An optimized multiplex flow cytometry protocol for the analysis of intracellular signaling in peripheral blood mononuclear cells

AbstractPhosphoflow cytometry is increasingly being used as a tool for the discovery of biomarkers used in the treatment and monitoring of disease and therapy. The ability to measure numerous phospho-protein targets simultaneously at a single cell level accurately and rapidly provides significant advantages over other methods. We here discuss important considerations required to successfully implement these methods. Three different blood collection tubes (lithium-heparin tubes, CPT with sodium citrate and CPT with sodium heparin) were evaluated, with PBMC isolated through lithium-heparin tubes/lymphoprep displaying reduced basal and increased stimulation induced phosphorylation compared to the other two methods. Further, we provide a protocol outlining an 8 color assay developed for the study of intracellular signaling in peripheral blood mononuclear cells. The assay allows for the quantitative measurement of the phospho-proteins ERK1/2, NF-κB p65, Stat1 (Y701), Stat1 (S727), Stat3 (Y705), Stat3 (S727), Stat4 (Y693), p38 and Stat5 (Y694), as well as the identification of T cells, B cells, natural killer cells and monocytes. The assay additionally incorporates fluorescent cell barcoding, reducing assay costs and increasing throughput while increasing data robustness. Inter-assay precision was assessed over a month long period for all experimental variables (phospho-protein measured, cell type and stimulant). Coefficient of variations (CVs) calculated from process triplicates of normalized median fluorescence intensity (MFI) of the phospho-proteins displayed median CVs under 10% when grouped according to cell type, stimulation agent and phospho-protein measured, while the CV for each triplicate did not exceed 20%

Deterministic enhancement of coherent photon generation from a nitrogen-vacancy center in ultrapure diamond

The nitrogen-vacancy (NV) center in diamond has an optically addressable, highly coherent spin. However, an NV center even in high quality single-crystalline material is a very poor source of single photons: extraction out of the high-index diamond is inefficient, the emission of coherent photons represents just a few per cent of the total emission, and the decay time is large. In principle, all three problems can be addressed with a resonant microcavity. In practice, it has proved difficult to implement this concept: photonic engineering hinges on nano-fabrication yet it is notoriously difficult to process diamond without degrading the NV centers. We present here a microcavity scheme which uses minimally processed diamond, thereby preserving the high quality of the starting material, and a tunable microcavity platform. We demonstrate a clear change in the lifetime for multiple individual NV centers on tuning both the cavity frequency and anti-node position, a Purcell effect. The overall Purcell factor $F_{\rm P}=2.0$ translates to a Purcell factor for the zero phonon line (ZPL) of $F_{\rm P}^{\rm ZPL}\sim30$ and an increase in the ZPL emission probability from $\sim 3 \%$ to $\sim 46 \%$. By making a step-change in the NV's optical properties in a deterministic way, these results pave the way for much enhanced spin-photon and spin-spin entanglement rates.Comment: 6 pages, 4 figure

Distribution of the North American Porcupine (Erethizon dorsatum) in Northern California

Western Wildlife 4:17–28, 2017: The North American Porcupine (Erethizon dorsatum) is one of the most widely distributed mammals in North America, but recent reports have suggested declines in parts of its range in the West. In California, little is known about the historical or current status of the porcupine, and maps of its distribution conflict considerably. Nevertheless, the species is of interest to natural resource managers. For much of the 1900s, foresters and others primarily treated porcupines as pests because of the undesirable damage they inflict feeding on trees and gnawing on manmade items in search of salt. More recently, porcupines have been recognized for their role in promoting forest structure and diversity, and as potential prey for the Fisher (Pekania pennanti). We collected records of porcupine occurrence in the northern part of California since the beginning of the 20th Century, relying on government and private databases, reports from the public, and other sources. These records confirm that porcupines may occur in most major regions and habitat types across northern California, in contrast to many published range maps. The contemporary distribution of porcupines in the state most closely resembles the California Wildlife Habitat Relationships System (CWHR) range map, which is based on projections of suitable habitat. We are unable to offer deeper insight into trends of abundance and possible changes in distribution because these records are likely spatiotemporally correlated with observer effort. This work is a first step and we recommend that a broader statewide effort be conducted to better understand the distribution, abundance, and ecology of North American Porcupines in California. See below or click here to view data

Tobacco, hypertension, and vascular disease: Risk factors for renal functional decline in an older population

Tobacco, hypertension, and vascular disease: Risk factors for renal functional decline in an older population.BackgroundA decline in renal function with age has been noted in some but not all individuals. The purpose of this study was to identify risk factors associated with a clinically significant increase in serum creatinine (of at least 0.3 mg/dL) in an older nondiabetic population.MethodsA retrospective case-control study was performed analyzing data obtained from 4142 nondiabetic participants of the Cardiovascular Health Study Cohort, all at least 65 years of age, who had two measurements of serum creatinine performed at least three years apart. Cases were identified as participants who developed an increase in serum creatinine of at least 0.3 mg/dL, with controls including participants who did not sustain such an increase.ResultsThere was an increase in the serum creatinine of at least 0.3 mg/dL in 2.8% of the population. In a multivariate “best-fit” model adjusted for gender, weight, black race, baseline serum creatinine, and age, the following factors were associated with an increase in serum creatinine: number of cigarettes smoked per day, systolic blood pressure, and maximum internal carotid artery intimal thickness.ConclusionsThese data suggest that three very preventable or treatable conditions—hypertension, smoking, and prevalent vascular disease, which are associated with large and small vessel disease—are highly associated with clinically important changes in renal function in an older population

The Mathematics of a Successful Deconvolution: A Quantitative Assessment of Mixture-Based Combinatorial Libraries Screened Against Two Formylpeptide Receptors

In the past 20 years, synthetic combinatorial methods have fundamentally advanced the ability to synthesize and screen large numbers of compounds for drug discovery and basic research. Mixture-based libraries and positional scanning deconvolution combine two approaches for the rapid identification of specific scaffolds and active ligands. Here we present a quantitative assessment of the screening of 32 positional scanning libraries in the identification of highly specific and selective ligands for two formylpeptide receptors. We also compare and contrast two mixture-based library approaches using a mathematical model to facilitate the selection of active scaffolds and libraries to be pursued for further evaluation. The flexibility demonstrated in the differently formatted mixture-based libraries allows for their screening in a wide range of assays

Impaired activation of STAT5 upon IL-2 stimulation in Tregs and elevated sIL-2R in Sjögren’s syndrome

Background Interleukin-2 (IL-2) and the high-affinity IL-2 receptor (IL-2R) are essential for the survival of regulatory T cells (Tregs) which are the main players in immune tolerance and prevention of autoimmune diseases. Sjögren’s syndrome (SS) is a chronic autoimmune disease predominantly affecting women and is characterised by sicca symptoms including oral and ocular dryness. The aim of this study was to investigate an association between IL-2R and Treg function in patients with SS of different severity defined by the salivary flow rate. Methods In a cross-sectional study, we determined plasma soluble IL-2R (sIL-2R) levels in women with SS (n=97) and healthy females (n=50) using ELISA. A subset of those (n=51) was screened for Treg function measured by the STAT5 signalling response to IL-2 using phospho-flow cytometry. Results We found that elevated plasma levels of sIL-2R were positively associated with the severity of SS reflected by a pathologically low salivary flow. Phospho-flow analysis revealed that patients with SS have a significantly lower frequency of pSTAT5+ Tregs upon IL-2 stimulation compared with healthy individuals, while the frequency of Tregs and pSTAT5 in conventional T cells remained unchanged. In addition, we observed more pSTAT5+ Tregs at baseline in patients with SS, which is significantly associated with seropositivity and elevated sIL-2R. Conclusions Our data indicates that Tregs have a weakened immunosuppressive function in patients with SS due to impaired IL-2/IL-2R signalling capacity. This could mediate lymphocytic infiltration into salivary glands inducing sicca symptoms. We believe that sIL-2R could act as a useful indicator for SS and disease severity.publishedVersio

Phase diagram of the chromatic polynomial on a torus

We study the zero-temperature partition function of the Potts antiferromagnet (i.e., the chromatic polynomial) on a torus using a transfer-matrix approach. We consider square- and triangular-lattice strips with fixed width L, arbitrary length N, and fully periodic boundary conditions. On the mathematical side, we obtain exact expressions for the chromatic polynomial of widths L=5,6,7 for the square and triangular lattices. On the physical side, we obtain the exact ``phase diagrams'' for these strips of width L and infinite length, and from these results we extract useful information about the infinite-volume phase diagram of this model: in particular, the number and position of the different phases.Comment: 72 pages (LaTeX2e). Includes tex file, three sty files, and 26 Postscript figures. Also included are Mathematica files transfer6_sq.m and transfer6_tri.m. Final version to appear in Nucl. Phys.

Is the five-flow conjecture almost false?

The number of nowhere zero Z_Q flows on a graph G can be shown to be a polynomial in Q, defining the flow polynomial \Phi_G(Q). According to Tutte's five-flow conjecture, \Phi_G(5) > 0 for any bridgeless G.A conjecture by Welsh that \Phi_G(Q) has no real roots for Q \in (4,\infty) was recently disproved by Haggard, Pearce and Royle. These authors conjectured the absence of roots for Q \in [5,\infty). We study the real roots of \Phi_G(Q) for a family of non-planar cubic graphs known as generalised Petersen graphs G(m,k). We show that the modified conjecture on real flow roots is also false, by exhibiting infinitely many real flow roots Q>5 within the class G(nk,k). In particular, we compute explicitly the flow polynomial of G(119,7), showing that it has real roots at Q\approx 5.0000197675 and Q\approx 5.1653424423. We moreover prove that the graph families G(6n,6) and G(7n,7) possess real flow roots that accumulate at Q=5 as n\to\infty (in the latter case from above and below); and that Q_c(7)\approx 5.2352605291 is an accumulation point of real zeros of the flow polynomials for G(7n,7) as n\to\infty.Comment: 44 pages (LaTeX2e). Includes tex file, three sty files, and a mathematica script polyG119_7.m. Many improvements from version 3, in particular Sections 3 and 4 have been mostly re-writen, and Sections 7 and 8 have been eliminated. (This material can now be found in arXiv:1303.5210.) Final version published in J. Combin. Theory