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.

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.

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.

A noninterventional management approach to endovascular coil migration into the sigmoid colon.

A 75-year-old man underwent endovascular treatment of a right internal iliac artery (IIA) aneurysm by placing coils in the distal IIA and occluding the inflow with a common iliac artery-to-external iliac artery stent graft. Surveillance computed tomography angiography discovered migration of an endovascular coil from the thrombosed right IIA into the sigmoid colon. Subsequent serial imaging demonstrated uncomplicated extracorporeal passage of the coil. We review the relevant literature and treatment rationale.

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.

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.

Asymmetric substitutions of 2-lithiated N-boc-piperidine and N-Boc-azepine by dynamic resolution.

Proton abstraction of N-tert-butoxycarbonyl-piperidine (N-Boc-piperidine) with sBuLi and TMEDA provides a racemic organolithium that can be resolved using a chiral ligand. The enantiomeric organolithiums can interconvert so that a dynamic resolution occurs. Two mechanisms for promoting enantioselectivity in the products are possible. Slow addition of an electrophile such as trimethylsilyl chloride allows dynamic resolution under kinetic control (DKR). This process occurs with high enantioselectivity and is successful by catalysis with substoichiometric chiral ligand (catalytic dynamic kinetic resolution). Alternatively, the two enantiomers of this organolithium can be resolved under thermodynamic control with good enantioselectivity (dynamic thermodynamic resolution, DTR). The best ligands found are based on chiral diamino-alkoxides. Using DTR, a variety of electrophiles can be used to provide an asymmetric synthesis of enantiomerically enriched 2-substituted piperidines, including (after Boc deprotection) the alkaloid (+)-beta-conhydrine. The chemistry was extended, albeit with lower yields, to the corresponding 2-substituted seven-membered azepine ring derivatives.

Volumetric diffusers: pseudorandom cylinder arrays on a periodic lattice.

Most conventional diffusers take the form of a surface based treatment, and as a result can only operate in hemispherical space. Placing a diffuser in the volume of a room might provide greater efficiency by allowing scattering into the whole space. A periodic cylinder array (or sonic crystal) produces periodicity lobes and uneven scattering. Introducing defects into an array, by removing or varying the size of some of the cylinders, can enhance their diffusing abilities. This paper applies number theoretic concepts to create cylinder arrays that have more even scattering. Predictions using a boundary element method are compared to measurements to verify the model, and suitable metrics are adopted to evaluate performance. Arrangements with good aperiodic autocorrelation properties tend to produce the best results. At low frequency power is controlled by object size and at high frequency diffusion is dominated by lattice spacing and structural similarity. Consequently the operational bandwidth is rather small. By using sparse arrays and varying cylinder sizes, a wider bandwidth can be achieved.

Endovascular coiling of an internal mammary artery pseudoaneurysm following placement of an internal jugular central venous catheter.

We discuss an interesting case of a right internal mammary artery pseudoaneurysm discovered after an attempted right internal jugular venous catheter placement. Such injury to the internal mammary artery is a rare complication of traumatic injuries to the chest, sternotomies, and central venous catheter placements. It has been reported after subclavian line placements, but not after an internal jugular catheterization.

Asymmetric lithiation-substitution of amines involving rearrangement of borates.

Asymmetric lithiation of substituted benzylamines, N-Boc-pyrrolidine, or N-Boc-indoline using Beak's methodology was followed by electrophilic quench with trialkylboranes. The resulting borate intermediates rearrange with concomitant C-N bond breakage to give, after oxidation, chiral secondary alcohols with high enantioselectivity.

Dynamic resolution of N-Boc-2-lithiopiperidine.

Dynamic thermodynamic resolution of N-Boc-2-lithiopiperidine is possible using a chiral ligand; the two enantiomers of this organolithium can be resolved with selectivities of up to 85 : 15 from a selection of 26 chiral diamino-alkoxide ligands screened.

Dynamic kinetic and kinetic resolution of N-Boc-2-lithiopiperidine.

Asymmetric substitution of 2-lithiopiperidines can be achieved by dynamic resolution; the organolithium is formed as a racemic mixture by proton abstraction (or tin-lithium exchange) and is resolved in the presence of a chiral ligand.

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.