Alternatively spliced, truncated GCSF receptor promotes leukemogenic properties and sensitivity to JAK inhibition.

Granulocyte colony-stimulating factor (GCSF) drives the production of myeloid progenitor and precursor cells toward neutrophils via the GCSF receptor (GCSFR, gene name CSF3R). Children with severe congenital neutropenia chronically receive pharmacologic doses of GCSF, and ∼30% will develop myelodysplasia/acute myeloid leukemia (AML) associated with GCSFR truncation mutations. In addition to mutations, multiple isoforms of CSF3R have also been reported. We found elevated expression of the alternatively spliced isoform, class IV CSF3R in adult myelodysplastic syndrome/AML patients. Aside from its association with monosomy 7 and higher rates of relapse in pediatric AML patients, little is known about the biology of the class IV isoform. We found developmental regulation of CSF3R isoforms with the class IV expression more representative of a progenitor cell stage. Striking differences were found in phosphoprotein signaling involving Janus kinase (JAK)-signal transducer and activator of transcription (STAT) and cell cycle gene expression. Enhanced proliferation by class IV GCSFR was associated with diminished STAT3 and STAT5 activation, yet showed sensitivity to JAK2 inhibitors. Alterations in the C-terminal domain of the GCSFR result in leukemic properties of enhanced growth, impaired differentiation and resistance to apoptosis, suggesting that they can behave as oncogenic drivers, sensitive to JAK2 inhibition.

The application of the acoustic spectrophonometer to biomolecular spectrometry: a step towards acoustic "fingerprinting".

A tunable acoustic biosensor for investigating the properties of biomolecules at the solid-liquid interfaces is described. In its current, format the device can be tuned to frequencies between 6.5 MHz and 1.1 GHz in order to provide a unique detection feature: a variable evanescent wave thickness at the sensor surface. The key to its successful implementation required the careful selection of antennae designs that could induce shear acoustic waves at the solid-liquid interface. This non-contact format makes it possible to recover resonant shear acoustic waves over 100 different harmonic frequencies as a result of the electrical characteristics of the spiral coil. For testing this multifrequency sensing concept the surface of a quartz disc was exposed to solutions of immunoglobulin G (IgG) to form an adsorbed monolayer, whence protein A and IgG were added again in order to form multilayers. Spectra at frequencies between 6 and 600 MHz were generated for each successive layer and revealed two characteristic phases: an initial phase at the low megahertz frequencies consistent with the conventional Sauerbrey relation, and a possible additional phase towards the high megahertz to gigahertz frequencies, that we believe relates to the structure of the biomolecular film. This two-phase behaviour evident from differences between high and low frequencies, rather than from any distinct frequency transition, was anticipated from the reduction in evanescent wave thickness down to nanometre dimensions, and thin film resonance phenomena that are known to occur for film and fluid systems. These measurements suggested that the single element acoustic biosensor we present here may form the basis from which to generate acoustic molecular spectra, or "acoustic fingerprints", in a manner akin to optical spectroscopy.

The acoustic spectrophonometer: a novel bioanalytical technique based on multifrequency acoustic devices.

A measurement technique similar to optical absorption spectroscopy but based on evanescent acoustic waves is described in this paper. This format employs a planar spiral coil to vibrate a single crystal of quartz from 6 to 400 MHz, in order to measure multifrequency acoustic spectra. Consistency with the defined Sauerbrey and Kanazawa terms K1 and K2 when applied to multiple frequencies was found for these specific operating conditions in terms of a significant fit between the measured and calculated values: For an IgG surface density of 13.5 ng mm(-2) the measured value of K1 is 22.5 x 10(-6) and the calculated value is 20.4 x 10(-6), whilst for glycerol viscous loadings of 5.131 cP the measured value of K2 is 0.47 and the calculated value is 0.54. Thus for these specific surface loadings the multifrequency data fits to the predictions of the Sauerbrey model to within 10% and to Kanazawa model within 13%. However collective frequency shifts for 5.131 cP solutions of sucrose, dextran and glucose were found to exhibit an unanticipated additional variability (R2 < 0.4) with frequency, but retained a square root of frequency dependency within a factor 2 of the interpolated K2 values. The response to the 5.131 cP dextran solution was found to be significantly below the other isoviscous solutions, with a substantially reduced frequency shift and K2 value than would be expected from its bulk viscosity. In comparison with these viscous solutions, IgG protein films consistently produced linear frequency shifts with little scatter (R2 > 0.96) that were proportional to the operating frequency, and fully consistent with the Sauerbrey model under these specific conditions. A t-test value of 14.52 was calculated from the variance and mean of the two groups, and demonstrates that the acoustic spectrophonometer can be used to distinguish between the acoustic impedance characteristics of two chemical systems that are not clearly differentiable at a single operating frequency.

Hypersonic evanescent waves generated with a planar spiral coil.

A planar spiral coil has been used to induce hypersonic evanescent waves in a quartz substrate with the unique ability to focus the acoustic wave down onto the chemical recognition layer. These special sensing conditions were achieved by investigating the application of a radio frequency current to a coaxial waveguide and spiral coil, so that wideband repeating electrical resonance conditions could be established over the MHz to GHz frequency range. At a selected operating frequency of 1.09 GHz, the evanescent wave depth of a quartz crystal hypersonic resonance is reduced to 17 nm, minimising unwanted coupling to the bulk fluid. Verification of the validity of the hypersonic resonance was carried out by characterising the system electrically and acoustically: Impedance calculations of the combined coil and coaxial waveguide demonstrated an excellent fit to the measured data, although above 400 MHz a transition zone was identified where unwanted impedance is parasitic of the coil influence efficiency, so the signal-to-noise ratio is reduced from 3000 to 300. Acoustic quartz crystal resonances at intervals of precisely 13.2138 MHz spacing, from the 6.6 MHz ultrasonic range and onto the desired hypersonic range above 1 GHz, were incrementally detected. Q factor measurements demonstrated that reductions in energy lost from the resonator to the fluid interface were consistent with the anticipated shrinkage of the evanescent wave with increasing operating frequency. Amplitude and frequency reduction in contact with a glucose solution was demonstrated at 1.09 GHz. The complex physical conditions arising at the solid-liquid interface under hypersonic entrainment are discussed with respect to acceleration induced slippage, rupture, longitudinal and shear radiation and multiphase relaxation affects.