GTx011, an Anti-Sickling Compound, Improves SS Blood Rheology By Reduction of HbS polymerization Via Allosteric Modulation of O2 Affinity

Sickle cell disease is caused by a mutation in the β-globin gene leading to production of hemoglobin S (HbS). HbS polymerizes under hypoxic conditions, leading to changes in cytoplasmic viscosity and formation of rigid, non-deformable sickled red blood cells (RBC). Loss of RBC deformability leads to abnormal blood rheology, diminished oxygen (O2) delivery and contributes to vaso-occlusion. GTx011 is a novel orally bioavailable small molecule that has been shown to inhibit HbS polymerization in vitro by maintaining oxygenated HbS under hypoxic conditions. We investigated the mechanism by which GTx011 modulates O2 affinity. Although it binds to the N-terminal α chain of Hb via a reversible Schiff base, 1D 1H NMR studies indicated that the compound allosterically influenced the intra-dimer interface of Hb (α122His and α103His), and the distal valine surrounding heme pockets for both the α and β chains, suggesting a unique solution phase structure. GTx011’s unique allosteric action upon the heme pocket allows it to improve O2 affinity without sterically blocking the release of O2. This modulation of O2 affinity delays the onset of HbS polymers and therefore we hypothesized will improve the abnormal blood rheology observed in SS blood. We report here the beneficial effect of GTx011 on SSRBC deformability using four independent whole cell systems: 1) the profile of SSRBC during passage through a gel filtration column, 2) the ability and corresponding pressure required for SSRBCs to pass through a polycarbonate filter under hypoxic conditions, 3) the tension required to aspirate SSRBCs into a micropipette under low O2 and 4) changes to blood viscosity during deoxygenation. Under deoxygenating conditions, GTx011 dose dependently enabled RBC deformability required for migration of SSRBCs through a gel filtration column. The pressure required to pass SSRBCs through a polycarbonate filter and the tension required to aspirate SSRBCs into a micropipette under hypoxic conditions were also reduced (Table 1). We have established a novel blood viscosity assay, which employs an enzymatic reaction (ascorbic acid/ascorbic oxidase) for deoxygenation of blood, followed by traditional measurements in cone plate viscometers. This protocol enabled us to reduce O2 levels without the technical limitations of using sodium dithionite, laser induced deoxygenation or passive deoxygenation via N2. Time dependent measures of viscosity, at a fixed shear rate, allowed us to quantify GTx011-induced delay in the onset of polymerization, and the increase in cytoplasmic viscosity. As in polymerization experiments, GTx011 dose dependently delayed the onset of hyperviscosity of SS blood under deoxygenating conditions. HbS modification ranging from 10-30%, was sufficient to achieve an improvement in SS blood hyperviscosity (Table 2) These data suggest that GTx011 is a promising agent that has the potential to improve SSRBC deformability and blood viscosity by inhibiting HbS polymerization, the pathophysiologic hallmark of sickle cell disease. Table 1 Assay Migration Filterability Deformability unit (% deformable cells) (mm Hg) (mDynes/cm2) [HbS] 1 mM 1 mM 1 mM [GTx011] (mM) 0 0.5 1 0 0.5 1 0 0.5 1 Readout 16 42 89 358 162 129 101 32 25 Table 2 Assay Polymerization Viscosity [HbS] 50 μM 1.5 mM GTx011 modified HbS (%) 0 10 30 0 10 30 Delay time (min) 13 18 33 21 26 31 Disclosures Patel: Global Blood Therapeutics: Employment, Equity Ownership. Cabrales:Global Blood Therapeutics: Consultancy. Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Metcalf:Global Blood Therapeutics: Consultancy, Equity Ownership. Sinha:Global Blood Therapeutics: Employment, Equity Ownership.

APPLICATION OF PIXE FOR A MULTI-ELEMENT ANALYSIS OF MARINE PHYTOPLANKTON TOWARDS THE DEVELOPMENT OF A NEW BIOLOGICAL ASSESSMENT METHOD FOR HEAVY METAL POLLUTION

Particle-induced X-ray emission (PIXE) was applied for the determination of heavy metals accumulated in natural phytoplankton. We selected four Japanese marine sites to collect phytoplankton: Toni Bay and Kamaishi Bay (Kamaishi, Iwate) as non-polluted areas; and Shimotsu port (Shimotsu, Wakayama) and Toyama new port (Shinminato, Toyama) as polluted areas. After removing the zooplankton and visible dust by passing the seawater through an NXX13 plankton net (95-μm mesh aperture), the phytoplankton were collected on a polycarbonate filter (25 mm diameter, 1.0 μm pore size) by suction filtration. The filter was dried and subjected to scanning electron microscopy (SEM) and a PIXE analysis. The phytoplankton consisted of diatoms, dinoflagellates and coccolithophores were observed by SEM analysis on the filter prepared from 100 ml of Toni Bay seawater. Heavy metals ( Pb , Ni , Cu , Cr , Zn , Mn , Hg and As ) could be detected by the PIXE analysis in almost all the cases tested, in which 50-200 ml each of respective seawater sample was filtered. There was a linear relationship between the amount of heavy metals on the polycarbonate filter (ng cm-2) and the volume of seawater (25-200 ml) filtered (i.e. the amount of phytoplankton collected on the filter). Likewise, the PIXE analysis revealed that significant differences in heavy metal contamination between the polluted and non-polluted areas, and also among sampling sites in a polluted area depending on the location and seawater movement. These results indicate that a multi-element analysis of marine phytoplankton by PIXE would be a promising technique for biologically assessing and monitoring heavy metal pollution in the first link of the aquatic food chain.

PIXE ANALYSIS OF SUSPENDED PARTICULATE MATTER ORIGINALLY COLLECTED FOR BETA-RAY ABSORPTION MASS MONITORING

A method of transferring suspended particulate matter (SPM) from the used filter of a beta-absorption mass monitor to a polycarbonate filter for subsequent PIXE analysis has been developed. The method allows determination of the relative elemental composition of SPM. It was demonstrated that PIXE analysis can detect S , Cl , Fe and Zn in SPM sampled over a one-hour period.

Evaluation of isolator caging systems for protection of mice against challenge with mouse hepatitis virus

Two isolator caging systems were evaluated against challenge with MHV-Y, an enterotropic strain of mouse hepatitis virus. The systems were similar in that they both used an identical shoebox cage equipped with a polycarbonate filter top incorporating a Reemay® filter. They differed in that one system supplied HEPA-filtered air through a grommet in the filter lid so that the cage was pressurized slightly. A rack holding 60 cages (30 front and back) was utilized. Thirty cages without filter tops housed one mouse each that had been infected orally with 19000 ID50 of MHV-Y and an uninfected cagemate. The remaining 30 cages, each housing 2 uninfected mice were divided into 3 groups of 10 cages. Group I cages (controls) had no filter top; Group II cages were equipped with filter tops; and Group III were equipped with filter tops and intracage HEPA-filtered air. The cages housing uninfected mice were interspersed between, above, below and behind cages housing infected mice. The uninfected mice were maintained in contact with the MHV-Y infected mice for 8 weeks. Transmission of MHV-Y was determined serologically by indirect ELISA. All mice housed within the Group I cages (control) seroconverted to MHV, while only 4 mice (2 cages) seroconverted in Group II, and no mice seroconverted in Group III

Is Precipitation of Aluminum Fast Enough to Explain Aluminum Deposition on Fish Gills?

A peristaltic pump was used to mix environmentally realistic concentrations of Al in soft water (14–15 °C) with 30 μM base (NH4OH) to simulate the alkalinization which occurs when acidic soft water passes over fish gills. Solutions were filtered under vacuum through 0.2 μm prerinsed filters using a polycarbonate filter apparatus. If final solution pH was [Formula: see text], only about 10% of total Al (159 μg∙L−1) was filtered from solution. If solution pH was raised from pH 5.3 to pH 5.7 with base, about 35% of Al was removed from solution, 1–4 s after the pH was raised. Similar results were obtained for 335 μg∙L−1 Al solutions. Production of additional Al retained by filters was fast enough to show that Al accumulation on fish gills may be partly due to polymerisation and precipitation of Al, as Al solubility is reduced in the more alkaline gill micro-environment.