Sequential Array Cytometry: Multi-Parameter Imaging with a Single Fluorescent Channel

Heterogeneity within the human population and within diseased tissues necessitates a personalized medicine approach to diagnostics and the treatment of diseases. Functional assays at the single-cell level can contribute to uncovering heterogeneity and ultimately assist in improved treatment decisions based on the presence of outlier cells. We aim to develop a platform for high-throughput, single-cell-based assays using well-characterized hydrodynamic cell isolation arrays which allow for precise cell and fluid handling. Here, we demonstrate the ability to extract spatial and temporal information about several intracellular components using a single fluorescent channel, eliminating the problem of overlapping fluorescence emission spectra. Integrated with imaging technologies such as wide field-of-view lens-free fluorescent imaging, fiber-optic array scanning technology, and microlens arrays, use of a single fluorescent channel will reduce the cost of reagents and optical components. Specifically, we sequentially stain hydrodynamically trapped cells with three biochemical labels all sharing the same fluorescence excitation and emission spectrum. These markers allow us to analyze the amount of DNA, and compare nucleus-to-cytoplasm ratio, as well as glycosylation of surface proteins. By imaging cells in real-time we enable measurements of temporal localization of cellular components and intracellular reaction kinetics, the latter is used as a measurement of multi-drug resistance. Demonstrating the efficacy of this single-cell analysis platform is the first step in designing and implementing more complete assays, aimed toward improving diagnosis and personalized treatments to complex diseases

p53 Target Gene SMAR1 Is Dysregulated in Breast Cancer: Its Role in Cancer Cell Migration and Invasion

Tumor suppressor SMAR1 interacts and stabilizes p53 through phosphorylation at its serine-15 residue. We show that SMAR1 transcription is regulated by p53 through its response element present in the SMAR1 promoter. Upon Doxorubicin induced DNA damage, acetylated p53 is recruited on SMAR1 promoter that allows activation of its transcription. Once SMAR1 is induced, cell cycle arrest is observed that is correlated to increased phospho-ser-15-p53 and decreased p53 acetylation. Further we demonstrate that SMAR1 expression is drastically reduced during advancement of human breast cancer. This was correlated with defective p53 expression in breast cancer where acetylated p53 is sequestered into the heterochromatin region and become inaccessible to activate SMAR1 promoter. In a recent report we have shown that SMAR1 represses Cyclin D1 transcription through recruitment of HDAC1 dependent repressor complex at the MAR site of Cyclin D1 promoter. Here we show that downmodulation of SMAR1 in high grade breast carcinoma is correlated with upregulated Cyclin D1 expression. We also established that SMAR1 inhibits tumor cell migration and metastases through inhibition of TGFβ signaling and its downstream target genes including cutl1 and various focal adhesion molecules. Thus, we report that SMAR1 plays a central role in coordinating p53 and TGFβ pathways in human breast cancer

Effective Rheology of Bubbles Moving in a Capillary Tube

We calculate the average volumetric flux versus pressure drop of bubbles moving in a single capillary tube with varying diameter, finding a square-root relation from mapping the flow equations onto that of a driven overdamped pendulum. The calculation is based on a derivation of the equation of motion of a bubble train from considering the capillary forces and the entropy production associated with the viscous flow. We also calculate the configurational probability of the positions of the bubbles.Comment: 4 pages, 1 figur

The interleukin (IL)-31/IL-31R axis contributes to tumor growth in human follicular lymphoma

Interleukin (IL)-31A binds to an heterodimer composed of IL-31 receptor A (IL-31RA) and Oncostatin M Receptor (OSMR). The IL-31/ IL-31R complex is involved in the pathogenesis of various skin diseases, including cutaneous T-cell lymphoma. No information is available on the relations between the IL-31/IL-31R complex and B-cell lymphoma. Here we have addressed this issue in follicular lymphoma (FL), a prototypic germinal center(GC)-derived B-cell malignancy. IL-31 enhanced primary FL cell proliferation through IL-31R-driven signal transducer and activator of transcription factor 1/3 (STAT1/3), extracellular signal–regulated kinase 1/2 (ERK1/2) and Akt phosphorylation. In contrast, GC B cells did not signal to IL-31 in spite of IL-31R expression. GC B cells expressed predominantly the inhibitory short IL-31RA isoform, whereas FL cells expressed predominantly the long signaling isoform. Moreover, GC B cells lacked expression of other IL-31RA isoforms potentially involved in the signaling pathway. IL-31 protein expression was significantly higher in surface membrane than in cytosol of both FL and GC B cells. IL-31 was detected in plasma membrane microvesicles from both cell types but not released in soluble form in culture supernatants. IL-31 and IL-31RA expression was higher in lymph nodes from FL patients with grade IIIa compared with grade I/II, suggesting a paracrine and/or autocrine role of IL-31/IL-31RA complex in tumor progression through microvesicle shedding

Superoxide Dismutase 1 and tgSOD1G93A Mouse Spinal Cord Seed Fibrils, Suggesting a Propagative Cell Death Mechanism in Amyotrophic Lateral Sclerosis

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that specifically affects motor neurons and leads to a progressive and ultimately fatal loss of function, resulting in death typically within 3 to 5 years of diagnosis. The disease starts with a focal centre of weakness, such as one limb, and appears to spread to other parts of the body. Mutations in superoxide dismutase 1 (SOD1) are known to cause disease and it is generally accepted they lead to pathology not by loss of enzymatic activity but by gain of some unknown toxic function(s). Although different mutations lead to varying tendencies of SOD1 to aggregate, we suggest abnormal proteins share a common misfolding pathway that leads to the formation of amyloid fibrils.Methodology/Principal Findings: Here we demonstrate that misfolding of superoxide dismutase 1 leads to the formation of amyloid fibrils associated with seeding activity, which can accelerate the formation of new fibrils in an autocatalytic cascade. The time limiting event is nucleation to form a stable protein "seed" before a rapid linear polymerisation results in amyloid fibrils analogous to other protein misfolding disorders. This phenomenon was not confined to fibrils of recombinant protein as here we show, for the first time, that spinal cord homogenates obtained from a transgenic mouse model that overexpresses mutant human superoxide dismutase 1 (the TgSOD1(G93A) mouse) also contain amyloid seeds that accelerate the formation of new fibrils in both wildtype and mutant SOD1 protein in vitro.Conclusions/Significance: These findings provide new insights into ALS disease mechanism and in particular a mechanism that could account for the spread of pathology throughout the nervous system. This model of disease spread, which has analogies to other protein misfolding disorders such as prion disease, also suggests it may be possible to design assays for therapeutics that can inhibit fibril propagation and hence, possibly, disease progression

PET Imaging of Soluble Yttrium-86-Labeled Carbon Nanotubes in Mice

The potential medical applications of nanomaterials are shaping the landscape of the nanobiotechnology field and driving it forward. A key factor in determining the suitability of these nanomaterials must be how they interface with biological systems. Single walled carbon nanotubes (CNT) are being investigated as platforms for the delivery of biological, radiological, and chemical payloads to target tissues. CNT are mechanically robust graphene cylinders comprised of sp(2)-bonded carbon atoms and possessing highly regular structures with defined periodicity. CNT exhibit unique mechanochemical properties that can be exploited for the development of novel drug delivery platforms. In order to evaluate the potential usefulness of this CNT scaffold, we undertook an imaging study to determine the tissue biodistribution and pharmacokinetics of prototypical DOTA-functionalized CNT labeled with yttrium-86 and indium-111 ((86)Y-CNT and (111)In-CNT, respectively) in a mouse model.The (86)Y-CNT construct was synthesized from amine-functionalized, water-soluble CNT by covalently attaching multiple copies of DOTA chelates and then radiolabeling with the positron-emitting metal-ion, yttrium-86. A gamma-emitting (111)In-CNT construct was similarly prepared and purified. The constructs were characterized spectroscopically, microscopically, and chromatographically. The whole-body distribution and clearance of yttrium-86 was characterized at 3 and 24 hours post-injection using positron emission tomography (PET). The yttrium-86 cleared the blood within 3 hours and distributed predominantly to the kidneys, liver, spleen and bone. Although the activity that accumulated in the kidney cleared with time, the whole-body clearance was slow. Differential uptake in these target tissues was observed following intravenous or intraperitoneal injection.The whole-body PET images indicated that the major sites of accumulation of activity resulting from the administration of (86)Y-CNT were the kidney, liver, spleen, and to a much less extent the bone. Blood clearance was rapid and could be beneficial in the use of short-lived radionuclides in diagnostic applications

Molecular cytogenetics (FISH, GISH) of Coccinia grandis: A ca. 3 myr-old species of Cucurbitaceae with the largest Y/autosome divergence in flowering plants

The independent evolution of heteromorphic sex chromosomes in 19 species from 4 families of flowering plants permits studying X/Y divergence after the initial recombination suppression. Here, we document autosome/Y divergence in the tropical Cucurbitaceae Coccinia grandis, which is ca. 3 myr old. Karyotyping and C-value measurements show that the C. grandis Y chromosome has twice the size of any of the other chromosomes, with a male/female C-value difference of 0.094 pg or 10% of the total genome. FISH staining revealed 5S and 45S rDNA sites on autosomes but not on the Y chromosome, making it unlikely that rDNA contributed to the elongation of the Y chromosome; recent end-to-end fusion also seems unlikely given the lack of interstitial telomeric signals. GISH with different concentrations of female blocking DNA detected a possible pseudo-autosomal region on the Y chromosome, and C-banding suggests that the entire Y chromosome in C. grandis is heterochromatic. During meiosis, there is an end-to-end connection between the X and the Y chromosome, but the X does not otherwise differ from the remaining chromosomes. These findings and a review of plants with heteromorphic sex chromosomes reveal no relationship between species age and degree of sex chromosome dimorphism. Its relatively small genome size (0.943 pg/2C in males), large Y chromosome, and phylogenetic proximity to the fully sequenced Cucumis sativus make C. grandis a promising model to study sex chromosome evolution. Copyright © 2012 S. Karger AG, Base

Alignment of the ALICE Inner Tracking System with cosmic-ray tracks

37 pages, 15 figures, revised version, accepted by JINSTALICE (A Large Ion Collider Experiment) is the LHC (Large Hadron Collider) experiment devoted to investigating the strongly interacting matter created in nucleus-nucleus collisions at the LHC energies. The ALICE ITS, Inner Tracking System, consists of six cylindrical layers of silicon detectors with three different technologies; in the outward direction: two layers of pixel detectors, two layers each of drift, and strip detectors. The number of parameters to be determined in the spatial alignment of the 2198 sensor modules of the ITS is about 13,000. The target alignment precision is well below 10 micron in some cases (pixels). The sources of alignment information include survey measurements, and the reconstructed tracks from cosmic rays and from proton-proton collisions. The main track-based alignment method uses the Millepede global approach. An iterative local method was developed and used as well. We present the results obtained for the ITS alignment using about 10^5 charged tracks from cosmic rays that have been collected during summer 2008, with the ALICE solenoidal magnet switched off.Peer reviewe