nature of the fluctuations. The structure functions can also be compared to values predicted by different turbulence models. We find that the limited range of velocity that can be measured by the Halley SuperDARN radar restricts our ability to calculate structure functions. We correct for this by using conditioning (removing velocity fluctuations with magnitudes larger than 3 standard deviations from our calculations). The resultant structure functions suggest that Kraichnan-Iroshnikov versions of P and log-normal models of turbulence best describe the velocity structure seen in the ionosphere.

In this paper we review some recent work investigating the turbulent structure of the ionospheric plasma velocity in the polar and auroral regions. The studies make use of spatially distributed measurements of the ionospheric plasma velocity made with the Halley Super Dual Auroral Radar Network (SuperDARN) radar between 1996 and 2003. The spatial structure is analyzed using structure functions, which provide a simple method for deriving information about the scaling and intermittency of the fluctuations that can be used to differentiate between different turbulence models. Evidence for scale‐free structure in regions of both open and closed magnetic field line topology is found, though with different power‐law exponents. It is suggested that these different exponents arise as a result of different origins of the scale‐free structure in the different regions, i.e., that seen on open magnetic field lines is a reflection of the spatial structure of the solar wind and that seen on closed magnetic field lines ...

To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified, biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK–up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes, a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts, including G6b, G6f, LRRC32, LAT2, and the G protein–coupled receptor SUCNR1, encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins, and flow cytometric analysis confirmed the expression of G6b, G6f, and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b, G6f, and LRRC32 are restricted to the platelet lineage, whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest, because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists.

Introduced by the late Per Bak and his colleagues, self-organized criticality (SOC) has been one of the most stimulating concepts to come out of statistical mechanics and condensed matter theory in the last few decades, and has played a significant role in the development of complexity science. SOC, and more generally fractals and power laws, have attracted much comment, ranging from the very positive to the polemical. The other papers (Aschwanden et al. in Space Sci. Rev., 2014, this issue; McAteer et al. in Space Sci. Rev., 2015, this issue; Sharma et al. in Space Sci. Rev. 2015, in preparation) in this special issue showcase the considerable body of observations in solar, magnetospheric and fusion plasma inspired by the SOC idea, and expose the fertile role the new paradigm has played in approaches to modeling and understanding multiscale plasma instabilities. This very broad impact, and the necessary process of adapting a scientific hypothesis to the conditions of a given physical system, has meant that SOC as studied in these fields has sometimes differed significantly from the definition originally given by its creators. In Bak’s own field of theoretical physics there are significant observational and theoretical open questions, even 25 years on (Pruessner 2012). One aim of the present review is to address the dichotomy between the great reception SOC has received in some areas, and its shortcomings, as they became manifest in the controversies it triggered. Our article tries to clear up what we think are misunderstandings of SOC in fields more remote from its origins in statistical mechanics, condensed matter and dynamical systems by revisiting Bak, Tang and Wiesenfeld’s original papers.

Published version, also available at http://dx.doi.org/10.1088/1748-9326/10/9/094018 Abstract Wedemonstrate how the fundamental timescales of anthropogenic climate change limit the identification of societally relevant aspects of changes in precipitation.Weshow that it is nevertheless possible to extract, solely from observations, some confident quantified assessments of change at certain thresholds and locations. Maps of such changes, for a variety of hydrologically-relevant, threshold-dependent metrics, are presented. In places in Scotland, for instance, the total precipitation on heavy rainfall days in winter has increased by more than 50%, but only in some locations has this been accompanied by a substantial increase in total seasonal precipitation; an important distinction for water and land management. These results are important for the presentation of scientific data by climate services, as a benchmark requirement for models which are used to provide projections on local scales, and for process-based climate and impacts research to understand local modulation of synoptic and global scale climate. They are a critical foundation for adaptation planning and for the scientific provision of locally relevant information about future climate.

The k-turn-binding protein 15.5K is unique in that it is essential for the hierarchical assembly of three RNP complexes distinct in both composition and function, namely, the U4/U6 snRNP, the box C/D snoRNP, and the RNP complex assembled on the U3 box B/C motif. 15.5K interacts with the cognate RNAs via an induced fit mechanism, which results in the folding of the surrounding RNA to create a binding site(s) for the RNP-specific proteins. However, it is possible that 15.5K also mediates RNP formation via protein-protein interactions with the complex-specific proteins. To investigate this possibility, we created a series of 15.5K mutations in which the surface properties of the protein had been changed. We assessed their ability to support the formation of the three distinct RNP complexes and found that the formation of each RNP requires a distinct set of regions on the surface of 15.5K. This implies that protein-protein contacts are essential for RNP formation in each complex. Further supporting this idea, direct protein-protein interaction could be observed between hU3-55K and 15.5K. In conclusion, our data suggest that the formation of each RNP involves the direct recognition of specific elements in both 15.5K protein and the specific RNA.

Summary. Background: Common genetic variants of cell surface receptors contribute to differences in functional responses and disease susceptibility. We have previously shown that single nucleotide polymorphisms (SNPs) in platelet glycoprotein VI (GP6) determine the extent of response to agonist. In addition, SNPs in the GP6 gene have been proposed as risk factors for coronary artery disease. Methods: To completely characterize genetic variation in the GP6 gene we generated a high-resolution SNP map by sequencing the promoter, exons and consensus splice sequences in 94 non-related Caucasoids. In addition, we sequenced DNA encoding the ligand-binding domains of GP6 from non-human primates to determine the level of evolutionary conservation. Results: Eighteen SNPs were identified, six of which encoded amino acid substitutions in the mature form of the protein. The single non-synonymous SNP identified in the exons encoding the ligand-binding domains, encoding for a 103Leu > Val substitution, resulted in reduced ligand binding. Two common protein isoforms were confirmed in Caucasoid with frequencies of 0.82 and 0.15. Variation at the GP6 locus was characterized further by determining SNP frequency in over 2000 individuals from different ethnic backgrounds. Conclusions: The SNPs were polymorphic in all populations studied although significant differences in allele frequencies were observed. Twelve additional GP6 protein isoforms were identified from the genotyping results and, despite extensive variation in GP6, the sequence of the ligand-binding domains is conserved. Sequences from non-human primates confirmed this observation. These data provide valuable information for the optimal selection of genetic variants for use in future association studies.

Cajal bodies (CBs) have been implicated in the nuclear phase of the biogenesis of spliceosomal U small nuclear ribonucleoproteins (U snRNPs). Here, we have investigated the distribution of the CB marker protein coilin, U snRNPs, and proteins present in C/D box small nucleolar (sno)RNPs in cells depleted of hTGS1, SMN, or PHAX. Knockdown of any of these three proteins by RNAi interferes with U snRNP maturation before the reentry of U snRNA Sm cores into the nucleus. Strikingly, CBs are lost in the absence of hTGS1, SMN, or PHAX and coilin is dispersed in the nucleoplasm into numerous small foci. This indicates that the integrity of canonical CBs is dependent on ongoing U snRNP biogenesis. Spliceosomal U snRNPs show no detectable concentration in nuclear foci and do not colocalize with coilin in cells lacking hTGS1, SMN, or PHAX. In contrast, C/D box snoRNP components concentrate into nuclear foci that partially colocalize with coilin after inhibition of U snRNP maturation. We demonstrate by siRNA-mediated depletion that coilin is required for the condensation of U snRNPs, but not C/D box snoRNP components, into nucleoplasmic foci, and also for merging these factors into canonical CBs. Altogether, our data suggest that CBs have a modular structure with distinct domains for spliceosomal U snRNPs and snoRNPs.

Single-amino-acid substitution Leu33Pro in the beta3-integrin is responsible for the formation of the human platelet antigen (HPA)-1. Alloimmunization against HPA-1a (beta3-Leu33) is the most frequent cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura.While HPA-1 genotyping a large cohort of patients with thromboembolic disease with a thermal cycler (LightCycler), one patient was identified with a unique HPA-1a melting curve.Sequence analysis revealed a C-to-G transversion at nucleotide 175 in the beta3-integrin (ITGB3) gene that alters the Leu33 codon to Val33. Further genotyping of healthy blood donors (n = 2950) identified one nonrelated Pro33Val33-positive individual. To examine whether the presence of Val33 affected the binding pattern of HPA-1 alloantibodies, transfectants were generated expressing recombinant beta3-Leu33 or beta3-Val33. Interestingly, differences in the reactivity of anti-HPA-1a were observed, with some HPA-1a alloantibodies showing diminished reactivity with beta3-Val33 compared to beta3-Leu33 and others reacting equally with both types. Similar findings were observed with recombinant human HPA-1a antibodies, with one of the three not binding to beta3-Val33.Our results demonstrate that the naturally occurring Leu33Val mutation in the beta3-integrin can disrupt some HPA-1a epitopes. These findings provide evidence for a heterogeneous humoral response against HPA-1a that may have potential clinical implications for alloimmune thrombocytopenia disorders.

Antibodies are a powerful tool for structure/function studies of platelet proteins. However, classic immunisation frequently elicits antibody responses against domains of minor functional interest. Robust strategies to generate antibodies against defined domains would be of significant interest in post-genome research. In this study, we report a new strategy using a combination of DNA vaccination and V gene phage display that allows the rapid generation of domain specific single-chain Fv antibodies (scFvs). This system was validated using the I-domain of alpha2 integrin as a model. The alpha2beta1 integrin, which is expressed on many cell types, is the dominant collagen attachment receptor on platelets, functioning in close interplay with the collagen signalling receptor glycoprotein VI. A novel set of I-domain specific antibodies was obtained by a DNA vaccination/V gene repertoire cloning approach. Mice were first immunised with a DNA vaccine in which the alpha2 I-domain is expressed as a fusion protein with fragment C of tetanus toxoid (FrC-TT). Then the heavy and kappa light chain variable gene repertoires were rescued from immune splenocytes using antibody phage display. A total of four alpha2 I-domain specific scFvs were isolated by selection on recombinant I-domain or native platelet alpha2beta1 integrin. Characterisation of the scFvs indicated that they recognised distinct epitopes that had profound differences in accessibility between native and recombinant I-domain. Our data suggest DNA immunisation and phage display represent versatile alternatives to protein immunisation and hybridoma-fusion techniques for the isolation of recombinant antibody reagents. This approach will be particularly useful for the generation of domain or splice-variant specific antibodies that recognise native protein.

Empirical determination of the scaling properties and exponents of time series presents a formidable challenge in testing, and developing, a theoretical understanding of turbulence and other out-of-equilibrium phenomena. We discuss the special case of self affine time series in the context of a stochastic process. We highlight two complementary approaches to the differenced variable of the data: i) attempting a scaling collapse of the Probability Density Functions which should then be well described by the solution of the corresponding Fokker-Planck equation and ii) using structure functions to determine the scaling properties of the higher order moments. Using the example of a L\'{e}vy flight, we demonstrate a method for conditioning the time series that recovers the underlying self affine scaling in a finite length time series., Comment: Submitted Nonlinear Processes in Geophysics, 18 pages including 8 figures

Abnormal haemodynamic conditions are implicated in the development of anastomotic myointimal hyperplasia (MIH). However, these conditions are difficult to determine in vivo, prompting research using ex vivo idealised models. To relate the understanding gained in idealised geometries to anatomically correct conditions we have investigated a reproducible approach to classify in vivo distal graft anastomoses and their inter-patient variability. In vivo distal anastomotic geometries were acquired by magnetic resonance (MR) angiography from 13 patients who had undergone infrageniculate autologous venous by-pass surgery. On average, the images were acquired 2 weeks post-operatively. Five patients also underwent repeat examinations 2 to 7 weeks later. For each geometry, the surface of the arterial lumen is represented by the zero level set of an implicit function constructed from radial basis functions that minimise curvature. The three-dimensional binary image created from the interpolated surface is processed using a skeletonisation algorithm to obtain the centreline of each branch in the geometry. This allows for the measurement of the branching angles between straight line approximations of the centrelines of each vessel, averaging them over a characteristic length of each anastomosis. The main finding in the application of the proposed classification methodology to this set of patients is that the spectrum of anastomoses can be reduced to a small subset of cases characterised by two angles: the angle between the graft and the plane of the host artery and the angle between the graft and the proximal branch of the artery.

The assembly and maturation of box C/D snoRNPs, factors essential for ribosome biogenesis, occur in the nucleoplasm. To investigate this process, we have analyzed non-snoRNP factors associated with the nucleoplasmic human U3 snoRNA. We show that both the precursor and mature length nucleoplasmic U3 snoRNAs are present in larger multiprotein complexes that contain the core box C/D proteins as well as many non-snoRNP factors linked to snoRNP assembly (TIP48, TIP49, Nopp140), RNA processing (TGS1, La, LSm4, hRrp46), and subcellular localization (CRM1, PHAX). Using RNAi, we show that most of these factors are essential for box C/D snoRNA accumulation. Furthermore, we demonstrate that the core proteins undergo a restructuring event that stabilizes their binding to the snoRNA. Importantly, restructuring, which may be mediated by the putative remodeling factor TIP49, appears to be linked to nucleolar localization. We believe that the assembly complex coordinates snoRNA processing, snoRNP assembly, restructuring, and localization.

The structure of alpha-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free alpha-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three alpha-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to alpha-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer alpha1beta1 interface in tetrameric HbA.

SummaryRecombinant HPA-1a antibodies with Fc, mutated to remove destructive effector functions, have been developed as a potential therapy for fetomaternal alloimmune thrombocytopenia (FMAIT), via blockade of binding of human HPA-1a polyclonal antibodies to fetal HPA-1a1b platelets. We have assessed the effect of the IgG1 HPA-1a antibody B2G1 and two mutated derivatives in various functional assays in resting and agoniststimulated platelets of the three HPA-1 genotypes. With HPA-1a1b platelets (fetal genotype), the antibodies did not activate signalling or CD62P expression in resting platelets, did not change in vitro bleeding time (IVBT), and did not inhibit platelet adhesion to collagen in flowing blood. Adhesion of HPA-1a1b platelets to fibrinogen was reduced by 20%, and aggregation induced by ADP by 50%, but collagen-related peptide (CRP-XL)-induced aggregation was normal. With HPA-1a1a platelets, aggregation to both ADP and CRP-XL was inhibited, with total blockade of adhesion to fibrinogen and of IVBT responses. Interestingly, a monovalent antibody fragment with identical specificity had no inhibitory effect on aggregation. In static adhesion assays using human αIIbβ3 or αVβ3 transfectants of HPA-1a (Leu33) phenotype, attachment to fibrinogen of the latter but not of the former was completely blocked by the HPA-1a antibodies. These observations are best explained by antibody-mediated blockade of the RGD binding site on β3 by a mechanism of steric hindrance. As the effect on platelet function is modest with HPA-1a1b (fetal type) platelets, the mutated HPA-1a antibodies described here could be developed further for FMAIT therapy.

Human leukocyte antigen (HLA) A2 is one of the most immunodominant HLA antigens. Through a process of light-chain variable domain (VL) shuffling, we analyzed the VL domains' role in anti-HLA-A2/A28-binding site diversity. This was achieved by combining a VH3-30-encoded HLA-A2/A28-specific heavy-chain variable domain with 10(4) non-immune VL domains. Twelve HLA-A2/A28-specific antibodies were subsequently identified. VL gene analysis demonstrated an absence of Vlambda domains and that all have VkappaI-encoded light chains. The affinities correlated with the VkappaI gene present, with the seven highest affinity antibodies using Vkappa domains encoded by the O18 gene segment. A 300-fold difference in affinity was observed between the 12 antibodies, and homology modeling demonstrated a correlation between electrostatic surface potential of the antigen-binding site and affinity for HLA. Overlap between the T-cell receptor-binding site and that of the antibodies was indicated by inhibition of cytotoxic T-lymphocyte killing of peptide-pulsed target cells. A model of antibody binding to HLA-A2 suggested contact with both alpha helices of the HLA molecule, such that the antigen-binding site spans the peptide-binding groove. These data increase the understanding of antibody recognition of HLA and may facilitate the production of clonotypic antibodies with peptide-specific binding.

We study scaling in fluctuations of the geomagnetic indices (AE, AU, and AL) that provide a measure of magnetospheric activity and of the epsilon parameter which is a measure of the solar wind driver. Generalized structure function (GSF) analysis shows that fluctuations exhibit self-similar scaling up to about 1 hour for the AU index and about 2 hours for AL, AE and epsilon when the most extreme fluctuations over 10 standard deviations are excluded. The scaling exponents of the GSF are found to be similar for the three AE indices, and to differ significantly from that of epsilon. This is corroborated by direct comparison of their rescaled probability density functions.