bluuebunny/crossref_metadata_2025 huggingface dataset:
KapI, a non-essential member of the Pse1p/Imp5 karyopherin family, controls colonial and asexual development in Aspergillus nidulans
Authors: Etxebeste Oier , Markina-Iñarrairaegui Ane , Garzia Aitor , Herrero-García Erika , Ugalde Unai , Espeso Eduardo A.
Published: November 2009

Asexual development in the filamentous fungus Aspergillus nidulans is governed by the timely expression and cellular localization of multiple transcription factors. Hence, factors mediating import and export across the nuclear pore complexes (karyopherins) are expected to play a key role in coordinating the developmental programme. Here we characterize KapI, a putative homologue of the Saccharomyces cerevisiae Kap121/Pse1p karyopherin. KapI is a non-essential importin-β-like protein located in the nucleus during vegetative growth and conidiophore development. The ΔkapI phenotype is aconidial with many aerial hyphae. This phenotype can be suppressed under abiotic stress. In this regard, it resembles that of the null allele of the bZIP transcription factor FlbB. However a ΔflbB; ΔkapI double mutant exhibited an additive phenotype with totally impaired conidiation, unresponsive to abiotic stress. In contrast to ΔflbB, the null kapI mutant is not a fluffy-low-bristle expression mutant. Taken together the findings indicate that KapI is required during asexual development, mediating the nuclear transport of factors acting in a different pathway(s) from those involving the upstream developmental activators.
Influence of type IV pilus retraction on the architecture of the Neisseria gonorrhoeae-infected cell cortex
Authors: Higashi Dustin L. , Zhang Gina H. , Biais Nicolas , Myers Lauren R. , Weyand Nathan J. , Elliott David A. , So Magdalene
Published: November 2009

Early in infection, Neisseria gonorrhoeae can be observed to attach to the epithelial cell surface as microcolonies and induce dramatic changes to the host cell cortex. We tested the hypothesis that type IV pili (Tfp) retraction plays a role in the ultrastructure of both the host cell cortex and the bacterial microcolony. Using serial ultrathin sectioning, transmission electron microscopy and 3D reconstruction of serial 2D images, we have obtained what we believe to be the first 3D reconstructions of the N. gonorrhoeae–host cell interface, and determined the architecture of infected cell microvilli as well as the attached microcolony. Tfp connect both wild-type (wt) and Tfp retraction-deficient bacteria with each other, and with the host cell membrane. Tfp fibres and microvilli form a lattice in the wt microcolony and at its periphery. Wt microcolonies induce microvilli formation and increases of surface area, leading to an approximately ninefold increase in the surface area of the host cell membrane at the site of attachment. In contrast, Tfp retraction-deficient microcolonies do not affect these parameters. Wt microcolonies had a symmetrical, dome-shaped structure with a circular ‘footprint’, while Tfp retraction-deficient microcolonies were notably less symmetrical. These findings support a major role for Tfp retraction in microvilli and microcolony architecture. They are consistent with the biophysical attributes of Tfp and the effects of Tfp retraction on epithelial cell signalling.
Vesicle-associated melanization in Cryptococcus neoformans
Authors: Eisenman Helene C. , Frases Susana , Nicola André M. , Rodrigues Marcio L. , Casadevall Arturo
Published: November 2009

Recently, several pathogenic fungi were shown to produce extracellular vesicles that contain various components associated with virulence. In the human pathogenic fungusCryptococcus neoformans, these components included laccase, an enzyme that catalyses melanin synthesis. Spherical melanin granules have been observed in the cell wall ofC. neoformans. Given that melanin granules have dimensions that are comparable to those of extracellular vesicles, and that metazoan organisms produce melanin in vesicular structures known as melanosomes, we investigated the role of vesicles in cryptococcal melanization. Extracellular vesicles melanized when incubated with the melanin precursorl-3,4-dihydroxyphenylalanine (l-DOPA). The kinetics of substrate incorporation into cells and vesicles was analysed using radiolabelledl-DOPA. The results indicated that substrate incorporation was different for cells and isolated vesicles. Acid-generated melanin ghosts stained with lipophilic dyes, implying the presence of associated lipid. A model forC. neoformansmelanization is proposed that accounts for these observations and provides a mechanism for the assembly of melanin into relatively uniform spherical particles stacked in an orderly arrangement in the cell wall.
Hydrophobic carboxy-terminal residues dramatically reduce protein levels in the haloarchaeon Haloferax volcanii
Authors: Reuter Christopher J. , Uthandi Sivakumar , Puentes Jose A. , Maupin-Furlow Julie A.
Published: November 2009

Proteolysis is important not only to cell physiology but also to the successful development of biocatalysts. While a wide-variety of signals are known to trigger protein degradation in bacteria and eukaryotes, these mechanisms are poorly understood in archaea, known for their ability to withstand harsh conditions. Here we present a systematic study in which single C-terminal amino acid residues were added to a reporter protein and shown to influence its levels in an archaeal cell. All 20 amino acid residues were examined for their impact on protein levels, using the reporter protein soluble modified red-shifted GFP (smRS-GFP) expressed in the haloarchaeon Haloferax volcanii as a model system. Addition of hydrophobic residues, including Leu, Cys, Met, Phe, Ala, Tyr, Ile and Val, gave the most pronounced reduction in smRS-GFP levels compared with the addition of either neutral or charged hydrophilic residues. In contrast to the altered protein levels, the C-terminal alterations had no influence on smRS-GFP-specific transcript levels, thus revealing that the effect is post-transcriptional.
New insights into the regulatory networks of paralogous genes in bacteria
Authors: Martínez-Núñez Mario A. , Pérez-Rueda Ernesto , Gutiérrez-Ríos Rosa María , Merino Enrique
Published: November 2009

Extensive genomic studies on gene duplication in model organisms such as Escherichia coli and Saccharomyces cerevisiae have recently been undertaken. In these models, it is commonly considered that a duplication event may include a transcription factor (TF), a target gene, or both. Following a gene duplication episode, varying scenarios have been postulated to describe the evolution of the regulatory network. However, in most of these, the TFs have emerged as the most important and in some cases the only factor shaping the regulatory network as the organism responds to a natural selection process, in order to fulfil its metabolic needs. Recent findings concerning the regulatory role played by elements other than TFs have indicated the need to reassess these early models. Thus, we performed an exhaustive review of paralogous gene regulation in E. coli and Bacillus subtilis based on published information, available in the NCBI PubMed database and in well-established regulatory databases. Our survey reinforces the notion that despite TFs being the most prominent components shaping the regulatory networks, other elements are also important. These include small RNAs, riboswitches, RNA-binding proteins, sigma factors, protein–protein interactions and DNA supercoiling, which modulate the expression of genes involved in particular metabolic processes or induce a more complex response in terms of the regulatory networks of paralogous genes in an integrated interplay with TFs.
Regulation of the dauBAR operon and characterization of d-amino acid dehydrogenase DauA in arginine and lysine catabolism of Pseudomonas aeruginosa PAO1
Authors: Li Congran , Yao Xiangyu , Lu Chung-Dar
Published: November 2009

A unique d-to-l racemization of arginine by coupled arginine dehydrogenases DauA and DauB encoded by the dauBAR operon has been recently reported as a prerequisite for d-arginine utilization as the sole source of carbon and nitrogen through l-arginine catabolic pathways in P. aeruginosa. In this study, enzymic properties of the catabolic FAD-dependent d-amino acid dehydrogenase DauA and the physiological functions of the dauBAR operon were further characterized with other d-amino acids. These results establish DauA as a d-amino acid dehydrogenase of broad substrate specificity, with d-Arg and d-Lys as the two most effective substrates, based on the kinetic parameters. In addition, expression of dauBAR is specifically induced by exogenous d-Arg and d-Lys, and mutations in the dauBAR operon affect utilization of these two amino acids alone. The function of DauR as a repressor in the control of the dauBAR operon was demonstrated by dauB promoter activity measurements in vivo and mobility shift assays with purified His-tagged protein in vitro. The potential effect of 2-ketoarginine (2-KA) derived from d-Arg deamination by DauA as a signal molecule in dauBAR induction was first revealed by mutation analysis and further supported by its in vitro effect on alleviation of DauR–DNA interactions. Through sequence analysis, putative DauR operators were identified and confirmed by mutation analysis. Induction of the dauBAR operon to the maximal level was found to require the l-arginine-responsive regulator ArgR, as supported by the loss of inductive effect by l-Arg on dauBAR expression in the argR mutant and binding of purified ArgR to the dauB regulatory region in vitro. In summary, this study establishes that optimal induction of the dauBAR operon requires relief of DauR repression by 2-KA and activation of ArgR by l-Arg as a result of d-Arg racemization by the encoded DauA and DauB.
Transcriptional and functional characterization of the gene encoding acyl carrier protein in Bacillus subtilis
Authors: Martinez Mariano A. , de Mendoza Diego , Schujman Gustavo E.
Published: November 2009

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid biosynthesis. The molecular mechanisms of regulation of theacpPstructural gene, as well as the function of its gene product, are poorly characterized inBacillus subtilisand other Gram-positive organisms. Here, we report that transcription ofacpPtakes place from two different promoters: PfapRand PacpP. Expression ofacpPfrom PfapRis coordinated with a cluster of genes involved in lipid synthesis (thefapRoperon); the operon consists offapR-plsX-fabD-fabG-acpP. PacpPis located immediately upstream of theacpPcoding sequence, and is necessary and sufficient for normal fatty acid synthesis. We also report thatacpPis essential for growth and differentiation, and that ACP localizes in the mother-cell compartment of the sporangium during spore formation. These results provide the first detailed characterization of the expression of the ACP-encoding gene in a Gram-positive bacterium, and highlight the importance of this protein inB. subtilisphysiology.
Degradation and aggresome formation of the Gn tail of the apathogenic Tula hantavirus
Authors: Wang Hao , Strandin Tomas , Hepojoki Jussi , Lankinen Hilkka , Vaheri Antti
Published: November 2009

The cytoplasmic tails of envelope glycoprotein Gn of pathogenic hantaviruses but not of the apathogenic Prospect Hill virus (PHV) were recently reported to be proteasomally degraded in simian COS7 cells. Here, we show that the cytoplasmic tails of the glycoproteins of the apathogenic hantaviruses Tula virus (TULV) and PHV are also degraded through the ubiquitin-proteasome pathway, both in human HEK-293 and in simian Vero E6 cells. TULV Gn tails formed aggresomes in cells with proteasomal inhibitors. We conclude that degradation upon aggregation of Gn tails, which may represent a general cellular response to misfolded protein used by hantaviruses to control maturation of virions, is unrelated to pathogenicity.
Modulation of alpha interferon anti-hepatitis C virus activity by ISG15
Authors: Chua Pong Kian , McCown Matthew F. , Rajyaguru Sonal , Kular Simran , Varma Ram , Symons Julian , Chiu Sophie S. , Cammack Nick , Nájera Isabel
Published: November 2009

ISG15 has recently been reported to possess antiviral properties against viruses, bothin vivoandin vitro. Knock-down of ISG15 gene expression by small interfering RNA followed by alpha interferon (IFN-α) treatment in Huh-7 cells resulted in an increased phenotypic sensitivity to IFN-α, as determined by measuring hepatitis C virus (HCV) RNA replication inhibition in stably transfected HCV replicon cells and in cells infected with genotype 1a HCVcc (infectious HCV). This IFN-α-specific effect, which was not observed with IFN-γ, correlated with an increase in expression of the IFN-α-inducible genes IFI6, IFITM3, OAS1 and MX1, whereas the expression of the non-IFN-α-inducible genes PTBP-1 and JAK1 remained unchanged. It has previously been reported that, unlike ISG15 knock-down, increased sensitivity to IFN-αafter knock-down of USP18 occurs through the prolonged phosphorylation of STAT-1. Combination knock-down of ISG15 and USP18 resulted in a moderate increase in IFN-α-inducible gene expression compared with single ISG15 or USP18 knock-down. Furthermore, the phenotype of increased gene expression after ISG15 knock-down and IFN-αtreatment was also observed in non-hepatic cell lines A549 and HeLa. Taken together, these results reveal a novel function for ISG15 in the regulation of the IFN-αpathway and its antiviral effect.
Establishment of an insect cell clone that harbours a partial baculoviral genome and is resistant to homologous virus infection
Authors: Weng Qingbei , Yang Kai , Xiao Wei , Yuan Meijin , Zhang Wenjun , Pang Yi
Published: November 2009

After serially undiluted passage of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), persistently infected Se301 cells were established. A cell strain, in which no polyhedra or viral particles were observed, was cloned and designated P8-Se301-C1. The P8-Se301-C1 cells are morphologically similar to but grow slower than Se301 cells and they can homologously interfere with SeMNPV. PCR analysis showed that SeMNPV ie-0 and polyhedrin genes were present but DNA polymerase and orf67 genes were absent in P8-Se301-C1, suggesting that the cells harbour incomplete SeMNPV genomes. Dot-blot analysis demonstrated that 0.32±0.16 ng SeMNPV DNA was present in 1.25×105 P8-Se301-C1 cells. A quantitative real-time PCR assay showed that there were 13.2±4.3 copies of the SeMNPV polyhedrin gene in each cell. Nested RT-PCR demonstrated the presence of SeMNPV polyhedrin transcripts in P8-Se301-C1 cells. The fact that P8-Se301-C1 cells carry low levels of partial viral genome but do not produce viral progeny suggests a latent-like viral infection in the cells.
Mutational analysis of eggplant latent viroid RNA processing in Chlamydomonas reinhardtii chloroplast
Authors: Martínez Fernando , Marqués Jorge , Salvador María L. , Daròs José-Antonio
Published: November 2009

Viroids of the family Avsunviroidae, such as eggplant latent viroid (ELVd), contain hammerhead ribozymes and replicate in the chloroplasts of the host plant through an RNA-based symmetrical rolling-circle mechanism in which oligomeric RNAs of both polarity are processed to monomeric linear RNAs (by cleavage) and to monomeric circular RNAs (by ligation). Using an experimental system consisting of transplastomic lines of the alga Chlamydomonas reinhardtii, a mutational analysis of sequence and structural elements in the ELVd molecule that are involved in transcript processing in vivo in a chloroplastic context was carried out. A collection of six insertion and three deletion ELVd mutants was created and expressed in C. reinhardtii chloroplast. All mutants cleaved efficiently except for the control with an insertion inside the hammerhead ribozyme domain, supporting the prediction that this domain is necessary and sufficient to mediate transcript cleavage in vivo. However, two deletion mutants that cleaved efficiently showed ligation defects, indicating that during RNA circularization, other parts of the molecule are involved in addition to the hammerhead ribozyme domain. This is probably a quasi double-stranded structure present in the central part of the molecule which contains the ligation site in an internal loop. However, the mutations prevented the viroid from infecting its natural host, eggplant, indicating that they affected other essential functions in ELVd infectious cycle. The insertion in the terminal loop of the right upper hairpin of ELVd did not have this effect; it was tolerated and partially maintained in the progeny.
Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2
Authors: Chambers Phil , Rima Bert K. , Duprex W. Paul
Published: November 2009

The Jeryl Lynn (JL) vaccine against mumps virus (MuV) contains two components, MuVJL5 and MuVJL2, which differ by over 400 nt. Due to the occurrence of bias in the direction of mutation, these differences and those found in nucleotide sequences of different isolates of the minor component in the vaccine (MuVJL2) might be due to the effect of ADAR-like deaminases on MuV grown in tissue-cultured cells. A molecular clone of MuVJL2 (pMuVJL2) and MuVJL2-specific helper plasmids were constructed in order to investigate molecular interactions between MuVJL5 and MuVJL2, to augment the existing molecular clone of MuVJL5 (pMuVJL5) and MuVJL5-specific helper plasmids. Genome and mRNA termini of MuVJL2 were characterized, and an unusual oligo-G insertion transcriptional editing event was detected near the F mRNA polyadenylation site of MuVJL2, but not of MuVJL5. Genes encoding glycoproteins of rMuVJL2 and rMuVJL5 have been exchanged to characterize the oligo-G insertion, which associated with the specific sequence of the F gene of MuVJL2 and not with any other genes or the RNA-dependent RNA polymerase of strain MuVJL2. The results indicate that a single G-to-A sequence change obliterates the co-transcriptional editing of the F mRNA and that this oligo-G insertion does not affect the growth of the virus.
Rat respiratory coronavirus infection: replication in airway and alveolar epithelial cells and the innate immune response
Authors: Funk C. Joel , Manzer Rizwan , Miura Tanya A. , Groshong Steve D. , Ito Yoko , Travanty Emily A. , Leete Jennifer , Holmes Kathryn V. , Mason Robert J.
Published: November 2009

The rat coronavirus sialodacryoadenitis virus (SDAV) causes respiratory infection and provides a system for investigating respiratory coronaviruses in a natural host. A viral suspension in the form of a microspray aerosol was delivered by intratracheal instillation into the distal lung of 6–8-week-old Fischer 344 rats. SDAV inoculation produced a 7 % body weight loss over a 5 day period that was followed by recovery over the next 7 days. SDAV caused focal lesions in the lung, which were most severe on day 4 post-inoculation (p.i.). Immunofluorescent staining showed that four cell types supported SDAV virus replication in the lower respiratory tract, namely Clara cells, ciliated cells in the bronchial airway and alveolar type I and type II cells in the lung parenchyma. In bronchial alveolar lavage fluid (BALF) a neutrophil influx increased the population of neutrophils to 45 % compared with 6 % of the cells in control samples on day 2 after mock inoculation. Virus infection induced an increase in surfactant protein SP-D levels in BALF of infected rats on days 4 and 8 p.i. that subsided by day 12. The concentrations of chemokines MCP-1, LIX and CINC-1 in BALF increased on day 4 p.i., but returned to control levels by day 8. Intratracheal instillation of rats with SDAV coronavirus caused an acute, self-limited infection that is a useful model for studying the early events of the innate immune response to respiratory coronavirus infections in lungs of the natural virus host.
Depletion of measles virus glycoprotein-specific antibodies from human sera reveals genotype-specific neutralizing antibodies
Authors: de Swart Rik L. , Yüksel Selma , Langerijs Carianne N. , Muller Claude P. , Osterhaus Albert D. M. E.
Published: November 2009

Measles virus (MV)-neutralizing antibodies in sera from vaccinated subjects are mainly directed against the haemagglutinin (H) protein. It has been shown previously that depletion of vaccination-induced H-specific antibodies by co-culture of sera with cells expressing the MV Edmonston strain H glycoprotein resulted in almost complete elimination of neutralizing activity. In the present study, MV H and/or fusion (F) protein-specific antibodies were depleted from sera of naturally immune subjects. Early convalescent samples were collected 1.5 years after a well-characterized measles outbreak in Luxembourg caused by a genotype C2 virus, whilst late convalescent samples were collected from healthy Dutch subjects born between 1960 and 1970. Depletion of both H- and F-specific antibodies completely eliminated virus-neutralizing (VN) activity against MV Edmonston. However, in the early convalescent samples, residual VN antibody against wild-type MV genotype C2 was detected. This demonstrated that, although the majority of MV-specific VN antibodies recognized epitopes conserved between different genotypes, genotype-specific VN epitopes were also induced. In sera depleted of H-specific antibodies only, VN activity against MV Edmonston was not completely eliminated, demonstrating the presence of F-specific VN antibodies. In conclusion, this study demonstrated that a fraction of VN antibodies induced by wild-type MV genotype C2 does not neutralize MV strain Edmonston. In addition, it was shown that, in sera from naturally immune donors, the majority of VN antibodies are specific for MV H protein, but up to 10 % of neutralizing antibodies are specific for MV F protein.
Eset partners with Oct4 to restrict extraembryonic trophoblast lineage potential in embryonic stem cells
Authors: Yuan Ping , Han Jianyong , Guo Guoji , Orlov Yuriy L. , Huss Mikael , Loh Yuin-Han , Yaw Lai-Ping , Robson Paul , Lim Bing , Ng Huck-Hui
Published: November 2009

The histone H3 Lys 9 (H3K9) methyltransferase Eset is an epigenetic regulator critical for the development of the inner cell mass (ICM). Although ICM-derived embryonic stem (ES) cells are normally unable to contribute to the trophectoderm (TE) in blastocysts, we find that depletion of Eset by shRNAs leads to differentiation with the formation of trophoblast-like cells and induction of trophoblast-associated gene expression. Using chromatin immmunoprecipitation (ChIP) and sequencing (ChIP-seq) analyses, we identified Eset target genes with Eset-dependent H3K9 trimethylation. We confirmed that genes that are preferentially expressed in the TE (Tcfap2a and Cdx2) are bound and repressed by Eset. Single-cell PCR analysis shows that the expression of Cdx2 and Tcfap2a is also induced in Eset-depleted morula cells. Importantly, Eset-depleted cells can incorporate into the TE of a blastocyst and, subsequently, placental tissues. Coimmunoprecipitation and ChIP assays further demonstrate that Eset interacts with Oct4, which in turn recruits Eset to silence these trophoblast-associated genes. Our results suggest that Eset restricts the extraembryonic trophoblast lineage potential of pluripotent cells and links an epigenetic regulator to key cell fate decision through a pluripotency factor.
Elucidation of the transcription network governing mammalian sex determination by exploiting strain-specific susceptibility to sex reversal
Authors: Munger Steven C. , Aylor David L. , Syed Haider Ali , Magwene Paul M. , Threadgill David W. , Capel Blanche
Published: November 2009

Despite the identification of some key genes that regulate sex determination, most cases of disorders of sexual development remain unexplained. Evidence suggests that the sexual fate decision in the developing gonad depends on a complex network of interacting factors that converge on a critical threshold. To elucidate the transcriptional network underlying sex determination, we took the first expression quantitative trait loci (eQTL) approach in a developing organ. We identified reproducible differences in the transcriptome of the embryonic day 11.5 (E11.5) XY gonad between C57BL/6J (B6) and 129S1/SvImJ (129S1), indicating that the reported sensitivity of B6 to sex reversal is consistent with a higher expression of a female-like transcriptome in B6. Gene expression is highly variable in F2 XY gonads from B6 and 129S1 intercrosses, yet strong correlations emerged. We estimated the F2 coexpression network and predicted roles for genes of unknown function based on their connectivity and position within the network. A genetic analysis of the F2 population detected autosomal regions that control the expression of many sex-related genes, includingSry(sex-determining region of the Y chromosome) andSox9(Sry-box containing gene 9), the key regulators of male sex determination. Our results reveal the complex transcription architecture underlying sex determination, and provide a mechanism by which individuals may be sensitized for sex reversal.
The dosage compensation complex shapes the conformation of the X chromosome in Drosophila
Authors: Grimaud Charlotte , Becker Peter B.
Published: November 2009

The dosage compensation complex (DCC) in Drosophila globally increases transcription from the X chromosome in males to compensate for its monosomy. We discovered a male-specific conformation of the X chromosome that depends on the associations of high-affinity binding sites (HAS) of the DCC. The core DCC subunits MSL1–MSL2 are responsible for this male-specific organization. Contrary to emerging concepts, we found that neither DCC assembly nor the conformation of the male X chromosome are influenced by nuclear pore components. We propose that nuclear organization of HAS is central to the faithful distribution of the DCC along the X chromosome.
A single-base resolution map of an archaeal transcriptome
Authors: Wurtzel Omri , Sapra Rajat , Chen Feng , Zhu Yiwen , Simmons Blake A. , Sorek Rotem
Published: November 2009

Organisms of the third domain of life, the Archaea, share molecular characteristics both with Bacteria and Eukarya. These organisms attract scientific attention as research models for regulation and evolution of processes such as transcription, translation, and RNA processing. We have reconstructed the primary transcriptome of Sulfolobus solfataricus P2, one of the most widely studied model archaeal organisms. Analysis of 625 million bases of sequenced cDNAs yielded a single-base-pair resolution map of transcription start sites and operon structures for more than 1000 transcriptional units. The analysis led to the discovery of 310 expressed noncoding RNAs, with an extensive expression of overlapping cis-antisense transcripts to a level unprecedented in any bacteria or archaea but resembling that of eukaryotes. As opposed to bacterial transcripts, most Sulfolobus transcripts completely lack 5′-UTR sequences, suggesting that mRNA/ncRNA interactions differ between Bacteria and Archaea. The data also reveal internal hotspots for transcript cleavage linked to RNA degradation and predict sequence motifs that promote RNA destabilization. This study highlights transcriptome sequencing as a key tool for understanding the mechanisms and extent of RNA-based regulation in Bacteria and Archaea.
Preparation of Insoluble GST Fusion Proteins
Authors: Rebay Ilaria , Fehon Richard G.
Published: November 2009

INTRODUCTION

A wide variety of plasmid vectors are commercially available for the production of fusion proteins in bacterial cells. Most are also designed to incorporate a tag that allows affinity purification of the expressed fusion protein from bacterial cell extracts. The most commonly used are vectors that incorporate a portion of the glutathione-S-transferase (GST) enzyme that is able to bind to immobilized glutathione and vectors that use a polyhistidine tag which binds immobilized nickel ions with high affinity. This protocol describes preparation of an insoluble GST fusion protein, isolated using glutathione agarose beads.
Confocal Immunocytochemistry of Embryonic and Juvenile Hawaiian Bobtail Squid (Euprymna scolopes) Tissues
Authors: Lee Patricia N. , McFall-Ngai Margaret J. , Callaerts Patrick , de Couet H. Gert
Published: November 2009

INTRODUCTION

The Hawaiian bobtail squid Euprymna scolopes is a cephalopod whose small size, short lifespan, rapid growth, and year-round availability make it suitable as a model organism. This protocol describes the preparation of whole juvenile squids by whole-mount immunocytochemistry for visualization by confocal microscopy.
Culturing Meristematic Tissue and Node Cuttings from Yams (Dioscorea)
Authors: Mignouna Hodeba D. , Abang Mathew M. , Asiedu Robert , Geeta R.
Published: November 2009

INTRODUCTION

This protocol describes how to produce whole yam (Dioscorea) plantlets in vitro through yam meristem culture and from yam node cuttings.
Extraction of DNA from Yam (Dioscorea) Leaves
Authors: Mignouna Hodeba D. , Abang Mathew M. , Asiedu Robert , Geeta R.
Published: November 2009

INTRODUCTION

This protocol describes how to extract DNA from the leaves of true yams (Dioscorea) for polymerase chain reaction (PCR) and other analyses.
Breastfeeding promotion for infants in neonatal units: a systematic review
Authors: Renfrew M. J. , Dyson L. , McCormick F. , Misso K. , Stenhouse E. , King S. E. , Williams A. F.
Published: November 2009

Background  Breastfeeding/breastmilk feeding of infants in neonatal units is vital to the preservation of short‐ and long‐term health, but rates are very low in many neonatal units internationally. The aim of this review was to evaluate the effectiveness of clinical, public health and health promotion interventions that may promote or inhibit breastfeeding/breastmilk feeding for infants admitted to neonatal units.

Methods  Systematic review with narrative synthesis. Studies were identified from structured searches of 19 electronic databases from inception to February 2008; hand searching of bibliographies; Advisory Group members helped identify additional sources. Inclusion criteria: controlled studies of interventions intended to increase breastfeeding/feeding with breastmilk that reported breastmilk feeding outcomes and included infants admitted to neonatal units, their mothers, families and caregivers. Data were extracted and appraised for quality using standard processes. Study selection, data extraction and quality assessment were independently checked. Study heterogeneity prevented meta‐analysis.

Results  Forty‐eight studies were identified, mainly measuring short‐term outcomes of single interventions in stable infants. We report here a sub‐set of 21 studies addressing interventions tested in at least one good‐quality or more than one moderate‐quality study. Effective interventions identified included kangaroo skin‐to‐skin contact, simultaneous milk expression, peer support in hospital and community, multidisciplinary staff training, and Unicef Baby Friendly accreditation of the associated maternity hospital.

Conclusions  Breastfeeding/breastmilk feeding is promoted by close, continuing skin‐to‐skin contact between mother and infant, effective breastmilk expression, peer support in hospital and community, and staff training. Evidence gaps include health outcomes and costs of intervening with less clinically stable infants, and maternal health and well‐being. Effects of public health and policy interventions and the organization of neonatal services remain unclear. Infant feeding in neonatal units should be included in public health surveillance and policy development; relevant definitions are proposed.
A longitudinal study of gastrointestinal diseases in individuals diagnosed with infantile autism as children
Authors: Mouridsen S. E. , Rich B. , Isager T.
Published: November 2009

Background  A number of studies have indicated a link between gastrointestinal (GI) diseases and autism spectrum disorders.

Method  The objective of this study was to compare the prevalence and types of GI diseases in a clinical sample of 118 individuals diagnosed as children with infantile autism (IA) with GI diseases in 336 matched controls from the general population, based on data from the nationwide Danish National Hospital Register (DNHR). The average observation time was 30.3 years (SD 0.4) (range 27–30 years), and mean age at the end of the observation period was 42.7 years (SD 7.7) (range between 27 and 57 years of age).

Results  Of the 118 individuals with IA, 97 (82.2%) had been in contact with a medical hospital (inpatient hospitalization or outpatient visits) during the observation period, compared with 312/336 (92.9%) in the control group (P= 0.001). A similar proportion of members from the case and comparison group had a diagnosis of any GI disease in the DNHR: 30.5% against 30.7%, but the nature of their diseases may be somewhat different. Only diseases of oral cavity were significantly associated with IA: 20.3% against 1.2%, P < 0.0001. Otherwise, specific GI diseases occurred with low frequency in both groups.

Conclusion  Overall, no evidence was found that patients with IA were more likely than control persons without IA to have defined GI diseases during the 30.3‐year observation period.
Pollen‐derived low‐molecular weight factors inhibit 6‐sulfo LacNAc+ dendritic cells' capacity to induce T‐helper type 1 responses
Authors: Gilles S. , Jacoby D. , Blume C. , Mueller M. J. , Jakob T. , Behrendt H. , Schaekel K. , Traidl‐Hoffmann C.
Published: November 2009

Background
Evidence is accumulating that the pollen exsudate contains an array of non‐allergenic, pro‐inflammatory and immunomodulatory substances acting on the innate and adaptive immune system. In this context, pollen‐associated E1‐phytoprostanes (PPE1) were shown to licence human monocyte‐derived dendritic cells for T‐helper type 2 (Th2) polarization of naïve T cells.

Objective
This study aims at analysing the impact of pollen‐associated lipid mediators on cytokine secretion and maturation of 6‐sulfo LacNAc+ dendritic cells (slanDCs), the most abundant native dendritic cell (DC) in human peripheral blood, and further dissecting the biologically active substance(s) within aqueous pollen extracts.

Results
Aqueous birch pollen extracts dose‐dependently inhibited the lipopolysaccharide (LPS)‐induced IL‐12 p70 production, while the levels of IL‐6 remained unaffected. PPE1 inhibited secretion of both IL‐12 p70 and IL‐6. Aqueous pollen extracts, but not PPE1 or F1‐phytoprostanes significantly reduced the LPS‐induced surface expression of the maturation markers CD80, CD83, CD40 and CCR‐7, an effect that was independent of proteins and that was still present in a 3 kDa cut‐off fraction of the pollen extract. These effects were observed irrespective of the atopy status of the donors. Finally, slanDCs exposed to aqueous pollen extracts were impaired in eliciting an IFN‐γ response in naïve CD4+ T cells.

Conclusion
Our data show that slanDCs, a subset of human blood DCs with constitutively high potency to induce Th1 responses, are susceptible to the Th2 polarizing effect of low molecular weight, non‐protein factors derived from pollen.

Cite this as: S. Gilles, D. Jacoby, C. Blume, M. J. Mueller, T. Jakob, H. Behrendt, K. Schaekel and C. Traidl‐Hoffmann, Clinical & Experimental Allergy, 2010 (40) 269–278.
The role of paracetamol in the pathogenesis of asthma
Authors: Farquhar H. , Stewart A. , Mitchell E. , Crane J. , Eyers S. , Weatherall M. , Beasley R.
Published: November 2009

Paracetamol use represents a putative risk factor for the development of asthma. There is convincing epidemiological evidence that the risk of asthma may be increased with exposure to paracetamol in the intrauterine environment, infancy, later childhood and adult life. A dose‐dependent association has also been observed in these different age groups in different populations world‐wide. An association has also been shown between paracetamol use in both rhinoconjunctivitis and eczema. There is biological plausibility with paracetamol use leading to decreased glutathione levels resulting in increased oxidant‐induced inflammation and potentially enhanced T‐helper type 2 responses. At the population level, patterns of paracetamol use might explain, to some extent, the world‐wide variation in the prevalence of asthma and related disorders, particularly the high rates in English‐speaking countries, which have high per capita prescription and over‐the‐counter use of paracetamol. A temporal association also exists between the international trends of increasing paracetamol use and increasing prevalence of asthma over recent decades. Further research is urgently required, in particular randomized‐controlled trials (RCTs) into the long‐term effects of frequent paracetamol use in childhood, to determine the magnitude and characteristics of any such risk. Importantly, RCTs will also enable evidence‐based guidelines for the recommended use of paracetamol to be developed.

Cite this as: H. Farquhar, A. Stewart, E. Mitchell, J. Crane, S. Eyers, M. Weatherall and R. Beasley,Clinical & Experimental Allergy, 2010 (40) 32–41.
Spring migration decisions in relation to weather are predicted by wing morphology among trans‐Mediterranean migratory birds
Authors: Saino Nicola , Rubolini Diego , Von Hardenberg Jost , Ambrosini Roberto , Provenzale Antonello , Romano Maria , Spina Fernando
Published: November 2009

1. Meteorological conditions are critical determinants of bird migration. However, response to weather should vary among species as well as between males and females, as phenotypic traits relevant to migration are also shaped by natural selection acting differentially on species and either sex in other life cycle phases.

2. We studied the covariation between capture frequencies and meteorological conditions at departure and at a stopover site during northward spring migration in 21 species of migratory birds. The stopover site is a small island located at the northern end of a wide ecological barrier (the Mediterranen Sea), that is crossed with non‐stop flights by migrants departing from North Africa.

3. Head and crosswinds at departure, and tail and crosswinds at stopover reduced the number of birds captured, although the strength of these effects varied among species. In the subset of 10 sexually dimorphic species, males and females were not differentially susceptible to weather conditions.

4. Because sexual and natural selection pressures on traits relevant to migration vary among species, we investigated whether body mass and wing morphology predicted species susceptibility to weather in analyses controlling for phylogenetic effects, using wing loading, wing aspect ratio, the ‘brachial index’, and wingtip roundedness and convexity as indicators of the morphology of the flight apparatus.

5. Captures of species whose wing morphology is more typical of long‐distance migrants were less affected by tail and crosswinds, suggesting that long‐distance migrants are less susceptible to weather.

6. Meteorological conditions at departure independently concurred with those at stopover in determining capture frequencies. The effect of weather on migration covaried with wing morphological traits that co‐evolved with migratory strategies. Changes in meteorological conditions driven by current climate change thus have the potential to differentially affect migration phenology in relation to migratory strategy and morphology of individual species.
Ultraviolet crown colouration affects contest outcomes among male blue tits, but only in the absence of prior encounters
Authors: Vedder Oscar , Schut Elske , Magrath Michael J. L. , Komdeur Jan
Published: November 2009

1. Static animal colour patches may function among competitors to minimize conflict escalation over resources, by serving as a signal of resource holding potential or aggressiveness. Empirical evidence for the use of colour patches in conflict resolution is largely restricted to pigment‐based colours (melanins and carotenoids) and rarely defines the context in which the signals are used.

2. Here we test whether structural‐based ultraviolet (UV) crown colouration functions in conflict resolution among dyads of first‐year male blue tits Cyanistes caeruleus in captivity, in a context that is known to favour aggressive individuals.

3. We found that on first encounter in a pairwise context, experimentally UV‐reduced males were significantly more likely to lose to control‐treated opponents than expected by chance. However, this disparity was less pronounced when conflicts were settled with physical fighting or when the opponent was considerably smaller in size.

4. When the same dyads were tested again several weeks later, but with the UV treatment reversed among opponents, none of the effects remained significant, but instead the winner was most likely to be the individual that won at their first encounter.

5. Our results suggest that structural‐based UV colouration can affect the outcome of an interaction, but that size differences and the outcome of initial interactions between opponents can override the influence of this signal in conflict resolution. Whether there is a functional basis to maintain a link between aggressiveness and colouration may thus be highly dependent on the general context under which individuals compete in the wild.
Spindle cell lesions of the bladder and urinary tract
Authors: Shanks Jonathan H , Iczkowski Kenneth A
Published: November 2009

Spindle cell lesions of the urinary tract encompass a variety of benign and malignant tumours as well as a group of lesions of controversial nomenclature that is the subject of ongoing debate. This review focuses on our current and evolving understanding of the lesion variably referred to as inflammatory myofibroblastic tumour, pseudosarcomatous myofibroblastic proliferation or inflammatory pseudotumour and the main differential diagnoses of sarcomatoid carcinoma and sarcoma. Other spindle cell lesions of the bladder are described.
Hypermethylation of APC promoter 1A is associated with moderate activation of Wnt signalling pathway in a subset  of colorectal serrated adenomas
Authors: Fu Xiangsheng , Li Jing , Li Kang , Tian Xiaoxiao , Zhang Yali
Published: November 2009

Aims:  The role of Wnt signalling pathway in serrated adenomas (SAs) remains to be identified. The aim of this study was to determine whether Wnt signalling plays a role in the pathogenesis of SAs, and to clarify the mechanism of Wnt signalling activation in SAs.

Methods and results:  This study investigated immunoreactivities of adenomatous polyposis coli (APC) and β‐catenin, mutations of APC and β‐catenin genes, methylation status of APC promoter 1A in 12 SAs, and compared the findings with normal colorectal mucosa, hyperplastic polyps, traditional adenomas (TAs) and colorectal cancers (CRCs). APC expression was moderately decreased in SAs. Cytoplasmic accumulation of β‐catenin was demonstrated in 41.7% (5/12) of SAs, but membranous immunoreactivity of β‐catenin was lost in only 8.3% (1/12) of SAs. No β‐catenin mutation was detected in any of 12 SAs, and only one SA was found to be positive for APC gene mutation. Complete methylation of APC promoter 1A was found in 41.7% (5/12) of SAs, but in no TAs or CRCs.

Conclusions:  Hypermethylation of APC promoter 1A, instead of mutations involving APC and β‐catenin, contributes to moderate activation of Wnt signalling in a subset of SAs.
Mucin extravasation in breast core biopsies – clinical significance and outcome correlation
Authors: Begum S M Khodeza Nahar , Jara‐Lazaro Ana Richelia , Thike Aye Aye , Tse Gary M‐K , Wong Jill S‐L , Ho Juliana T‐S , Tan Puay Hoon
Published: November 2009

Aims:  To document the spectrum of lesions associated with mucin extravasation (ME) in breast core biopsy specimens, and to correlate with open surgical excisions.

Methods and results:  Thirty‐nine lesions in 37 women with ME on core biopsies constituted the study group. Fibrocystic change (FC), atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS) were found in 21 (53.8%), 13 (33.3%) and four (10.3%) core biopsy specimens, respectively, with one (2.6%) consisting only of mucin pools. Except for the latter, all disclosed mucocoele‐like lesions (MLL) accompanying ME. Columnar cell lesions (CCL) were frequently observed (84.6%). On open biopsy, three cases underdiagnosed on core biopsy included FC that later disclosed ADH; one ADH lesion on core later upgraded to DCIS; and a case of mucin pools that revealed mucinous carcinoma on excision. The extent of CCL on core biopsy appeared to predict sinister lesions on open excision. For calcified lesions that were completely removed on core biopsy, there were no malignant lesions discovered on open excision that had not already been diagnosed preoperatively.

Conclusions:  ME and MLL on core biopsy warrant close radiological–pathological correlation. When the entire radiological abnormality has been removed with large core mammotome biopsy specimens, surgery may potentially be avoided in histologically benign lesions, although such an approach requires further validation.
Clinicopathological study of a series of 92 adrenocortical carcinomas: from a proposal of simplified diagnostic algorithm to prognostic stratification
Authors: Volante Marco , Bollito Enrico , Sperone Paola , Tavaglione Veronica , Daffara Fulvia , Porpiglia Francesco , Terzolo Massimo , Berruti Alfredo , Papotti Mauro
Published: November 2009

Aims:  Pathological diagnosis of adrenocortical carcinoma relies on several microscopic features commonly used in combination in different scoring systems that are sometimes subjective and/or time consuming. The aim was to investigate the impact of individual pathological parameters in the diagnosis and prognosis of adrenocortical carcinoma.

Methods and results:  The series included 92 malignant cases and a control series of 47 adenomas, all classified according to Weiss score criteria. The presence of disruption of the reticular network, as highlighted by histochemical staining, was present in all adrenocortical carcinomas and the inclusion of at least one of the three following additional parameters – mitotic index >5/50 high‐power fields (HPF), presence of necrosis and presence of vascular invasion – gave an algorithm with 100% sensitivity and specificity to recognize malignant tumours according to the Weiss system, with easier and more practical applicability. Moreover, on multivariate analysis, stage III/IV and mitotic count >9/50 HPF showed a strong adverse impact on disease‐free and overall survival, leading to the identification of three risk groups affected by a significantly different prognosis.

Conclusions:  We have defined an easy‐to‐perform and highly specific and sensitive algorithm for the diagnosis and prognostic categorization of adrenocortical tumours.
Biological role of NHERF1 protein expression in breast cancer
Authors: Mangia Anita , Chiriatti Annalisa , Bellizzi Antonia , Malfettone Andrea , Stea Baldassarre , Zito Francesso Alfredo , Reshkin Stephen Joel , Simone Giovanni , Paradiso Angelo
Published: November 2009

Aims:  To determine the role of Na+/H+ exchanger regulatory factor (NHERF1) in breast cancerogenesis and progression.

Methods and results:  NHERF1 expression was examined in normal tissue, ductal carcinoma in situ (DCIS), invasive carcinoma (IBC), synchronous metastatic lymph node and metachronous distant metastases of a retrospective series of breast cancers. Fifty‐one IBC, 42 DCIS and normal tissues were examined immunohistochemically, and the colocalization between NHERF1 and HER2/neu was studied by immunofluorescence. NHERF1 showed a different localization and pattern of expression in the different compartments of the breast. The mean value of cytoplasmic NHERF1 expression in paired samples was significantly higher in DCIS, IBC, distant metastases and metastatic lymph nodes with respect to normal tissues. Moreover, in metastatic lymph nodes NHERF1 was exclusively cytoplasmic. In the membrane NHERF1 was colocalized with overexpressed HER2/neu in DCIS, IBC and distant metastases.

Conclusions:  Breast cancerogenesis is characterized by increased cytoplasmic expression of NHERF1 as the tumour progresses, suggesting a role in this process. The switch from apical membranous to cytoplasmic expression is compatible with a dual role for NHERF1 as a tumour suppressor or tumour promoter dependent on its subcellular localization.