Spanning Region Research Articles

‘Byron and the Mediterranean’ 10th International Student Byron Conference Messolonghi, 22–27 May 2015

The Tenth International Student Byron Conference, held from 22-27 May 2015 in Messolonghi, Greece, appropriately acknowledged tenth anniversary of such an ambitious and successful conference by choosing broad theme 'Byron and Mediterranean'. As in past, conference-which as always was flawlessly organised and managed by President of Messolonghi Byron Society. Mrs. Rosa Florou- drew participants from around world for nearly a week of insightful scholarship regarding Byron's life and works, travel spanning region, and intellectual companionship. This year's participants were undergraduate and graduate students, professors and local enthusiasts. They traveled from such places as United States, Canada, United Kingdom, Taiwan, Lebanon, and Greece.Because conference officially began with registration at four o'clock on 22 May, that first Friday was an opportunity for many participants to explore parts of Athens (where most travellers first arrived from their home countries), beautiful coastline and mountains that separate Athens and Messolonghi and that include such famous sites as Corinth Canal, or charming town of Messolonghi itself. Once everyone arrived and checked in at Theoxenia Hotel, participants boarded a coach for Byron House containing administrative office of Messolonghi Byron Society and library of Byron Research Center. There they were greeted by delicious homemade desserts and coffee and had an opportunity to mingle before being welcomed by conference directors Mrs. Rosa Florou, Professor Peter Graham, Director of International Relations for Messolonghi Byron center, and Joint President of International Association of Byron Societies, Professor Naji Oueijan. After refreshments, group was treated to a tour of a variety of significant sites in Messolonghi, including Cathedral of Agios Spyridon, which Byron visited during his time living in 'sacred city', and Gallery of Christos and Sophia Moschandreou to see an exhibition of modern paintings relating to Byron and to local area. Both were beautiful experiences that built nicely towards final sightseeing opportunity at Municipal Museum of History and Art Municipal Gallery, which provided useful historical background information of area. After perusing art in Municipal Gallery, participants were welcomed by Mayor of Messolonghi, Mr. Nikos Karapanos, who gave a warm and passionate speech in Greek, translated into English by several lively impromptu translators. The evening concluded with a welcoming dinner at nearby Archontiko restaurant, followed by (for many of those jet lagged) an early bedtime or else (for those more rested) a night of dancing and socialising in heart of Messolonghi.The academic portion of conference began on Saturday 23 May, at Regional building. There, Rosa Florou, Peter Graham, Jonathan Gross, Naji Oueijan, and Peter Myrian greeted participants but also, more significantly, reflected on life and scholarship of recently deceased English Byron scholar Peter Cochran. Following a moment of silence, first session, chaired by Peter Myrian, began. The first paper, given by Stephen Minta (York), revisited Stephen Cheeke's Byron and Place: History, Translation, and Nostalgia and paid particular attention to Byron's complicated relationship with Greece of past and of his present day using Childe Harold's Pilgrimage as a foundation for his argument. Perhaps even more interestingly, Minta addressed questions regarding 'authenticity of traveller's engagement with what Byron calls the truth of history [...] and of place.' Elli Karampela (Aristotle University of Thessaloniki) gave next paper, which made use of Benedict Anderson's theoretical framework 'imagined communities' to examine Byron's own complex relationship with place of England versus place of Greece. …

Hybrid de novo tandem repeat detection using short and long reads.

BackgroundAs one of the most studied genome rearrangements, tandem repeats have a considerable impact on genetic backgrounds of inherited diseases. Many methods designed for tandem repeat detection on reference sequences obtain high quality results. However, in the case of a de novo context, where no reference sequence is available, tandem repeat detection remains a difficult problem. The short reads obtained with the second-generation sequencing methods are not long enough to span regions that contain long repeats. This length limitation was tackled by the long reads obtained with the third-generation sequencing platforms such as Pacific Biosciences technologies. Nevertheless, the gain on the read length came with a significant increase of the error rate. The main objective of nowadays studies on long reads is to handle the high error rate up to 16%.MethodsIn this paper we present MixTaR, the first de novo method for tandem repeat detection that combines the high-quality of short reads and the large length of long reads. Our hybrid algorithm uses the set of short reads for tandem repeat pattern detection based on a de Bruijn graph. These patterns are then validated using the long reads, and the tandem repeat sequences are constructed using local greedy assemblies.ResultsMixTaR is tested with both simulated and real reads from complex organisms. For a complete analysis of its robustness to errors, we use short and long reads with different error rates. The results are then analysed in terms of number of tandem repeats detected and the length of their patterns.ConclusionsOur method shows high precision and sensitivity. With low false positive rates even for highly erroneous reads, MixTaR is able to detect accurate tandem repeats with pattern lengths varying within a significant interval.

Loss of 4q21.23-22.1 is a prognostic marker for disease free and overall survival in non-small cell lung cancer.

This study was performed to assess the prognostic relevance of genomic aberrations at chromosome 4q in NSCLC patients. We have previously identified copy number changes at 4q12-q32 to be significantly associated with the early hematogenous dissemination of non-small cell lung cancer (NSCLC), and now aim to narrow down potential hot-spots within this 107 Mb spanning region. Using eight microsatellite markers at position 4q12-35, allelic imbalance (AI) analyses were performed on a preliminary study cohort (n = 86). Positions indicating clinicopathological and prognostic associations in AI analyses were further validated in a larger study cohort using fluorescence in situ hybridization (FISH) in 209 NSCLC patients. Losses at positions 4q21.23 and 4q22.1 were shown to be associated with advanced clinicopathological characteristics as well as with shortened disease free (DFS) and overall survival (OS) (DFS: P = 0.019; OS: P = 0.002). Multivariate analyses identified the losses of 4q21.23-22.1 to be an independent prognostic marker for both DFS and OS in NSCLC (HR 1.64–2.20, all P<0.04), and especially in squamous cell lung cancer (P<0.05). A case report study of a lung cancer patient further revealed a loss of 4q21.23 in disseminated tumor cells (DTCs). Neither gains at the latter positions, nor genomic aberrations at 4q12, 4q31.2 and 4q35.1, indicated a prognostic relevance. In conclusion, our data indicate that loss at 4q21.23-22.1 in NSCLC is of prognostic relevance in NSCLC patients and thus, includes potential new tumor suppressor genes with clinical relevance.

Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly.

Membrane fusion is essential for human health, playing a vital role in processes as diverse as neurotransmission and blood glucose control. Two protein families are key: (1) the Sec1p/Munc18 (SM) and (2) the soluble N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins. Whilst the essential nature of these proteins is irrefutable, their exact regulatory roles in membrane fusion remain controversial. In particular, whether SM proteins promote and/or inhibit the SNARE-complex formation required for membrane fusion is not resolved. Crystal structures of SM proteins alone and in complex with their cognate SNARE proteins have provided some insight, however, these structures lack the transmembrane spanning regions of the SNARE proteins and may not accurately reflect the native state. Here, we review the literature surrounding the regulatory role of mammalian Munc18 SM proteins required for exocytosis in eukaryotes. Our analysis suggests that the conflicting roles reported for these SM proteins may reflect differences in experimental design. SNARE proteins appear to require C-terminal immobilization or anchoring, for example through a transmembrane domain, to form a functional fusion complex in the presence of Munc18 proteins.

Differential regulation of non-protein coding RNAs from Prader-Willi Syndrome locus.

Prader-Willi Syndrome (PWS) is a neurogenetic disorder caused by the deletion of imprinted genes on the paternally inherited human chromosome 15q11-q13. This locus harbours a long non-protein-coding RNA (U-UBE3A-ATS) that contains six intron-encoded snoRNAs, including the SNORD116 and SNORD115 repetitive clusters. The 3′-region of U-UBE3A-ATS is transcribed in the cis-antisense direction to the ubiquitin-protein ligase E3A (UBE3A) gene. Deletion of the SNORD116 region causes key characteristics of PWS. There are few indications that SNORD115 might regulate serotonin receptor (5HT2C) pre-mRNA processing. Here we performed quantitative real-time expression analyses of RNAs from the PWS locus across 20 human tissues and combined it with deep-sequencing data derived from Cap Analysis of Gene Expression (CAGE-seq) libraries. We found that the expression profiles of SNORD64, SNORD107, SNORD108 and SNORD116 are similar across analyzed tissues and correlate well with SNORD116 embedded U-UBE3A-ATS exons (IPW116). Notable differences in expressions between the aforementioned RNAs and SNORD115 together with the host IPW115 and UBE3A cis-antisense exons were observed. CAGE-seq analysis revealed the presence of potential transcriptional start sites originated from the U-UBE3A-ATS spanning region. Our findings indicate novel aspects for the expression regulation in the PWS locus.

Antigenic and 3D structural characterization of soluble X4 and hybrid X4-R5 HIV-1 Env trimers.

BackgroundHIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4. The variable loop 3 (V3) of the HIV-1 envelope protein (Env) is the main determinant for coreceptor usage. The predominant CCR5 using (R5) HIV-1 Env has been intensively studied in function and structure, whereas the trimeric architecture of the less frequent, but more cytopathic CXCR4 using (X4) HIV-1 Env is largely unknown, as are the consequences of sequence changes in and near V3 on antigenicity and trimeric Env structure.ResultsSoluble trimeric gp140 Env constructs were used as immunogenic mimics of the native spikes to analyze their antigenic properties in the context of their overall 3D structure. We generated soluble, uncleaved, gp140 trimers from a prototypic T-cell line-adapted (TCLA) X4 HIV-1 strain (NL4-3) and a hybrid (NL4-3/ADA), in which the V3 spanning region was substituted with that from the primary R5 isolate ADA. Compared to an ADA (R5) gp140, the NL4-3 (X4) construct revealed an overall higher antibody accessibility, which was most pronounced for the CD4 binding site (CD4bs), but also observed for mAbs against CD4 induced (CD4i) epitopes and gp41 mAbs. V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis. Of interest, the NL4-3/ADA construct with the hybrid NL4-3/ADA CD4bs showed impaired CD4 and CD4bs mAb reactivity despite the presence of the essential elements of the CD4bs epitope. We obtained 3D reconstructions of the NL4-3 and the NL4-3/ADA gp140 trimers via electron microscopy and single particle analysis, which indicates that both constructs inherit a propeller-like architecture. The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env. Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction.Conclusions3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA.

Transverse Analysis of Box-Girder for Continuous Beam Arch Composite Railway Bridge

A comparative study was carried out by establishing an overall bridge spatial finite element model and a transverse frame model on transverse structural characteristics of Kunyang Bridge on Wenzhou-Fuzhou Railway line. The longitudinal variation of transverse stress in box-girder shows peaks distribution. The absolute values of transverse stress decrease gradually at peaks and valleys in overall tendency as it’s close to the mid span. The calculation results of the rigid support frame method can envelop the calculation results of the overall bridge spatial finite element model in side span region without suspenders.It verifies the reasonability of the rigid support frame method to calculate transverse stress of this region. And the width of transverse frame should designate one third of suspender spacing or more in transverse stress calculations of central span region with suspenders.

Impact of Clinically Relevant Mutations on the Pharmacoregulation and Signaling Bias of the Calcium-Sensing Receptor by Positive and Negative Allosteric Modulators

Cinacalcet is predominantly used to treat secondary hyperparathyroidism due to end-stage renal failure, but, more recently, its potential clinical efficacy in treating patients with loss-of-function mutations in the calcium-sensing receptor (CaSR) has been recognized. Many clinically relevant CaSR mutations are located in the heptahelical membrane spanning and extracellular loop regions of the receptor, where allosteric modulators are predicted to bind. The aim of the present study was to investigate the impact of such mutations on the pharmacoregulation of the CaSR by the positive and negative allosteric modulators, cinacalcet and NPS-2143, respectively. Both cinacalcet and NPS-2143 effectively rescued mutants whose cell surface expression was substantially impaired, suggesting that both classes of drug can stabilize a receptor conformation that is trafficked more effectively to the cell surface. In addition, functional impairments in almost all mutant CaSRs were rescued by either cinacalcet or NPS-2143 via restoration of intracellular signaling. There was a significantly greater ability of both compounds to modulate agonist-stimulated intracellular Ca(2+) mobilization than ERK1/2 phosphorylation, indicating that the allosteric modulators engender bias in agonist-stimulated CaSR signaling to different pathways. Three mutations (G(670)R, P(748)R, and L(773)R) altered the binding affinity of allosteric modulators to the CaSR, and 3 mutations (V(817)I, L(773)R, and E(767)K) altered the cooperativity between the allosteric modulator and Ca(2+)(o). These findings have important implications for the treatment of diseases associated with CaSR mutations using allosteric CaSR modulators and for analyzing the effects of mutations on the function and pharmacoregulation of the CaSR.

J052012 フィン付き円柱周りの非定常流れと空力音に及ぼすフィン間隔の影響(〔J052-01〕流体関連の騒音と振動(1))

Finned tube that is used to the heat exchanger sometimes induced the air resonance phenomenon to avoid it, the fin pitch is varied to be wide. We analyzed the flow around the finned tube with various fin pitches by the simulation. It is learned Karman vortices one generated behind the finned tube and makes the aeolian tone large. The wake vortices are generated at the same time in large span region at the narrow fin-spacing. The wake width become large at the narrow fin tube, the influence of fin pitch to an aeolian tone and the unsteady flow characteristic.

Clonal Chromosomal Anomalies Similar to CLL and Other Hematologic Malignancies Can Be Found in “Normal” Individuals

AbstractAbstract 873 Introduction:Clonal chromosomal anomalies have been well-characterized in the blood cells of patients with Chronic Lymphocytic Leukemia (CLL) and other hematological malignancies. However, the frequencies and types of chromosomal anomalies found in “normal” people (i.e. no history of hematological cancer) are much less well known. Here we compare chromosomal anomalies in untreated CLL patients with those found in a large sample of people (n>50,000) recruited for genome-wide association studies in the Gene Environment Association Studies (GENEVA) consortium. This comparison provides information about the extent to which people without a prior history of hematological cancer have chromosomal anomalies that are characteristic of CLL. Methods and Results:The anomaly detection method focused on acquired, rather than inherited, chromosomal anomalies identified in whole blood samples. The method and results for the GENEVA subjects were published recently (Laurie et al. 2012, Nat. Gen. 44:642–650). The same method was applied to pre-treatment samples from 220 CLL patients from US Intergroup Trial E2997 of the Eastern Cooperative Oncology Group (ECOG). Acquired anomalies were detected as mosaic mixtures of normal disomic cells along with cells containing autosomal anomalies consisting of large (generally > 1 Mb) deletions, duplications and/or acquired uniparental disomy (UPD). Detection requires a large fraction of cells with the same abnormal karyotype (>5–10%), which is presumably of clonal origin. The subjects (of all ages) came from 12 different GENEVA studies, which addressed a variety of health conditions not directly related to hematological cancer (e.g. addiction and glaucoma). DNA was extracted mainly from blood samples (or, in 8% of subjects, from buccal/saliva samples) and genotyped on Illumina SNP mircroarrays. Clonal mosaic anomalies were detected from change points in intensity and genotype data (B Allele Frequency and Log R Ratio). We found 514 anomalies in 404 GENEVA subjects out of 50,222 examined (0.8% of subjects), although the prevalence increased with age. Clinical data from about 75% of subjects with a clonal chromosomal abnormality revealed that only a small fraction (2.8%, 95% CI=1.0 – 4.7%) had a record of prior hematological cancer.In the ECOG study, blood samples from 220 CLL patients enrolled in protocol E2997 were genotyped on the Illumina HumanOmni1-Quad_v1 array and clonal mosaic chromosome anomalies were detected using the GENEVA method. Chromosomal anomalies typical of CLL also were identified using interphase FISH (i.e. 6q23.3-, 11q22-, 12q15+, 13q14-, 17p13-) as previously reported (Grever, JCO 25:799-804). We found 489 anomalies in 148 patients of the 220 examined (67% of patients). The anomalies detected by FISH and SNP microarray were highly concordant (>98%). Although, the most common recurrent anomalies occurred on chromosomes 6, 11, 12, 13 and 17 in regions that span the FISH probes (except for chromosome 6), a majority of the anomalies detected by SNP data (74% of 489) were not detected by the CLL FISH panel.The frequency of large chromosomal anomalies was much higher in CLL patients than in GENEVA (67% versus 0.8% of subjects) and the overall composition of anomaly types found was also different. The fraction of anomalies consisting of acquired UPD was substantially higher in GENEVA subjects than in ECOG (34.0% versus 10.6%), while deletions were less common in GENEVA (50.1% versus 72.4%) and duplications were similar (15.6% versus 17.0%) (Chi-squared test p-value=3 × 10−18). We compared the most commonly deleted regions (CDR) on each chromosome across studies. In the ECOG CLL patients, four chromosomes each had 10 or more overlapping deletions in their top CDR (6, 11, 13 and 17), which span the FISH probes (except for chromosome 6). The GENEVA subjects also had CDR on chromosomes 11, 13 and 17, which overlap those in ECOG. In addition, GENEVA had CDR spanning genes or regions implicated in myeloid malignancies, including DNMT3a on 2p, TET2 on 4q, PRAME on 22q, and a region on 20q13. Conclusions:These genotyping results of >50,000 subjects suggest clonal chromosomal anomalies may represent precursor states, or perhaps undiagnosed or unrecorded cases, of a variety of hematological malignancies. Further studies to validate these findings and explore the natural history of these genetic defects in individuals without hematologic malignancy are warranted. Disclosures:Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract 3974: A comprehensive resequence-analysis of 250kb region of 8q24 in Africans

Abstract To date, genome-wide association studies (GWAS) have identified more than 45 prostate cancer susceptibility variants, of which at least 5 independent loci were identified within a 654kb region on 8q24. This region also harbors additional loci associated with cancers of the breast, colon, bladder, and chronic lymphocytic leukemia. Previously, the linkage disequilibrium (LD) pattern and recombination hotspots (chr8:128050768-128300801, hg19) defined a 250kb span (region 2) harboring multiple prostate cancer susceptibility loci identified in multiple distinct populations, as well as a locus rs2456449 associated with chronic lymphocytic leukemia in Europeans. Because GWAS identify genetic markers, fine-mapping using sequence analysis in diverse populations is necessary for defining the optimal variants for biological follow-up. Sequence analysis in populations of distinct ancestry can provide important insights into the differences of LD patterns and thus narrow down high interest variants. In the present study, we have resequenced the region in 125 men of African ancestry, which included 78 individuals from a population-based study in Ghana and 47 African-American individuals from the Johns Hopkins Hospital. Upon comparison with 1000 Genomes data (Oct 2011 release), we have identified 365 novel loci and additional surrogates of known susceptibility loci which could be important in establishing priorities for future functional work, designed to explain the biological basis of the association signal for both prostate cancer and perhaps, chronic lymphocytic leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3974. doi:1538-7445.AM2012-3974

2008 LC18: a potentially unstable Neptune Trojan

The recent discovery of the first Neptune Trojan at the planet's trailing (L5) Lagrange point, 2008 LC18, offers an opportunity to confirm the formation mechanism of a member of this important tracer population for the Solar system's dynamical history. We tested the stability of 2008 LC18's orbit through a detailed dynamical study, using test particles spread across the orbital uncertainties in a, e, i and {\Omega}. This showed that the wide uncertainties of the published orbit span regions of both extreme dynamical instability, with lifetimes < 100 Myr, and with significant stability (> 1 Gyr lifetimes). The stability of 2008 LC18's clones is greatly dependent on their semi-major axis and only weakly correlated with their eccentricity. Test particles on orbits with an initial semi-major axis less than 29.91 AU have dynamical half-lives shorter than 100 Myr; in contrast, particles with an initial semi-major axis greater than 29.91 AU exhibit such strong dynamical stability that almost all are retained over the 1 Gyr of our simulations. More observations of this object are necessary to improve the orbit. If 2008 LC18 is in the unstable region, then our simulations imply that it is either a temporary Trojan capture, or a representative of a slowly decaying Trojan population (like its sibling the L4 Neptunian Trojan 2001 QR322), and that it may not be primordial. Alternatively, if the orbit falls into the larger, stable region, then 2008 LC18 is a primordial member of the highly stable and highly inclined component of the Neptune Trojan population, joining 2005 TN53 and 2007 VL305. We attempted to recover 2008 LC18 using the 2.3m telescope at Siding Spring Observatory to provide this astrometry, but were unsuccessful due to the high stellar density of its current sky location near the galactic centre. The recovery of this object will require a telescope in the 8m class.

A Back-to-Front Fragment-Based Drug Design Search Strategy Targeting the DFG-Out Pocket of Protein Tyrosine Kinases.

We present a straightforward process for the discovery of novel back pocket-binding fragment molecules against protein tyrosine kinases. The approach begins by screening against the nonphosphorylated target kinase with subsequent counterscreening of hits against the phosphorylated enzyme. Back pocket-binding fragments are inactive against the phosphorylated kinase. Fragment molecules are of insufficient size to span both regions of the ATP binding pocket; thus, the outcome is binary (back pocket-binding or hinge-binding). Next, fragments with the appropriate binding profile are assayed in combination with a known hinge-binding fragment and subsequently with a known back pocket-binding fragment. Confirmation of back pocket-binding by Yonetani-Theorell plot analysis progresses candidate fragments to crystallization trials. The method is exemplified by a fragment screening campaign against vascular endothelial growth factor receptor 2, and a novel back pocket-binding fragment is presented.

Fatigue Crack Initiation and Propagation Behavior of a Nickel Alloy ; A Study of Structural Integrity Assessment Method Based on Polycrystalline FE Analysis

Evaluation of fatigue crack nucleation and propagation behavior in anisotropic metals is necessary for improving an accuracy of structural integrity assessment. In this paper, high cycle fatigue tests and polycrystalline FE analyses were carried out to investigate a relationship between crack nucleation positions and stress distribution. Fatigue tests of a rectangular specimen were conducted by four-point bending under load control. Fatigue crack nucleation and propagation in the gauge region was carefully observed during the tests. Grain morphology and crystallographic orientation of tensile surface were characterized using electron beam backscattering diffraction pattern (EBSP) analysis. The size of characterized area was determined to sufficiently cover the gauge region of four-point bending inner span. Stress distribution of the specimen was computed by polycrystalline finite element analysis. Resolved shear stresses were calculated in expected twelve slip systems. Fatigue cracks were tend to be initiated in areas indicating relatively high resolved shear stresses in several slip systems.

J051032 小型遠心ブロワ用ディフューザの内部流れに関する研究

Compact centrifugal blowers with vaned diffusers are often used in home electric vacuum cleaners. To achieve high efficiency and low noise, it is important to improve the performance of impellers and diffusers. In this study, we used a large solidity diffuser whose flow duct is low in height. We studied the influence of internal flow on the diffuser performance by using PIV and pressure measurement and visualizing the flow field in detail. From our measurement results, low velocity was observed in the middle span section and the pressure side in the middle of the overlapped region. Area of the low velocity region became larger with the decrease of flow rate. Pressure recovery increased gradually at the downstream of the low velocity region. From our simulation results, we found that in the low velocity area, low-energy fluid from shroud and hub regions accumulated to the middle span region, which led to low velocity in the middle of the overlapped region.

Membrane-associated RING-CH 10 (MARCH10 Protein) Is a Microtubule-associated E3 Ubiquitin Ligase of the Spermatid Flagella

Spermiogenesis is a complex and dynamic process of the metamorphosis of spermatids into spermatozoa. There is a great deal that is still unknown regarding the regulatory mechanisms for the formation of the sperm flagellum. In this study, we determined that the membrane-associated RING-CH 10 (March10) gene is predominantly expressed in rat testis. We isolated two March10 isoforms encoding MARCH10a and MARCH10b, which are generated by alternative splicing. MARCH10a is a long RING finger protein, and MARCH10b is a short RING finger-less protein. Immunohistochemical staining revealed that the MARCH10 proteins are specifically expressed in elongating and elongated spermatids, and the expression is absent in epididymal spermatozoa. MARCH10 immunoreactivity was observed in the cytoplasmic lobes as well as the principal piece and annulus of the flagella. When overexpressed in COS7 cells, MARCH10a was localized along the microtubules, whereas MARCH10b was distributed throughout the cytoplasm. An in vitro microtubule cosedimentation assay showed that MARCH10a is directly associated with microtubules. An in vitro ubiquitination assay demonstrated that the RING finger domain of MARCH10a exhibits an E3 ubiquitin ligase activity along with the E2 ubiquitin-conjugating enzyme UBE2B. Moreover, MARCH10a undergoes proteasomal degradation by autoubiquitination in transfected COS7 cells, but this activity was abolished upon microtubule disassembly. These results suggest that MARCH10 is involved in spermiogenesis by regulating the formation and maintenance of the flagella in developing spermatids.

Evidence for bovine besnoitiosis being endemic in Italy—First in vitro isolation of Besnoitia besnoiti from cattle born in Italy

Until 2009, bovine besnoitiosis had never been considered endemic in Italy and the only report on the disease in this country referred to animals imported from France shortly before. However, recently, an autochthonous outbreak of bovine besnoitiosis was reported in four herds located at the intersection of the borders between Emilia-Romagna, Toscana and Marche (Northern Apennine Mountains), which has led to an increased awareness concerning this disease. The present study describes a further outbreak of bovine besnoitiosis in Italy. The afflicted herd was a dairy herd with no evidence for contact with cattle from regions known to be endemic for bovine besnoitiosis. The farm investigation was initiated after a three-year old Holstein Friesian dairy cow with generalized thickening and lichenification of the skin was diagnosed with bovine besnoitiosis. The clinical diagnosis was confirmed by gross pathology, histopathology, serology and PCR. Bradyzoites released from tissue cysts obtained from the skin of this animal enabled the first in vitro isolation of Besnoitia besnoiti in Italy. This isolate was named Bb-Italy1. Sequencing of a 2118bp spanning region including the complete internal transcribed spacer 1 and parts of the 18S and the 5.8S rRNA gene from DNA extracted from skin-derived zoites revealed a 99.9% identity to sequences known for other B. besnoiti isolated from cattle in Europe. Two GKO mice which had been inoculated intraperitoneally with bovine skin-derived bradyzoites became ill 7 days post inoculation. Parasitophorous vacuoles with multiplying zoites were observed in the cell culture inoculated with peritoneal fluids of these mice and a B. besnoiti infection in the mice and in the cell culture could be confirmed by real-time PCR. A serological investigation in the afflicted herd using immunoblots and an immunofluorescent antibody test (IFAT) revealed an overall herd seroprevalence of 9.7% (31/321), whereas within the female animals older than 2years 17.0% (29/171) of the dams were tested positive. With one exception, an imported cow from Germany, all the seropositive animals were born in Italy. In connection with previously described autochthonous cases of bovine besnoitiosis the case described herein suggests that bovine besnoitiosis should be considered endemic in Italy.

Large eddy simulation of a compressor cascade and the influence of spanwise domain

A controlled diffusion compressor cascade is studied using large eddy simulation (LES). The aim of this study is to assess the capability of LES to be used in an industrial context. The Reynolds number is approximately 700 000 based on chord length and inlet velocity. A ‘thin-slice’ representation of the cascade is used as the reference grid, and the influence of a narrow span is studied by comparison simulations with a domain that has a span five times larger than the thin-slice grid. While the instantaneous flow-fields of the thin-slice and wide-domain simulations are qualitatively similar, the thin-slice simulations suffer from flow confinement problems caused by the imposition of the narrow span. The non-unity axial velocity density ratio of the flow enforces the use of inviscid wall spanwise boundaries, which have a parasitic influence on the development of the flow in the thin-slice simulations. The resultant data obtained from the thin-slice simulations are therefore compromised and the computed loss estimation is considered unreliable. However, when comparing mean quantities such as surface pressure and boundary layer growth the narrow does give reasonable predictions. While the inviscid spanwise walls also affect the flow near the boundaries in the wide domain simulations, there is sufficient region of span from which reliable flow data and loss estimations can be obtained. For blade flows at off-design conditions, a span of 20 per cent of the blade chord is sufficient to give good agreement with experimental data. This incurs a computational cost that may be too high to incorporate parametric LES studies into the design cycle of turbomachinery components with current computers.

Membrane proteomic signatures of karyotypically normal and abnormal human embryonic stem cell lines and derivatives

Cultured human embryonic stem cells (hESCs) and derived derivatives contain heterogeneous cell populations with varying degrees of differentiation and karyotypic stability. The inability to isolate homogenous population presents a challenge toward cell-based applications and therapies. A proteomics approach was utilized to discover novel membrane proteins able to distinguish between the hESC lines BG01, WA09, and abBG02 (trisomy 12, 14, 17 and an extra copy of the X chromosome), along with WA09-derived human neural progenitor (hNP) cells. Membrane protein signatures were developed using sucrose-gradient isolation, 1-D gel electrophoresis followed by in-gel digestion and analysis by reverse phase chromatography coupled to ion trap-FT-ICR. At a ≤1.0% false discovery rate, 1918 proteins were identified; 775 were annotated as membrane proteins and 720 predicted to contain transmembrane spanning regions. Flow cytometry was used to validate cell surface expression of selected proteins. Junctional adhesion molecule 1 expression was shared by BG01, BG02 and abBG02 hESC lines. Dysferlin expression was specific to the WA09 hESC line and not the derived neural or mesenchymal progenitors. Ciliary neurotrophic factor receptor distinguished WA09-derived human neural progenitor cells from the parent hESC population, and WA09-derived mesenchymal progenitor cells. This study expands the current membrane protein data set for hESCs.

Development and use of genic molecular markers (GMMs) for construction of a transcript map of chickpea (Cicer arietinum L.).

A transcript map has been constructed by the development and integration of genic molecular markers (GMMs) including single nucleotide polymorphism (SNP), genic microsatellite or simple sequence repeat (SSR) and intron spanning region (ISR)-based markers, on an inter-specific mapping population of chickpea, the third food legume crop of the world and the first food legume crop of India. For SNP discovery through allele re-sequencing, primer pairs were designed for 688 genes/expressed sequence tags (ESTs) of chickpea and 657 genes/ESTs of closely related species of chickpea. High-quality sequence data obtained for 220 candidate genic regions on 2–20 genotypes representing 9 Cicer species provided 1,893 SNPs with an average frequency of 1/35.83 bp and 0.34 PIC (polymorphism information content) value. On an average 2.9 haplotypes were present in 220 candidate genic regions with an average haplotype diversity of 0.6326. SNP2CAPS analysis of 220 sequence alignments, as mentioned above, provided a total of 192 CAPS candidates. Experimental analysis of these 192 CAPS candidates together with 87 CAPS candidates identified earlier through in silico mining of ESTs provided scorable amplification in 173 (62.01%) cases of which predicted assays were validated in 143 (82.66%) cases (CGMM). Alignments of chickpea unigenes with Medicago truncatula genome were used to develop 121 intron spanning region (CISR) markers of which 87 yielded scorable products. In addition, optimization of 77 EST-derived SSR (ICCeM) markers provided 51 scorable markers. Screening of easily assayable 281 markers including 143 CGMMs, 87 CISRs and 51 ICCeMs on 5 parental genotypes of three mapping populations identified 104 polymorphic markers including 90 markers on the inter-specific mapping population. Sixty-two of these GMMs together with 218 earlier published markers (including 64 GMM loci) and 20 other unpublished markers could be integrated into this genetic map. A genetic map developed here, therefore, has a total of 300 loci including 126 GMM loci and spans 766.56 cM, with an average inter-marker distance of 2.55 cM. In summary, this is the first report on the development of large-scale genic markers including development of easily assayable markers and a transcript map of chickpea. These resources should be useful not only for genome analysis and genetics and breeding applications of chickpea, but also for comparative legume genomics.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-011-1556-1) contains supplementary material, which is available to authorized users.