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author:("halperin, S")
1.  North Atlantic Migratory Bird Flyways Provide Routes for Intercontinental Movement of Avian Influenza Viruses 
PLoS ONE  2014;9(3):e92075.
Avian influenza virus (AIV) in wild birds has been of increasing interest over the last decade due to the emergence of AIVs that cause significant disease and mortality in both poultry and humans. While research clearly demonstrates that AIVs can move across the Pacific or Atlantic Ocean, there has been no data to support the mechanism of how this occurs. In spring and autumn of 2010 and autumn of 2011 we obtained cloacal swab samples from 1078 waterfowl, gulls, and shorebirds of various species in southwest and west Iceland and tested them for AIV. From these, we isolated and fully sequenced the genomes of 29 AIVs from wild caught gulls (Charadriiformes) and waterfowl (Anseriformes) in Iceland. We detected viruses that were entirely (8 of 8 genomic segments) of American lineage, viruses that were entirely of Eurasian lineage, and viruses with mixed American-Eurasian lineage. Prior to this work only 2 AIVs had been reported from wild birds in Iceland and only the sequence from one segment was available in GenBank. This is the first report of finding AIVs of entirely American lineage and Eurasian lineage, as well as reassortant viruses, together in the same geographic location. Our study demonstrates the importance of the North Atlantic as a corridor for the movement of AIVs between Europe and North America.
PMCID: PMC3960164  PMID: 24647410
2.  Shifts in Geographic Distribution and Antimicrobial Resistance during a Prolonged Typhoid Fever Outbreak — Bundibugyo and Kasese Districts, Uganda, 2009–2011 
Salmonella enterica serovar Typhi is transmitted by fecally contaminated food and water and causes approximately 22 million typhoid fever infections worldwide each year. Most cases occur in developing countries, where approximately 4% of patients develop intestinal perforation (IP). In Kasese District, Uganda, a typhoid fever outbreak notable for a high IP rate began in 2008. We report that this outbreak continued through 2011, when it spread to the neighboring district of Bundibugyo.
Methodology/Principal Findings
A suspected typhoid fever case was defined as IP or symptoms of fever, abdominal pain, and ≥1 of the following: gastrointestinal disruptions, body weakness, joint pain, headache, clinically suspected IP, or non-responsiveness to antimalarial medications. Cases were identified retrospectively via medical record reviews and prospectively through laboratory-enhanced case finding. Among Kasese residents, 709 cases were identified from August 1, 2009–December 31, 2011; of these, 149 were identified during the prospective period beginning November 1, 2011. Among Bundibugyo residents, 333 cases were identified from January 1–December 31, 2011, including 128 cases identified during the prospective period beginning October 28, 2011. IP was reported for 507 (82%) and 59 (20%) of Kasese and Bundibugyo cases, respectively. Blood and stool cultures performed for 154 patients during the prospective period yielded isolates from 24 (16%) patients. Three pulsed-field gel electrophoresis pattern combinations, including one observed in a Kasese isolate in 2009, were shared among Kasese and Bundibugyo isolates. Antimicrobial susceptibility was assessed for 18 isolates; among these 15 (83%) were multidrug-resistant (MDR), compared to 5% of 2009 isolates.
Molecular and epidemiological evidence suggest that during a prolonged outbreak, typhoid spread from Kasese to Bundibugyo. MDR strains became prevalent. Lasting interventions, such as typhoid vaccination and improvements in drinking water infrastructure, should be considered to minimize the risk of prolonged outbreaks in the future.
Author Summary
Typhoid fever is an acute febrile illness caused by the bacteria Salmonella Typhi and transmitted through food and water contaminated with the feces of typhoid fever patients or carriers. We investigated typhoid fever outbreaks in two neighboring Ugandan districts, Kasese and Bundibugyo, where typhoid fever outbreaks began in 2008 and 2011, respectively. In Kasese from August 2009–December 2011, we documented 709 cases of typhoid fever. In Bundibugyo from January–December 2011, we documented 333 cases. Salmonella Typhi from Bundibugyo and Kasese had indistinguishable molecular fingerprints; laboratory and epidemiological evidence indicate that the outbreak spread from Kasese to Bundibugyo. Salmonella Typhi isolated during our investigation were resistant to more antibiotics than isolates obtained from Kasese in 2009. Drinking water in both districts was fecally contaminated and the likely vehicle for the outbreaks. Our investigation highlights that in unchecked typhoid fever outbreaks, illness can become geographically dispersed and outbreak strains can become increasingly resistant to antibiotics. Lasting interventions, including investments in drinking water infrastructure and typhoid vaccination, are needed to control these outbreaks and prevent future outbreaks.
PMCID: PMC3945727  PMID: 24603860
3.  The Barley Genome Sequence Assembly Reveals Three Additional Members of the CslF (1,3;1,4)-β-Glucan Synthase Gene Family 
PLoS ONE  2014;9(3):e90888.
An important component of barley cell walls, particularly in the endosperm, is (1,3;1,4)-β- glucan, a polymer that has proven health benefits in humans and that influences processability in the brewing industry. Genes of the cellulose synthase-like (Csl) F gene family have been shown to be involved in (1,3;1,4)-β-glucan synthesis but many aspects of the biosynthesis are still unclear. Examination of the sequence assembly of the barley genome has revealed the presence of an additional three HvCslF genes (HvCslF11, HvCslF12 and HvCslF13) which may be involved in (1,3;1,4)-β-glucan synthesis. Transcripts of HvCslF11 and HvCslF12 mRNA were found in roots and young leaves, respectively. Transient expression of these genes in Nicotiana benthamiana resulted in phenotypic changes in the infiltrated leaves, although no authentic (1,3;1,4)-β-glucan was detected. Comparisons of the CslF gene families in cereals revealed evidence of intergenic recombination, gene duplications and translocation events. This significant divergence within the gene family might be related to multiple functions of (1,3;1,4)-β-glucans in the Poaceae. Emerging genomic and global expression data for barley and other cereals is a powerful resource for characterising the evolution and dynamics of complete gene families. In the case of the CslF gene family, the results will contribute to a more thorough understanding of carbohydrate metabolism in grass cell walls.
PMCID: PMC3940952  PMID: 24595438
4.  Integration of Multiple Components in Polystyrene-based Microfluidic Devices Part 1: Fabrication and Characterization 
The Analyst  2012;138(1):129-136.
In Part I of a two-part series, we describe a simple, and inexpensive approach to fabricate polystyrene devices that is based upon melting polystyrene (from either a Petri dish or powder form) against PDMS molds or around electrode materials. The ability to incorporate microchannels in polystyrene and integrate the resulting device with standard laboratory equipment such as an optical plate reader for analyte readout and micropipettors for fluid propulsion is first described. A simple approach for sample and reagent delivery to the device channels using a standard, multi-channel micropipette and a PDMS-based injection block is detailed. Integration of the microfluidic device with these off-chip functions (sample delivery and readout) enables high throughput screens and analyses. An approach to fabricate polystyrene-based devices with embedded electrodes is also demonstrated, thereby enabling the integration of microchip electrophoresis with electrochemical detection through the use of a palladium electrode (for a decoupler) and carbon-fiber bundle (for detection). The device was sealed against a PDMS-based microchannel and used for the electrophoretic separation and amperometric detection of dopamine, epinephrine, catechol, and 3,4-dihydroxyphenylacetic acid. Finally, these devices were compared against PDMS-based microchips in terms of their optical transparency and absorption of an anti-platelet drug, clopidogrel. Part I of this series lays the foundation for Part II, where these devices were utilized for various on-chip cellular analysis.
PMCID: PMC3523208  PMID: 23120747
5.  Integration of Multiple Components in Polystyrene-based Microfluidic Devices Part 2: Cellular Analysis 
The Analyst  2012;138(1):137-143.
In Part II of this series describing the use of polystyrene (PS) devices for microfluidic-based cellular assays, various cellular types and detection strategies are employed to determine three fundamental assays often associated with cells. Specifically, using either integrated electrochemical sensing or optical measurements with a standard multi-well plate reader, cellular uptake, production, or release of important cellular analytes are determined on a PS-based device. One experiment involved the fluorescence measurement of nitric oxide (NO) produced within an endothelial cell line following stimulation with ATP. The result was a four-fold increase in NO production (as compared to a control), with this receptor-based mechanism of NO production verifying the maintenance of cell receptors following immobilization onto the PS substrate. The ability to monitor cellular uptake was also demonstrated by optical determination of Ca2+ into endothelial cells following stimulation with the Ca2+ ionophore A20317. The result was a significant increase (42%) in the calcium uptake in the presence of the ionophore, as compared to a control (17%) (p < 0.05). Finally, the release of catecholamines from a dopaminergic cell line (PC 12 cells) was electrochemically monitored, with the electrodes being embedded into the PS-based device. The PC 12 cells had better adherence on the PS devices, as compared to use of PDMS. Potassium-stimulation resulted in the release of 114 ± 11 µM catecholamines, a significant increase (p < 0.05) over the release from cells that had been exposed to an inhibitor (reserpine, 20 ± 2 µM of catecholamines). The ability to successfully measure multiple analytes, generated in different means from various cells under investigation, suggests that PS may be a useful material for microfluidic device fabrication, especially considering the enhanced cell adhesion to PS, its enhanced rigidity/amenability to automation, and its ability to enable a wider range of analytes to be investigated, even analytes with a high degree of hydrophobicity.
PMCID: PMC3523209  PMID: 23120748
6.  Sequence Analysis of In Vivo Defective Interfering-Like RNA of Influenza A H1N1 Pandemic Virus 
Journal of Virology  2013;87(14):8064-8074.
Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are recognized for the role they play in inhibiting viral replication and for the impact they have on the production of infectious virions. To date, influenza virus DI particles have been reported primarily as a phenomenon of cell culture and in experimentally infected embryonated chicken eggs. They have also been isolated from a respiratory infection of chickens. Using a sequencing approach, we characterize several subgenomic viral RNAs from human nasopharyngeal specimens infected with the influenza A(H1N1)pdm09 virus. The distribution of these in vivo-derived DI-like RNAs was similar to that of in vitro DIs, with the majority of the defective RNAs generated from the PB2 (segment 1) of the polymerase complex, followed by PB1 and PA. The lengths of the in vivo-derived DI-like segments also are similar to those of known in vitro DIs, and the in vivo-derived DI-like segments share internal deletions of the same segments. The presence of identical DI-like RNAs in patients linked by direct contact is compatible with transmission between them. The functional role of DI-like RNAs in natural infections remains to be established.
PMCID: PMC3700204  PMID: 23678180
7.  Identification of crop cultivars with consistently high lignocellulosic sugar release requires the use of appropriate statistical design and modelling 
In this study, a multi-parent population of barley cultivars was grown in the field for two consecutive years and then straw saccharification (sugar release by enzymes) was subsequently analysed in the laboratory to identify the cultivars with the highest consistent sugar yield. This experiment was used to assess the benefit of accounting for both the multi-phase and multi-environment aspects of large-scale phenotyping experiments with field-grown germplasm through sound statistical design and analysis.
Complementary designs at both the field and laboratory phases of the experiment ensured that non-genetic sources of variation could be separated from the genetic variation of cultivars, which was the main target of the study. The field phase included biological replication and plot randomisation. The laboratory phase employed re-randomisation and technical replication of samples within a batch, with a subset of cultivars chosen as duplicates that were randomly allocated across batches. The resulting data was analysed using a linear mixed model that incorporated field and laboratory variation and a cultivar by trial interaction, and ensured that the cultivar means were more accurately represented than if the non-genetic variation was ignored. The heritability detected was more than doubled in each year of the trial by accounting for the non-genetic variation in the analysis, clearly showing the benefit of this design and approach.
The importance of accounting for both field and laboratory variation, as well as the cultivar by trial interaction, by fitting a single statistical model (multi-environment trial, MET, model), was evidenced by the changes in list of the top 40 cultivars showing the highest sugar yields. Failure to account for this interaction resulted in only eight cultivars that were consistently in the top 40 in different years. The correspondence between the rankings of cultivars was much higher at 25 in the MET model. This approach is suited to any multi-phase and multi-environment population-based genetic experiment.
PMCID: PMC3878416  PMID: 24359577
Multi-phase experiment; Multi-environment trial; Saccharification; Barley; Phenotyping; Second generation biofuels
8.  Acute cholecystitis 
Clinical Evidence  2011;2011:0411.
Of people admitted to hospital for biliary tract disease, 20% have acute cholecystitis. Up to the age of 50 years, acute calculous cholecystitis is three times more common in women than in men, and about 1.5 times more common in women than in men thereafter. About 95% of people with acute cholecystitis have gallstones. Optimal therapy for acute cholecystitis, based on timing and severity of presentation, remains controversial.
Methods and outcomes
We conducted a systematic review and aimed to answer the following clinical question: What are the effects of treatments for acute cholecystitis? We searched: Medline, Embase, The Cochrane Library, and other important databases up to April 2011 (Clinical Evidence reviews are updated periodically; please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA).
We found 17 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions.
In this systematic review we present information relating to the effectiveness and safety of the following interventions: early cholecystectomy, laparoscopic cholecystectomy, minilaparoscopic cholecystectomy, observation alone, open cholecystectomy, and percutaneous cholecystostomy.
Key Points
Acute cholecystitis causes unremitting right upper quadrant pain, anorexia, nausea, vomiting, and fever, and if untreated can lead to perforations, abscess formation, or fistulae. About 95% of people with acute cholecystitis have gallstones.It is thought that blockage of the bile duct by a gallstone or local inflammation can lead to acute cholecystitis, but we don't know whether bacterial infection is also necessary.
Early cholecystectomy within 7 days of onset of symptoms is the treatment of choice for acute cholecystitis. Early surgery reduces the duration of hospital admission compared with delayed surgery, but does not reduce mortality or complications.Up to one quarter of people scheduled for delayed surgery may require urgent operations because of recurrent or worsening symptoms.
Laparoscopic cholecystectomy may reduce the duration of hospital admission and improve intra-operative and postoperative outcomes compared with open cholecystectomy, but it may increase the risk of bile duct injury. Up to one quarter of people having laparoscopic cholecystectomy may need conversion to open surgery because of risks of complications or uncontrolled bleeding. Minilaparoscopic surgery may be associated with slightly longer operative times than laparoscopic surgery, although it may reduce pain scores and the need for postoperative analgesia.
Routine abdominal drainage in both uncomplicated laparoscopic and open cholecystectomy is associated with an increase in wound infections compared with no drainage.
PMCID: PMC3275134  PMID: 22186260
9.  Possible increased potency of current levothyroxine 
PMCID: PMC3505392  PMID: 23211241
10.  Migration and Persistence of Human Influenza A Viruses, Vietnam, 2001–2008 
Emerging Infectious Diseases  2013;19(11):1756-1765.
Understanding global influenza migration and persistence is crucial for vaccine strain selection. Using 240 new human influenza A virus whole genomes collected in Vietnam during 2001–2008, we looked for persistence patterns and migratory connections between Vietnam and other countries. We found that viruses in Vietnam migrate to and from China, Hong Kong, Taiwan, Cambodia, Japan, South Korea, and the United States. We attempted to reduce geographic bias by generating phylogenies subsampled at the year and country levels. However, migration events in these phylogenies were still driven by the presence or absence of sequence data, indicating that an epidemiologic study design that controls for prevalence is required for robust migration analysis. With whole-genome data, most migration events are not detectable from the phylogeny of the hemagglutinin segment alone, although general migratory relationships between Vietnam and other countries are visible in the hemagglutinin phylogeny. It is possible that virus lineages in Vietnam persisted for >1 year.
PMCID: PMC3837676  PMID: 24188643
influenza; tropics; migration; persistence; phylogeography; viruses; Vietnam
11.  Third generation bisphosphonates for treatment of sensorineural hearing loss in otosclerosis 
Otosclerosis is a disease of abnormal bone remodeling in the otic capsule. Although several drugs that inhibit bone remodeling, including sodium fluoride and first generation bisphosphonates, have been tried, there remains no clearly effective treatment for otosclerosis-related sensorineural hearing loss (SNHL). Over the past several years, third generation bisphosphonates, with more powerful anti-resorptive properties and increased bone affinity, have been developed and have demonstrated effectiveness in the treatment of osteoporosis and other metabolic bone diseases. We hypothesized that newer generation bisphosphonates, such as risedronate and zoledronate, would be effective in slowing the progression of SNHL in patients with otosclerosis.
Study design
Retrospective review
Tertiary referral center, ambulatory care
Risedronate or zoledronate administration
Main outcome measures
Bone conduction pure tone threshold averages (PTA) and word recognition (WR) scores were examined for each ear before and after bisphosphonate treatment. Criteria for significant change were defined as greater than 10 decibels in PTA or between 4 to 18% in WR based on binomial variance.
All 10 patients had audiometric progression of SNHL in the pre-treatment monitoring interval and 12 ears met criteria for significant progression. All 10 patients (19 ears) showed at least no significant progression of SNHL (i.e. stabilization) at an average follow up of 13 months. Two patients (3 ears) showed improvement by defined audiometric criteria. There were no major complications.
Treatment with zoledronate or risedronate stabilized progressive SNHL related to otosclerosis in this small group of patients. Further evaluation of third generation bisphosphonate treatments is warranted.
PMCID: PMC3442123  PMID: 22935809
12.  Ambient temperature influences birds' decisions to eat toxic prey☆ 
Animal Behaviour  2013;86(4):733-740.
Aposematic prey warn predators of their toxicity using conspicuous signals. However, predators regularly include aposematic prey in their diets, particularly when they are in a poor energetic state and in need of nutrients. We investigated whether or not an environmental factor, ambient temperature, could change the energetic state of predators and lead to an increased intake of prey that they know to contain toxins. We found that European starlings, Sturnus vulgaris, increased their consumption of mealworm, Tenebrio molitor, prey containing quinine (a mild toxin) when the ambient temperature was reduced below their thermoneutral zone from 20 °C to 6 °C. The birds differed in their sensitivity to changes in ambient temperature, with heavier birds increasing the number of toxic prey they ate more rapidly with decreasing temperature compared to birds with lower body mass. This could have been the result of their requiring more nutrients at lower temperatures or being better able to detoxify quinine. Taken together, our results suggest that conspicuous coloration may be more costly at lower temperatures, and that aposematic prey may need to invest more in chemical defences as temperatures decline. Our study also provides novel insights into what factors affect birds' decisions to eat toxic prey, and demonstrates that selection pressures acting on prey defences can vary with changing temperature across days, seasons, climes, and potentially in response to climate change.
•We investigated the effect of temperature on birds' decisions to eat toxic prey.•As it got cooler, birds were more likely to eat prey containing toxins.•Heavier birds were more sensitive to changes in temperature.•Selection pressures on prey defences will change over days, seasons and climes.
PMCID: PMC3791422  PMID: 24109148
aposematism; decision making; dietary cognition; mimicry; nutrient–toxin trade-off; state-dependent foraging
13.  Murine Recombinant ACE2: Effect on Angiotensin II Dependent Hypertension and Distinctive ACE2 Inhibitor Characteristics on rodent and human ACE2 
Hypertension  2012;60(3):730-740.
A newly produced murine recombinant ACE2 was characterized in vivo and in vitro. The effects of available ACE2 inhibitors (MLN-4760 and two conformational variants of DX600 –linear and cyclic) were also examined. When murine ACE2 was given to mice for 4 weeks, a marked increase in serum ACE2 activity was sustainable. In acute studies, mouse ACE2 (1mg/kg) obliterated hypertension induced by angiotensin II infusion by rapidly decreasing plasma angiotensin II. These effects were blocked by MLN-4760 but not by either form of DX600. In vitro, conversion from angiotensin II to angiotensin-(1–7) by mouse ACE2 was blocked by MLN-4760 (10−6M) but not by either form of DX600 (10−5M). Quantitative analysis of multiple angiotensin peptides in plasma ex vivo revealed formation of angiotensin-(1–9) from angiotensin I by human but not by mouse ACE2. Both human and mouse ACE2 led to the dissipation of angiotensin II with formation of angiotensin-(1–7). By contrast, mouse ACE2-driven angiotensin-(1–7) formation from angiotensin II was blocked by MLN-4760 but not by either linear or cyclic DX600.
In conclusion, sustained elevations in serum ACE2 activity can be accomplished with murine ACE2 administration thereby providing a strategy for ACE2 amplification in chronic studies using rodent models of hypertension and cardiovascular disease. Human, but not mouse ACE2, degrades angiotensin I to form angiotensin-(1–9). There are also species differences regarding rodent and human ACE2 inhibition by known inhibitors such that MLN-4760 inhibits both human and mouse ACE2 whereas DX600 only blocks human ACE2 activity.
PMCID: PMC3426447  PMID: 22777933
ACE2; Recombinant; Mouse; Human; Hypertension; Angiotensin; Inhibitor
14.  Influenza A Virus Migration and Persistence in North American Wild Birds 
PLoS Pathogens  2013;9(8):e1003570.
Wild birds have been implicated in the emergence of human and livestock influenza. The successful prediction of viral spread and disease emergence, as well as formulation of preparedness plans have been hampered by a critical lack of knowledge of viral movements between different host populations. The patterns of viral spread and subsequent risk posed by wild bird viruses therefore remain unpredictable. Here we analyze genomic data, including 287 newly sequenced avian influenza A virus (AIV) samples isolated over a 34-year period of continuous systematic surveillance of North American migratory birds. We use a Bayesian statistical framework to test hypotheses of viral migration, population structure and patterns of genetic reassortment. Our results reveal that despite the high prevalence of Charadriiformes infected in Delaware Bay this host population does not appear to significantly contribute to the North American AIV diversity sampled in Anseriformes. In contrast, influenza viruses sampled from Anseriformes in Alberta are representative of the AIV diversity circulating in North American Anseriformes. While AIV may be restricted to specific migratory flyways over short time frames, our large-scale analysis showed that the long-term persistence of AIV was independent of bird flyways with migration between populations throughout North America. Analysis of long-term surveillance data provides vital insights to develop appropriately informed predictive models critical for pandemic preparedness and livestock protection.
Author Summary
Despite continuous virological surveillance (1976–2009) in wild waterfowl (Anseriformes) and shorebirds (Charadriiformes), the ecological and evolutionary dynamics of avian influenza A virus (AIV) in these hosts is poorly understood. Comparative genomic analysis of AIV data revealed that the high prevalence of Charadriiformes infected in Delaware Bay is a reservoir of AIV that is phylogenetically distinct from AIV sampled from most North American Anseriformes. In contrast, influenza viruses sampled from Anseriformes in Alberta are representative of the remaining AIV diversity sampled across North America. While AIV may be restricted to specific migratory flyways over short time frames, our large-scale analysis showed that this population genetic structure was transient and the long-term persistence of AIV was independent of bird flyways. These results suggest an introduced virus lineage may initially be restricted to one flyway, but migration to a major congregation site such as Alberta could occur followed by subsequent spread across flyways. These generalized predictions for virus movement will be critical to assess the associated risk for widespread diffusion and inform surveillance for pandemic preparedness.
PMCID: PMC3757048  PMID: 24009503
15.  Audiology In The Sudden Hearing Loss Clinical Trial 
To report the pre- and post treatment population characteristics, and the overall stability of the audiologic outcomes found during the Sudden Hearing Loss Clinical Trial [ Identifier NCT00097448].
Study Design
Multi-center, prospective randomized non-inferiority trial of oral v. intratympanic (IT) steroid treatment of sudden sensorineural hearing loss (SSNHL).
Fifteen academically-based otology practices
250 patients with unilateral SSNHL presenting within 14 days of onset with ≥50 dBHL pure tone average hearing threshold in the affected ear.
Either 60 mg/day oral prednisone for 14 days with a 5-day taper (121 patients) or four IT doses over 14 days of 40mg/ml methylprednisolone (129 patients).
Main Outcome Measure
Primary endpoint was change in hearing [dB PTA] at 2 months after treatment. Non-inferiority was defined as <10 dB difference in hearing outcome between treatments. In this article, pre- and post treatment hearing findings will be reported in detail.
A general (and stable) effect of treatment, and a specific effect of greater improvement at low frequencies was found in both treatment groups.
Hearing improvements are stable and a significantly greater improvement occurs wit lower frequency following either oral or IT steroid treatment of SSNHL.
PMCID: PMC3400088  PMID: 22805100
16.  Complete Genome Sequence of a Reassortant H14N2 Avian Influenza Virus from California 
Genome Announcements  2013;1(4):e00543-13.
We report the complete genome sequence of a reassortant H14N2 avian influenza virus isolated in 2011 from a northern shoveler in California. This introduced Eurasian subtype acquired seven segments from North American viruses and circulated in the Pacific Flyway 1 year after its detection in the Mississippi Flyway.
PMCID: PMC3731840  PMID: 23908286
17.  Predicting sites of new hemorrhage with amyloid imaging in cerebral amyloid angiopathy 
Neurology  2012;79(4):320-326.
We aimed to determine whether amyloid imaging can help predict the location and number of future hemorrhages in cerebral amyloid angiopathy (CAA).
We performed a longitudinal cohort study of 11 patients with CAA without dementia who underwent serial brain MRIs after baseline amyloid imaging with Pittsburgh compound B (PiB). Mean distribution volume ratio (DVR) of PiB was determined at the sites of new micro/macrobleeds identified on follow-up MRI and compared with PiB retention at “simulated” hemorrhages, randomly placed in the same subjects using a probability distribution map of CAA-hemorrhage location. Mean PiB retention at the sites of observed new bleeds was also compared to that in shells concentrically surrounding the bleeds. Finally the association between number of incident bleeds and 3 regional amyloid measures were obtained.
Nine of 11 subjects had at least one new microbleed on follow-up MRI (median 4, interquartile range [IQR] 1–9) and 2 had 5 new intracerebral hemorrhages. Mean DVR was greater at the sites of incident bleeds (1.34, 95% confidence interval [CI] 1.23–1.46) than simulated lesions (1.14, 95% CI 1.07–1.22, p < 0.0001) in multivariable models. PiB retention decreased with increasing distance from sites of observed bleeds (p < 0.0001). Mean DVR in a superior frontal/parasagittal region of interest correlated independently with number of future hemorrhages after adjustment for relevant covariates (p = 0.003).
Our results provide direct evidence that new CAA-related hemorrhages occur preferentially at sites of increased amyloid deposition and suggest that PiB-PET imaging may be a useful tool in prediction of incident hemorrhages in patients with CAA.
PMCID: PMC3400097  PMID: 22786597
18.  Functional MRI Detection of Vascular Reactivity in Cerebral Amyloid Angiopathy 
Annals of neurology  2012;72(1):76-81.
In addition to its role in hemorrhagic stroke, advanced cerebral amyloid angiopathy (CAA) is also associated with ischemic lesions and vascular cognitive impairment. We used functional MRI techniques to identify CAA-associated vascular dysfunction.
Functional MRI was performed on 25 nondemented subjects with probable CAA (mean ± standard deviation age 70.2±7.8) and 12 healthy elderly controls (age 75.3±6.2). Parameters measured were reactivity to visual stimulation (quantified as blood oxygen level-dependent [BOLD] response amplitude, time to peak response, and time to return to baseline after stimulus cessation) and resting absolute cerebral blood flow in the visually activated region (measured by arterial spin labeling).
CAA subjects demonstrated reduced response amplitude (percent change in BOLD signal 0.65±0.28 vs 0.89±0.14, p<0.01), prolonged time to peak (11.1±5.1 vs 6.4±1.8 sec, p<0.001) and prolonged time to baseline (16.5±6.7 vs 11.6±3.1 sec, p<0.001) relative to controls. These differences were independent of age, sex, and hypertension in multivariable analysis and were also present in secondary analyses excluding nonresponsive voxels or voxels containing chronic blood products. Within the CAA group, longer time to peak correlated with overall volume of white matter T2 hyperintensity (Pearson correlation 0.53, p=0.007). Absolute resting blood flow in visual cortex, in contrast, was essentially identical between the groups (44.0±12.6 vs 45.0±10.0 ml/100g/min, p=0.8).
Functional MRI identifies robust differences in both amplitude and timing of the response to visual stimulation in advanced CAA. These findings point to potentially powerful approaches for identifying the mechanistic links between vascular amyloid deposits, vascular dysfunction, and CAA-related brain injury.
PMCID: PMC3408630  PMID: 22829269
19.  Resistance to TRAIL in non-transformed cells is due to multiple redundant pathways 
Cell Death & Disease  2013;4(7):e702-.
Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine and a selective inducer of apoptosis in a range of tumour cells, but not in normal, untransformed cells. A large number of chemotherapeutics as well as biological agents are being tested for their potential to sensitise resistant tumour cells to TRAIL as a means to broaden the range of tumours treatable with TRAIL. However, because of the incomplete understanding of the mechanism(s) underlying TRAIL resistance in non-malignant cells, it is unpredictable whether the effect of these sensitisers will be restricted to tumour cells or they would also sensitise non-transformed cells causing unwanted toxicity. In this study, we carried out a systematic analysis of the mechanisms driving TRAIL resistance in non-transformed cells. We found that cellular FLICE-like inhibitory protein, anti-apoptotic B-cell lymphoma 2 proteins, and X-linked inhibitor of apoptosis protein were independently able to provide resistance to TRAIL. Deficiency of only one of these proteins was not sufficient to elicit TRAIL sensitivity, demonstrating that in non-transformed cells multiple pathways control TRAIL resistance and they act in a redundant manner. This is contrary to the resistance mechanisms found in tumour cell types, many of them tend to rely on a single mechanism of resistance. Supporting this notion we found that 76% of TRAIL-resistant cell lines (13 out of 17) expressed only one of the above-identified anti-apoptotic proteins at a high level (≥1.2-fold higher than the mean expression across all cell lines). Furthermore, inhibition or knockdown of the single overexpressed protein in these tumour cells was sufficient to trigger TRAIL sensitivity. Therefore, the redundancy in resistance pathways in non-transformed cells may offer a safe therapeutic window for TRAIL-based combination therapies where selective sensitisation of the tumour to TRAIL can be achieved by targeting the single non-redundant resistance pathway.
PMCID: PMC3730397  PMID: 23828565
non-transformed primary cells; TRAIL; apoptosis; c-FLIP; XIAP; Bcl-2
20.  Molecular Signature of High Yield (Growth) Influenza A Virus Reassortants Prepared as Candidate Vaccine Seeds 
PLoS ONE  2013;8(6):e65955.
Human influenza virus isolates generally grow poorly in embryonated chicken eggs. Hence, gene reassortment of influenza A wild type (wt) viruses is performed with a highly egg adapted donor virus, A/Puerto Rico/8/1934 (PR8), to provide the high yield reassortant (HYR) viral ‘seeds’ for vaccine production. HYR must contain the hemagglutinin (HA) and neuraminidase (NA) genes of wt virus and one to six ‘internal’ genes from PR8. Most studies of influenza wt and HYRs have focused on the HA gene. The main objective of this study is the identification of the molecular signature in all eight gene segments of influenza A HYR candidate vaccine seeds associated with high growth in ovo.
The genomes of 14 wt parental viruses, 23 HYRs (5 H1N1; 2, 1976 H1N1-SOIV; 2, 2009 H1N1pdm; 2 H2N2 and 12 H3N2) and PR8 were sequenced using the high-throughput sequencing pipeline with big dye terminator chemistry.
Silent and coding mutations were found in all internal genes derived from PR8 with the exception of the M gene. The M gene derived from PR8 was invariant in all 23 HYRs underlining the critical role of PR8 M in high yield phenotype. None of the wt virus derived internal genes had any silent change(s) except the PB1 gene in X-157. The highest number of recurrent silent and coding mutations was found in NS. With respect to the surface antigens, the majority of HYRs had coding mutations in HA; only 2 HYRs had coding mutations in NA.
In the era of application of reverse genetics to alter influenza A virus genomes, the mutations identified in the HYR gene segments associated with high growth in ovo may be of great practical benefit to modify PR8 and/or wt virus gene sequences for improved growth of vaccine ‘seed’ viruses.
PMCID: PMC3679156  PMID: 23776579
21.  Characterization of SiGe thin films using a laboratory X-ray instrument 
Journal of Applied Crystallography  2013;46(Pt 4):898-902.
This article reports the characterization of thin SiGe/Si(100) epilayers using reciprocal space maps measured by a laboratory X-ray instrument and a high-resolution X-ray diffraction study of partially relaxed SiGe/Si thin films.
The technique of reciprocal space mapping using X-rays is a recognized tool for the nondestructive characterization of epitaxial films. X-ray scattering from epitaxial Si0.4Ge0.6 films on Si(100) substrates using a laboratory X-ray source was investigated. It is shown that a laboratory source with a rotating anode makes it possible to investigate the material parameters of the super-thin 2–6 nm layers. For another set of partially relaxed layers, 50–200 nm thick, it is shown that from a high-resolution reciprocal space map, conditioned from diffuse scattering on dislocations, it is possible to determine quantitatively from the shape of a diffraction peak (possessing no thickness fringes) additional parameters such as misfit dislocation density and layer thickness as well as concentration and relaxation.
PMCID: PMC3769059  PMID: 24046495
thin films; high-resolution reciprocal space mapping; partly relaxed epitaxial films; misfit dislocation
22.  Evolution of a reassortant North American gull influenza virus lineage: drift, shift and stability 
Virology Journal  2013;10:179.
The role of gulls in the ecology of avian influenza (AI) is different than that of waterfowl. Different constellations of subtypes circulate within the two groups of birds and AI viruses isolated from North American gulls frequently possess reassortant genomes with genetic elements from both North America and Eurasian lineages. A 2008 isolate from a Newfoundland Great Black-backed Gull contained a mix of North American waterfowl, North American gull and Eurasian lineage genes.
We isolated, sequenced and phylogenetically compared avian influenza viruses from 2009 Canadian wild birds.
We analyzed six 2009 virus isolates from Canada and found the same phylogenetic lineage had persisted over a larger geographic area, with an expanded host range that included dabbling and diving ducks as well as gulls. All of the 2009 virus isolates contained an internal protein coding set of genes of the same Eurasian lineage genes except PB1 that was from a North American lineage, and these genes continued to evolve by genetic drift. We show evidence that the 2008 Great Black-backed Gull virus was derived from this lineage with a reassortment of a North American PA gene into the more stable core set of internal protein coding genes that has circulated in avian populations for at least 2 years. From this core, the surface glycoprotein genes have switched several times creating H13N6, H13N2, and H16N3 subtypes. These gene segments were from North American lineages except for the H16 and N3 vRNAs.
This process appears similar to genetic shifts seen with swine influenza where a stable “triple reassortant internal gene” core has circulated in swine populations with genetic shifts occurring with hemaggluttinin and neuraminidase proteins getting periodically switched. Thus gulls may serve as genetic mixing vessels for different lineages of avian influenza, similar to the role of swine with regards to human influenza. These findings illustrate the need for continued surveillance in gull and waterfowl populations, both on the Pacific and especially Atlantic coasts of North America, to document virus intercontinental movement and the role of gull species in the evolution and epidemiology of AI.
PMCID: PMC3706275  PMID: 23742717
23.  Variable Expression of O:61 in Salmonella Group C2 
Journal of Clinical Microbiology  2012;50(12):4098-4099.
According to the Kauffmann-White scheme, 39 pairs of serovars in Salmonella serogroup C2 differ only by the minor antigen O:61. We characterized strains from 10 serovars representing five Salmonella serogroup C2 pairs. All strains demonstrated variable expression of O:61. These results indicate that these pairs are not distinct serovars.
PMCID: PMC3502993  PMID: 23015665
24.  Predators' decisions to eat defended prey depend on the size of undefended prey☆ 
Animal Behaviour  2013;85(6):1315-1321.
Predators that have learned to associate warning coloration with toxicity often continue to include aposematic prey in their diet in order to gain the nutrients and energy that they contain. As body size is widely reported to correlate with energetic content, we predicted that prey size would affect predators' decisions to eat aposematic prey. We used a well-established system of wild-caught European starlings, Sturnus vulgaris, foraging on mealworms, Tenebrio molitor, to test how the size of undefended (water-injected) and defended (quinine-injected) prey, on different coloured backgrounds, affected birds’ decisions to eat defended prey. We found that birds ate fewer defended prey, and less quinine, when undefended prey were large compared with when they were small, but that the size of the defended prey had no effect on the numbers eaten. Consequently, we found no evidence that the mass of the defended prey or the overall mass of prey ingested affected the amount of toxin that a predator was willing to ingest, and instead the mass of undefended prey eaten was more important. This is a surprising finding, challenging the assumptions of state-dependent models of aposematism and mimicry, and highlighting the need to understand better the mechanisms of predator decision making. In addition, the birds did not learn to discriminate visually between defended and undefended prey based on size, but only on the basis of colour. This suggests that colour signals may be more salient to predators than size differences, allowing Batesian mimics to benefit from aposematic models even when they differ in size.
•The size of toxic prey did not affect the amount of toxin ingested by birds.•Total prey mass eaten did not affect the amount of toxin ingested by birds.•The amount of toxin ingested by birds depended on the mass of nontoxic prey.•Colour signals may be more salient to predators than size differences.•Findings have implications for the selection pressures acting on aposematic prey.
PMCID: PMC3693033  PMID: 23814280
aposematism; educated predator; energy; European starling; foraging; mimicry; prey size; Sturnus vulgaris; toxic prey
25.  Migratory Flyway and Geographical Distance are Barriers to the Gene Flow of Influenza Virus among North American Birds 
Ecology letters  2011;15(1):24-33.
Despite the importance of migratory birds in the ecology and evolution of avian influenza virus (AIV), there is a lack of information on the patterns of AIV spread at the intra-continental scale. We applied a variety of statistical phylogeographic techniques to a plethora of viral genome sequence data to determine the strength, pattern, and determinants of gene flow in AIV sampled from wild birds in North America. These analyses revealed a clear isolation-by-distance of AIV among sampling localities. In addition, we show that phylogeographic models incorporating information on the avian flyway of sampling proved a better fit to the observed sequence data than those specifying homogeneous or random rates of gene flow among localities. In sum, these data strongly suggest that the intra-continental spread of AIV by migratory birds is subject to major ecological barriers, including spatial distance and avian flyway.
PMCID: PMC3228906  PMID: 22008513
avian influenza; phylogeography; evolution; gene flow; ecological barriers; flyways; spatial distance

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