An efficient strategy for cell-based antibody library selection using an integrated vector system

BACKGROUND: Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9. RESULTS: A mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb) suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9. CONCLUSIONS: This study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets

The ancient phosphatidylinositol 3-kinase signaling system is a master regulator of energy and carbon metabolism in algae

Algae undergo a complete metabolic transformation under stress by arresting cell growth, inducing autophagy and hyperaccumulating biofuel precursors such as triacylglycerols and starch. However, the regulatory mechanisms behind this stress-induced transformation are still unclear. Here, we use biochemical, mutational, and “omics” approaches to demonstrate that PI3K signaling mediates the homeostasis of energy molecules and influences carbon metabolism in algae. In Chlamydomonas reinhardtii, the inhibition and knockdown (KD) of algal class III PI3K led to significantly decreased cell growth, altered cell morphology, and higher lipid and starch contents. Lipid profiling of wild-type and PI3K KD lines showed significantly reduced membrane lipid breakdown under nitrogen starvation (-N) in the KD. RNA-seq and network analyses showed that under -N conditions, the KD line carried out lipogenesis rather than lipid hydrolysis by initiating de novo fatty acid biosynthesis, which was supported by tricarboxylic acid cycle down-regulation and via acetyl-CoA synthesis from glycolysis. Remarkably, autophagic responses did not have primacy over inositide signaling in algae, unlike in mammals and vascular plants. The mutant displayed a fundamental shift in intracellular energy flux, analogous to that in tumor cells. The high free fatty acid levels and reduced mitochondrial ATP generation led to decreased cell viability. These results indicate that the PI3K signal transduction pathway is the metabolic gatekeeper restraining biofuel yields, thus maintaining fitness and viability under stress in algae. This study demonstrates the existence of homeostasis between starch and lipid synthesis controlled by lipid signaling in algae and expands our understanding of such processes, with biotechnological and evolutionary implications.Ministry of Science, ICT and Future Planning 2015M3A6A2065697Ministry of Oceans and Fisheries 2015018

Cyclic AMP Concentrations in Follicular Fluids: Relationship to Outcomes of In Vitro Fertilization Cycles

Follicular fluids and their matched oocytes were obtained from 16 follicles of 7 women who conceived after in vitro fertilization(IVF), and 49 follicles of 17 women who failed to conceive after the procedure. Follicular development was induced with a combination of follicle-stimulating hormone and human menopausal gonadotropin. There was no significant difference in follicular cyclic AMP concentrations between preovulatory and immature oocytes. However, follicles of preovulatory oocytes obtained from women who conceived after IVF contained lower cyclic AMP concentrations than those from women who failed to conceive after the procedure. Follicles of cleaved oocytes from pregnant women contained lower cyclic AMP concentrations than those of cleaved oocytes from nonpregnant women or all uncleaved oocytes. These results suggest that lower cyclic AMP concentrations in follicular fluids are associated with successful pregnancies in IVF cycles

Causes and effects of 2008 financial crisis

Beginning in the mid 2007’s the US financial market started to slide into the “worst financial crisis since the Great Depression of the early 1930’s” (Thakor, 2015: p.156). The domino effect of several events and occasions were leading first to a countrywide recession in the USA then later spreading globally. In the following this term paper will deal with the main causes and effects of 2008 financial crisis. Unlike other topics in literature there is no consensus about the question of guilt in this sense. Among economists there are different approaches to explain the main causes of the financial crisis

Anti-inflammatory effects of isoketocharbroic acid from brown alga, Sargassum micracanthum

During our on-going screening program designed to isolate natural compounds from marine environments, we isolated isoketochabrolic acid (IKCA) from Sargassum micracanthum, an important brown algae distributed in Jeju Island, Korea. Furthermore, we evaluated the inhibitory effects of IKCA on nitric oxide (NO) production in lipopolysaccharide (LPS)-triggered macrophages. IKCA strongly inhibited NO production, with an IC50 value of 58.31 μM. Subsequent studies demonstrated that IKCA potently and concentration-dependently reduced prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6 cytokine production. In conclusion, to the best of our knowledge, this is the first study to show that IKCA isolated from S. micracanthum has a potent anti-inflammatory activity. Therefore, IKCA might be useful as an anti-inflammatory health supplement or functional cosmetics

6,7-Dimethoxy-4-methylcoumarin suppresses pro-inflammatory mediator expression through inactivation of the NF-kappaB and MAPK pathways in LPS-induced RAW 264.7 cells

In this study, we investigated the ability of 6,7-dimethoxy-4-methylcoumarin (DMC) to inhibit lipopolysaccharide (LPS)-induced expression of pro-inflammatory mediators in mouse macrophage (RAW 264.7) cells, and the molecular mechanism through which this inhibition occurred. Our results indicated that DMC down regulated LPS-induced nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, thereby reducing the production of NO and prostaglandin E2 (PGE2) in LPS-activated RAW 264.7 cells. Furthermore, DMC suppressed LPS-induced production of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. To elucidate the mechanism underlying the anti- inflammatory activity of DMC, we assessed its effects on the mitogen-activated protein kinase (MAPK) pathway and the activity and expression of nuclear transcription factor kappa-B (NF-κB). The experiments demonstrated that DMC inhibited LPS-induced phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38. In addition, it attenuated LPS-induced NF-κB activation via the inhibition of IκB-α phosphorylation. Taken together, these data suggest that DMC exerts its anti-inflammatory effects in RAW 264.7 cells through the inhibition of LPS-stimulated NF-κB and MAPK signaling, thereby downregulating the expression of pro-inflammatory mediators

KMT-2016-BLG-1107: A New Hollywood-Planet Close/Wide Degeneracy

We show that microlensing event KMT-2016-BLG-1107 displays a new type of degeneracy between wide-binary and close-binary Hollywood events in which a giant-star source envelops the planetary caustic. The planetary anomaly takes the form of a smooth, two-day "bump" far out on the falling wing of the light curve, which can be interpreted either as the source completely enveloping a minor-image caustic due to a close companion with mass ratio $q=0.036$, or partially enveloping a major-image caustic due to a wide companion with $q=0.004$. The best estimates of the companion masses are both in the planetary regime ($3.3^{+3.5}_{-1.8}\,M_{\rm jup}$ and $0.090^{+0.096}_{-0.037}\,M_{\rm jup}$) but differ by an even larger factor than the mass ratios due to different inferred host masses. We show that the two solutions can be distinguished by high-resolution imaging at first light on next-generation ("30m") telescopes. We provide analytic guidance to understand the conditions under which this new type of degeneracy can appear.Comment: 23 pages, 7 figures, accepted for publication in A