Correlated libration in liquid water.

The libration spectrum of liquid H2O is resolved into an octupolar twisting libration band at 485 cm-1 and dipolar rocking-wagging libration bands at 707 and 743 cm-1 using polarization analysis of the hyper-Raman scattering (HRS) spectrum. Dipole interactions and orientation correlation over distances less than 2 nm account for the 36 cm-1 splitting of the longitudinal and transverse polarized bands of the dipolar rocking-wagging libration mode, while the intensity difference observed for the bands is the result of libration correlation over distances larger than 200 nm. The coupled rock and wag libration in water is similar to libration modes in ice. The libration relaxation time determined from the width of the spectrum is 36-54 fs. Polarization analysis of the HRS spectrum also shows long range correlation for molecular orientation and hindered translation, bending and stretching vibrations in water.

Long-range correlation of intra-molecular and inter-molecular vibration in liquid CCl4.

Experiments measuring the polarization dependence of hyper-Raman light scattering reveal long-range correlation of molecular vibrations in liquid CCl4. The ν3 and ν1 + ν4 intra-molecular vibrations at about 770 cm-1 are strongly polarized transverse to the scattering wavevector. Weaker transverse polarization is exhibited by the ν1, ν2, and ν4 intra-molecular vibrations and by the inter-molecular collision-induced band around 0 cm-1. The observed polarization dependence is due to the correlation of the vibrations on molecules separated by about 200 nm. The strength of the observed correlation increases with the transition dipole moment for the vibration mode and is consistent with dipole-dipole coupling.

Validation of ROS1 by immunohistochemistry against fluorescent in situ hybridisation on cytology and small biopsy samples in a large teaching hospital.

OBJECTIVE: Rearranged ROS1, present in 1%-2% of non-small cell lung cancer (NSCLC) patients, usually young, never or light smokers, is assessed by fluorescence in situ hybridization (FISH) to determine eligibility for tyrosine kinase inhibitors (TKI). Immunohistochemistry (IHC) for the protein product of ROS1 rearrangement, a cost-effective alternative, is validated on cytology and small biopsy samples. METHODS: From 1 March to 31 December 2019, cytology cell blocks and small biopsy samples from a selected cohort of NSCLC patients were concurrently tested for ROS1 gene rearrangement by Vysis 6q22 Break Apart FISH probe and IHC using Cell Signalling D4D6 antibody. Mismatch cases were tested by an RNA fusion next generation sequencing (NGS) panel. RESULTS: In a prospective population of 95 cases, 91 were negative and two were positive by both FISH and IHC. Both dual positive cases were female never smokers and benefited from TKI treatment. Another two cases were positive by FISH but negative by IHC and repeat by NGS showed one to be negative but one failed. Turnaround time for IHC was 0 to 8 days from request to authorisation, whilst that of FISH was 9 to 42 days at a cost of £51 and £159 respectively. CONCLUSION: IHC to assess for the protein product of ROS1 gene rearrangement on cytology cell blocks and small biopsy samples in a routine setting is a promising screening method to assess eligibility for TKI treatment with positive and indeterminate cases confirmed by FISH or NGS as it has good negative predictive value, faster turnaround time and is cost effective, with proven technical and clinical validation.

Vibration overtone hyperpolarizability measured for H2.

The second hyperpolarizability (γ) of the H2 molecule was measured by gas-phase electric field induced second harmonic generation at the frequencies of the one-photon resonance for the 3-0 Q(J) overtone transitions (v, J = 0, J → 3, J for J = 0, 1, 2, and 3). The magnitude of the resonant contribution to γ was measured with 2% accuracy using the previously determined non-resonant γ for calibration. Pressure broadening and frequency shift for the transitions were also measured. A theoretical expression for the resonant vibrational γ contribution in terms of transition polarizabilities is compared to the observations. The measured γ resonance strength is 4%-14% larger than the results obtained from this theoretical expression evaluated using ab initio transition polarizabilities.

Effects of Monoclonal Antibodies against Nerve Growth Factor on Healthy Bone and Joint Tissues in Mice, Rats, and Monkeys: Histopathologic, Biomarker, and Microcomputed Tomographic Assessments.

Tanezumab, an anti-nerve growth factor (NGF) antibody, is in development for management of chronic pain. During clinical trials of anti-NGF antibodies, some patients reported unexpected adverse events requiring total joint replacements, resulting in a partial clinical hold on all NGF inhibitors. Three nonclinical toxicology studies were conducted to evaluate the effects of tanezumab or the murine precursor muMab911 on selected bone and joint endpoints and biomarkers in cynomolgus monkeys, Sprague-Dawley rats, and C57BL/6 mice. Joint and bone endpoints included histology, immunohistochemistry, microcomputed tomography (mCT) imaging, and serum biomarkers of bone physiology. Responses of bone endpoints to tanezumab were evaluated in monkeys at 4 to 30 mg/kg/week for 26 weeks and in rats at 0.2 to 10 mg/kg twice weekly for 28 days. The effects of muMab911 at 10 mg/kg/week for 12 weeks on selected bone endpoints were determined in mice. Tanezumab and muMab911 had no adverse effects on any bone or joint parameter. There were no test article-related effects on bone or joint histology, immunohistochemistry, or structure. Reversible, higher osteocalcin concentrations occurred only in the rat study. No deleterious effects were observed in joints or bones in monkeys, rats, or mice administered high doses of tanezumab or muMab911.

Response to "Comment on 'Water-water correlations in electrolyte solutions probed by hyper-Rayleigh scattering'" [J. Chem. Phys. 149, 167101 (2018)].

The Comment proposes that the discrepancy between two second harmonic scattering (SHS) experiments for D2O electrolyte solutions with 100 ns and 190 fs laser pulses is due to unexplained processes for 100 ns pulses that give non-quadratic power dependence for the second harmonic signal. However, the different power dependence of the second harmonic scattering signals measured with 100 ns and 190 fs laser pulses is due to changes in laser beam propagation and focal intensity caused by the combined effect of thermal defocusing and Kerr lens self-focusing. Non-quadratic power dependence does not explain the discrepancy in the second harmonic scattering results.

Third harmonic scattering in liquids.

Third harmonic scattering (THS) from liquids has been observed and analyzed in several recent papers. It is considered to be analogous to second harmonic scattering (hyper-Rayleigh scattering) and to provide a means for measuring the second hyperpolarizability tensor of molecules in a liquid. However, the observed signal for a pure solvent is in fact mainly due to coherent third harmonic generation followed by Rayleigh scattering and direct incoherent THS (direct THS) makes only a small contribution (<2% for parallel polarized THS). This invalidates the internal reference method and the polarization analysis that has been applied for pure liquids. Theoretical comparison for the two processes, extensive experimental measurements for CCl4 liquid, SiO2 glass, and CCl2F2 gas and survey measurements for D2O, CDCl3, CD3CN, and (CD3)2SO liquid, are presented.

What is measured by hyper-Rayleigh scattering from a liquid?

Polarization and angle dependence of hyper-Rayleigh scattering (HRS) measured for liquid acetonitrile and dimethyl sulfoxide (DMSO) is analyzed in terms of contributions from randomly oriented molecules and additional contributions produced during intermolecular collisions and induced by the electric field of dissolved ions. All three contributions show the effect of long-range correlation, and the correlation functions are determined using the HRS observations combined with the results of molecular dynamics simulations. HRS from acetonitrile is polarized transverse to the scattering vector. This is due to long-range molecular orientation correlation produced by the dipole-dipole interaction, and correlation at distances r > 100 nm must be included to account for the HRS observations. Analysis of the HRS measurements for acetonitrile determines the length scale a = 0.185 nm for the long-range longitudinal and transverse orientation correlation functions BL=-2BT=a3/r3. Transverse polarized collision-induced HRS is also observed for acetonitrile, indicating long-range correlation of intermolecular modes. Strong longitudinal HRS is induced by the radial electric field of dissolved ions in acetonitrile. For DMSO, the angle between the molecular dipole and the vector part of the first hyperpolarizability tensor is about 100°. As a result, HRS from the randomly oriented molecules in DMSO is nearly unaffected by dipole correlation, and ion-induced HRS is weak. The strong longitudinal polarized HRS observed for DMSO is due to the collision-induced contribution, indicating long-range correlation of intermolecular modes. The HRS observations require correlation that has r-3 long-range asymptotic form, for molecular orientation and for intermolecular vibration and libration, for both acetonitrile and DMSO.

Engineering Highly Potent and Selective Microproteins against Nav1.7 Sodium Channel for Treatment of Pain.

The prominent role of voltage-gated sodium channel 1.7 (Nav1.7) in nociception was revealed by remarkable human clinical and genetic evidence. Development of potent and subtype-selective inhibitors of this ion channel is crucial for obtaining therapeutically useful analgesic compounds. Microproteins isolated from animal venoms have been identified as promising therapeutic leads for ion channels, because they naturally evolved to be potent ion channel blockers. Here, we report the engineering of highly potent and selective inhibitors of the Nav1.7 channel based on tarantula ceratotoxin-1 (CcoTx1). We utilized a combination of directed evolution, saturation mutagenesis, chemical modification, and rational drug design to obtain higher potency and selectivity to the Nav1.7 channel. The resulting microproteins are highly potent (IC50 to Nav1.7 of 2.5 nm) and selective. We achieved 80- and 20-fold selectivity over the closely related Nav1.2 and Nav1.6 channels, respectively, and the IC50 on skeletal (Nav1.4) and cardiac (Nav1.5) sodium channels is above 3000 nm The lead molecules have the potential for future clinical development as novel therapeutics in the treatment of pain.

Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies.

Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.

Nerve growth factor inhibition with tanezumab influences weight-bearing and subsequent cartilage damage in the rat medial meniscal tear model.

OBJECTIVE: To investigate whether the effects of nerve growth factor (NGF) inhibition with tanezumab on rats with medial meniscal tear (MMT) effectively model rapidly progressive osteoarthritis (RPOA) observed in clinical trials. METHODS: Male Lewis rats underwent MMT surgery and were treated weekly with tanezumab (0.1, 1 or 10 mg/kg), isotype control or vehicle for 7, 14 or 28 days. Gait deficiency was measured to assess weight-bearing on the operated limb. Joint damage was assessed via histopathology. A second arm, delayed onset of treatment (starting 3-8 weeks after MMT surgery) was used to control for analgesia early in the disease process. A third arm, mid-tibial amputation, evaluated the dependency of the model on weight-bearing. RESULTS: Gait deficiency in untreated rats was present 3-7 days after MMT surgery, with a return to normal weight-bearing by days 14-28. Prophylactic treatment with tanezumab prevented gait deficiency and resulted in more severe cartilage damage. When onset of treatment with tanezumab was delayed to 3-8 weeks after MMT surgery, there was no increase in cartilage damage. Mid-tibial amputation completely prevented cartilage damage in untreated MMT rats. CONCLUSIONS: These data suggest that analgesia due to NGF inhibition during the acute injury phase is responsible for increased voluntary weight-bearing and subsequent cartilage damage in the rat MMT model. This model failed to replicate the hypotrophic bone response observed in tanezumab-treated patients with RPOA.

Improved Lysosomal Trafficking Can Modulate the Potency of Antibody Drug Conjugates.

Antibody drug conjugates (ADCs) provide an efficacious and relatively safe means by which chemotherapeutic agents can be specifically targeted to cancer cells. In addition to the selection of antibody targets, ADCs offer a modular design that allows selection of ADC characteristics through the choice of linker chemistries, toxins, and conjugation sites. Many studies have indicated that release of toxins bound to antibodies via noncleavable linker chemistries relies on the internalization and intracellular trafficking of the ADC. While this can make noncleavable ADCs more stable in the serum, it can also result in lower efficacy when their respective targets are not internalized efficiently or are recycled back to the cell surface following internalization. Here, we show that a lysosomally targeted ADC against the protein APLP2 mediates cell killing, both in vitro and in vivo, more effectively than an ADC against Trop2, a protein with less efficient lysosomal targeting. We also engineered a bispecific ADC with one arm targeting HER2 for the purpose of directing the ADC to tumors, and the other arm targeting APLP2, whose purpose is to direct the ADC to lysosomes for toxin release. This proof-of-concept bispecific ADC demonstrates that this technology can be used to shift the intracellular trafficking of a constitutively recycled target by directing one arm of the antibody against a lysosomally delivered protein. Our data also show limitations of this approach and potential future directions for development.

Structural correlation in water probed by hyper-Rayleigh scattering.

Second-harmonic or hyper-Rayleigh scattering (HRS) is sensitive to molecular interactions and correlations, and there is a large coherent HRS contribution for water. This work shows that the distinctive angle and polarization dependence observed for HRS from water is due to the long-range orientation correlation of the molecules. The results of HRS experiments for water are analyzed in combination with a molecular dynamics simulation to determine the molecular hyperpolarizability and the short-range and long-range orientation correlation functions for the molecules. At long range, the longitudinal and transverse dipole-dipole orientation correlation functions are BL(r) = -2BT(r) = a3/r3 with a = 0.166 nm. Molecular correlation at distances r > 100 nm must be included to account for the HRS observations.

Water-water correlations in electrolyte solutions probed by hyper-Rayleigh scattering.

Long-range ion-induced correlations between water molecules have been observed by second-harmonic or hyper-Rayleigh scattering experiments with conflicting results. The most recent work observed a large difference between the results for H2O and D2O, and large discrepancies with the previously proposed theory. However, the present observations are in quantitative agreement with the model where the ion electric field induces second harmonic generation by the water molecules, and ion-ion correlations given by the Debye-Huckel theory account for intensity saturation at high ion concentration. This work compares experimental results with theory and addresses the apparent discrepancies with previous experiments.

Orientation correlation and local field in liquid nitrobenzene.

Hyper-Rayleigh scattering (HRS) is sensitive to long-range molecular orientation correlation in isotropic liquids composed of dipolar molecules. Measurements of the polarization, angle, and spectral dependence for HRS from liquid nitrobenzene (NB) are analyzed to determine the NB molecular orientation correlations at long range. The longitudinal and transverse orientation correlation functions for r > 3 nm are BL(r) = (a/r)(3) and BT(r) = - BL(r)/2, where a = 0.20 ± 0.01 nm. Measurements of HRS induced by dissolved ions are also analyzed and combined with molecular dynamics simulation and dielectric response results, to determine the molecular dipole moment µ = 3.90 ± 0.04 D, Kirkwood orientation correlation factor gK = 0.68 ± 0.02, and local field factor f(0) = 0.85 ± 0.04 × Onsager local field factor in liquid nitrobenzene.

Dual band sensitivity enhancements of a VO(x) microbolometer array using a patterned gold black absorber.

Infrared-absorbing gold black has been selectively patterned onto the active surfaces of a vanadium-oxide-based infrared bolometer array. Patterning by metal lift-off relies on protection of the fragile gold black with an evaporated oxide, which preserves much of gold black's high absorptance. This patterned gold black also survives the dry-etch removal of the sacrificial polyimide used to fabricate the air-bridge bolometers. For our fabricated devices, infrared responsivity is improved 22% in the long-wave IR and 70% in the mid-wave IR by the gold black coating, with no significant change in detector noise, using a 300°C blackbody and 80 Hz chopping rate. The increase in the time constant caused by the additional mass of gold black is ∼15%.

Effect of attachment site on stability of cleavable antibody drug conjugates.

The systemic stability of the antibody-drug linker is crucial for delivery of an intact antibody-drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-p-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell.

Long-range orientation correlation in dipolar liquids probed by hyper-Rayleigh scattering.

Hyper-Rayleigh scattering (HRS) is sensitive to long-range molecular orientation correlation in isotropic liquids composed of dipolar molecules. The correlation functions that appear in the calculation of HRS mediated by the vector part of the first hyperpolarizability ß are the same as the correlation functions for the homogeneous isotropic random vector fields that appear in the description of fluid turbulence. Recent experiments measuring the angle and polarization dependence of HRS from water find a dominant transverse mode contribution with amplitude independent of the scattering wavevector, and this observation of transverse mode HRS strongly constrains the form of the orientation correlation function. Analysis of these HRS results for water determines that the long-range molecular orientation correlation function varies as r(-3±Îµ) with |ε| < 0.03 on spatial scales up to 2000 nm.

Hyperpolarizability dispersion measured for (CH3)2O.

The third-order nonlinear-optical susceptibility of dimethyl ether, (CH3)2O, has been measured in the gas phase over the wavelength range 488 nm < λ < 1064 nm using the technique of gas-phase electric-field-induced second-harmonic generation with periodic phase matching and with N2 as the reference gas. Measurements span a range of temperature, which allows for separation of the temperature-independent second hyperpolarizability term from the temperature-dependent first hyperpolarizability term. The dispersion curves of the isotropically averaged first and second hyperpolarizabilities (ß and γ) are deduced.