Seeing is believing; tracking metalloproteins by fluorescent probe in vivo and in vitro

Abstract no. EuAsC2S-12/S1-OP22Extensive genome research has shown that around 1/4 to 1/3 proteins are metalloproteins (or metal-binding proteins) with various metal ions incorporated with proteins for either structural or functional purposes. Thus, metalloproteomics/metallomics are developed to investigate the molecular mechanism of metal-related biological processes and the entirety of metal/metalloid species within a cell or tissue type[1]. Fluorescence labeling is probably the best method in view of its capability in providing rapid and sensitive identification in living biological systems. In spite of the development of fluorescent proteins, synthetic small-molecule fluorescence agents have been utilized to identify specific targets in cells, while metal-chelation methodology has been extensively applied to the study of metal-oriented biological process[2]. Although different types of metal-responsive sensors have been developed to label cellular metals[3], tracking of metal-binding proteins in living cells by fluorescence is still highly anticipated. In this work, novel fluorescent probe was designed to label metalloproteins both in vivo and in vitro. The protein partners of several metal ions such as Ni2+ (Histidine-rich proteins in particular), Bi3+, Cr3+ have been identified by the agent. The fluorescent agent exhibited “turnon” response to the targets in SDS-PAGE, and its excellent permeability enabled “lighting up” of targeted proteins in living cells, providing valuable information on metalloprotein spatial distribution in biology.postprintThe 12th EuroAsia Conference on Chemical Sciences (EuAsC2S-12), Corfu, Greece, 16-21 April 2012

Integrative approach for the analysis of the proteome-wide response to bismuth drugs in Helicobacter pylori

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Melt blending and characterization of carbon nanoparticles-filled thermoplastic polyurethane elastomers

In this work, thermoplastic polyurethane (TPU) elastomers reinforced with carbon nanosized particles were produced by a special melt blending technique. A TPU was melt blended with high-structured carbon black and carbon nanofibres (1 wt%). A miniature asymmetric batch mixer, which applies high shear levels to the melt, ensured good particles dispersion. The TPU material systems were then thoroughly characterized using thermogravimetric analysis, differential scanning calorimetry, tensile mechanical testing, electrical resistance measurements and flammability tests. The different nanofillers exhibited different influences on the TPU properties, these materials featuring interesting and improved multifunctional behaviours, with high propensity for large deformation sensors applications.This work was supported by FCT – Portuguese Foundation for Science and Technology through projects NANOSens – PTDC/CTM/73465/2006

UBR2 of the N-End Rule Pathway Is Required for Chromosome Stability via Histone Ubiquitylation in Spermatocytes and Somatic Cells

The N-end rule pathway is a proteolytic system in which its recognition components (N-recognins) recognize destabilizing N-terminal residues of short-lived proteins as an essential element of specific degrons, called N-degrons. The RING E3 ligases UBR2 and UBR1 are major N-recognins that share size (200 kDa), conserved domains and substrate specificities to N-degrons. Despite the known function of the N-end rule pathway in degradation of cytosolic proteins, the major phenotype of UBR2-deficient male mice is infertility caused by arrest of spermatocytes at meiotic prophase I. UBR2-deficient spermatocytes are impaired in transcriptional silencing of sex chromosome-linked genes and ubiquitylation of histone H2A. In this study we show that the recruitment of UBR2 to meiotic chromosomes spatiotemporally correlates to the induction of chromatin-associated ubiquitylation, which is significantly impaired in UBR2-deficient spermatocytes. UBR2 functions as a scaffold E3 that promotes HR6B/UbcH2-dependent ubiquitylation of H2A and H2B but not H3 and H4, through a mechanism distinct from typical polyubiquitylation. The E3 activity of UBR2 in histone ubiquitylation is allosterically activated by dipeptides bearing destabilizing N-terminal residues. Insufficient monoubiquitylation and polyubiquitylation on UBR2-deficient meiotic chromosomes correlate to defects in double strand break (DSB) repair and other meiotic processes, resulting in pachytene arrest at stage IV and apoptosis. Some of these functions of UBR2 are observed in somatic cells, in which UBR2 is a chromatin-binding protein involved in chromatin-associated ubiquitylation upon DNA damage. UBR2-deficient somatic cells show an array of chromosomal abnormalities, including hyperproliferation, chromosome instability, and hypersensitivity to DNA damage-inducing reagents. UBR2-deficient mice enriched in C57 background die upon birth with defects in lung expansion and neural development. Thus, UBR2, known as the recognition component of a major cellular proteolytic system, is associated with chromatin and controls chromatin dynamics and gene expression in both germ cells and somatic cells

A major genetic locus controlling natural Plasmodium falciparum infection is shared by East and West African Anopheles gambiae

Background: Genetic linkage mapping identified a region of chromosome 2L in the Anopheles gambiae genome that exerts major control over natural infection by Plasmodium falciparum. This 2L Plasmodium-resistance interval was mapped in mosquitoes from a natural population in Mali, West Africa, and controls the numbers of P. falciparum oocysts that develop on the vector midgut. An important question is whether genetic variation with respect to Plasmodium-resistance exists across Africa, and if so whether the same or multiple geographically distinct resistance mechanisms are responsible for the trait. Methods: To identify P falciparum resistance loci in pedigrees generated and infected in Kenya, East Africa, 28 microsatellite loci were typed across the mosquito genome. Genetic linkage mapping was used to detect significant linkage between genotype and numbers of midgut oocysts surviving to 7–8 days post-infection. Results: A major malaria-control locus was identified on chromosome 2L in East African mosquitoes, in the same apparent position originally identified from the West African population. Presence of this resistance locus explains 75% of parasite free mosquitoes. The Kenyan resistance locus is named EA_Pfin1 (East Africa_ Plasmodium falciparum Infection Intensity). Conclusion: Detection of a malaria-control locus at the same chromosomal location in both East and West African mosquitoes indicates that, to the level of genetic resolution of the analysis, the same mechanism of Plasmodium-resistance, or a mechanism controlled by the same genomic region, is found across Africa, and thus probably operates in A. gambiae throughout its entire range

A Novel Liquid Multi-Phytonutrient Supplement Demonstrates DNA-Protective Effects

This study explored the DNA protective (anti-mutagenic) effects of an oral, liquid, multi-phytonutrient dietary supplement containing a proprietary blend of fruits, vegetables and aloe vera concentrated components in addition to a proprietary catechin complex from green tea (VIBE Cardiac & Life, Eniva Nutraceuticals, Anoka, MN; herein described as “VIBE”). This study tested the hypothesis that VIBE would reduce DNA damage in skin cells exposed to UVR. Human epidermal cells, from the cell line A431NS, were treated with 0% (control), 0.125%, 0.5%, 1% and 2% VIBE, and then exposed to 240 J/m2 UVR. The amount of DNA damage was assessed using the COMET assay. At each concentration tested, a significantly smaller amount of DNA damage was measured by the COMET assay for the VIBE treated cells compared to the control cells exposed to UVR without VIBE. The dose response curves showed a maximal response at 0.5% VIBE with a threefold reduction in COMET tail density compared to the control samples without VIBE (p < 0.001). Additional research is warranted in human clinical trials to further explore the results of this study which demonstrated the DNA protective and anti-mutagenic effects of VIBE for human skin cells exposed to UVR-induced DNA damage

Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer

<p>Abstract</p> <p>Background</p> <p><it>MAP2K4 </it>is a putative tumor and metastasis suppressor gene frequently found to be deleted in various cancer types. We aimed to conduct a comprehensive analysis of this gene to assess its involvement in ovarian cancer.</p> <p>Methods</p> <p>We screened for mutations in <it>MAP2K4 </it>using High Resolution Melt analysis of 149 primary ovarian tumors and methylation at the promoter using Methylation-Specific Single-Stranded Conformation Polymorphism analysis of 39 tumors. We also considered the clinical impact of changes in <it>MAP2K4 </it>using publicly available expression and copy number array data. Finally, we used siRNA to measure the effect of reducing <it>MAP2K4 </it>expression in cell lines.</p> <p>Results</p> <p>In addition to 4 previously detected homozygous deletions, we identified a homozygous 16 bp truncating deletion and a heterozygous 4 bp deletion, each in one ovarian tumor. No promoter methylation was detected. The frequency of <it>MAP2K4 </it>homozygous inactivation was 5.6% overall, and 9.8% in high-grade serous cases. Hemizygous deletion of <it>MAP2K4 </it>was observed in 38% of samples. There were significant correlations of copy number and expression in three microarray data sets. There was a significant correlation between <it>MAP2K4 </it>expression and overall survival in one expression array data set, but this was not confirmed in an independent set. Treatment of JAM and HOSE6.3 cell lines with <it>MAP2K4 </it>siRNA showed some reduction in proliferation.</p> <p>Conclusions</p> <p><it>MAP2K4 </it>is targeted by genetic inactivation in ovarian cancer and restricted to high grade serous and endometrioid carcinomas in our cohort.</p

Biomimetic transferable surface for a real time control over wettability and photoerasable writing with water drop lens

We demonstrate a transferable device that can turn wettability of surfaces to sticky or slippy, as per requirement. It is composed of polymeric yarn with a fibrous structure, which can be lifted and placed on any surface to render it the unique wettability properties. We introduce Polyvinylidenefluoride (PVDF) random fiber as biomimetic rose petal surface. When it is decorated with PVDF nanofibers yarns, the random mesh transform from rose petal sticky state into grass leaf slippy state. When it is placed on sticky, hydrophilic metal coin, it converts the surface of the coin to super hydrophobic. Adjustments in the yarn system, like interyarn spacing, can be done in real time to influence its wettability, which is a unique feature. Next, we load the polymer with a photochromic compound for chemical restructuring. It affects the sliding angle of water drop and makes the fibers optically active. We also demonstrate a “water droplets lens” concept that enables erasable writing on photochromic rose petal sticky fibrous surface. The droplet on a highly hydrophobic surface acts as a ball lens to concentrate light onto a hot spot; thereby we demonstrate UV light writing with water lenses and visible light erasing

Berberine Improves Glucose Metabolism in Diabetic Rats by Inhibition of Hepatic Gluconeogenesis

Berberine (BBR) is a compound originally identified in a Chinese herbal medicine Huanglian (Coptis chinensis French). It improves glucose metabolism in type 2 diabetic patients. The mechanisms involve in activation of adenosine monophosphate activated protein kinase (AMPK) and improvement of insulin sensitivity. However, it is not clear if BBR reduces blood glucose through other mechanism. In this study, we addressed this issue by examining liver response to BBR in diabetic rats, in which hyperglycemia was induced in Sprague-Dawley rats by high fat diet. We observed that BBR decreased fasting glucose significantly. Gluconeogenic genes, Phosphoenolpyruvate carboxykinase (PEPCK) and Glucose-6-phosphatase (G6Pase), were decreased in liver by BBR. Hepatic steatosis was also reduced by BBR and expression of fatty acid synthase (FAS) was inhibited in liver. Activities of transcription factors including Forkhead transcription factor O1 (FoxO1), sterol regulatory element-binding protein 1c (SREBP1) and carbohydrate responsive element-binding protein (ChREBP) were decreased. Insulin signaling pathway was not altered in the liver. In cultured hepatocytes, BBR inhibited oxygen consumption and reduced intracellular adenosine triphosphate (ATP) level. The data suggest that BBR improves fasting blood glucose by direct inhibition of gluconeogenesis in liver. This activity is not dependent on insulin action. The gluconeogenic inhibition is likely a result of mitochondria inhibition by BBR. The observation supports that BBR improves glucose metabolism through an insulin-independent pathway

Applying Harmonic Optical Microscopy for Spatial Alignment of Atrial Collagen Fibers

BACKGROUND: Atrial fibrosis creates a vulnerable tissue for atrial fibrillation (AF), but the spatial disarray of collagen fibers underlying atrial fibrosis is not fully elucidated. OBJECTIVE: This study hypothesizes that harmonics optical microscopy can illuminate the spatial mal-alignment of collagen fibers in AF via a layer-by-layer approach. PATIENTS AND METHODS: Atrial tissues taken from patients who underwent open-heart surgery were examined by harmonics optical microscopy. Using the two-dimensional Fourier transformation method, a spectral-energy description of image texture was constituted and its entropy was used to quantify the mal-alignment of collagen fibers. The amount of collagen fiber was derived from its area ratio to total atrial tissue in each image. Serum C-terminal pro-collagen pro-peptide (CICP), pro-matrix metalloproteinase-1 (pro-MMP-1), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) were also evaluated. RESULTS: 46 patients were evaluated, including 20 with normal sinus rhythm and 26 with AF. The entropy of spectral-energy distribution of collagen alignment was significantly higher in AF than that in sinus rhythm (3.97 ± 0.33 vs. 2.80 ± 0.18, p<0.005). This difference was more significant in the permanent AF group. The amount of collagen was also significantly higher in AF patients (0.39 ± 0.13 vs. 0.18 ± 0.06, p<0.005) but serum markers of cardiac fibrosis were not significantly different between the two groups. CONCLUSIONS: Harmonics optical microscopy can quantify the spatial mal-alignment of collagen fibers in AF. The entropy of spectral-energy distribution of collagen alignment is a potential tool for research in atrial remodeling