Robust LC3B lipidation analysis by precisely adjusting autophagic flux

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.

Optimization of the Guide Design of MIRACLES, the Neutron Time-of-Flight Backscattering Spectrometer at the European Spallation Source

To boost the science case of MIRACLES, the time-of-flight backscattering spectrometer at the European Spallation Source (ESS), an optimized neutron guide system, is proposed. This systematic study resulted in an enhancement in the transport of cold neutrons, compared with the previous conceptual design, with wavelengths ranging from λ = 2 Å to 20 Å along the 162.5-m distance from source to sample. This maintained the undisturbed main focus of the instrument, viz, to carry out quasielastic and inelastic neutron scattering (QENS and INS) experiments on a large dynamic range and for both energy-gain and energy-loss sides. To improve the collection of cold neutrons from the source and direct them to the sample position, the vertical geometry was adjusted to an adapted version of a ballistic elliptical profile. Its horizontal geometry was conceived to: (i) keep the high-resolution performance of the instrument, and (ii) minimize the background originating from fast and thermal neutrons. To comply with the first requirement, a narrow guide section at the pulse shaping chopper position has been implemented. To fulfil the second, a curved guide segment has been chosen to suppress neutrons with wavelengths λ < 2 Å. Subsequent tailoring of the phase space provided an efficient transport of cold neutrons along the beamline to reach a 3 × 3 cm2 sample. Finally, additional calculations were performed to present a potential upgrade, with the exchange of the final segment, to focus on samples of approximately 1 × 1 cm2; the proposal anticipates a flux increase of 70% in this 1 cm2 sample area.

Responses of Net Anthropogenic N Inputs and Export Fluxes in the Megacity of Chengdu, China

Anthropogenic N inputs have become progressively more problematic and have profoundly affected the water quality in megacities throughout China. Thus, to design and implement appropriate megalopolis watershed management, it is important to understand the relationship between N inputs and exports and to identify the N pollution sources. To that end, in this work, the net anthropogenic N inputs (NANI) in Chengdu City were estimated based on statistical data collected between 1970 and 2019. N input fluxes and pollution sources were estimated through sample collection and field measurements that were performed between 2017 and 2019, while nitrate (NO3−) was identified using stable isotope and Bayesian model (SIAR) analysis. The NANI was found to be affected primarily by livestock and poultry consumption of N rich feed. Moreover, the N export fluxes and runoff showed a high degree of correlation. Notably, NO3− fluxes exhibited a significant increase over the course of the study period, such that, by 2019, the total N fluxes (18,883.85 N kg/km2) exceeded the NANI (17,093.87 N kg/km2). The results indicate that although livestock and poultry farming were the original primary sources of NANI, their contributions declined on an annual basis. Moreover, with the emphasis placed on point source management in Chengdu City, domestic sewage discharge has been significantly reduced. Therefore, N retention in groundwater is thought to be the factor driving the N flux increase. These findings are pivotal to solving the N pollution problem in megacities like Chengdu (China).

The climatic trends of temperature and salinity in the Bering straight in the last decades

Представлены результаты расчетов линейных климатических трендов температуры и солености поверхностных вод Берингова пролива и прилегающих акваторий за временной период 1950 – 2020 гг., отдельно за август и февраль месяцы. Отмечается нагрев поступающей тихоокеанской воды в Чукотское море в летний период на +0,120С/10лет, в зимний период +0,140С/10лет. Одновременно происходит временной тренд в сторону уменьшения солености поступающих вод, это ‒0,060/00/10лет в летний сезон и ‒0,03 S0/00/10лет в зимний сезон. Таким образом в случае инерционного изменения климата через 100 лет вода в Беринговом проливе будет на 1,30С теплее и на 0,450/00 менее соленой. Сделаны оценки роста потока тепла через Берингов пролив в Чукотское море вследствие климатического тренда, который составляет +2,4*1019Дж/10лет. Отмечено, что направления трендов температуры и солености в Беринговом море и Беринговом проливе в сторону нагрева и уменьшения солености совпадают, а в Чукотском море климатические тенденции противоположные. The calculation of the climatic linear trends of the surface waters temperature and salinity in the Bering Strait and nearby water areas are presented. The time period is 1950 – 2020 years. The Time data for the warm season - August and the cold season - February series are shown separately. The heating of the incoming Pacific water into the Chukchi Sea is note. There is the summer period +0.120C/decade, in the winter period +0.140C/decade. At the same time, there is some trend towards decrease in salinity the straight water, this is ‒0.06psu/decade in the summer season and this is ‒0.03psu/decade in the winter season. Following of an inertial climate change in 100 years there is the Bering Strait water will be +1.30Cwarmer and ‒0.45psu/decade salty less. The estimate of the heat flux increase through the Bering Strait to the Chukchi Sea due to the climatic trend is +2.4*1019J/decade. There is a peculiarity that the time trends of temperature and salinity in the Bering Sea and the Bering Strait have the same direction to the heating and the salinity decrease, but at the same time the Chukchi Sea has the opposite tendency. An explanation of this discrepancy is given.

A mini-module with embedded spacers for high-throughput ultrafiltration

Ultrafiltration membrane modules suffer from performance losses that arise during filtration from concentration polarization and fouling. Such performance losses are frequently mitigated by controlling the hydrodynamic conditions at the membrane/fluid interface. For instance, the hydrodynamic conditions are manipulated using mesh spacers that act as a static mixer. The design of such spacers is rarely optimized to effectively maintain mass transport through the membrane. Also, the spacer is an additional part added to the feed channel of the membrane module, improving mass transport in general, yet accepting less transport in dead zones.Here, we present a mini module with spacers embedded in the module housing of a flat-sheet ultrafiltration membrane to attain high permeation rates. The performance of two new embedded spacer geometries – staggered herringbone and sinusoidal corrugation – prove experimentally that indeed a CFD-simulated flux increase can be realized during bovine serum albumin (BSA) filtration. The flow characteristics inside the mini module are further investigated using magnetic resonance velocity imaging. The new embedded sinusoidal corrugation spacers outper- form conventional mesh spacer inlays. The fabrication of such module-embedded spacers has been conceptually implemented through an in-silico design and a 3D-printing production process. The latter can be easily realized using injection molding processes, as is now done for the Sartorius ambra(R) crossflow product line.

Performance Characterization of Hollow Fiber Vacuum Membrane Distillation Module for Desalination

3D laminar flow simulations were conducted using OpenFOAM to resolve the temperature, concentration, velocity, and pressure field for two hollow fiber vacuum membrane distillation configurations with feed solution flowing either inside or outside a single hollow fiber. The fiber has a circular cross-section, a fixed length of 120mm, and an inner diameter of 2 mm. The wall thickness was varied from 150 to 450μm, and the pore diameter was varied from 0.1 to 0.3μm based on commercial fibers. The feed solution is an aqueous solution of water and NaCl. The feed flow was simulated at a Reynolds number of 500 and vacuum pressure of 5,000 Pascals. It was found that there was a 75% increase in flux, from 9.58 to 41.41 kg/m2h, between the worst and the best case in membrane properties. Increasing the pore diameter or wall thickness while the other value was fixed resulted in a 45–57% flux increase depending on the fixed value. The module with the feed solution flowing outside a hollow fiber yields 24% higher flux than the module with the feed solution flowing inside the hollow fiber at the same conditions.

Effect of Osmotic Pressure on Whey Protein Concentration in Forward Osmosis

Forward osmosis (FO) is an emerging process to dewater whey streams energy efficiently. The driving force for the process is the concentration gradient between the feed (FS) and the concentrated draw (DS) solution. Here we investigate not only the effect of the DS concentration on the performance, but also that of the FS is varied to maintain equal driving force at different absolute concentrations. Experiments with clean water as feed reveal a flux increase at higher osmotic pressure. When high product purities and thus low reverse salt fluxes are required, operation at lower DS concentrations is preferred. Whey as FS induces severe initial flux decline due to instantaneous protein fouling of the membrane. This is mostly due to reversible fouling, and to a smaller extent to irreversible fouling. Concentration factors in the range of 1.2–1.3 are obtained. When 0.5 M NaCl is added to whey as FS, clearly lower fluxes are obtained due to more severe concentration polarization. Multiple runs over longer times show though that irreversible fouling is fully suppressed due to salting in/out effects and flux decline is the result of reversible fouling only.

Creating a bottleneck: Robust LC3B lipidation analysis by adjusting autophagic flux with low concentrations of Bafilomycin A1

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.

Carbon export and fate beneath a dynamic upwelled filament off the California coast

To understand the vertical variations in carbon fluxes in biologically productive waters, four autonomous carbon flux explorers (CFEs), ship-lowered CTD-interfaced particle-sensitive transmissometer and scattering sensors, and surface-drogued sediment traps were deployed in a filament of offshore flowing, recently upwelled water, during the June 2017 California Current Ecosystem – Long Term Ecological Research process study. The Lagrangian CFEs operating at depths from 100–500 m yielded carbon flux and its partitioning with size from 30 µm–1 cm at three intensive study locations within the filament and in waters outside the filament. Size analysis codes intended to enable long-term CFE operations independent of ships are described. Different particle classes (anchovy pellets, copepod pellets, and > 1000 µm aggregates) dominated the 100–150 m fluxes during successive stages of the filament evolution as it progressed offshore. Fluxes were very high at all locations in the filament; below 150 m, flux was invariant or increased with depth at the two locations closer to the coast. Martin curve b factors (± denotes 95 % confidence intervals) for total particulate carbon flux were +0.37 ± 0.59, +0.85 ± 0.31, −0.24 ± 0.68, and −0.45 ± 0.70 at the three successively occupied locations within the plume, and in transitional waters. Interestingly, the flux profiles for all particles < 400 µm were a much closer fit to the canonical Martin profile (b−0.86); however, most (typically > 90 %) of the particle flux was carried by > 1000 µm sized aggregates which increased with depth. Mechanisms to explain the factor of 3 flux increase between 150 and 500 m at the mid-plume location are investigated.

Modeling the broadband emission of 3C 454.3

The results of a long-term multiwavelength study of the powerful flat spectrum radio quasar 3C 454.3 using Fermi-LAT and Swift XRT/UVOT data are reported. In the γ-ray band, Fermi-LAT observations show several major flares when the source flux was &gt;10−5 photon cm−2 s−1; the peak γ-ray flux above 141.6 MeV, (9.22 ± 1.96) × 10−5 photon cm−2 s−1 observed on MJD 55519.33, corresponds to 2.15 × 1050 erg s−1 isotropic γ-ray luminosity. The analysis of Swift XRT and UVOT data revealed a flux increase, although with smaller amplitudes, also in the X-ray and optical/UV bands. The X-ray emission of 3C 454.3 is with a hard spectral index of ΓX = 1.16–1.75, and the flux in the flaring states increased up to (1.80 ± 0.18) × 10−10 erg cm−2 s−1. Through combining the analyzed data, it was possible to assemble 362 high-quality and quasi-simultaneous spectral energy distributions of 3C 454.3 in 2008-2018 which all were modeled within a one-zone leptonic scenario assuming the emission region is within the broad line region, involving synchrotron, synchrotron self-Compton and external Compton mechanisms. Such an extensive modeling is the key for constraining the underlying emission mechanisms in the 3C 454.3 jet and allows to derive the physical parameters of the jet and investigate their evolution in time. The modeling suggests that during the flares, along with the variation of emitting electron parameters, the Doppler boosting factor increased substantially implying that the emission in these periods has most likely originated in a faster moving region.