bluuebunny/crossref_metadata_2025_split huggingface dataset:
Overexpressing Tagged Proteins in Plants Using a Modified Gateway Cloning Strategy: Figure 1.
Authors: Dubin Manu J. , Bowler Chris , Benvenuto Giovanna 
Published: March 2010

INTRODUCTIONIn recent years, sequence-specific recombination cloning methods such as the Gateway system have become increasingly popular for (over)expressing tagged proteins in high-throughput investigations in many different organisms, including plants. Because of their versatility and ease of use, these methods have gained favor in low- and medium-throughput investigations as well. However, due to the recombination step, the resulting fusion proteins contain long and often highly charged polylinker sequences that can interfere with their physiological function. Furthermore, in some cases the gene of interest must be cloned twice (once with and once without a stop codon) for N- and C-terminal tagging. Here, we present a hybrid combinatorial cloning strategy that overcomes many of these limitations. In the first step, the gene of interest is cloned into an entry vector containing standardized cloning sites with the desired N- or C-terminal tag and an optimized polylinker sequence. A Gateway recombination reaction is used to transfer the protein-tag fusion from the entry clone to a Gateway destination vector with the desired promoter and selectable marker for the organism of interest. As experimental requirements evolve, constructs for expressing the protein of interest with the desired tag, promoter, and selectable marker or other features can rapidly and easily be created.
Baculovirus-Based Display and Gene Delivery Systems: Figure 1.
Authors: Mäkelä Anna R. , Ernst Wolfgang , Grabherr Reingard , Oker-Blom Christian 
Published: March 2010

INTRODUCTIONThe baculovirus expression vector system has been used extensively to produce numerous proteins originating from both prokaryotic and eukaryotic sources. In addition to easy cloning techniques and abundant viral propagation, the system’s insect cell environment provides eukaryotic post-translational modification machinery. The recently established eukaryotic molecular biology tool, the baculovirus display vector system (BDVS), allows the combination of genotype with phenotype, enabling presentation of foreign peptides or even complex proteins on the baculoviral envelope or capsid. This strategy is important because it can be used to enhance viral binding and entry to mammalian cells as well as to produce antibodies against the displayed antigen. In addition, the technology should enable modifications of intracellular behavior, that is, trafficking of recombinant “nanoparticles,” a highly relevant feature for studies of targeted gene or protein delivery. This article discusses the design and potential uses of insect-derived baculoviral display vectors.
Management of drooling in disabled patients with scopolamine patches
Authors: Mato Abigail , Limeres Jacobo , Tomás Inmaculada , Muñoz Maria , Abuín Concepción , Feijoo Javier F. , Diz Pedro 
Published: March 2010

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Drooling is a common problem in disabled individuals and has significant physical, psychosocial and aesthetic repercussions. Scopolamine has been used to treat drooling in a number of medical specialities, including otorhinolaryngology, neurology and palliative care. This study evaluates the efficacy of this treatment in severely disabled patients.WHAT THIS STUDY ADDS• Transdermal scopolamine can be a therapeutic option to control drooling in unco‐operative patients. It is an effective and safe method, although it requires appropriate patient selection and is not free from adverse effects.AIMTo evaluate the efficacy of scopolamine administered transdermally for the treatment of drooling in severely disabled patients.METHODSA prospective, randomized, double‐blind, crossover, placebo‐controlled clinical trial was designed. The study group consisted of 30 handicapped patients with persistent drooling. The exclusion criteria were the specific contra‐indications of scopolamine. Severity of drooling was quantified using a modified Thomas‐Stonell and Greenberg visual scale simplified into three grades: 1 = dry; 2 = mild/moderate; 3 = severe/fulsome. The frequency of drooling was estimated using the number of bibs used each day. The baseline observational phase was followed by the application of a 1.5 mg scopolamine (Scopoderm TTS; Novartis Consumer Healthcare, UK) or placebo patch every 72 h for a fortnight. This was followed by a 1 week washout period and then crossover of assignments for 2 weeks.RESULTSAt baseline, 77% of patients showed grade 3 of drooling. The placebo administration showed no significant reduction in drooling. We found a significant drooling reduction (P< 0.005) in the scopolamine group in the 1 and 2 week controls (69% and 80% respectively ≤ grade 3). The mean number of bibs/day decreased during the scopolamine phase from 6/day at baseline to 3/day at the 2 week control. Four patients (13.3%) dropped out because of scopolamine side effects and minor adverse reactions were observed in three other patients. No blood alterations were found during the study period.CONCLUSIONScopolamine can be useful to control drooling in severely disabled patients although it requires appropriate patient selection and is not free from adverse effects.
High immunohistochemical expression of p‐AKT predicts inferior survival in patients with diffuse large B‐cell lymphoma treated with immunochemotherapy
Authors: Hasselblom Sverker , Hansson Ulrika , Olsson Markus , Torén Leif , Bergström Anders , Nilsson‐Ehle Herman , Andersson Per‐Ola 
Published: March 2010

SummaryChemotherapy and rituximab (R) is current standard therapy in diffuse large B‐cell lymphoma (DLBCL), but a substantial proportion of patients still fail to reach sustained remission. In vitro studies have indicated that rituximab resistance could be accompanied by dysregulated apoptotic pathways, such as the phosphatidylinositol 3‐kinase (PI3K)/AKT signaling pathway, which can be constitutively activated in DLBCL. In this retrospective, immunohistochemical study on 106 patients treated with R‐CHO(E)P (cyclophosphamide, doxorubicin, vincristine, prednisone, rituximab [+etoposide]), we investigated the prognostic role of proteins involved in different apoptotic pathways; phosphorylated AKT (p‐AKT), bcl‐2, MCL1, bcl‐xL, Bax and Bak. High p‐AKT expression (>108 cells/mm2, highest quartile, n = 27) predicted worse progression‐free (PFS) (P = 0·02) and overall (OS) (P = 0·01) survival, independent of International Prognostic Index and sex. Also bcl‐2+ (cut‐off 50%) predicted worse PFS (P = 0·005) and OS (P = 0·05) but after adjustment for clinical factors only the influence on PFS (P = 0·03) remained significant. The prognostic impact of p‐AKT overexpression was independent of bcl‐2 status. MCL1, bcl‐xL, Bax and Bak expression did not add any prognostic information. Our results suggest that high p‐AKT expression predicts worse outcome, possibly indicating that inhibition of the activated PI3K/AKT pathway could be of clinical interest in DLBCL patients. In addition, bcl‐2 status could have prognostic importance also in the era of immunochemotherapy.
Cross‐sectional survey on immunoglobulin E reactivity in 23 077 subjects using an allergenic molecule‐based microarray detection system
Authors: Scala E. , Alessandri C. , Bernardi M. L. , Ferrara R. , Palazzo P. , Pomponi D. , Quaratino D. , Rasi C. , Zaffiro A. , Zennaro D. , Mari A. 
Published: March 2010

SummaryBackground
The availability of allergenic molecules and high‐throughput microtechnologies allow the collection of a large number of IgE results at the same time in a single test. This can be carried out applying the test in the routine diagnostic work‐up.Objective
The aim of this study was to make a cross‐sectional evaluation of the raw prevalence of IgE reactivity to allergenic molecules in serum samples from a cohort of Italian patients using an innovative tool.Methods
The ISAC, a microarray system, has been used for specific IgE detection using 75 different allergenic molecules. Sera were collected from 23 077 unselected consecutive individuals complaining about any allergic disease.Results
Sixteen thousand four hundred and eight of 23 077 patients had IgE to at least one of 75 allergenic molecules. The top‐ranked molecules in this cohort were Cup a 1 (42.7%), Der f 2 (38.7%), and Phl p 1 (37.9%), whereas all the other allergens tested scored in a range between 36.8% and 0.04%, including the first food allergen, Pru p 3, ranked 15th (9.79%). Prevalence varied quite markedly depending on the age range considered, and showing a different behaviour in the lifetime sensitization process. Unsupervised two‐way hierarchical clustering analysis generated distinctive patterns of reactivity as the result of IgE recognition of either homologous allergens belonging to different biological sources or non‐homologous belonging to the same biological source.Conclusions
Allergen‐based microarray is a tool for the detection of IgE‐related sensitization to panels of allergens and gives a more precise and comprehensive evaluation for an IgE‐based epidemiology. This insight brings data for better understanding of the sensitization process.
Effect of plant species loss on aphid–parasitoid communities
Authors: Petermann Jana S. , Müller Christine B. , Weigelt Alexandra , Weisser Wolfgang W. , Schmid Bernhard 
Published: March 2010

Summary1.The consequences of species loss on ecosystem functioning within a single trophic level have been extensively studied. However, the loss of basal species is likely to have profound impacts on the abundance, richness and ecosystem functioning of species at higher trophic levels.2.Here, we used experimentally established plant communities with a species richness gradient to study the effects of plant species loss on a multi‐trophic insect community in the field. We measured densities and species richness of aphids and parasitic wasps (primary, secondary and facultative tertiary parasitoids of aphids) that naturally colonized the grassland plots.3.Furthermore, we calculated two ecosystem functions: aphid load (the number of aphid individuals per host plant biomass used as a proxy for herbivory) and parasitism rate. We used structural equation models to explore pathways of direct and indirect effects of plant species richness on higher trophic levels.4.We found that the densities and richness of species at all trophic levels were influenced by changes in plant species richness. The effects were rarely direct, but instead mediated by the abundance and species richness of aphid host plants and subsequent trophic levels.5.The herbivore and primary parasitoid levels were most directly affected by changes in plant species richness, with highest insect densities and species richness occurring at intermediate plant species richness. The densities and species richness of secondary parasitoids declined linearly with plant species richness owing to sparser resources, resulting in shorter food chains in communities with the highest plant species richness.6.Aphid load was highest at intermediate plant species richness and negatively affected by both host plant biomass and host plant species richness. Parasitism rate was mostly affected indirectly via aphid density and overall only weakly negatively related to plant species richness.7.Our results demonstrate that plant species richness can have strong cascading effects up to high trophic levels. However, their direction may differ at the lower and higher ends of the plant species richness spectrum, cautioning against simplifying consequences of plant species loss for specialist food webs that may become limited by sparse resources at high plant richness.
Septum formation is regulated by the RHO4‐specific exchange factors BUD3 and RGF3 and by the landmark protein BUD4 in Neurospora crassa
Authors: Justa‐Schuch Daniela , Heilig Yvonne , Richthammer Corinna , Seiler Stephan 
Published: March 2010

SummaryRho GTPases have multiple, yet poorly defined functions during cytokinesis. By screening a Neurospora crassa knock‐out collection for Rho guanine nucleotide exchange factor (GEF) mutants that phenocopy rho‐4 defects (i.e. lack of septa, slow growth, abnormal branching and cytoplasmic leakage), we identified two strains defective in homologues of Bud3p and Rgf3 of budding and fission yeast respectively. The function of these proteins as RHO4‐specific GEFs was determined by in vitro assays. In vivo microscopy suggested that the two GEFs and their target GTPase act as two independent modules during the selection of the septation site and the actual septation process. Furthermore, we determined that the N. crassa homologue of the anillinrelated protein BUD4 is required for septum initiation and that its deficiency leads to typical rho4 defects. Localization of BUD4 as a cortical ring prior to septation initiation was independent of functional BUD3 or RGF3. These data position BUD4 upstream of both RHO4 functions in the septation process and make BUD4 a prime candidate for a cortical marker protein involved in the selection of future septation sites. The persistence of both BUD proteins and of RHO4 at the septal pore suggests additional functions of these proteins at mature septa.
Distinct roles of ppGpp and DksA in Legionella pneumophila differentiation
Authors: Dalebroux Zachary D. , Yagi Brian F. , Sahr Tobias , Buchrieser Carmen , Swanson Michele S. 
Published: March 2010

SummaryTo transit between hosts, intracellular Legionella pneumophila transform into a motile, infectious, transmissive state. Here we exploit the pathogen's life cycle to examine how guanosine tetraphosphate (ppGpp) and DksA cooperate to govern bacterial differentiation. Transcriptional profiling revealed that during transmission alarmone accumulation increases the mRNA for flagellar and Type IV‐secretion components, secreted host effectors and regulators, and decreases transcripts for translation, membrane modification and ATP synthesis machinery. DksA is critical for differentiation, since mutants are defective for stationary phase survival, flagellar gene activation, lysosome avoidance and macrophage cytotoxicity. The roles of ppGpp and DksA depend on the context. For macrophage transmission, ppGpp is essential, whereas DksA is dispensable, indicating that ppGpp can act autonomously. In broth, DksA promotes differentiation when ppGpp levels increase, or during fatty acid stress, as judged by flaA expression and evasion of degradation by macrophages. For flagella morphogenesis, DksA is required for basal fliA (σ28) promoter activity. When alarmone levels increase, DksA cooperates with ppGpp to generate a pulse of Class II rod RNA or to amplify the Class III sigma factor and Class IV flagellin RNAs. Thus, DksA responds to the level of ppGpp and other stress signals to co‐ordinate L. pneumophila differentiation.
Structure of the cytoplasmic domain of FlhA and implication for flagellar type III protein export
Authors: Saijo‐Hamano Yumiko , Imada Katsumi , Minamino Tohru , Kihara May , Shimada Masafumi , Kitao Akio , Namba Keiichi 
Published: March 2010

SummaryFlhA is the largest integral membrane component of the flagellar type III protein export apparatus of Salmonella and is composed of an N‐terminal transmembrane domain (FlhATM) and a C‐terminal cytoplasmic domain (FlhAC). FlhAC is thought to form a platform of the export gate for the soluble components to bind to for efficient delivery of export substrates to the gate. Here, we report a structure of FlhAC at 2.8 Å resolution. FlhAC consists of four subdomains (ACD1, ACD2, ACD3 and ACD4) and a linker connecting FlhAC to FlhATM. The sites of temperature‐sensitive (ts) mutations that impair protein export are distributed to all four domains, with half of them at subdomain interfaces. Analyses of the ts mutations and four suppressor mutations to the G368C ts mutation suggested that FlhAC changes its conformation for its function. Molecular dynamics simulation demonstrated an open‐close motion with a 5–10 ns oscillation in the distance between ACD2 and ACD4. These results along with further mutation analyses suggest that a dynamic domain motion of FlhAC is essential for protein export.
Identification of residues within ligand‐binding domain 1 (LBD1) of the Borrelia burgdorferi OspC protein required for function in the mammalian environment
Authors: Earnhart Christopher G. , LeBlanc DeLacy V. , Alix Katie E. , Desrosiers Daniel C. , Radolf Justin D. , Marconi Richard T. 
Published: March 2010

Summary Borrelia burgdorferi outer surface protein C (ospC) is required for the establishment of infection in mammals. However, its precise function remains controversial. The biologically active form of OspC appears to be a homodimer. Alpha helix 1 and 1′ of the apposing monomers form a solvent‐accessible pocket at the dimeric interface that presents a putative ligand‐binding domain (LBD1). Here we employ site‐directed and allelic‐exchange mutagenesis to test the hypothesis that LBD1 is a determinant of OspC function in the mammalian environment. Substitution of residues K60, E61 and E63 which line LBD1 resulted in the loss of infectivity or influenced dissemination. Analyses of the corresponding recombinant proteins demonstrated that the loss of function was not due to structural perturbation, impaired dimer formation or the loss of plasminogen binding. This study is the first to assess the involvement of individual residues and domains of OspC in its in vivo function. The data support the hypothesis that OspC interacts with a mammalian derived ligand that is critical for survival during early infection. These results shed new light on the structure–functions relationships of OspC and challenge existing hypotheses regarding OspC function in mammals.
Cuticular hydrocarbons influence female attractiveness to males in the Australian field cricket, Teleogryllus oceanicus
Authors: THOMAS MELISSA L. , SIMMONS LEIGH W. 
Published: March 2010

AbstractSexual dimorphism is thought to result from directional sexual selection acting on male signal traits, with female signal traits given little, if any, attention. Here, we examine male mating preferences in the Australian field cricket, Teleogryllus oceanicus. Using a multivariate selection analysis approach, we found that male preferences have the potential to exert selection on female cuticular hydrocarbons, chemical compounds widely used as sexual signals in insects. In addition to finding both stabilizing and disruptive preference gradients, we also found weak negative directional preference for female cuticular hydrocarbons. We contrast our results with a recent study examining sexual selection via female choice on male T. oceanicus cuticular hydrocarbons and suggest that differences in the form and intensity of sexual selection between the genders may provide part of the net selection differential necessary for the evolution of sexual dimorphism in this species.
Motivating medicine: Why money is not enough
Authors: Dalton Sarah 
Published: March 2010

AbstractThe public health system in Australia currently faces unprecedented challenges managing an ageing population with a rising burden of illness. Health professionals employed in the area are under constant pressure to do more with less, leading to high levels of employee dissatisfaction. Physician satisfaction is a critical aspect of quality health care, allowing the development of a motivated workforce committed to improving patient outcomes. Herzberg has identified several factors that contribute to employee motivation, including financial remuneration, work–life balance and the recognition and reward of good performance. Some strategies are more effective than others; here we discuss their relative advantages, the role of financial rewards, and propose other interventions to ‘switch on’ motivation.
Ghrelin and adiponectin levels in colostrum, cord blood and maternal serum
Authors: Dündar Nihal O. , Dündar Bumin , Cesur Gökhan , Yılmaz Nigar , Sütçu Recep , Özgüner Fehmi 
Published: March 2010

AbstractBackground:  Ghrelin and adiponectin, which are considered to take part in the regulation of energy metabolism, have been found in breast milk and cord blood. The aims of this study were to determine ghrelin and adiponectin levels in colostrum, cord blood and maternal serum and to investigate the correlations between colostrum and cord blood levels of these peptides and the anthropometry of newborn infants and their mothers.Methods:  Total ghrelin (TGHR), free ghrelin (FGHR) and adiponectin levels were studied in colostrum and the serum samples of 25 healthy lactating women and the cord blood of their healthy full‐term infants.Results:  No significant differences could be found among TGHR and adiponectin levels in colostrum, cord blood and maternal serum. The median FGHR level in colostrum was significantly higher than that of maternal serum and cord blood. The colostrum TGHR was negatively correlated with body mass index (BMI) and weight of the infants at birth. TGHR and FGHR levels in colostrum were found to be positively correlated with those of maternal TGHR and FGHR concentrations, respectively. Adiponectin levels in colostrum were not correlated with BMI or birthweight of the infants or BMI of the mothers.Conclusion:  These findings suggest that the source of ghrelin in breast milk is probably both breast tissue itself and the serum of the mother. Ghrelin in colostrum seems to be related to the anthropometry of infants even at birth, unlike adiponectin.
Long‐term stability of thyroid hormones and DNA in blood spots kept under varying storage conditions
Authors: EL Ezzi Asmahan A. , El‐Saidi Mohammed A. , Kuddus Ruhul H. 
Published: March 2010

AbstractBackground:  Congenital hypothyroidism is screened using blood spotted on filter paper that may be transported from remote areas to central testing facilities. However, storage conditions and transportation may affect sample quality.Methods:  We examined long‐term stability of thyroid‐stimulating hormone (TSH) and thyroxin (TT4) in blood spotted on filter paper, which was stored at room temperature (RT), 4°C and −20°C under continuous or intermittent power supply (six hours on and six hours off around the clock.) Hormone levels in the discs were measured periodically for up to ten years. Extraction of DNA from blood spots and polymerase chain reaction were performed.Results:  Our results showed that TT4 was stable for up to 6.1, 5.34 and 5.16 years when stored at −20°C, 4°C and RT, respectively. TSH was stable for up to 2.7 years at RT, and for up to 6.5 and 4.1 years when stored at −20°C and 4°C, respectively, under continuous power supply. However, under intermittent power supply, TSH was stable for up to 3.8 and 2.5 years when stored at 4°C and −20°C, respectively. Mitochondrial cytochrome oxidase and sex‐determining region of Y chromosome genes were successfully amplified from DNA extracted from the blood spots.Conclusion:  Our data indicate that TT4 and TSH are most stable in blood spots stored at −20°C under continuous power supply. However, they can be stored at RT or at 4°C and −20°C under interrupted power supply for at least 2.5 years. Moreover, the DNA extracted from the blood spots was intact and suitable for genetic studies.