bluuebunny/crossref_metadata_2025 huggingface dataset:
Determining Single-Molecule Intensity as a Function of Power Density: Figure 1.
Authors: Reck-Peterson Samara L. , Derr Nathan D. , Stuurman Nico
Published: March 2010

INTRODUCTION

When performing analysis using total internal reflection fluorescence microscopy (TIRFM), fluorescence intensity depends linearly on excitation intensity, until the dye spends most of its time in the excited state (i.e., saturation is reached). How much light is seen by the dyes in the sample depends not only on the amount of light exiting the objective but also on the area over which it is spread out. The parameter of interest is the power density, measured in W/cm2. The highest signal-to-noise ratios will be reached when the illumination power just approaches saturation. To determine at what power density a dye is saturated, one should measure fluorescence intensity as a function of power density, as described in this protocol.
Imaging Single Molecular Motor Motility with Total Internal Reflection Fluorescence Microscopy (TIRFM): Movie 1.
Authors: Reck-Peterson Samara L. , Derr Nathan D. , Stuurman Nico
Published: March 2010

INTRODUCTION

Total internal reflection fluorescence microscopy (TIRFM) allows fluorescent molecules on or near the plasma membrane to be visualized with a very high signal-to-noise ratio. This strategy has been very successful for imaging molecular machines as they move and do work. We provide here a general protocol for imaging single molecular motors as they move along microtubule tracks. Our protocol is designed for the study of cytoplasmic dynein purified from Saccharomyces cerevisiae, but it represents a general framework for any in vitro single-molecule assay.
Unbiased Identification of Protein-Bait Interactions Using Biochemical Enrichment and Quantitative Proteomics
Authors: Ong Shao-En
Published: March 2010

INTRODUCTION

The use of recombinant proteins, antibodies, small molecules, or nucleic acids as affinity reagents is a simple yet powerful strategy to study the protein-bait interactions that drive biological processes. However, such experiments are often analyzed by Western blotting, limiting the ability to detect novel protein interactors. Unbiased protein identification by mass spectrometry (MS) extends these experiments beyond the study of pairwise interactions, allowing analyses of whole networks of protein-bait interactions. With the latest advances in MS, it is not uncommon to identify thousands of proteins from complex mixtures. Paradoxically, the improved sensitivity of proteomic analyses can make it more difficult to distinguish bait-specific interactions from the large background of identified proteins. In quantitative proteomics, MS signals from protein populations labeled with stable isotopes such as 13C and 15N can be identified and quantified relative to unlabeled counterparts. Using quantitative proteomics to compare biochemical enrichments with the bait of interest against those obtained with control baits allows sensitive detection and discrimination of specific protein-bait interactions among the large number of nonspecific interactions with beads. Ad hoc optimization of enrichment conditions is minimized, and mild purification conditions preserve secondary or high-order protein-protein interactions. The combination of biochemical enrichment and quantitative proteomics allows rapid characterization of molecular baits with their interacting proteins, providing tremendous insight into their biological mechanisms of action.
Overexpressing Tagged Proteins in Plants Using a Modified Gateway Cloning Strategy: Figure 1.
Authors: Dubin Manu J. , Bowler Chris , Benvenuto Giovanna
Published: March 2010

INTRODUCTION

In 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

INTRODUCTION

The 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.
Differential quantitative analysis of background structure in energy-filtered convergent-beam electron diffraction patterns
Authors: Nakashima Philip N. H. , Muddle Barrington C.
Published: March 2010

Measurements of electronic structure in solids by quantitative convergent-beam electron diffraction (QCBED) will not reach their ultimate accuracy or precision until the contribution of the background to the reflections in energy-filtered CBED patterns is fully accounted for. Apart from the well known diffuse background that arises from thermal diffuse scattering of electrons, there is a component that has a much higher angular frequency. The present work reports experimental evidence that this component mimics the angular distribution of the elastically scattered electrons within each reflection. A differential approach to QCBED is suggested as a means of quantitatively accounting for the background in energy-filtered CBED data.
Accelerated computation of diffuse scattering patterns and application to magnetic neutron scattering
Authors: Gutmann Matthias J.
Published: March 2010

The original method of Butler & Welberry [J. Appl. Cryst.(1992),25, 391–399] to compute diffuse scattering patterns from a simulated model crystal is extended to include magnetic scattering. In addition, it is shown that these computations can be accelerated by at least one order of magnitude using modern consumer graphics cards.
The crystal structure of perdeuterated methanol hemiammoniate (CD3OD·0.5ND3) determined from neutron powder diffraction data at 4.2 and 180 K
Authors: Fortes A. D. , Wood I. G. , Knight K. S.
Published: March 2010

The crystal structure of perdeuterated methanol hemiammoniate, CD3OD·0.5ND3, has been solved from neutron powder diffraction data collected at 4.2 and 180 K. The structure is orthorhombic, space groupPn21a(Z= 4), with unit-cell dimensionsa= 12.70615 (16),b= 8.84589 (9),c= 4.73876 (4) Å,V= 532.623 (8) Å3[ρcalc= 1149.57 (2) kg m−3] at 4.2 K, anda= 12.90413 (16),b= 8.96975 (8),c= 4.79198 (4) Å,V= 554.656 (7) Å3[ρcalc= 1103.90 (1) kg m−3] at 180 K. The crystal structure was determined byab initiomethods from the powder data; atomic coordinates and isotropic displacement parameters were subsequently refined by the Rietveld method toRp≃ 2% at both temperatures. The crystal structure comprises a three-dimensionally hydrogen-bonded network in which the ND3molecules are tetrahedrally coordinated by the hydroxy moieties of the methanol molecule. This connectivity leads to the formation of zigzag chains of ammonia–hydroxy groups extending along thecaxis, formedviaN—D···O hydrogen bonds; these chains are cross-linked along theaaxis through the hydroxy moiety of the second methanol moleculeviaN—D···O and O—D···O hydrogen bonds. This `bridging' hydroxy group in turn donates an O—D···N hydrogen bond to ammonia in adjacent chains stacked along thebaxis. The methyl deuterons in methanol hemiammoniate, unlike those in methanol monoammoniate, do not participate in hydrogen bonding and reveal evidence of orientational disorder at 180 K. The relative volume change on warming from 4.2 to 180 K, ΔV/V, is + 4.14%, which is comparable to, but more nearly isotropic (as determined from the relative change in axial lengths,e.g.Δa/a) than, that observed in deuterated methanol monohydrate, and very similar to what is observed in methanol monoammoniate.
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.
Drug‐ and non‐drug‐associated QT interval prolongation
Authors: Van Noord Charlotte , Eijgelsheim Mark , Stricker Bruno H. Ch.
Published: March 2010

Sudden cardiac death is among the most common causes of cardiovascular death in developed countries. The majority of sudden cardiac deaths are caused by acute ventricular arrhythmia following repolarization disturbances. An important risk factor for repolarization disturbances is use of QT prolonging drugs, probably partly explained by gene–drug interactions. In this review, we will summarize QT interval physiology, known risk factors for QT prolongation, including drugs and the contribution of pharmacogenetics. The long QT syndrome can be congenital or acquired. The congenital long QT syndrome is caused by mutations in ion channel subunits or regulatory protein coding genes and is a rare monogenic disorder with a mendelian pattern of inheritance. Apart from that, several common genetic variants that are associated with QT interval duration have been identified. Acquired QT prolongation is more prevalent than the congenital form. Several risk factors have been identified with use of QT prolonging drugs as the most frequent cause. Most drugs that prolong the QT interval act by blocking hERG‐encoded potassium channels, although some drugs mainly modify sodium channels. Both pharmacodynamic as well as pharmacokinetic mechanisms may be responsible for QT prolongation. Pharmacokinetic interactions often involve drugs that are metabolized by cytochrome P450 enzymes. Pharmacodynamic gene–drug interactions are due to genetic variants that potentiate the QT prolonging effect of drugs. QT prolongation, often due to use of QT prolonging drugs, is a major public health issue. Recently, common genetic variants associated with QT prolongation have been identified. Few pharmacogenetic studies have been performed to establish the genetic background of acquired QT prolongation but additional studies in this newly developing field are warranted.
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

Chemotherapy 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

Background
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

1.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

Rho 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

To 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

FlhA 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

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

Sexual 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

The 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

Background:  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

Background:  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.
Does plant height determine the freezing resistance in the páramo plants?
Authors: SKLENÁŘ PETR , KUČEROVÁ ANDREA , MACEK PETR , MACKOVÁ JANA
Published: March 2010

Neotropical ecosystems between treeline and snowline, called páramos, stretch along Andean ranges from Costa Rica to northern Peru. The páramo climate is characterized by regular night frosts occurring throughout the year. Páramo plants use two strategies to deal with freezing temperatures. They either avoid ice formation in the tissues or tolerate extracellular ice formation. We tested the microclimate hypothesis, which suggests that the freezing resistance of the páramo plants is determined by plant height, that is, that taller plants experience a milder microclimate and avoid freezing, whereas smaller plants are exposed to the more extreme thermal conditions near the ground and tolerate them. We measured the temperature at which ice formed inside the plants (the ‘exotherm’), and compared it with the temperature at which 50% damage to the tissue occurred (Lt50); a significant difference between the exotherm and Lt50 would indicate freezing tolerance whereas the absence of a difference would indicate avoidance by supercooling. We analysed the freezing resistance of 38 common Ecuadorian páramo species. We found no correlation between plant height and freezing resistance mechanism or injury temperature and reject the microclimate hypothesis. Tolerant plants reach higher altitudes than avoidant plants, but their altitudinal ranges largely overlap and the Lt50 does not differ between them. These results suggest that there is no qualitative difference between the two strategies to survive the páramo frosts. Shrub leaves were injured at significantly lower temperatures than other life forms, such as herbs, which may reflect leaf anatomical differences among the plants.
Does decomposition of standard materials differ among grassland patches maintained by livestock?
Authors: VAIERETTI MARÍA VICTORIA , CINGOLANI ANA M. , HARGUINDEGUY NATALIA PÉREZ , GURVICH DIEGO E. , CABIDO MARCELO
Published: March 2010

Grazing can modify vegetation structure and species composition through selective consumption, modifying plant litter quality and hence decomposability. In most grasslands, moderate stocking rates maintain a mosaic of high‐quality patches, preferentially used by herbivores (‘grazing lawns’), and low‐quality tall patches, which are avoided. In grazing lawns decomposition rates can be accelerated because of the higher litter quality of its component species and, besides, through the indirect effect of increased nutrient availability in soil. We aimed at testing this indirect effect using standard materials, comparing their decomposition in grazing lawns, open and closed tall tussock grasslands. We selected 10 patches of each type and sampled floristic composition, soil variables and cattle dung deposition. Standard materials were filter paper and Poa stuckertii litter. We prepared litterbags of 0.3 mm (thin mesh) and 1 mm mesh size (coarse mesh). Samples were incubated for 65 days in two ways: above‐ground (thin and coarse mesh) and below‐ground (only thin mesh), aiming at analysing the conditions for decomposition for surface litter and buried litter or dead roots, respectively. Physical and chemical soil variables did not differ among patch types, despite the differences in species composition. Closed tussock grasslands showed the lowest dung deposition, confirming the less intense use of these patches. Soil nitrogen availability (N‐NO3‐ and N‐NH4+) was not significantly different among patch types. Each standard material followed a different decomposition pattern across patch types. For above‐ground incubated samples, Poa litter decomposed significantly faster in lawns, and slower in open tussock grasslands. Filter paper decomposed significantly faster in closed tussock grasslands than in the other two patch types. Decomposition of below‐ground incubated samples did not significantly differ among patch types, in line with results for soil variables. Above‐ground differences in decomposition may be associated with differences in microclimatic conditions resulting from differences in vegetation structure.
Trajectories of change: riparian vegetation and soil conditions following livestock removal and replanting
Authors: BURGER B. , REICH P. , CAVAGNARO T. R.
Published: March 2010

Riparian zones provide critically important ecological functions, including the interception of nutrients and sediments before they enter waterways. Consequently, riparian zones, and the vegetation they support, are often considered as an important ‘final buffer’ between waterways and adjacent land. In agricultural ecosystems, riparian zones are therefore increasingly recognized as an important component of strategies aimed at minimizing the flow of nutrients and sediments into waterways. Accordingly, riparian zones are increasingly afforded protection and are targeted for restoration. Here we present results of a study in which we aimed to identify patterns of change in soil and vegetation properties in riparian zones, under different management regimes, adjacent to tributary streams in one of south‐eastern Australia's main agricultural regions. We compared riparia that were heavily impacted by agricultural activities, were in remnant condition or had undergone some restoration activities and were thus in a transitional state. There was an increase in plant cover and soil C concentration between impacted through to remnant sites, with transitional sites intermediate, suggesting that improvements in soil conditions were becoming evident following restoration activities. In our assessment of soil physicochemical properties we investigated the relationships between riparian condition and soil properties, taking into account the influence of adjacent land use on these relationships. Importantly, the concentrations of NO3‐ and plant available P in riparian surface soils were more or less influenced by concentrations in the adjacent land depending upon riparian condition. This will, in turn, have consequences for nutrient inputs into streams. This study emphasizes that riparian zones need to be managed within their wider landscape context. Furthermore, the results of this study will inform efforts seeking to minimize impacts of agricultural activities on waterways, through the conservation and/or restoration of riparian ecosystems.
Raman spectral imaging of prostate cancer: can Raman molecular imaging be used to augment standard histopathology?
Authors: Tollefson Matthew , Magera James , Sebo Thomas , Cohen Jeffrey , Drauch Amy , Maier John , Frank Igor
Published: March 2010

Study Type – Diagnostic (exploratory cohort) Level of Evidence 2b

OBJECTIVE

To evaluate whether Raman molecular imaging (RMI, which combines digital imaging and analytical spectroscopy to evaluate the biochemical composition of interrogated material) can be used to identify biochemical differences in patients with Gleason 7 prostate cancer who progress to metastatic disease and die from prostate cancer.

PATIENTS AND METHODS

We identified 38 patients who had a radical prostatectomy for Gleason 7 adenocarcinoma of the prostate. Half progressed to metastatic disease and half had no evidence of disease after treatment. Patients were matched for preoperative prostate‐specific antigen level, surgical margin status, pathological stage, tumour volume, age at surgery, year of surgery and DNA ploidy. Sequential 5 µm sections were obtained from paraffin‐embedded tissue and one genitourinary pathologist selected areas of tumour for study. Principal component analysis was used to investigate the correlation between spectral response and clinical outcome.

RESULTS

The analysis was able to distinguish between those with progressive disease and those with no evidence of disease, most notably within the Gleason 3 regions when evaluating the epithelium and stroma as separate histological elements. A two‐sample t‐test gave P < 0.01 for both the Gleason 3 and 4 epithelium and stroma classes.

CONCLUSIONS

RMI is a novel technique that shows promise for identifying patients at risk of progression by visualizing molecular information not seen using other current methods. In Gleason 7 disease, RMI shows distinctive chemical differences in patients who progress to metastatic disease in both Gleason pattern 3 and 4 regions. This preliminary work lays the foundation for the further study of RMI for evaluating prostate tissue.
Long‐term efficacy of the bone‐anchored male sling for moderate and severe stress urinary incontinence
Authors: Carmel Maude , Hage Bechir , Hanna Samer , Schmutz Gerard , Tu Le Mai
Published: March 2010

Study Type – Therapy (outcomes research) Level of Evidence 2c

OBJECTIVE

To present a prospective long‐term evaluation of the bone‐anchored male sling (InVanceTM, American Medical Systems, Minnetonka, MN, USA) for patients with moderate to severe stress urinary incontinence (SUI) after prostate surgery

PATIENTS AND METHODS

Forty‐five patients had a male sling implanted for SUI after prostate surgery. The evaluation before and after surgery included a complete history and physical examination, various questionnaires (American Urological Association Symptom Score, University of California Los Angeles/RAND prostate index), cysto‐urethroscopy, International Continence Society (ICS) 1‐h pad test and a urodynamic study. The follow‐up assessments were at 2, 6 and 12 months after surgery, and yearly thereafter. The success rate was defined as the percentage of patients ‘dry’ and ‘improved’.

RESULTS

The median (range) follow‐up was 36 (2–64) months. Twelve patients (27%) had previous adjuvant radiotherapy. Eighteen patients (40%) had moderate SUI (two or three pads/day) and 27 (60%) had severe SUI (more than three pads/day). All preoperative ICS 1‐h pad tests were positive. The success rate was 76%; 16 patients were dry (36%), 18 were improved (one or two pads/day, 40%) and in 11 the sling was a failure (24%); 76% of ICS 1‐h pad tests were negative after surgery. The postoperative urodynamic study showed no signs of bladder outlet obstruction. In all, 72% of patients were satisfied/very satisfied with the surgery and 86% considered themselves cured/almost cured. The success rate was not affected by the presence of previous radiotherapy or the severity of SUI. Ten patients had perineal numbness, but in all it resolved within 1–3 months. There was one mesh infection which required its removal. There was no urethral erosion.

CONCLUSION

Compared to previous studies, the InVance male sling had a good success rate for moderate and severe SUI, with a median follow‐up of 36 months. The results did not differ for moderate or severe SUI or with the presence of previous radiotherapy.
Corticotropin‐releasing hormone‐receptor 2 is required for acute stress‐induced bladder vascular permeability and release of vascular endothelial growth factor
Authors: Boucher William , Kempuraj Duraisamy , Michaelian Margaret , Theoharides Theoharis C.
Published: March 2010

OBJECTIVE

To investigate the corticotropin‐releasing hormone (CRH) receptor (CRH‐R) requirement for the effect of acute stress on bladder vascular permeability and release of vascular endothelial growth factor (VEGF), as increasing evidence indicates that acute stress worsens certain inflammatory disorders, including interstitial cystitis/painful bladder syndrome (IC/PBlS), which is characterized by pain, variable bladder inflammation, increased expression of bladder vascular endothelial growth factor (VEGF), and many detrusor mast cells.

MATERIALS AND METHODS

Bladders of normal C57BL/6, and C57BL/6‐ derived CRH‐R1, CRH‐R2 or double CRH‐R1 + 2 knockout (−/−) female mice (10–12 weeks old) were catheterized under anaesthesia. After emptying the urine, normal saline was instilled with or without intravesical CRH‐R antagonists in C57BL/6 mice before they were stressed by placing them in a restrainer for 30 min. Evans blue was injected in the tail vein before stress for the permeability experiments. The bladders from C57BL/6 or CRH‐R −/− mice were then removed, minced into 1 mm2 pieces and cultured overnight. Culture media were collected 24 h later for VEGF assay. C57BL/6 bladder was processed for CRH‐R immunohistochemistry.

RESULTS

Acute stress increased bladder vascular permeability in control C57BL/6 and CRH‐R1 −/− mice, but not CRH‐R2 −/− or CRH‐R1+2 −/− mice. The CRH‐R2 antagonist Astressin 2B, but not the CRH‐R1 antagonist Antalarmin, inhibited stress‐induced VEGF release from C57BL/6 mouse bladder explants. Stress could not induce a VEGF increase from bladder explants of CRH‐R2 −/− or CRH‐R1+2 −/− mice, but did so in CRH‐R1 −/− mice. Bladder CRH‐R2 immunoreactivity was detected in C57BL/6 bladders.

CONCLUSIONS

Acute stress induces bladder vascular permeability and VEGF release that is dependent on CRH‐R2. These findings suggest that CRH and VEGF might participate in the pathogenesis of IC/PBlS and provide for new therapeutic targets.
Radical prostatectomy in older men: survival outcomes in septuagenarians and octogenarians
Authors: Pierorazio Phillip M. , Humphreys Elizabeth , Walsh Patrick C. , Partin Alan W. , Han Misop
Published: March 2010

Study Type – Therapy (case series) Level of Evidence 4

OBJECTIVE

To examine the survival outcomes of septuagenarians and octogenarians (aged ≥80 years) who underwent radical prostatectomy (RP) at our institution, as the US Preventive Services Task Force recently released recommendations that men aged ≥75 years should not be screened for prostate cancer.

PATIENTS AND METHODS

Our institutional RP database (1982–2008) was queried for men aged ≥70 years at the time of surgery to evaluate actuarial survival after RP; 386 aged 70–81 years (median 71) underwent RP. The median (range) follow‐up was 6.5 (1–22) years. Clinicopathological characteristics and mortality data were evaluated; mortality data were gathered through Social Security Administration Death Index and causes of death were confirmed with the Center for Disease Control National Death Index information. Kaplan‐Meier analysis was used to evaluate cause‐specific survival.

RESULTS

Ten patients (2.6%) had clinical stage T1a‐b, 213 (55.3%) had T1c, and 143 (37.1%) had T2 prostate cancer. The median (range) preoperative prostate‐specific antigen (PSA) level was 6.2 (0.2–49.9) ng/mL, and the biopsy and pathological Gleason sum was 6 (3–9) and 7 (4–9), respectively. Causes of death included prostate cancer (17), other malignancies (14), cardiovascular causes (14), neurological disease (four), pneumonia (two) and accident (one). The prostate cancer‐specific survival rate was 97.6%, 94.0% and 90.2% at 5, 10 and 15 years after RP, respectively; the respective cardiovascular survival rate was 99.5%, 97.6% and 92.5%, and the overall survival rate was 93.1%, 82.5% and 68.9%, respectively.

CONCLUSIONS

If appropriately selected, older men have excellent overall and prostate‐cancer specific survival after RP. The benefits of surgery should be weighed against the increased risks of surgical and anaesthetic complications.