HPLC Plasma Assay of a Novel Anti-MRSA Compound, Kaempferol-3-O-Alpha-L-(2 \u27\u27,3 \u27\u27-di-p-coumaroyl)rhamnoside, from Sycamore Leaves

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious pathogen that is resistant to current antibiotic therapy. Thus, there is an urgent need for novel antimicrobial agents that can effectively combat these new strains of drug-resistant superbugs . Recently, fractionation of an extract from Platanus occidentalis (American sycamore) leaves produced an active kaempferol molecule, 3-O-alpha-L-(2 ,3 -di-p-coumaroyl)rhamnoside (KCR), in four isomeric forms; all four isomers exhibit potent anti-MRSA activity. In order to further the preclinical development of KCR as a new antibiotic class, we developed and validated a simple analytical method for assaying KCR plasma concentration. Because KCR will be developed as a new drug, although comprising four stereoisomers, the analytical method was devised to assay the total amount of all four isomers. In the present work, both a plasma processing procedure and an HPLC method have been developed and validated. Mouse plasma containing KCR was first treated with ethanol and then centrifuged. The supernatant was dried, suspended in ethanol, centrifuged, and the supernatant was injected into an HPLC system comprising a Waters C18, a mobile phase composing methanol, acetonitrile, and trifluoroacetic acid and monitored at 313 nm. The method was validated by parameters including a good linear correlation, a limit of quantification of 0.27 μg/mL, and high accuracy. In summary, this method allows a rapid analysis of KCR in the plasma samples for pharmacokinetics studies

Ptychographic electron microscopy using high-angle dark-field scattering for sub-nanometre resolution imaging

Diffractive imaging, in which image-forming optics are replaced by an inverse computation using scattered intensity data, could, in principle, realize wavelength-scale resolution in a transmission electron microscope. However, to date all implementations of this approach have suffered from various experimental restrictions. Here we demonstrate a form of diffractive imaging that unshackles the image formation process from the constraints of electron optics, improving resolution over that of the lens used by a factor of five and showing for the first time that it is possible to recover the complex exit wave (in modulus and phase) at atomic resolution, over an unlimited field of view, using low-energy (30 keV) electrons. Our method, called electron ptychography, has no fundamental experimental boundaries: further development of this proof-of-principle could revolutionize sub-atomic scale transmission imaging

Identification of a Novel Variant in EARS2 Associated with a Severe Clinical Phenotype Expands the Clinical Spectrum of LTBL

The EARS2 nuclear gene encodes mitochondrial glutamyl-tRNA synthetase, a member of the class I family of aminoacyl-tRNA synthetases (aaRSs) that plays a crucial role in mitochondrial protein biosynthesis by catalyzing the charging of glutamate to mitochondrial tRNA(Glu). Pathogenic EARS2 variants have been associated with a rare mitochondrial disorder known as leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The targeted sequencing of 150 nuclear genes encoding respiratory chain complex subunits and proteins implicated in the oxidative phosphorylation (OXPHOS) function was performed. The oxygen consumption rate (OCR), and the extracellular acidification rate (ECAR), were measured. The enzymatic activities of Complexes I-V were analyzed spectrophotometrically. We describe a patient carrying two heterozygous EARS2 variants, c.376C>T (p.Gln126*) and c.670G>A (p.Gly224Ser), with infantile-onset disease and a severe clinical presentation. We demonstrate a clear defect in mitochondrial function in the patient's fibroblasts, suggesting the molecular mechanism underlying the pathogenicity of these EARS2 variants. Experimental validation using patient-derived fibroblasts allowed an accurate characterization of the disease-causing variants, and by comparing our patient's clinical presentation with that of previously reported cases, new clinical and radiological features of LTBL were identified, expanding the clinical spectrum of this disease

Willingness to pay for multifunctional megaprojects: A stated preference analysis among firms in the Amsterdam Zuidas area

http://hdl.handle.net/1871/1621

Gene Expression Profiling in a Mouse Model Identifies Fetal Liver- and Placenta-Derived Potential Biomarkers for Down Syndrome Screening

BACKGROUND: As a first step to identify novel potential biomarkers for prenatal Down Syndrome screening, we analyzed gene expression in embryos of wild type mice and the Down Syndrome model Ts1Cje. Since current Down Syndrome screening markers are derived from placenta and fetal liver, these tissues were chosen as target. METHODOLOGY/PRINCIPAL FINDINGS: Placenta and fetal liver at 15.5 days gestation were analyzed by microarray profiling. We confirmed increased expression of genes located at the trisomic chromosomal region. Overall, between the two genotypes more differentially expressed genes were found in fetal liver than in placenta. Furthermore, the fetal liver data are in line with the hematological aberrations found in humans with Down Syndrome as well as Ts1Cje mice. Together, we found 25 targets that are predicted (by Gene Ontology, UniProt, or the Human Plasma Proteome project) to be detectable in human serum. CONCLUSIONS/SIGNIFICANCE: Fetal liver might harbor more promising targets for Down Syndrome screening studies. We expect these new targets will help focus further experimental studies on identifying and validating human maternal serum biomarkers for Down Syndrome screening

Stabilin 1 and 2 are important regulators for cellular uptake of apolipoprotein B-containing lipoproteins in zebrafish

BACKGROUND AND AIMS\nMETHODS\nRESULTS\nCONCLUSIONS\nScavenger receptors form a superfamily of membrane-bound receptors that bind and internalize different types of ligands, including pro-atherogenic oxidized low-density lipoproteins (oxLDLs). In vitro studies have indicated a role for the liver sinusoidal endothelial cell receptors stabilin 1 (stab1) and 2 (stab2) in oxLDL clearance. In this study, we evaluated the potential role of stab1 and stab2 in lipoprotein uptake in zebrafish, an upcoming model for studying cholesterol metabolism and atherosclerosis.\nLipoproteins were injected in the duct of Cuvier of wild-type (ABTL) or stab1 and stab2 mutant (stab1-/-stab2-/-) zebrafish larvae at 3 days post-fertilization. To examine the effect of stabilin deficiency on lipoprotein and cholesterol metabolism, zebrafish larvae were challenged with a high cholesterol diet (HCD; 4% w/w) for 10 days.\nLipoprotein injections showed impaired uptake of both LDL and oxLDL into the vessel wall of caudal veins of stab1-/-stab2-/- zebrafish, which was paralleled by redistribution to tissue macrophages. Total body cholesterol levels did not differ between HCD-fed stab1-/-stab2-/- and ABTL zebrafish. However, stab1-/-stab2-/- larvae exhibited 1.4-fold higher mRNA expression levels of ldlra involved in (modified) LDL uptake, whereas the expression levels of scavenger receptors scarb1 and cd36 were significantly decreased.\nWe have shown that stabilins 1 and 2 have an important scavenging function for apolipoprotein B-containing lipoproteins in zebrafish and that combined deficiency of these two proteins strongly upregulates the clearance of lipoproteins by macrophages within the caudal vein. Our current study highlights the use of zebrafish as model to study lipoprotein metabolism and liver sinusoidal endothelial cell function.Drug Delivery Technolog