Aptamer-based multiplexed proteomic technology for biomarker discovery

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine

Differences in genotype and virulence among four multidrug-resistant <i>Streptococcus pneumoniae</i> isolates belonging to the PMEN1 clone

We report on the comparative genomics and characterization of the virulence phenotypes of four &lt;i&gt;S. pneumoniae&lt;/i&gt; strains that belong to the multidrug resistant clone PMEN1 (Spain&lt;sup&gt;23F&lt;/sup&gt; ST81). Strains SV35-T23 and SV36-T3 were recovered in 1996 from the nasopharynx of patients at an AIDS hospice in New York. Strain SV36-T3 expressed capsule type 3 which is unusual for this clone and represents the product of an in vivo capsular switch event. A third PMEN1 isolate - PN4595-T23 - was recovered in 1996 from the nasopharynx of a child attending day care in Portugal, and a fourth strain - ATCC700669 - was originally isolated from a patient with pneumococcal disease in Spain in 1984. We compared the genomes among four PMEN1 strains and 47 previously sequenced pneumococcal isolates for gene possession differences and allelic variations within core genes. In contrast to the 47 strains - representing a variety of clonal types - the four PMEN1 strains grouped closely together, demonstrating high genomic conservation within this lineage relative to the rest of the species. In the four PMEN1 strains allelic and gene possession differences were clustered into 18 genomic regions including the capsule, the blp bacteriocins, erythromycin resistance, the MM1-2008 prophage and multiple cell wall anchored proteins. In spite of their genomic similarity, the high resolution chinchilla model was able to detect variations in virulence properties of the PMEN1 strains highlighting how small genic or allelic variation can lead to significant changes in pathogenicity and making this set of strains ideal for the identification of novel virulence determinant

Hematopoietic Cell Transplantation in Patients With Primary Immune Regulatory Disorders (PIRD): A Primary Immune Deficiency Treatment Consortium (PIDTC) Survey.

Primary Immune Regulatory Disorders (PIRD) are an expanding group of diseases caused by gene defects in several different immune pathways, such as regulatory T cell function. Patients with PIRD develop clinical manifestations associated with diminished and exaggerated immune responses. Management of these patients is complicated; oftentimes immunosuppressive therapies are insufficient, and patients may require hematopoietic cell transplant (HCT) for treatment. Analysis of HCT data in PIRD patients have previously focused on a single gene defect. This study surveyed transplanted patients with a phenotypic clinical picture consistent with PIRD treated in 33 Primary Immune Deficiency Treatment Consortium centers and European centers. Our data showed that PIRD patients often had immunodeficient and autoimmune features affecting multiple organ systems. Transplantation resulted in resolution of disease manifestations in more than half of the patients with an overall 5-years survival of 67%. This study, the first to encompass disorders across the PIRD spectrum, highlights the need for further research in PIRD management

Characterising extratropical near‐tropopause analysis humidity biases and their radiative effects on temperature forecasts

A cold bias in the extratropical lowermost stratosphere in forecasts is one of the most prominent systematic temperature errors in numerical weather prediction models. Hypothesized causes of this bias include radiative effects from a collocated moist bias in model analyses. Such biases would be expected to affect extratropical dynamics and result in the misrepresentation of wave propagation at tropopause level. Here the extent to which these humidity and temperature biases are connected is quantified. Observations from radiosondes are compared to operational analyses and forecasts from the European Centre for Medium-Range Weather Forecasts (ECMWF) Integrated Forecasting System (IFS) and Met Office Unified Model (MetUM) to determine the magnitude and vertical structure of these biases. Both operational models over-estimate lowermost stratospheric specific humidity, with a maximum moist bias around 1 km above the tropopause where humidities are around 170% of the observed values on average. This moist bias is already present in the initial conditions and changes little in forecasts over the first five days. Though temperatures are represented well in the analyses, the IFS forecasts anomalously cool in the lower stratosphere, relative to verifying radiosonde observations, by 0.2 K day –1. The IFS single column model is used to show this temperature change can be attributed to increased long-wave radiative cooling due to the lowermost stratospheric moist bias in the initial conditions. However, the MetUM temperature biases cannot be entirely attributed to the moist bias, and another significant factor must be present. These results highlight the importance of improving the humidity analysis to reduce the extratropical lowermost stratospheric cold bias in forecast models and the need to understand and mitigate the causes of the moist bias in these models

Defining discovery:is Google Scholar a discovery platform? An essay on the need for a new approach to scholarly discovery

This essay discusses the concept of discovery, intended as content discovery, and defines it in the new context of Open Science, with a focus on Social Sciences and Humanities (SSH). Starting from the example of Google Scholar, the authors show that this well established service does not address the current needs, practices, and variety of discovery. Alternatives in terms of technical choices, features, and governance, do however exist, offering richer and more open discovery. The paper presents in particular the implementations and research work of the H2020 project TRIPLE (Transforming Research through Innovative Practices for Linked Interdisciplinary Exploration). Dedicated to the building of a discovery platform for the SSH, the project is meant to address the specificities and evolution of discovery in this field. Prevailing scholarly resource platforms like Google Scholar limit discovery by focussing only on publications, and favouring through their algorithm well-cited papers, English content, and discipline-specific resources. A limitation in the context of cross-disciplinary and collaborative Open Science, such a service more specifically hinders discovery in the SSH. Characterized by a fragmented landscape, a variety of languages, data types, and outputs, research in the SSH requires services that fully exploit discovery potentialities. Moreover, a survey conducted within the TRIPLE project showed that most SSH researchers use Google Scholar as their starting point, and that they recognise the lack of control they have with this system. Beyond the extension of features and content, transparency is the other important criterion for the building of an Open Infrastructure actually serving the research community. In light of this, we present in some detail the GoTriple platform, which exploits today's technological potential and incorporates the best known functionalities in order to unveil more and innovative scholarly outputs and lead to international and interdisciplinary research project collaborations

Virulence phenotypes of low-passage clinical isolates of Nontypeable Haemophilus influenzae assessed using the chinchilla laniger model of otitis media

<p>Abstract</p> <p>Background</p> <p>The nontypeable Haemophilus influenzae (NTHi) are associated with a spectrum of respiratory mucosal infections including: acute otitis media (AOM); chronic otitis media with effusion (COME); otorrhea; locally invasive diseases such as mastoiditis; as well as a range of systemic disease states, suggesting a wide range of virulence phenotypes. Genomic studies have demonstrated that each clinical strain contains a unique genic distribution from a population-based supragenome, the distributed genome hypothesis. These diverse clinical and genotypic findings suggest that each NTHi strain possesses a unique set of virulence factors that contributes to the course of the disease.</p> <p>Results</p> <p>The local and systemic virulence patterns of ten genomically characterized low-passage clinical NTHi strains (PittAA – PittJJ) obtained from children with COME or otorrhea were stratified using the chinchilla model of otitis media (OM). Each isolate was used to bilaterally inoculate six animals and thereafter clinical assessments were carried out daily for 8 days by blinded observers. There was no statistical difference in the time it took for any of the 10 NTHi strains to induce otologic (local) disease with respect to any or all of the other strains, however the differences in time to maximal local disease and the severity of local disease were both significant between the strains. Parameters of systemic disease indicated that the strains were not all equivalent: time to development of the systemic disease, maximal systemic scores and mortality were all statistically different among the strains. PittGG induced 100% mortality while PittBB, PittCC, and PittEE produced no mortality. Overall Pitt GG, PittII, and Pitt FF produced the most rapid and most severe local and systemic disease. A post hoc determination of the clinical origins of the 10 NTHi strains revealed that these three strains were of otorrheic origin, whereas the other 7 were from patients with COME.</p> <p>Conclusion</p> <p>Collectively these data suggest that the chinchilla OM model is useful for discriminating between otorrheic and COME NTHi strains as to their disease-producing potential in humans, and combined with whole genome analyses, point the way towards identifying classes of virulence genes.</p

Performance characteristics of an instrument-free point-of-care CD4 test (VISITECTVR CD4) for use in resource-limited settings

Objective: CD4þ T lymphocyte count remains the most common biomarker of immune status and disease progression in human immunodeficiency virus (HIV)-positive individuals. VISITECTVR CD4 is an instrument-free, low-cost point-of-care CD4 test with a cut-off of 350 CD4 cells/lL. This study aimed to evaluate VISITECTVR CD4 test’s diagnostic accuracy. Methods: Two hundred HIV-positive patients attending a tertiary HIV centre in South India were recruited. Patients provided venous blood for reference and VISITECTVR CD4 tests. An additional finger-prick blood sample was obtained for VISITECTVR CD4. VISITECTVR CD4’s diagnostic performance in identifying individuals with CD4 counts 350 cells/lL was assessed by calculating sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) taking flow cytometry as the reference. Results: The overall agreement between VISITECTVR CD4 and flow cytometry was 89.5% using venous blood and 81.5% using finger-prick blood. VISITECTVR CD4 showed better performance using venous blood [sensitivity: 96.6% (95% confidence interval: 92.1%–98.9%), specificity: 70.9% (57.1%–82.4%), PPV: 89.7% (83.9%–94.0%) and NPV: 88.6% (75.4%–96.2%)] than using fingerprick blood [sensitivity: 84.8% (77.9%–90.2%), specificity: 72.7% (59.0%–83.9%), PPV: 89.1% (82.7%–93.8%) and NPV: 64.5% (51.3%–76.3%)]. Conclusion: VISITECTVR CD4 performed well using venous blood, demonstrating its potential utility in decentralization of CD4 testing services in resource-constrained settings

Field Performance and Diagnostic Accuracy of a Low-Cost Instrument-Free Point-of-Care CD4 Test (Visitect CD4) Performed by Different Health Worker Cadres among Pregnant Women

Measuring CD4 counts remains an important component of HIV care. The Visitect CD4 is the first instrument-free low-cost point-of-care CD4 test with results interpreted visually after 40 min, providing a result of 350 CD4 cells/mm3. The field performance and diagnostic accuracy of the test was assessed among HIVinfected pregnant women in South Africa. A nurse performed testing at the point-ofcare using both venous and finger-prick blood, and a counselor and laboratory staff tested venous blood in the clinic laboratory (four Visitect CD4 tests/participant). Performance was compared to the mean CD4 count from duplicate flow cytometry tests on venous blood (FACSCalibur Trucount). In 2017, 156 patients were enrolled, providing a total of 624 Visitect CD4 tests (468 venous and 156 finger-prick samples). Of 624 tests, 28 (4.5%) were inconclusive. Generalized linear mixed modeling showed better performance of the test on venous blood (sensitivity 81.7%; 95% confidence interval [CI] 72.3 to 91.1]; specificity 82.6%, 95% CI 77.1 to 88.1) than on finger-prick specimens (sensitivity 60.7%; 95% CI 45.0 to 76.3; specificity 89.5%, 95% CI 83.2 to 95.8; P 0.001). No difference in performance was detected by cadre of health worker (P 0.113) or between point-of-care versus laboratory-based testing (P 0.108). Adequate performance of Visitect CD4 with different operators and at the point of care, with no need of electricity or instrument, shows the potential utility of this device, especially for facilitating decentralization of CD4 testing services in rural areas