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This study aims to address and improve the low physical activity levels among people with mild dementia by implementing a novel shared decision-making and motivational support program, named "Changing the Focus". It will utilise a pre-post mixed methods approach, aiming to recruit 60 community living older people with mild dementia and their care-partners. The shared decision-making process will involve the person living with dementia, their care-partner, and a research therapist, using a purpose-designed discussion tool including factors such as preferred physical activities, health status, local opportunities and program accessibility. This process aims to identify personalised local physical activity opportunities. Participants will be supported with the help of a research therapist to engage in targeted community-based physical activities for 12-months, to progress towards the recommended physical activity guidelines of 150 minutes per week. The intervention provided by the research therapist will include three home visits (baseline, 6- and 12-months) and seven motivational support phone calls (within the first six months). Research therapists may provide additional home visits and support calls as needed. Primary outcomes include program participation (participants living with dementia continuing with the program after 12-months), total physical activity time per week (measured using the Active Australia Survey at baseline, 6- and 12- months) and program acceptability (assessed through semi-structured interviews with participants, care-partners, referrers, and physical activity providers). Secondary outcomes include physical performance, mental health, wellbeing measures, and impact on care-partners (evaluated through physical tests or validated scales at baseline, 6- and 12-months). Other implementation aspects include reach, maintenance, safety (falls, other adverse events) and an economic evaluation. Results will inform feasibility, potential benefits, and challenges associated with this innovative shared decision-making and supported physical activity program for people living with mild dementia. Findings will guide future large-scale studies and contribute to enhancing physical activity opportunities for this population. [ABSTRACT FROM AUTHOR]

BackgroundVarious rescue techniques are used for the prehospital transport of trauma patients. This study compares different techniques in terms of immobilization of the cervical spine and the rescue time.MethodsA wireless motion capture system (Xsens Technologies, Enschede, The Netherlands) was used to record motion in three-dimensional space and the rescue time in a standardized environment. Immobilization was performed by applying different techniques through different teams of trained paramedics and physicians. All tests were performed on the set course, starting with the test person lying on the floor and ending with the test person lying on an ambulance cot ready to be loaded into an ambulance. Six different settings for rescue techniques were examined: rescue sheet with/without rigid cervical collar (P1S1, P1S0), vacuum mattress and scoop stretcher with/without rigid cervical collar (P2S1, P2S0), and long spinal board with/without rigid cervical collar (P3S1, P3S0). Four time intervals were defined: the time interval in which the rigid cervical collar is applied (T0), the time interval in which the test person is positioned on rescue sheet, vacuum mattress and scoop stretcher, or long spinal board (T1), the time interval in which the test person is carried to the ambulance cot (T2), and the time interval in which the ambulance cot is rolled to the ambulance (T3). An ANOVA was performed to compare the different techniques.ResultsDuring the simulated extrication procedures, a rigid cervical collar provided biomechanical stability at all angles with hardly any loss of time (mean angle ranges during T1: axial rotation P1S0 vs P1S1 p

Diaphanous related formins are regulatory cytoskeletal protein involved in actin elongation and microtubule stabilization. In humans, defects in two of the three diaphanous genes (DIAPH1 and DIAPH3) have been associated with different types of hearing loss. Here, we investigate the role of the third member of the family, DIAPH2, in nonsyndromic hearing loss, prompted by the identification, by exome sequencing, of a predicted pathogenic missense variant in DIAPH2. This variant occurs at a conserved site and segregated with nonsyndromic X-linked hearing loss in an Italian family. Our immunohistochemical studies indicated that the mouse ortholog protein Diaph2 is expressed during development in the cochlea, specifically in the actin-rich stereocilia of the sensory outer hair cells. In-vitro studies showed a functional impairment of the mutant DIAPH2 protein upon RhoA-dependent activation. Finally, Diaph2 knock-out and knock-in mice were generated by CRISPR/Cas9 technology and auditory brainstem response measurements performed at 4, 8 and 14 weeks. However, no hearing impairment was detected. Our findings indicate that DIAPH2 may play a role in the inner ear; further studies are however needed to clarify the contribution of DIAPH2 to deafness.

In Burundi most small-scale farmers still grow traditional cassava landraces that are adapted to local conditions and have been selected for consumer preferred attributes. They tend to be susceptible, in varying degrees, to devastating cassava viral diseases such as Cassava Brown Streak Disease (CBSD) and Cassava Mosaic Disease (CMD) with annual production losses of US$1 billion. For long term resistance to the disease, several breeding strategies have been proposed. A sound basis for a breeding program is to understand the genetic diversity of both landraces and elite introduced breeding cultivars. This will also assist in efforts to conserve landraces ahead of the broad distribution of improved varieties which have the possibility of replacing landraces. Our study aimed at determining the genetic diversity and relationships within and between local landraces and introduced elite germplasm using morphological and single nucleotide polymorphism (SNP) markers. A total of 118 cultivars were characterized for morphological trait variation based on leaf, stem and root traits, and genetic variation using SNP markers. Results of morphological characterization based on Ward’s Method revealed three main clusters and five accessions sharing similar characteristics. Molecular characterization identified over 18,000 SNPs and six main clusters and three pairs of duplicates which should be pooled together as one cultivar to avoid redundancy. Results of population genetic analysis showed low genetic distance between populations and between local landraces and elite germplasm. Accessions that shared similar morphological traits were divergent at the molecular level indicating that clustering using morphological traits was inconsistent. Despite the variabilities found within the collection, it was observed that cassava germplasm in Burundi have a narrow genetic base.

Tasmanian devil (tde) mice are deaf and exhibit circling behaviour. Sensory hair cells of mutants show disorganised hair bundles with abnormally thin stereocilia. The origin of this mutation is the insertion of a transgene which disrupts expression of the Grxcr1 (glutaredoxin cysteine rich 1) gene. We report here that Grxcr1 exons and transcript sequences are not affected by the transgene insertion in tde homozygous (tde/tde) mice. Furthermore, 5'RACE PCR experiments showed the presence of two different transcripts of the Grxcr1 gene, expressed in both tde/tde and in wild-type controls. However, quantitative analysis of Grxcr1 transcripts revealed a significantly decreased mRNA level in tde/tde mice. The key stereociliary proteins ESPN, MYO7A, EPS8 and PTPRQ were distributed in hair bundles of homozygous tde mutants in a similar pattern compared with control mice. We found that the abnormal morphology of the stereociliary bundle was associated with a reduction in the size and Ca2+-sensitivity of the mechanoelectrical transducer (MET) current. We propose that GRXCR1 is key for the normal growth of the stereociliary bundle prior to the onset of hearing, and in its absence hair cells are unable to mature into fully functional sensory receptors.

BackgroundThe National Health Service (NHS) abdominal aortic aneurysm (AAA) screening programme (NAAASP) in England screens 65-year-old men. The programme monitors those with an aneurysm, and early intervention for large aneurysms reduces ruptures and AAA-related mortality. AAA screening services have been disrupted following COVID-19 but it is not known how this may impact AAA-related mortality, or where efforts should be focussed as services resume.MethodsWe repurposed a previously validated discrete event simulation model to investigate the impact of COVID-19-related service disruption on key outcomes. This model was used to explore the impact of delayed invitation and reduced attendance in men invited to screening. Additionally, we investigated the impact of temporarily suspending scans, increasing the threshold for elective surgery to 7cm and increasing drop-out in the AAA cohort under surveillance, using data from NAAASP to inform the population.FindingsDelaying invitation to primary screening up to two years had little impact on key outcomes whereas a 10% reduction in attendance could lead to a 2% lifetime increase in AAA-related deaths. In surveillance patients, a 1-year suspension of surveillance or increase in the elective threshold resulted in a 0.4% increase in excess AAA-related deaths (8% in those 5-5.4cm at the start). Longer suspensions or a doubling of drop-out from surveillance would have a pronounced impact on outcomes.InterpretationEfforts should be directed towards encouraging men to attend AAA screening service appointments post-COVID-19. Those with AAAs on surveillance should be prioritised as the screening programme resumes, as changes to these services beyond one year are likely to have a larger impact on surgical burden and AAA-related mortality.

Given the negative environmental impacts of intensive agriculture, there is an urgent need to reduce the impact of food production on biodiversity. Ecological restoration of farmland could potentially contribute to this goal. While the positive impacts of ecological restoration on biodiversity are well established, less evidence is available regarding impacts on economic development and employment. Potentially, prospects for economic development could be enhanced by ecological restoration though increased provision of ecosystem services, on which some economic activity depends. Here we examined this issue through the development of contrasting land use scenarios for the county of Dorset, southern England. Two scenarios of future agricultural expansion were compared with two scenarios of landscape-scale ecological restoration and the current situation. Impacts on provision of multiple ecosystem services (ES) were explored using InVEST models and proxy values for different land cover types. Impacts on economic employment were examined using an economic input-output model, which was adjusted for variation in ES flows using empirically determined ES dependency values for different economic sectors. Using the unadjusted input-output model, the scenarios had only a slight economic impact (≤ 0.3% Gross Value Added, GVA). Conversely, when the input-output model was adjusted to take account of ES flows, GVA increased by up to 5.4% in the restoration scenarios, whereas under the scenario with greatest agricultural expansion, GVA was reduced by -4.5%. Similarly, employment increased by up to 6.7% following restoration, compared to declines of up to -5.6% following maximum agricultural expansion. These results show that the economic contribution of rural land is far greater than that attributable to agricultural production alone. Landscape-scale restoration of agricultural land can potentially increase the contribution of farmland to economic development and employment, by increasing flows of multiple ES to the many economic sectors that depend on them.

Age-related hearing loss in humans (presbycusis) typically involves impairment of high frequency sensitivity before becoming progressively more severe at lower frequencies. Pathologies initially affecting lower frequency regions of hearing are less common. Here we describe a progressive, predominantly low-frequency recessive hearing impairment in two mutant mouse lines carrying different mutant alleles of the Klhl18 gene: a spontaneous missense mutation (Klhl18lowf) and a targeted mutation (Klhl18tm1a(KOMP)Wtsi). Both males and females were studied, and the two mutant lines showed similar phenotypes. Threshold for auditory brainstem responses (ABR; a measure of auditory nerve and brainstem neural activity) were normal at 3 weeks old but showed progressive increases from 4 weeks onwards. In contrast, distortion product otoacoustic emission (DPOAE) sensitivity and amplitudes (a reflection of cochlear outer hair cell function) remained normal in mutants. Electrophysiological recordings from the round window of Klhl18lowf mutants at 6 weeks old revealed 1) raised compound action potential thresholds that were similar to ABR thresholds, 2) cochlear microphonic potentials that were normal compared with wildtype and heterozygous control mice and 3) summating potentials that were reduced in amplitude compared to control mice. Scanning electron microscopy showed that Klhl18lowf mutant mice had abnormally tapering of the tips of inner hair cell stereocilia in the apical half of the cochlea while their synapses appeared normal. These results suggest that Klhl18 is necessary to maintain inner hair cell stereocilia and normal inner hair cell function at low frequencies.

In Sub-Saharan Africa cassava (Manihot esculenta Crantz) is one of the most important food crops where more than 40% of the population relies on it as their staple carbohydrate source. Biotic constraints such as viral diseases, mainly Cassava Mosaic Disease (CMD) and Cassava Brown Streak Disease (CBSD), and arthropod pests, particularly Cassava Green Mite (CGM), are major constraints to the realization of cassava's full production potential in Africa. To address these problems, we aimed to map the quantitative trait loci (QTL) associated with resistance to CBSD foliar and root necrosis symptoms, foliar CMD and CGM symptoms in a full-sib mapping population derived from the genotypes AR40-6 and Albert. A high-density linkage map was constructed with 2,125 SNP markers using a genotyping-by-sequencing approach. For phenotyping, clonal evaluation trials were conducted with 120 F1 individuals for two consecutive field seasons using an alpha-lattice design at Chambezi and Naliendele, Tanzania. Previously identified QTL for resistance to CBSD foliar symptoms were corroborated, and a new putative QTL for CBSD root necrosis identified (qCBSDRNc14AR) from AR40-6. Two QTL were identified within the region of the previously recognized CMD2 locus from this population in which both parents are thought to possess the CMD2 locus. Interestingly, a minor but consistent QTL, qCGM18AR, for CGM resistance at 3 months after planting stage was also detected and co-localized with a previously identified SSR marker, NS346, linked with CGM resistance. Markers underlying these QTL may be used to increase efficiencies in cassava breeding programs.

Chlamydia abortus is one of the most commonly diagnosed causes of infectious abortion in small ruminants worldwide. Control of the disease (Enzootic Abortion of Ewes or EAE) is achieved using the commercial live, attenuated C. abortus 1B vaccine strain, which can be distinguished from virulent wild-type (wt) strains by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Published studies applying this typing method and whole-genome sequence analyses to cases of EAE in vaccinated and non-vaccinated animals have provided strong evidence that the 1B strain is not attenuated and can infect the placenta causing disease in some ewes. Therefore, the objective of this study was to characterise the lesions found in the placentas of ewes vaccinated with the 1B strain and to compare these to those resulting from a wt infection. A C. abortus-free flock of multiparous adult ewes was vaccinated twice, over three breeding seasons, each before mating, with the commercial C. abortus 1B vaccine strain (Cevac® Chlamydia, Ceva Animal Health Ltd.). In the second lambing season following vaccination, placentas (n = 117) were collected at parturition and analysed by C. abortus-specific real-time quantitative PCR (qPCR). Two placentas, from a single ewe, which gave birth to live twin lambs, were found to be positive by qPCR and viable organisms were recovered and identified as vaccine type (vt) by PCR-RFLP, with no evidence of any wt strain being present. All cotyledons from the vt-infected placentas were analysed by histopathology and immunohistochemistry and compared to those from wt-infected placentas. Both vt-infected placentas showed lesions typical of those found in a wt infection in terms of their severity, distribution, and associated intensity of antigen labelling. These results conclusively demonstrate that the 1B strain can infect the placenta, producing typical EAE placental lesions that are indistinguishable from those found in wt infected animals.

PurposeTo examine the use of Normalisation Process Theory (NPT) to establish if, and in what ways, the AMBER care bundle can be successfully normalised into acute hospital practice, and to identify necessary modifications to optimise its implementation.MethodMulti-method process evaluation embedded within a mixed-method feasibility cluster randomised controlled trial in two district general hospitals in England. Data were collected using (i) focus groups with health professionals (HPs), (ii) semi-structured interviews with patients and/or carers, (iii) non-participant observations of multi-disciplinary team meetings and (iv) patient clinical note review. Thematic analysis and descriptive statistics, with interpretation guided by NPT components (coherence; cognitive participation; collective action; reflexive monitoring). Data triangulated across sources.ResultsTwo focus groups (26 HPs), nine non-participant observations, 12 interviews (two patients, 10 relatives), 29 clinical note reviews were conducted. While coherence was evident, with HPs recognising the value of the AMBER care bundle, cognitive participation and collective action presented challenges. Specifically: (1) HPs were unable and unwilling to operationalise the concept of 'risk of dying' intervention eligibility criteria (2) integration relied on a 'champion' to drive participation and ensure sustainability; and (3) differing skills and confidence led to variable engagement with difficult conversations with patients and families about, for example, nearness to end of life. Opportunities for reflexive monitoring were not routinely embedded within the intervention. Reflections on the use of the AMBER care bundle from HPs and patients and families, including recommended modifications became evident through this NPT-driven analysis.ConclusionTo be successfully normalised, new clinical practices, such as the AMBER care bundle, must be studied within the wider context in which they operate. NPT can be used to the aid identification of practical strategies to assist in normalisation of complex interventions where the focus of care is on clinical uncertainty in acute hospital settings.

This study sought to determine the prevalence of significant liver disease in those subjects with serum alanine aminotransferase levels in the range between the current and the newly suggested upper limit of normal (termed the delta range). The files of the previous study subjects (who underwent at least one alanine aminotransferase measurement in 2002 and followed to 2012) were reviewed for a diagnosis of chronic liver disease; aspartate aminotransferase/platelet ratio index, FIB-4 and alanine aminotransferase/aspartate aminotransferase ratio were used to evaluate liver fibrosis. The prevalence of significant liver disease, by diagnoses and fibrosis scores was compared between subjects with alanine aminotransferase levels in the delta range (men, 42-45 IU/L; women, 26-34 IU/L) and in the newly suggested normal range (men, 15-42 IU/L; women, 10-26 IU/L). The cohort included 49,634 subjects (41% male, mean age 83±6 years) of whom 2022 were diagnosed with chronic liver disease including 366 with cirrhosis. Compared to subjects with alanine aminotransferase levels in the newly suggested normal range, subjects with alanine aminotransferase levels in the delta range had a significantly higher rate of chronic liver disease (men, 15.3% vs. 4.9%; women, 7.8% vs. 3.3%) and of cirrhosis specifically (men, 4.2% vs. 0.9%; women, 1.5% vs. 0.4%) and also had higher mean fibrosis scores (P

Nearshore marine ecosystems can benefit from their interaction with adjacent ecosystems, especially if they alleviate nutrient limitations in nutrient poor areas. This was the case in our oligo- to mesotrophic study area, the KwaZulu-Natal Bight on the South African subtropical east coast, which is bordered by the Agulhas current. We built stoichiometric, multitrophic ecosystem networks depicting biomass and material flows of carbon, nitrogen and phosphorus in three subsystems of the bight. The networks were analysed to investigate whether the southern, middle and northern bight function similarly in terms of their productivity, transfer efficiency between trophic levels, material cycling, and nutrient limitations. The middle region of the bight was clearly influenced by nutrient additions from the Thukela River, as it had the highest ecosystem productivity, lower transfer efficiencies and degree of cycling. Most nodes in the networks were limited by phosphorus, followed by nitrogen. The middle region adjacent to the Thukela River showed a lower proportion of P limitation especially in summer. Interestingly, there was a clear distinction in sensitivities to nutrient limitations between lower and higher trophic level organisms. This is a reflection of their discrepant nutrient turnover times that are either higher, or lower, than that of the systems, and which might provide a balance to the system through this antagonistic influence. Furthermore, by tracking the stoichiometry through entire food webs it appeared how important the role of lower trophic level organisms was to regulate stoichiometry to more suitable ratios for higher trophic level requirements. Although we gained good insight into the behaviour of the three subsystems in the KZN Bight and the role of terrestrial influence on their functioning, a merged approach of incorporating data on metabolic constraints derived from experiments could further improve the representativeness of multitrophic stoichiometric ecosystem networks.

Although numerous studies of diversity have been conducted in cassava, there is no comprehensive assessment of global genetic diversity. Here we draw on previous studies and breeders' knowledge to select diversity sets from the International Institute of Tropical Agriculture (IITA) and the International Center for Tropical Agriculture (CIAT) genebanks and breeders' germplasm, as well as elite germplasm and landraces from eastern, southern and central (ESC) Africa to make a global assessment of diversity in cassava, using a SNP based GoldenGate (Illumina Inc.) assay. A synthesis of results from genetic distance and ADMIXTURE analysis essentially revealed four populations (i) South American germplasm characterised by relatively higher genetic diversity with hypothetical ancestral founder genotypes from Brazil, (ii) a smaller group of African introduction germplasm which is more distantly related to all other germplasm, (iii) West Africa germplasm dominated by IITA breeding lines, containing sources of cassava mosaic disease resistance, and IITA genebank accessions from West Africa, both characterised by slightly lower diversity, and (iv) a less cohesive group of African germplasm, termed 'Other', with moderate levels of diversity and a majority of germplasm from ESC Africa. This study highlights opportunities for heterosis breeding, purging of duplicates in genebanks and the need for conservation of ESC Africa landraces.

Data are presented on the identification and partial characterisation of proteins comprising the chlamydial outer membrane complex (COMC) fraction of Chlamydia abortus (C. abortus)-the aetiological agent of ovine enzootic abortion. Inoculation with the COMC fraction is known to be highly effective in protecting sheep against experimental challenge and its constituent proteins are therefore of interest as potential vaccine candidates. Sodium N-lauroylsarcosine (sarkosyl) insoluble COMC proteins resolved by SDS-PAGE were interrogated by mass spectrometry using combined rapid monolithic column liquid chromatography and fast MS/MS scanning. Downstream database mining of processed tandem MS data revealed the presence of 67 proteins in total, including putative membrane associated proteins (n = 36), such as porins, polymorphic membrane proteins (Pmps), chaperonins and hypothetical membrane proteins, in addition to others (n = 22) that appear more likely to have originated from other subcellular compartments. Electrophoretic mobility data combined with detailed amino acid sequence information derived from secondary fragmentation spectra for 8 Pmps enabled peptides originating from protein cleavage fragments to be mapped to corresponding regions of parent precursor molecules yielding preliminary evidence in support of endogenous post-translational processing of outer membrane proteins in C. abortus. The data presented here will facilitate a deeper understanding of the pathogenesis of C. abortus infection and represent an important step towards the elucidation of the mechanisms of immunoprotection against C. abortus infection and the identification of potential target vaccine candidate antigens.

BACKGROUND:We explored understanding and experiences of health advocacy among psychiatry and family medicine residents and faculty and the implications for clinical care and teaching through the lens of relationship-centred care. METHODS:This qualitative study was conducted in the psychiatry and family medicine departments at a large urban university. We interviewed 19 faculty members and conducted two focus groups with 18 residents. Semi-structured questions explored the relational meaning of health advocacy, how residents and faculty learned about the role and ethical considerations involved in incorporating advocacy work into clinical practice within a relationship-centred care framework. RESULTS:Four themes emerged from the interviews and focus groups: 1) health advocacy as an extension of the relationship to self; 2) health advocacy and professional boundaries in the physician-patient relationship; 3) health advocacy within a team-based approach; and 4) health advocacy and the physician-community/organization relationship. Participants described implications for practice of the challenges of health advocacy, including perceived institutional risks, professional boundaries and the appropriation of patient voice. CONCLUSIONS:Our study provides insights into the relational complexities of the health advocate role in residency curriculum and clinical practice. All participants described health advocacy as a broad spectrum of actions that are guided by relationships among patients, health care professionals and communities. Our analysis revealed that some challenges that participants identified with a health advocacy role could be addressed by anchoring the role within a specific theoretical framework. This would better enable us to create a culture of advocacy in the training and development of physicians.

This study investigated the pathogenesis of two variant strains (LLG and POS) of Chlamydia abortus, in comparison to a typical wild-type strain (S26/3) which is known to be responsible for late term abortion in small ruminants. Challenge with the three strains at mid-gestation resulted in similar pregnancy outcomes, with abortion occurring in approximately 50-60% of ewes with the mean gestational lengths also being similar. However, differences were observed in the severity of placental pathology, with infection appearing milder for strain LLG, which was reflected in the lower number of organisms shed in vaginal swabs post-partum and less gross pathology and organisms present in placental smears. Results for strain POS were somewhat different than LLG with a more focal restriction of infection observed. Post-abortion antibody responses revealed prominent differences in seropositivity to the major outer membrane protein (MOMP) present in elementary body (EB) preparations under denaturing conditions, most notably with anti-LLG and anti-POS convalescent sera where there was no or reduced detection of MOMP present in EBs derived from the three strains. These results and additional analysis of whole EB and chlamydial outer membrane complex preparations suggest that there are conformational differences in MOMP for the three strains. Overall, the results suggest that gross placental pathology and clinical outcome is not indicative of bacterial colonization and the severity of infection. The results also highlight potential conformational differences in MOMP epitopes that perhaps impact on disease diagnosis and the development of new vaccines.

This work focuses on the design of transmitting coils in weakly coupled magnetic induction communication systems. We propose several optimization methods that reduce the active, reactive and apparent power consumption of the coil. These problems are formulated as minimization problems, in which the power consumed by the transmitting coil is minimized, under the constraint of providing a required magnetic field at the receiver location. We develop efficient numeric and analytic methods to solve the resulting problems, which are of high dimension, and in certain cases non-convex. For the objective of minimal reactive power an analytic solution for the optimal current distribution in flat disc transmitting coils is provided. This problem is extended to general three-dimensional coils, for which we develop an expression for the optimal current distribution. Considering the objective of minimal apparent power, a method is developed to reduce the computational complexity of the problem by transforming it to an equivalent problem of lower dimension, allowing a quick and accurate numeric solution. These results are verified experimentally by testing a number of coil geometries. The results obtained allow reduced power consumption and increased performances in magnetic induction communication systems. Specifically, for wideband systems, an optimal design of the transmitter coil reduces the peak instantaneous power provided by the transmitter circuitry, and thus reduces its size, complexity and cost.

Since implementation of the 13-valent polyvalent conjugate vaccine (PCV13) in Canada during 2010, the proportion of PCV13 serotypes causing invasive pneumococcal disease (IPD) has declined from 55% (n = 1492) in 2010 to 31% (n = 764) in 2014. A concurrent increase of non-PCV13 serotypes has occurred and 22F has become the most prevalent serotype in Canada increasing from 7% (n = 183) to 11% (n = 283). Core single nucleotide variant phylogenetic analysis was performed on 137 Streptococcus pneumoniae serotype 22F isolates collected across Canada from 2005-2015. Six phylogenetic lineages (n = 117) were identified among a serotype 22F/ST433 clonal complex (CC), including a recently expanding erythromycin-resistant clone. Erythromycin-resistance was observed in 25 isolates possessing ermB, mef or a 23S rRNA A2061G point mutation; 2 penicillin-resistant isolates had recombinant pbp1a, pbp2a and/or pbp2x; 3 tetracycline-resistant isolates contained tetM; and 1 isolate was multidrug-resistant. Virulence factor analysis indicated a high level of homogeneity among the 22F/ST433 clonal complex strains. A group of 6 phylogenetic outlier strains had differing MLST, antimicrobial resistance and molecular profiles suggestive of capsule switching events. While capsule switch events among S. pneumoniae serotype 22F has been observed, increasing prevalence of S. pneumoniae serotype 22F can be attributed to an evolving homogenous clone expanding nationally through local transmission events.

Cassava (Manihot esculenta) is an important tropical subsistence crop that is severely affected by cassava brown streak disease (CBSD) in East Africa. The disease is caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Both have a (+)-sense single-stranded RNA genome with a 5' covalently-linked viral protein, which functionally resembles the cap structure of mRNA, binds to eukaryotic translation initiation factor 4E (eIF4E) or its analogues, and then enable the translation of viral genomic RNA in host cells. To characterize cassava eIF4Es and their potential role in CBSD tolerance and susceptibility, we cloned five eIF4E transcripts from cassava (accession TMS60444). Sequence analysis indicated that the cassava eIF4E family of proteins consisted of one eIF4E, two eIF(iso)4E, and two divergent copies of novel cap-binding proteins (nCBPs). Our data demonstrated experimentally the coding of these five genes as annotated in the published cassava genome and provided additional evidence for refined annotations. Illumina resequencing data of the five eIF4E genes were analyzed from 14 cassava lines tolerant or susceptible to CBSD. Abundant single nucleotide polymorphisms (SNP) and biallelic variations were observed in the eIF4E genes; however, most of the SNPs were located in the introns and non-coding regions of the exons. Association studies of non-synonymous SNPs revealed no significant association between any SNP of the five eIF4E genes and the tolerance or susceptibility to CBSD. However, two SNPs in two genes were weakly associated with the CBSD responses but had no direct causal-effect relationship. SNPs in an intergenic region upstream of eIF4E_me showed a surprising strong association with CBSD responses. Digital expression profile analysis showed differential expression of different eIF4E genes but no significant difference in gene expression was found between susceptible and tolerant cassava accessions despite the association of the intergenic SNPs with CBSD responses.

Canada has one of the lowest rates of tuberculosis (TB) in the world, however, among certain sub-populations, disease incidence rates approach those observed in sub-Saharan Africa, and other high incidence regions. In this study, we applied mycobacterial interspersed repetitive unit (MIRU) variable number of tandem repeat (VNTR) and whole genome sequencing (WGS) to the analysis of Mycobacterium tuberculosis isolates obtained from Northern communities in the territory of Nunavut. WGS was carried out using the Illumina MiSeq, with identified variants used to infer phylogenetic relationships and annotated to infer functional implications. Additionally, the sequencing data from these isolates were augmented with publically available WGS to evaluate data from the Nunavut outbreak in the broader Canadian context. In this study, isolates could be classified into four major clusters by MIRU-VNTR analysis. These could be further resolved into sub-clusters using WGS. No evidence for antimicrobial resistance, either genetic or phenotypic, was observed in this cohort. Among most subjects with multiple samples, reactivation/incomplete treatment likely contributed to recurrence. However, isolates from two subjects appeared more likely to have occurred via reinfection, based on the large number of genomic single nucleotide variants detected. Finally, although quite distinct from previously reported Canadian MTB strains, isolates obtained from Nunavut clustered most closely with a cohort of samples originating in the Nunavik region of Northern Quebec. This study demonstrates the benefit of using WGS for discriminatory analysis of MTB in Canada, especially in high incidence regions. It further emphasizes the importance of focusing epidemiological intervention efforts on interrupting transmission chains of endemic TB throughout Northern communities, rather than relying on strategies applied in regions where the majority of TB cases result from importation of foreign strains.

INTRODUCTION:Breathlessness is a common symptom of advanced disease placing a huge burden on patients, health systems and informal carers (families and friends providing daily help and support). It causes distress and isolation. Carers provide complex personal, practical and emotional support yet often feel ill-prepared to care. They lack knowledge and confidence in their caring role. The need to educate carers and families about breathlessness is established, yet we lack robustly developed carer-targeted educational interventions to meet their needs. METHODS:We conducted a qualitative interview study with twenty five purposively-sampled patient-carer dyads living with breathlessness in advanced disease (half living with advanced cancer and half with advanced chronic obstructive pulmonary disease (COPD). We sought to identify carers' educational needs (including what they wanted to learn about) and explore differences by diagnostic group in order to inform an educational intervention for carers of patients with breathlessness in advanced disease. RESULTS:There was a strong desire among carers for an educational intervention on breathlessness. Six key topics emerged as salient for them: 1) understanding breathlessness, 2) managing anxiety, panic and breathlessness, 3) managing infections, 4) keeping active, 5) living positively and 6) knowing what to expect in the future. A cross-cutting theme was relationship management: there were tensions within dyads resulting from mismatched expectations related to most topics. Carers felt that knowledge-gains would not only help them to support the patient better, but also help them to manage their own frustrations, anxieties, and quality of life. Different drivers for education need were identified by diagnostic group, possibly related to differences in caring role duration and resulting impacts. CONCLUSION:Meeting the educational needs of carers requires robustly developed and evaluated interventions. This study provides the evidence-base for the content of an educational intervention for carers of patients with breathlessness in advanced disease.

Whereas the infant gut microbiome is the subject of intense study, relatively little is known regarding the nares microbiome in newborns and during early life. This study aimed to survey the typical composition and diversity of human anterior nare microflora for developing infants over time, and to explore how these correlate to their primary caregivers. Single nare swabs were collected at five time points over a one-year period for each subject from infant-caregiver pairs. Our study comprised of 50 infants (recruited at 2 weeks, post delivery) and their 50 primary caregivers. Applying the chaperonin-60 (cpn60) universal target (UT) amplicon as our molecular barcoding marker to census survey the microbial communities, we longitudinally surveyed infant nares microbiota at 5 time points over the course of the first year of life. The inter- and intra-subject diversity was catalogued and compared, both longitudinally and relative to their adult primary caregivers. Although within-subject variability over time and inter-subject variability were both observed, the assessment detected only one or two predominant genera for individual infant samples, belonging mainly to phyla Actinobacteria, Firmicutes, and Proteobacteria. Consistent with previously observed microbial population dynamics in other body sites, the diversity of nares microflora increased over the first year of life and infants showed differential operational taxonomic units (OTUs) relative to their matched primary caregiver. The collected evidence also support that both temporal and seasonal changes occur with respect to carriage of potentially pathogenic bacteria (PPBs), which may influence host predisposition to infection. This pilot study surveying paired infant/caregiver nare microbiomes provides novel longitudinal diversity information that is pertinent to better understanding nare microbiome development in infants.

The advent and widespread application of next-generation sequencing (NGS) technologies to the study of microbial genomes has led to a substantial increase in the number of studies in which whole genome sequencing (WGS) is applied to the analysis of microbial genomic epidemiology. However, microorganisms such as Mycobacterium tuberculosis (MTB) present unique problems for sequencing and downstream analysis based on their unique physiology and the composition of their genomes. In this study, we compare the quality of sequence data generated using the Nextera and TruSeq isolate preparation kits for library construction prior to Illumina sequencing-by-synthesis. Our results confirm that MTB NGS data quality is highly dependent on the purity of the DNA sample submitted for sequencing and its guanine-cytosine content (or GC-content). Our data additionally demonstrate that the choice of library preparation method plays an important role in mitigating downstream sequencing quality issues. Importantly for MTB, the Illumina TruSeq library preparation kit produces more uniform data quality than the Nextera XT method, regardless of the quality of the input DNA. Furthermore, specific genomic sequence motifs are commonly missed by the Nextera XT method, as are regions of especially high GC-content relative to the rest of the MTB genome. As coverage bias is highly undesirable, this study illustrates the importance of appropriate protocol selection when performing NGS studies in order to ensure that sound inferences can be made regarding mycobacterial genomes.

Macrophage-mediated inflammation has been implicated in various kidney diseases. We previously reported that Rac1, a Rho family small GTP-binding protein, was overactivated in several chronic kidney disease models, and that Rac1 inhibitors ameliorated renal injury, in part via inhibition of inflammation, but the detailed mechanisms have not been clarified. In the present study, we examined whether Rac1 in macrophages effects cytokine production and the inflammatory mechanisms contributing to kidney derangement. Myeloid-selective Rac1 flox control (M-Rac1 FC) and knockout (M-Rac1 KO) mice were generated using the cre-loxP system. Renal function under basal conditions did not differ between M-Rac1 FC and KO mice. Accordingly, lipopolysaccharide (LPS)-evoked kidney injury model was created. LPS elevated blood urea nitrogen and serum creatinine, enhanced expressions of kidney injury biomarkers, Kim-1 and Ngal, and promoted tubular injury in M-Rac1 FC mice. By contrast, deletion of myeloid Rac1 almost completely prevented the LPS-mediated renal impairment. LPS triggered a marked induction of macrophage-derived inflammatory cytokines, IL-6 and TNFα, in M-Rac1 FC mice, which was accompanied by Rac1 activation, stimulation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and reactive oxygen species overproduction. These changes were inhibited in M-Rac1 KO mice. LPS evoked F4/80-positive macrophages accumulation in the kidney, which was not affected by myeloid Rac1 deficiency. We further tested the role of Rac1 signaling in cytokine production using macrophage cell line, RAW264.7. Exposure to LPS increased IL-6 and TNFα mRNA expression. The LPS-driven cytokine induction was dose-dependently blocked by the Rac1 inhibitor EHT1864, NADPH oxidase inhibitor diphenyleneiodonium, and NF-κB inhibitor BAY11-7082. In conclusion, genetic ablation of Rac1 in the myeloid lineage protected against LPS-induced renal inflammation and injury, by suppressing macrophage-derived cytokines, IL-6 and TNFα, without blocking recruitment. Our data suggest that Rac1 in macrophage is a novel target for the treatment of kidney disease through inhibition of cytokine production.

INTRODUCTION:Increasing longevity means more people will be dying in very old age, but little is known about the preferences of the 'oldest old' regarding their care at the end of life. AIMS:To understand very old people's preferences regarding care towards the end of life and attitudes towards dying, to inform policy and practice. METHODS:Qualitative data collection for n = 42 population-based cohort study participants aged 95-101 (88% women, 42% in long-term-care): topic-guided interviews with n = 33 participants and n = 39 proxy informants, most with both (n = 30: 4 jointly + separate interviews for 26 dyads). RESULTS:Death was a part of life: these very old people mainly live day-to-day. Most were ready to die, reflecting their concerns regarding quality of life, being a nuisance, having nothing to live for and having lived long enough. Contrasting views were rare exceptions but voiced firmly. Most were not worried about death itself, but concerned more about the dying process and impacts on those left behind; a peaceful and pain-free death was a common ideal. Attitudes ranged from not wanting to think about death, through accepting its inevitable approach to longing for its release. Preferring to be made comfortable rather than have life-saving treatment if seriously ill, and wishing to avoid hospital, were commonly expressed views. There was little or no future planning, some consciously choosing not to. Uncertainty hampered end-of-life planning even when death was expected soon. Some stressed circumstances, such as severe dependency and others' likely decision-making roles, would influence choices. Carers found these issues harder to raise but felt they would know their older relatives' preferences, usually palliative care, although we found two discrepant views. CONCLUSIONS:This study's rare data show ≥95-year-olds are willing to discuss dying and end-of-life care but seldom do. Formal documentation of wishes is extremely rare and may not be welcome. Although being "ready to die" and preferring a palliative approach predominated, these preferences cannot be assumed.

Tight regulation of receptor tyrosine kinases (RTKs) is crucial for normal development and homeostasis. Dysregulation of RTKs signaling is associated with diverse pathological conditions including cancer. The Met RTK is the receptor for hepatocyte growth factor (HGF) and is dysregulated in numerous human tumors. Here we show that Abl family of non-receptor tyrosine kinases, comprised of Abl (ABL1) and Arg (ABL2), are activated downstream of the Met receptor, and that inhibition of Abl kinases dramatically suppresses HGF-induced cell scattering and tubulogenesis. We uncover a critical role for Abl kinases in the regulation of HGF/Met-dependent RhoA activation and RhoA-mediated actomyosin contractility and actin cytoskeleton remodeling in epithelial cells. Moreover, treatment of breast cancer cells with Abl inhibitors markedly decreases Met-driven cell migration and invasion. Notably, expression of a transforming mutant of the Met receptor in the mouse mammary epithelium results in hyper-activation of both Abl and Arg kinases. Together these data demonstrate that Abl kinases link Met activation to Rho signaling and Abl kinases are required for Met-dependent cell scattering, tubulogenesis, migration, and invasion. Thus, inhibition of Abl kinases might be exploited for the treatment of cancers driven by hyperactivation of HGF/Met signaling.

This study aimed to investigate whether preschool children can learn how to interpret dogs' behaviours, with the purpose of helping avoid dog bites. Three- to five-year-old children (N = 70) were tested on their ability to answer questions about dogs' emotional states before and after participating in either an educational intervention about dog behaviour (intervention group) or an activity about wild animals (control group). Children who had received training about dog behaviour (intervention group) were significantly better at judging the dogs' emotional states after the intervention compared to before. The frequency with which they referred to relevant behaviours in justifying their judgements also increased significantly. In contrast, the control group's performance did not differ significantly between the two testing times. These results indicate that preschool children can be taught how to correctly interpret dogs' behaviours. This implies that incorporating such training into prevention programmes may contribute to reducing dog bite incidents.

UnlabelledAnxiety is common in children with autism spectrum disorders (ASD), with specific fears and phobias one of the most frequent subtypes. Specific fears and phobias can have a serious impact on young people with ASD and their families. In this study we developed and evaluated a unique treatment combining cognitive behaviour therapy (CBT) with graduated exposure in a virtual reality environment (VRE). Nine verbally fluent boys with an ASD diagnosis and no reported learning disability, aged 7 to 13 years old, were recruited. Each had anxiety around a specific situation (e.g. crowded buses) or stimulus (e.g. pigeons). An individualised scene was recreated in our 'wrap-around' VRE. In the VRE participants were coached by a psychologist in cognitive and behavioural techniques (e.g. relaxation and breathing exercises) while the exposure to the phobia/fear stimulus was gradually increased as the child felt ready. Each child received four 20-30 minute sessions. After participating in the study, eight of the nine children were able to tackle their phobia situation. Four of the participants completely overcame their phobia. Treatment effects were maintained at 12 months. These results provide evidence that CBT with VRE can be a highly effective treatment for specific phobia/fear for some young people with ASD.Trial registrationControlled-Trials.com ISRCTN58483069.

Considerable progress has been made in identifying deafness genes, but still little is known about the genetic basis of normal variation in hearing function. We recently carried out a Genome Wide Association Study (GWAS) of quantitative hearing traits in southern European populations and found several SNPs with suggestive but none with significant association. In the current study, we followed up these SNPs to investigate which of them might show a genuine association with auditory function using alternative approaches. Firstly, we generated a shortlist of 19 genes from the published GWAS results. Secondly, we carried out immunocytochemistry to examine expression of these 19 genes in the mouse inner ear. Twelve of them showed distinctive cochlear expression patterns. Four showed expression restricted to sensory hair cells (Csmd1, Arsg, Slc16a6 and Gabrg3), one only in marginal cells of the stria vascularis (Dclk1) while the others (Ptprd, Grm8, GlyBP, Evi5, Rimbp2, Ank2, Cdh13) in multiple cochlear cell types. In the third step, we tested these 12 genes for replication of association in an independent set of samples from the Caucasus and Central Asia. Nine out of them showed nominally significant association (p

BACKGROUND: Clostridium difficile are gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15-35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Genotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production. CONCLUSIONS/SIGNIFICANCE: This is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described.

Latency is a key feature of the animal pathogen Chlamydia abortus, where infection remains inapparent in the non-pregnant animal and only becomes evident during a subsequent pregnancy. Often the first sign that an animal is infected is abortion occurring late in gestation. Despite this, little is understood of the underlying mechanisms that control latency or the recrudescence of infection that occurs during subsequent pregnancy. The aim of this study was to develop an experimental model of latency by mimicking the natural route of infection through the intranasal inoculation of non-pregnant sheep with C. abortus.Three groups of sheep (groups 1, 2 and 3) were experimentally infected with different doses of C. abortus (5×10(3), 5×10(5) and 5×10(7) inclusion forming units (IFU), respectively) prior to mating and monitored over 2 breeding cycles for clinical, microbiological, pathological, immunological and serological outcomes. Two further groups received either negative control inoculum (group 4a,b) or were inoculated subcutaneously on day 70 of gestation with 2×10(6) IFU C. abortus (group 5). Animals in groups 1, 2 and 5 experienced an abortion rate of 50-67%, while only one animal aborted in group 3 and none in group 4a,b. Pathological, microbiological, immunological and serological analyses support the view that the maternal protective immune response is influenced by initial exposure to the bacterium.The results show that intranasal administration of non-pregnant sheep with a low/medium dose of C. abortus results in a latent infection that leads in a subsequent pregnancy to infection of the placenta and abortion. In contrast a high dose stimulates protective immunity, resulting in a much lower abortion rate. This model will be useful in understanding the mechanisms of infection underlying latency and onset of disease, as well as in the development of novel therapeutics and vaccines for controlling infection.

Tumor fibroblasts are active partners in tumor progression, but the genes and pathways that mediate this collaboration are ill-defined. Previous work demonstrates that Ets2 function in stromal cells significantly contributes to breast tumor progression. Conditional mouse models were used to study the function of Ets2 in both mammary stromal fibroblasts and epithelial cells. Conditional inactivation of Ets2 in stromal fibroblasts in PyMT and ErbB2 driven tumors significantly reduced tumor growth, however deletion of Ets2 in epithelial cells in the PyMT model had no significant effect. Analysis of gene expression in fibroblasts revealed a tumor- and Ets2-dependent gene signature that was enriched in genes important for ECM remodeling, cell migration, and angiogenesis in both PyMT and ErbB2 driven-tumors. Consistent with these results, PyMT and ErbB2 tumors lacking Ets2 in fibroblasts had fewer functional blood vessels, and Ets2 in fibroblasts elicited changes in gene expression in tumor endothelial cells consistent with this phenotype. An in vivo angiogenesis assay revealed the ability of Ets2 in fibroblasts to promote blood vessel formation in the absence of tumor cells. Importantly, the Ets2-dependent gene expression signatures from both mouse models were able to distinguish human breast tumor stroma from normal stroma, and correlated with patient outcomes in two whole tumor breast cancer data sets. The data reveals a key function for Ets2 in tumor fibroblasts in signaling to endothelial cells to promote tumor angiogenesis. The results highlight the collaborative networks that orchestrate communication between stromal cells and tumor cells, and suggest that targeting tumor fibroblasts may be an effective strategy for developing novel anti-angiogenic therapies.

Dried blood spots (DBS) are an alternative specimen collection format for HIV-1 genotyping. DBS produce HIV genotyping results that are robust and equivalent to plasma when using conventional sequencing methods. However, using tagged, pooled pyrosequencing, we demonstrate that concordance between plasma and DBS is not absolute and varies according to viral load (VL), duration of HIV infection and antiretroviral therapy (ART) status. The plasma/DBS concordance is the highest when VL is ≥5,000 copies/ml and/or the patient has no ART exposure and/or when the duration of HIV infection is ≤2 years. Stepwise regression analysis revealed that VL is most important independent predictor for concordance of DBS with plasma genotypes. This is the first study to use next generation sequencing to identify discordance between DBS and plasma genotypes. Consideration should be given to VL, duration of infection, and ART exposure when interpreting DBS genotypes produced using next generation sequencing. These findings are of particular significance when DBS are to be used for clinical monitoring purposes.

Chlamydia possess a unique family of autotransporter proteins known as the Polymorphic membrane proteins (Pmps). While the total number of pmp genes varies between Chlamydia species, all encode a single pmpD gene. In both Chlamydia trachomatis (C. trachomatis) and C. pneumoniae, the PmpD protein is proteolytically cleaved on the cell surface. The current study was carried out to determine the cleavage patterns of the PmpD protein in the animal pathogen C. abortus (termed Pmp18D).Using antibodies directed against different regions of Pmp18D, proteomic techniques revealed that the mature protein was cleaved on the cell surface, resulting in a100 kDa N-terminal product and a 60 kDa carboxy-terminal protein. The N-terminal protein was further processed into 84, 76 and 73 kDa products. Clustering analysis resolved PmpD proteins into three distinct clades with C. abortus Pmp18D, being most similar to those originating from C. psittaci, C. felis and C. caviae.This study indicates that C. abortus Pmp18D is proteolytically processed at the cell surface similar to the proteins of C. trachomatis and C. pneumoniae. However, patterns of cleavage are species-specific, with low sequence conservation of PmpD across the genus. The absence of conserved domains indicates that the function of the PmpD molecule in chlamydia remains to be elucidated.

Culicoides oxystoma (Diptera: Ceratopogonidae) is an important vector species, reported mainly from Asia, with high potential to transmit viral diseases affecting livestock. In Japan, many arboviruses have been isolated from C. oxystoma, suggesting it as a key player in the epidemiology of several Culicoides-borne diseases. Over the years, C. oxystoma has also been reported in the Middle East region, including Israel. In this region, however, C. oxystoma cannot be easily distinguished morphologically from its sibling species included in the Culicoides schultzei complex. We therefore used genomic data for species identification and phylogeny resolution. Phylogenetic analyses based on internal transcribed spacer 1 (ITS-1) of ribosomal DNA and the mitochondrial gene encoding cytochrome oxidase subunit I (COI) showed that C. oxystoma from Israel is closely related to C. oxystoma from Japan. Using differential probing PCR, we showed that C. oxystoma is distributed all over the country, especially in Mediterranean climate regions. Culicoides oxystoma is less common or even absent in arid regions, while the other genetic cluster of C. schultzei complex was found only in the east of the country (mostly arid and semiarid regions). The molecular finding of C. oxystoma in wide geographical regions, together with its high proportion in the general Culicoides population and its vectoring potential, imply that it may be an important vector species in the Middle East.

BackgroundIn April 2009, a novel triple-reassortant swine influenza A H1N1 virus ("A/H1N1pdm"; also known as SOIV) was detected and spread globally as the first influenza pandemic of the 21(st) century. Sequencing has since been conducted at an unprecedented rate globally in order to monitor the diversification of this emergent virus and to track mutations that may affect virus behavior.Methodology/principal findingsBy Sanger sequencing, we determined consensus whole-genome sequences for A/H1N1pdm viruses sampled nationwide in Canada over 33 weeks during the 2009 first and second pandemic waves. A total of 235 virus genomes sampled from unique subjects were analyzed, providing insight into the temporal and spatial trajectory of A/H1N1pdm lineages within Canada. Three clades (2, 3, and 7) were identifiable within the first two weeks of A/H1N1pdm appearance, with clades 5 and 6 appearing thereafter; further diversification was not apparent. Only two viral sites displayed evidence of adaptive evolution, located in hemagglutinin (HA) corresponding to D222 in the HA receptor-binding site, and to E374 at HA2-subunit position 47. Among the Canadian sampled viruses, we observed notable genetic diversity (1.47 x 10⁻³ amino acid substitutions per site) in the gene encoding PB1, particularly within the viral genomic RNA (vRNA)-binding domain (residues 493-757). This genome data set supports the conclusion that A/H1N1pdm is evolving but not excessively relative to other H1N1 influenza A viruses. Entropy analysis was used to investigate whether any mutated A/H1N1pdm protein residues were associated with infection severity; however no virus genotypes were observed to trend with infection severity. One virus that harboured heterozygote coding mutations, including PB2 D567D/G, was attributed to a severe and potentially mixed infection; yet the functional significance of this PB2 mutation remains unknown.Conclusions/significanceThese findings contribute to enhanced understanding of Influenza A/H1N1pdm viral dynamics.

Pyrosequencing technology has the potential to rapidly sequence HIV-1 viral quasispecies without requiring the traditional approach of cloning. In this study, we investigated the utility of ultra-deep pyrosequencing to characterize genetic diversity of the HIV-1 gag quasispecies and assessed the possible contribution of pyrosequencing technology in studying HIV-1 biology and evolution.HIV-1 gag gene was amplified from 96 patients using nested PCR. The PCR products were cloned and sequenced using capillary based Sanger fluorescent dideoxy termination sequencing. The same PCR products were also directly sequenced using the 454 pyrosequencing technology. The two sequencing methods were evaluated for their ability to characterize quasispecies variation, and to reveal sites under host immune pressure for their putative functional significance. A total of 14,034 variations were identified by 454 pyrosequencing versus 3,632 variations by Sanger clone-based (SCB) sequencing. 11,050 of these variations were detected only by pyrosequencing. These undetected variations were located in the HIV-1 Gag region which is known to contain putative cytotoxic T lymphocyte (CTL) and neutralizing antibody epitopes, and sites related to virus assembly and packaging. Analysis of the positively selected sites derived by the two sequencing methods identified several differences. All of them were located within the CTL epitope regions.Ultra-deep pyrosequencing has proven to be a powerful tool for characterization of HIV-1 genetic diversity with enhanced sensitivity, efficiency, and accuracy. It also improved reliability of downstream evolutionary and functional analysis of HIV-1 quasispecies.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a leading cause of death, particularly in developing countries. Little is known about the effects of economic development on COPD mortality, although economic development may potentially have positive and negative influences over the life course on COPD. We took advantage of a unique population whose rapid and recent economic development is marked by changes at clearly delineated and identifiable time points, and where few women smoke, to examine the effect of macro-level events on COPD mortality. METHODS: We used Poisson regression to decompose sex-specific COPD mortality rates in Hong Kong from 1981 to 2005 into the effects of age, period and cohort. RESULTS: COPD mortality declined strongly over generations for people born from the early to mid 20th century, which was particularly evident for the first generation to grow up in a more economically developed environment for both sexes. Population wide COPD mortality decreased when air quality improved and increased with increasing air pollution. COPD mortality increased with age, particularly after menopause among women. CONCLUSIONS: Economic development may reduce vulnerability to COPD by reducing long-lasting insults to the respiratory system, such as infections, poor nutrition and indoor air pollution. However, some of these gains may be offset if economic development results in increasing air pollution or increasing smoking.

Glycoprotein non-metastatic melanoma protein B (GPNMB)/Osteoactivin (OA) is a transmembrane protein expressed in approximately 40-75% of breast cancers. GPNMB/OA promotes the migration, invasion and metastasis of breast cancer cells; it is commonly expressed in basal/triple-negative breast tumors and is associated with shorter recurrence-free and overall survival times in patients with breast cancer. Thus, GPNMB/OA represents an attractive target for therapeutic intervention in breast cancer; however, little is known about the functions of GPNMB/OA within the primary tumor microenvironment.We have employed mouse and human breast cancer cells to investigate the effects of GPNMB/OA on tumor growth and angiogenesis. GPNMB/OA-expressing tumors display elevated endothelial recruitment and reduced apoptosis when compared to vector control-derived tumors. Primary human breast cancers characterized by high vascular density also display elevated levels of GPNMB/OA when compared to those with low vascular density. Using immunoblot and ELISA assays, we demonstrate the GPNMB/OA ectodomain is shed from the surface of breast cancer cells. Transient siRNA-mediated knockdown studies of known sheddases identified ADAM10 as the protease responsible for GPNMB/OA processing. Finally, we demonstrate that the shed extracellular domain (ECD) of GPNMB/OA can promote endothelial migration in vitro.GPNMB/OA expression promotes tumor growth, which is associated with enhanced endothelial recruitment. We identify ADAM10 as a sheddase capable of releasing the GPNMB/OA ectodomain from the surface of breast cancer cells, which induces endothelial cell migration. Thus, ectodomain shedding may serve as a novel mechanism by which GPNMB/OA promotes angiogenesis in breast cancer.

Breast tumors consist of several different tissue components. Despite the heterogeneity, most gene expression analyses have traditionally been performed without prior microdissection of the tissue sample. Thus, the gene expression profiles obtained reflect the mRNA contribution from the various tissue components. We utilized histopathological estimations of area fractions of tumor and stromal tissue components in 198 fresh-frozen breast tumor tissue samples for a cell type-associated gene expression analysis associated with distant metastasis. Sets of differentially expressed gene-probes were identified in tumors from patients who developed distant metastasis compared with those who did not, by weighing the contribution from each tumor with the relative content of stromal and tumor epithelial cells in their individual tumor specimen. The analyses were performed under various assumptions of mRNA transcription level from tumor epithelial cells compared with stromal cells. A set of 30 differentially expressed gene-probes was ascribed solely to carcinoma cells. Furthermore, two sets of 38 and five differentially expressed gene-probes were mostly associated to tumor epithelial and stromal cells, respectively. Finally, a set of 26 differentially expressed gene-probes was identified independently of cell type focus. The differentially expressed genes were validated in independent gene expression data from a set of laser capture microdissected invasive ductal carcinomas. We present a method for identifying and ascribing differentially expressed genes to tumor epithelial and/or stromal cells, by utilizing pathologic information and weighted t-statistics. Although a transcriptional contribution from the stromal cell fraction is detectable in microarray experiments performed on bulk tumor, the gene expression differences between the distant metastasis and no distant metastasis group were mostly ascribed to the tumor epithelial cells of the primary breast tumors. However, the gene PIP5K2A was found significantly elevated in stroma cells in distant metastasis group, compared to stroma in no distant metastasis group. These findings were confirmed in gene expression data from the representative compartments from microdissected breast tissue. The method described was also found to be robust to different histopathological procedures.