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1.  Frataxin-bypassing Isu1: characterization of the bypass activity in cells and mitochondria 
The Biochemical journal  2014;459(1):71-81.
Frataxin is a conserved mitochondrial protein, and deficiency underlies the neurodegenerative disease Friedreich’s ataxia. Frataxin interacts with the core machinery for Fe–S cluster assembly in mitochondria. Recently we reported that in frataxin-deleted yeast strains, a spontaneously occurring mutation in one of two genes encoding redundant Isu scaffold proteins, bypassed the mutant phenotypes. In the present study we created strains expressing a single scaffold protein, either Isu1 or the bypass mutant M107I Isu1. Our results show that in the frataxin-deletion strain expressing the bypass mutant Isu1, cell growth, Fe–S cluster protein activities, haem proteins and iron homoeostasis were restored to normal or close to normal. The bypass effects were not mediated by changes in Isu1 expression level. The persulfide-forming activity of the cysteine desulfurase was diminished in the frataxin deletion (Δyfh1 ISU1) and was improved by expression of the bypass Isu1 (Δyfh1 M107I ISU1). The addition of purified bypass M107I Isu1 protein to a Δyfh1 lysate conferred similar enhancement of cysteine desulfurase as did frataxin, suggesting that this effect contributed to the bypass mechanism. Fe–S cluster-forming activity in isolated mitochondria was stimulated by the bypass Isu1, albeit at a lower rate. The rescuing effects of the bypass Isu1 point to ways that the core defects in Friedreich’s ataxia mitochondria can be restored.
doi:10.1042/BJ20131273
PMCID: PMC4021491  PMID: 24433162
cysteine desulfurase; eukaryote; frataxin; iron–sulfur; Isu1 scaffold; mitochondrion
2.  Mutation in the Fe–S scaffold protein Isu bypasses frataxin deletion 
The Biochemical journal  2012;441(1):473-480.
Frataxin is a conserved mitochondrial protein deficient in patients with Friedreich’s ataxia. Frataxin has been implicated in control of iron homoeostasis and Fe–S cluster assembly. In yeast or human mitochondria, frataxin interacts with components of the Fe–S cluster synthesis machinery, including the cysteine desulfurase Nfs1, accessory protein Isd11 and scaffold protein Isu. In the present paper, we report that a single amino acid substitution (methionine to isoleucine) at position 107 in the mature form of Isu1 restored many deficient functions in Δyfh1 or frataxin-depleted yeast cells. Iron homoeostasis was improved such that soluble/usable mitochondrial iron was increased and accumulation of insoluble/non-usable iron within mitochondria was largely prevented. Cytochromes were returned to normal and haem synthesis was restored. In mitochondria carrying the mutant Isu1 and no frataxin, Fe–S cluster enzyme activities were improved. The efficiency of newFe–S cluster synthesis in isolated mitochondria was markedly increased compared with frataxin-negative cells, although the response to added iron was minimal. The M107I substitution in the highly conserved Isu scaffold protein is typically found in bacterial orthologues, suggesting that a unique feature of the bacterial Fe–S cluster machinery may be involved. The mechanism by which the mutant Isu bypasses the absence of frataxin remains to be determined, but could be related to direct effects on Fe–S cluster assembly and/or indirect effects on mitochondrial iron availability.
doi:10.1042/BJ20111637
PMCID: PMC4018837  PMID: 21936771
frataxin; haem; iron; iron–sulfur cluster (Fe–S cluster); mitochondrion
3.  Recombinations in Staphylococcal Cassette Chromosome mec Elements Compromise the Molecular Detection of Methicillin Resistance in Staphylococcus aureus 
PLoS ONE  2014;9(6):e101419.
Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (∼4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates.
doi:10.1371/journal.pone.0101419
PMCID: PMC4074205  PMID: 24972080
4.  Short Communication: Effect of Short-Course Antenatal Zidovudine and Single-Dose Nevirapine on the BED Capture Enzyme Immunoassay Levels in HIV Type 1 Subtype C Infection 
Abstract
Cross-sectional prevalence studies based on immunoassays that discriminate between recent and long-term infections, such as the BED assay, have been widely used to estimate HIV incidence. However, individuals receiving highly active antiretroviral therapy tend to have lower BED levels and are associated with a higher risk for being mistakenly classified as recent infections. To assess the effect of short-term antenatal zidovudine (ZDV) and single-dose nevirapine (sdNVP) on the BED levels in HIV-1C infection, we measured longitudinal BED normalized optical density (OD-n) levels using stored plasma samples collected prenatally and postnatally from 159 pregnant HIV-infected women in Botswana who participated in the randomized clinical Mother-to-Child-Prevention study, the Mashi study. All women received ZDV from 34 weeks gestation through delivery and were randomized to receive either sdNVP or placebo during labor. Among 159 subjects, the OD-n levels decreased from baseline to delivery in 93 subjects (p=0.039), suggesting that short-course ZDV may decrease OD-n levels. sdNVP at delivery did not affect longitudinal BED OD-n levels postdelivery. However, sdNVP appeared to modify the association between CD4 count at delivery and OD-n levels postdelivery. When estimating HIV incidence with the BED assay, special care may be required regarding women who received short-term ZDV for prevention of mother-to-child transmission.
doi:10.1089/aid.2012.0294
PMCID: PMC3653368  PMID: 23521375
6.  The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host–parasite interaction 
Nucleic Acids Research  2014;42(11):7113-7131.
Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5′ ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct.
doi:10.1093/nar/gku322
PMCID: PMC4066756  PMID: 24799432
7.  PolyTB: A genomic variation map for Mycobacterium tuberculosis 
Summary
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second major cause of death from an infectious disease worldwide. Recent advances in DNA sequencing are leading to the ability to generate whole genome information in clinical isolates of M. tuberculosis complex (MTBC). The identification of informative genetic variants such as phylogenetic markers and those associated with drug resistance or virulence will help barcode Mtb in the context of epidemiological, diagnostic and clinical studies. Mtb genomic datasets are increasingly available as raw sequences, which are potentially difficult and computer intensive to process, and compare across studies. Here we have processed the raw sequence data (>1500 isolates, eight studies) to compile a catalogue of SNPs (n = 74,039, 63% non-synonymous, 51.1% in more than one isolate, i.e. non-private), small indels (n = 4810) and larger structural variants (n = 800). We have developed the PolyTB web-based tool (http://pathogenseq.lshtm.ac.uk/polytb) to visualise the resulting variation and important meta-data (e.g. in silico inferred strain-types, location) within geographical map and phylogenetic views. This resource will allow researchers to identify polymorphisms within candidate genes of interest, as well as examine the genomic diversity and distribution of strains. PolyTB source code is freely available to researchers wishing to develop similar tools for their pathogen of interest.
doi:10.1016/j.tube.2014.02.005
PMCID: PMC4066953  PMID: 24637013
Mycobacterium tuberculosis; Database; Genomics; Software; Molecular epidemiology; Whole-genome sequencing
8.  Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury 
Cancer letters  2011;327(0):48-60.
Cellular exposure to ionizing radiation leads to oxidizing events that alter atomic structure through direct interactions of radiation with target macromolecules or via products of water radiolysis. Further, the oxidative damage may spread from the targeted to neighboring, non-targeted bystander cells through redox-modulated intercellular communication mechanisms. To cope with the induced stress and the changes in the redox environment, organisms elicit transient responses at the molecular, cellular and tissue levels to counteract toxic effects of radiation. Metabolic pathways are induced during and shortly after the exposure. Depending on radiation dose, dose-rate and quality, these protective mechanisms may or may not be sufficient to cope with the stress. When the harmful effects exceed those of homeostatic biochemical processes, induced biological changes persist and may be propagated to progeny cells. Physiological levels of reactive oxygen and nitrogen species play critical roles in many cellular functions. In irradiated cells, levels of these reactive species may be increased due to perturbations in oxidative metabolism and chronic inflammatory responses, thereby contributing to the long-term effects of exposure to ionizing radiation on genomic stability. Here, in addition to immediate biological effects of water radiolysis on DNA damage, we also discuss the role of mitochondria in the delayed outcomes of ionization radiation. Defects in mitochondrial functions lead to accelerated aging and numerous pathological conditions. Different types of radiation vary in their linear energy transfer (LET) properties, and we discuss their effects on various aspects of mitochondrial physiology. These include short and long-term in vitro and in vivo effects on mitochondrial DNA, mitochondrial protein import and metabolic and antioxidant enzymes.
doi:10.1016/j.canlet.2011.12.012
PMCID: PMC3980444  PMID: 22182453
Ionizing radiation; Reactive oxygen/nitrogen species; Oxidative metabolism; Mitochondria; Genomic instability; adaptive responses; bystander effects
9.  SVAMP: sequence variation analysis, maps and phylogeny 
Bioinformatics  2014;30(15):2227-2229.
Summary: SVAMP is a stand-alone desktop application to visualize genomic variants (in variant call format) in the context of geographical metadata. Users of SVAMP are able to generate phylogenetic trees and perform principal coordinate analysis in real time from variant call format (VCF) and associated metadata files. Allele frequency map, geographical map of isolates, Tajima’s D metric, single nucleotide polymorphism density, GC and variation density are also available for visualization in real time. We demonstrate the utility of SVAMP in tracking a methicillin-resistant Staphylococcus aureus outbreak from published next-generation sequencing data across 15 countries. We also demonstrate the scalability and accuracy of our software on 245 Plasmodium falciparum malaria isolates from three continents.
Availability and implementation: The Qt/C++ software code, binaries, user manual and example datasets are available at http://cbrc.kaust.edu.sa/svamp
Contact: arnab.pain@kaust.edu.sa or arnab.pain@cantab.net
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btu176
PMCID: PMC4103593  PMID: 24700318
10.  The genome and life-stage specific transcriptomes of Globodera pallida elucidate key aspects of plant parasitism by a cyst nematode 
Genome Biology  2014;15(3):R43.
Background
Globodera pallida is a devastating pathogen of potato crops, making it one of the most economically important plant parasitic nematodes. It is also an important model for the biology of cyst nematodes. Cyst nematodes and root-knot nematodes are the two most important plant parasitic nematode groups and together represent a global threat to food security.
Results
We present the complete genome sequence of G. pallida, together with transcriptomic data from most of the nematode life cycle, particularly focusing on the life cycle stages involved in root invasion and establishment of the biotrophic feeding site. Despite the relatively close phylogenetic relationship with root-knot nematodes, we describe a very different gene family content between the two groups and in particular extensive differences in the repertoire of effectors, including an enormous expansion of the SPRY domain protein family in G. pallida, which includes the SPRYSEC family of effectors. This highlights the distinct biology of cyst nematodes compared to the root-knot nematodes that were, until now, the only sedentary plant parasitic nematodes for which genome information was available. We also present in-depth descriptions of the repertoires of other genes likely to be important in understanding the unique biology of cyst nematodes and of potential drug targets and other targets for their control.
Conclusions
The data and analyses we present will be central in exploiting post-genomic approaches in the development of much-needed novel strategies for the control of G. pallida and related pathogens.
doi:10.1186/gb-2014-15-3-r43
PMCID: PMC4054857  PMID: 24580726
11.  Pilomyxoid astrocytoma of the cerebellar vermis in an elderly patient 
Background:
Pilomyxoid astrocytoma (PMA) has recently been accepted as an aggressive variant of pilocytic astrocytoma with distinct histopathological features. PMAs have been frequently described in the pediatric population with a predilection for the hypothalamic/chiasmatic region.
Case Description:
A 72-year-old African American male presented with 6 months of memory loss, difficulty expressing himself, and a progressively worsening gait. Magnetic resonance imaging of the brain demonstrated a heterogeneously enhancing cystic mass centered within the cerebellar vermis with mass effect on the fourth ventricle and ventriculomegaly. The patient underwent placement of a ventriculoperitoneal shunt followed by a surgical resection of the lesion, which after immunohistopathologic evaluation, was diagnosed as a World Health Organization grade II PMA. The patient refused further treatment of the lesion and expired 11 months after initial symptom presentation and 4 months after surgery.
Conclusion:
To our knowledge, this is the first report of PMA of the cerebellar vermis in a previously unreported age group. This case report describes the natural history of this type of tumor in a patient who refused adjuvant therapy following surgical resection.
doi:10.4103/2152-7806.127968
PMCID: PMC3994688  PMID: 24778917
Cerebellar vermis; elderly patient; natural history; pilomyxoid astrocytoma
12.  Assembly of the Genome of the Disease Vector Aedes aegypti onto a Genetic Linkage Map Allows Mapping of Genes Affecting Disease Transmission 
The mosquito Aedes aegypti transmits some of the most important human arboviruses, including dengue, yellow fever and chikungunya viruses. It has a large genome containing many repetitive sequences, which has resulted in the genome being poorly assembled — there are 4,758 scaffolds, few of which have been assigned to a chromosome. To allow the mapping of genes affecting disease transmission, we have improved the genome assembly by scoring a large number of SNPs in recombinant progeny from a cross between two strains of Ae. aegypti, and used these to generate a genetic map. This revealed a high rate of misassemblies in the current genome, where, for example, sequences from different chromosomes were found on the same scaffold. Once these were corrected, we were able to assign 60% of the genome sequence to chromosomes and approximately order the scaffolds along the chromosome. We found that there are very large regions of suppressed recombination around the centromeres, which can extend to as much as 47% of the chromosome. To illustrate the utility of this new genome assembly, we mapped a gene that makes Ae. aegypti resistant to the human parasite Brugia malayi, and generated a list of candidate genes that could be affecting the trait.
Author Summary
Mosquitoes are important for transmission of human diseases including dengue and yellow fever. The sequencing of the genomes of key mosquito species including Aedes aegypti has helped us to understand the factors that allow mosquitoes to vector disease. While the genome for Ae. aegypti has been sequenced, it is in many pieces which have not yet been arranged on chromosomes. To this end, we have created a genetic linkage map and measured the distance between genetic markers, which allows us to assign them to regions of the genome. Using this method, we also detected errors in the current genome sequences. We used our genetic map to find regions of the mosquito genome associated with the development of Brugia malayi, a nematode that causes lymphatic filariasis in humans. A better genome assembly will be important for the development of novel methods for controlling disease transmission.
doi:10.1371/journal.pntd.0002652
PMCID: PMC3907309  PMID: 24498447
15.  Mitochondrial Two-Component Signaling Systems in Candida albicans 
Eukaryotic Cell  2013;12(6):913-922.
Two-component signal transduction pathways are one of the primary means by which microorganisms respond to environmental signals. These signaling cascades originated in prokaryotes and were inherited by eukaryotes via endosymbiotic lateral gene transfer from ancestral cyanobacteria. We report here that the nuclear genome of the pathogenic fungus Candida albicans contains elements of a two-component signaling pathway that seem to be targeted to the mitochondria. The C. albicans two-component response regulator protein Srr1 (stress response regulator 1) contains a mitochondrial targeting sequence at the N terminus, and fluorescence microscopy reveals mitochondrial localization of green fluorescent protein-tagged Srr1. Moreover, phylogenetic analysis indicates that C. albicans Srr1 is more closely related to histidine kinases and response regulators found in marine bacteria than are other two-component proteins present in the fungi. These data suggest conservation of this protein during the evolutionary transition from endosymbiont to a subcellular organelle. We used microarray analysis to determine whether the phenotypes observed with a srr1Δ/Δ mutant could be correlated with gene transcriptional changes. The expression of mitochondrial genes was altered in the srr1Δ/Δ null mutant in comparison to their expression in the wild type. Furthermore, apoptosis increased significantly in the srr1Δ/Δ mutant strain compared to the level of apoptosis in the wild type, suggesting the activation of a mitochondrion-dependent apoptotic cell death pathway in the srr1Δ/Δ mutant. Collectively, this study shows for the first time that a lower eukaryote like C. albicans possesses a two-component response regulator protein that has survived in mitochondria and regulates a subset of genes whose functions are associated with the oxidative stress response and programmed cell death (apoptosis).
doi:10.1128/EC.00048-13
PMCID: PMC3675996  PMID: 23584995
16.  Genome sequence of the human malaria parasite Plasmodium falciparum 
Nature  2002;419(6906):10.1038/nature01097.
The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host–parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.
doi:10.1038/nature01097
PMCID: PMC3836256  PMID: 12368864
17.  Bimodal Targeting of Microsomal CYP2E1 to Mitochondria through Activation of an N-terminal Chimeric Signal by cAMP-mediated Phosphorylation* 
The Journal of biological chemistry  2002;277(43):40583-40593.
Cytochrome P450 2E1 (CYP2E1) plays an important role in alcohol-induced toxicity and oxidative stress. Recently, we showed that this predominantly microsomal protein is also localized in rat hepatic mitochondria. In this report, we show that the N-terminal 30 amino acids of CYP2E1 contain a chimeric signal for bimodal targeting of the apoprotein to endoplasmic reticulum (ER) and mitochondria. We demonstrate that the cryptic mitochondrial targeting signal at sequence 21–31 of the protein is activated by cAMP-dependent phosphorylation at Ser-129. S129A mutation resulted in lower affinity for binding to cytoplasmic Hsp70, mitochondrial translocases (TOM40 and TIM44) and reduced mitochondrial import. S129A mutation, however, did not affect the extent of binding to the signal recognition particle and association with ER membrane translocator protein Sec61. Addition of saturating levels of signal recognition particle caused only a partial inhibition of CYP2E1 translation under in vitro conditions, and saturating levels of ER resulted only in partial membrane integration. cAMP enhanced the mitochondrial CYP2E1 (referred to as P450MT5) level but did not affect its level in the ER. Our results provide new insights on the mechanism of cAMP-mediated activation of a cryptic mitochondrial targeting signal and regulation of P450MT5 targeting to mitochondria.
doi:10.1074/jbc.M203292200
PMCID: PMC3800117  PMID: 12191992
18.  Natural product disaccharide engineering through tandem glycosyltransferase catalysis reversibility and neoglycosylation 
Organic letters  2012;14(19):5086-5089.
A two-step strategy for disaccharide modulation using vancomycin as a model is reported. The strategy relies upon a glycosyltransferase-catalyzed ‘reverse’ reaction to enable the facile attachment of an alkoxyamine-bearing sugar to the vancomycin core. Neoglycosylation of the corresponding aglycon led to a novel set of vancomycin 1,6-disaccharide variants. While the in vitro antibacterial properties of corresponding vancomycin 1,6-disaccharide analogs were equipotent to the parent antibiotic, the chemoenzymatic method presented is expected to be broadly applicable.
doi:10.1021/ol3023374
PMCID: PMC3489467  PMID: 22984807
19.  Identification of a Nfs1p-bound persulfide intermediate in Fe-S cluster synthesis by intact mitochondria 
Mitochondrion  2012;12(5):539-549.
Cysteine desulfurases generate a covalent persulfide intermediate from cysteine, and this activated form of sulfur is essential for the synthesis of iron-sulfur (Fe-S) clusters. In yeast mitochondria, there is a complete machinery for Fe-S cluster synthesis, including a cysteine desulfurase, Nfs1p. Here we show that following supplementation of isolated mitochondria with [35S]cysteine, a radiolabeled persulfide could be detected on Nfs1p. The persulfide persisted under conditions that did not permit Fe-S cluster formation, such as nucleotide and/or iron depletion of mitochondria. By contrast, under permissive conditions, the radiolabeled Nfs1p persulfide was greatly reduced and radiolabeled aconitase was formed, indicating transfer of persulfide to downstream Fe-S cluster recipients. Nfs1p in mitochondria was found to be relatively more resistant to inactivation by N-ethylmaleimide (NEM) as compared with a prokaryotic cysteine desulfurase. Mitochondria treated with NEM (1 mM) formed the persulfide on Nfs1p but failed to generate Fe-S clusters on aconitase, likely due to inactivation of downstream recipient(s) of the Nfs1p persulfide. Thus the Nfs1p-bound persulfide as described here represents a precursor en route to Fe-S cluster synthesis in mitochondria.
doi:10.1016/j.mito.2012.07.103
PMCID: PMC3462219  PMID: 22813754
Cysteine desulfurase; Nfs1p•Isd11p complex; persulfide; Fe-S clusters; aconitase; yeast
20.  The agr Locus Regulates Virulence and Colonization Genes in Clostridium difficile 027 
Journal of Bacteriology  2013;195(16):3672-3681.
The transcriptional regulator AgrA, a member of the LytTR family of proteins, plays a key role in controlling gene expression in some Gram-positive pathogens, including Staphylococcus aureus and Enterococcus faecalis. AgrA is encoded by the agrACDB global regulatory locus, and orthologues are found within the genome of most Clostridium difficile isolates, including the epidemic lineage 027/BI/NAP1. Comparative RNA sequencing of the wild type and otherwise isogenic agrA null mutant derivatives of C. difficile R20291 revealed a network of approximately 75 differentially regulated transcripts at late exponential growth phase, including many genes associated with flagellar assembly and function, such as the major structural subunit, FliC. Other differentially regulated genes include several involved in bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) synthesis and toxin A expression. C. difficile 027 R20291 agrA mutant derivatives were poorly flagellated and exhibited reduced levels of colonization and relapses in the murine infection model. Thus, the agr locus likely plays a contributory role in the fitness and virulence potential of C. difficile strains in the 027/BI/NAP1 lineage.
doi:10.1128/JB.00473-13
PMCID: PMC3754575  PMID: 23772065
21.  Improved Bacterial Mutagenesis by High-Frequency Allele Exchange, Demonstrated in Clostridium difficile and Streptococcus suis 
Applied and Environmental Microbiology  2013;79(15):4768-4771.
Here we show that the frequency of mutant isolation by two-step allele exchange can be improved by increasing the length of homologous DNA and the opportunity for recombination, obviating the need for counterselection markers. These principles are demonstrated in Clostridium difficile and Streptococcus suis but are likely to be generally applicable.
doi:10.1128/AEM.01195-13
PMCID: PMC3719504  PMID: 23728809
22.  Epigenetics and phenotypic variability: some interesting insights from birds 
Little is known about epigenetic mechanisms in birds with the exception of the phenomenon of dosage compensation of sex chromosomes, although such mechanisms could be involved in the phenotypic variability of birds, as in several livestock species. This paper reviews the literature on epigenetic mechanisms that could contribute significantly to trait variability in birds, and compares the results to the existing knowledge of epigenetic mechanisms in mammals. The main issues addressed in this paper are: (1) Does genomic imprinting exist in birds? (2) How does the embryonic environment influence the adult phenotype in avian species? (3) Does the embryonic environment have an impact on phenotypic variability across several successive generations? The potential for epigenetic studies to improve the performance of individual animals through the implementation of limited changes in breeding conditions or the addition of new parameters in selection models is still an open question.
doi:10.1186/1297-9686-45-16
PMCID: PMC3693910  PMID: 23758635
23.  Contraction type influences the human ability to use the available torque capacity of skeletal muscle during explosive efforts 
The influence of contraction type on the human ability to use the torque capacity of skeletal muscle during explosive efforts has not been documented. Fourteen male participants completed explosive voluntary contractions of the knee extensors in four separate conditions: concentric (CON) and eccentric (ECC); and isometric at two knee angles (101°, ISO101 and 155°, ISO155). In each condition, torque was measured at 25 ms intervals up to 150 ms from torque onset, and then normalized to the maximum voluntary torque (MVT) specific to that joint angle and angular velocity. Explosive voluntary torque after 50 ms in each condition was also expressed as a percentage of torque generated after 50 ms during a supramaximal 300 Hz electrically evoked octet in the same condition. Explosive voluntary torque normalized to MVT was more than 60 per cent larger in CON than any other condition after the initial 25 ms. The percentage of evoked torque expressed after 50 ms of the explosive voluntary contractions was also greatest in CON (ANOVA; p < 0.001), suggesting higher concentric volitional activation. This was confirmed by greater agonist electromyography normalized to Mmax (recorded during the explosive voluntary contractions) in CON. These results provide novel evidence that the ability to use the muscle's torque capacity explosively is influenced by contraction type, with concentric contractions being more conducive to explosive performance due to a more effective neural strategy.
doi:10.1098/rspb.2011.2109
PMCID: PMC3321696  PMID: 22258636
rate of torque development; neural activation; concentric contractions; eccentric contractions; isometric contractions
24.  Establishing a surgical partnership between Addis Ababa, Ethiopia, and Toronto, Canada 
Canadian Journal of Surgery  2013;56(3):E19-E23.
Background
Academic partnerships between high-and low/middle-income countries can improve the quality of surgical education and health care delivery in each setting. We report the perceived needs related to collaborative surgical education in a resource-limited setting.
Methods
We used qualitative methods to elicit the opinions of surgical faculty members and surgical residents and quantitative methods to outline surgical procedure type and volume.
Results
Ethiopian faculty members identified the management of trauma and emergency surgical care as a priority. They identified supervision in the operating room (OR), topic-specific lectures and supervising resident assessments in the clinic as appropriate roles for partners. Residents were in agreement with faculty members, highlighting a desire for supervision in the OR and topic-specific lectures.
Conclusion
We present specific experiences and needs of a surgical teaching unit in a low-income country, paving the way to form a meaningful and responsive relationship between 2 surgical departments in 2 universities.
doi:10.1503/cjs.027011
PMCID: PMC3672439  PMID: 23706853
25.  Recruitment of EB1, a Master Regulator of Microtubule Dynamics, to the Surface of the Theileria annulata Schizont 
PLoS Pathogens  2013;9(5):e1003346.
The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.
Author Summary
The apicomplexan parasite Theileria can reprogram the cell it infects, inducing uncontrolled proliferation and clonal expansion. This is brought about by the schizont, which resides free in the host cell cytoplasm. As the schizont never leaves the cell to infect other cells, it can only persist provided it is distributed over the two daughter cells each time the host cell divides. This is achieved by interacting dynamically with microtubules (MTs) that form part of the host cell mitotic apparatus. How MTs are recruited to the schizont surface is not known. MTs are highly dynamic, undergoing continuous cycles of growth and shrinkage that is regulated to a large extent by an array of proteins, called +TIPs, that associate with the free plus-ends of MTs. End-binding protein 1 (EB1) is a master regulator and central adaptor that mediates MT plus-end tracking of potentially all other +TIPs. We established that a schizont surface protein, p104, provides a docking site for EB1, which critically depends on a consensus SxIP motif, present in p104. These finding provides important new insight into the complex interaction of the transforming schizont with host cell MTs. To our knowledge, p104 is the first pathogen-derived protein identified so far to join the SxIP family of EB1-binding proteins.
doi:10.1371/journal.ppat.1003346
PMCID: PMC3649978  PMID: 23675298

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