bluuebunny/crossref_metadata_2025 huggingface dataset:
Resolutions of the Coulomb operator
Authors: Varganov Sergey A. , Gilbert Andrew T. B. , Deplazes Evelyne , Gill Peter M. W.
Published: June 2008

We discuss a generalization of the resolution of the identity by considering one-body resolutions of two-body operators, with particular emphasis on the Coulomb operator. We introduce a set of functions that are orthonormal with respect to 1∕r12 and propose that the resulting “resolution of the Coulomb operator,” r12−1=∣ϕi⟩⟨ϕi∣, may be useful for the treatment of large systems due to the separation of two-body interactions. We validate our approach by using it to compute the Coulomb energy of large systems of point charges.
The Limits of Performance Assessments of Public Bodies: External Constraints in English Local Government
Authors: Gutiérrez-Romero Roxana , Haubrich Dirk , McLean Iain
Published: June 2008

Policy makers have an interest in evaluating what government and taxpayers get for their money. In the United Kingdom, central government has developed elaborate oversight mechanisms involving a plethora of indicators, targets, and inspections to assess public sector performance across a wide range of services. The underlying assumption is that auditees are ultimately responsible for the performance on which they are measured. This rationale might neglect the fact that they face external constraints not influenced easily by their decisions. The research presented here scrutinises one such evaluation approach, the Comprehensive Performance Assessment (CPA) of local government in England, for the significance of such factors. Using panel-data analysis involving the Index of Multiple Deprivation 2004 and data from the first three CPA rounds in 2002–04, the research finds that deprivation in education, crime, and living environment had a significant negative effect on local authorities' overall CPA scores, while discretionary expenditure had a positive but not significant effect. However, the effects differ widely across the five types of authorities that exist. The model also reveals that there is no statistically significant evidence to suggest that authorities controlled by a single party perform better than those without, and that, although Conservative councils have higher CPA scores in simple bivariate analysis than those controlled by other parties or with no overall control, these differences disappear in multivariate analysis.
Thinking the Event: Badiou's Philosophy of the Event and the Example of the Paris Commune
Authors: Bassett Keith
Published: June 2008

The problem of theorising the ‘event’ as a moment of major time–space transformation has become a focus of enquiry in philosophy, history, and the social sciences. In this paper I explore the theory of the event to be found in the recent work of Alain Badiou, relating his ideas to the particular case of the 1871 Paris Commune. I focus on particular problems with his approach, using an alternative formulation of the event found in the work of William Sewell Jr, which for purposes of comparison and critical debate I also relate to the event of the Paris Commune. I conclude by raising and discussing a range of wider issues that arise from the comparison.
The Effects of Curvature on Haptic Judgments of Extent in Sighted and Blind People
Authors: Heller Morton A , Kappers Astrid M L , McCarthy Melissa , Clark Ashley , Riddle Tara , Fulkerson Erin , Wemple Lindsay , Walk Anne McClure , Basso Andreana , Wanek Crystal , Russler Kristen
Published: June 2008

A series of experiments was carried out to examine the effect of curvature on haptic judgments of extent in sighted and blind individuals. Experiment 1 showed that diameters connecting the endpoints of semicircular lines were underestimated with respect to straight lines, but failed to show an effect of visual experience on length judgments. In experiment 2 we tested arc lengths. The effects of curvature on perceived path length were weaker, but were still present in this experiment. Visual experience had no effect on path length judgments. Another experiment was performed to examine the effect of repeated tracing (1, 5, 9, or unlimited number of traces) on judgments of the lengths of straight lines and diameters of semicircles. Judgments of extent were more accurate when subjects engaged in larger numbers of traces. There was no effect of number of traces on curve-height judgments, suggesting that subjects were not using height estimates to judge diameters of semicircles. In a further experiment we tested the effect of number of traces on curves that varied in height. Restricting subjects to a single trace magnified the effect of path length on judgments of the distance between the endpoints of curves. Additional experiments showed that curvature effects on diameter judgments were not eliminated when stimuli were in the frontal plane or when the curves were explored with the use of two hands. Arm support had no effect on judged length in experiment 7. A final experiment showed a robust horizontal – vertical illusion in haptic perception of convex curves, with overestimation of the heights of the curves compared with their widths. The practical and theoretical implications of the results are discussed.
Corrigendum to: Clinical significance of questionnaire-elicited or clinically reported anorectal symptoms for rectal Neisseria gonorrhoeae and Chlamydia trachomatis amongst men who have sex with men
Authors: Lister Nichole A. , Chaves Nadia J. , Pang Chee W. , Smith Anthony , Fairley Christopher K.
Published: June 2008

Background: Although Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are common infections in men who have sex with men, it is unclear from previous studies whether anorectal symptoms are reliable clinical indicators of infection. Aim: The objective of the study was to investigate the clinical significance of questionnaire-elicited or clinically reported anal symptoms for rectal Ng and Ct. Methods: During 2002 to 2003, men who have sex with men (MSM) screened or tested for Ng or Ct according to the national guidelines were invited to participate in a questionnaire. Results: During the study period, 366 MSM were enrolled into the study (88% recruitment rate), of whom 20 (5%) and 25 (7%) were diagnosed with rectal Ng or Ct, respectively. Overall, 'any' anorectal symptoms on a questionnaire were reported equally by those with and without rectal Ng (75 v. 74%, P�=�0.69), but heavy anal discharge (P�<�0.01) and anal pain (P�=�0.04) were more common in those with rectal Ng. Symptoms on the questionnaire were not different among those with and without Ct. Any anal symptoms were reported substantially more often via questionnaire than in a clinical consultation (75 v. 16%, P�<�0.01) and symptoms reported in a clinical consultation were not associated with Ng or Ct detection. Conclusion: The weak or absent association between symptoms and the presence of Ct or Ng highlights the importance of annual sexually transmitted infection screening in MSM regardless of symptoms.
Homosexual men in Australia: population, distributionand HIV prevalence
Authors: Prestage Garrett , Ferris Jason , Grierson Jeffrey , Thorpe Rachel , Zablotska Iryna , Imrie John , Smith Anthony , Grulich Andrew E.
Published: June 2008

Objectives: To assess the size, distribution and changes in the population of homosexual and bisexual men in Australia, and the capacity of available measures to make this estimation. Methods: We used data from five sources: the Australian Study of Health and Relationships, the Gay Community Periodic Surveys, HIV Futures, the Health in Men cohort study, the Australian National HIV and AIDS Registries and the Australian Household Census. Results: We estimated that in 2001 there were ~74 000 homosexual and bisexual men in New South Wales (NSW), ~42 000 in Victoria and ~37 000 in Queensland. There was, however, some discrepancy between datasets in the estimates of the overall proportions and distribution of homosexual and bisexual men across states. We also estimated HIV prevalence rates among homosexual and bisexual men in 2001 at ~8% in NSW, 5% in Victoria and 4% in Queensland. There were insufficient data to estimate whether the state-specific populations of homosexual men were changing with time. Conclusion: There are ~75% more homosexual and bisexual men in NSW than in Victoria and about twice as many as in Queensland. There are about two-thirds as many HIV-positive men in NSW as in Victoria and Queensland combined. Improved collection of population-based data on homosexuality are required.
Sexually transmissible infection testing guidelines for men who have sex with men
Authors: Bourne Chris , Edwards Barry , Shaw Miranda , Gowers Andrew , Rodgers Craig , Ferson Mark
Published: June 2008

Since 2002, biennial production of sexually transmissible infection testing guidelines for men who have sex with men has supported sexually transmitted infection control efforts in inner Sydney, Australia.
Sexually transmissible infection and HIV management among men who have sex with men with and without HIV: survey of medical practitioners who are members of the Australasian Society for HIV Medicine
Authors: Fairley Christopher K. , Fehler Glenda , Lewin Sharon R. , Pitts Marian , Chen Marcus Y. , Bradshaw Catriona S. , Hocking Jane S.
Published: June 2008

Background: Rates of HIV in New South Wales (NSW) have been stable, but have increased significantly in other Australian states. The reasons for this are unknown and may be associated with differences in the management of sexually transmissible infections (STI) and HIV in different states in Australia. Our aim was to determine if the use of suppressive treatment for genital herpes, the treatment of HIV or STI screening practices were different between states in Australia. Methods: This study was a cross-sectional survey of medical practitioners who are S100 prescribers and members of the Australian Society for HIV Medicine. Results: In general, there were no differences between the clinical practices of practitioners in NSW and other states except that in NSW fewer practitioners tested HIV-positive men who have sex with men (MSM) for syphilis annually (NSW 78% v. others 87%, P = 0.04) or treated MSM with advanced HIV disease (CD4 < 150 × 106 cells L–1) with acicylovir in the absence of herpes simplex virus (HSV) (NSW 4% v. others 13%, P = 0.03), and more practitioners in NSW tested HIV-negative MSM for HSV type-specific serology (NSW 21% v. others 11%, P = 0.02). Conclusions: It is unlikely that the minor differences in HSV and HIV treatment or STI screening practices among practitioners in NSW and other Australian states explains the differences in HIV notifications between these two areas.
Using mathematical modelling to help explain the differential increase in HIV incidence in New South Wales, Victoria and Queensland: importance of other sexually transmissible infections
Authors: Hoare Alexander , Wilson David P. , Regan David G. , Kaldor John , Law Matthew G.
Published: June 2008

Background: Since 1999 there has been an increase in the number of HIV diagnoses in Australia, predominantly among men who have sex with men (MSM), but the magnitude of increase differs between states: ~7% rise in New South Wales, ~96% rise in Victoria, and ~68% rise in Queensland. Methods: Epidemiological, clinical, behavioural and biological data were collated into a mechanistic mathematical model to explore possible reasons for this increase in HIV notifications in MSM. The model was then used to make projections to 2015 under various scenarios. Results: The model suggests that trends in clinical and behavioural parameters, including increases in unprotected anal intercourse, cannot explain the magnitude of the observed rise in HIV notifications, without a substantial increase in a ‘transmission-increasing’ factor. We suggest that a highly plausible biological factor is an increase in the prevalence of other sexually transmissible infections (STI). It was found that New South Wales required an ~2-fold increase in other STI to match the data, Victoria needed an ~11-fold increase, and Queensland required an ~9-fold increase. This is consistent with observed trends in Australia for some STI in recent years. Future projections also indicate that the best way to control the current rise in HIV notifications is to reduce the prevalence of other STI and to promote condom use, testing for HIV, and initiation of early treatment in MSM diagnosed during primary infection. Conclusions: Our model can explain the recent rise in HIV notifications with an increase in the prevalence of other STI. This analysis highlights that further investigation into the causes and impact of other STI is warranted in Australia, particularly in Victoria.
Interaction between human and robot
Authors: Agrawal Pramila , Liu Changchun , Sarkar Nilanjan
Published: June 2008

This paper presents a human–robot interaction framework where a robot can infer implicit affective cues of a human and respond to them appropriately. Affective cues are inferred by the robot in real-time from physiological signals. A robot-based basketball game is designed where a robotic “coach” monitors the human participant’s anxiety to dynamically reconfigure game parameters to allow skill improvement while maintaining desired anxiety levels. The results of the above-mentioned anxiety-based sessions are compared with performance-based sessions where in the latter sessions, the game is adapted only according to the player’s performance. It was observed that 79% of the participants showed lower anxiety during anxiety-based session than in the performance-based session, 64% showed a greater improvement in performance after the anxiety-based session and 71% of the participants reported greater overall satisfaction during the anxiety-based sessions. This is the first time, to our knowledge, that the impact of real-time affective communication between a robot and a human has been demonstrated experimentally.
Desmin mediates TNF-α–induced aggregate formation and intercalated disk reorganization in heart failure
Authors: Panagopoulou Panagiota , Davos Constantinos H. , Milner Derek J. , Varela Emily , Cameron JoAnn , Mann Douglas L. , Capetanaki Yassemi
Published: June 2008

We explored the involvement of the muscle-specific intermediate filament protein desmin in the model of tumor necrosis factor α (TNF-α)–induced cardiomyopathy. We demonstrate that in mice overexpressing TNF-α in the heart (α–myosin heavy chain promoter-driven secretable TNF-α [MHCsTNF]), desmin is modified, loses its intercalated disk (ID) localization, and forms aggregates that colocalize with heat shock protein 25 and ubiquitin. Additionally, other ID proteins such as desmoplakin and β-catenin show similar localization changes in a desmin-dependent fashion. To address underlying mechanisms, we examined whether desmin is a substrate for caspase-6 in vivo as well as the implications of desmin cleavage in MHCsTNF mice. We generated transgenic mice with cardiac-restricted expression of a desmin mutant (D263E) and proved that it is resistant to caspase cleavage in the MHCsTNF myocardium. The aggregates are diminished in these mice, and D263E desmin, desmoplakin, and β-catenin largely retain their proper ID localization. Importantly, D263E desmin expression attenuated cardiomyocyte apoptosis, prevented left ventricular wall thinning, and improved the function of MHCsTNF hearts.
Differential Correlation Between Rates of Antimicrobial Drug Consumption and Prevalence of Antimicrobial Resistance in a Tertiary Care Hospital in Greece
Authors: Iosifidis Elias , Antachopoulos Charalampos , Tsivitanidou Maria , Katragkou Aspasia , Farmaki Evagelia , Tsiakou Maria , Kyriazi Theano , Sofianou Danai , Roilides Emmanuel
Published: June 2008

Objective.

To investigate whether there is a correlation between the rates of antimicrobial drug consumption in hospital departments and the prevalence of antimicrobial resistance among clinically important bacteria recovered in the hospital.

Design.

Retrospective study.

Setting.

Tertiary care hospital in Greece.

Methods.

Data on antimicrobial consumption (from January 2001 through December 2004) were expressed as defined daily doses per 100 bed-days. The prevalence of antimicrobial resistance among isolates of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterococcusfaecium recovered during the same time period were calculated by the microbiology department. We then performed the following analyses: (1) a comparison of the consumption rates for different antimicrobial groups in individual hospital departments, (2) a comparison of the prevalence of resistance to different antimicrobials, and (3) a correlation analysis of antimicrobial consumption rates and the prevalence of antimicrobial resistance.

Results.

The rates of antimicrobial consumption and the prevalence of resistance varied substantially among the hospital's departments. The annual rate of consumption for carbapenems correlated with the rate of consumption for glycopeptides and third-generation cephalosporins (P < .05). Among P. aeruginosa isolates, the prevalence of imipenem resistance correlated with the prevalence of resistance to amikacin, ciprofloxacin, and ceftazidime (P < .05). The rate of carbapenem consumption correlated with the prevalence of imipenem resistance among P. aeruginosa and A. baumannii isolates (P < .05). The rate of aminoglycoside consumption correlated with the prevalence of amikacin resistance among P. aeruginosa, K. pneumoniae, and E. coli isolates (P < .05). However, the rate of consumption for fluoroquinolones and glycopeptides had no correlation with the prevalence of ciprofloxacin resistance among gram-negative bacteria or vancomycin resistance among E. faecium isolates.

Conclusions.

These data are suggestive of a differential relationship between antimicrobial consumption and the prevalence of antimicrobial resistance among various species and for various antimicrobial agents. These findings may help to optimize antimicrobial prescription policies in the hospital, especially in departments that have both high rates of antimicrobial consumption and a high prevalence of antimicrobial resistance.
Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging
Authors: Morimoto Richard I.
Published: June 2008

The long-term health of the cell is inextricably linked to protein quality control. Under optimal conditions this is accomplished by protein homeostasis, a highly complex network of molecular interactions that balances protein biosynthesis, folding, translocation, assembly/disassembly, and clearance. This review will examine the consequences of an imbalance in homeostasis on the flux of misfolded proteins that, if unattended, can result in severe molecular damage to the cell. Adaptation and survival requires the ability to sense damaged proteins and to coordinate the activities of protective stress response pathways and chaperone networks. Yet, despite the abundance and apparent capacity of chaperones and other components of homeostasis to restore folding equilibrium, the cell appears poorly adapted for chronic proteotoxic stress when conformationally challenged aggregation-prone proteins are expressed in cancer, metabolic disease, and neurodegenerative disease. The decline in biosynthetic and repair activities that compromises the integrity of the proteome is influenced strongly by genes that control aging, thus linking stress and protein homeostasis with the health and life span of the organism.
TopBP1 activates ATR through ATRIP and a PIKK regulatory domain
Authors: Mordes Daniel A. , Glick Gloria G. , Zhao Runxiang , Cortez David
Published: June 2008

The ATR (ATM and Rad3-related) kinase and its regulatory partner ATRIP (ATR-interacting protein) coordinate checkpoint responses to DNA damage and replication stress. TopBP1 functions as a general activator of ATR. However, the mechanism by which TopBP1 activates ATR is unknown. Here, we show that ATRIP contains a TopBP1-interacting region that is necessary for the association of TopBP1 and ATR, for TopBP1-mediated activation of ATR, and for cells to survive and recover DNA synthesis following replication stress. We demonstrate that this region is functionally conserved in the Saccharomyces cerevisiae ATRIP ortholog Ddc2, suggesting a conserved mechanism of regulation. In addition, we identify a domain of ATR that is critical for its activation by TopBP1. Mutations of the ATR PRD (PIKK [phosphoinositide 3-kinase related kinase] Regulatory Domain) do not affect the basal kinase activity of ATR but prevent its activation. Cellular complementation experiments demonstrate that TopBP1-mediated ATR activation is required for checkpoint signaling and cellular viability. The PRDs of ATM and mTOR (mammalian target of rapamycin) were shown previously to regulate the activities of these kinases, and our data indicate that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) PRD is important for DNA-PKcs regulation. Therefore, divergent amino acid sequences within the PRD and a unique protein partner allow each of these PIK kinases to respond to distinct cellular events.
DLC1 is a chromosome 8p tumor suppressor whose loss promotes hepatocellular carcinoma
Authors: Xue Wen , Krasnitz Alexander , Lucito Robert , Sordella Raffaella , VanAelst Linda , Cordon-Cardo Carlos , Singer Stephan , Kuehnel Florian , Wigler Michael , Powers Scott , Zender Lars , Lowe Scott W.
Published: June 2008

Deletions on chromosome 8p are common in human tumors, suggesting that one or more tumor suppressor genes reside in this region. Deleted in Liver Cancer 1 (DLC1) encodes a Rho-GTPase activating protein and is a candidate 8p tumor suppressor. We show that DLC1 knockdown cooperates with Myc to promote hepatocellular carcinoma in mice, and that reintroduction of wild-type DLC1 into hepatoma cells with low DLC1 levels suppresses tumor growth in situ. Cells with reduced DLC1 protein contain increased GTP-bound RhoA, and enforced expression a constitutively activated RhoA allele mimics DLC1 loss in promoting hepatocellular carcinogenesis. Conversely, down-regulation of RhoA selectively inhibits tumor growth of hepatoma cells with disabled DLC1. Our data validate DLC1 as a potent tumor suppressor gene and suggest that its loss creates a dependence on the RhoA pathway that may be targeted therapeutically.
Failure of CDKN2A/B (INK4A/B–ARF)-mediated tumor suppression and resistance to targeted therapy in acute lymphoblastic leukemia induced by BCR-ABL
Authors: Mullighan Charles G. , Williams Richard T. , Downing James R. , Sherr Charles J.
Published: June 2008

Deletions of the CDKN2A/B tumor suppressor locus and of the IKAROS and PAX5 genes that promote B-lineage development occur frequently in lymphoid, but not myeloid leukemias initiated by the BCR-ABL tyrosine kinase. Why is this the case, and how do these genetic lesions contribute to an aggressive disease that fails to durably respond to targeted kinase inhibitors?
Trans-regulation of the expression of the transcription factor MtHAP2-1 by a uORF controls root nodule development
Authors: Combier Jean Philippe , de Billy Françoise , Gamas Pascal , Niebel Andreas , Rivas Susana
Published: June 2008

MtHAP2-1 is a CCAAT-binding transcription factor from the model legume Medicago truncatula. We previously showed that MtHAP2-1 expression is regulated both spatially and temporally by microRNA169. Here we present a novel regulatory mechanism controlling MtHAP2-1 expression. Alternative splicing of an intron in the MtHAP2-1 5′leader sequence (LS) becomes predominant during the development of root nodules, leading to the production of a small peptide, uORF1p. Our results indicate that binding of uORF1p to MtHAP2-1 5′LS mRNA leads to reduced accumulation of the MtHAP2-1 transcript and may contribute to spatial restriction of MtHAP2-1 expression within the nodule. We propose that miR169 and uORF1p play essential, sequential, and nonredundant roles in regulating MtHAP2-1 expression. Importantly, in contrast to previously described cis-acting uORFs, uORF1p is able to act in trans to down-regulate gene expression. Our work thus contributes to a better understanding of the action of upstream ORFs (uORFs) in the regulation of gene expression.
Maternal noncoding transcripts antagonize the targeting of DNA elimination by scanRNAs in Paramecium tetraurelia
Authors: Lepère Gersende , Bétermier Mireille , Meyer Eric , Duharcourt Sandra
Published: June 2008

The germline genome of ciliates is extensively rearranged during the development of a new somatic macronucleus from the germline micronucleus, after sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) are precisely excised from coding sequences and intergenic regions. For a subset of IESs, introduction of the IES sequence into the maternal macronucleus specifically inhibits excision of the homologous IES in the developing zygotic macronucleus, suggesting that epigenetic regulation of excision involves a global comparison of germline and somatic genomes. ScanRNAs (scnRNAs) produced during micronuclear meiosis by a developmentally regulated RNAi pathway have been proposed to mediate this transnuclear cross-talk. In this study, microinjection experiments provide direct evidence that 25-nucleotide (nt) scnRNAs promote IES excision. We further show that noncoding RNAs are produced from the somatic maternal genome, both during vegetative growth and during sexual events. Maternal inhibition of IES excision is abolished when maternal somatic transcripts containing an IES are targeted for degradation by a distinct RNAi pathway involving 23-nt siRNAs. The results strongly support a scnRNA/macronuclear RNA scanning model in which a natural genomic subtraction, occurring during meiosis between deletion-inducing scnRNAs and antagonistic transcripts from the maternal macronucleus, regulates rearrangements of the zygotic genome.
The new paradigm of flow cell sequencing: Table 1.
Authors: Holt Robert A. , Jones Steven J.M.
Published: June 2008

DNA sequencing is in a period of rapid change, in which capillary sequencing is no longer the technology of choice for most ultra-high-throughput applications. A new generation of instruments that utilize primed synthesis in flow cells to obtain, simultaneously, the sequence of millions of different DNA templates has changed the field. We compare and contrast these new sequencing platforms in terms of stage of development, instrument configuration, template format, sequencing chemistry, throughput capability, operating cost, data handling issues, and error models. While these platforms outperform capillary instruments in terms of bases per day and cost per base, the short length of sequence reads obtained from most instruments and the limited number of samples that can be run simultaneously imposes some practical constraints on sequencing applications. However, recently developed methods for paired-end sequencing and for array-based direct selection of desired templates from complex mixtures extend the utility of these platforms for genome analysis. Given the ever increasing demand for DNA sequence information, we can expect continuous improvement of this new generation of instruments and their eventual replacement by even more powerful technology.
A Test of the Markov Model of Evolution in Proteins
Authors: Mount David W.
Published: June 2008

INTRODUCTION

The percent accepted mutation (PAM) scoring matrix is based on the Dayhoff model of protein evolution, which is a Markov process. In the Markov model of amino acid change, the probability of mutation at each site is independent of the previous history of mutations. Use of this model makes it possible to extrapolate amino acid substitutions observed over a relatively short period of evolutionary time to longer periods of evolutionary time. One criticism of the PAM scoring matrix is that the frequency of amino acid changes that require two nucleotide changes is higher than would be expected by chance. This article describes a test of the Markov model of protein evolution, which shows that the model can be valid if certain changes are made in the way that PAM matrices are calculated.
Comparison of the PAM and BLOSUM Amino Acid Substitution Matrices
Authors: Mount David W.
Published: June 2008

INTRODUCTION

The choice of a scoring system including scores for matches, mismatches, substitutions, insertions, and deletions influences the alignment of both DNA and protein sequences. To score matches and mismatches in alignments of proteins, it is necessary to know how often one amino acid is substituted for another in related proteins. Percent accepted mutation (PAM) matrices list the likelihood of change from one amino acid to another in homologous protein sequences during evolution and thus are focused on tracking the evolutionary origins of proteins. In contrast, the blocks amino acid substitution matrices (BLOSUM) are based on scoring substitutions found over a range of evolutionary periods. There are important differences in the ways that the PAM and BLOSUM scoring matrices were derived. These differences, which are discussed in this article, should be appreciated when interpreting the results of protein sequence alignments obtained with these matrices.
In-Gel S-Pyridylethylation of Gel-Resolved Proteins: Individual Spot Method
Authors: Simpson Richard J.
Published: June 2008

INTRODUCTION

This protocol describes a method for performing reduction and S-alkylation of Coomassie-blue-stained proteins within individually excised, stained protein spots or bands. Reduction is performed with dithiothreitol, and alkylation with 4-vinylpyridine. (Treatment of free cysteines with 4-vinylpyridine yields the S-β-(4-pyridylethyl) cysteinyl derivative.) S-β-(4-pyridylethyl) cysteine-containing peptides can be readily identified during RP-HPLC by their characteristic absorbance at 254 nm and during electrospray ionization tandem mass spectrometry by the appearance of a characteristic pyridylethyl fragment ion of 106 Da. The position of cysteine residues in a polypeptide sequence can be determined either by mass spectrometry or as phenylthiohydantoin S-β-(4-pyridylethyl) cysteine during Edman degradation.
Isolation and Whole-Cell Patch Clamping of Arabidopsis Guard Cell Protoplasts
Authors: Zhang Wei , Nilson Sarah E. , Assmann Sarah M.
Published: June 2008

INTRODUCTION

The flux of ions across membranes via ion channels is vital to cellular responses to internal and external stimuli, and therefore to cellular survival in changing circumstances. Patch clamping is a powerful technique for ion channel investigation, because it enables measurement of both net ion fluxes across the entire surface area of a cell and ion currents flowing through a single open channel. However, unlike animal cells, plant cells are surrounded by cell walls that prevent the physical contact between the patch pipette and the plasma membrane necessary for the patch clamp technique. To demonstrate how patch clamping can be applied to plant physiology research, we describe a protocol used to record potassium ion (K+) channel currents in Arabidopsis guard cell protoplasts (a widely studied model cell type in plant biology). The protocol requires a two-step cellulase and pectinase digestion to isolate high quality Arabidopsis guard cell protoplasts (i.e., plant cells lacking their cell walls), preparation of suitable glass capillary microelectrodes, and formation of the whole-cell configuration with a gigaohm (GΩ) seal. We also describe the history of the protocol and list other types of plant cells from which successful patch clamp recordings have been obtained.
Immunogold Staining Following Freeze Substitution and Low Temperature Embedding after Chemical Fixation or after Cryoimmobilization for Transmission Electron Microscopy (TEM): Figure 1.
Authors: Skepper Jeremy N. , Powell Janet M.
Published: June 2008

INTRODUCTION

In this method for freeze substitution and low-temperature embedding in resin prior to immunogold staining, lightly fixed tissue pieces are cryoprotected by immersion in polypropylene glycol. The cryoprotected tissues are quench frozen and transferred under liquid nitrogen to vials containing frozen methanol or methanol containing uranyl acetate. The vials are transferred to a substitution vessel where the temperature can be controlled and a nitrogen atmosphere maintained. The temperature is raised (typically at 5°C/h to -90°C) and maintained for 24 h. This temperature is cold enough to prevent recrystallization of water and thus tissue disruption, but high enough for movement of water to occur and allow substitution with the liquid methanol. After 24 h, ~90% of the water has been substituted. The substitution medium is replaced and the temperature is raised to -70°C for 24 h. The substitution medium is changed again and the temperature is raised to -50°C. The tissue is impregnated with Lowicryl HM20 over a period of 1-5 d and the resin is polymerized by UV irradiation. Tissue is then sectioned and stained immunochemically with primary antibodies raised against antigens exposed on the surface of the sections, and primary antibodies are visualized by staining with secondary antibodies conjugated to colloidal gold particles. The immunochemically stained sections are contrast stained with uranyl acetate and lead citrate to reveal the ultrastructure of the cells, and are finally viewed by transmission electron microscopy (TEM). This is the simplest and most versatile of the post-embedding procedures.
Immunogold Staining of Ultrathin Thawed Cryosections for Transmission Electron Microscopy (TEM): Figure 1.
Authors: Skepper Jeremy N. , Powell Janet M.
Published: June 2008

INTRODUCTION

A pre-embedding method of immunochemical staining is used if antigens are damaged by resin embedding, or if the best preservation of membranes is required. Applying immunogold reagents to sections of lightly fixed tissue, free of embedding medium, can be a very sensitive method of immunochemical staining. Cells or tissues are fixed as strongly as possible and then treated with a cryoprotectant, which is usually a mixture of sucrose and polyvinylpyrrolidone (PVP). They are frozen onto pins in liquid nitrogen and sectioned at approximately -100°C. The frozen sections are thaw-mounted on to Formvar/nickel film grids and the cryoprotectant is removed by floating the grids on drops of phosphate-buffered saline (PBS). The immunogold staining is performed on the unembedded sections, which are subsequently contrast counterstained and infiltrated with a mixture of methylcellulose and uranyl acetate. In this protocol, samples are sectioned at low temperature, thaw-mounted onto film grids, immunochemically stained, contrast counterstained, and embedded/encapsulated in situ on the grid before viewing by transmission electron microscopy (TEM).
High-Resolution Fluorescent In Situ Hybridization of Drosophila Embryos and Tissues: Figure 1.
Authors: Lécuyer Eric , Nećakov Aleksandar S. , Cáceres Lucía , Krause Henry M.
Published: June 2008

INTRODUCTION

Fluorescent in situ hybridization (FISH) is commonly used to analyze the three-dimensional distribution of RNAs in intact embryos and tissues. Tyramide signal amplification (TSA) significantly increases the sensitivity and resolution of FISH probe signals. This protocol includes optimized TSA-FISH procedures for Drosophila embryos, ovaries, and larval tissues. Instructions are given for the preparation of RNA probes, the collection and fixation of tissues, and the hybridization and TSA-mediated detection of probes, including options for high-throughput processing in 96-well plates. Variations of the procedure for RNA-RNA and RNA-protein costaining are also described.
Using PAM Matrices in Sequence Alignments
Authors: Mount David W.
Published: June 2008

INTRODUCTION

Certain amino acid substitutions commonly occur in related proteins from different species. Because a protein still functions with these substitutions, the substituted amino acids are compatible with protein structure and function. Knowing the types of changes that are most and least common in a large number of proteins can assist with predicting alignments for any set of protein sequences. If related protein sequences are quite similar, they are easy to align, and one can readily determine the single-step amino acid changes. If ancestor relationships among a group of proteins are assessed, the most likely amino acid changes that occurred during evolution can be predicted. This type of analysis was pioneered by Margaret Dayhoff and used by her to produce a type of scoring matrix called a percent accepted mutation (PAM) matrix. This article introduces Dayhoff PAM matrices, explains how they are constructed and how they can be used for sequence alignments, and highlights their strengths and limitations.
Copy Number Variation Detection via High-Density SNP Genotyping
Authors: Wang Kai , Bucan Maja
Published: June 2008

INTRODUCTION

High-density single nucleotide polymorphism (SNP) genotyping arrays recently have been used for copy number variation (CNV) detection and analysis, because the arrays can serve a dual role for SNP- and CNV-based association studies. They also can provide considerably higher precision and resolution than traditional techniques. Here we describe PennCNV, a computational protocol designed for CNV detection from high-density SNP genotyping data. This protocol extracts allele-specific signal intensities from genotyping arrays, and then integrates information on SNP spacing and SNP allele frequencies to generate CNV calls by a hidden Markov model (HMM) algorithm. Analyses of CNVs from SNP genotyping arrays will provide a more comprehensive view of genome variation, and complement current genome-wide association studies in identifying disease susceptibility loci.
Concurrent Graves disease thyrotoxicosis and myasthenia gravis: the treatment of the former may dangerously reveal the latter
Authors: Lakhal K. , Blel Y. , Fysekidis M. , Mohammedi K. , Bouadma L.
Published: June 2008

Autoimmune thyroxicosis and myasthenia gravis are often associated. In both diseases, clinical features may include neuromuscular weakness, making their distinction challenging. We report a patient with known Graves disease who presented with generalised fatigue, initially attributed solely to thyrotoxicosis, and who experienced severe respiratory failure linked to associated myasthenia gravis that was unmasked by medication used in the perioperative management of his thyroxicosis. Anaesthetists should always consider myasthenia gravis in cases of hyperthyroidism presenting with neuromuscular features.
Pre‐oxygenation in pregnancy: the effect of fresh gas flow rates within a circle breathing system*
Authors: Russell E. C. , Wrench I. , Feast M. , Mohammed F.
Published: June 2008

We have investigated the effect of oxygen flow rate on pre‐oxygenation in pregnant patients at term using a circle system. Twenty patients presenting for elective Caesarean section maintained tidal volume breathing through a standard circle system for 3 min. Subjects were pre‐oxygenated using oxygen flow rates of 5 l.min−1, 10 l.min−1 and 15 l.min−1 presented in random order. The mean (SD) fractional end‐tidal oxygen at the end of 3 min was 0.86 (0.07) for 5 l.min−1, 0.92 (0.05) for 10 l.min−1and 0.90 (0.09) for 15 l.min−1 (p < 0.001). Entrainment of air occurred in 22% of pre‐oxygenation sessions. Oxygen flow rates of 10 l.min−1 or above provide optimal pre‐oxygenation using a circle system in term parturients. In our study, entrainment of air occurred in a surprisingly high percentage of cases.
Diagnosis and treatment of post‐transplantation lymphoproliferative disorder in pediatric heart transplant patients
Authors: Schubert S. , Abdul‐Khaliq H. , Lehmkuhl H. B. , Yegitbasi M. , Reinke P. , Kebelmann‐Betzig C. , Hauptmann K. , Gross‐Wieltsch U. , Hetzer R. , Berger F.
Published: June 2008

Abstract:  PTLD is a severe complication in transplant recipients. Detection of increased EBV load in the peripheral blood acts as a surrogate marker for increased risk of PTLD development. We analyzed the time course of the disease, its severitiy, the organs involved, and mortality rates in our institutional experience of pediatric heart transplantation. This paper identifies risk factors for PTLD and describes the different ways of diagnosing and treating the disease. PTLD was screened for in 146 pediatric heart transplant patients using a retrospective analysis in patients who received transplantation before 1998. Prospective determination was performed in 72/146 patients transplanted after 1998 within the post‐transplant follow‐up. The occurrence of PTLD with all interventions, including tapering of immunosuppression, surgery, viral monitoring, and antiviral interventions, was recorded. PTLD was diagnosed in 12/147 (8.2%) children at a mean age of 7.2 ± 3.3 yr after a mean post‐transplant period of 3.2 ± 2.2 yr. PTLD manifested in: lymph nodes (n = 4), intestine (n = 3), tonsils and adenoids (n = 2), eye (n = 2), and lung (n = 1). It was diagnosed in 7/12 as a monomorphic B‐cell lymphoma and in four patients as a monomorphic Burkitt lymphoma, a polymorphic B‐cell lymphoma, a T‐cell rich or angiocentric lymphoma (Liebow) and as reactive plasmacytic hyperplasia (early lesion), respectively. Histology was not possible in one patient with ocular manifestation. EBV association was 83%. Risk factors in the comparison with patients without PTLD were age at time of Tx, primary EBV infection after Tx, use of Azathioprine and ≥3 doses of ATG. CMV mismatch and CMV infection, rejection episodes and steroids were not risk factors. Despite reduction of immunosuppression, treatment consisted of surgical procedures to remove tumor masses (n = 6), Rituximab (n = 5), polychemotherapy (n = 3), antiviral (n = 1) and autologous T‐cell therapy (n = 1). All patients demonstrated full remission without death related to PTLD or treatment at 3.9 (1.3–6.2) yr median follow‐up time. The manifestation of PTLD in pediatric heart transplant recipients is associated with EBV infection and is predominantly in the form of a B‐cell lymphoma. A tight and specific follow‐up including early assessment of immunity status and specific therapeutic intervention to improve cellular immunity is warranted and may contribute to a significant reduction of PTLD‐related morbidity and mortality.
The application of ethephon (an ethylene releaser) increases growth, photosynthesis and nitrogen accumulation in mustard (Brassica juncea L.) under high nitrogen levels
Authors: Khan N. A. , Mir M. R. , Nazar R. , Singh S.
Published: June 2008

Ethephon (2‐chloroethyl phosphonic acid), an ethylene‐releasing compound, influences growth and photosynthesis of mustard (Brassica juncea L. Czern & Coss.). We show the effect of nitrogen availability on ethylene evolution and how this affects growth, photosynthesis and nitrogen accumulation. Ethylene evolution in the control with low N (100 mg N kg−1 soil) was two‐times higher than with high N (200 mg N kg−1 soil). The application of 100–400 μl·l−1 ethephon post‐flowering, i.e. 60 days after sowing, on plants receiving low or high N further increased ethylene evolution. Leaf area, relative growth rate (RGR), photosynthesis, leaf nitrate reductase (NR) activity and leaf N reached a maximum with application of 200 μl·l−1 ethephon and high N. The results suggest that the application of ethephon influences growth, photosynthesis and N accumulation, depending on the amount of nitrogen in the soil.
QTL analysis of leaf morphological characters in a Quercus robur full‐sib family (Q. robur × Q. robur ssp. slavonica)
Authors: Gailing O.
Published: June 2008

The distinction between white oak species (section Quercus sensu stricto) is largely based on leaf morphological characters. There is, however, considerable within‐species variation and no single species‐diagnostic character, possibly due to phenotypic plasticity and/or underlying genetic variation. The aim of the present study was to identify quantitative trait loci (QTL) underlying the high within‐species variation for leaf morphological characters in an F1 full‐sib family derived from a cross between Q. robur and Q. robur ssp. slavonica. In accordance with an earlier QTL mapping study in an intraspecific Q. robur full‐sib family, polygenic inheritance was detected for leaf morphological characters that are used to discriminate between the species Quercus robur and Q. petraea. QTLs were distributed over ten linkage groups, showed a moderate effect in terms of phenotypic variance explained (PVE) in the mapping pedigree (3.6–9.6%), but accounted for a considerable amount of the parental differences. Co‐localisation of QTLs on the same linkage group in different genetic backgrounds was found for the number and percentage of intercalary veins (NV, PV) on linkage group 3 and for NV on linkage group 5, revealing a high congruence in the relative QTL positions. The generally low correspondence of the other QTLs in the different mapping pedigrees may be an effect of the genetic background and of the environment. In conclusion, leaf morphological characters were found to be under polygenic control, and a comparison to earlier published results led to the identification of two QTLs that were stable across different genetic backgrounds.
Morphological patterns of extrafloral nectaries in woody plant species of the Braziliancerrado
Authors: Machado S. R. , Morellato L. P. C. , Sajo M. G. , Oliveira P. S.
Published: June 2008

Extrafloral nectaries are nectar‐secreting structures that are especially common among the woody flora of the Braziliancerrado, a savanna‐like vegetation. In this study, we provide morphological and anatomical descriptions of extrafloral nectaries (EFNs) occurring on vegetative and reproductive organs of several plant species from thecerrado, and discuss their function and ecological relevance. We describe the morphology and anatomy of EFNs of 40 species belonging to 15 woody families using scanning electron microscopy and light microscopy. We categorise EFNs following a structural–topographical classification, and characterise the vascularised and complex nectaries, amorphous nectaries and secretory trichomes. Fabaceae, Bignoniaceae, Malpighiaceae and Vochysiaceae were the plant families with the majority of species having EFNs. Ten species possess more than one morphotype of gland structure. Observations and experimental field studies in thecerradosupport the anti‐herbivore role of EFN‐gathering ants in this habitat. Additional morphological studies of EFNs‐bearing plants, including other growth forms (e.g.herbs and lianas), are being undertaken and will hopefully cast further light on the ecological relevance of these glands in thecerrado, especially with respect to their attractiveness to multiple visitors.