Advances in the Specificity of Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy Based Structural Characterisation Methods for Synthetic Oligonucleotides.

Identity testing is a critical part in the development of a therapeutic synthetic oligonucleotide. Tandem Mass Spectrometry (MS/MS) is commonly used for the analysis of oligonucleotides to obtain structural and sequence information, however there are challenges resulting from chemical modifications introduced to improve their pharmacokinetics and stability. For these structurally complex oligonucleotides, Nuclear Magnetic Resonance (NMR) Spectroscopy has found limited use for characterisation and identity testing, as only partial NMR resonance assignment for oligonucleotides is achieved without isotopic labelling methodologies. Regardless of the choice of method used for oligonucleotide analysis, the specificity is of critical importance. In this work, in-source dissociation mass spectrometry and proton (1H) and carbon (13C) NMR at high temperature were used to analyse danvatirsen, a 16 nucleotide phosphorothioate antisense oligonucleotide, and its closely related switch sequences. Both approaches have shown specificity to distinguish danvatirsen from these similar sequences.

Engineering Chiral Light-Matter Interactions in a Waveguide-Coupled Nanocavity.

Spin-dependent, directional light-matter interactions form the basis of chiral quantum networks. In the solid state, quantum emitters commonly possess circularly polarized optical transitions with spin-dependent handedness. We demonstrate numerically that spin-dependent chiral coupling can be realized by embedding such an emitter in a waveguide-coupled nanocavity, which supports two near-degenerate, orthogonally polarized cavity modes. The chiral behavior arises due to direction-dependent interference between the cavity modes upon coupling to two single-mode output waveguides. Notably, an experimentally realistic cavity design simultaneously supports near-unity chiral contrast, efficient (>95%) cavity-waveguide coupling and enhanced light-matter interaction strength (Purcell factor F P > 70). In combination, these parameters enable the development of highly coherent spin-photon interfaces ready for integration into nanophotonic circuits.

Accidental self-inoculation of a veterinary field worker with Calf Paratyphoid Vaccine.

No abstract available.

Unconventionally Acquired Brain Injury: Guidance and Instruction About an Emerging Challenge to Warfighter Brain Health.

Special Operations Forces have made brain health a medical priority in recent years, and new guidance identified a new challenge-unconventionally acquired brain injury (UBI). Although this emerging condition is described as a cluster of neurosensory and cognitive symptoms with unknown etiology/ origin, there remain critical questions about how this diagnosis differs from other brain injuries. More importantly, there are limited recommendations about how medical personnel should approach the problem. The current discussion will provide context and information about UBI based on higher guidance and will also review the scant literature to provide context. Foremost, UBI can be distinguished from traumatic brain injury (TBI) largely due to an unknown point of injury. The described symptoms otherwise appear to be largely the same as TBI. Likewise, the recommended course of treatment is to follow the Clinical Practice Guidelines for mild TBI/TBI even if the injury is an actual or suspected UBI. Personnel must be careful to avoid entering sensitive information into the medical record, which may be particularly challenging if identifying the cause involves classified information about an unconventional weapon. Finally, we briefly discuss the literature about several suspected incidents fitting UBI diagnostic criteria, and we conclude with five primary takeaways for medical personnel to follow.

Monitoring apoptosis in intact cells by high-resolution magic angle spinning 1 H NMR spectroscopy.

Apoptosis maintains an equilibrium between cell proliferation and cell death. Many diseases, including cancer, develop because of defects in apoptosis. A known metabolic marker of apoptosis is a notable increase in 1 H NMR-observable resonances associated with lipids stored in lipid droplets. However, standard one-dimensional NMR experiments allow the quantification of lipid concentration only, without providing information about physical characteristics such as the size of lipid droplets, viscosity of the cytosol, or cytoskeletal rigidity. This additional information can improve monitoring of apoptosis-based cancer treatments in intact cells and provide us with mechanistic insight into why these changes occur. In this paper, we use high-resolution magic angle spinning (HRMAS) 1 H NMR spectroscopy to monitor lipid concentrations and apparent diffusion coefficients of mobile lipid in intact cells treated with the apoptotic agents cisplatin or etoposide. We also use solution-state NMR spectroscopy to study changes in lipid profiles of organic solvent cell extracts. Both NMR techniques show an increase in the concentration of lipids but the relative changes are 10 times larger by HRMAS 1 H NMR spectroscopy. Moreover, the apparent diffusion rates of lipids in apoptotic cells measured by HRMAS 1 H NMR spectroscopy decrease significantly as compared with control cells. Slower diffusion rates of mobile lipids in apoptotic cells correlate well with the formation of larger lipid droplets as observed by microscopy. We also compared the mean lipid droplet displacement values calculated from the two methods. Both methods showed shorter displacements of lipid droplets in apoptotic cells. Our results demonstrate that the NMR-based diffusion experiments on intact cells discriminate between control and apoptotic cells. Apparent diffusion measurements in conjunction with 1 H NMR spectroscopy-derived lipid signals provide a novel means of following apoptosis in intact cells. This method could have potential application in enhancing drug discovery by monitoring drug treatments in vitro, particularly for agents that cause portioning of lipids such as apoptosis.

NMR and Thermal Studies for the Characterization of Mass Transport and Phase Separation in Paracetamol/Copovidone Hot-Melt Extrusion Formulations.

The formulation of drug/polymer amorphous solid dispersions (ASDs) is one of the most successful strategies for improving the oral bioavailability of poorly soluble active pharmaceutical ingredients (APIs). Hot-melt extrusion (HME) is one method for preparing ASDs that is growing in importance in the pharmaceutical industry, but there are still substantial gaps in our understanding regarding the dynamics of drug dissolution and dispersion in viscous polymers and the physical stability of the final formulations. Furthermore, computational models have been built to predict optimal processing conditions, but they are limited by the lack of experimental data for key mass transport parameters, such as the diffusion coefficient. The work presented here reports direct measurements of API diffusion in pharmaceutical polymer melts, using high-temperature pulsed-field gradient NMR. The diffusion coefficient of a model drug/polymer system (paracetamol/copovidone) was determined for different drug loadings and at temperatures relevant to the HME process. The mechanisms of the diffusion process are also explored with the Stokes-Einstein and Arrhenius models. The results show that diffusivity is linked exponentially to temperature. Furthermore, this study includes rheological characterization, differential scanning calorimetry (DSC), and 1H ssNMR T1 and T1ρ measurements to give additional insights into the physical state, phase separation, and API/polymer interactions in paracetamol/copovidone ASD formulations.

Online monitoring of a photocatalytic reaction by real-time high resolution FlowNMR spectroscopy.

We demonstrate how FlowNMR spectroscopy can readily be applied to investigate photochemical reactions that require sustained input of light and air to yield mechanistic insight under realistic conditions. The Eosin Y mediated photo-oxidation of N-allylbenzylamine is shown to produce imines as primary reaction products from which undesired aldehydes form after longer reaction times. Facile variation of reaction conditions during the reaction in flow allows for probe experiments that give information about the mode of action of the photocatalyst.

Control of the Porous Structure of Polystyrene Particles Obtained by Nonsolvent Induced Phase Separation.

Porous polystyrene microspheres were produced by a process of nonsolvent induced phase separation (NIPS) from ternary polymer-solvent-nonsolvent (polystyrene-toluene-ethanol) systems and characterized by scanning electron microscopy (SEM) and small-angle X-ray scattering (SAXS) techniques. This study provides evidence for a link between the structural morphology of the porous polystyrene particles and the polystyrene concentration in the initial solutions. A reciprocal relationship between pore diameter and polymer concentration was observed for the systems with the polymer amount below the critical chain overlap concentration, C*. Above C*, this relationship breaks down. The reciprocal relationship between porosity and polymer concentration can be used to facilitate the fine control of the void size. We demonstrate that the observed reciprocal relationship between pore diameter and polymer concentration correlates well with the relative amount of nonsolvent present in the system at the onset of the phase separation process. The pore size can be reduced and, consequently, the pore surface area can be increased either by reducing the polymer concentration in the initial solution or by decreasing the polymer molecular weight in the sample composition.

Optical source of individual pairs of colour-conjugated photons.

We theoretically demonstrate that Kerr nonlinearity in optical circuits can lead to both resonant four-wave mixing and photon blockade, which can be used for high-yield generation of high-fidelity individual photon pairs with conjugated frequencies. We propose an optical circuit, which, in the optimal pulsed-drive regime, would produce photon pairs at the rate up to 5 × 105 s -1 (0.5 pairs per pulse) with [Formula: see text] for one of the conjugated frequencies. We show that such a scheme can be utilised to generate colour-entangled photons.

Discovery of Pyrazolo[1,5-a]pyrimidine B-Cell Lymphoma 6 (BCL6) Binders and Optimization to High Affinity Macrocyclic Inhibitors.

Inhibition of the protein-protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo[1,5-a]pyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand-protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven.

Potent and selective bivalent inhibitors of BET bromodomains.

Proteins of the bromodomain and extraterminal (BET) family, in particular bromodomain-containing protein 4 (BRD4), are of great interest as biological targets. BET proteins contain two separate bromodomains, and existing inhibitors bind to them monovalently. Here we describe the discovery and characterization of probe compound biBET, capable of engaging both bromodomains simultaneously in a bivalent, in cis binding mode. The evidence provided here was obtained in a variety of biophysical and cellular experiments. The bivalent binding results in very high cellular potency for BRD4 binding and pharmacological responses such as disruption of BRD4-mediator complex subunit 1 foci with an EC50 of 100 pM. These compounds will be of considerable utility as BET/BRD4 chemical probes. This work illustrates a novel concept in ligand design-simultaneous targeting of two separate domains with a drug-like small molecule-providing precedent for a potentially more effective paradigm for developing ligands for other multi-domain proteins.

Optimization of a Series of Bivalent Triazolopyridazine Based Bromodomain and Extraterminal Inhibitors: The Discovery of (3R)-4-[2-[4-[1-(3-Methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)-4-piperidyl]phenoxy]ethyl]-1,3-dimethyl-piperazin-2-one (AZD5153).

Here we report the discovery and optimization of a series of bivalent bromodomain and extraterminal inhibitors. Starting with the observation of BRD4 activity of compounds from a previous program, the compounds were optimized for BRD4 potency and physical properties. The optimized compound from this campaign exhibited excellent pharmacokinetic profile and exhibited high potency in vitro and in vivo effecting c-Myc downregulation and tumor growth inhibition in xenograft studies. This compound was selected as the development candidate, AZD5153. The series showed enhanced potency as a result of bivalent binding and a clear correlation between BRD4 activity and cellular potency.

Spatially modulated structural colour in bird feathers.

Eurasian Jay (Garrulus glandarius) feathers display periodic variations in the reflected colour from white through light blue, dark blue and black. We find the structures responsible for the colour are continuous in their size and spatially controlled by the degree of spinodal phase separation in the corresponding region of the feather barb. Blue structures have a well-defined broadband ultra-violet (UV) to blue wavelength distribution; the corresponding nanostructure has characteristic spinodal morphology with a lengthscale of order 150 nm. White regions have a larger 200 nm nanostructure, consistent with a spinodal process that has coarsened further, yielding broader wavelength white reflectance. Our analysis shows that nanostructure in single bird feather barbs can be varied continuously by controlling the time the keratin network is allowed to phase separate before mobility in the system is arrested. Dynamic scaling analysis of the single barb scattering data implies that the phase separation arrest mechanism is rapid and also distinct from the spinodal phase separation mechanism i.e. it is not gelation or intermolecular re-association. Any growing lengthscale using this spinodal phase separation approach must first traverse the UV and blue wavelength regions, growing the structure by coarsening, resulting in a broad distribution of domain sizes.

Honeybee Colony Vibrational Measurements to Highlight the Brood Cycle.

Insect pollination is of great importance to crop production worldwide and honey bees are amongst its chief facilitators. Because of the decline of managed colonies, the use of sensor technology is growing in popularity and it is of interest to develop new methods which can more accurately and less invasively assess honey bee colony status. Our approach is to use accelerometers to measure vibrations in order to provide information on colony activity and development. The accelerometers provide amplitude and frequency information which is recorded every three minutes and analysed for night time only. Vibrational data were validated by comparison to visual inspection data, particularly the brood development. We show a strong correlation between vibrational amplitude data and the brood cycle in the vicinity of the sensor. We have further explored the minimum data that is required, when frequency information is also included, to accurately predict the current point in the brood cycle. Such a technique should enable beekeepers to reduce the frequency with which visual inspections are required, reducing the stress this places on the colony and saving the beekeeper time.

Discovery and optimization of a novel series of Dyrk1B kinase inhibitors to explore a MEK resistance hypothesis.

Potent and selective inhibitors of Dyrk1B kinase were developed to explore the hypothesis, based on siRNA studies, that Dyrk1B may be a resistance mechanism in cells undergoing a stress response.

Symptomatic lumbar osteochondroma treated via a multidisciplinary military surgical team: case report and review of the literature.

The authors describe the case of a giant osteochondroma emanating from the L5 vertebral body and extending into the retroperitoneum of a 40-year-old man, causing low back pain. Osteochondromas are benign bony tumors that typically occur within the appendicular skeleton, although in the sporadic form, up to 4% occur in the spine. A review of the English language literature has returned 44 cases of lumbar osteochondroma, including the present example. The lesions were sporadic in 81% of cases. Mean age of presentation overall is 39.5 years, with a mean age of 18.4 years (range 8-34 years) for hereditary cases and 45.7 years (range 11-81 years) for solitary lesions. Of the instances where gender was reported, 64% were male. The most common level of origin was L4 (38%). The most common anatomic site of origin was the inferior articular process (one-third). Of those lesions treated operatively, 46% underwent simple decompression, with 22% requiring decompression and fusion. This particular lesion was resected via a transperitoneal approach performed by a multidisciplinary team of neurosurgeons, vascular surgeons, and urologists. The bony tumor measured 6.1 × 7.8 × 7.7 cm. Removal of the lesion resulted in a significant improvement of the patient's symptoms.

Intraoperative duplex ultrasound criteria for performing interposition bypass in the treatment of popliteal artery entrapment syndrome.

We present a case series of 3 surgical procedures (2 patients) in which intraoperative duplex ultrasound (IDUS) was used to determine whether the chronic compression of the popliteal artery caused by popliteal artery entrapment syndrome had injured the artery to such a degree that interposition bypass was required. Patients initially underwent standard clinical evaluation including history and physical examination and noninvasive diagnostic testing including postexercise ankle-brachial indexes and angiography with evocative maneuvers before surgery. IDUS was performed. Doppler was used to calculate peak systolic velocities (PSVs) and velocity ratios (VRs) across areas of suspected injury. B-mode was used to assess arterial wall thickness (AWT) and sclerotic changes. Patients were followed in the postoperative period with surveillance duplex ultrasound (US). Three limbs (2 patients) underwent IDUS evaluation after popliteal decompression. Limb 1 demonstrated an elevated intraoperative PSV of 295 cm/sec with an elevated VR of 2.52 (295/117 cm/sec) and AWT of 1.1 mm. Interposition bypass was performed after popliteal decompression. Postoperative surveillance duplex US revealed a reduction of the PSV to 90 cm/sec. Limb 2 showed a mildly elevated intraoperative PSV of 211.5 cm/sec with a VR of 1.86 (211.5/114 cm/sec) and AWT of 0.8 mm. An interposition bypass was not performed. Limb 3 demonstrated an elevated intraoperative PSV of 300 cm/sec with an elevated VR of 2.51 (300/119.5 cm/sec) and AWT of 1.0. Interposition bypass was performed. Postoperative surveillance duplex US revealed a reduction of the PSV to 115 cm/sec. IDUS was very helpful in the operative management and intraoperative decision making process for popliteal artery entrapment. An elevated PSV of 250-275 cm/sec or greater on IDUS and a VR of 2.0 or greater, in conjunction with B-mode demonstration of arterial wall injury, was useful in identifying severely injured popliteal arterial segments. Additional prospective studies are warranted to further investigate objective criteria that indicate the need for bypass.