Replication protein A-directed unloading of PCNA by the Ctf18 cohesion establishment complex

The replication clamp PCNA is loaded around DNA by replication factor C (RFC) and functions in DNA replication and repair. Regulated unloading of PCNA during the progression and termination of DNA replication may require additional factors. Here we show that a Saccharomyces cerevisiae complex required for the establishment of sister chromatid cohesion functions as an efficient unloader of PCNA. Unloading requires ATP hydrolysis. This seven-subunit Ctf18-RFC complex consists of the four small subunits of RFC, together with Ctf18, Dcc1, and Ctf8. Ctf18-RFC was also a weak loader of PCNA onto naked template-primer DNA. However, when the single-stranded DNA template was coated by the yeast single-stranded DNA binding protein replication protein A (RPA) but not by a mutant form of RPA or a heterologous single-stranded DNA binding protein, both binding of Ctf18-RFC to substrate DNA and loading of PCNA were strongly inhibited, and unloading predominated. Neither yeast RFC itself nor two other related clamp loaders, containing either Rad24 or Elg1, catalyzed significant unloading of PCNA. The Dcc1 and Ctf8 subunits of Ctf18-RFC, while required for establishing sister chromatid cohesion in vivo, did not function specifically in PCNA unloading in vitro, thereby separating the functionality of the Ctf18-RFC complex into two distinct paths.     

A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production

A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted. The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A. vadensis were much higher than those of A. niger. These characteristics make A. vadensis a very promising candidate for homologous, and possibly heterologous, protein production.     

Lineage-specific gene duplication and loss in human and great ape evolution

Given that gene duplication is a major driving force of evolutionary change and the key mechanism underlying the emergence of new genes and biological processes, this study sought to use a novel genome-wide approach to identify genes that have undergone lineage-specific duplications or contractions among several hominoid lineages. Interspecies cDNA array-based comparative genomic hybridization was used to individually compare copy number variation for 39,711 cDNAs, representing 29,619 human genes, across five hominoid species, including human. We identified 1,005 genes, either as isolated genes or in clusters positionally biased toward rearrangement-prone genomic regions, that produced relative hybridization signals unique to one or more of the hominoid lineages. Measured as a function of the evolutionary age of each lineage, genes showing copy number expansions were most pronounced in human (134) and include a number of genes thought to be involved in the structure and function of the brain. This work represents, to our knowledge, the first genome-wide gene-based survey of gene duplication across hominoid species. The genes identified here likely represent a significant majority of the major gene copy number changes that have occurred over the past 15 million years of human and great ape evolution and are likely to underlie some of the key phenotypic characteristics that distinguish these species.     

The fidelity of DNA replication,particularly on GC-rich templates,is reduced by defects of the Fe–S cluster in DNA polymerase δ

Iron-sulfur clusters (4Fe–4S) exist in many enzymes concerned with DNA replication and repair. The contribution of these clusters to enzymatic activity is not fully understood. We identified the MET18 (MMS19) gene of Saccharomyces cerevisiae as a strong mutator on GC-rich genes. Met18p is required for the efficient insertion of iron-sulfur clusters into various proteins. met18 mutants have an elevated rate of deletions between short flanking repeats, consistent with increased DNA polymerase slippage. This phenotype is very similar to that observed in mutants of POL3 (encoding the catalytic subunit of Pol δ) that weaken binding of the iron-sulfur cluster. Comparable mutants of POL2 (Pol ϵ) do not elevate deletions. Further support for the conclusion that met18 strains result in impaired DNA synthesis by Pol δ are the observations that Pol δ isolated from met18 strains has less bound iron and is less processive in vitro than the wild-type holoenzyme.     

人呼吸道合胞病毒兰州株基因组全长cDNA克隆的构建及鉴定

目的构建用于呼吸道合胞病毒(respiratory syncytial virus,RSV)体外拯救的RSV基因组全长cDNA克隆,并进行鉴定。方法根据RSV Long株基因组序列设计并合成引物,利用RT-PCR技术分6段扩增RSV LZ01/09基因组序列并构建克隆载体;测序后,利用重叠PCR与酶切连接技术,根据基因组序列选择特异性酶切位点,引入Kpn I、Xma I和Sal I酶切位点,构建成4个亚克隆载体;将亚克隆载体的插入片段连接至经过改造且包含T7启动子、锤头状核酶、多克隆位点、丁肝核酶、T7终止子的p RSV1载体中,构建RSV基因组全长cDNA克隆;对克隆全长cDNA序列进行测定,与亲本RSV LZ01/09基因组进行同源性比对分析,并与RSV实验参比株进行系统进化树分析。结果测序结果显示,RSV LZ 01/09的基因组全长为15 204 bp,与GenBank公布的RSV基因组序列长度相当,将完整的序列提交GenBank,登录号为KY782635;酶切及测序结果显示,用于RSV全长cDNA克隆构建的基本载体p BSKS-MCS(简称p RSV1)与预期相符,RSV全长基因组cDNA克隆质粒(简称转录载体p RSV1-4F)酶切片段大小与预期一致;同源性比对结果显示,全长cDNA序列与亲本RSV LZ01/09基因组序列同源性高达99.83%;系统进化树分析结果显示,其与RSV-A亚型序列同属于一个分支。结论测序及酶切分析结果表明已成功构建RSVLZ01/09基因组全长cDNA克隆,为建立拯救RSV重组病毒的反向遗传学系统平台奠定了基础。     

Substructural specificity and polyvalent carbohydrate recognition by the Entamoeba histolytica and rat hepatic N- acetylgalactosamine/galactose lectins

Both the Entamoeba histolytica lectin, a virulence factor for the causative agent of amebiasis, and the mammalian hepatic lectin bind to N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on oligosaccharides, with preference for GalNAc. Polyvalent GalNAc- derivatized neoglycoproteins have >1000-fold enhanced binding affinity for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr. and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were required for binding to both lectins, whereas only the E.histolytica lectin required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to the E.histolytica lectin than to the mammalian hepatic lectin, galactosamine and N-benzoyl galactosamine bind with higher affinity to the E. histolytica lectin. Therefore, a synthetic scheme for converting polyamine carriers to poly-N-acyl galactosamine derivatives (linked through the galactosamine primary amino group) was developed to test whether such ligands would bind the E.histolytica lectin with high specificity and high affinity. Contrary to expectations, polyvalent derivatives including GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the mammalian hepatic lectin but little or no enhancement of binding to the E.histolytica lectin. We propose that the mammalian hepatic lectin binds with greatest affinity to GalNAc "miniclusters," which mimic branched termini of N-linked oligosaccharides, whereas the E.histolytica lectin binds most effectively to "maxiclusters," which may mimic more widely spaced GalNAc residues on intestinal mucins.      

Normalized lift: an energy interpretation of the lift coefficient simplifies comparisons of the lifting ability of rotating and flapping surfaces

For a century, researchers have used the standard lift coefficient C(L) to evaluate the lift, L, generated by fixed wings over an area S against dynamic pressure, ?ρv(2), where v is the effective velocity of the wing. Because the lift coefficient was developed initially for fixed wings in steady flow, its application to other lifting systems requires either simplifying assumptions or complex adjustments as is the case for flapping wings and rotating cylinders.This paper interprets the standard lift coefficient of a fixed wing slightly differently, as the work exerted by the wing on the surrounding flow field (L/ρ·S), compared against the total kinetic energy required for generating said lift, ?v(2). This reinterpreted coefficient, the normalized lift, is derived from the work-energy theorem and compares the lifting capabilities of dissimilar lift systems on a similar energy footing. The normalized lift is the same as the standard lift coefficient for fixed wings, but differs for wings with more complex motions; it also accounts for such complex motions explicitly and without complex modifications or adjustments. We compare the normalized lift with the previously-reported values of lift coefficient for a rotating cylinder in Magnus effect, a bat during hovering and forward flight, and a hovering dipteran.The maximum standard lift coefficient for a fixed wing without flaps in steady flow is around 1.5, yet for a rotating cylinder it may exceed 9.0, a value that implies that a rotating cylinder generates nearly 6 times the maximum lift of a wing. The maximum normalized lift for a rotating cylinder is 1.5. We suggest that the normalized lift can be used to evaluate propellers, rotors, flapping wings of animals and micro air vehicles, and underwater thrust-generating fins in the same way the lift coefficient is currently used to evaluate fixed wings.     

Automated determination of bone age and bone mineral density in patients with juvenile idiopathic arthritis: a feasibility study

Introduction

Chronic inflammation combined with glucocorticoid treatment and immobilization puts juvenile idiopathic arthritis (JIA) patients at risk of impaired growth and reduced bone mineral density (BMD). Conventional methods for evaluating bone age and BMD are time-consuming or come with additional costs and radiation exposure. In addition, an automated measurement of bone age and BMD is likely to be more consistent than visual evaluation. In this study, we aimed to evaluate the feasibility of an automated method for determination of bone age and (cortical) bone mineral density (cBMD) in severely affected JIA patients. A secondary objective was to describe bone age and cBMD in this specific JIA population eligible for biologic treatment.

Methods

In total, 69 patients with standard hand radiographs at the start of etanercept treatment and of calendar age within the reliability ranges (2.5 to 17 years for boys and 2 to 15 years for girls) were extracted from the Dutch Arthritis and Biologicals in Children register. Radiographs were analyzed using the BoneXpert method, thus automatically determining bone age and cBMD expressed as bone health index (BHI). Agreement between measurements of the left- and right-hand radiographs and a repeated measurement of the left hand were assessed with the intraclass correlation coefficient (ICC). Regression analysis was used to identify variables associated with Z-scores of bone age and BHI.

Results

The BoneXpert method was reliable in the evaluation of radiographs of 67 patients (radiographs of 2 patients were rejected because of poor image quality). Agreement between left- and right-hand radiographs (ICC = 0.838 to 0.996) and repeated measurements (ICC = 0.999 to 1.000) was good. Mean Z-scores of bone age (−0.36, P = 0.051) and BHI (−0.85, P < 0.001) were lower compared to the healthy population. Glucocorticoid use was associated with delayed bone age (0.79 standard deviation (SD), P = 0.028), and male gender was associated with a lower Z-score of BHI (0.65 SD, P = 0.021).

Conclusions

BoneXpert is an easy-to-use method for assessing bone age and cBMD in patients with JIA, provided that radiographs are of reasonable quality and patients’ bone age lies within the age ranges of the program. The population investigated had delayed bone maturation and lower cBMD than healthy children.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0424-1) contains supplementary material, which is available to authorized users.     

Peptide profiling of cerebrospinal fluid by mass spectrometry

The search for biomarkers is driven by the increasing clinical importance of early diagnosis. Reliable biomarkers can also assist in directing therapy, monitoring disease activity and the efficacy of treatment. In addition, the discovery of novel biomarkers might provide clues to the pathogenesis of a disease. The dynamic range of protein concentrations in body fluids exceeds 10 orders of magnitude. These huge differences in concentrations complicate the detection of proteins with low expression levels. Since all classical biomarkers have low expression levels (e.g., prostate-specific antigen: 2-4 microg/l; and CA125: 20-35 U/ml), new developments with respect to identification and validation techniques of the low-abundance proteins are required. This review will discuss the current status of profiling cerebrospinal fluid using mass spectrometry-based techniques, and new developments in this area.