text:
In humans, Cidea (cell death-inducing DNA fragmentation factor alpha-like effector A) is highly but variably expressed in white fat, and expression correlates with metabolic health. Here we generate transgenic mice expressing human Cidea in adipose tissues (aP2-hCidea mice) and show that Cidea is mechanistically associated with a robust increase in adipose tissue expandability. Under humanized conditions (thermoneutrality, mature age and prolonged exposure to high-fat diet), aP2-hCidea mice develop a much more pronounced obesity than their wild-type littermates. Remarkably, the malfunctioning of visceral fat normally caused by massive obesity is fully overcome-perilipin 1 and Akt expression are preserved, tissue degradation is prevented, macrophage accumulation is decreased and adiponectin expression remains high. Importantly, the aP2-hCidea mice display enhanced insulin sensitivity. Our data establish a functional role for Cidea and suggest that, in humans, the association between Cidea levels in white fat and metabolic health is not only correlative but also causative.
Several mutational events are involved in the multistage process of cancer. Genetic instability of tumor DNA is shown in a variety of genetic changes such as point mutations, chromosomal aberrations, aneuploidy, DNA amplification and somatic recombination. Highly repetitive sequences, minisatellite DNA, have been changed in tumors, most probably by DNA amplification.
To better predict carcinogens all these genetic changes should be considered. The present investigation has focused on the study of genetic changes especially DNA amplification with the use of genetic instability of minisatellites. DNA alterations in human bladder tumors were examined. A new in vitro test system was developed to study DNA amplification.
In this study DNA fingerprint analysis, with the use of minisatellite DNA, of 22 bladder tumor patients is performed. DNA alterations were determined in approximately 50% of the examined bladder tumor patients. In eight of these ten patients a loss of bands was demonstrated.
To study DNA amplifications an in vitro test system was developed. A haploid yeast strain was constructed, the TR(MS1)-1, which carries a chromosomal integration of the human minisatellite sequence MSI. The spontaneous frequency of new MSI length alleles was approximately 30%. Both amplifications and deamplifications can be detected in this system without selection. A plasmid pop-out frequency can also be meausured.
A number of model compounds was used during this study, namely three alkylating agents, a nongenotoxic carcinogen and several nitrogen heterocyclic compounds. To compare genotoxicity and the mechanism of action, these compounds are tested in several in vitro test systems. Ethylene oxide (EO) was demonstrated to be more potent than propylene oxide (PO) in several of the measured endpoints in yeast. EO induced changes in the amplification spectrum of new MSI length alleles in TR(MS1)-1 while PO increased the plasmid pop-out frequency. 2,3,7,8-Tetrachlorodibenzo(p)dioxin (TCDD) was also seen to induce changes in the amplification spectrum of new MSI length alleles, thus demonstrating an effect of TCDD at the DNA level. Among the N-heterocyclic compounds, camptothecin increased the plasmid pop-out frequency in TR(MS1)-1. Two tryptophan photoproducts, "284" and "312", were shown to bind to the A A-receptor. They were demonstrated to be nongenotoxic and antimutagenic in bacteria and slightly genotoxic in yeast. These two compounds are antimutagenic by inhibiting the cytochrome P450IA1. "284" increased cell survival or cell division. These two tryptophan photoproducts, "284" and "312", are postulated to be biological signal substances by being the endogenous ligand to the Ah -receptor.
Grp170 and Hsp110 proteins constitute two evolutionary distinct branches of the Hsp70 family that share the ability to function as nucleotide exchange factors (NEFs) for canonical Hsp70s. Although the NEF mechanism of the cytoplasmic Hsp110s is well understood, little is known regarding the mechanism used by Grp170s in the endoplasmic reticulum. In this study, we compare the yeast Grp170 Lhs1 with the yeast Hsp110 Sse1. We find that residues important for Sse1 NEF activity are conserved in Lhs1 and that mutations in these residues in Lhs1 compromise NEF activity. As previously reported for Sse1, Lhs1 requires ATP to trigger nucleotide exchange in its cognate Hsp70 partner Kar2. Using site-specific cross-linking, we show that the nucleotide-binding domain (NBD) of Lhs1 interacts with the NBD of Kar2 face to face, and that Lhs1 contacts the side of the Kar2 NBD via its protruding C-terminal alpha-helical domain. To directly address the mechanism of nucleotide exchange, we have compared the hydrogen-exchange characteristics of a yeast Hsp70 NBD (Ssa1) in complex with either Sse1 or Lhs1. We find that Lhs1 and Sse1 induce very similar changes in the conformational dynamics in the Hsp70. Thus, our findings demonstrate that despite some differences between Hsp110 and Grp170 proteins, they use a similar mechanism to trigger nucleotide exchange.
Mitochondrial dysfunction is a common feature of many neurodegenerative diseases, including proteinopathies such as Alzheimer's or Parkinson's disease, which are characterized by the deposition of aggregated proteins in the form of insoluble fibrils or plaques. The distinct molecular processes that eventually result in mitochondrial dysfunction during neurodegeneration are well studied but still not fully understood. However, defects in mitochondrial fission and fusion, mitophagy, oxidative phosphorylation and mitochondrial bioenergetics have been linked to cellular demise. These processes are influenced by the lipid environment within mitochondrial membranes as, besides membrane structure and curvature, recruitment and activity of different proteins also largely depend on the respective lipid composition. Hence, the interaction of neurotoxic proteins with certain lipids and the modification of lipid composition in different cell compartments, in particular mitochondria, decisively impact cell death associated with neurodegeneration. Here, we discuss the relevance of mitochondrial lipids in the pathological alterations that result in neuronal demise, focussing on proteinopathies.
Animal models of human anxiety often invoke a conflict between approach and avoidance [1, 2]. In these, a key behavioral assay comprises passive avoidance of potential threat and inhibition, both thought to be controlled by ventral hippocampus [2-6]. Efforts to translate these approaches to clinical contexts [7, 8] are hampered by the fact that it is not known whether humans manifest analogous approach-avoidance dispositions and, if so, whether they share a homologous neurobiological substrate [9]. Here, we developed a paradigm to investigate the role of human hippocampus in arbitrating an approach-avoidance conflict under varying levels of potential threat. Across four experiments, subjects showed analogous behavior by adapting both passive avoidance behavior and behavioral inhibition to threat level. Using functional magnetic resonance imaging (fMRI), we observe that threat level engages the anterior hippocampus, the human homolog of rodent ventral hippocampus [10]. Testing patients with selective hippocampal lesions, we demonstrate a causal role for the hippocampus with patients showing reduced passive avoidance behavior and inhibition across all threat levels. Our data provide the first human assay for approach-avoidance conflict akin to that of animal anxiety models. The findings bridge rodent and human research on passive avoidance and behavioral inhibition and furnish a framework for addressing the neuronal underpinnings of human anxiety disorders, where our data indicate a major role for the hippocampus.
The main aim of this thesis was to study the role of the indirect actions of γ-rays and α-particles on the complexity of primary DNA damages and the repair fidelity of major DNA repair pathways: non-homologous end joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER). The complexity of radiation-induced damages increases and the proximity between damages decreases with increasing LET due to formation of ionization clusters along the particle track. The complexity of damages formed can be modified by the free radical scavenger dimethyl sulfoxide (DMSO). In addition, the effects of low doses of low dose rate γ-radiation on cellular response in terms of differentiation were investigated.
Paper I investigates the role of the indirect effect of radiation on repair fidelity of HRR, NHEJ and BER when damages of different complexity were induced by radiation or by potassium bromate. We found that potassium bromate induces complex DNA damages through processing of base modifications and that the indirect effect of radiation has a high impact on the NHEJ pathway. Results in paper II confirmed our conclusions in paper I that the indirect effect from both γ-rays and α-particles has an impact on all three repair pathways studied and NHEJ benefits the most when the indirect effect of radiation is removed.
In paper III we investigated the effects of low dose/dose rate γ-radiation on the developmental process of neural cells by using cell models for neurons and astrocytes. Our results suggest that low dose/dose rate γ-radiation attenuates differentiation and down-regulates proteins involved in the differentiation process of neural cells by an epigenetic rather than cytotoxic mechanism.
In order to investigate the relative involvement of the different DNA repair pathways NHEJ, HRR and BER in repair of DNA lesions of different complexity, we have compared clonogenic survival and induction of micronuclei in a panel of repair-deficient CHO cell lines after exposure to γ-rays and α-particle radiation. The complexity of the DNA lesions formed was also modified by exposures to 2 M DMSO, a potent radical scavenger, which is known to interact with the lesions produced by direct hits on DNA.
The NHEJ pathway gained the most from scavenging of the free radicals after irradiation to γ-rays or α-particles as evaluated by cell survival and the yields of MN. Results presented here also implicate that clustered base damages were induced by α-radiation and contributed to the yield of DNA double-strand breaks.
The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO3).
CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulphoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay.
The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied.
The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.
Synthesis of proteins is performed by the ribosome, a large ribonucleoprotein complex. Apart from the ribosome, numerous protein factors participate in this process. Elongation factor 2 (eEF2) is one of these factors. eEF2 is an essential protein with a mol. mass of about 100 kDa. The amino acid sequence of eEF2 is highly conserved in different organisms. eEF2 from S. cerevisiae contains 842 amino acids. The role of eEF2 in protein synthesis is to participate in the translocation of tRNAs from the A- and P-sites on the ribosome to the P- and E-sites. This movement of tRNAs is accompanied by a simultaneous movement of mRNA by one codon. eEF2 consists of six domains referred to as domains G, G′ and II-V, belongs to the G-protein super-family and possesses all structural motifs characterizing proteins in this family. eEF2 binds to the ribosome in complex with GTP. After GTP hydrolysis and translocation, it leaves the ribosome bound to GDP. The rate of protein synthesis in the cell can be regulated by phosphorylation of eEF2. Phosphorylation occurs on two threonine residues, situated in the G domain of the factor. Phosphorylation of eEF2 is catalysed by Rck2-kinase in yeast which is activated in response to osmotic stress. Despite the high degree of conservation of the threonine residues, they are not essential for yeast cell under normal growth conditions. However, under mild osmotic stress the growth rate of the cells lacking threonine residues was decreased. Region where threonine residues are located, called Switch I. Cryo-EM reconstruction shows that this region adopts ordered conformation when the eEF2•GTP complex is bound to the ribosome but became structurally disordered upon GTP hydrolysis. Mutagenesis of individual amino acids in Switch I resulted in both functional and non-functional eEF2 depending on the site of mutation and the substituting amino acid. Both functional and non-functional Switch I mutants were able to bind to the ribosome, indicating that mutations did not abolish the capacity of the factor to bind GTP. Yeast eEF2 with Switch I region from E. coli was able to substitute the wild type protein in vivo, though the growth rate of these cells was severely impaired. The eEF2-dependent GTP hydrolysis can be activated by ribosome from heterologous sources as seen in vitro. However, eEF2 from A. thaliana, D. melanogaster and S. solfataricus could not substi-tute yeast eEF2 in vivo. This may indicate additional roles of eEF2 in the yeast cell, apart from translocation itself.
Yeast elongation factor 2 is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress. The importance of residues T56 and T58 for elongation factor 2 function in yeast was studied using site directed mutagenesis and functional complementation. Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor 2 whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional. Expression of mutants T56C, T56S and T58S was associated with reduced growth rate. The double mutants T56M/T58W and T56M/T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate. The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions. Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures. Similarly, cells expressing mutants T56M and T56M/T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally. All mutants tested were retained their ability to bind to ribosomes in vivo. However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor 2 were less capable of competing with wild-type elongation factor 2 in ribosome binding. The presence of nonfunctional but ribosome binding forms of elongation factor 2 did not affect the growth rate of yeast cells also expressing wild-type elongation factor 2.
Biogenesis of respiratory chain complexes depends on the expression of mitochondrial-encoded subunits. Their synthesis occurs on membrane-associated ribosomes and is probably coupled to their membrane insertion. Defects in expression of mitochondrial translation products are among the major causes of mitochondrial disorders. Mdm38 is related to Letm1, a protein affected in Wolf-Hirschhorn syndrome patients. Like Mba1 and Oxa1, Mdm38 is an inner membrane protein that interacts with ribosomes and is involved in respiratory chain biogenesis. We find that simultaneous loss of Mba1 and Mdm38 causes severe synthetic defects in the biogenesis of cytochrome reductase and cytochrome oxidase. These defects are not due to a compromised membrane binding of ribosomes but the consequence of a mis-regulation in the synthesis of Cox1 and cytochrome b. Cox1 expression is restored by replacing Cox1-specific regulatory regions in the mRNA. We conclude, that Mdm38 and Mba1 exhibit overlapping regulatory functions in translation of selected mitochondrial mRNAs.
The cardiovascular system is a key route of exposure to nanoparticles (NPs). The exposure could costendothelial cell dysfunction and impairment in blood circulation that could lead to cardiovascular diseasessuch as atherosclerosis. Currently, ultra-small nanoparticles (USNPs) at 1-3 nm, are receiving growingattention due to their unique properties. Emerging application for rutile TiO2-USNPs in medicine areexploring due to their insoluble nature, lack of oxidative activity and strong luminescence not observed inlarger NPs. On organic nanoparticle side, single walled carbon nanotubes (SWCNTs) are candidatemolecules for drug delivery from the chemical perspective. However their potential applications arehindered by their high oxidative activity and potential toxicity. Here we used transcriptome sequencing(RNA-seq) to evaluate the effects of exposure to sub-lethal concentration of TiO2-USNPs, TiO2-NPs andSWCNTs on human dermal microvascular endothelial cells. Specific toxicological effects were inferredfrom the functions of genes whose transcripts either increased or decreased. Our results show that TiO2-USNPs mostly induced the up-regulation of transcripts involved in lipid and cholesterol metabolism.TiO2-NPs induced the highest number of differentially expressed transcripts involved in cellularsenescence, endoplasmic reticulum (ER) stress, and heat shock responses as well lipid metabolism.Finally, SWCNTs affected to those genes involved in early stress and inflammatory responses.
Functionalization of super paramagnetic iron oxide NPs (SPIONs) with different coatings renders them with unique physicochemicalproperties that allow them to be used in a broad range of applications such as drug targeting and water purification. However, it is required tofill the gap between the promises of any new functionalized SPIONs and the effects of these coatings on the NPs safety. Nanotoxicology isoffering diverse strategies to assess the effect of exposure to SPIONs in a case-by-case manner but an integrated nanoimpact scale has notbeen developed yet. We have implemented the classical integrated biological response (IBR) into an integrated nanoimpact index (INI) as anearly warning scale of nano-impact based on a combination of toxicological end points such as cell proliferation, oxidative stress, apoptosisand genotoxicity. Here, the effect of SPIONs functionalized with tri-sodium citrate (TSC), polyethylenimine (PEI), aminopropyltriethoxysilane(APTES) and Chitosan (chitosan) were assessed on human keratinocytes and endothelial cells. Our results show thatendothelial cells were more sensitive to exposure than keratinocytes and the initial cell culture density modulated the toxicity. PEI-SPIONshad the strongest effects in both cell types while TSC-SPIONS were the most biocompatible. This study emphasizes not only the importanceof surface coatings but also the cell type and the initial cell density on the selection of toxicity assays. The INI developed here could offer aninitial rationale to choose either modifying SPIONs properties to reduce its nanoimpact or performing a complete risk assessment to definethe risk boundaries.
The cell nucleus of eukaryotic organisms contains the genome surrounded by a nuclear envelope consisting of a double-lipid membrane with embedded nuclear pores and an underlying nuclear lamina. The uniformity in size and density makes it possible to isolate pure intact nuclei at high yields from tissue homogenates by centrifugation through a sucrose cushion. Nuclear envelopes can be prepared from isolated nuclei by enzymatic degradation of their nucleic acid content. The resulting nuclear envelope preparations contain structurally well-conserved inner and outer nuclear membranes with attached ribosomes, nuclear pore complexes and nuclear lamina. Reliable methods for preparation of nuclei and nuclear envelopes play an important role in the successful identification of components that are located in nuclei and in nuclear subcompartments.
The first ever 'Cellular Dynamics' meeting on the membrane-cytoskeleton interface took place in Southbridge, MA on May 21-24, 2017 and was co-organized by Michael Way, Elizabeth Chen, Margaret Gardel and Jennifer Lippincott-Schwarz. Investigators from around the world studying a broad range of related topics shared their insights into the function and regulation of the cytoskeleton and membrane compartments. This provided great opportunities to learn about key questions in various cellular processes, from the basic organization and operation of the cell to higher-order interactions in adhesion, migration, metastasis, division and immune cell interactions in different model organisms. This unique and diverse mix of research interests created a stimulating and educational meeting that will hopefully continue to be a successful meeting for years to come.
The correlation coefficient r is a measure of similarity used to compare regions of interest in image pairs. In fluorescence microscopy there is a basic tradeoff between the degree of image noise and the frequency with which images can be acquired and therefore the ability to follow dynamic events. The correlation coefficient r is commonly used in fluorescence microscopy for colocalization measurements, when the relative distributions of two fluorophores are of interest. Unfortunately, r is known to be biased understating the true correlation when noise is present. A better measure of correlation is needed. This article analyses the expected value of r and comes up with a procedure for evaluating the bias of r, expected value formulas. A Taylor series of so-called invariant factors is analyzed in detail. These formulas indicate ways to correct r and thereby obtain a corrected value free from the influence of noise that is on average accurate (unbiased). One possible correction is the attenuated corrected correlation coefficient R, introduced heuristically by Spearman (in Am. J. Psychol. 15:72-101, 1904). An ideal correction formula in terms of expected values is derived. For large samples R tends towards the ideal correction formula and the true noise-free correlation. Correlation measurements using simulation based on the types of noise found in fluorescence microscopy images illustrate both the power of the method and the variance of R. We conclude that the correction formula is valid and is particularly useful for making correct analyses from very noisy datasets.
The nuclear envelope, consisting of an outer and an inner nuclear membrane, surrounds the genomic material. The genomic material (chromatin) is highly structured with (transcriptionally inactive) heterochromatin mostly found in the nuclear periphery and (transcriptionally active) euchromatin mostly found in the nuclear interior. Underlying the nuclear envelope is the nuclear lamina that consists of lamin proteins and nuclear envelope transmembrane proteins (NETs), which organize chromatin in the nuclear periphery. There are several hundred uncharacterized tissue-specific NETs, with only a few linked to cellular differentiation. Induced pluripotent stem cells (iPSCs) enable studies of early differentiation and are a promising tool for cell replacement therapies.
In this licentiate thesis, we have focused on investigating the role of the inner nuclear membrane protein Samp1 in chromatin organization and cell differentiation. Overexpression of Samp1 induced a fast differentiation of iPSCs, suggesting that Samp1 may be involved in the differentiation process. We have also developed a novel image analysis method to be able to monitor chromatin organization in live cells. Depletion of Samp1 affected chromatin distribution and resulted in increased formation of peripheral heterochromatin, contradictory to what is expected of other characterized NETs. It is possible that Samp1 might have a role in both differentiation and chromatin organization and that future studies might link these two processes together.
The ability of iPSCs (induced pluripotent stem cells) to generate any cell type in the body makes them valuable tools for cell replacement therapies. However, differentiation of iPSCs can be demanding, slowand variable. During differentiation chromatin is re-organized and silent dense heterochromatin becomes tethered to the nuclear periphery by processes involving the nuclear lamina and proteins of the INM(inner nuclearmembrane). The INM protein, Samp1 (Spindle AssociatedMembrane Protein 1) interacts with Lamin A/C and the INMprotein Emerin, which has a chromatin binding LEM(Lap2-Emerin-Man1)-domain. In this paperweinvestigate if Samp1 can play a role in the differentiation of iPSCs. Samp1 levels increased as differentiating iPSCs started to express Lamin A/C. Interestingly, even under pluripotent culturing conditions, ectopic expression of Samp1 induced a rapid differentiation of iPSCs, ofwhich some expressed the neuronal marker beta III-tubulin already after 6 days. This suggests that Samp1 is involved in early differentiation of iPSCs and could potentially be explored as a tool to promote progression of the differentiation process.
In most cells, transcriptionally inactive heterochromatin is preferentially localized in the nuclear periphery and transcriptionally active euchromatin is localized in the nuclear interior. Different cell types display characteristic chromatin distribution patterns, which change dramatically during cell differentiation, proliferation, senescence and different pathological conditions. Chromatin organization has been extensively studied on a cell population level, but there is a need to understand dynamic reorganization of chromatin at the single cell level, especially in live cells. We have developed a novel image analysis tool that we term Fluorescence Ratiometric Imaging of Chromatin (FRIC) to quantitatively monitor dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation. We also found that peripheral heterochromatin depended on the levels of the inner nuclear membrane protein Samp1, suggesting an important role in promoting peripheral heterochromatin. Taken together, FRIC is a powerful and robust new tool to study dynamic chromatin redistribution in live cells.
When a cell is stimulated by signalling molecules such as growth factors, one immediate response is an increased motile activity of the cell. Membrane lamella and spikes appear at the cell surface and perform undulating movements. This is caused by the activation and reorganisation of the highly dynamic microfilament system, present beneath the cell membrane. The major component of the microfilament system is actin, a protein that can polymerise into filaments -microfilaments-, and several actin-binding proteins which act in concert to organise actin into different supramolecular arrangements. To understand how a signal is transmitted form the exterior of the cell to the microfilament system, it is important to reveal how the activities of different actin-binding proteins are regulated.
This thesis focus on the actin-binding protein profilin, which binds actin monomers and regulates actin polymerisation. Profilin also interacts with polyphosphoinositides, which are important signalling molecules, and this association appears to regulate both the function of profilin and the turnover of polyphosphoinositides. Furthermore, profilin binds poly(L-proline) and proline-rich proteins. The latter interaction seems to be involved in the positioning of profilin within a cell, and may also be important for signal transduction-related processes. In the current investigation site-directed mutagenesis was combined with biochemical methods to further investigate the function of profilin.
The replacement of two amino acid residues located in a hydrophobic patch on the surface of the profilin molecule was shown to abolish the interaction with poly(L-proline), and enabled the localisation of the poly(L-proline) binding-site to this part of profilin. A new purification method was developed for these profilin mutants and further biochemical characterisation showed that the two mutations also decreased the affinity of profilin for actin. As the poly(L-proline)-binding site is separated from the actin-binding surface of profilin this showed that these amino acid replacements in the poly(L-proline)-binding site also introduced long-range alterations in the profilin molecule.
Profilin was shown to be phosphorylated on a serine and a tyrosine residue by an epidermal growth factor receptor complex isolated from stimulated cells. The phosphorylated residues were located to the poly(L-proline) binding site of profilin, and the phosphorylation interfered with the binding of profilin to poly(L-proline). This suggests that this phosphorylation regulate interactions between profilin and proline-rich proteins.
The simultaneous deletion of two amino acid residues adjacent to the actin-binding site of profilin was shown to reduce the affinity of profilin for actin, without affecting the interaction of profilin with other ligands. The effect of this mutant profilin on the organisation of the microfilament system in living cells was compared to that of wild type profilin in microinjection experiments. While wild type profilin caused disruption of actin bundles within the cell, the profilin mutant had no apparent effect, showing that the derangement of the actin bundles by profilin is dependent on the stability of the profilin:actin complex.
The nuclear envelope consists of inner and outer nuclear membranes. Whereas the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane (INM) represents a unique membranous environment containing specific proteins. The mechanisms of integral INM protein degradation are unknown. Here, we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome independently of the vacuole and that it exhibited a half-life of similar to 45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistent with these data, Asi2 is post-translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus.
Pentameric ligand-gated ion channels (pLGICs) are similar in structure but either inhibited or potentiated by alcohols and anesthetics. This dual modulation has previously not been understood, but the determination of X-ray structures of prokaryotic GLIC provides an ideal model system. Here, we show that a single-site mutation at the F14' site in the GLIC transmembrane domain turns desflurane and chloroform from inhibitors to potentiators, and that this is explained by competing allosteric sites. The F14'A mutation opens an intersubunit site lined by N239 (15'), 1240 (16'), and Y263. Free energy calculations confirm this site is the preferred binding location for desflurane and chloroform in GLIC F14'A. In contrast, both anesthetics prefer an intrasubunit site in wild-type GLIC. Modulation is therefore the net effect of competitive binding between the intersubunit potentiating site and an intrasubunit inhibitory site. This provides direct evidence for a dual-site model of allosteric regulation of pLGICs.
Several lines of evidence indicate that 15-lipoxygenase type 1 (15-LO-1) plays a pathophysiological role in asthma. The aim for this study was to investigate the 15-LO-1 expression and activity in primary human airway epithelial cells cultivated on micro-porous filters at air liquid interface. Incubation of human airway epithelial cells with arachidonic acid led to the formation of 15(S)-hydroxy-eicosatetraenoic acid (15-HETE) and exposing the cells to bacteria or physical injury markedly increased their production of 15-HETE. The cells were also found to convert arachidonic acid to eoxin C-4 (EXC4). Subcellular fractionation revealed that the conversion of EXA(4) to EXC4 was catalyzed by a soluble glutathione transferase (GST). The GST P1-1 enzyme was found to possess the highest activity of the investigated soluble GSTs. Following IL-4 treatment of airway epithelial cells, microarray analysis confirmed high expression of 15-LO-1 and GST P1-1, and immunohistochemical staining of bronchial biopsies revealed co-localization of 15-LO-1 and GST P1-1 in airway epithelial cells. These results indicate that respiratory infection and cell injury may activate the 15-LO pathway in airway epithelial cells. Furthermore, we also demonstrate that airway epithelial cells have the capacity to produce EXC4.
Cell growth and proliferation are processes in the cell that must be tightly regulated. Transcription of ribosomal RNA and ribosomal biogenesis are directly linked to cell growth and proliferation, since the ribosomal RNA encodes for the majority of transcription in a cell and ribosomal biogenesis influences directly the number of proteins that are synthesized.
In the work presented in this thesis, we have investigated the ribosomal RNA genes, namely the ribosomal DNA genes and the 5S rRNA genes, and their transcriptional regulation. One protein complex that is involved in RNA polymerase I and III transcription is the chromatin remodelling complex B‑WICH (WSTF, SNF2h, NM1). RNA polymerase I transcribes the rDNA gene, while RNA polymerase III transcribes the 5S rRNA gene, among others. In Study I we determined the mechanism by which B‑WICH is involved in regulating RNA polymerase I transcription. B‑WICH is associated with the rDNA gene and was able to create a more open chromatin structure, thereby facilitating the binding of HATs and the subsequent histone acetylation. This resulted in a more active transcription of the ribosomal DNA gene. In Study II we wanted to specify the role of NM1 in RNA polymerase I transcription. We found that NM1 is not capable of remodelling chromatin in the same way as B‑WICH, but we demonstrated also that NM1 is needed for active RNA polymerase I transcription and is able to attract the HAT PCAF. In Study III we investigated the intergenic part of the ribosomal DNA gene. We detected non-coding RNAs transcribed from the intergenic region that are transcribed by different RNA polymerases and that are regulated differently in different stress situations. Furthermore, these ncRNAs are distributed at different locations in the cell, suggesting that they have different functions. In Study IV we showed the involvement of B‑WICH in RNA Pol III transcription and, as we previously had shown in Study I, that B‑WICH is able to create a more open chromatin structure, in this case by acting as a licensing factor for c-Myc and the Myc/Max/Mxd network.
Taken together, we have revealed the mechanism by which the B‑WICH complex is able to regulate RNA Pol I and Pol III transcription and we have determined the role of NM1 in the B‑WICH complex. We conclude that B‑WICH is an important factor in the regulation of cell growth and proliferation. Furthermore, we found that the intergenic spacer of the rDNA gene is actively transcribed, producing ncRNAs. Different cellular locations suggest that the ncRNAs have different functions.
Four forms of short neuropeptide F (sNPF1-4), derived from the gene snpf, have been identified in Drosophila and are known to act on a single G-protein-coupled receptor (sNPFR). Several functions have been suggested for sNPFs in Drosophila, including the regulation of feeding and growth in larvae, the control of insulin signalling and the modulation of neuronal circuits in adult flies. Furthermore, sNPF has been shown to act as a nutritional state-dependent neuromodulator in the olfactory system. The role of sNPF in the larval nervous system is less well known. To analyse sites of action of sNPF in the larva, we mapped the distribution of sNPF- and sNPFR-expressing neurons. In particular, we studied circuits associated with chemosensory inputs and systems involved in the regulation of feeding, including neurosecretory cell systems and the hypocerebral ganglion. We employed a combination of immunocytochemistry and enhancer trap and promoter Gal4 lines to drive green fluorescent protein. We found a good match between the distribution of the receptor and its ligand. However, several differences between the larval and adult systems were observed. Thus, neither sNPF nor its receptor was found in the olfactory (or other sensory) systems in the larva and cells producing insulin-like peptides did not co-express sNPFR, as opposed to results from adults. Moreover, sNPF was expressed in a subpopulation of Hugin cells (second-order gustatory neurons) only in adult flies. We propose that the differences in sNPF signalling between the developmental stages is explained by differences in their feeding behaviour.
Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research.
Compaction of chromosomes in the eukaryotic cell is due to interactions between DNA and proteins and interactions between proteins. These two types of interaction form a dynamic structure, known as "chromatin". The condensation of chromatin must be carefully regulated, since the structure is an obstacle for factors that need access to the DNA. An extensive range of components, one group of which is the ATP-dependent chromatin remodel-ling complexes, controls the accessibility of DNA. These complexes have been studied in a variety of eukaryotic systems, and their functions in major events in the cell, such as replication, DNA-repair and transcription have been established, as have their roles in the assembly and maintenance of chromatin. All of the complexes contain a highly conserved ATPase, which belongs to the SWI2/SNF2 family of proteins, one group of which is known as the ISWI proteins. There are two forms of ISWI in human, known as "SNF2h" and "SNF2l".
We have identified a human SNF2h-assembly, B-WICH, that consists of SNF2h, William's syndrome transcription factor (WSTF), nuclear myosin (NM1), and a number of additional nuclear proteins including the Myb-binding protein 1a (Myb bp1a), SF3b155/SAP155, the RNA helicase II/Guα, the proto-oncogene Dek, and the Cockayne Syndrome protein B (CSB). The 45S rRNA, 5S rRNA and 7SL RNA are all parts of the B-WICH assembly. The formation of B-WICH depends on active transcription, and is implicated in the regulation of both RNA transcription by both pol I and pol III. The B-WICH provides a link between RNA and the chromatin structure.
The biological effectiveness of ionising radiation is related to the ionisation density which is defined by the linear energy transfer LET. Radiation quality factors are applied to calculate the equivalent dose in the field of radiation protection and the biologically effective dose in the field of radiotherapy. Additivity is assumed in exposure scenarios where radiations of different qualities are mixed. We have carried out a series of studies on the cytogenetic effect of exposing human peripheral blood lymphocytes to a mixed beam of the high LET alpha radiation and low LET X-rays and could demonstrate that both radiations interact in producing more chromosomal aberrations than expected based on additivity. The aim of the present investigation was to look at the mechanism of the interaction, especially with respect to the question if it is due to an augmented level of initial damage or impaired DNA repair. The level of DNA damage and the kinetics of damage repair was quantified by the alkaline comet assay. The levels of phosphorylated, key DNA damage response (DDR) proteins were also measured by Western blotting. The results revealed that alpha particles and X-rays interact in inducing DNA damage above the level predicted by assuming additivity and that the repair of damage occurs with a delay. Moreover, the activation levels of the key DDR proteins ATM, p53 and DNA PK were highest in cells exposed to mixed beams substantiating the idea exposure to mixed beams presents a challenge to the cellular DNA damage response system.
Accumulating evidence suggests a synergistic effect in cells simultaneously exposed to different types of clustered and dispersed DNA damage. We aimed to analyse the effect of mixed beams of alpha particles and X-rays (1:1 dose of each) on DNA damage response genes in human peripheral blood lymphocytes isolated from four donors. Two donors were compared upon inhibition of ATM or DNA-PK and at different sampling times. qPCR was used to measure mRNA levels of FDXR, GADD45A, BBC3, MDM2, CDKN1A, and XPC 24 h following exposure. Generally, alpha particles and mixed beams were stronger inducers of gene expression compared to X-rays, displaying saturated versus linear dose-response curves, respectively. Three out of four donors responded synergistically to mixed beams. When two donors were sampled again one year later, the former additive effect in one donor was now synergistic and no significant difference in intrinsic radiosensitivity was displayed, as determined by gamma-radiation-induced micronuclei. ATM, but not DNA-PK inhibition, reduced the radiation-induced gene expression, but differently for alpha radiation between the two donors. In conclusion, synergy was present for all donors, but the results suggest individual variability in the response to mixed beams, most likely due to lifestyle changes.
Purpose: Recent studies have shown that low temperature (hypothermia) at exposure can act in a radioprotective manner at the level of cytogenetic damage. The mechanisms of this phenomenon are not understood, but it was suggested to be due to hypothermia-induced perturbations of the cell cycle. The purpose of the present study was to detect whether a reduced frequency of micronuclei is observed in peripheral blood lymphocytes (PBL) irradiated at low temperature and harvested sequentially at 3 time points. Additionally, the level of apoptosis was estimated by microscopic analysis of the MN slides. Materials and methods: Experiments were carried out with blood drawn from three donors at the Stockholm University and from three donors at the Jan Kochanowski University. Prior to irradiation, blood samples were incubated for 20 mm and irradiated at the respective temperature (0 degrees C and 37 degrees C) with gamma rays. Whole blood cultures were set up, cytochalasin B was added after 44h of irradiation and the samples were harvested after 72,96 and 120 h of incubation time. Results and conclusions: The frequency of micronuclei was markedly lower in PBL harvested at 72h, 96 h and 120 h following irradiation at 0 degrees C as compared to 37 degrees C. This indicates that the temperature effect observed in peripheral blood lymphocytes after irradiation is not related to a temporary perturbation of the cell cycle. Also, it is not due to selective elimination of damaged cells by apoptosis.
Purpose: Low temperature (hypothermia) during irradiation of cells has been reported to have a radioprotective effect. The mechanisms are not fully understood. This study further investigates the possible mechanisms behind hypothermia-mediated radioprotection. Materials and methods: Human lymphoblastoid TK6 cells were incubated for 20 min at 0.8 or 37 degrees C and subsequently exposed to 1 Gy of gamma- or X-rays. The influence of ataxia telangiectasia mutated (ATM)-mediated double-strand break signalling and histone deacetylase-dependent chromatin condensation was investigated using the micronucleus assay. Furthermore, the effect of hypothermia was investigated at the level of phosphorylated histone 2AX (gamma H2AX) foci, clonogenic cell survival and micronuclei in sequentially-harvested cells. Results: The radioprotective effect of hypothermia (called the temperature effect [TE]) was evident only at the level of micronuclei at a single fixation time, was not influenced by the inhibition of ATM kinase activity and completely abolished by the histone deacetylase inhibition. No TE was seen at the level of gamma H2AX foci and cell survival. Conclusions: We suggest that low temperature during irradiation can induce a temporary cell cycle shift, which could lead to a reduced micronucleus frequency. Future experiments focused on cell cycle progression are needed to confirm this hypothesis.
Background: Osteoradionecrosis (ORN) of the mandible is a severe complication of head and neck radiotherapy (RT) treatment, where the impact of individual radiosensitivity has been a suggested explanation. Methods: A cohort of patients with stage II/III ORN was compared to matched controls. Blood was collected and irradiated in vitro to study the capacity to handle radiation-induced oxidative stress. Patients were also genotyped for 8 single-nucleotide polymorphisms (SNPs) in genes involved in the oxidative stress response. Results: A difference in 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dG) levels was found between the patient cohorts (p = 0.01). The SNP rs1695 in glutathione s-transferase p1 (GSTP1) was also found to be more frequent in the patients with ORN (p = .02). Multivariate analysis of the clinical and biological factors revealed concomitant brachytherapy plus the 2 biomarkers to be significant factors which influense risk of mandibular osteoradionecrosis after radiotherapy of head and neck cancer. Conclusion: The current study indicates that oxidative stress response contributes to individual radiosensitivity and healthy tissue damage caused by RT and may be predicted by biomarker analysis.
The transformation of normal cells to malignant, metastatic tumor cells is a multistep process caused by the sequential acquirement of genetic changes. To identify these changes, we compared the transcriptomes and levels and distribution of proteins in a four-stage cell model of isogenically matched normal, immortalized, transformed, and metastatic human cells, using deep transcriptome sequencing and immunofluorescence microscopy. The data show that similar to 6% (n = 1,357) of the human protein-coding genes are differentially expressed across the stages in the model. Interestingly, the majority of these genes are down-regulated, linking malignant transformation to dedifferentiation. The up-regulated genes are mainly components that control cellular proliferation, whereas the down-regulated genes consist of proteins exposed on or secreted from the cell surface. As many of the identified gene products control basic cellular functions that are defective in cancers, the data provide candidates for follow-up studies to investigate their functional roles in tumor formation. When we further compared the expression levels of four of the identified proteins in clinical cancer cohorts, similar differences were observed between benign and cancer cells, as in the cell model. This shows that this comprehensive demonstration of the molecular changes underlying malignant transformation is a relevant model to study the process of tumor formation.
The fungal pathogen Candida albicans is a common cause of opportunistic infections in humans. We report that wild-type Drosophila melanogaster (OrR) flies are susceptible to virulent C. albicans infections and have established experimental conditions that enable OrR flies to serve as model hosts for studying C. albicans virulence. After injection into the thorax, wild-type C. albicans cells disseminate and invade tissues throughout the fly, leading to lethality. Similar to results obtained monitoring systemic infections in mice, well-characterized cph1Δ efg1Δ and csh3Δ fungal mutants exhibit attenuated virulence in flies. Using the OrR fly host model, we assessed the virulence of C. albicans strains individually lacking functional components of the SPS sensing pathway. In response to extracellular amino acids, the plasma membrane localized SPS-sensor (Ssy1, Ptr3, and Ssy5) activates two transcription factors (Stp1 and Stp2) to differentially control two distinct modes of nitrogen acquisition (host protein catabolism and amino acid uptake, respectively). Our results indicate that a functional SPS-sensor and Stp1 controlled genes required for host protein catabolism and utilization, including the major secreted aspartyl protease SAP2, are required to establish virulent infections. By contrast, Stp2, which activates genes required for amino acid uptake, is dispensable for virulence. These results indicate that nutrient availability within infected hosts directly influences C. albicans virulence.
The nature of brown adipose tissue in humans is presently debated: whether it is classical brown or of brite/beige nature. The dissimilar developmental origins and proposed distinct functions of the brown and brite/beige tissues make it essential to ascertain the identity of human depots with the perspective of recruiting and activating them for the treatment of obesity and type 2 diabetes. For identification of the tissues, a number of marker genes have been proposed, but the validity of the markers has not been well documented. We used established brown (interscapular), brite (inguinal), and white (epididymal) mouse adipose tissues and corresponding primary cell cultures as validators and examined the informative value of a series of suggested markers earlier used in the discussion considering the nature of human brown adipose tissue. Most of these markers unexpectedly turned out to be noninformative concerning tissue classification (Car4, Cited1, Ebf3, Eva1, Fbxo31, Fgf21, Lhx8, Hoxc8, and Hoxc9). Only Zic1 (brown), Cd137, Epsti1, Tbx1, Tmem26 (brite), and Tcf21 (white) proved to be informative in these three tissues. However, the expression of the brite markers was not maintained in cell culture. In a more extensive set of adipose depots, these validated markers provide new information about depot identity. Principal component analysis supported our single-gene conclusions. Furthermore, Zic1, Hoxc8, Hoxc9, and Tcf21 displayed anteroposterior expression patterns, indicating a relationship between anatomic localization and adipose tissue identity (and possibly function). Together, the observed expression patterns of these validated marker genes necessitates reconsideration of adipose depot identity in mice and humans.
Human and rodent brown adipose tissues (BAT) appear morphologically and molecularly different. Here we compare human BAT with both classical brown and brite/beige adipose tissues of 'physiologically humanized' mice: middle-aged mice living under conditions approaching human thermal and nutritional conditions, that is, prolonged exposure to thermoneutral temperature (approximately 30 degrees C) and to an energy-rich (high-fat, high-sugar) diet. We find that the morphological, cellular and molecular characteristics (both marker and adipose-selective gene expression) of classical brown fat, but not of brite/beige fat, of these physiologically humanized mice are notably similar to human BAT. We also demonstrate, both in silico and experimentally, that in physiologically humanized mice only classical BAT possesses a high thermogenic potential. These observations suggest that classical rodent BAT is the tissue of choice for translational studies aimed at recruiting human BAT to counteract the development of obesity and its comorbidities.
Brown and brite/beige adipocytes are attractive therapeutic targets to treat metabolic diseases. To maximally utilize their functional potential, further understanding is required about their identities and their functional differences. Recent studies with β3-adrenergic receptor knockout mice reported that brite/beige adipocytes, but not classical brown adipocytes, require the β3-adrenergic receptor for cold-induced transcriptional activation of thermogenic genes. We aimed to further characterize this requirement of the β3-adrenergic receptor as a functional distinction between classical brown and brite/beige adipocytes. However, when comparing wild-type and β3-adrenergic receptor knockout mice, we observed no differences in cold-induced thermogenic gene expression (Ucp1, Pgc1a, Dio2 and Cidea) in brown or white (brite/beige) adipose tissues. Irrespective of the duration of the cold exposure or the sex of the mice, we observed no effect of the absence of the β3-adrenergic receptor. Experiments with the β3-adrenergic receptor agonist CL-316,243 verified the functional absence of β3-adrenergic signaling in these knockout mice. The β3-adrenergic receptor knockout model in the present study was maintained on a FVB/N background, whereas earlier reports used C57BL/6 and 129Sv mice. Thus, our data imply background-dependent differences in adrenergic signaling mechanisms in response to cold exposure. Nonetheless, the present data indicate that the β3-adrenergic receptor is dispensable for cold-induced transcriptional activation in both classical brown and, as opposed to earlier studies, brite/beige cells. This should be taken into account in the increasing number of studies on the induction of browning and their extrapolation to human physiology.
Most research concerning the effects of air pollutants on human health focuses on urban centers and on the role of vehicular and industrial emissions as major sources of pollution. However, approximately 3 billion people world-wide are exposed to air pollution from biomass burning [1]. Herein, particulate matter (PM) emitted from artisanal cashew nut roasting, an important economic and social activity worldwide [2,3], was investigated. This study focused on: i) chemical characterization of polycyclic aromatic hydrocarbons (PAHs) and their oxy-PAH derivatives; ii) time-dependent activation of DNA damage signaling and genotoxic effects, and iii) differential expression of genes involved in xenobiotic metabolism, inflammation, cell cycle arrest and DNA repair using A549 lung cells. Among the PAHs, chrysene, benzo[a]pyrene (B[a]P), benzo[b]fluoranthene, and benz[a]anthracene showed the highest concentrations (7.8-10 ng/m3), while among oxy-PAHs, benzanthrone and 9,10-anthraquinone were the most abundant. Testing of PM extracts was based on B[a]P equivalent doses (B[a]Peq). IC50 values for viability was 5.7 and 3.0 nM B[a]Peq at 24 h and 48 h, respectively. Based on this, all other experiments were conducted at doses up to 2 nM B[a]Peq. At these low doses, we observed a dose-dependent activation of DNA damage signaling (phosphorylation of Chk1) and genotoxicity (double strand breaks). In comparison, effects of B[a]P alone was observed at micromolar range. To our knowledge, no other study has demonstrated an activation of pChk1, a biomarker used to estimate the carcinogenic potency of PAHs in vitro [4], in lung cells exposed to biomass burning extracts. Persistent increased gene expression of several important stress response mediators of xenobiotic metabolism (CYP1A1, CYP1B1), inflammation (IL-8, TNF-α), cell cycle arrest (CDKN1A), and DNA repair (DDB2) was also identified. In conclusion, our data show high potency of biomass burning PM to induce cellular stress including genotoxicity, and more potently so when compared to B[a]P alone. Our study provides new data that will help elucidate the mechanism of lung cancer development associated with biomass burning. In addition, the results of this study support the establishment of new guidelines for human health protection in regions strongly impacted by biomass burning.
Approximately 3 billion people world-wide are exposed to air pollution from biomass burning. Herein, particulate matter(PM) emitted from artisanal cashew nut roasting, an important economic activity worldwide, was investigated. This study focused on: i) chemical characterization of polycyclic aromatic hydrocarbons (PAHs) and oxygenated (oxy-) PAHs; ii) intracellular levels of reactive oxygen species (ROS); iii) genotoxic effects and time- and dose-dependent activation of DNA damage signaling, and iv) differential expression of genes involved in xenobiotic metabolism, inflammation, cell cycle arrest and DNA repair, using A549 lung cells. Among the PAHs, chrysene, benzo[a]pyrene (B[a]P), benzo[b]fluoranthene, and benz[a]anthracene showed the highest concentrations (7.8–10 ng/m3), while benzanthrone and 9,10-anthraquinone were the most abundant oxy-PAHs. Testing of PM extracts was based on B[a]P equivalent doses (B[a]Peq). IC50 values for viability were 5.7 and 3.0 nM B[a]Peq at 24 h and 48 h, respectively. At these low doses, we observed a time- and dose-dependent increase in intracellular levels of ROS, genotoxicity (DNA strand breaks) and DNA damage signaling (phosphorylation of the protein checkpoint kinase 1 – Chk1). In comparison, effects of B[a]P alone was observed at micromolar range. To our knowledge, no previous study has demonstrated an activation of pChk1, a biomarker used to estimate the carcinogenic potency of PAHs in vitro, in lung cells exposed to cashew nut roasting extracts. Sustained induction of expression of several important stress response mediators of xenobiotic metabolism (CYP1A1, CYP1B1), ROS and pro-inflammatory response (IL-8, TNF-α, IL-2,COX2), and DNA damage response (CDKN1A and DDB2) was also identified. In conclusion, our data show high potency of cashew nut roasting PM to induce cellular stress including genotoxicity, and more potently when compared to B[a]P alone. Our study provides new data that will help elucidate the toxic effects of low-levels of PAH mixtures from air PM generated by cashew nut roasting.
The connection between T cell activation, plasma membrane order and actin filament dynamics was the main focus of this study. Laurdan and di-4-ANEPPDHQ, membrane order sensing probes, were shown to report only on lipid packing rather than being influenced by the presence of membrane-inserted peptides justifying their use in membrane order studies. These dyes were used to follow plasma membrane order in live cells at 37°C. Disrupting actin filaments had a disordering effect while stabilizing actin filaments had an ordering effect on the plasma membrane, indicating there is a basal level of ordered domains in resting cells. Lowering PI(4,5)P2 levels decreased the proportion of ordered domains strongly suggesting that the connection of actin filaments to the plasma membrane is responsible for the maintaining the level of ordered membrane domains. Membrane blebs, which are detached from the underlying actin filaments, contained a low fraction of ordered domains. Aggregation of membrane components resulted in a higher proportion of ordered plasma membrane domains and an increase in cell peripheral actin polymerization. This strongly suggests that the attachment of actin filaments to the plasma membrane induces the formation of ordered domains. Limited cholesterol depletion with methyl-beta-cyclodextrin triggered peripheral actin polymerization. Cholesterol depleted cells showed an increase in plasma membrane order as a result of actin filament accumulation underneath the membrane. Moderate cholesterol depletion also induced membrane domain aggregation and activation of T cell signaling events. The T cell receptor (TCR) aggregation caused redistribution of domains resulting in TCR patches of higher order and the bulk membrane correspondingly depleted of ordered domains. This suggests the preexistence of small ordered membrane domains in resting T cells that aggregate upon cell activation. Increased actin polymerization at the TCR aggregation sites showed that actin polymerization is strongly correlated with the changes in the distribution of ordered domains. The distribution of the TCR in resting cells and its colocalization with actin filaments is cell cycle dependent. We conclude that actin filament attachment to the plasma membrane, which is regulated via PI(4,5)P2, plays a crucial role in the formation of ordered domains.


text:
Check additional details and price.
Original WildCat
This is copy of Skier design. Not cheap.
Very good feedback.
Looks good guys. I think I'll pull the trigger if it gets available through PV deals again.
Thanks feha!
@miko i had pay about $40 usd in import tax ... item is not posted from UK ...
I also just received my Ifootage Wild Cat. First I should say I am not an expert, but have used an Indie half-moon type of stabilizer before that was around $80 and I was not happy with it. This Small Carbon stabilizer is absolutely great, albeit at almost 5 times the cost; but as they say you get what you pay for. I am in the USA. Shipment was fast, came via Los Angeles with DHL, and I had to pay no fees when DHL delivered it to me just a couple of hours ago.
I have made a pretty amateurish video of my unboxing and my first attempt at balancing my GH3 with the PL 25mm/f1.4 lens without reading the manual. After reading the manual I was able to do much better with the balancing and it works pretty sweet for the GH3.
I have no hesitation recommending this stabilizer for the GHx cameras. It is easy to balance. Here are hints: 1) each of the three legs have three weights, a large, a medium, and a small; use the medium and the small for the GH3. 2) Place the column height at around 40 or at a point where the stabilizer legs barely hang down; this setting makes the balancing of the camera very sensitive so you can adjust its position to balance it really well. Then 3) Increase the size of the column until the drop time is 1 to 2 seconds. My GH3 is in perfect balance now.
Here's the link to the vimeo unboxing and initial test video Ifootage Wild Cat
Feha, one question: how much you pay for import taxes? or the pack come from inside Europe (UK ?)?
Will make a video review when i have more time , here comes some images ...
VK, do you expect to have a deal on this again? This is looking like a great stabilizer for use with the BMD Pocket Cam.
edit - nevermind I'm an idiot, it's in CatchIt right now!
@tinyrobot , yes, but i need some more free time ... This is nice piece of work, well build, however I'm not much expert of this kin stabilizer so if footage gets bouncing I's is my fault ...
@feha I look forward to your review. Please include test footage.
I got my IFootage WildCat today :-) Excellent quality. Need to make some tests and right balancing ...
@Nayche , I have ordered one and waiting ... Will let you know ...
Has anyone used this yet? I am quite intrigued.
It'll must be available on tomorrow deals.
@Vitaliy_Kiselev , thank you very much for making it available for us on PV. (i might wait for carbon one ...)
We have alu already available for good price on deals.
Soon we'll have carbon one. But it is far from cheap.
One day they'll make a pink version. Then I'll get one.
Aluminium version with slightly more simple legs design
@vicharris , i understand :-) I like the construction and quality, hope we will have this on deals, I'm curios to test this one. I's very compact. You are right, the product demo video should show the full potential of this product. As they do it, makes one suspicious if it will work OK ...
I understand it takes practice but if you can't operate your own product properly and show a rough demo video, why show one at all? The guys over at Wonderland make great promo videos, why can't these guys get someone to come in and use it properly? That's what I meant. Trust me, I'm sure as soon as I get my Glidecam in my footage will look like a cruise liner listing left and right but I'm not selling a product. So far that video and product look no better than some youtube video I saw where a guy was using the wonderfully crafted Opteka Steady thing. :)
Maybe it's just what @Rambo said. All the running around stuff instead of slow, controlled movements. I'd like to see some better, more controlled footage because it looks like it's built very well. I'd drop the fanny pack though ;)
@vicharris , I think this steadyCam is OK but as any of them it requires a bit of experience/practice to get desired results. :-)
Is it me or does this rock back and forth pretty bad? I felt kind of sick watching it. The guys over at Wonderland or however you spell it have much better demo videos IMO.
The actual floating is what makes most people feel ill not the slight amount of rolling. Ideally, steadyCam footage should be slow not fast, why people feel they have to run or shoot up-close subjects is anyone's guess. Most camera motion in film is slow, whether that be from sliders, jibs, dollies or steadyCams.
Is it me or does this rock back and forth pretty bad? I felt kind of sick watching it. The guys over at Wonderland or however you spell it have much better demo videos IMO.
@Vitaliy_Kiselev , hope you get deals for this one. I like it very much. Found this sample:
Wow the size is just phenomenal...it's too bad it's quite expensive. I recently got the Wieldy Carbon fiber and it's quite big compared to this Wild Cat.
It looks like you're new here. If you want to get involved, click one of these buttons!

subset: commoncrawl


text:
The European Cup is an annual baseball tournament, sanctioned and created by the Confederation of European Baseball (CEB). The tournament features the top teams of the professional baseball leagues in Europe.
Starting in 2008, the tournament is now held in two locations. The best two teams from each tournament then compete in the European Champion Cup "Final Four", with the winner receiving the European Champion Cup.
In 2013, the "Final Four" format was replaced with a best-of-three series between the winners of each tournament.
The first season of the tournament was held in 1963 and was won by the Picadero Jockey Club Barcelona.
External linksEdit
Community content is available under CC-BY-SA unless otherwise noted.


text:
- You are here
- Home
- [Magnetopia] Magnetic Felt Series(Animal/Fruit/Traffic) Select 1
The Best Online Store for Fans of Korea
This is soft and comfortable. As it has the magnet in it, it can be easily stuck onto the board. It is the magnetic felt series.
1. Components of product : Animals (10 pieces), Fruits (10 pieces), Traffics (9 pieces)
2. As the soft felt has the magnet in it, that it can easily be attached onto the board.
3. As it has various colors, soft touch and refined design, the children can grow their perception capability of things naturally through the role playing.
4. It is designed to store the pieces easily after play. It is easy to carry as it has the strap.
▷ Notes ◁
- Keep the product away from source of fire.
- Keep the product away from the mouth of children.
- Avoid the product from the direct sunlight.
- Make sure that it is not wrinkled by hand.
- Keep the product away from the credit card or any magnetic things.
|Volume Weight


text:
RYA Go Cruising - A young crew's guide
RYA Go Cruising! covers all the essentials required to help young people become interested and valuable members of the crew on either sail or motor cruisers.
It will prove very useful for the cruising family as its beautifully illustrated storyboard format makes learning this sometimes complicated subject both accessible and fun.
Produced by celebrated artist and experienced sailor Claudia Myatt, RYA Go Cruising is ideal for 7 -12 year olds.
We Also Recommend


text:
Note to self: Pamela Isley's cheap and a lousy tipper.
This guide to public transporation has been sponsored by Gotham City Sirens #11 (July 2010) by Paul Dini, Andres Guinaldo, and Raul Fernandez.
Tags: Catwoman, comic, comic book, Comics, comics books, DC, DC Comics, DC Universe, Doctor Pamela Lillian Isley, Dr. Pamela Isley, Dr. Pamela Lillian Isley, Pamela Isley, Pamela Lillian Isley, Poison Ivy, Selina Kyle


text:
2 hrs of Video Content
In 18 episodes, Forrest breaks down his mindset & approach to commercial photography, creating concepts and being yourself on social media.
4 All-New Presets
Get access to Forrest's newest Lightroom presets. You won't find them anywhere else.
Workshop PDF
A downloadable PDF accompanies the workshop with module recaps, notes, and links to Forrest's favorite tools.
"With the right mindset, you can turn an obstacle into an opportunity."
Forrest Mankins
Meet Forrest
Take a look through even just a handful of his photographs, and it immediately becomes apparent that Forrest has developed a distinctly personal style. While our previous workshops have all focused on the essential skills needed to capture a moment, this one sets out to show you how to actually create one.
In this deep dive into the way Forrest views the world, you'll learn his strategies and high-level vision that will help you elevate your work to the next level.
From forming a creative concept from scratch, to finding shoot locations near your home, to confidently directing subjects — you'll learn how to take the vision you have in your head and put it into your photography.
This is where developing your own unique style as an artist begins.
Watch A Preview
Enroll Now
Enroll now to get lifetime access to the workshop content.
Workshop Curriculum
Alex introduces one of his favorite creators, Forrest Mankins, and gives some backstory to their relationship and the workshop.
Alex and Forrest have tea and kickoff the workshop.
You can do an amazing job at planning out an entire shoot, but by choosing a less than amazing image as a final result you've wasted the idea and your own time.
With the right mindset, you can turn an obstacle into an opportunity. Learn from Forrest's experience with burnout and find out what brought him back into creativity.
Forrest explains how taking the time to write down brief outlines of your ideas can help you come back to photography with a new passion and direction.
It's important to think about the point or the substance of a photo as your primary goal first, with the location being secondary. We don't need everything to be perfect, just the elements we're choosing to put in our frame.
Forrest helps you find/identify people to shoot with, even if you've never done it before. He explains how and why to properly communicate everything from your concept to logistics.
Go above and beyond with how you treat people, and you'll make new creative relationships that put you on the path to creating better work than you've ever made.
Forrest demystifies "building a team" and suggests practical and easy ways to do so.


text:
The City of Helotes submitted a Public Information Act Catastrophe Notice and an Extension Notice to the Office of the Attorney General. The requirements of the PIA were suspended during the following period: Friday, March 27, 2020 - Friday, April 10, 2020. Additionally, the Office of the Attorney General issued a Clarification Notice that provides governmental bodies with guidelines when calculating business days.
Public Information Act Requests:
Requests for public information must be submitted in writing and delivered to the City Secretary's Office in person, via email, via fax, or by completing and submitting the form below.
General Request for Public Information Form
To submit a public information request by mail, please mail to: City Secretary P O Box 507, Helotes, TX 78023. A request for public information may also be sent via fax to 210-695-2123, delivered in person at City Hall, 12951 Bandera Road, or sent via email to email@example.com.
The City responds to Requests for Public Information within ten (10) business days of the initial request. Once notified by the City Secretary's Office that the report is ready, please pick up the report at the Development Services window at City Hall. If the requestor elects, the City Secretary's Office may also email or mail the report to the requestor. Requestors should provide a stamped, self-addressed envelope with the Request for Public Information form when requesting the documents to be mailed.
A fee of ten cents ($0.10) per page applies to all General Public Information and Incident Reports. A fee of six dollars ($6.00) applies to all Police Accident Reports. Additional certification fees may apply. Please contact the City Secretary's Office for additional information.
EMS Reports:
For EMS Incident reports, picture identification of the patient listed in the report must be provided when picking up the report. If you are not the patient listed in the report, a valid "Power of Attorney" document authorizing the release of said records to you must be provided before the City will provide you the report.
EMS Incident reports may also be mailed to the requestor. When submitting the initial Authorization Form, please attach a copy of your picture identification, a check in the amount of all requisite fees, and a stamped, self-addressed envelope.
Questions regarding EMS billing should be directed to the City's billing / collection agency, Business and Professional Service, by telephone at (800) 284-0485 between 8:00 A.M. and 5:00 P.M. CST.
Helotes Fire /EMS Protected Health Information Notice
Fire / EMS Incident Report Request Form


text:
Beauty at Tesco? So I've seen the aisles lined with Rimmel and Maybelline but I confess, it's not the first place I think of going to for my beauty fix. I normally consider Boots as my beauty Mecca.
That was until I tried BD Trade Secrets, Tesco's own beauty range.
Evolved from the original Barbara Daly range, BD Trade Secrets is a collection of sleek looking essentials at very affordable prices – prices start from £2.99.
We've been brought up to think that expensive means better but is that the case? Check out my highlights from the range and decide for yourself!
I used the limited edition BD Love Lips Love Cheeks palette as part of the Valentines Day Makeup Tutorial I did for Yahoo.
Let's be honest, it looks like something that I once got free with Bliss magazine – but the content quality is great! Such a shame that the lid is transparent, if it was a glossy black it would just look ten times more expensive.
The formulations are well pigmented but easily blendable and the colours are great – I just wish it didn't look so cheap as the results are very good!
The star of the show is the Va Va Boom mascara – top name, well done Tesco.
I've never seen a brush like it, the Va Va Boom is curved in the middle and fans out at both ends, what is this!!!? What is it? Genius for curling lashes, that's what.
It gives absolutely heaps of volume from the first coat, so fans of the "natural look" and those with really fair eyelashes might want to steer clear. However, if like me you like your lashes full on and FAT, then this mascara does the job.
It reminds me a bit of Rimmel Volume Flash Mascara but with a double curve not just one curve.
Finally, this BDTrade Secrets lipstick is also worth a mention. The quality and the coverage are decent and it doesn't taste cheap like some cheap lipsticks do but it's not breaking any ground here.
I did really like the colour though, it's called COUTURE and it's a dusky pink/brown shade aka perfect for Kylie Jenner lips. I rubbed my lips together and it created this great lip liner effect – boom.
All in all, a very respectable range – with prices reflecting the lack of investment in packaging rather than the product itself.
I would definitely look into buying these, such a shame that my local Tesco doesn't stock the BD Trade Secrets Range! Thank god for Online Shopping!
What are your thoughts on supermarket beauty?Do you have any favourite BD Trade Secrets products? You'll have tweet me or let me know via Facebook!
By Zoe Dubs


text:
By Design has two staff members who have close family members currently fighting Cancer and we want to show our support to them as well as countless others in the community who are fighting cancer, and their caregivers. We know the fight is not easy, and By Design Ltd want to do our part to help so we have entered a team in this year's Relay for Life on May 17th and 18th
Please consider supporting our team "Design for Life" with a donation or let us know if you would like to join our team on the track. Stay tuned for our additional fund raising initiatives as well!


text:
During our initial exploratory visit we took special note of the intergenerational program. The positive impression it gave us was a strong incentive in having Rui-yu attend. From the time she joined the program she has enjoyed her activities with the children tremendously.
We are pleased that the center has a recliner room since Rui-yu often naps in the afternoon. Also, the frequency and variety of the field trips provide opportunities that she might not otherwise be able to enjoy.
We especially appreciate the accommodations by the staff due to her mid-stage Alzheimer's and limited knowledge of English. She has benefited from a stimulating environment and the gentle, caring attention of the staff.
We are very happy with her experience at the Senior Day Center; this rich and robust program has it all and we give it high marks. Please feel free to share our comments with the staff.


text:
As we end the first month of the year, we are no seeing several invites for classes and workshops exclusive for sewing enthusiasts. Some of these workshops come at a hefty rate, while some are also conducted within the community with people you already know. As a sewing hobbyist myself, I have also come across with these invites, and had my own apprehensions at first. I've been so used to learning things by myself, so why would I need to learn it from someone else, instead?
However, I realized that much of my learning were also through the experiences of other people. After one sewing workshop, I have been a regular attendee of these workshops already, both paid and free. Here are the things that you can expect and get from attending sewing classes and workshops:
1) Personalized techniques
When we are simply hopping from one online pattern to another, we tend to also follow the sewing style of the previous person who did it. When you go to these classes, you'll realize that there are actually more sewing techniques that you can try, that are more appropriate for your personal choices, body type, and goals. Sewing gurus from the workshop will be more than willing to help you with this.
2) Expert unabashed tips on fabric selection
Fabric choice as always been my weakness. If you are like me, then you will definitely greatly benefit from these classes as the guru, as well as your fellow learners have all the right experience in selecting the perfect fabric for a certain sewing project. Some workshops even have their own fabric choices for you to try.
3) Feedback
This for me is one of the most precious takeaway from any sewing workshop, which is something that i certainly cannot get by simply learning at home. You get constructive criticisms and feedback that will only improve your skills and confidence in the craft.
4) Sewing friends
With sewing workshops, you take a break from merely connecting with your fellow sewing enthusiasts virtually. It is refreshing to meet new people, who may have different skill levels as you, but still share the same passion in sewing.
Another year is about to start, and the fashionistas in us are naturally already panicking about the...
This post is certainly long overdue. Over the course of sewing and blogging about it, I've been as...
For those people who have strong admiration towards sewing, the holiday season is actually a perfect...
There is no better apparel piece that can easily spell power dressing than a good blazer. It can be ...
We've seen a lot of them from boutique shops and at some point fashion runways, and somehow those ...


text:
Even a Virat Kohli century couldn't take India past the Aussie total, resulting in the series coming to an interesting point where Ravi Shastri would say all three results are possible. However, Australians not finding a way out in the middle-overs would be a huge concern for the visitors.
Can Australia find a way out of the debris?
Justin Langer knows a thing or two about pumping his wards. During his time as the coach of Western Australia and Perth Scorchers, his man-management was one of the primary reasons why the teams from WA had so much success to boast. In 2018, as a member of the West Coast Eagles footy, an AFL club in Australia, he gave a pep talk to the team which echoed in the players' ears in such a way that it ended up West Coast winning the title. Not even getting into elite honesty, it is the thing that he failed to inculcate into the Australian side.
After the visitors got up a great start by scoring 193 runs for the opening wicket between Usman Khawaja and Aaron Finch, they failed to accelerate the gear and despite a short cameo by Glenn Maxwell, they ended up scoring only 313 runs in 50 overs. They have a good enough pace bowling unit which can be as effective as any in the world on their day, but their batting is failing to pick up the debris at the right time. The spin strangulation worked against their plans and their failure to play any good pace bowlers resulted in India winning the middle-over exchange.
The problem is not with the way Australia are operating their batsmen in the middle-over, but it has something to do with the fact that they are not finding a way to tackle once a wicket falls. With the wicket being a road in the first few overs, it was only apparent that once the effect of heat goes down, it would start turning and the cracks would come into play. Instead of trying to play out the only spinner gamely, they seemed clueless to end up with a score which, despite the result, was not a safe one.
It was supposed to be a cricket match, not an athletic meet, JSCA
Playing cricket in India during early summer is an interesting proposition. The ball comes to the bat nicely and with the cracks opening and dew coming into effect, it becomes interesting towards the end in an ODI run-chase. However, taking a
In Hyderabad, the ball moved with a pace that would make Usain Bolt proud. The lack of moisture added to it as most of the balls had literally become
When the surface becomes so fast, a fielder knows that he wouldn't be able to get close to the ball. The less fast - not a slow one for sure - ensures an even field and the small joys like saving a couple of runs for the team, or the fielder stretching himself for a couple of yards adds meaning to the game. Could Mohali and Delhi take a clue from it?
Where was the intent, Jadeja?
One of the joys of watching Ravindra Jadeja on a cricket field is that even on the bad days, he never shied away from taking the challenge head-on. His quest to prove himself and taking the high-ground has been insatiable and there is a genuine leap of joy whenever he does manage to get what he always wanted. Since returning to the limited-overs set-up during India's Asia Cup campaign last year, it is very hard to find a moment where the Saurashtra boy hasn't given his all.
The ability to run faster than usual to save the boundaries, the intent to throw to effect that odd run-out or
When Jadeja came out to bat, just after the dismissal of Virat Kohli, India were 219/6 in 37.3 overs, needing 95 runs off 75 balls. With Vijay Shankar at the other end, Jadeja could have played the second fiddle perfectly in order to take the bounce off his shoulders. However, instead of playing gamely, he rocked back and found
Cricket FootBall Kabaddi
Basketball Hockey
Perennially waiting for next holiday on the beach, I found a job which will pay the bill. It is an easy job. I just watch and blabber about cricket all day and act like I can write. Easy money.


text:
About us
Our Clients
Contacts us
Connecting YOU to Great Careers
Connect with us for great careers in Sales/Marketing & Pre Sales
Testimonials: "Great Service! Connect Takes the pain of understanding the needs and identifying the more suitable candidates - they take that extra step" -- Neraj Sharma, MD Lenovo India | Connect has ben vey succesful in connecting us with several top notch professionals across the country, who aspire a career in EMC! -- Pramod Fernandes, Dir HR EMC | "Connect has played an important role in fulfilling our top talent search and staffing requirements. To match the right person he right job is the key to our business success and over the past two years they have partnered with us to achive this" -- Shaily, MD BEA India | "Connect has been an invaluable partner for us in India. Our company's presence in India is only recent but we have been able to execute on aggressive growth plans with the support of connect, who have helped not only by leading the recruiting effort in three cities for us, but by guiding us on variety of human resource issues." -- Jim Lenox, GM South Asia VMware
Powered by


text:
Morocco produced record 128,000 tons of dates in 2016, a 16 pc increase compared to 2015, according to the Ministry of Agriculture and Fisheries.
This record production was achieved on an area of 50,000 ha of palm groves, compared to an average annual production of 90,000 tons until 2009, the ministry pointed out in a statement.
The date sector contributes 40-60 pc to the agricultural income for more than 2 million people, creates 1.6 million working days per year for the rural population in the most fragile regions of Morocco, and represent nearly 40 pc of the national territory.


text:
UVA Chamber Music Series - I-Jen Fang, percussion
On Wednesday, March 28th at 8:00 pm, the University of Virginia Chamber Music Series presents a concert by I-Jen Fang. Ms Fang will be joined by Joanna Beaver, viola; Julianna Lee, percussion; Mark Panetti, percussion and Kelly Sulick, flute. I-Jen Fang will perform a panoply of percussion delights. Her program features Lou Harrison's haunting Ariadne for Flute and Percussion alongside John Cage's Child of Tree for percussion and amplified plant materials, Start Dancing by Zae Munn for viola and percussion (with Johanna Beaver) and Crumbs by Leah Reid. The program will also feature Mark Panetti's To Live to Be Free for three percussionists.
Described as an "intrepid percussionist" by Fanfare Magazine, I-Jen Fang has a career as a solo performer, chamber musician, orchestral player, and teacher. She joined the faculty of the McIntire Department of Music at the University of Virginia in 2005 and as Principal Timpanist and Percussionist of the Charlottesville Symphony.
As a soloist, I-Jen has performed as a marimba soloist in Taiwan, U.S., Austria, France, Hungary, Romania, and South Africa. She was also the featured marimba soloist with the Charlottesville Symphony in 2006 and 2010. As a chamber musician, I-Jen has performed or recorded with artists such as Keiko Abe, William Cahn, Christopher Deane, Mark Ford, Heini Kärkkäinen, Mike Mainieri, Jan Müller-Szeraws, Diane Pascal, Carsten Schmidt, Ed Smith, Michael Spiro, NanikWenton, Nyoman Wenton, Attacca Percussion Group, and DaCapo Chamber Players. She has appeared in Heritage Theater Festival, Staunton Music Festival, University of Virginia Chamber Music Series, Percussive Arts Society International Convention and Regional PAS Day of Percussion.
Ariadne for Flute and Percussion (1987)
|Lou Harrison (1917-2003)
Child of Tree (1975)
|John Cage (1912-1992)
Start Dancing (1999)
|Zae Munn (b. 1953)
On Points (2017)*
|Christopher Deane (b. 1957)
Crumbs (2017)
|Leah Reid (b. 1985)
To Live to Be Free (2016)*
|Mark Panetti (b. 1996)
|*World Premiere
An advocate of New Music, I-Jen is currently the director of the UVA New Music Ensemble. The ensemble has recently collaborated with composer/improviser George Lewis, and performed for composers such as Phillip Glass and Christian Wolff. She has also commissioned and/or premiered works by Matthew Burtner, Ted Coffey, Kevin Davis, Christopher Deane, Erik DeLuca, Aurie Hsu, Sarah O'Halloran, Chris Peck, Judith Shatin, Brian Simalchik, Ed Smith, and D.J. Sparr.
Born in Taipei, Taiwan, I-Jen began her musical education at age six taking piano. Taking up percussion at the age of nine, she came to the United States at age fifteen to pursue her Bachelor of Fine Arts degree in Percussion Performance at Carnegie Mellon University. She received her Master of Music degree from Northwestern University and her Doctor of Musical Arts degree from the University of North Texas where she served as a teaching fellow.
I-Jen is an Innovative Percussion artist.
Johanna Beaver began teaching in 1993 while pursuing her Master of Music degree in violin performance at the North Carolina School of the Arts. Since then, she has served on the faculties of the University of Virginia, University of Mary Washington, Interlochen Arts Camp, Ann Arbor Summer Arts Camp, Opus 118 Harlem School of Music, and Stony Brook Pre-college.
Johanna enjoys playing all genres of music but her favorite is chamber music. As a founding member of the Afton String Quartet with Wintergreen violinist Alison Hall, Ms. Beaver has helped to create a series of educational concerts called ASQ Away!, reaching under-resourced schools in South-Central Virginia.
As an orchestral violist, Ms. Beaver performs regularly in the Roanoke and Richmond Symphonies, The Oratorio Society of Virginia, and the Wintergreen Festival Orchestra. Currently, Ms. Beaver is faculty at Mary Baldwin College, Charlottesville Day School, and teaches private lessons from her studio at home in Charlottesville. A frequent guest artist, Ms. Beaver gives master-classes and recitals throughout the eastern U.S. Ms. Beaver holds a Doctor of Musical Arts degree in viola performance from the State University of New York at Stony Brook where her mentors were Mitchell Stern, Katherine Murdock, and the Emerson String Quartet.
Kelly Sulick currently teaches at the University of Virginia and serves as Principal Flute in the Charlottesville Symphony. Prior to her appointment, she served as Principal Flute with the Evansville Philharmonic Orchestra and as Consortium Instructor of Flute at the University of Evansville for three years. She earned her Master of Music degree in Flute Performance from the University of Southern California; prior to her graduate studies, she earned a Bachelor of Music degree in Flute Performance and a Bachelor of Arts degree in English Language and Literature from the University of Michigan, where she graduated with highest honors and was named a James B. Angell Scholar for her academic achievements.
An active orchestral musician, Ms. Sulick has performed with the Richmond Symphony Orchestra, the Ash Lawn Opera Orchestra, the Owensboro Symphony Orchestra, Orchestra Kentucky, and the Livingston (MI) Symphony. She completed three seasons as principal flute with the Young Musicians Foundation Debut Orchestra in Los Angeles, California. Hailed as "flawless" by the Evansville Courier Press for a concerto performance with the Evansville Philharmonic Orchestra, Sulick has also appeared as a concerto soloist with ensembles throughout the country, including the Southern Illinois Music Festival Orchestra, the Charlottesville Symphony, and most recently the Ann Arbor Symphony Orchestra and alongside Sir James Galway at the Kennedy Center.
A champion of new music, she has commissioned and recorded dozens of works for solo flute and flute with electronics, and has premiered several additional works for solo flute and chamber ensemble. She has performed at the SEAMUS National Conference, the Atlas INTERSECTIONS festival, the TomTom Founders Festival, the Technosonics Festival, and the Minimalist Jukebox series, a music festival curated by John Adams. She has worked with such notable composers as Leslie Bassett, William Bolcom, Matthew Burtner, Evan Chambers, Michael Daugherty, Karel Husa, Daniel Kessner, Lowell Liebermann, Judith Shatin, and Frank Ticheli, among others.
Ms. Sulick won second place in the 2010 National Flute Association's Young Artist Competition, and was awarded the prize for the best performance of Kristin P. Kuster's "Perpetual Afternoon." She can be heard on several compact discs, including William Bolcom's "Songs of Innocence and of Experience" under Leonard Slatkin, a Naxos release that received four Grammy awards including Best Classical Album. Her principal teachers include Marina Piccinini, Amy Porter, and Jim Walker.
Julianna Lee is a fourth year student at the University of Virginia. She will graduate this Spring with a degree in Music with a Performance Concentration in percussion. She became interested in percussion in the fifth grade and quickly developed a love for the art. In high school, she made All District Band three times, specifically third chair percussion junior year and second chair senior year. She hopes to pursue a career in the music industry as a member of a military band, an orchestra, or a percussion group. In the spring of 2017, she was one of two percussionists selected from a pool of applicants across the nation to perform with the U.S. Air Force Band in their Collegiate Symposium. She has thoroughly enjoyed furthering her musical studies here at UVA, namely in courses such as Music Ethnography and History of Jazz. She is also a member of the UVA Percussion Ensemble, Wind Ensemble, Concert Band, and Marching Band. She performed with The Oratorio Society of Virginia in their Christmas at the Paramount performance this December 2017. In addition, she is the Dance Captain of a group named AKAdeMiX, a hip-hop dance crew at UVA, and gets to travel across the east coast to compete and perform.
Mark Panetti is a third year UVA student. He will graduate this Spring with a double major in math and computer science. He has been playing percussion since middle school and has played in numerous ensembles such as marching band, jazz band, symphonic band, indoor percussion, and pit orchestra. In high school, he was first chair on trap set in VBODA District 16 jazz and first chair on percussion in the District band his freshman and senior years, respectively. He started composing towards the end of his junior year, taking an AP Music Theory course the following year. He continues to enjoy studying theory and composing for percussion here at UVA, and he is currently a member of both the UVA Percussion Ensemble and the Charlottesville Symphony. He particularly enjoys progressive rock and progressive metal music and incorporates a similar musical style into his compositions.
Tickets for all concerts are $15 general admission, $13 for UVA Faculty and Staff, $8 for students, and are free for UVA students who reserve in advance and for those under 18. Tickets can be purchased by calling the Arts Box Office at 434.924.3376, or by visiting artsboxoffice.virginia.edu.
For additional information, please visit http://music.virginia.edu/uvacms.
Old Cabell Hall is located on the south end of UVA's historic lawn, directly opposite the Rotunda (map). Parking is available in the Central Grounds parking garage on Emmet Street, in the C1 parking lot off McCormick Road, and in the parking lots at the UVA Corner.
Please call the Music Department at 434.924.3052 for more information.
All events are subject to change.