Detection and Differentiation of Two Koala Gammaherpesviruses by Use of High-Resolution Melt (HRM) Analysis Reveals Differences in Viral Prevalence and Clinical Associations in a Large Study of Free-Ranging Koalas

ABSTRACTThe iconic koala (Phascolarctos cinereus) is host to two divergent gammaherpesviruses, phascolarctid gammaherpesviruses 1 and 2 (PhaHV-1 and -2), but the clinical significance of the individual viruses is unknown and current diagnostic methods are unsuitable for differentiating between the viruses in large-scale studies. To address this, we modified a pan-herpesvirus nested PCR to incorporate high-resolution melt analysis. We applied this assay in a molecular epidemiological study of 810 koalas from disparate populations across Victoria, Australia, including isolated island populations. Animal and clinical data recorded at sampling were analyzed and compared to infection status. Between populations, the prevalence of PhaHV-1 and -2 varied significantly, ranging from 1% to 55%. Adult and older animals were 5 to 13 times more likely to be positive for PhaHV-1 than juveniles (P< 0.001), whereas PhaHV-2 detection did not change with age, suggesting differences in how these two viruses are acquired over the life of the animal. PhaHV-1 detection was uniquely associated with the detection of koala retrovirus, particularly in females (P= 0.008). Both viruses were significantly associated (P< 0.05) with the presence of genital tract abnormalities (uterine/ovarian cysts and testicular malformation), reduced fertility in females, urinary incontinence, and detection ofChlamydia pecorum, although the strength of these associations varied by sex and virus. Understanding the clinical significance of these viruses and how they interact with other pathogens will inform future management of threatened koala populations.

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Parallel Distributed Networks Resolved at High Resolution Reveal Close Juxtaposition of Distinct Regions

10.1101/475806 ◽

2018 ◽

Author(s):

Rodrigo M. Braga ◽

Koene R. A. Van Dijk ◽

Jonathan R. Polimeni ◽

Mark C. Eldaief ◽

Randy L. Buckner

Keyword(s):

High Resolution ◽

Large Scale ◽

Spatial Separation ◽

Distributed Networks ◽

Association Cortex ◽

Default Network ◽

Network Organization ◽

Parahippocampal Cortex ◽

The Individual ◽

Insight Into

Examination of large-scale distributed networks within the individual reveals details of cortical network organization that are absent in group-averaged studies. One recent discovery is that a distributed transmodal network, often referred to as the ‘default network’, is comprised of two separate but closely interdigitated networks, only one of which is coupled to posterior parahippocampal cortex. Not all studies of individuals have identified the same networks and questions remain about the degree to which the two networks are separate, particularly within regions hypothesized to be interconnected hubs. Here we replicate the observation of network separation across analytical (seed-based connectivity and parcellation) and data projection (volume and surface) methods in 2 individuals each scanned 31 times. Additionally, 3 individuals were examined with high-resolution fMRI to gain further insight into the anatomical details. The two networks were identified with separate regions localized to adjacent portions of the cortical ribbon, sometimes inside the same sulcus. Midline regions previously implicated as hubs revealed near complete spatial separation of the two networks, displaying a complex spatial topography in the posterior cingulate and precuneus. The network coupled to parahippocampal cortex also revealed a separate region directly within the hippocampus at or near the subiculum. These collective results support that the default network is composed of at least two spatially juxtaposed networks. Fine spatial details and juxta-positions of the two networks can be identified within individuals at high resolution, providing insight into the network organization of association cortex and placing further constraints on interpretation of group-averaged neuroimaging data.

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Differentiation of Community-Associated and Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates and Identification of spa Types by Use of PCR and High-Resolution Melt Curve Analysis

Journal of Clinical Microbiology ◽

10.1128/jcm.02088-19 ◽

2020 ◽

Vol 58 (5) ◽

Cited By ~ 1

Author(s):

Seyed A. Ghorashi ◽

Jane Heller ◽

Quincy Zhang ◽

Shafi Sahibzada

Keyword(s):

Staphylococcus Aureus ◽

High Resolution ◽

Curve Analysis ◽

Health Concern ◽

Staphylococcal Protein A ◽

Visual Interpretation ◽

High Resolution Melt ◽

Methicillin Resistant ◽

Content Type ◽

Mrsa Strains

ABSTRACT Infections due to methicillin-resistant Staphylococcus aureus (MRSA) are present worldwide and represent a major public health concern. The capability of PCR followed by high-resolution melt (HRM) curve analysis for the detection of community-associated and livestock-associated MRSA strains and the identification of staphylococcal protein A (spa) locus was evaluated in 74 MRSA samples which were isolated from the environment, humans, and pigs on a single piggery. PCR-HRM curve analysis identified four spa types among MRSA samples and differentiated MRSA strains accordingly. A nonsubjective differentiation model was developed according to genetic confidence percentage values produced by tested samples, which did not require visual interpretation of HRM curve results. The test was carried out at different settings, and result data were reanalyzed and confirmed with DNA sequencing. PCR-HRM curve analysis proved to be a robust and reliable test for spa typing and can be used as a tool in epidemiological studies.

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Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

Journal of Clinical Microbiology ◽

10.1128/jcm.02104-16 ◽

2017 ◽

Vol 55 (6) ◽

pp. 1755-1766 ◽

Cited By ~ 9

Author(s):

Divya Anthwal ◽

Rakesh Kumar Gupta ◽

Manpreet Bhalla ◽

Shinjini Bhatnagar ◽

Jaya Sivaswami Tyagi ◽

...

Keyword(s):

High Resolution ◽

Direct Detection ◽

Melting Curve ◽

Curve Analysis ◽

High Resolution Melt ◽

Melt Curve Analysis ◽

Content Type ◽

Plasmid Library ◽

Mdr Tb ◽

Smear Negative

ABSTRACT Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB , katG , and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH- katG , and INH- inhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.

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High-Resolution Analysis of Gut Environment and Bacterial Microbiota Reveals Functional Compartmentation of the Gut in Wood-Feeding Higher Termites (Nasutitermes spp.)

Applied and Environmental Microbiology ◽

10.1128/aem.00683-12 ◽

2012 ◽

Vol 78 (13) ◽

pp. 4691-4701 ◽

Cited By ~ 142

Author(s):

Tim Köhler ◽

Carsten Dietrich ◽

Rudolf H. Scheffrahn ◽

Andreas Brune

Keyword(s):

High Resolution ◽

16S Rrna Genes ◽

Rrna Genes ◽

Metagenomic Data ◽

Redox Potentials ◽

Fermentation Products ◽

Content Type ◽

Bacterial Microbiota ◽

High Resolution Analysis ◽

The Individual

ABSTRACTHigher termites are characterized by a purely prokaryotic gut microbiota and an increased compartmentation of their intestinal tract. In soil-feeding species, each gut compartment has different physicochemical conditions and is colonized by a specific microbial community. Although considerable information has accumulated also for wood-feeding species of the genusNasutitermes, including cellulase activities and metagenomic data, a comprehensive study linking physicochemical gut conditions with the structure of the microbial communities in the different gut compartments is lacking. In this study, we measured high-resolution profiles of H2, O2, pH, and redox potential in the gut ofNasutitermes cornigertermites, determined the fermentation products accumulating in the individual gut compartments, and analyzed the bacterial communities in detail by pyrotag sequencing of the V3-V4 region of the 16S rRNA genes. The dilated hindgut paunch (P3 compartment) was the only anoxic gut region, showed the highest density of bacteria, and accumulated H2to high partial pressures (up to 12 kPa). Molecular hydrogen is apparently produced by a dense community ofSpirochaetesandFibrobacteres, which also dominate the gut of otherNasutitermesspecies. All other compartments, such as the alkaline P1 compartment (average pH, 10.0), showed high redox potentials and comprised small but distinct populations characteristic for each gut region. In the crop and the posterior hindgut compartments, the community was even more diverse than in the paunch. Similarities in the communities of the posterior hindgut and crop suggested that proctodeal trophallaxis or coprophagy also occurs in higher termites. The large sampling depths of pyrotag sequencing in combination with the determination of important physicochemical parameters allow cautious conclusions concerning the functions of particular bacterial lineages in the respective gut sections to be drawn.

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Innovative real estate development finance – evidence from Europe

Journal of Financial Management of Property and Construction ◽

10.1108/jfmpc-09-2015-0036 ◽

2016 ◽

Vol 21 (1) ◽

pp. 54-72 ◽

Cited By ~ 3

Author(s):

Graham Squires ◽

Norman Hutchison ◽

Alastair Adair ◽

Jim Berry ◽

Stanley McGreal ◽

...

Keyword(s):

Real Estate ◽

Large Scale ◽

Real Estate Development ◽

Equity Financing ◽

Private Debt ◽

Content Type ◽

Development Finance ◽

Fund Managers ◽

The Individual ◽

Multi Stakeholder

Purpose – This research aims to provide an insight into large-scale real estate projects in Europe and how they are using a more innovative blend of finance. Design/methodology/approach – The methodology involved a mix of desk-based study, interviews and case studies. Interviews were held with financiers, policymakers, developers, investors, fund managers and academics. The specific case projects were Battersea Power Station Development in London; Leipziger Platz site in Berlin; and the Lammenschans site in the city of Leiden, The Netherlands. Findings – The research found that there is growth in the blend of financial products used in real estate development within large-scale mixed-use projects. This new blend is set with greater equity financing, often from domestic and foreign consortiums generating institutional funds – alongside private debt financing – that utilise a mix of large-scale multi-bank finance. Practical implications – The scale of the challenge in financing real estate development allied with capital budget constraints has meant that the appetite for innovative finance mechanisms has gained considerable momentum in practice and policy. This research investigates current examples in development finance and provides a discussion of the opinion of key multi-stakeholder participants in the individual cases, and trends more strategically at a broader level. Originality/value – This detailed study of three major development sites and at a more broader strategic level is significant, in that it provides a better understanding of the differing blends of finance that are being used.

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Barriers to smart services for manufacturing companies – an exploratory study in the capital goods industry

Journal of Business and Industrial Marketing ◽

10.1108/jbim-10-2015-0204 ◽

2018 ◽

Vol 33 (6) ◽

pp. 846-856 ◽

Cited By ~ 10

Author(s):

Maximilian Michael Klein ◽

Sebastian Simon Biehl ◽

Thomas Friedli

Keyword(s):

Quantitative Research ◽

Large Scale ◽

Holistic Approach ◽

Manufacturing Companies ◽

Capital Goods ◽

Smart Services ◽

Content Type ◽

Smart Service ◽

The Individual ◽

Service Businesses

Purpose The purpose of this paper is to identify and investigate non-technical barriers for smart services in the capital goods industry. Design/methodology/approach A multiple methodology approach is adopted. First, qualitative workshops and interviews were conducted with 14 experts from five companies. The findings generated subsequently provided a basis for a large-scale quantitative survey of manufacturing company service representatives in the capital goods industry, the data from which were analyzed using explorative factor analysis. Findings In total, 25 items that represent barriers to smart service businesses were identified, using qualitative research. Large-scale quantitative research revealed 24 items structured into four factors. Additionally, the respondents’ assessment of the individual barriers’ impact on their smart service businesses is presented. Research limitations/implications The study focuses on manufacturing companies in the capital goods industry, mainly, in the European countries. Caution should be exercised in seeking to generalize the results to other industries. The findings should be confirmed with subsequent confirmatory analyses using additional data. Practical implications The authors’ findings provide a comprehensive list and classification of barriers, as well as an assessment of their severity, serving as a practical guideline for managers. Originality/value This paper explores the barriers to smart services from a provider’s perspective. Its holistic approach and use of large-scale quantitative data qualify it as one of the first studies of this kind.

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Trainable High Resolution Melt Curve Machine Learning Classifier for Large-Scale Reliable Genotyping of Sequence Variants

PLoS ONE ◽

10.1371/journal.pone.0109094 ◽

2014 ◽

Vol 9 (10) ◽

pp. e109094 ◽

Cited By ~ 22

Author(s):

Pornpat Athamanolap ◽

Vishwa Parekh ◽

Stephanie I. Fraley ◽

Vatsal Agarwal ◽

Dong J. Shin ◽

...

Keyword(s):

Machine Learning ◽

High Resolution ◽

Large Scale ◽

Sequence Variants ◽

High Resolution Melt ◽

Learning Classifier

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Rapid High-Resolution Melt Analysis of Cytauxzoon felis CytochromebTo Aid in the Prognosis of Cytauxzoonosis

Journal of Clinical Microbiology ◽

10.1128/jcm.00635-15 ◽

2015 ◽

Vol 53 (8) ◽

pp. 2517-2524 ◽

Cited By ~ 3

Author(s):

Megan E. Schreeg ◽

Henry S. Marr ◽

Jaime L. Tarigo ◽

Leah A. Cohn ◽

Michael G. Levy ◽

...

Keyword(s):

High Resolution ◽

Survival Rates ◽

Protozoan Parasite ◽

Nucleotide Polymorphisms ◽

High Resolution Melt ◽

Prognostic Information ◽

Single Nucleotide ◽

Content Type ◽

High Resolution Melt Analysis ◽

Cytauxzoon Felis

Cytauxzoon felisis a virulent, tick-transmitted, protozoan parasite that infects felines. Cytauxzoonosis was previously thought to be uniformly fatal in domestic cats. Treatment combining atovaquone and azithromycin (A&A) has been associated with survival rates of over 60%. Atovaquone, a ubiquinone analogue, targetsC. feliscytochromeb(cytb), of which 30 unique genotypes have been identified. TheC. feliscytbgenotypecytb1is associated with increased survival rates in cats treated with A&A. The purpose of this study was to design a PCR panel that could distinguishC. felis cytb1from other cytochromebgenotypes. Primer pairs were designed to span five different nucleotide positions at which single-nucleotide polymorphisms in theC. felis cytbgene had been identified. Through the use of high-resolution melt analysis, this panel was predicted to distinguishcytb1from othercytbgenotypes. Assays were validated using samples from 69 cats with cytauxzoonosis for which theC. felis cytbgenotypes had been characterized previously. The PCR panel identifiedC. felis cytb1with 100% sensitivity and 98.2% specificity. High-resolution melt analysis can rapidly provide prognostic information for clients considering A&A treatment in cats with cytauxzoonosis.

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Organization of Organelles within Hyphae of Ashbya gossypii Revealed by Electron Tomography

Eukaryotic Cell ◽

10.1128/ec.00106-13 ◽

2013 ◽

Vol 12 (11) ◽

pp. 1423-1432 ◽

Cited By ~ 8

Author(s):

Romain Gibeaux ◽

Dominic Hoepfner ◽

Ivan Schlatter ◽

Claude Antony ◽

Peter Philippsen

Keyword(s):

High Resolution ◽

Large Scale ◽

Electron Tomography ◽

Three Dimensional ◽

Multivesicular Bodies ◽

Ashbya Gossypii ◽

Space Filling ◽

Content Type ◽

Hyphal Tip ◽

Dense Vesicles

ABSTRACT Ashbya gossypii grows as multinucleated and constantly elongating hyphae. Nuclei are in continuous forward and backward motion, also move during mitosis, and frequently bypass each other. Whereas these nuclear movements are well documented, comparatively little is known about the density and morphology of organelles which very likely influence these movements. To understand the three-dimensional subcellular organization of hyphae at high resolution, we performed large-scale electron tomography of the tip regions in A. gossypii . Here, we present a comprehensive space-filling model in which most membrane-limited organelles including nuclei, mitochondria, endosomes, multivesicular bodies, vacuoles, autophagosomes, peroxisomes, and vesicles are modeled. Nuclei revealed different morphologies and protrusions filled by the nucleolus. Mitochondria are very abundant and form a tubular network with a polarized spherical fraction. The organelles of the degradative pathways show a clustered organization. By analyzing vesicle-like bodies, we identified three size classes of electron-dense vesicles (∼200, ∼150, and ∼100 nm) homogeneously distributed in the cytoplasm which most likely represent peroxisomes. Finally, coated and uncoated vesicles with approximately 40-nm diameters show a polarized distribution toward the hyphal tip with the coated vesicles preferentially localizing at the hyphal periphery.

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