Evaluation of two high-throughput proteomic technologies for plasma biomarker discovery in immunotherapy-treated melanoma patients

Background: Selective kinase and immune checkpoint inhibitors, and their combinations, have significantly improved the survival of patients with advanced metastatic melanoma. Not all patients will respond to treatment however, and some patients will present with significant toxicities. Hence, the identification of biomarkers is critical for the selection and management of patients receiving treatment. Biomarker discovery often involves proteomic techniques that simultaneously profile multiple proteins but few studies have compared these platforms. Methods: In this study, we used the multiplex bead-based Eve Technologies Discovery assay and the aptamer-based SomaLogic SOMAscan assay to identify circulating proteins predictive of response to immunotherapy in melanoma patients treated with combination immune checkpoint inhibitors. Expression of four plasma proteins were further validated using the bead-based Millipore Milliplex assay. Results: Both the Discovery and the SOMAscan assays detected circulating plasma proteins in immunotherapy-treated melanoma patients. However, these widely used assays showed limited correlation in relative protein quantification, due to differences in specificity and the dynamic range of protein detection. Protein data derived from the Discovery and Milliplex bead-based assays were highly correlated. Conclusions: Our study highlights significant limitations imposed by inconsistent sensitivity and specificity due to differences in the detection antibodies or aptamers of these widespread biomarker discovery approaches. Our findings emphasize the need to improve these technologies for the accurate identification of biomarkers

Androgen receptor signalling in Vascular Endothelial cells is dispensable for spermatogenesis and male fertility

<p>Abstract</p> <p>Background</p> <p>Androgen signalling is essential both for male development and function of the male reproductive system in adulthood. Within the adult testis, Germ cells (GC) do not express androgen receptor (AR) suggesting androgen-mediated promotion of spermatogenesis must act via AR-expressing somatic cell-types. Several recent studies have exploited the Cre/lox system of conditional gene-targeting to ablate AR function from key somatic cell-types in order to establish the cell-specific role of AR in promotion of male fertility. In this study, we have used a similar approach to specifically ablate AR-signalling from Vascular Endothelial (VE) cells, with a view to defining the significance of androgen signalling within this cell-type on spermatogenesis.</p> <p>Findings</p> <p>AR expression in VE cells of the testicular vasculature was confirmed using an antibody against AR. A Cre-inducible fluorescent reporter line was used to empirically establish the utility of a mouse line expressing Cre Recombinase driven by the Tie2-Promoter, for targeting VE cells. Immunofluorescent detection revealed expression of YFP (and therefore Cre Recombinase function) limited to VE cells and an interstitial population of cells, believed to be macrophages, that did not express AR. Mating of Tie2-Cre males to females carrying a floxed AR gene produced Vascular Endothelial Androgen Receptor Knockout (VEARKO) mice and littermate controls. Ablation of AR from all VE cells was confirmed; however, no significant differences in bodyweight or reproductive tissue weights could be detected in VEARKO animals and spermatogenesis and fertility was unaffected.</p> <p>Conclusions</p> <p>We demonstrate the successful generation and empirical validation of a cell-specific knockout of AR from VE cells, and conclude that AR expression in VE cells is not essential for spermatogenesis or male fertility.</p

Repeatability and sensitivity of T2* measurements in patients with head and neck squamous cell carcinoma at 3T.

Purpose To determine whether quantitation of T2* is sufficiently repeatable and sensitive to detect clinically relevant oxygenation levels in head and neck squamous cell carcinoma (HNSCC) at 3T.Materials and methods Ten patients with newly diagnosed locally advanced HNSCC underwent two magnetic resonance imaging (MRI) scans between 24 and 168 hours apart prior to chemoradiotherapy treatment. A multiple gradient echo sequence was used to calculate T2* maps. A quadratic function was used to model the blood transverse relaxation rate as a function of blood oxygenation. A set of published coefficients measured at 3T were incorporated to account for tissue hematocrit levels and used to plot the dependence of fractional blood oxygenation (Y) on T2* values, together with the corresponding repeatability range. Repeatability of T2* using Bland-Altman analysis, and calculation of limits of agreement (LoA), was used to assess the sensitivity, defined as the minimum difference in fractional blood oxygenation that can be confidently detected.Results T2* LoA for 22 outlined tumor volumes were 13%. The T2* dependence of fractional blood oxygenation increases monotonically, resulting in increasing sensitivity of the method with increasing blood oxygenation. For fractional blood oxygenation values above 0.11, changes in T2* were sufficient to detect differences in blood oxygenation greater than 10% (Δ T2* > LoA for ΔY > 0.1).Conclusion Quantitation of T2* at 3T can detect clinically relevant changes in tumor oxygenation within a wide range of blood volumes and oxygen tensions, including levels reported in HNSCC. J. Magn. Reson. Imaging 2016;44:72-80

25-hydroxyvitamin D is lower in deprived groups, but is not associated with carotid intima media thickness or plaques: results from pSoBid

Objective: The association of the circulating serum vitamin D metabolite 25-hydroxyvitamin D (25OHD) with atherosclerotic burden is unclear, with previous studies reporting disparate results. &lt;p/&gt;Method: Psychological, social and biological determinants of ill health (pSoBid) is a study of participants aged 35–64 years from Glasgow who live at extremes of the socioeconomic spectrum. Vitamin D deficiency was defined as 25OHD &#60; 25nmol/L, as per convention. Cross-sectional associations between circulating 25OHD concentrations and a range of socioeconomic, lifestyle, and biochemistry factors, as well as carotid intima media thickness (cIMT) and plaque presence were assessed in 625 participants. &lt;p/&gt;Results: Geometric mean levels of circulating 25OHD were higher among the least deprived (45.6 nmol/L, 1-SD range 24.4–85.5) versus most deprived (34.2 nmol/L, 1-SD range 16.9–69.2; p &#60; 0.0001). In the least deprived group 15% were “deficient” in circulating 25OHD versus 30.8% in the most deprived (χ2p &#60; 0.0001). Log 25OHD was 27% lower among smokers (p &#60; 0.0001), 20% higher among the physically active versus inactive (p = 0.01), 2% lower per 1 kg/m2 increase in body mass index (BMI) (p &#60; 0.0001), and showed expected seasonal variation (χ2p &#60; 0.0001). Log 25OHD was 13% lower in the most versus least deprived independent of the aforementioned lifestyle confounding factors (p = 0.03). One unit increase in log 25OHD was not associated with atherosclerotic burden in univariable models; cIMT (effect estimate 0.000 mm [95% CI −0.011, 0.012]); plaque presence (OR 0.88 [0.75, 1.03]), or in multivariable models. &lt;p/&gt;Conclusion: There is no strong association of 25OHD with cIMT or plaque presence, despite strong evidence 25OHD associates with lifestyle factors and socioeconomic deprivation

Subclinical thyroid dysfunction and cognitive decline in old age

&lt;p&gt;Background: Subclinical thyroid dysfunction has been implicated as a risk factor for cognitive decline in old age, but results are inconsistent. We investigated the association between subclinical thyroid dysfunction and cognitive decline in the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER).&lt;/p&gt; &lt;p&gt;Methods: Prospective longitudinal study of men and women aged 70–82 years with pre-existing vascular disease or more than one risk factor to develop this condition (N = 5,154). Participants taking antithyroid medications, thyroid hormone supplementation and/or amiodarone were excluded. Thyroid function was measured at baseline: subclinical hyper- and hypothyroidism were defined as thyroid stimulating hormones (TSH) &#60;0.45 mU/L or &#62;4.50 mU/L respectively, with normal levels of free thyroxine (FT4). Cognitive performance was tested at baseline and at four subsequent time points during a mean follow-up of 3 years, using five neuropsychological performance tests.&lt;/p&gt; &lt;p&gt;Results: Subclinical hyperthyroidism and hypothyroidism were found in 65 and 161 participants, respectively. We found no consistent association of subclinical hyper- or hypothyroidism with altered cognitive performance compared to euthyroid participants on the individual cognitive tests. Similarly, there was no association with rate of cognitive decline during follow-up.&lt;/p&gt; &lt;p&gt;Conclusion: We found no consistent evidence that subclinical hyper- or hypothyroidism contribute to cognitive impairment or decline in old age. Although our data are not in support of treatment of subclinical thyroid dysfunction to prevent cognitive dysfunction in later life, only large randomized controlled trials can provide definitive evidence.&lt;/p&gt

Subclinical thyroid dysfunction and cognitive decline in old age

&lt;p&gt;Background: Subclinical thyroid dysfunction has been implicated as a risk factor for cognitive decline in old age, but results are inconsistent. We investigated the association between subclinical thyroid dysfunction and cognitive decline in the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER).&lt;/p&gt; &lt;p&gt;Methods: Prospective longitudinal study of men and women aged 70–82 years with pre-existing vascular disease or more than one risk factor to develop this condition (N = 5,154). Participants taking antithyroid medications, thyroid hormone supplementation and/or amiodarone were excluded. Thyroid function was measured at baseline: subclinical hyper- and hypothyroidism were defined as thyroid stimulating hormones (TSH) &#60;0.45 mU/L or &#62;4.50 mU/L respectively, with normal levels of free thyroxine (FT4). Cognitive performance was tested at baseline and at four subsequent time points during a mean follow-up of 3 years, using five neuropsychological performance tests.&lt;/p&gt; &lt;p&gt;Results: Subclinical hyperthyroidism and hypothyroidism were found in 65 and 161 participants, respectively. We found no consistent association of subclinical hyper- or hypothyroidism with altered cognitive performance compared to euthyroid participants on the individual cognitive tests. Similarly, there was no association with rate of cognitive decline during follow-up.&lt;/p&gt; &lt;p&gt;Conclusion: We found no consistent evidence that subclinical hyper- or hypothyroidism contribute to cognitive impairment or decline in old age. Although our data are not in support of treatment of subclinical thyroid dysfunction to prevent cognitive dysfunction in later life, only large randomized controlled trials can provide definitive evidence.&lt;/p&gt

Accelerated and Improved Quantification of Lymphocytic Choriomeningitis Virus (LCMV) Titers by Flow Cytometry

Lymphocytic choriomeningitis virus (LCMV), a natural murine pathogen, is a member of the Arenavirus family, may cause atypical meningitis in humans, and has been utilized extensively as a model pathogen for the study of virus-induced disease and immune responses. Historically, viral titers have been quantified by a standard plaque assay, but for non-cytopathic viruses including LCMV this requires lengthy incubation, so results cannot be obtained rapidly. Additionally, due to specific technical constraints of the plaque assay including the visual detection format, it has an element of subjectivity along with limited sensitivity. In this study, we describe the development of a FACS-based assay that utilizes detection of LCMV nucleoprotein (NP) expression in infected cells to determine viral titers, and that exhibits several advantages over the standard plaque assay. We show that the LCMV-NP FACS assay is an objective and reproducible detection method that requires smaller sample volumes, exhibits a ∼20-fold increase in sensitivity to and produces results three times faster than the plaque assay. Importantly, when applied to models of acute and chronic LCMV infection, the LCMV-NP FACS assay revealed the presence of infectious virus in samples that were determined to be negative by plaque assay. Therefore, this technique represents an accelerated, enhanced and objective alternative method for detection of infectious LCMV that is amenable to adaptation for other viral infections as well as high throughput diagnostic platforms

Visual Search Strategies of Soccer Players Executing a Power vs. Placement Penalty Kick

Introduction: When taking a soccer penalty kick, there are two distinct kicking techniques that can be adopted; a ‘power’ penalty or a ‘placement’ penalty. The current study investigated how the type of penalty kick being taken affected the kicker’s visual search strategy and where the ball hit the goal (end ball location). Method: Wearing a portable eye tracker, 12 university footballers executed 2 power and placement penalty kicks, indoors, both with and without the presence of a goalkeeper. Video cameras were used to determine initial ball velocity and end ball location. Results: When taking the power penalty, the football was kicked significantly harder and more centrally in the goal compared to the placement penalty. During the power penalty, players fixated on the football for longer and more often at the goalkeeper (and by implication the middle of the goal), whereas in the placement penalty, fixated longer at the goal, specifically the edges. Findings remained consistent irrespective of goalkeeper presence. Discussion/conclusion: Findings indicate differences in visual search strategy and end ball location as a function of type of penalty kick. When taking the placement penalty, players fixated and kicked the football to the edges of the goal in an attempt to direct the ball to an area that the goalkeeper would have difficulty reaching and saving. Fixating significantly longer on the football when taking the power compared to placement penalty indicates a greater importance of obtaining visual information from the football. This can be attributed to ensuring accurate foot-to-ball contact and subsequent generation of ball velocity. Aligning gaze and kicking the football centrally in the goal when executing the power compared to placement penalty may have been a strategy to reduce the risk of kicking wide of the goal altogether

Substrate Cooperativity in Marine Luciferases

Marine luciferases are increasingly used as reporters to study gene regulation. These luciferases have utility in bioluminescent assay development, although little has been reported on their catalytic properties in response to substrate concentration. Here, we report that the two marine luciferases from the copepods, Gaussia princeps (GLuc) and Metridia longa (MLuc) were found, surprisingly, to produce light in a cooperative manner with respect to their luciferin substrate concentration; as the substrate concentration was decreased 10 fold the rate of light production decreased 1000 fold. This positive cooperative effect is likely a result of allostery between the two proposed catalytic domains found in Gaussia and Metridia. In contrast, the marine luciferases from Renilla reniformis (RLuc) and Cypridina noctiluca (CLuc) demonstrate a linear relationship between the concentration of their respective luciferin and the rate of light produced. The consequences of these enzyme responses are discussed

Error Rates, Decisive Outcomes and Publication Bias with Several Inferential Methods

Correction to this article published Hopkins, W.G. & Batterham, A.M. Sports Med (2016) 46: 923. doi:10.1007/s40279-016-0530-