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TPG Credit Arm Chasing More Spotify-Like Deals With Goldman Hire
(Bloomberg) -- TPG Sixth Street Partners, the $21 billion credit arm of TPG, has tapped Mike McGinn to expand its business tied to financing growing companies, according to a person with knowledge of the matter.
McGinn was one of the co-heads of the private-capital investing team at Goldman Sachs Group Inc. and will help lead a similar group at TSSP along with Bo Stanley, the person said, asking not to be identified as the information isn’t public. The TPG unit was part of an investment team that helped music-streaming service Spotify Ltd. raise $1 billion in convertible debt in 2016.
A representative for TSSP declined to comment.
TSSP along with another unit within TPG and Dragoneer Investment Group participated in the Spotify deal in March 2016. The investors converted a portion of the Spotify debt deal into equity and sold the shares to Chinese internet giant Tencent Holdings Ltd., according to a regulatory filing. The rest of the debt was converted to shares, the filing shows.
TSSP was formed in 2009 and is led by former Goldman partner Alan Waxman. It accounts for more than a quarter of TPG’s assets under management. Waxman had previously led Goldman’s special situations group, which houses the private-capital investing arm.
Waxman’s credit platform is also seeking to raise more than $8 billion for funds to pursue direct-lending deals and other credit opportunities, according to a memo seen by Bloomberg. One of the funds is a so-called contingent vehicle that would deploy capital when there’s an uptick in market turbulence leading to new distressed opportunities.
The financing for growth companies would include deals across the capital structure, from equity to debt deals. TSSP was also one of the lead backers for a $400 million capital raise by data-center provider AirTrunk.
©2018 Bloomberg L.P.


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- Vrbo Property
- Beautiful 1 Bedroom Unit at Resort Sleeps 4
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domain: travel


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Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the β2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both β2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.
26 Related JoVE Articles!
Detection of Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation
Institutions: Queen's University - Kingston, Ontario.
Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2
. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3
, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4
. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR Gαi proteins and MMP-9 5
. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl α-2,3-linked β-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6
. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.
Cellular Biology, Issue 43, Neu1 sialidase, TOLL-like receptors, macrophages, sialidase substrate, fluorescence microscopy, cell signaling, receptor activation
High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles
Institutions: Iowa State University, Iowa State University.
Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1
. Several biodegradable polymers have been studied as vaccine delivery vehicles1
; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13
C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15
. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10
. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11
and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a
Bioengineering, Issue 65, Chemical Engineering, High-throughput, Automation, Carbohydrates, Synthesis, Polyanhydrides, Nanoparticles, Functionalization, Targeting, Fluorous Solid Phase Extraction
Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro
Institutions: University of Rochester.
The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1
. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2
. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3
. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro
system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 μm/min. T lymphocyte migration can be inhibited by integrin blockade 1
or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2
Immunology, Issue 40, T lymphocyte, Migration, Integrin, LFA-1, ICAM-1, Chemokine
In Vivo Modeling of the Morbid Human Genome using Danio rerio
Institutions: Duke University Medical Center, Duke University, Duke University Medical Center.
Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo
complementation in zebrafish. Zebrafish (Danio rerio
) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo,
and can be genetically manipulated.1
These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.
Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model
Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
Institutions: Institut Pasteur .
RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3
. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7
Immunology, Issue 52, Polymerase fidelity, RNA virus, mutation frequency, mutagen, RNA polymerase, viral evolution
gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair
Institutions: Centre Georges-François Leclerc.
The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM
, and TP53
) from promoter to 3'-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.
Genetics, Issue 92, gDNA enrichment, Nextera, NGS, DNA damage, BRCA1, BRCA2
Infinium Assay for Large-scale SNP Genotyping Applications
Institutions: Oklahoma Medical Research Foundation.
Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina's panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.
Basic Protocol, Issue 81, genomics, SNP, Genotyping, Infinium, iScan, HiScan, Illumina
Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models
Institutions: Perelman School of Medicine at the University of Pennsylvania.
Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae
. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and α-synuclein, implicated in Parkinson’s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and α-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.
Microbiology, Issue 93, Protein-misfolding disorders, yeast proteinopathy models, Hsp104, proteotoxicity, amyloid, disaggregation
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER
Institutions: Washington University School of Medicine, Washington University School of Medicine, Washington University School of Medicine.
As DNA sequencing technology has markedly advanced in recent years2
, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3
. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5
. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8
. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9
and a novel software package1
for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (https://www.ibridgenetwork.org/wustl/splinter). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.
Genetics, Issue 64, Genomics, Cancer Biology, Bioinformatics, Pooled DNA sequencing, SPLINTER, rare genetic variants, genetic screening, phenotype, high throughput, computational analysis, DNA, PCR, primers
Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease
Institutions: SRI International, University of California-Santa Cruz.
Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer's disease, but its effects in Parkinson's disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone. To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e.
5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder. Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD. The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates. This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.
Medicine, Issue 83, MPTP, dopamine, Iba1, TH, GFAP, lipoxygenase, transgenic, gene-environment interactions, mouse, Parkinson's disease, neurodegeneration, neuroinflammation
Isolation of Mouse Lung Dendritic Cells
Institutions: Louisiana State University .
Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2
as well as in the control of tolerogenic responses 3
in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4
, plasmacytoid DC (pDC) 5
and the IFN-producing killer DC (IKDC) 6,7
. The cDC subset is the most prominent DC subset in the lung 8
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (β2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9
. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+
cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4/80, an splenic macrophage marker, the CD11chi
lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.
Immunology, Issue 57, Lung, dendritic cells, classical, conventional, isolation, mouse, innate immunity, pulmonary
Cecal Ligation Puncture Procedure
Institutions: Temple University , Temple University .
Human sepsis is characterized by a set of systemic reactions in response to intensive and massive infection that failed to be locally contained by the host. Currently, sepsis ranks among the top ten causes of mortality in the USA intensive care units 1
. During sepsis there are two established haemodynamic phases that may overlap. The initial phase (hyperdynamic) is defined as a massive production of proinflammatory cytokines and reactive oxygen species by macrophages and neutrophils that affects vascular permeability (leading to hypotension), cardiac function and induces metabolic changes culminating in tissue necrosis and organ failure. Consequently, the most common cause of mortality is acute kidney injury. The second phase (hypodynamic) is an anti-inflammatory process involving altered monocyte antigen presentation, decreased lymphocyte proliferation and function and increased apoptosis. This state known as immunosuppression or immune depression sharply increases the risk of nocosomial infections and ultimately, death. The mechanisms of these pathophysiological processes are not well characterized. Because both phases of sepsis may cause irreversible and irreparable damage, it is essential to determine the immunological and physiological status of the patient. This is the main reason why many therapeutic drugs have failed. The same drug given at different stages of sepsis may be therapeutic or otherwise harmful or have no effect 2,3
. To understand sepsis at various levels it is crucial to have a suitable and comprehensive animal model that reproduces the clinical course of the disease. It is important to characterize the pathophysiological mechanisms occurring during sepsis and control the model conditions for testing potential therapeutic agents.
To study the etiology of human sepsis researchers have developed different animal models. The most widely used clinical model is cecal ligation and puncture (CLP). The CLP model consists of the perforation of the cecum allowing the release of fecal material into the peritoneal cavity to generate an exacerbated immune response induced by polymicrobial infection. This model fulfills the human condition that is clinically relevant. As in humans, mice that undergo CLP with fluid resuscitation show the first (early) hyperdynamic phase that in time progresses to the second (late) hypodynamic phase. In addition, the cytokine profile is similar to that seen in human sepsis where there is increased lymphocyte apoptosis (reviewed in 4,5
). Due to the multiple and overlapping mechanisms involved in sepsis, researchers need a suitable sepsis model of controlled severity in order to obtain consistent and reproducible results.
Medicine, Issue 51, sepsis, systemic inflammation, infection, septic shock, animal model
Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure
Institutions: Monash University.
Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2
and heart failure4
. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5
. In vitro
methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro
studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6
however, similar to many in vitro
assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo
assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9
. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo
studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10
. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo
vessel chamber perfusion model9
which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11
as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.
Immunology, Issue 54, Leukocyte adhesion, intraluminal pressure, endothelial dysfunction, inflammation, hypertension
Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells
Institutions: University of Michigan Comprehensive Cancer Center, University of Michigan Comprehensive Cancer Center.
Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1
. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex vivo
. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4
. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3
. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7
. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.
Medicine, Issue 86, pathological conditions, signs and symptoms, neoplasms, three dimensional cultures, Matrigel, breast cells, malignant phenotype, signaling
A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites
Institutions: Boston University School of Medicine, Boston University School of Medicine.
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis
, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis
accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
Immunology, Issue 90,
Pathogen-Induced Chronic Inflammation; Porphyromonas gingivalis; Oral Bone Loss; Periodontal Disease; Atherosclerosis; Chronic Inflammation; Host-Pathogen Interaction; microCT; MRI
Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons
Institutions: Oregon Health & Science University, Oregon Health & Science University.
Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+
concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.
Neuroscience, Issue 82, Synaptic Transmission, Action Potentials, Circadian Rhythm, Excitatory Postsynaptic Potentials, Life Sciences (General), circadian rhythm, suprachiasmatic nucleus, membrane potential, patch clamp recording, fluorescent probe, intracellular calcium
Tangential Flow Ultrafiltration: A “Green” Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles
Institutions: Wright State University, Wright State University.
Nowadays, AgNPs are extensively used in the manufacture of consumer products,1
and biomedical devices4
due to their powerful antimicrobial properties.3-6
These nanoparticle applications are strongly influenced by the AgNP size and aggregation state. Many challenges exist in the controlled fabrication7
and size-based isolation4,8
of unfunctionalized, homogenous AgNPs that are free from chemically aggressive capping/stabilizing agents or organic solvents.7-13
Limitations emerge from the toxicity of reagents, high costs or reduced efficiency of the AgNP synthesis or isolation methods (e.g.
, centrifugation, size-dependent solubility, size-exclusion chromatography, etc.).10,14-18
To overcome this, we recently showed that TFU permits greater control over the size, concentration and aggregation state of Creighton AgNPs (300 ml of 15.3 μg ml-1
down to 10 ml of 198.7 μg ml-1
) than conventional methods of isolation such as ultracentrifugation.19
TFU is a recirculation method commonly used for the weight-based isolation of proteins, viruses and cells.20,21
Briefly, the liquid sample is passed through a series of hollow fiber membranes with pore size ranging from 1,000 kD to 10 kD. Smaller suspended or dissolved constituents in the sample will pass through the porous barrier together with the solvent (filtrate), while the larger constituents are retained (retentate). TFU may be considered a "green" method as it neither damages the sample nor requires additional solvent to eliminate toxic excess reagents and byproducts. Furthermore, TFU may be applied to a large variety of nanoparticles as both hydrophobic and hydrophilic filters are available.
The two main objectives of this study were: 1) to illustrate the experimental aspects of the TFU approach through an invited video experience and 2) to demonstrate the feasibility of the TFU method for larger volumes of colloidal nanoparticles and smaller volumes of retentate. First, unfuctionalized AgNPs (4 L, 15.2 μg ml-1
) were synthesized using the well-established Creighton method22,23
by the reduction of AgNO3
. AgNP polydispersity was then minimized via a 3-step TFU using a 50-nm filter (460 cm2
) to remove AgNPs and AgNP-aggregates larger than 50 nm, followed by two 100-kD (200 cm2
and 20 cm2
) filters to concentrate the AgNPs. Representative samples were characterized using transmission electron microscopy, UV-Vis absorption spectrophotometry, Raman spectroscopy, and inductively coupled plasma optical emission spectroscopy. The final retentate consisted of highly concentrated (4 ml, 8,539.9 μg ml-1
) yet lowly aggregated and homogeneous AgNPs of 1-20 nm in diameter. This corresponds to a silver concentration yield of about 62%.
Chemistry, Issue 68, Biomedical Engineering, Chemical Engineering, Nanotechnology, silver nanoparticles, size selection, concentration, tangential flow ultrafiltration
Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
Institutions: University of Exeter.
A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.
Biophysics, Issue 91, differential scanning fluorimetry, dissociation constant, protein-ligand interactions, StepOne, cooperativity, WcbI.
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
Institutions: The University of Memphis.
In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2
). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2
. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4
, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7
. Compared to traditional studies of locomotor activity in vivo
and SCN explants ex vivo
, cell-based in vitro
assays allow for discovery of cell-autonomous circadian defects5,8
. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase
as a reporter has become a common technique for studying circadian rhythms in mammals14,15
, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17
or stable transduction5,10,18,19
. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20
. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2
reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.
Genetics, Issue 67, Molecular Biology, Cellular Biology, Chemical Biology, Circadian clock, firefly luciferase, real-time bioluminescence technology, cell-autonomous model, lentiviral vector, RNA interference (RNAi), high-throughput screening (HTS)
Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE
Institutions: University of Cambridge, UK, University of Cambridge, UK.
Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via
functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v
.) or intracerebroventricular (i.c.v.
) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.
) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo
Here we describe the methods that we have developed for the i.v
. and i.c.v.
delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.
Immunology, Issue 86, Somatic neural stem/precursor cells, neurodegenerative disorders, regenerative medicine, multiple sclerosis, experimental autoimmune encephalomyelitis, systemic delivery, intravenous, intracerebroventricular
DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat
Institutions: BC Children's Hospital.
Inflammatory Bowel Diseases (IBD), including Crohn's Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48 hr and rats fed SO by 72 hr. Weight gain resumed after 72 hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5 days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.
Medicine, Issue 84, Chemical colitis, Inflammatory Bowel Disease, intra rectal administration, intestinal inflammation, transmural inflammation, myeloperoxidase activity
Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method
Institutions: University of California, University of California, University of California, University of California, University of California, University of California.
Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck & Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47
), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.
Immunology, Issue 50, spinal cord injury, cellular inflammation, neuroinflammation, OptiPrep, central nervous system, neutrophils, macrophages, microglia, T-cells, flow cytometry
Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model
Institutions: University of Kentucky College of Medicine, University of Kentucky College of Medicine, University of Kentucky College of Medicine, University of Kentucky College of Medicine.
Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs
-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf
transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf
animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1
. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf
fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.
Medicine, Issue 79, Skin, Inflammation, Photometry, Ultraviolet Rays, Skin Pigmentation, melanocortin 1 receptor, Mc1r, forskolin, cAMP, mean erythematous dose, skin pigmentation, melanocyte, melanin, sunburn, UV, inflammation
Pyrosequencing: A Simple Method for Accurate Genotyping
Institutions: Washington University in St. Louis.
Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.
Cellular Biology, Issue 11, Springer Protocols, Pyrosequencing, genotype, polymorphism, SNP, pharmacogenetics, pharmacogenomics, PCR

domain: medical


text:
Dunn, Oslin named to Nashville Songwriters Hall
Tuesday, August 7, 2018
– Ronnie Dunn, K.T. Oslin, Byron Hill, Wayne Kirkpatrick and Joe Melson will be inducted into the Nashville Songwriters Hall of Fame in October, it was announced today.
The five new inductees will join the 208 existing members of the organization when they are officially inducted during the 48th Anniversary Nashville Songwriters Hall of Fame Gala on Oct. 28.
"This time of year, as board chair of the Nashville Songwriters Hall of Fame Foundation, I am always reminded of the broad variety and high quality of the songwriting talent we are so fortunate to be able to celebrate," said Pat Alger. "The musical trends might change through the years, but for us it always comes down to great songs and legendary songwriters - the bedrock of the town that continues to be hailed as Music City. This year's nominees for the Nashville Songwriters Hall of Fame were inspiring and impressive as always, each one deserving recognition for the impact they made. Today it's my great honor to welcome the Nashville Songwriters Hall of Fame class of 2018: Byron Hill and Wayne Kirkpatrick in the songwriter category; Joe Melson in the veteran songwriter category; Ronnie Dunn as our songwriter/artist and K.T. Oslin as our veteran songwriter/ artist."
Hill's songwriter credits include "Pickin' Up Strangers" (Johnny Lee), "Fool Hearted Memory" (George Strait) and "Nothing On But The Radio" (Gary Allan).
Kirkpatrick's resume is known for the Grammy-winning "Change The World" (Eric Clapton) and "Little White Church" (Little Big Town) and the Broadway musical Something Rotten!.
Melson is the co-writer of the Roy Orbison hits "Only The Lonely (Know The Way I Feel)," "Crying" and "Blue Bayou."
Dunn popularized many of his own compositions, including the Brooks & Dunn hits "Neon Moon," "Boot Scootin' Boogie" and "Believe."
Oslin recorded many of her self-penned hits, including "80s Ladies," "Hold Me" and "Come Next Monday."
More news for Ronnie Dunn
Editorial: Walking the talk
When names like Hank Williams, Johnny Cash, Waylon and the Hag are invoked, you're talking hard core country. These are the touchstones of country , the guys who made country music what it was and still is (or maybe can be). When these folks would sing about being down-and-out and the rough-and-tumble, they knew of what they were singing about. Fast forward a few years to the country singers of today. »»»
Concert Review: McLean shows far more than two good songs
If you believe one overheard remark, that a Don McLean concert features only two good songs ("American Pie," "Vincent"), you would have missed a show that strongly contradicts such an assumption. McLean's performance offered an enjoyable mixture of career highlights and favorite oldies and is by no means a two-hit wonder.... »»»
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Elsewhere in the news
Currently at the CST blogs
The Long Ryders have come a long way since they were initially associated with other Los Angeles relatively retro acts collected under the Paisley Underground umbrella. Even back during the mid to late '80s,... »»»
Suffice it to say that the past has always loomed large throughout Chip Taylor's career. That's all the more obvious if only for the fact that Taylor wrote some of the biggest pop hits of the '60s, "Wild Thing"... »»»
Dan Tyminski (known simply as "Tyminski" on his 2017 release "Southern Gothic") has traditional music roots and unassailable bluegrass street cred especially given his membership in Alison Krauss' Union Station. He is also a powerful songwriter and has been writing songs for himself and others for years now.... »»»
Love and Revelation
Some might say all you need to know about Over The Rhine is its primary lead vocalist, Karin Bergquist. And that's true; her angelic singing - alone throughout "Love & Revelation" - makes the album well worth your ear time. »»»
Maren Morris, who had a huge pop hit with "The Middle," is never going to be anybody's ideal traditional country singer. Although, thankfully, there isn't anything that mind-numbingly beat-driven on her "GIRL" album, »»»
As a genre, Americana has become a massive umbrella, and yet, the actual definition remains elusive at best. Still, for those that relate to its former manifestation as "roots rock" or "country crossover," the Vandoliers come remarkably close to identifying with a definitive sound. »»»

domain: artistic


Welcome to the SP Z-RACE Collection. Here you will find racing inspired apparel derived from our signature series. This collection represents the brand! All purchases and earnings are 100% invested directly back into the YouTube Channel. Thank you for being part of the community, and for helping us continue to share what these amazing performance cars are all about!If you're looking for a top-quality, instant-favorite sweatshirt, you've come to the right place! Our Premium Hoodie is everything you could ask for: it's warm and cozy, heavyweight and roomy, and built to last.
- 80% cotton / 20% polyester (heather gray is 85% cotton / 15% rayon, heather denim and charcoal gray are 60% cotton, 40% polyester)| Fabric weight: 8.3 oz (midweight)
- Contrasting, complementary-colored drawstring and inner necktape
- Stitched slit inside kangaroo-style pocket and a small loop inside the hood for headphone usage
- Reinforced cuffs and waist
- Combed-cotton exterior, fuzzy fleece interior
- Imported garment, processed and printed in the U.S.A.
|S||27.48 in||20.98 in||23.98 in|
|M||28.98 in||21.97 in||25.00 in|
|L||29.49 in||23.46 in||25.47 in|
|XL||30.47 in||24.80 in||26.50 in|
|2XL||30.98 in||25.00 in||27.48 in|


text:
These photos are from 2 years ago when we went on our honeymoon to the Nordic countries. Loved all the spots but Copenhagen was our favorite of the larger cities.
There is so much variety on this planet for every group of animals. Every single one is beautiful in their own way.
Respect the sacred site. Happy to see that the trail to the top is now closed. We were one of the last ones to see tourists desecrating the trail that is to only be used by the Aboriginal people.

domain: geography


OCEANSIDE (NEWS 8) – A pilot was killed and a passenger seriously injured when a single-engine plane crashed into a hillside near state Route 76 late Monday night in Oceanside.
Dispatchers received a call shortly before 7:15 a.m., Tuesday from a woman reporting she saw the wreckage of a plane on a hillside near SR-76 and Canyon Drive, Oceanside police spokesman Tom Bussey said.
According to Oceanside police the pilot was already dead upon their arrival and the passenger who survived had been waiting for over ten hours to be rescued.
The passenger was extricated and transported by air ambulance to Scripps Memorial Hospital La Jolla for treatment of serious injuries, Bussey said.
The crash happened around two miles west of the Oceanside Municipal Airport.
Frank Lanza lives in a neighborhood close to the crash site. “I didn’t hear anything. It’s terrible. I mean, another ten feet and he would have made it.”
Lanza and other area residents told News 8 the conditions at the time made it very difficult to see. “[It was] very foggy. I could not see out much past my street. I could not see anything. It was solid white,” said Lanza.
Investigators were working to determine whether the plane was taking off or attempting to land at the time it went down.
Jared Foti is the assistant manager at the Oceanside Municipal Airport. He told News 8 he does not know either of the two men onboard the aircraft. According to Foti, the airport is an uncontrolled airport – meaning there are no air traffic controllers - airplanes fly in and out without pilots checking in all the time. “They do not request to take off. They do not request to land. They announce their intentions for the aircraft that may be in the area,” Foti said.
“We’re trying to find out if they tried to make an approach back to land once they may have realized that the weather was you know, not suitable. Based off of our video footage, it looked like it was very foggy at the that time. We could not even see the other end of our parking lot,” said Foti.
Oceanside Municipal Airport would not release or share surveillance video out of fear the public would know where the cameras are located, but said surveillance cameras show three men were onboard the plane on Monday.
A third individual was dropped off before the final flight at 9 p.m., on Monday.
Timeline of events:
- 4:30 p.m. – The plane left the airport safely.
- 6:30 p.m. – The plane returns to the airport and remains on the ground for about an hour. The third individual is dropped off.
- 8:30 p.m. – Surveillance video shows the men in the parking lot.
- 8:53 p.m. – The rented piper fixed-wing single engine aircraft takes off for a final time with two men onboard. The third individual sees the aircraft depart.
- 9:00 p.m. – Oceanside Police receive a call of someone hearing a loud crash noise off Highway 76. Officers search for hours in the dark but do not find anything.
- 7:00 a.m. – Police discover the plane wreckage.
“We don’t know if they were capable of flying in the weather conditions,” said Foti. He said to fly in fog, it is required that pilots and planes be certified to do so. “We don’t know if that is the case. We will have to find out from the FAA.”
News 8 has learned the plane is registered to a man in Vista. That man’s wife told News 8 the plane had been rented out and she had no idea who was onboard.
The National Transportation Safety Board and the Federal Aviation Administration are continuing their investigation into the cause of the crash.
Chopper 8 flew over the scene of the crash
The lone survivor of the crash was airlifted to Oceanside Airport and then to the hospital


text:
Join us on the Upper Level, by Zara for some Springtime Family fun in partnership with @WhereToMommy and Jam with Jamie.
Create yummy treats using marshmallows & hop to the beat of the Bunny Hop.
Our friends from Peppermint Park will be treating us to delicious bunny-shaped cotton candy.
Pose for a photo moment in our whimsical secret garden and Springtime spots.
Come dressed in your cutest bunny outfits for a chance to win a prize.


text:
Super cute stationery, stickers, and pins (particularly a lot of cat-themed goodies!).
Beautiful, handmade woven baskets for stunning, minimal storage.
Shop all of my products that are £10 and under. Budget-friendly finds that anyone will love!
Shop Gifts Under £10
Fun and quirky illustrated gifts from Jess. Shop art prints, accessories, stationery, and apparel.
Fun stamped pencils in super cute colours.
Small-batch handmakers of natural skincare products that are plastic-free and palm oil free too!
Delicious and personalisable handmade chocolates in the heart of Kent!
Delicious flavoured popcorn treats that are perfect as a stocking stuffer or tree hanging gift, yum!
So many stunning pieces, from paintings to embroidery, but I love these cute little hand-embroidered decorations
Incredible original laser cut brooches made from plywood, and layered using paint and wood stains.
If you're interested in sustainable fashion, Socko is a great sock brand for you. Each pack also contains a darning kit to repair them!


The average air conditioning coil assembler salary in Colorado, United States is $33,353 or an equivalent hourly rate of $16. This is 0% lower (+$51) than the average air conditioning coil assembler salary in the United States. In addition, they earn an average bonus of $477. Salary estimates based on salary survey data collected directly from employers and anonymous employees in Colorado, United States.
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A convenient, highly effective flea and tick spray for dogs and cats. It does not contain potentially toxic insecticides. A good choice for rapid activity in severe flea infestations.Spray provides fast-acting, long-lasting, waterproof treatment and control of fleas, ticks and chewing lice for adult dogs and cats and for puppies and kittens 8 weeks of age or older. Limited supply- 1 left
Can be used with monthly topical flea control like Frontline. Only needs to be sprayed once a month over the body to give your pet superior flea and tick protection.
ACTIVE INGREDIENT: fipronil: 0.29%
INERT INGREDIENTS: 99.71%
- Kills fleas
- Kills newly emerged adult fleas before they lay eggs
- Stops existing infestations and prevents establishment of new infestations
- Kills fleas which may cause flea allergy dermatitis
- Rapidly eliminates chewing lice infestations
- Aids in control of sarcoptic mange infestations in dogs
- Kills all life stages of ticks (larva, nymph and adult), including brown dog ticks (Rhipicephalus sanguineus), American dog ticks (Dermacentor variabilis), lone star ticks (Amblyomma americanum) and deer ticks (Ixodes scapularis).
- Kills ticks that may transmit Lyme disease, Rocky Mountain spotted fever, ehrlichiosis, babesiosis, anaplasmosis, haemobartonellosis and other tick-borne diseases
FRONTLINE Spray stops existing infestations and prevents establishment of new infestations by killing fleas, ticks, and chewing lice for at least 30 days.
FRONTLINE Spray may protect dogs against fleas for up to 90 days.
Directions for Use:
1. Wear household latex gloves.
2: Hold bottle in upright position. Ruffle the animal’s coat with one hand while applying spray mist to the animal’s back, sides, abdomen, legs, shoulders and neck.
3: For head and eye area, spray FRONTLINE on a gloved hand and rub gently into animal’s hair.
Do not get this product in your pet’s eyes or mouth. Apply spray mist until animal’s hair is damp to thoroughly wet. Approximately 1 to 2 pumps per pound of the animal’s body weight will be required. Pets with long or dense coats will require the higher rate.
As with any flea control product, wash hands and exposed skin thoroughly with soap and water after use.
To prevent flea build-up or reinfestation, use FRONTLINE Spray prior to the onset of flea season and monthly thereafter. Ticks do not need to receive spray directly upon their body for complete control. Multiple monthly treatments are recommended for the elimination of mites. Do not reapply spray for 30 days.
FRONTLINE Spray remains effective even after bathing, water immersion, or exposure to sunlight.

domain: pet


text:
A list of the top attractions of Luxor, and the must-see places...
Just to save you time digging around to know where to go, or what to visit.
Luxor attractions are in two diffirent sides of the Nile, so they are listed below according to the location. You can visit each side in one single day.
1-Luxor East bank of the Nile:
2-Luxor West bank of the Nile:
*The valley of the Kings
*The valley of the Queens
*The Temple of Queen Hatshepseut
*The Temple of Medeinet Habu (King Ramses III)
*The Nobles tombs
*The colossi of Memnon


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It’s only been a few days, and Newcastle United are already being linked with every man and his dog on the transfer market.
As we’ve said previously, it was bound to happen, but the reality of the takeover has sent certain members of the Italian member in something of a frenzy when it comes to the Magpies.
Thrown into the races for the likes of Marcelo Brozovic or even Federico Chiesa, Newcastle are now said to be a team to watch out for when it comes to Dusan Vlahovic.
The Serbian will not be renewing his contract at Fiorentina, and it’s clear he will be sold sooner rather than later, with teams such as Tottenham and Manchester City already after him.
While Fabio Paratici has said Spurs aren’t keen on Vlahovic, that hasn’t stopped agents interviewed by Tuttosport to mention them, and the St James’ Park residents, when it comes to who could buy the prolific goalscorer.
Italian intermediary Vincenzo Morabito was the first to mention the Magpies, saying: “I expect a change of team already in January: beware of Tottenham and even Newcastle. In Italy, I’d say more Milan than Juventus”.
A bit later, Gianluca Di Domenico, the agent of Christian Fassnacht at Young Boys, added: “The English are rich and now, and now Newcastle will join the big teams. Commisso, the great businessman that he is, will do everything to sell the player in the Premier League”.
All this is somewhat backed up by local newspaper Il Tirreno, who hint that the Magpies are the latest ‘in chronological order’ to ‘follow him’ and his situation.
Whether Steve Bruce’s side do turn up remains to be seen, but they will likely have to be very convincing, since Vlahovic will likely want to make a step up in his career on a football level as well as earn more money.
It’s no slight to what the Magpies might achieve with their new riches down the line, but, right now, in January, joining a team who are currently 19th in the Premier League might not be what he’s looking for.

domain: news


text:
Has the Warning Bell Sounded for Asset Protection Planners?
Probate & Property Magazine 2010, March/April, Volume 24, Number 2
By David J. Slenn
David J. Slenn is an associate in the Naples, Florida, office of Porter Wright Morris & Arthur and the vice-chair of the Trust & Estate Asset Protection Planning Committee.
A recent case out of the Sunshine State should give pause to the way some attorneys engage in asset protection planning. In
re Harwell, 414 B.R. 770 (M.D. Fla. 2009), involved a debtor who directed sums of money into his attorney"s trust account, despite the presence of a judgment.

domain: finance


text:
Three Twins Sundae Cones, Organic, Triple Chocolate
Bittersweet chocolate sundae cones topped with mini chocolate chips. USDA Organic. 1% for the planet member. Ice cream for acres. Inconceivably delicious. Three Twins made the world a little more delicious in 2016, when we introduced the first-ever organic sundae cone featuring the classic combination of vanilla ice cream topped with chocolate and roasted peanuts. Unlike the big boys, who use ingredients like whey and corn syrup, were not afraid to use pure ingredients like organic milk and organic cane sugar. Three Twins is now upping the ante by bringing Triple Chocolate Sundae Cones from our factory to your taste buds. And, I think that youll thank us. These barbarians of flavor slay the competition with their robust chocolate ice cream topped by dark chocolate chips and drizzled with organic chocolate. Plus, Three Twins proudly funds the purchase of six square feet of land with the purchase of each three-pack through our land conservation initiative, Ice Cream For Acres. Enjoy, - Neal Gottlieb, Founding twin. 6 sq feet. Your purchase protect at least 6 square feet of land through ice cream for acres. Certified organic by CCOF. threetwins.com.

domain: food


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lyon stainless is flatware silverware 3 stainless soup spoons international silver black.
24 inch folding stool folding stool inch folding stool incredible counter height folding chairs and bar stools high top bar height folding chairs plan folding bar stool.
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make water fountain recirculating water fountain actually looks really easy and cheap unlike all the ones that just say they are.
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solar garden pole lights outdoor solar lamp post the next generation in outdoor solar lights solar garden lamp post lights.

domain: design


Coronavirus infection rates are continuing to rocket in every single part of Sussex.
The continuing rise in COVID-19 cases does not bode well for the county’s near future under Tier 2 restrictions.
Hastings (507.2) and neighbouring Rother (339.3) are the only districts that are above the national average - however many other areas seeing huge week-on-week increases.
While West Sussex has a much lower overall average infection rate, infection rates are more than doubling in many areas.
Hastings, which has the highest highest infection rate in Sussex, is the 15th worst affected district in England.
In the seven days to December 12, the district recorded 470 cases, taking the infection rate up to a staggering 507.2.
Out of the top 30 worst affected districts in England, Hastings is the only district in Tier 2, with every other area already under Tier 3 restrictions.
When the Tier system was first announced, there had been hopes some areas would be putting forward a case to be lowered to Tier 1.
But the recent pattern of infection rates suggests that not only is Tier 1 no longer a realistic possibility, the prospect of being plunged into Tier 3 ahead of Christmas is a likely outcome, particularly for East Sussex.
The government has confirmed the Tier review will be taking place today (December 16).
The announcement on any locations switching Tiers is expected to be made tomorrow, although this has not been confirmed yet.
Sussex's latest coronavirus infection rates in full
Sussex was broken down into three areas for the purpose of the Tier system last time around at the end of November - those are East Sussex, West Sussex and Brighton and Hove.
Here is the list in full. From left to right, it reads: name of local authority; the tier of Covid-19 restrictions in which the local authority is currently placed; rate of new cases in the seven days to December 12; number (in brackets) of new cases recorded in the seven days to December 12; rate of new cases in the seven days to December 5; number (in brackets) of new cases recorded in the seven days to December 5.
Hastings: Tier 2, 507.2, (470), 210.4, (195)
Rother: Tier 2, 339.3, (326), 167.6, (161)
Wealden: Tier 2, 167.2, (270), 115.8, (187)
Eastbourne: Tier 2, 132.1, (137), 74.2, (77)
Lewes: Tier 2, 110.4, (114), 42.6, (44)
Mid Sussex: Tier 2, 150.3, (227), 87.4, (132)
Crawley: Tier 2, 143.2, (161), 64.1, (72)
Arun: Tier 2, 120.7, (194), 61.0, (98)
Adur: Tier 2, 98.0, (63), 34.2, (22)
Horsham: Tier 2, 97.4, (140), 80.7, (116)
Chichester: Tier 2, 83.4, (101), 53.7, (65)
Worthing: Tier 2, 80.5, (89), 31.7, (35)
Brighton and Hove
Brighton and Hove: Tier 2, 101.1, (294), 62.2, (181)


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Greetings fellow tentacle freaks!
This month we look at the 1988 supernatural erotic thriller Gedō Gakuen! Some of you dedicated Maeda fans might recognize this work but know it by its U.S anime title “Nightmare Campus” instead though! Since the original manga was never licensed or released overseas, I decided to give you all a full plot summary of this forgotten gem right here so please be sure to check it out! Many thanks and see you all again in a few weeks!


BUFFALO, N.Y. — The NFL announced that Buffalo Bills linebacker Lorenzo Alexander has been named a nominee for the Walter Payton Man of the Year award for the third straight year.
Each team has one nominee for the award, and the winner will be announced shortly before Super Bowl LIV in Miami. Each nominee will be given a donation up to $50,000 to a charity of their choice.
The award is one of the highest honors players can receive for their work off the field. Alexander and the other 31 nominees around the league will wear a special decal on the back of their helmet for the rest of the season.
Alexander has been active in the community since arriving in Buffalo in 2016.


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An exciting day of UEFA World Cup qualifiers provided its fair share of surprises, the biggest of which was France's meltdown in a 2-0 loss to Ukraine in Kiev.
Elsewhere, the much-anticipated Cristiano Ronaldo-Zlatan Ibrahimovic matchup was largely a disappointment, save for Ronaldo's winner; Iceland kept its unlikely World Cup story alive (barely), and Greece and Romania gave us more excitement than we bargained for.
Here are some quick thoughts on all four of the UEFA playoff first legs:
Les Bleus have the blues - Forget Ronaldo vs. Ibra for a second. What if all the talent available on the French national team missed out on the 2014 World Cup? That is looking like a distinct possibility now, after Les Bleus dropped the first leg of their playoff against Ukraine 2-0 in Kiev.
Though France had more than enough chances to get itself into the game (France had 14 shots, six of which were on target), it was still Ukraine that seemed to have the run of things in its home stadium. Time and time again, the unsung Ukrainians were able to snuff out the attacking threats of Franck Ribery, Samir Nasri and Olivier Giroud with tough tackling and a commitment to breaking forward that kept the French defense on edge.
In a way, each half was exactly the same. France would start out controlling tempo and creating most of the chances, only for Ukraine to grab a foothold in the match as France got flustered. The difference was, in the first half Ukraine couldn't take advantage of its momentum. In the second half, the hosts had no such problems.
Roman Zozulya was the hero in the 60th minute, as the burly striker worked a nice one-two combination with Edmar at the top of the box, easily shoved off the attentions of Mathieu Debuchy, and placed his slow-rolling finish through a crowd of feet and over the line for the game's first goal.
France was under pressure, and it clearly got to the favored visitors. Nasri put his shot from the center of the box right into the hands of the Ukraine goalkeeper. Ribéry and Blaise Matuidi won free kicks, but they came to nothing. Then, the killer: A complete meltdown by defender Laurent Koscielny.
It was bad enough that the defender allowed Zozulya to get past him so easily in the penalty area in the 82nd minute. His follow-up challnege was even worse -- connecting with all of the Ukranian's knees and none of the ball, setting up a penalty kick that Andiy Yarmolenko powered past Hugo Lloris, despite the French goalkeeper making contact with his palm on the way through.
Amazingly enough it would get worse: Koscielny was shown a straight red card for picking a needless fight away from the ball - an ejection that will rule him out for the second leg and put France's already-thin backline to the ultimate test in Paris.
Ukraine will be down a defender as well. Veteran Aleksandr Kucher received a red of his own in the game's closing moments for a bad foul on Ribéry. But at least Ukraine will have a lead to defend, while France will need to go all-out in hopes of making up the two-goal advantage.
If there's a plus side for Les Bleus, it is their immense talent up front. In Nasri, Ribéry, and Loïc Remy, the French have capable midfielders who can break down a bunkered side, and strikers Giroud and Karim Benzema are more than capable of dealing with Ukraine's physicality and finishing off chances when they are on their game.
So, the French test will be mostly mental. Can Les Bleus, with all their recent history of internal squabbles and on-field meltdowns, avoid yet another international disappointment in the second leg? Or will the 2014 World Cup go on without not just one of the most talented individuals in the world in Ribery, but also one of the most talented teams?
Clutch Cristiano, Invisible Ibra -- In a Portugal-Sweden match that was hyped up as a clash between two of the most inventive attacking players in the game, the match itself seemed to revolve around everyone except those two players.
Until the last 10 minutes, that is.
After 80 minutes of back-and-forth soccer in which neither Cristiano Ronaldo nor Zlatan Ibrahimovic could do much to lift his respective, otherwise mediocre side, it was Ronaldo that found an extra gear in Portugal's 1-0 victory over Sweden in Lisbon. The Real Madrid man was peripheral to almost every effective attack Portugal was able to mount, and as the second half drew to a close he looked clearly frustrated at his side's inability to break through.
Then, like many great players, he popped up when it mattered. Miguel Veloso's cross from the left seemed close enough to goal for Swedish goalkeeper Andreas Isaksson to come out and claim for himself, but instead he let it go and Ronaldo swooped low to head in the game's only goal in the 82nd minute.
It was far and away as involved as he had been in the whole game. In the preceeding 80 minutes, Ronaldo either occupied his standard wing forward role, where his passes were inaccurate or well-defended by the Swedish fullbacks. On occasion he would drift inside and act as a center forward, but there his teammates starved him of service, and one of the world's best players was reduced to a bystander.
Ibrahimovic had similar problems. The big Swede had little to no impact on the game, primarily due to a lack of service. Of Sweden's six total shots in the game, Ibrahimovic had just one of them, a 24th minute effort from distance that was easily saved by Portugal's Rui Patricio.
However, there is still plenty of reason for Sweden to be confident going into their home leg despite the loss. Ronaldo's goal and his header that hit the crossbar soon after, were perhaps the only clear-cut chances Portugal had all game. If the Swedes can defend similarly well in Stockholm and improve their service to Ibrahimovic even a little bit, they could easily grab an equalizing goal. After that, given the attacking players involved, anything can happen (and probably will).
Greece rides the wave - With all four games running simultaneously, international soccer fans were given a tough choice of which one to follow from 2:45-3:30 p.m. (Eastern time). It's unlikely most neutrals picked Greece-Romania, and it's too bad -- in terms of pure entertainment, the first stanza of that one was far beyond anything the others had to offer with a flurry of goals midway through.
Kostas Mitroglou got started on what would be a man-of-the-match performance for Greece, finishing a lovely goal on the full volley after a lofted ball over the Romanian defense by Dimitrios Salpingidis in the 14th minute. Bogdan Stancu responded for the Romanians, heading a freekick into the back of the net so close to the near post he was lucky he didn't bang his head against the woodwork in the process.
Romania had the away goal and a tie game at its feet, but the visitors' defense let them down just as momentum was starting to build in their favor. The Romanian back four allowed Vasilis Torosidis space on the wing, with his cross finding Salpingidis unmarked in the box. The finish was easy, and once again Greece was on top. After three goals in the space of seven minutes, this tie appeared to be on fire.
The fire died down, though, as both defenses toughened up and mired the match in midfield to close the first half. Greece continued to press for a third after the break, and got one after Mitroglou scored his second well-taken goal of the game. Check out the flicked header from Kostas Katsouranis.
Romania will need to get better offensively in the second leg in Bucharest if its is to maintain any hope of going through, as the side managed only one shot on goal all game.
Iceland is still alive, but missed an opportunity -- The day's first match kicked off at the home of the playoff round's most unlikely participants, as Iceland hosted Croatia at the 10,000 capacity Laugardalsvöllur arena in Reykjavík.
Those 10,000 sounded like easily twice that on the broadcast, though, as Iceland's faithful came out in force to see their team try to secure a World Cup berth for the first time in their history. And though they may not have been rewarded with a victory, there were plenty of positives to take into the second leg.
Perhaps the biggest mismatch on the field was between the Croatian attack, featuring master string-puller Luka Modric in midfield and deadly finisher Mario Mandzukic up top, and the Icelandic defense, a unit that contains nowhere near the same international pedigree.
The gulf in class didn't seem to matter on Thursday, just as it hadn't throughout the whole of World Cup qualifying for the Nordic side. Time and time again, Iceland's deep-lying, compact back four repelled Croatian attacks, while its potent attacking unit featuring Tottenham's Gylfi Sigurdsson and Ajax striker Kolbeinn Sigthorson tried to catch Croatia on the counter.
Those plans were dealt a blow just after halftime, when Olafor Skulason was given a straight red card in the 50th minute for a rough challenge on Ivan Perisic. From there, the Icelandic gameplan got even more defensive, with even Sigurdsson chipping in on multiple occasions to clear the lines.

domain: sports