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1.  Mapping Antigenic Sites of an Immunodominant Surface Lipoprotein of Mycoplasma agalactiae, AvgC, with the Use of Synthetic Peptides  
Infection and Immunity  2002;70(1):171-176.
As a first step toward the design of an epitope vaccine to prevent contagious agalactia, the strongly immunogenic 55-kDa protein of Mycoplasma agalactiae was studied and found to correspond to the AvgC protein encoded by the avgC gene. The avg genes of M. agalactiae, which encode four variable surface lipoproteins, display a significant homology to the vsp (variable membrane surface lipoproteins) genes of the bovine pathogen Mycoplasma bovis at their promoter region as well as their N-terminus-encoding regions. Some members of the Vsp family are known to be involved in cytoadhesion to host cells. In order to localize immunogenic peptides in the AvgC antigen, the protein sequence was submitted to epitope prediction analysis, and five sets of overlapping peptides, corresponding to five selected regions, were synthesized by Spot synthesis. Reactive peptides were selected by immunobinding assay with sera from infected sheep. The three most immunogenic epitopes were shown to be surface exposed by immunoprecipitation assays, and one of these was specifically recognized by all tested sera. Our study indicates that selected epitopes of the AvgC lipoprotein may be used to develop a peptide-based vaccine which is effective against M. agalactiae infection.
PMCID: PMC127643  PMID: 11748179
2.  Phenotypic switching of variable surface lipoproteins in Mycoplasma bovis involves high-frequency chromosomal rearrangements. 
Journal of Bacteriology  1996;178(18):5395-5401.
Mycoplasma bovis, an important pathogen of cattle, was recently shown to possess a family of phase- and size-variable membrane surface lipoprotein antigens (Vsps). These proteins spontaneously undergo noncoordinate phase variation between ON and OFF expression states, generating surface antigenic variation. In the present study, we show that the spontaneously high rate of Vsp phenotypic switching involves DNA rearrangements that occur at high frequency in the M. bovis chromosome. A 1.5-kb HindIII genomic fragment carrying the vspA gene from M. bovis PG45 was cloned and sequenced. The deduced VspA amino acid sequence revealed that 80% of the VspA molecule is composed of reiterated intragenic coding sequences, creating a periodic polypeptide structure. Four distinct internal regions of repetitive sequences in the form of in-tandem blocks extending from the N-terminal to the C-terminal portion of the Vsp product were identified. Southern blot analysis of phenotypically switched isogenic lineages representing ON or OFF phase states of Vsp products suggested that changes in the Vsp expression profile were associated with detectable changes at the DNA level. By using a synthetic oligonucleotide representing a sequence complementary to the repetitive vspA gene region as a probe, we could identify the vspA-bearing restriction fragment undergoing high-frequency reversible rearrangements during oscillating phase transition of vspA. The 1.5-kb HindIII fragment carrying the vspA gene (on state) rearranged and produced a 2.3-kb HindIII fragment (OFF state) and vice versa. Two newly discovered vsp genes (vspE and vspF) were localized on two HindIII fragments flanking the vsp gene upstream and downstream. Southern blot hybridization with vspE- and vspF-specific oligonucleotides as probes against genomic DNA of VspA phase variants showed that the organization and size of the fragments adjacent to the vspA gene remained unchanged during VspA ON-OFF switching. The mechanisms regulating the vsp genes are yet unknown; our findings suggest that a recombinative mechanism possibly involving DNA inversions, DNA insertion, or mobile genetic elements may play a role in generating the observed high-frequency DNA rearrangements.
PMCID: PMC178356  PMID: 8808927
3.  Surface Diversity in Mycoplasma agalactiae Is Driven by Site-Specific DNA Inversions within the vpma Multigene Locus 
Journal of Bacteriology  2002;184(21):5987-5998.
The ruminant pathogen Mycoplasma agalactiae possesses a family of abundantly expressed variable surface lipoproteins called Vpmas. Phenotypic switches between Vpma members have previously been correlated with DNA rearrangements within a locus of vpma genes and are proposed to play an important role in disease pathogenesis. In this study, six vpma genes were characterized in the M. agalactiae type strain PG2. All vpma genes clustered within an 8-kb region and shared highly conserved 5′ untranslated regions, lipoprotein signal sequences, and short N-terminal sequences. Analyses of the vpma loci from consecutive clonal isolates showed that vpma DNA rearrangements were site specific and that cleavage and strand exchange occurred within a minimal region of 21 bp located within the 5′ untranslated region of all vpma genes. This process controlled expression of vpma genes by effectively linking the open reading frame (ORF) of a silent gene to a unique active promoter sequence within the locus. An ORF (xer1) immediately adjacent to one end of the vpma locus did not undergo rearrangement and had significant homology to a distinct subset of genes belonging to the λ integrase family of site-specific xer recombinases. It is proposed that xer1 codes for a site-specific recombinase that is not involved in chromosome dimer resolution but rather is responsible for the observed vpma-specific recombination in M. agalactiae.
PMCID: PMC135373  PMID: 12374833
4.  Juxtaposition of an Active Promoter to vsp Genes via Site-Specific DNA Inversions Generates Antigenic Variation in Mycoplasma bovis 
Journal of Bacteriology  2001;183(19):5698-5708.
Mycoplasma bovis, the most important etiological agent of bovine mycoplasmosis, undergoes extensive antigenic variation of major and highly immunogenic surface lipoprotein antigens (Vsps). A family of 13 related but divergent vsp genes, which occur as single chromosomal copies, was recently found in the chromosome of M. bovis. In the present study, the molecular mechanism mediating the high-frequency phase variation of two Vsps (VspA and VspC) as representatives of the Vsp family was investigated. Analysis of clonal isolates exhibiting phase transitions of VspA or of VspC (i.e., ON→OFF→ON) has shown that DNA inversions occur during Vsp phase variation. The upstream region of each vsp gene contains two sequence cassettes. The first (cassette no. 1), a 71-bp region upstream of the ATG initiation codon, exhibits 98% homology among all vsp genes, while the second (cassette no. 2), upstream of cassette no. 1, ranges in size from 50 to 180 bp and is more divergent. Examination of the ends of the inverted fragments during VspA or VspC phase variation revealed that in both cases, a change in the organization of vsp upstream cassettes involving three vsp genes had occurred. Primer extension and Northern blot analysis have shown that a specific cassette no. 2, designated A2, is an active promoter and that juxtaposition of this regulatory element to a silent vsp gene by DNA inversions allows transcription initiation of the recipient gene. Further genetic analysis revealed that phase variation of VspA or of VspC involves two site-specific DNA inversions occurring between inverted copies of a specific 35-bp sequence present within the conserved cassette no. 1. A model for the control of Vsp phase variation is proposed.
PMCID: PMC95462  PMID: 11544233
5.  Intrachromosomal Recombination within the vsp Locus of Mycoplasma bovis Generates a Chimeric Variable Surface Lipoprotein Antigen 
Infection and Immunity  2001;69(6):3703-3712.
A family of 13 related but divergent vsp genes was recently found in the chromosome of the bovine pathogen Mycoplasma bovis. The vsp genomic locus was shown to undergo high-frequency rearrangements and to mediate phenotypic switching of variable lipoprotein antigens (Vsps) on the mycoplasma cell surface. Here we report that the vsp gene repertoire is subject to changes. Genetic analysis of M. bovis clonal isolates displaying distinct Vsp phenotypes showed that an intergenic recombination event between two closely related members of the vsp gene family, the formerly expressed vspA gene and the vspO gene, led to the formation of a new chimeric and functional vsp gene, vspC. The 5′ end of the recombination event was identified within the highly conserved vsp-upstream region, while the 3′ end was localized within the first repetitive domain (RA1) present in both vspA and vspO structural genes. As a result, the vspC gene is an embodiment of the following domains: an N-terminus-encoding region linked to the highly conserved vsp-upstream region provided by the vspO gene; and a C-terminus-encoding region and the more distal and divergent vsp-upstream region acquired from the vspA gene. The generation of chimeric genes encoding surface antigens may provide an important element of genetic variation and an additional source of antigenic diversification within the mycoplasma population.
PMCID: PMC98374  PMID: 11349034
6.  Characterization of a Multigene Family Undergoing High-Frequency DNA Rearrangements and Coding for Abundant Variable Surface Proteins in Mycoplasma agalactiae 
Infection and Immunity  2000;68(8):4539-4548.
A family of abundant surface proteins (Vpmas [variable proteins of Mycoplasma agalactiae]) undergoing phase variation in M. agalactiae has been characterized using monoclonal antibodies and specific polyclonal sera. Two expressed members of 39 kDa (Vpma39) and 34 kDa (Vpma34), which varied in expression between clones of a lineage, shared a common amino-terminal sequence but were immunologically distinct. An amino-terminal oligonucleotide probe identified multiple vpma genes which were clustered within a 14-kb ClaI genomic fragment. Rearrangements were found to have occurred within the vpma locus between clones which correlated with changes in their Vpma phenotype. Two neighboring vpma genes were cloned and sequenced from one M. agalactiae clonal variant expressing Vpma39. The two genes, vpmaX and vpmaY, were orientated divergently and shared highly homologous 5′ untranslated regions, 25-amino-acid (aa) lipoprotein leader sequences, and amino-terminal sequences. The vpmaY gene coded for 346 aa and 84% of the open reading frame, comprised of 1.5 units of a large repeat of 186 aa. Although the sequence for an entire second vpmaY repeat was present, it was prematurely terminated by insertion of two nucleotides. The vpmaX gene encoded 221 aa and possessed 102 aa of the 186-aa repeat of vpmaY. Many of the features in common between the vpma genes were also found to be shared by the vsp genes of M. bovis, which also undergo DNA rearrangements concomitant with phenotypic changes. Since M. bovis is the closest phylogenetic relative to M. agalactiae, the vpma and vsp gene families most probably represent homologous systems.
PMCID: PMC98368  PMID: 10899853
7.  The vsp Locus of Mycoplasma bovis: Gene Organization and Structural Features 
Journal of Bacteriology  1999;181(18):5734-5741.
Major lipoprotein antigens, known as variable membrane surface lipoproteins (Vsps), on the surface of the bovine pathogen Mycoplasma bovis were shown to spontaneously undergo noncoordinate phase variation between ON and OFF expression states. The high rate of Vsp phenotypic switching was also shown to be linked with DNA rearrangements that occur at high frequency in the M. bovis chromosome (I. Lysnyansky, R. Rosengarten, and D. Yogev, J. Bacteriol. 178:5395–5401, 1996). In the present study, 13 single-copy vsp genes organized in a chromosomal cluster were identified and characterized. All vsp genes encode highly conserved N-terminal domains for membrane insertion and lipoprotein processing but divergent mature Vsp proteins. About 80% of each vsp coding region is composed of reiterated coding sequences that create a periodic polypeptide structure. Eighteen distinct repetitive domains of different lengths and amino acid sequences are distributed within the products of the various vsp genes that are subject to size variation due to spontaneous insertions or deletions of these periodic units. Some of these repeats were found to be present in only one Vsp family member, whereas other repeats recurred at variable locations in several Vsps. Each vsp gene is also 5′ linked to a highly homologous upstream region composed of two internal cassettes. The findings that rearrangement events are associated with Vsp phenotypic switching and that multiple regions of high sequence similarity are present upstream of the vsp genes and within the vsp coding regions suggest that modulation of the Vsp antigenic repertoire is determined by recombination processes that occur at a high frequency within the vsp locus of M. bovis.
PMCID: PMC94094  PMID: 10482515
8.  Correlation of Pre-existing Vascular Pathology With Arteriovenous Graft Outcomes in Hemodialysis Patients 
Arteriovenous grafts (AVGs) are prone to neointimal hyperplasia leading to AVG failure. We hypothesized that pre-existing pathologic abnormalities of the vessels used to create AVG (including venous intimal hyperplasia, arterial intimal hyperplasia, arterial medial fibrosis, and arterial calcification) are associated with inferior AVG survival.
Study Design
Prospective observational study.
Setting & Participants
Patients with chronic kidney disease undergoing placement of a new AVG at a large medical center who had vascular specimens obtained at the time of surgery (n=76)
Maximal intimal thickness of the arterial and venous intima, arterial medial fibrosis, and arterial medial calcification.
Outcome & Measurements
Unassisted primary AVG survival (time to first intervention) and frequency of AVG interventions.
55 patients (72%) underwent interventions and 148 graft interventions occurred during 89.9 years of follow-up (1.65 interventions per graft-year). Unassisted primary AVG survival was not significantly associated with arterial intimal thickness (HR, 0.72; 95% CI, 0.40-1.27; p=0.3), venous intimal thickness (HR, 0.64; 95% CI, 0.37-1.10; p=0.1), severe arterial medial fibrosis (HR, 0.58; 95% CI, 0.32-1.06; p=0.6), or severe arterial calcification (HR, 0.68; 95% CI, 0.37-1.31; p=0.3). The frequency of AVG interventions per year was inversely associated with arterial intimal thickness (relative risk [RR], 1.99; 95% CI, 1.16-3.42; p<0.001 for thickness <10 vs >25 μm); venous intimal thickness (RR, 2.11; 95% CI, 1.39-3.20; p<0.001 for thickness <5 vs >10 μm); arterial medial fibrosis (RR, 3.17; 95% CI, 1.96-5.13; p<0.001 for fibrosis <70% vs ≥70%), and arterial calcification (RR, 2.12; 95% CI, 1.31-3.43; p=0.001 for <10% vs ≥10% calcification).
Single center study. Study may be underpowered to demonstrate differences in unassisted primary AVG survival.
Pre-existing vascular pathologic abnormalities in CKD patients may not be significantly associated with unassisted primary AVG survival. However, vascular intimal hyperplasia, arterial medial fibrosis, and arterial calcification may be associated with a decreased frequency of AVG interventions.
PMCID: PMC3778052  PMID: 23746379
9.  Active Video Games and Health Indicators in Children and Youth: A Systematic Review 
PLoS ONE  2013;8(6):e65351.
Active video games (AVGs) have gained interest as a way to increase physical activity in children and youth. The effect of AVGs on acute energy expenditure (EE) has previously been reported; however, the influence of AVGs on other health-related lifestyle indicators remains unclear.
This systematic review aimed to explain the relationship between AVGs and nine health and behavioural indicators in the pediatric population (aged 0–17 years).
Data sources
Online databases (MEDLINE, EMBASE, psycINFO, SPORTDiscus and Cochrane Central Database) and personal libraries were searched and content experts were consulted for additional material.
Data selection
Included articles were required to have a measure of AVG and at least one relevant health or behaviour indicator: EE (both habitual and acute), adherence and appeal (i.e., participation and enjoyment), opportunity cost (both time and financial considerations, and adverse events), adiposity, cardiometabolic health, energy intake, adaptation (effects of continued play), learning and rehabilitation, and video game evolution (i.e., sustainability of AVG technology).
51 unique studies, represented in 52 articles were included in the review. Data were available from 1992 participants, aged 3–17 years, from 8 countries, and published from 2006–2012. Overall, AVGs are associated with acute increases in EE, but effects on habitual physical activity are not clear. Further, AVGs show promise when used for learning and rehabilitation within special populations. Evidence related to other indicators was limited and inconclusive.
Controlled studies show that AVGs acutely increase light- to moderate-intensity physical activity; however, the findings about if or how AVG lead to increases in habitual physical activity or decreases in sedentary behaviour are less clear. Although AVGs may elicit some health benefits in special populations, there is not sufficient evidence to recommend AVGs as a means of increasing daily physical activity.
PMCID: PMC3683002  PMID: 23799008
10.  A family of phase- and size-variant membrane surface lipoprotein antigens (Vsps) of Mycoplasma bovis. 
Infection and Immunity  1994;62(11):5075-5084.
A set of strain- and size-variant highly immunogenic membrane surface protein antigens of Mycoplasma bovis, which has been identified by a monoclonal antibody, is shown in this report to make up a family of antigenically and structurally related lipid-modified proteins, designated Vsps (variable surface proteins). By systematic analysis of several isogenic clonal lineages of the type strain PG45, three members of this family have been identified, VspA, VspB, and VspC, each of which was shown to undergo independent high-frequency changes in size as well as noncoordinate phase variation between ON and OFF expression states. The monoclonal antibody-defined epitope common to VspA, VspB, and VspC was accessible on the cell surface in most, but not all, of the clonal populations analyzed and was present on a C-terminal limit tryptic fragment of each Vsp variant that was released from the membrane surface. VspA and VspC were distinguished from VspB by their selective detection with colloidal gold and by their distinctive reaction with a polyclonal antibody against M. bovis D490. VspA, VspB, and VspC were further distinguishable from one another by their characteristic patterns of degradation at carboxypeptidase Y pause sites. While these Vsp-specific structural fingerprints with an irregular periodic spacing were constant for similarly sized variants of a defined Vsp product, they showed distinct differences among variants differing in size. This variability included gain or loss of individual bands within distinct subsets of bands, as well as shifts of the entire banding patterns up- or downwards, indicating that insertions or deletions underlying Vsp size variation can occur at various locations either within the C-terminal domain or within other regions of these proteins. This was similarly confirmed by comparative epitope mapping analysis of tryptic cleavage products generated from different Vsp size variants. The Vsp family of M. bovis described in this study represents a newly discovered system of surface antigenic variation in mycoplasmas displaying features which closely resemble but are also different from the characteristics reported for the Vlp (variable lipoprotein) system of M. hyorhinis. The isogenic lineages established here provide key populations for subsequent analysis of corresponding genes to further elucidate Vsp structure and variation, which may have important relevance for a better understanding of the pathogenicity of this agent.
PMCID: PMC303228  PMID: 7927790
11.  Xer1-Mediated Site-Specific DNA Inversions and Excisions in Mycoplasma agalactiae▿ ‡  
Journal of Bacteriology  2010;192(17):4462-4473.
Surface antigen variation in Mycoplasma agalactiae, the etiologic agent of contagious agalactia in sheep and goats, is governed by site-specific recombination within the vpma multigene locus encoding the Vpma family of variable surface lipoproteins. This high-frequency Vpma phase switching was previously shown to be mediated by a Xer1 recombinase encoded adjacent to the vpma locus. In this study, it was demonstrated in Escherichia coli that the Xer1 recombinase is responsible for catalyzing vpma gene inversions between recombination sites (RS) located in the 5′-untranslated region (UTR) in all six vpma genes, causing cleavage and strand exchange within a 21-bp conserved region that serves as a recognition sequence. It was further shown that the outcome of the site-specific recombination event depends on the orientation of the two vpma RS, as direct or inverted repeats. While recombination between inverted vpma RS led to inversions, recombination between direct repeat vpma RS led to excisions. Using a newly developed excision assay based on the lacZ reporter system, we were able to successfully demonstrate under native conditions that such Xer1-mediated excisions can indeed also occur in the M. agalactiae type strain PG2, whereas they were not observed in the control xer1-disrupted VpmaY phase-locked mutant (PLMY), which lacks Xer1 recombinase. Unless there are specific regulatory mechanisms preventing such excisions, this might be the cost that the pathogen has to render at the population level for maintaining this high-frequency phase variation machinery.
PMCID: PMC2937384  PMID: 20562305
12.  Accumulation of retained nonfunctional arteriovenous grafts correlates with severity of inflammation in asymptomatic ESRD patients 
The contribution of multiple retained nonfunctional arteriovenous grafts (AVGs) to the burden of chronic inflammation in chronic hemodialysis patients has not been well studied. Here, we sought to evaluate the association between plasma levels of C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and albumin and the number of retained nonfunctional AVGs.
This cross-sectional study enrolled 91 prevalent patients undergoing in-center hemodialysis without evidence of infection or inflammation. A baseline blood sample was obtained at study enrollment. A general linear model (GLM) was used to compare levels of biomarkers of systemic inflammation across groups defined by the number of retained, nonfunctional AVGs.
A total of 43 patients had one or more retained thrombosed AVG and had significantly greater plasma log-CRP levels compared with patients without a previous AVG (P= 0.036), regardless of the current AV access type. Using a GLM, we found that for every additional retained thrombosed AVG, plasma log-CRP, log-IL-6 and TNF-alpha concentrations increased significantly by 0.30 mg/L (P= 0.011), 0.18 pg/mL (P= 0.046) and 0.72 pg/mL (P= 0.046), respectively, following adjustment.
Hence, the severity of inflammation increases with the number of retained nonfunctional AVG's, suggesting that AVG accumulation may contribute to the cardiovascular morbidity and mortality associated with chronic inflammation in asymptomatic end-stage renal disease (ESRD) patients. Further study is indicated to determine whether patients with one or more thrombosed, retained AVG may benefit from periodic screening with CRP monitoring to identify those patients who may benefit from AVG resection.
PMCID: PMC3611890  PMID: 23090982
AVG; ESRD; hemodialysis; inflammation
13.  Adventitial Endothelial Implants Reduce Matrix Metalloproteinase-2 Expression and Increase Luminal Diameter in Porcine Arteriovenous Grafts 
Vascular access dysfunction is a major problem in hemodialysis patients, only 50% of arteriovenous grafts (AVG) will remain patent 1 year after surgery. AVG frequently develop stenoses and occlusions at the venous anastomoses, in the venous outflow tract. Lumen diameter is determined not only by intimal thickening but is also influenced by remodeling of the vessel wall. Vascular remodeling requires degradation and reorganization of the extracellular matrix by the degradation enzymes, matrix metalloproteinases (MMPs). In this study, we aimed to provide further insight into the mechanism of endothelial regulation of vascular remodeling and luminal narrowing in AVG.
End-to-side carotid artery-jugular vein polytetrafluoroethylene grafts were created in twenty domestic swine. The anastomoses and outflow vein were treated with Gelfoam® matrices containing allogeneic porcine aortic endothelial cells (PAE, n=10) or control matrices without cells (n=10) and the biological responses to PAE implants investigated 3 and 28-days postoperatively. Pre-sacrifice angiograms were evaluated in comparison to baseline angiograms. Tissue sections were stained with hematoxylin & eosin, Verhoeff’s elastin as well and antibodies specific to MMP-9 and MMP-2 and subjected to histopathological, morphometric and immunohistochemical analysis.
Veins treated with PAE implants had a 2.8-fold increase in venous lumen diameter compared to baseline (P<.05), a 2.3-fold increase in lumen diameter compared to control and an 81% decrease in stenosis (P<.05) compared to control at 28-days. The increase in lumen diameter by angiographic analysis correlated with morphometric analysis of tissue sections. PAE implants increased venous lumen area 2.3 fold (P < .05), decreased venous luminal occlusion 66%, and increased positive venous remodeling 1.9 fold (P< .05) compared to control at 28-days. PAE implants reduced MMP-2 expression at 3 and 28 days, neovascularization at 3 and 28-days and adventitial fibrosis at 28-days, suggesting a role of the implants in controlling the affects of medial and adventitial cells in the response to vascular injury.
These results demonstrate that the adventitial application of endothelial implants significantly reduced MMP-2 expression within the venous wall, increased venous lumen diameter and positive remodeling in a porcine arteriovenous graft model. Adventitial endothelial implants may be useful in decreasing luminal narrowing in a clinical setting.
Clinical Relevance
Vascular access dysfunction is the leading cause of hospitalization and morbidity in patients receiving hemodialysis for end-stage renal disease. Data indicate that the majority of AVG fail due to the formation of stenosis at the vein-graft anastomotic and venous outflow sites. The stenoses result in luminal narrowing and subsequent graft thrombosis and failure. A satisfactory long term pharmacologic means of preventing stenosis due to intimal hyperplasia in hemodialysis grafts has yet to be found. In a large animal model of AVG we show that placement of an adventitial endothelial implant significantly increased venous lumen diameter at 28-days.
PMCID: PMC2702136  PMID: 17826244
14.  Rapid detection of pandemic influenza in the presence of seasonal influenza 
BMC Public Health  2010;10:726.
Key to the control of pandemic influenza are surveillance systems that raise alarms rapidly and sensitively. In addition, they must minimise false alarms during a normal influenza season. We develop a method that uses historical syndromic influenza data from the existing surveillance system 'SERVIS' (Scottish Enhanced Respiratory Virus Infection Surveillance) for influenza-like illness (ILI) in Scotland.
We develop an algorithm based on the weekly case ratio (WCR) of reported ILI cases to generate an alarm for pandemic influenza. From the seasonal influenza data from 13 Scottish health boards, we estimate the joint probability distribution of the country-level WCR and the number of health boards showing synchronous increases in reported influenza cases over the previous week. Pandemic cases are sampled with various case reporting rates from simulated pandemic influenza infections and overlaid with seasonal SERVIS data from 2001 to 2007. Using this combined time series we test our method for speed of detection, sensitivity and specificity. Also, the 2008-09 SERVIS ILI cases are used for testing detection performances of the three methods with a real pandemic data.
We compare our method, based on our simulation study, to the moving-average Cumulative Sums (Mov-Avg Cusum) and ILI rate threshold methods and find it to be more sensitive and rapid. For 1% case reporting and detection specificity of 95%, our method is 100% sensitive and has median detection time (MDT) of 4 weeks while the Mov-Avg Cusum and ILI rate threshold methods are, respectively, 97% and 100% sensitive with MDT of 5 weeks. At 99% specificity, our method remains 100% sensitive with MDT of 5 weeks. Although the threshold method maintains its sensitivity of 100% with MDT of 5 weeks, sensitivity of Mov-Avg Cusum declines to 92% with increased MDT of 6 weeks. For a two-fold decrease in the case reporting rate (0.5%) and 99% specificity, the WCR and threshold methods, respectively, have MDT of 5 and 6 weeks with both having sensitivity close to 100% while the Mov-Avg Cusum method can only manage sensitivity of 77% with MDT of 6 weeks. However, the WCR and Mov-Avg Cusum methods outperform the ILI threshold method by 1 week in retrospective detection of the 2009 pandemic in Scotland.
While computationally and statistically simple to implement, the WCR algorithm is capable of raising alarms, rapidly and sensitively, for influenza pandemics against a background of seasonal influenza. Although the algorithm was developed using the SERVIS data, it has the capacity to be used at other geographic scales and for different disease systems where buying some early extra time is critical.
PMCID: PMC3001734  PMID: 21106071
15.  Phase-locked mutants of Mycoplasma agalactiae: defining the molecular switch of high-frequency Vpma antigenic variation 
Molecular Microbiology  2008;67(6):1196-1210.
Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits antigenic diversity by switching the expression of multiple surface lipoproteins called Vpmas (Variable proteins of M. agalactiae). Although phase variation has been shown to play important roles in many host–pathogen interactions, the biological significance and the mechanism of Vpma oscillations remain largely unclear. Here, we demonstrate that all six Vpma proteins are expressed in the type strain PG2 and all undergo phase variation at an unusually high frequency. Furthermore, targeted gene disruption of the xer1 gene encoding a putative site-specific recombinase adjacent to the vpma locus was accomplished via homologous recombination using a replicon-based vector. Inactivation of xer1 abolished further Vpma switching and the ‘phase-locked’ mutants (PLMs) continued to steadily express only a single Vpma product. Complementation of the wild-type xer1 gene in PLMs restored Vpma phase variation thereby proving that Xer1 is essential for vpma inversions. The study is not only instrumental in enhancing our ability to understand the role of Vpmas in M. agalactiae infections but also provides useful molecular approaches to study potential disease factors in other ‘difficult-to-manipulate’ mycoplasmas.
PMCID: PMC2268961  PMID: 18248580
16.  Activation of Stress Response Pathways Promotes Formation of Antiviral Granules and Restricts Virus Replication 
Molecular and Cellular Biology  2014;34(11):2003-2016.
The formation of protein-RNA granules is a part of both natural cellular function (P-bodies and nuclear HNRNPs) and the response to cellular stress (stress granules and ND10 bodies). To better understand the role of stress-induced granules in viral infection, we have studied the ability of cells to restrict poxvirus replication through the formation of antiviral granules (AVGs). Of cells infected with a wild-type poxvirus, a small number spontaneously formed AVGs. In these AVG-positive cells, viral gene expression was inhibited. The addition of compounds that altered RNA helicase activity, induced oxidative stress, or stimulated translation initiation factor phosphorylation significantly increased the number of AVG-positive cells. When AVGs formed, both viral translation and titers were decreased even when host translation persisted. Treatment with the antiviral compound isatin β-thiosemicarbazone (IBT), a compound that was used to treat smallpox infections, induced AVGs, suggesting a role for these structures in the pharmacological inhibition of poxvirus replication. These findings provide evidence that AVGs are an innate host response that can be exogenously stimulated to combat virus infection. Since small molecules are able to stimulate AVG formation, it is a potential target for new antiviral development.
PMCID: PMC4019067  PMID: 24662051
17.  Biologically induced mineralization of dypingite by cyanobacteria from an alkaline wetland near Atlin, British Columbia, Canada 
This study provides experimental evidence for biologically induced precipitation of magnesium carbonates, specifically dypingite (Mg5(CO3)4(OH)2·5H2O), by cyanobacteria from an alkaline wetland near Atlin, British Columbia. This wetland is part of a larger hydromagnesite (Mg5(CO3)4(OH)2·4H2O) playa. Abiotic and biotic processes for magnesium carbonate precipitation in this environment are compared.
Field observations show that evaporation of wetland water produces carbonate films of nesquehonite (MgCO3·3H2O) on the water surface and crusts on exposed surfaces. In contrast, benthic microbial mats possessing filamentous cyanobacteria (Lyngbya sp.) contain platy dypingite (Mg5(CO3)4(OH)2·5H2O) and aragonite. Bulk carbonates in the benthic mats (δ13C avg. = 6.7‰, δ18O avg. = 17.2‰) were isotopically distinguishable from abiotically formed nesquehonite (δ13C avg. = 9.3‰, δ18O avg. = 24.9‰). Field and laboratory experiments, which emulated natural conditions, were conducted to provide insight into the processes for magnesium carbonate precipitation in this environment. Field microcosm experiments included an abiotic control and two microbial systems, one containing ambient wetland water and one amended with nutrients to simulate eutrophic conditions. The abiotic control developed an extensive crust of nesquehonite on its bottom surface during which [Mg2+] decreased by 16.7% relative to the starting concentration. In the microbial systems, precipitation occurred within the mats and was not simply due to the capturing of mineral grains settling out of the water column. Magnesium concentrations decreased by 22.2% and 38.7% in the microbial systems, respectively. Laboratory experiments using natural waters from the Atlin site produced rosettes and flakey globular aggregates of dypingite precipitated in association with filamentous cyanobacteria dominated biofilms cultured from the site, whereas the abiotic control again precipitated nesquehonite.
Microbial mats in the Atlin wetland create ideal conditions for biologically induced precipitation of dypingite and have presumably played a significant role in the development of this natural Mg-carbonate playa. This biogeochemical process represents an important link between the biosphere and the inorganic carbon pool.
PMCID: PMC2213640  PMID: 18053262
18.  Infected Prosthetic Dialysis Arteriovenous Grafts: A Single Dialysis Center Study 
Surgical Infections  2012;13(6):366-370.
The prosthetic arteriovenous grafts (AVG) being used increasingly to create hemodialysis access are prone to infections that pose potentially life-threatening infectious and bleeding complications, as well as loss of dialysis access. In this study, we identified the bacteriologic agents of infected AVGs by site swab, blood culture, and prosthesis cultures, and to evaluate the role of microbiological findings in the management of the infection.
We focused on 51 patients with 53 AVGs operated on in our clinic from January 2006 to December 2009. An infected AVG was identified by clinical, ultrasound, and microbiological findings. Sensitivity to antibiotics was determined for all bacterial strains. Isolates were identified by pulsed-field gel electrophoresis (PFGE) of bacterial DNA. In a few cases, positron emission tomography-computed tomography (PET-CT) examination was performed.
Strains of Staphylococcus spp., especially S. aureus, were the most frequent cause of infected AVG. All S. aureus strains were sensitive to methicillin. With the exception of a single case, isolates obtained simultaneously from the skin site and the vascular prosthesis were identical genetically.
Our results suggest that bacterial infectious agents detected in site swab, blood, or graft culture confirm a suspicion of AVG infection. A PET-CT examination can provide confirmation. The combination of microbiologic and radionuclide findings can improve the management of the AVG infection, but surgery remains essential.
PMCID: PMC3532001  PMID: 23216527
19.  Are videogame training gains specific or general? 
Many recent studies using healthy adults document enhancements in perception and cognition from playing commercial action videogames (AVGs). Playing action games (e.g., Call of Duty, Medal of Honor) is associated with improved bottom-up lower-level information processing skills like visual-perceptual and attentional processes. One proposal states a general improvement in the ability to interpret and gather statistical information to predict future actions which then leads to better performance across different perceptual/attentional tasks. Another proposal claims all the tasks are separately trained in the AVGs because the AVGs and laboratory tasks contain similar demands. We review studies of action and non-AVGs to show support for the latter proposal. To explain transfer in AVGs, we argue that the perceptual and attention tasks share common demands with the trained videogames (e.g., multiple object tracking (MOT), rapid attentional switches, and peripheral vision). In non-AVGs, several studies also demonstrate specific, limited transfer. One instance of specific transfer is the specific enhancement to mental rotation after training in games with a spatial emphasis (e.g., Tetris). In contrast, the evidence for transfer is equivocal where the game and task do not share common demands (e.g., executive functioning). Thus, the “common demands” hypothesis of transfer not only characterizes transfer effects in AVGs, but also non-action games. Furthermore, such a theory provides specific predictions, which can help in the selection of games to train human cognition as well as in the design of videogames purposed for human cognitive and perceptual enhancement. Finally this hypothesis is consistent with the cognitive training literature where most post-training gains are for tasks similar to the training rather than general, non-specific improvements.
PMCID: PMC3986546  PMID: 24782722
video games; transfer (psychology); cognition; perception; learning
20.  Occurrence, Plasticity, and Evolution of the vpma Gene Family, a Genetic System Devoted to High-Frequency Surface Variation in Mycoplasma agalactiae▿ † 
Journal of Bacteriology  2009;191(13):4111-4121.
Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits a very versatile surface architecture by switching multiple, related lipoproteins (Vpmas) on and off. In the type strain, PG2, Vpma phase variation is generated by a cluster of six vpma genes that undergo frequent DNA rearrangements via site-specific recombination. To further comprehend the degree of diversity that can be generated at the M. agalactiae surface, the vpma gene repertoire of a field strain, 5632, was analyzed and shown to contain an extended repertoire of 23 vpma genes distributed between two loci located 250 kbp apart. Loci I and II include 16 and 7 vpma genes, respectively, with all vpma genes of locus II being duplicated at locus I. Several Vpmas displayed a chimeric structure suggestive of homologous recombination, and a global proteomic analysis further indicated that at least 13 of the 16 Vpmas can be expressed by the 5632 strain. Because a single promoter is present in each vpma locus, concomitant Vpma expression can occur in a strain with duplicated loci. Consequently, the number of possible surface combinations is much higher for strain 5632 than for the type strain. Finally, our data suggested that insertion sequences are likely to be involved in 5632 vpma locus duplication at a remote chromosomal position. The role of such mobile genetic elements in chromosomal shuffling of genes encoding major surface components may have important evolutionary and epidemiological consequences for pathogens, such as mycoplasmas, that have a reduced genome and no cell wall.
PMCID: PMC2698505  PMID: 19376859
21.  Active Healthy Kids Canada’s Position on Active Video Games for Children and Youth 
Paediatrics & Child Health  2013;18(10):529-532.
The effect of active video games (AVGs) on acute energy expenditure has previously been reported; however, the influence of AVGs on other health-related lifestyle indicators remains unclear. To address this knowledge gap, Active Healthy Kids Canada (AHKC) convened an international group of researchers to conduct a systematic review to understand whether AVGs should be promoted to increase physical activity and improve health indicators in children and youth (zero to 17 years of age). The present article outlines the process and outcomes of the development of the AHKC’s position on active video games for children and youth. In light of the available evidence, AHKC does not recommend AVGs as a strategy to help children be more physically active. However, AVGs may exchange some sedentary time for light- to moderate-intensity physical activity, and there may be specific situations in which AVGs provide benefit (eg, motor skill development in special populations and rehabilitation).
PMCID: PMC3907348  PMID: 24497779
Active video games; Children; Exergaming; Physical activity; Position statement
22.  An animal paired crossover ePTFE arteriovenous graft model 
Previously, we developed a porcine model for Arterio Venous Graft (AVG) failure to allow assessment of new access strategies. This model was limited concerning graft length. In the present technical report, we describe a modification of our model allowing the assessment of long AVGs.
In 4 pigs, AVGs of 15 cm length were created bilaterally in a cross-over fashion between the carotid artery and the contralateral jugular vein. Two days (2 pigs) and two weeks (2 pigs) after AV shunting, graft patency was evaluated by angiography, showing all four grafts to be patent, with no sign of angiographic or macroscopic narrowing at the anastomoses sites.
In this modified pig AVG failure model, implantation of a bilateral cross-over long AVG is a feasible approach. The present model offers a suitable tool to study local interventions or compare various long graft designs aimed at improvement of AVG patency.
PMCID: PMC3006397  PMID: 21110903
23.  The relationship between mitral annular systolic velocity and ejection fraction in patients with preserved global systolic function of the left ventricle 
The aim of the study was to investigate the relationship between the ejection fraction (EF) and the mitral annular systolic velocity (Sm) in patients with preserved left ventricular systolic function (EF>55%). The study task was to evaluate whether the assessment of Sm(avg) can be used as an alternative to the Simpson’s method in assessment of the EF. The expected benefit was that Sm could be used to predict EF, when EF is difficult to assess due to poor image quality (IQ).
Sm was obtained by spectral pulse wave Tissue Doppler Imaging (pwTDI) from the lateral and septal sites of the mitral annulus (MA) and an averaged value was calculated - Sm(avg). EF was assessed using Simpson’s rule. Participants were divided into controls (n=70), hypertensive (HTN, n=56), HTN with diastolic dysfunction (HTN/DD, n=65), HTN with diabetes mellitus (HTN/DM, n=52) and HTN with DD and DM (HTN/DD/DM, n=65).
Sm(avg) showed strong correlation with EF (r=0.978; p<0.0001). There were no significant differences between the correlation coefficients between the subgroups and the controls. The mathematical model that the study recommended to assess the EF is: EF=45.0 + 2 × Sm(avg).
The assessment of Sm(avg) could be used as an alternative to EF. This approach may be useful especially when the IQ is poor. The method maintains high accuracy and reproducibility in prediction of the EF.
PMCID: PMC4231345  PMID: 24160570
Mitral annulus velocity; Ejection fraction; EF; Systolic function; Sm; TDI
24.  Epitope Mapping of Immunogenic and Adhesive Structures in Repetitive Domains of Mycoplasma bovis Variable Surface Lipoproteins 
Infection and Immunity  2000;68(2):680-687.
The family of variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis includes some of the most immunogenic antigens of this microorganism. Vsps were shown to undergo high-frequency phase and size variations and to possess extensive reiterated coding sequences extending from the N-terminal end to the C-terminal end of the Vsp molecule. In the present study, mapping experiments were conducted to detect regions with immunogenicity and/or adhesion sites in repetitive domains of four Vsp antigens of M. bovis, VspA, VspB, VspE, and VspF. In enzyme-linked immunosorbent assay experiments, sera obtained from naturally infected cattle showed antibodies to different repeating peptide units of the Vsps, particularly to units RA1, RA2, RA4.1, RB2.1, RE1, and RF1, all of which were found to contain immunodominant epitopes of three to seven amino acids. Competitive adherence trials revealed that a number of oligopeptides derived from various repeating units of VspA, VspB, VspE, and VspF partially inhibited cytoadhesion of M. bovis PG45 to embryonic bovine lung cells. Consequently, putative adherence sites were identified in the same repeating units (RA1, RA2, RA4.1, RB2.1, RE1, and RF1) and in RF2. The positions and lengths of the antigenic determinants were mostly identical to those of adhesion-mediating sites in all short repeating units, whereas in the considerably longer RF1 unit (84 amino acid residues), there was only one case of identity among four immunogenic epitopes and six adherence sites. The identification of epitopes and adhesive structures in repetitive domains of Vsp molecules is consistent with the highly immunogenic nature observed for several members of the Vsp family and suggests a possible function for these Vsp molecules as complex adherence-mediating regions in pathogenesis.
PMCID: PMC97192  PMID: 10639433
25.  Reproducibility of measurements and variability of the classification algorithm of Stratus OCT in normal, hypertensive, and glaucomatous patients 
To assess the reproducibility of retinal nerve fiber layer (RNFL) measurements and the variability of the probabilistic classification algorithm in normal, hypertensive and glaucomatous eyes using Stratus optical coherence tomography (OCT).
Forty-nine eyes (13 normal, 17 ocular hypertensive [OHT] and 19 glaucomatous) of 49 subjects were included in this study. RNFL was determined with Stratus OCT using the standard protocol RNFL thickness 3.4. Three different images of each eye were taken consecutively during the same session. To evaluate OCT reproducibility, coefficient of variation (COV) and intraclass correlation coefficient (ICC) were calculated for average thickness (AvgT), superior average thickness (Savg), and inferior average thickness (Iavg) parameters.
The variability of the results of the probabilistic classification algorithm, based on the OCT normative database, was also analyzed. The percentage of eyes with changes in the category assigned was calculated for each group.
The 50th percentile of COV was 2.96%, 4.00%, and 4.31% for AvgT, Savg, and Iavg, respectively. Glaucoma group presented the largest COV for all three parameters (3.87%, 5.55%, 7.82%). ICC were greater than 0.75 for almost all measures (except from the inferior thickness parameter in the normal group; ICC = 0.64, 95% CI 0.334–0.857).
Regarding the probabilistic classification algorithm for the three parameters (AvgT, Savg, Iavg), the percentage of eyes without color-code category changes among the three images was as follows: normal group, 100%, 84.6% and 92%; OHT group, 89.5%, 52.7%, 79%; and Glaucoma group, 82%, 70.6%, and 76.5%, respectively. A probabilistic category switch from pathologic to normal or vice versa was observed in three eyes (15.8%) of the glaucomatous group for the Savg parameter and in two eyes of the OHT group: one eye (5,9%) for the AvgT and one eye (5.9%) for the Savg parameter.
OCT RNFL measurements showed a good reproducibility in normal, OHT, and glaucoma eyes. The probabilistic classification for the three main parameters showed certain variability, especially in glaucoma group and OHT group. Therefore, one isolated category result should be interpreted with caution before clinical classification of the patient.
PMCID: PMC2709007  PMID: 19668558
optical coherence tomography; glaucoma; nerve fiber layer; algorithm classification; reproducibility

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