Impact of Metabolic Regulators on the Expression of the Obesity Associated Genes FTO and NAMPT in Human Preadipocytes and Adipocytes

FTO and NAMPT/PBEF/visfatin are thought to play a role in obesity but their transcriptional regulation in adipocytes is not fully understood. In this study, we evaluated the transcriptional regulation of FTO and NAMPT in preadipocytes and adipocytes by metabolic regulators.We assessed FTO mRNA expression during human adipocyte differentiation of Simpson-Golabi-Behmel syndrome (SGBS) cells and primary subcutaneous preadipocytes in vitro and evaluated the effect of the metabolic regulators glucose, insulin, dexamethasone, IGF-1 and isoproterenol on FTO and NAMPT mRNA expression in SGBS preadipocytes and adipocytes. FTO mRNA levels were not significantly modulated during adipocyte differentiation. Also, metabolic regulators had no impact on FTO expression in preadipocytes or adipocytes. In SGBS preadipocytes NAMPT expression was more than 3fold induced by dexamethasone and isoproterenol and 1.6fold by dexamethasone in adipocytes. Complete glucose restriction caused an increase in NAMPT mRNA expression by more than 5fold and 1.4fold in SGBS preadipocytes and adipocytes, respectively.FTO mRNA expression is not significantly affected by differentiation or metabolic regulators in human adipocytes. The stimulation of NAMPT expression by dexamethasone, isoproterenol and complete glucose restriction may indicate a regulation of NAMPT by metabolic stress, which was more pronounced in preadipocytes compared to mature adipocytes

Breaking the interface:efficient extraction of magnetic beads from nanoliter-droplets for automated sequential immunoassays

Droplet-based microfluidic systems offer a high potential for miniaturization and automation. Therefore, they are becoming an increasingly important tool in analytical chemistry, biosciences, and medicine. Heterogeneous assays commonly utilize magnetic beads as a solid phase. However, the sensitivity of state of the art microfluidic systems is limited by the high bead concentrations required for efficient extraction across the water-oil interface. Furthermore, current systems suffer from a lack of technical solutions for sequential measurements of multiple samples, limiting their throughput and capacity for automation. Taking advantage of the different wetting properties of hydrophilic and hydrophobic areas in the channels, we improve the extraction efficiency of magnetic beads from aqueous nanoliter-sized droplets by 2 orders of magnitude to the low μg/mL range. Furthermore, the introduction of a switchable magnetic trap enables repetitive capture and release of magnetic particles for sequential analysis of multiple samples, enhancing the throughput. In comparison to conventional ELISA-based sandwich immunoassays on microtiter plates, our microfluidic setup offers a 25-50-fold reduction of sample and reagent consumption with up to 50 technical replicates per sample. The enhanced sensitivity and throughput of this system open avenues for the development of automated detection of biomolecules at the nanoliter scale

Crystallization conditions and petrogenesis of the lava dome from the ∼900 years BP eruption of Cerro Machín Volcano, Colombia

Highlights: • Whole-rock, mineral and isotope chemical data of Cerro Machín Volcano. • Mixing of distinct mafic and silic melts produced hybrid magma. • Amphibole thermobarometry indicates resident magma reservoir at 13 ± 2 km depth. • Dacites have adakitic geochemical signature. The last known eruption at Cerro Machín Volcano (CMV) in the Central Cordillera of Colombia occurred ∼900 years BP and ended with the formation of a dacitic lava dome. The dome rocks contain both normally and reversely zoned plagioclase (An24–54), unzoned and reversely zoned amphiboles of dominantly tschermakite and pargasite/magnesio-hastingsite composition and olivine xenocrysts (Fo = 85–88) with amphibole/clinopyroxene overgrowth, all suggesting interaction with mafic magma at depth. Plagioclase additionally exhibits complex oscillatory zoning patterns reflecting repeated replenishment, fractionation and changes in intrinsic conditions in the magma reservoir. Unzoned amphiboles and cores of the reversely zoned amphiboles give identical crystallization conditions of 910 ± 30 °C and 360 ± 70 MPa, corresponding to a depth of about 13 ± 2 km, at moderately oxidized conditions (fO2 = +0.5 ± 0.2 ΔNNO). The water content in the melt, calculated based on amphibole chemistry, is 7.1 ± 0.4 wt.%. Rims of the reversely zoned amphiboles are relatively enriched in MgO and yield higher crystallization temperatures (T = 970 ± 25 °C), slightly lower melt H2O contents (6.1 ± 0.7 wt.%) and overlapping pressures (410 ± 100 MPa). We suggest that these rims crystallized following an influx of mafic melt into a resident magma reservoir at mid-crustal depths, further supported by the occurrence of xenocrystic olivine. Crystallization of biotite, albite-rich plagioclase and quartz occurred at comparatively low temperatures (probably <800 °C) during early stages of ascent or storage at shallower levels. Based on amphibole mineral chemistry, the felsic resident melt had a rhyolitic composition (71 ± 2 wt.% SiO2), whereas the hybrid magma, from which the amphibole rims crystallized, was dacitic (64 ± 3 wt.% SiO2). The bulk rock chemistry of the CMV lava dome dacites is homogenous. They have elevated (La/Nb)N ratios of 3.8–4.5, typical for convergent margin magmas, and display several geochemical characteristics of adakites. Both Sr and Nd isotope compositions (87Sr/86Sr ∼0.70497, 143Nd/144Nd ∼0.51267) are among the most radiogenic observed for the Northern Volcanic Zone of the Andes. They are distinct from oceanic crust that has been subducted in the region, pointing to a continental crustal control on the isotope composition and hence the adakitic signature, possibly in a crustal “hot zone

Expansion of NKG2A−LIR1− Natural Killer Cells in HLA-Matched, Killer Cell Immunoglobulin-Like Receptors/HLA-Ligand Mismatched Patients following Hematopoietic Cell Transplantation

The prognosis after hematopoietic cell transplantation (HCT) for the treatment of leukemia or lymphoma in humans is influenced by donor-derived natural killer (NK) cells, which enhance the graft-versus-leukemia (GVL) effect. Such alloreactive killer cells can be generated in vivo after HCT if the donor expresses killer cell immunoglobulin-like receptors (KIRs), such as KIR2DL1, KIR2DL2/3, or KIR3DL1, for which the recipient lacks HLA class I ligands. We studied effector cells from 22 KIR/HLA-ligand mismatched and 14 KIR/HLA-ligand matched, primarily HLA-matched patient-donor pairs after allogeneic HCT. A novel 8-color flow cytometry panel allowed us to characterize effector-cell populations without “broadly reactive” inhibitory receptors such as CD94/NKG2A or LIR1. The numbers of such NKG2A– LIR1– NK cells increased following HCT in patients transplanted by KIR/HLA-ligand mismatched grafts, compared to KIR/HLA-ligand matched grafts, and in patients transplanted from donors of the A/B, compared to A/A, KIR haplotypes. NKG2A–LIR1– NK cells expressing only those inhibitory KIRs for which the patient had no HLA class I ligands could be stimulated by HLA class I-deficient cells to express CD107a. Thus, NKG2A–LIR1– NK cells may be important GVL effector cells following HCT, even in patients transplanted from HLA-matched donors

<i>FTO</i> expression in response to metabolic regulators.

<p>Preadipocytes (<b>A+C</b>) and adipocytes (day 10 of differentiation) (<b>B+D</b>) were stimulated with 100 nM insulin (Ins), 100 nM dexamethasone (Dex), 100 nM IGF-1 and 100 nM isoproterenol (Iso) for 24 h and with increasing concentrations of glucose for 48 h as indicated. Expression in untreated cells (C) and at 17.5 mM glucose was set = 1. Data are shown for 3 independent cell experiments. Statistical significance was assessed by student's t-test.</p

Sequences of primer and probes.

<p>Sequences of primer and probes.</p

<i>FTO</i> and <i>RBL2</i> expression during adipogenesis.

<p><i>FTO</i> and <i>RBL2</i> expression was not modulated during <i>in vitro</i> differentiation of SGBS (<b>A</b>) and primary subcutaneous (<b>B</b>) preadipocytes to mature adipocytes (expression in preadipocytes at day 0 was set = 1). An expected increase in mRNA expression of <i>PPARγ</i> confirmed efficient adipogenesis. Data are shown for at least 3 independent cell experiments. Statistical significance was assessed by ANOVA with repeated measurements and Dunnett's post test. Data are mean±SEM.</p

<i>NAMPT</i> expression in response metabolic regulators.

<p>Preadipocytes (<b>A+C</b>) and adipocytes (day 10 of differentiation) (<b>B+D</b>) were stimulated with 100 nM insulin (Ins), 100 nM dexamethasone (Dex), 100 nM IGF-1and 100 nM isoproterenol (Iso) for 24 h and with increasing concentrations of glucose for 48 h as indicated. Expression in untreated cells (C) and at 17.5 mM glucose was set = 1. Data are shown for 3 independent cell experiments. Statistical significance was assessed by student's t-test.</p