Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM₆), octasaccharide (HM₈), and decasaccharide (HM₁₀), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM₈ and HM₁₀ are significantly more potent than HM₆ in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM₈ relative to HM₆ in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM₈ to bind and dimerize FGF2. Notably FGF2·HM₈ exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.     

Human Adipose-Derived Mesenchymal Stem Cells in Cell Therapy: Safety and Feasibility in Different Hospital Exemption Clinical Applications

Based on immunomodulatory, osteogenic, and pro-angiogenic properties of adipose-derived stem cells (ASCs), this study aims to assess the safety and efficacy of ASC-derived cell therapies for clinical indications. Two autologous ASC-derived products were proposed to 17 patients who had not experienced any success with conventional therapies: (1) a scaffold-free osteogenic three-dimensional graft for the treatment of bone non-union and (2) a biological dressing for dermal reconstruction of non-healing chronic wounds. Safety was studied using the quality control of the final product (genetic stability, microbiological/mycoplasma/endotoxin contamination) and the in vivo evaluation of adverse events after transplantation. Feasibility was assessed by the ability to reproducibly obtain the final ASC-based product with specific characteristics, the time necessary for graft manufacturing, the capacity to produce enough material to treat the lesion, the surgical handling of the graft, and the ability to manufacture the graft in line with hospital exemption regulations. For 16 patients (one patient did not undergo grafting because of spontaneous bone healing), in-process controls found no microbiological/mycoplasma/endotoxin contamination, no obvious deleterious genomic anomalies, and optimal ASC purity. Each type of graft was reproducibly obtained without significant delay for implantation and surgical handling was always according to the surgical procedure and the implantation site. No serious adverse events were noted for up to 54 months. We demonstrated that autologous ASC transplantation can be considered a safe and feasible therapy tool for extreme clinical indications of ASC properties and physiopathology of disease.     

Does contamination buildup limit throughput for automated cryoEM?

The development of automated systems for data acquisition in cryo electron microscopy has enabled the possibility of acquiring very large number of images from a single specimen grid. We have demonstrated that over images of 250,000 single particles can be acquired in a 24 h period. This has raised questions as to whether contamination buildup on the specimen limits the quality of the data that can be acquired during these long duration experiments and also whether the data acquisition session could be extended to allow acquisition of more than 1,000,000 particles. We report here a systematic characterization of contamination of specimens maintained for long periods of time at liquid nitrogen temperatures using standard side entry cryo stages. As part of this characterization we developed a more reliable method for accurately estimating specimen ice thickness. Using the method, we were able to calibrate image contrast against ice thickness under a variety of magnifications, objective aperture positions, and defoci, and demonstrated the strong dependence of the calibration curve on these parameters. The results show the anti-contamination aperture is, as expected, critical to the prevention of contamination and that loading film into the microscope dramatically increases the contamination rate, particularly in the first 3 h after the insertion of the film box. In the absence of film, we were able to reproducibly demonstrate that the contamination rate can be limited to a rate of approximately 1 angstrom/h providing reassurance that contamination will not be a major limiting factor for long term cryoEM experiments if a CCD camera is used for the imaging.     

cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells

We have investigated the direct effect of arachidonic acid on cholesterol transport in intact cells or isolated mitochondria from steroidogenic cells and the effect of cyclic-AMP on the specific release of this fatty acid inside the mitochondria. We show for the first time that cyclic-AMP can regulate the release of arachidonic acid in a specialized compartment of MA-10 Leydig cells, e.g. the mitochondria, and that the fatty acid induces cholesterol transport through a mechanism different from the classical pathway. Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol transport in isolated mitochondria from nonstimulated cells. The effect of arachidonoyl-CoA is inhibited by the reduction in the expression or in the activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a substrate to release arachidonic acid. cAMP-induced arachidonic acid accumulation into the mitochondria is also reduced when the mitochondrial thioesterase activity or expression is blocked. This new feature in the regulation of cholesterol transport by arachidonic acid and the release of arachidonic acid in specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis but also would be important in other situations such as neuropathological disorders or oncology disorders, where cholesterol transport plays an important role.     

A physical map of large segments of pig Chromosome 7q11–q14: comparative analysis with human Chromosome 6p21

The aim of this study was to establish a porcine physical map along the chromosome SSC7q by construction of BAC contigs between microsatellites Sw1409 and S0102. The SLA class II contig, located on SSC7q, was lengthened. Four major BAC contigs and 10 short contigs span a region equivalent to 800 cR measured by IMpRH7000 mapping. The BAC contigs were initiated by PCR screening with primers derived from human orthologous segments, extended by chromosome walking, and controlled and oriented by RH mapping with the two available panels, IMpRH7000Rad and IMNpRH12000Rad. The location of 43 genes was revealed by sequenced segments, either from BAC ends or PCR products from BAC clones. The 220 BAC end sequences (BES) were also used to analyze the different marks of evolution. Comparative mapping analysis between pigs and humans demonstrated that the gene organization on HSA6p21 and on SSC7p11 and q11-q14 segments was conserved during evolution, with the exception of long fragments of HSA6p12 which shuffled and spliced the SLA extended class II region. Additional punctual variations (unique gene insertion/deletion) were observed, even within conserved segments, revealing the evolutionary complexity of this region. In addition, 18 new polymorphic microsatellites have been selected in order to cover the entire SSC7p11-q14 region.     

Interleukin-6 is used as a growth factor by virulent Mycobacterium avium: presence of specific receptors.

In this paper, we examined the contribution of the lymphokine interleukin-6 (IL-6) to the growth of four virulent strains of Mycobacterium avium and the nature of the binding moieties on the mycobacteria. First, we showed that human or mouse recombinant interleukin-6 are potent growth factors for four strains of virulent M. avium. This was shown to occur in tissue culture medium, which does not support maximal growth of M. avium. Bioactive IL-6 was required, inasmuch as heat-activating IL-6 or adding an antibody against IL-6 blocked this growth-enhancing ability. The rapid uptake of IL-6 by M. avium was indicated by the fact that the incubation of IL-6 with the four M. avium strains led to a rapid removal of the bioactivity from the culture medium and a rapid removal of radiolabeled IL-6. Scatchard analysis of receptor interaction showed that the M. avium strains had a single receptor species with a Kd of 50 nM and the number of receptor sites was approximately 15,000 bacterium. Blocking experiments showed that the binding of radiolabeled IL-6 was fully displaceable with cold IL-6, but not with other lymphokines. These data suggest that IL-6 may play an important role in the pathogenesis of M. avium infections, notably by promoting growth of M. avium, and that some virulent M. avium strains bind IL-6 in a specific manner.     

Exenatide does not evoke pancreatitis and attenuates chemically induced pancreatitis in normal and diabetic rodents

The risk of developing pancreatitis is elevated in type 2 diabetes and obesity. Cases of pancreatitis have been reported in type 2 diabetes patients treated with GLP-1 (GLP-1R) receptor agonists. To examine whether the GLP-1R agonist exenatide potentially induces or modulates pancreatitis, the effect of exenatide was evaluated in normal or diabetic rodents. Normal and diabetic rats received a single exenatide dose (0.072, 0.24, and 0.72 nmol/kg) or vehicle. Diabetic ob/ob or HF-STZ mice were infused with exenatide (1.2 and 7.2 nmol·kg(-1)·day(-1)) or vehicle for 4 wk. Post-exenatide treatment, pancreatitis was induced with caerulein (CRN) or sodium taurocholate (ST), and changes in plasma amylase and lipase were measured. In ob/ob mice, plasma cytokines (IL-1β, IL-2, IL-6, MCP-1, IFNγ, and TNFα) and pancreatitis-associated genes were assessed. Pancreata were weighed and examined histologically. Exenatide treatment alone did not modify plasma amylase or lipase in any models tested. Exenatide attenuated CRN-induced release of amylase and lipase in normal rats and ob/ob mice but did not modify the response to ST infusion. Plasma cytokines and pancreatic weight were unaffected by exenatide. Exenatide upregulated Reg3b but not Il6, Ccl2, Nfkb1, or Vamp8 expression. Histological analysis revealed that the highest doses of exenatide decreased CRN- or ST-induced acute inflammation, vacuolation, and acinar single cell necrosis in mice and rats, respectively. Ductal cell proliferation rates were low and similar across all groups of ob/ob mice. In conclusion, exenatide did not modify plasma amylase and lipase concentrations in rodents without pancreatitis and improved chemically induced pancreatitis in normal and diabetic rodents.