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As a step towards implementing non-aversive handling techniques at a big mouse breeding facility in Germany, tunnel handling was introduced in a breeding unit comprising three inbred mouse strains. To assess whether tunnel handling would be feasible for the animal technicians in their everyday work and beneficial for the mice when being handled during weekly cage change only, the behaviour of tunnel- and tail-handled animals of both sexes was examined before, during and after the handling events over a period of nine weeks. Moreover, the time expenditure was compared between both handling techniques. It was possible to use the tunnel in all three mouse strains. However, the impact of the handling techniques on the behavioural parameters investigated in the present study were strain-specific. All behavioural parameters suggested that NZW mice benefited the most from tunnel handling. The results obtained from Hello Kitty and WNK mice were ambiguous, which may suggest that a brief handling session during the cage clean may have not been sufficient to habituate them to the process of handling. It took the animal technicians approximately 3 seconds longer per mouse when using a tunnel. The strain-specific results should encourage researchers to share their experiences with non-aversive handling techniques in different mouse strains, for example, along with their research articles., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Commonly used media for the differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) contain high concentrations of proteins, in particular albumin, which is prone to quality variations and presents a substantial cost factor, hampering the clinical translation of in vitro-generated cardiomyocytes for heart repair. To overcome these limitations, we have developed chemically defined, entirely protein-free media based on RPMI, supplemented with L-ascorbic acid 2-phosphate (AA-2P) and either the non-ionic surfactant Pluronic F-68 or a specific polyvinyl alcohol (PVA)., Methods and Results: Both media compositions enable the efficient, directed differentiation of embryonic and induced hPSCs, matching the cell yields and cardiomyocyte purity ranging from 85 to 99% achieved with the widely used protein-based CDM3 medium. The protein-free differentiation approach was readily up-scaled to a 2000 mL process scale in a fully controlled stirred tank bioreactor in suspension culture, producing > 1.3 × 10 9 cardiomyocytes in a single process run. Transcriptome analysis, flow cytometry, electrophysiology, and contractile force measurements revealed that the mass-produced cardiomyocytes differentiated in protein-free medium exhibit the expected ventricular-like properties equivalent to the well-established characteristics of CDM3-control cells., Conclusions: This study promotes the robustness and upscaling of the cardiomyogenic differentiation process, substantially reduces media costs, and provides an important step toward the clinical translation of hPSC-CMs for heart regeneration., (© 2024. The Author(s).)

New particle formation in the free troposphere is a major source of cloud condensation nuclei globally. The prevailing view is that in the free troposphere, new particles are formed predominantly in convective cloud outflows. We present another mechanism using global observations. We find that during stratospheric air intrusion events, the mixing of descending ozone-rich stratospheric air with more moist free tropospheric background results in elevated hydroxyl radical (OH) concentrations. Such mixing is most prevalent near the tropopause where the sulfur dioxide (SO 2 ) mixing ratios are high. The combination of elevated SO 2 and OH levels leads to enhanced sulfuric acid concentrations, promoting particle formation. Such new particle formation occurs frequently and over large geographic regions, representing an important particle source in the midlatitude free troposphere.

A promising cell-therapy approach for heart failure aims at differentiating human pluripotent stem cells (hPSCs) into functional cardiomyocytes (CMs) in vitro to replace the disease-induced loss of patients' heart muscle cells in vivo. But many challenges remain for the routine clinical application of hPSC-derived CMs (hPSC-CMs), including good manufacturing practice (GMP)-compliant production strategies. This protocol describes the efficient generation of hPSC-CM aggregates in suspension culture, emphasizing process simplicity, robustness and GMP compliance. The strategy promotes clinical translation and other applications that require large numbers of CMs. Using a simple spinner-flask platform, this protocol is applicable to a broad range of users with general experience in handling hPSCs without extensive know-how in biotechnology. hPSCs are expanded in monolayer to generate the required cell numbers for process inoculation in suspension culture, followed by stirring-controlled formation of cell-only aggregates at a 300-ml scale. After 48 h at checkpoint (CP) 0, chemically defined cardiac differentiation is induced by WNT-pathway modulation through use of the glycogen-synthase kinase-3 inhibitor CHIR99021 (WNT agonist), which is replaced 24 h later by the chemical WNT-pathway inhibitor IWP-2. The exact application of the described process parameters is important to ensure process efficiency and robustness. After 10 d of differentiation (CP I), the production of ≥100 × 10 6 CMs is expected. Moreover, to 'uncouple' cell production from downstream applications, continuous maintenance of CM aggregates for up to 35 d in culture (CP II) is demonstrated without a reduction in CM content, supporting downstream logistics while potentially overcoming the requirement for cryopreservation., (© 2024. Springer Nature Limited.)

Background: Human pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) has enormous potential for fuelling these cell demands. In this study, the efficient long-term matrix-free suspension culture of hPSC aggregates is shown., Methods and Results: STBR-controlled, chemical aggregate dissociation and optimized passage duration of 3 or 4 days promotes exponential hPSC proliferation, process efficiency and upscaling by a seed train approach. Intermediate high-density cryopreservation of suspension-derived hPSCs followed by direct STBR inoculation enabled complete omission of matrix-dependent 2D (two-dimensional) culture. Optimized 3D cultivation over 8 passages (32 days) cumulatively yielded ≈4.7 × 10 15 cells, while maintaining hPSCs' pluripotency, differentiation potential and karyotype stability. Gene expression profiling reveals novel insights into the adaption of hPSCs to continuous 3D culture compared to conventional 2D controls., Conclusions: Together, an entirely matrix-free, highly efficient, flexible and automation-friendly hPSC expansion strategy is demonstrated, facilitating the development of good manufacturing practice-compliant closed-system manufacturing in large scale., (© 2024. The Author(s).)

External cycling regenerating nitrogen oxides (NO x  ≡ NO + NO 2 ) from their oxidative reservoir, NO z , is proposed to reshape the temporal-spatial distribution of NO x and consequently hydroxyl radical (OH), the most important oxidant in the atmosphere. Here we verify the in situ external cycling of NO x in various environments with nitrous acid (HONO) as an intermediate based on synthesized field evidence collected onboard aircraft platform at daytime. External cycling helps to reconcile stubborn underestimation on observed ratios of HONO/NO 2 and NO 2 /NO z by current chemical model schemes and rationalize atypical diurnal concentration profiles of HONO and NO 2 lacking noontime valleys specially observed in low-NO x atmospheres. Perturbation on the budget of HONO and NO x by external cycling is also found to increase as NO x concentration decreases. Consequently, model underestimation of OH observations by up to 41% in low NO x atmospheres is attributed to the omission of external cycling in models., (© 2023. The Author(s).)

Phospholipids (PLs) are widely used in the pharma industry and a better understanding of their behavior under different conditions is helpful for applications such as their use as medical transporters. The transition temperature T m affects the lipid conformation and the interfacial tension between p er f luoroperhydro p henanthrene (PFP) and an aqueous suspension of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), as well as a mixture of these PLs with cholesterol. Interfacial tensions were measured with the Du Noüy ring at quasi-equilibrium; the area per molecule was calculated according to the Gibbsian approach and a time-dependent tension gradient. Results show that the time t ε to reach quasi-equilibrium was shorter when the temperature was above T m , indicating a faster adsorption process ( t ε ,DPPC , 36 °C = 48 h, t ε ,DPPC,48 °C = 24 h) for PL in the liquid crystalline state than in the gel state (T < T m ). In addition, concentration-dependent results of the interfacial tension revealed that above the respective T m and at all concentrations c > 0.1 mM, the average minimum interfacial tension for DPPC and DSPC (14.1 mN/m and 15.3 mN/m) does not differ significantly between those two lipids. Equilibrium between monolayers and bilayers shows that for T < T m , surface pressures ∏ ≈ 31 mN/m are reached while for T > T m , ∏ ≈ 41 mN/m. Mixtures with cholesterol only reach ∏ ≤ 31 mN/m T m , with no significant difference between the two PLs. The higher interfacial tension of the mixture indicates stabilization of the liposomal conformation in the aqueous phase by the addition of cholesterol. The high diffusion coefficients show that adsorption is mainly based on liposomes.

Background: Reconstruction of continuity defects following osteonecrosis in multimorbid patients is challenging. In all cases of the predescribed palliative treatment method for alloplastic mandible reconstruction, plate fractures were detected in follow-up. We hypothesized that a modification could avoid these fractures, leading to stable long-term results., Methods: This retrospective study compares the original method with a modification using single, laser-sintered CAD-CAM plates instead of manually bent miniplates. The predescribed shuttering technique was used to reconstruct the mandible in its original shape with bone cement., Results: Uneventful wound healing was observed in 86% of the cases after modification of the method. No implant or plate fracture occurred., Conclusions: The presented method should be considered as a treatment option for mandible reconstruction in elderly, multimorbid patients in palliative situations. The results of this study suggest that the modification of the method leads to significantly improved long-term stability. Donor site morbidity is avoided with this method of palliative surgery., (© 2023 The Authors. Head & Neck published by Wiley Periodicals LLC.)

The hydroxyl radical (OH) fuels atmospheric chemical cycling as the main sink for methane and a driver of the formation and loss of many air pollutants, but direct OH observations are sparse. We develop and evaluate an observation-based proxy for short-term, spatial variations in OH (Proxy OH ) in the remote marine troposphere using comprehensive measurements from the NASA Atmospheric Tomography (ATom) airborne campaign. Proxy OH is a reduced form of the OH steady-state equation representing the dominant OH production and loss pathways in the remote marine troposphere, according to box model simulations of OH constrained with ATom observations. Proxy OH comprises only eight variables that are generally observed by routine ground- or satellite-based instruments. Proxy OH scales linearly with in situ [OH] spatial variations along the ATom flight tracks (median r 2 = 0.90, interquartile range = 0.80 to 0.94 across 2-km altitude by 20° latitudinal regions). We deconstruct spatial variations in Proxy OH as a first-order approximation of the sensitivity of OH variations to individual terms. Two terms modulate within-region Proxy OH variations-water vapor (H 2 O) and, to a lesser extent, nitric oxide (NO). This implies that a limited set of observations could offer an avenue for observation-based mapping of OH spatial variations over much of the remote marine troposphere. Both H 2 O and NO are expected to change with climate, while NO also varies strongly with human activities. We also illustrate the utility of Proxy OH as a process-based approach for evaluating intermodel differences in remote marine tropospheric OH.

A new configuration of the Community Earth System Model (CESM)/Community Atmosphere Model with full chemistry (CAM-chem) supporting the capability of horizontal mesh refinement through the use of the spectral element (SE) dynamical core is developed and called CESM/CAM-chem-SE. Horizontal mesh refinement in CESM/CAM-chem-SE is unique and novel in that pollutants such as ozone are accurately represented at human exposure relevant scales while also directly including global feedbacks. CESM/CAM-chem-SE with mesh refinement down to ∼14 km over the conterminous US (CONUS) is the beginning of the Multi-Scale Infrastructure for Chemistry and Aerosols (MUSICAv0). Here, MUSICAv0 is evaluated and used to better understand how horizontal resolution and chemical complexity impact ozone and ozone precursors over CONUS as compared to measurements from five aircraft campaigns, which occurred in 2013. This field campaign analysis demonstrates the importance of using finer horizontal resolution to accurately simulate ozone precursors such as nitrogen oxides and carbon monoxide. In general, the impact of using more complex chemistry on ozone and other oxidation products is more pronounced when using finer horizontal resolution where a larger number of chemical regimes are resolved. Large model biases for ozone near the surface remain in the Southeast US as compared to the aircraft observations even with updated chemistry and finer horizontal resolution. This suggests a need for adding the capability of replacing sections of global emission inventories with regional inventories, increasing the vertical resolution in the planetary boundary layer, and reducing model biases in meteorological variables such as temperature and clouds., (© 2022. The Authors. Journal of Advances in Modeling Earth Systems published by Wiley Periodicals LLC on behalf of American Geophysical Union.)