Characterization of a high throughput approach for large scale retention measurement in liquid chromatography

Many contemporary challenges in liquid chromatography—such as the need for “smarter” method development tools, and deeper understanding of chromatographic phenomena—could be addressed more efficiently and effectively with larger volumes of experimental retention data than are available. The paucity of publicly accessible, high-quality measurements needed for the development of retention models and simulation tools has largely been due to the high cost in time and resources associated with traditional retention measurement approaches. Recently we described an approach to improve the throughput of such measurements by using very short columns (typically 5 mm), while maintaining measurement accuracy. In this paper we present a perspective on the characteristics of a dataset containing about 13,000 retention measurements obtained using this approach, and describe a different sample introduction method that is better suited to this application than the approach we used in prior work. The dataset comprises results for 35 different small molecules, nine different stationary phases, and several mobile phase compositions for each analyte/phase combination. During the acquisition of these data, we have interspersed repeated measurements of a small number of compounds for quality control purposes. The data from these measurements not only enable detection of outliers but also assessment of the repeatability and reproducibility of retention measurements over time. For retention factors greater than 1, the mean relative standard deviation (RSD) of replicate (typically n=5) measurements is 0.4%, and the standard deviation of RSDs is 0.4%. Most differences between selectivity values measured six months apart for 15 non-ionogenic compounds were in the range of +/- 1%, indicating good reproducibility. A critically important observation from these analyses is that selectivity defined as retention of a given analyte relative to the retention of a reference compound (kx/kref) is a much more consistent measure of retention over a time span of months compared to the retention factor alone. While this work and dataset also highlight the importance of stationary phase stability over time for achieving reliable retention measurements, we are nevertheless optimistic that this approach will enable the compilation of large databases (>> 10,000 measurements) of retention values over long time periods (years), which can in turn be leveraged to address some of the most important contemporary challenges in liquid chromatography. All the data discussed in the manuscript are provided as Supplemental Information

miR-23a-3p causes cellular senescence by targeting hyaluronan synthase2: Possible implication for skin aging.

Even though aging and cellular senescence appear to be linked the biological mechanisms interconnecting these two processes remain to be unravelled. Therefore, miRNA profiles were analyzed ex vivo by gene array in fibroblasts isolated from young and old human donors. Expression of several miRNAs was positively correlated with donor age. Among those miR-23a-3p was shown to target hyaluronan-synthase 2. Hyaluronan (HA) is a polysaccharide of the extracellular matrix that critically regulates the phenotype of fibroblasts. Indeed, both aged and senescent fibroblasts showed increased miR-23a-3p expression and secreted significantly lower amounts of HA compared to young and non senescent fibroblasts. Ectopic overexpression of miR-23a-3p in non senescent fibroblasts led to decreased HAS2 mediated HA-synthesis, upregulation of senescence associated markers and decreased proliferation. In addition, siRNA mediated downregulation of HAS2 and pharmacologic inhibition of HA-synthesis by 4-methylumbelliferone mimicked the effects of miR-23a-3p. In vivo, miR-23a-3p was upregulated and HAS2 was downregulated in the skin of old mice versus young mice. Inhibition of HA-synthesis by 4-methylumbelliferone in mice reduced dermal hydration and viscoelasticity thereby mimicking an aged skin phenotype. Taken together, these findings appear to link miR-23a-3p and the HA-microenvironment as effector mechanisms in both dermal aging and senescence

Immunoproteasomal inhibition with ONX-0914 attenuates atherosclerosis and reduces white adipose tissue mass and metabolic syndrome in mice

Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects.We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit β5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit β1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr-/- and APOE*3-Leiden.CETP mice.ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose.We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.Biopharmaceutic

Immunoediting role for major vault protein in apoptotic signaling induced by bacterial N-acyl homoserine lactones

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa. Here, we report the use of photoactive probes to identify MVP as a target of the N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N-acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL)