A high-throughput screening RT-qPCR assay for quantifying surrogate markers of immunity from PBMCs

Immunoassays that quantitate cytokines and other surrogate markers of immunity from peripheral blood mononuclear cells (PBMCs), such as flow cytometry or Enzyme-Linked Immunosorbent Spot (ELIspot), allow highly sensitive measurements of immune effector function. However, those assays consume relatively high numbers of cells and expensive reagents, precluding comprehensive analyses and high-throughput screening (HTS). To address this issue, we developed a sensitive and specific reverse transcription-quantitative PCR (RT-qPCR)-based HTS assay, specifically designed to quantify surrogate markers of immunity from very low numbers of PBMCs. We systematically evaluated the volumes and concentrations of critical reagents within the RT-qPCR protocol, miniaturizing the assay and ultimately reducing the cost by almost 90% compared to current standard practice. We assessed the suitability of this cost-optimized RT-qPCR protocol as an HTS tool and determined the assay exceeds HTS uniformity and signal variance testing standards. Furthermore, we demonstrate this technique can effectively delineate a hierarchy of responses from as little as 50,000 PBMCs stimulated with CD4+ or CD8+ T cell peptide epitopes. Finally, we establish that this HTS-optimized protocol has single-cell analytical sensitivity and a diagnostic sensitivity equivalent to detecting 1:10,000 responding cells (i.e., 100 Spot Forming Cells/106 PBMCs by ELIspot) with over 90% accuracy. We anticipate this assay will have widespread applicability in preclinical and clinical studies, especially when samples are limited, and cost is an important consideration

Phase Evolution and Li Diffusion in LATP Solid-State Electrolyte Synthesized via a Direct Heat-Cycling Method

Herein, the direct synthesis of phase-pure lithium aluminum titanium phosphate (Li_{1.3}Al_{0.3}Ti_{1.7}(PO_{4})_{3}, LATP) solid-electrolyte powder in 220 min and relatively low temperatures (850 °C) is achieved via a new (cyclic) fast heat treatment (c-FHT) route. The complex structural evolution highlights rate-limited lithium incorporation of intermediate metal phosphates formed prior to the final phase-pure LATP. The prepared LATP product powder displays similar bulk (2 × 10^{−10} cm^{2} s^{−1}) and local (3 × 10^{−10} cm^{2} s^{−1}) values for lithium diffusion coefficients (D_{Li}) characterized by electrochemical impedance spectroscopy and muon spin relaxation (μSR), respectively. The similarity between both D_{Li} values suggests excellent retention of inter- and intraparticle lithium diffusion, which is attributed to the absence of deleterious surface impurities such as AlPO4. A low-energy barrier (E_{a} = 73 meV) of lithium diffusion is also estimated from the μSR data

Effect of scandium triflate on the RAFT copolymerization of methyl acrylate and vinyl acetate controlled by an acid/base “switchable” chain transfer agent

Modulation of the activity of an acid/base switchable dithiocarbamate RAFT agent, cyanomethyl (4-fluorophenyl)(pyridin-4-yl)carbamodithioate, with the Lewis acid scandium triflate (Sc(OTf)3) was investigated to examine the ability to deliver improved control over RAFT copolymerizations involving both more-activated and less-activated monomers—specifically the copolymerization of methyl acrylate (MA) and vinyl acetate (VAc). The introduction of either 0.5 or 1 mol equiv of Sc(OTf)3, with respect to RAFT agent, into a RAFT copolymerization of MA and VAc provides substantially improved control resulting in significantly reduced molar mass dispersities (Đ) (∼1.1–1.3) than achieved in its absence (Đ ∼ 1.3–1.4). Furthermore, similar introduction of Sc(OTf)3 into MA homopolymerization mediated by the same RAFT agent also delivered polymers of very low Đ (∼1.15). Sc(OTf)3 was also found to lower the rate of polymerization and alter the copolymerization reactivity ratios for MA and VAc. Increasing the Lewis acid concentration provides enhanced incorporation of the less active monomer, VAc, into the copolymers ([Sc(OTf)3]/[RAFT] = 0, rMA = 4.04, rVAc = 0.032; [Sc(OTf)3]/[RAFT] = 0.5, rMA = 3.08, rVAc = 0.17; [Sc(OTf)3]/[RAFT] = 1, rMA = 2.68, rVAc = 0.62). Carbon nuclear magnetic resonance (13C NMR) and differential scanning calorimetry (DSC) analysis of preparative samples confirm the enhanced VAc incorporation with increased levels of Sc(OTf)3. Importantly the inclusion of Sc(OTf)3 does not deleteriously affect the thiocarbonylthio end-groups of the RAFT polymers, with high end-group fidelity being observed in all copolymerizations

Soil-related habitat specialization in dipterocarp rain forest tree species in Borneo

Summary 1 We conducted a field experiment to test whether aggregated spatial distributions were related to soil variation in locally sympatric tree species in the rain forests of Sarawak, Malaysia. Dryobalanops aromatica , Shorea laxa , and Swintonia schwenkii are naturally aggregated on low-fertility humult ultisols, Dryobalanops lanceolata and Hopea dryobalanoides on moderate-fertility udult ultisols and Shorea balanocarpoides is found on both soil types. 2 Seedlings of all six species were grown in a nested-factorial experiment for 20 months in humult and udult soils in gaps and in the understorey to test for soil-specific differences in performance. Phosphorus addition was used to test for effects due to P-limitation. 3 Four species showed significantly higher growth on their natural soils, but one humultsoil species ( D. aromatica ) and the broadly distributed species were not significantly affected by soil type. 4 One udult-soil species, D. lanceolata , had both lower relative growth rate and lower mycorrhizal colonization on humult soil. However, humult soils also had lower levels of Ca, Mg, K, N and probably water availability. 5 The overall ranking of growth rates among species was similar on the two soils. Growth rates were strongly positively correlated with leaf area ratio and specific leaf area among species in both soils. With the exception of D. aromatica , species of the higher-nutrient soils had higher growth rates on both soils. 6 Although P addition led to elevated soil-P concentrations, elevated root-and leaf-tissue P concentrations on both soils, there was no significant growth enhancement and therefore no evidence that P availability limits the growth or constrains the distribution of any of the six species in the field. Differences in soil water availability between soils may be more important. 7 Our results suggest that habitat-mediated differences in seedling performance strongly influence the spatial distributions of tropical trees and are therefore likely to play a key role in structuring tropical rain forest communities

The Effect of Tropical Temperatures on the Quality of RNA Extracted from Stabilized Whole-Blood Samples

Whole-blood-derived transcriptional profiling is widely used in biomarker discovery, immunological research, and therapeutic development. Traditional molecular and high-throughput transcriptomic platforms, including molecular assays with quantitative PCR (qPCR) and RNAsequencing (RNA-seq), are dependent upon high-quality and intact RNA. However, collecting high-quality RNA from field studies in remote tropical locations can be challenging due to resource restrictions and logistics of post-collection processing. The current study tested the relative performance of the two most widely used whole-blood RNA collection systems, PAXgene® and Tempus™, in optimal laboratory conditions as well as suboptimal conditions in tropical field sites, including the effects of extended storage times and high storage temperatures. We found that Tempus™ tubes maintained a slightly higher RNA quantity and integrity relative to PAXgene® tubes at suboptimal tropical conditions. Both PAXgene® and Tempus™ tubes gave similar RNA purity (A260/A280). Additionally, Tempus™ tubes preferentially maintained the stability of mRNA transcripts for two reference genes tested, Succinate dehydrogenase complex, subunit A (SDHA) and TATA-box-binding protein (TBP), even when RNA quality decreased with storage length and temperature. Both tube types preserved the rRNA transcript 18S ribosomal RNA (18S) equally. Our results suggest that Tempus™ blood RNA collection tubes are preferable to PAXgene® for whole-blood collection in suboptimal tropical conditions for RNA-based studies in resource-limited settings

Improving pulsar-timing solutions through dynamic pulse fitting

Precision pulsar timing is integral to the detection of the nanohertz stochastic gravitational-wave background as well as understanding the physics of neutron stars. Conventional pulsar timing often uses fixed time and frequency-averaged templates to determine the pulse times of arrival, which can lead to reduced accuracy when the pulse profile evolves over time. We illustrate a dynamic timing method that fits each observing epoch using basis functions. By fitting each epoch separately, we allow for the evolution of the pulse shape epoch to epoch. We apply our method to PSR J1103$-$5403 and demonstrate that it undergoes mode changing, making it the fourth millisecond pulsar to exhibit such behaviour. Our method, which is able to identify and time a single mode, yields a timing solution with a root-mean-square error of 1.343 $\mu \mathrm{s}$, a factor of 1.78 improvement over template fitting on both modes. In addition, the white-noise amplitude is reduced 4.3 times, suggesting that fitting the full data set causes the mode changing to be incorrectly classified as white noise. This reduction in white noise boosts the signal-to-noise ratio of a gravitational-wave background signal for this particular pulsar by 32%. We discuss the possible applications for this method of timing to study pulsar magnetospheres and further improve the sensitivity of searches for nanohertz gravitational waves.Comment: Accepted in MNRAS, 8 pages, 8 figure

The first GCT camera for the Cherenkov Telescope Array

The Gamma Cherenkov Telescope (GCT) is proposed to be part of the Small Size Telescope (SST) array of the Cherenkov Telescope Array (CTA). The GCT dual-mirror optical design allows the use of a compact camera of diameter roughly 0.4 m. The curved focal plane is equipped with 2048 pixels of ~0.2{\deg} angular size, resulting in a field of view of ~9{\deg}. The GCT camera is designed to record the flashes of Cherenkov light from electromagnetic cascades, which last only a few tens of nanoseconds. Modules based on custom ASICs provide the required fast electronics, facilitating sampling and digitisation as well as first level of triggering. The first GCT camera prototype is currently being commissioned in the UK. On-telescope tests are planned later this year. Here we give a detailed description of the camera prototype and present recent progress with testing and commissioning.Comment: In Proceedings of the 34th International Cosmic Ray Conference (ICRC2015), The Hague, The Netherlands. All CTA contributions at arXiv:1508.0589

Incidence of community-acquired lower respiratory tract infections and pneumonia among older adults in the United Kingdom: a population-based study.

Community-acquired lower respiratory tract infections (LRTI) and pneumonia (CAP) are common causes of morbidity and mortality among those aged ≥65 years; a growing population in many countries. Detailed incidence estimates for these infections among older adults in the United Kingdom (UK) are lacking. We used electronic general practice records from the Clinical Practice Research Data link, linked to Hospital Episode Statistics inpatient data, to estimate incidence of community-acquired LRTI and CAP among UK older adults between April 1997-March 2011, by age, sex, region and deprivation quintile. Levels of antibiotic prescribing were also assessed. LRTI incidence increased with fluctuations over time, was higher in men than women aged ≥70 and increased with age from 92.21 episodes/1000 person-years (65-69 years) to 187.91/1000 (85-89 years). CAP incidence increased more markedly with age, from 2.81 to 21.81 episodes/1000 person-years respectively, and was higher among men. For both infection groups, increases over time were attenuated after age-standardisation, indicating that these rises were largely due to population aging. Rates among those in the most deprived quintile were around 70% higher than the least deprived and were generally higher in the North of England. GP antibiotic prescribing rates were high for LRTI but lower for CAP (mostly due to immediate hospitalisation). This is the first study to provide long-term detailed incidence estimates of community-acquired LRTI and CAP in UK older individuals, taking person-time at risk into account. The summary incidence commonly presented for the ≥65 age group considerably underestimates LRTI/CAP rates, particularly among older individuals within this group. Our methodology and findings are likely to be highly relevant to health planners and researchers in other countries with aging populations

Ultra-Early Differential Diagnosis of Acute Cerebral Ischemia and Hemorrhagic Stroke by Measuring the Prehospital Release Rate of GFAP

BACKGROUND: Plasma glial fibrillary acidic protein (GFAP) and tau are promising markers for differentiating acute cerebral ischemia (ACI) and hemorrhagic stroke (HS), but their prehospital dynamics and usefulness are unknown. METHODS: We performed ultra-sensitivite single-molecule array (Simoa((R))) measurements of plasma GFAP and total tau in a stroke code patient cohort with cardinal stroke symptoms [National Institutes of Health Stroke Scale (NIHSS) >= 3]. Sequential sampling included 2 ultra-early samples, and a follow-up sample on the next morning. RESULTS: We included 272 cases (203 ACI, 60 HS, and 9 stroke mimics). Median (IQR) last-known-well to sampling time was 53 (35-90) minutes for initial prehospital samples, 90 (67-130) minutes for secondary acute samples, and 21 (16-24) hours for next morning samples. Plasma GFAP was significantly higher in patients with HS than ACI (P410pg/mL, or prehospital GFAP 90-410pg/mL together with GFAP release >0.6pg/mL/minute) enabled ruling out HS with high certainty (NPV 98.4%) in 68% of patients with ACI (sensitivity for HS 96.6%, specificity 68%, PPV 50%). CONCLUSIONS: In comparison to single-point measurement, monitoring the prehospital GFAP release rate improves ultra-early differentiation of stroke subtypes. With serial measurement GFAP has potential to improve future prehospital stroke diagnostics.Peer reviewe