Conservative Black Hole Scattering at Fifth Post-Minkowskian and First Self-Force Order.

We compute the fifth post-Minkowskian (5PM) order contributions to the scattering angle and impulse of classical black hole scattering in the conservative sector at first self-force order using the worldline quantum field theory formalism. This challenging four-loop computation required the use of advanced integration-by-parts and differential equation technology implemented on high-performance computing systems. Use of partial fraction identities allowed us to render the complete integrand in a fully planar form. The resulting function space is simpler than expected: In the scattering angle, we see only multiple polylogarithms up to weight three and a total absence of the elliptic integrals that appeared at 4PM order. All checks on our result, both internal-cancellation of dimensional regularization poles and preservation of the on-shell condition-and external-matching the slow-velocity limit with the post-Newtonian (PN) literature up to 5PN order and matching the tail terms to the 4PM loss of energy-are passed.

Conservative Scattering of Spinning Black Holes at Fourth Post-Minkowskian Order.

Using the N=1 supersymmetric, spinning worldline quantum field theory formalism, we compute the conservative spin-orbit part of the momentum impulse Δp_{i}^{µ}, spin kick ΔS_{i}^{µ}, and scattering angle θ from the scattering of two spinning massive bodies (black holes or neutron stars) up to fourth post-Minkowskian (PM) order. These three-loop results extend the state of the art for generically spinning binaries from 3PM to 4PM. They are obtained by employing recursion relations for the integrand construction and advanced multiloop Feynman integral technology in the causal (in-in) worldline quantum field theory framework to directly produce classical observables. We focus on the conservative contribution (including tail effects) and outline the computations for the dissipative contributions as well. Our spin-orbit results agree with next-to-next-to-next-to-leading-order post-Newtonian and test-body data in the respective limits. We also reconfirm the conservative 4PM nonspinning results.

Dissipative Scattering of Spinning Black Holes at Fourth Post-Minkowskian Order.

We compute the radiation reacted momentum impulse Δp_{i}^{µ}, spin kick ΔS_{i}^{µ}, and scattering angle θ between two scattered spinning massive bodies (black holes or neutron stars) using the N=1 supersymmetric worldline quantum field theory formalism up to fourth post-Minkowskian (4PM) order. Our calculation confirms the state-of-the-art nonspinning results, and extends them to include spin-orbit effects. Advanced multiloop Feynman integral technology including differential equations and the method of regions are applied and extended to deal with the retarded propagators arising in a causal description of the scattering dynamics. From these results we determine a complete set of radiative fluxes at subleading PM order: the 4PM radiated four-momentum and, via linear response, the 3PM radiated angular momentum, both again including spin-orbit effects.

Application of X-ray fluorescence spectrometry for screening pharmaceutical products for Elemental Impurities according to ICH guideline Q3D.

Energy-dispersive X-ray fluorescence spectrometry (EDXRF) is a suitable analytical procedure for screening drug products for Elemental Impurities (EI) according to ICH guideline Q3D. EDXRF represents a cost-efficient, robust and standard-free alternative compared to other methodologies for trace analysis, and therefore utilization of this application should be encouraged. This study demonstrates the capability of EDXRF for EI screening of oral solid dosage drug products (OSD products) within a defined matrix range. Method development and validation focused on class 1 (Cd, Pb, As, Hg) and class 2A (Co, V, Ni) elements, as defined by ICH guideline Q3D. In order to limit validation activities, a novel cluster approach was applied, based on matrix properties. This included comprehensive characterization of method performance parameters for exemplary pharmaceutical matrices and demonstration of LOQ independence from matrix effects by using a set of limit samples representing typical matrix variations of OSD products. The methodology can be used as a limit test for class 1 and class 2A elements and is fully compliant with method validation requirements according to the European Pharmacopeia. The novelty of the present work is the application of EDXRF for a routine screening of OSD products for Elemental Impurities within the pharmaceutical industry beyond previously published feasibility studies for a limited number of pharmaceutical raw materials or products.

Comparison of biomechanical properties in ascending aortic aneurysms of patients with congenital bicuspid aortic valve and Marfan syndrome.

BACKGROUND: In patients with ascending aortic aneurysms (AscAA), biomechanical differences are seen among patients with congenital bicuspid aortic valves (BAV), Marfan syndrome (MFS), and tricuspid aortic valves (TAV). We examined the hemodynamic profiles and ultrastructures of aneurysmal specimens, focusing on vascular remodelling to better understand AscAA pathogenesis. METHODS: A total of 795 patients with BAV (43.97 ±â€¯0.51 years; 93.2% male), 69 with MFS (34.43 ±â€¯1.44 years; 86.2% male), and 90 with TAV (67.27 ±â€¯0.58 years; 60% male) were enrolled, primarily upon admission with AscAA. The biomechanical properties of the aortic root were assessed and intraoperative specimens were analyzed by light-microscopy and two-photon autofluorescence microscopy. RESULTS: Patients with BAV had significantly greater distension of the aortic root, irrespective of age or aneurysmal widening (R2 = 0.543, p < 0.05). This was associated with significantly increase in the size of the tunica media. Patients with MFS displayed significant stiffness in the sinuses that worsened with age (R2 = 0.752, p < 0.001), similar to patients with TAV (R2 = 0.626, p < 0.05). Patients with MFS showed significant root elasticity with aneurysmal growth (R2 = 0.596, p < 0.05) and increased medial degeneration. Patients with TAV maintained biomechanical properties, apart from aneurysmal dimensions and high levels of inflammation. CONCLUSIONS: Among patients with AscAA, those with BAV maintain tissue elasticity in the aortic root, regardless of age. Patients with MFS demonstrate increased sinus stiffness with medial degeneration, both during aging and with aneurysmal growth. Patients with TAV and AscAA present with increased inflammation.

PKCα diffusion and translocation are independent of an intact cytoskeleton.

Translocation of cytosolic cPKC to the plasma membrane is a key event in their activation process but its exact nature is still unclear with particular dispute whether sole diffusion or additional active transport along the cell's cytoskeleton contributes to cPKC's dynamics. This was addressed by analyzing the recruitment behavior of PKCα while manipulating the cytoskeleton. Photolytic Ca2+ uncaging allowed us to quantify the kinetics of PKCα redistribution to the plasma membrane when fused to monomeric, dimeric and tetrameric fluorescence proteins. Results indicated that translocation kinetics were modulated by the state of oligomerization as expected for varying Stokes' radii of the participating proteins. Following depolymerization of the microtubules and the actin filaments we found that Ca2+ induced membrane accumulation of PKCα was independent of the filamentous state of the cytoskeleton. Fusion of PKCα to the photo-convertible fluorescent protein Dendra2 enabled the investigation of PKCα-cytoskeleton interactions under resting conditions. Redistribution following spatially restricted photoconversion showed that the mobility of the fusion protein was independent of the state of the cytoskeleton. Our data demonstrated that in living cells neither actin filaments nor microtubules contribute to PKCα's cytosolic mobility or Ca2+-induced translocation to the plasma membrane. Instead translocation is a solely diffusion-driven process.

Two-photon microscopy using fiber-based nanosecond excitation.

Two-photon excitation fluorescence (TPEF) microscopy is a powerful technique for sensitive tissue imaging at depths of up to 1000 micrometers. However, due to the shallow penetration, for in vivo imaging of internal organs in patients beam delivery by an endoscope is crucial. Until today, this is hindered by linear and non-linear pulse broadening of the femtosecond pulses in the optical fibers of the endoscopes. Here we present an endoscope-ready, fiber-based TPEF microscope, using nanosecond pulses at low repetition rates instead of femtosecond pulses. These nanosecond pulses lack most of the problems connected with femtosecond pulses but are equally suited for TPEF imaging. We derive and demonstrate that at given cw-power the TPEF signal only depends on the duty cycle of the laser source. Due to the higher pulse energy at the same peak power we can also demonstrate single shot two-photon fluorescence lifetime measurements.

Two-photon photolysis combined with a kilobeam array scanner to probe calcium signaling in cardiomyocytes.

Photorelease of caged compounds allows a fast and defined intracellular increase in calcium (Ca(2+)) or other biologically relevant substances or molecules without impinging on other cellular functions. In particular, two-photon photolysis (2PP) allows a spatially restricted uncaging in sub-femtoliter volumes. Here, we describe how to combine 2PP with a confocal kilobeam array scanner and provide an example where Ca(2+) is released in an isolated cardiac myocyte.

Confocal FLIM of genetically encoded FRET sensors for quantitative Ca2+ imaging.

Fluorescence lifetime imaging (FLIM) is a powerful imaging mode that can be combined with confocal imaging. Changes in the fluorescence decay time of a donor in an intramolecular Förster resonance energy transfer (FRET)-based biosensor provide intrinsic quantitative data. Here, we describe a protocol using both the Ca(2+) sensor TN-XL, which uses troponin C, as the Ca(2+)-sensing unit, and the FLIM technology based on time-correlated single-photon counting.

Gαq and Gα11 contribute to the maintenance of cellular electrophysiology and Ca2+ handling in ventricular cardiomyocytes.

AIMS: Gα(q) and Gα(11) signalling pathways contribute to cardiac diseases such as hypertrophy and arrhythmia, but their role in cardiac myocytes from healthy hearts has remained unclear. We aimed to investigate the contribution of Gα(q) and Gα(11) signalling to the basal properties of ventricular myocytes. METHODS AND RESULTS: We created a conditional Gα(q) knockout (KO) after tamoxifen injection into gnaq(flox/flox) gna11(-/-) α-MHC Cre(tg/0) mice and found alterations in the electrophysiological and Ca(2+) handling properties of ventricular myocytes using patch-clamp and Fura-2 video imaging. To reveal the genuine effects of protein KO, we investigated the individual contributions of (i) tamoxifen injection, (ii) Cre recombinase expression, (iii) Gα(11) KO, and (iv) Gα(q) KO. Profound and persistent alterations in myocyte properties occurred following the tamoxifen injection alone. Consequently, we used the presence or absence of Cre recombinase expression as the determinant for the Gα(q) KO. Myocytes from the Gα(q) and/or Gα(11) KO mice displayed genuine alterations in the action potentials, membrane capacitance, membrane currents, and Ca(2+) handling (amplitude, post-rest behaviour, and Ca(2+) removal processes). CONCLUSIONS: We conclude that, in a transgenic model, the role of Gα(q) can be best studied using Cre recombinase expression as the molecular determinant for Gα(q) KO rather than tamoxifen/miglyol injection. While excessive hormonal stimulation of the Gα(q)/Gα(11) signalling pathways plays an essential role in cardiac diseases, we propose that the persistent low-level stimulation of these pathways by Gα(q)/Gα(11) activation is instrumental in the physiological behaviour of ventricular myocytes.

Norcantharidin analogues: synthesis, anticancer activity and protein phosphatase 1 and 2A inhibition.

Cantharidin (1) and its derivatives are of significant interest as serine/threonine protein phosphatase 1 and 2A inhibitors. Additionally, compounds of this type have displayed growth inhibition of various tumour cell lines. To further explore both of these inhibition pathways, a number of amide-acid norcantharidin analogues (15-26) were prepared. Compounds 23 and 24, containing two carboxylic acid residues, showed good PP1 and PP2A activity, with IC(50) values of approximately 15 and approximately 3 mum, respectively. Substituted aromatic amide analogues 45, 48, 49, 52, 53, and 54 also displayed good PP1 and PP2A inhibition, with IC(50) values in the range of 15-10 microM (PP1) and 11-5 microM (PP2A). However, bulky ortho substituents on the aromatic ring caused the aromatic ring to be skewed from the NCO planarity, leading to a decrease in PP1 and PP2A inhibition. A number of analogues, 20, 22, 25 and 46, showed excellent tumour growth inhibition, with 46 in particular being more potent than the lead, norcantharidin 2.

Norcantharimides, synthesis and anticancer activity: Synthesis of new norcantharidin analogues and their anticancer evaluation.

A range of amines was reacted with norcantharidin (2) to provide the corresponding norcantharimides (9-43). Treatment of norcantharidin with allylamine afforded the corresponding allyl-norcantharimide (20) which was amenable to epoxidation (mCPBA, 22) and subsequent ring opening (MeOH/H(+); 23) or alternatively, osmylation (OsO(4)/NMO; 24). These simple synthetic modifications of 2 facilitated the development of a novel series of norcantharimides displaying modest to good broad spectrum cytotoxicity against HT29 and SW480 (colorectal carcinoma); MCF-7 (breast adenocarcinoma); A2780 (ovarian carcinoma); H460 (lung carcinoma); A431 (epidermoid carcinoma); DU145 (prostate carcinoma); BE2-C (neuroblastoma); and SJ-G2 (glioblastoma). Analogues possessing a C(10), C(12) or C(14) alkyl chain or a C(12) linked bis-norcantharimide displayed the highest levels of cytotoxicity.

Heterocyclic substituted cantharidin and norcantharidin analogues--synthesis, protein phosphatase (1 and 2A) inhibition, and anti-cancer activity.

Norcantharidin (3) is a potent PP1 (IC(50)=9.0+/-1.4 microM) and PP2A (IC(50)=3.0+/-0.4 microM) inhibitor with 3-fold PP2A selectivity and induces growth inhibition (GI(50) approximately 45 microM) across a range of human cancer cell lines including those of colorectal (HT29, SW480), breast (MCF-7), ovarian (A2780), lung (H460), skin (A431), prostate (DU145), neuroblastoma (BE2-C), and glioblastoma (SJ-G2) origin. Until now limited modifications to the parent compound have been tolerated. Surprisingly, simple heterocyclic half-acid norcantharidin analogues are more active than the original lead compound, with the morphilino-substituted (9) being a more potent (IC(50)=2.8+/-0.10 microM) and selective (4.6-fold) PP2A inhibitor with greater in vitro cytotoxicity (GI(50) approximately 9.6 microM) relative to norcantharidin. The analogous thiomorpholine-substituted (10) displays increased PP1 inhibition (IC(50)=3.2+/-0 microM) and reduced PP2A inhibition (IC(50)=5.1+/-0.41 microM), to norcantharidin. Synthesis of the analogous cantharidin analogue (19) with incorporation of the amine nitrogen into the heterocycle further increases PP1 (IC(50)=5.9+/-2.2 microM) and PP2A (IC(50)=0.79+/-0.1 microM) inhibition and cell cytotoxicity (GI(50) approximately 3.3 microM). These analogues represent the most potent cantharidin analogues thus reported.