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Biological drug substance (DS) is often frozen to enhance storage stability, prolong shelf life, and increase flexibility during manufacturing. However, the freezing and thawing (F/T) of bulk DS at the manufacturing scale can impact product quality as a result of various critical conditions, including cryo-concentration during freezing, which are influenced, among other things, by product-independent process parameters (e.g., container type, fill level, F/T equipment, and protocols). In this article, we report the optimization of two major methodologies to study product-independent process parameters in DS bottles at the manufacturing scale, namely the recording of temperature profiles and liquid sampling after thawing to quantify the concentration gradients in the solution. We report experimentally justified measuring positions for temperature recordings, especially for the selection of the last point to freeze position, and highlight the implementation of camera-assisted inspection to determine the last point to thaw and the actual thawing time. In particular, we provide, for the first time, a detailed description of the technical implementation of these two measuring set-ups. Based on the reported case studies, we recommend choosing relevant measuring positions as a result of initial equipment characterization, resulting in a resource-conscious study set-up.
Wiechmann, Svenja ; Maisonneuve, Pierre ; Grebbin, Britta Moyo ; Hoffmeister, Meike ; Kaulich, Manuel ; Clevers, Hans ; Rajalingam, Krishnaraj ; Kurinov, Igor ; Farin, Henner ; Sicheri, Frank ; Ernst, Andreas
The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.
Highlights
• Executive functions contribute to problem-solving in six- to eight-year-olds.
• Working memory and cognitive flexibility contribute to problem-solving.
•Inhibition does not significantly contribute to problem-solving.
Abstract
Previous research has shown that executive functions can contribute to successful problem-solving in preschool and elementary school children. However, most studies did not simultaneously assess the role of different specific aspects of executive functions. Therefore, the aim of our study was to investigate the individual contribution of inhibition, working memory, and cognitive flexibility to science problem-solving performance in elementary school children. A total of 478 children from first and second grades (Mage = 7.44 years) participated in our study. They performed a Go/No-go task (inhibition), a Corsi blocks backward task (working memory), a flexible item selection task (cognitive flexibility), and three science problem-solving tasks, including two gear turning tasks and one stabilization task. Structural equation modeling showed that working memory and cognitive flexibility individually contributed to problem-solving performance, whereas inhibition did not. We conclude that maintaining task requirements and dynamic object relations (working memory) and switching between different problem-solving phases (cognitive flexibility) are essential components of successful science problem-solving in elementary school children. Inhibitory processes may be more relevant in tasks involving a higher degree of interference at the task or response level.
Delegated online search
(2024)
In a delegation problem, a principal P with commitment power tries to pick one out of 𝑛 options. Each option is drawn independently from a known distribution. Instead of inspecting the options herself, P delegates the information acquisition to a rational and self-interested agent A. After inspection, A proposes one of the options, and P can accept or reject. Delegation is a classic setting in economic information design with many prominent applications, but the computational problems are only poorly understood. In this paper, we study a natural online variant of delegation, in which the agent searches through the options in an online fashion. For each option, he has to irrevocably decide if he wants to propose the current option or discard it, before seeing information on the next option(s). How can we design algorithms for P that approximate the utility of her best option in hindsight? We show that in general P can obtain a Θ(1∕𝑛)-approximation and extend this result to ratios of Θ(𝑘∕𝑛) in case (1) A has a lookahead of 𝑘 rounds, or (2) A can propose up to 𝑘 different options. We provide fine-grained bounds independent of 𝑛 based on three parameters. If the ratio of maximum and minimum utility for A is bounded by a factor 𝛼, we obtain an Ω(loglog 𝛼∕ log 𝛼)- approximation algorithm, and we show that this is best possible. Additionally, if P cannot distinguish options with the same value for herself, we show that ratios polynomial in 1∕𝛼 cannot be avoided. If there are at most 𝛽 different utility values for A, we show a Θ(1∕𝛽)-approximation. If the utilities of P and A for each option are related by a factor 𝛾, we obtain an Ω(1∕ log 𝛾)- approximation, where 𝑂(log log 𝛾∕ log 𝛾) is best possible.
Highlights
• Extreme weather events (floods, droughts, extreme heat) impact freshwater ecosystems.
• Effects of extreme events are site-specific, varying by organism traits and site hydrography.
• Cumulative impact of events is more significant than single events' magnitude.
• Temporal dynamics and biological characteristics are crucial for evaluating impacts.
• Freshwater ecosystem resilience depends on complex factors, not just event severity.
Abstract
Understanding the impacts of extreme weather events on freshwater ecosystems is imperative during a time when a multitude of challenges compromises these environments' health. Exploring how such events affect macroinvertebrate communities in rivers sheds light on the resilience of freshwater ecosystems, which is essential for human well-being and biodiversity conservation. In this study, long-term time series of benthic macroinvertebrate communities from four sites along three freshwater streams within the Rhine-Main-Observatory Long-Term Ecological Research site in Germany were analyzed. Each of them was sampled annually over a span of ~20 years to assess the impacts of extreme weather events (floods, droughts, and extreme heat) on macroinvertebrate communities. The findings reveal that the effects of extreme events are site-specific, suggesting that the impacts of an extreme event can vary based on several potential factors, including the life history traits of the organisms within the community and, among others, the hydrography of the site. Moreover, the analysis highlights that the cumulative impact of these events over time is more significant than the impact of a single event's magnitude, while following distinct temporal dynamics. This underscores the importance of considering both the temporal dynamics and the biological characteristics of communities when evaluating the consequences of extreme weather events on biodiversity, illustrating that the resilience of freshwater ecosystems and their biodiversity under such conditions depends on a complex interplay of factors rather than the severity of individual events.
By linearly parameterizing the QCD Landau free energy near the critical point in the baryon chemical potential and temperature plane, we study the fluctuations of the QCD chiral order parameter field (the σ field) in the equilibrium case and dynamical phase transition, respectively. By setting the system size to the typical size of the QGP fireball (≈103 fm3), we show that in the equilibrium case, the discontinuity of the order parameter in the first order phase transition region is replaced by smooth crossover, and the corresponding fluctuations are broadened. Meanwhile, the quartic cumulant κ4 of the σ field is generally negative near the phase transition line. We further derive the dynamical evolution of the QCD Landau free energy in the Fokker-Plank framework, based on which we deduce the dynamical cumulants of the σ field. Assuming the temperature decreases as a known function of time, we numerically evaluate the dynamical cumulants and confirm that the cumulants present clear memory effects. Moreover, the memory effects on the first order phase transition side is stronger than that on the crossover side, and the dynamical cumulants at the hypothetical freeze-out line present rich non-monotonic structures.
Darragh, Kathy ; Orteu, Anna ; Black, Daniella ; Byers, Kelsey J. R. P. ; Szczerbowski, Daiane ; Warren, Ian A. ; Rastas, Pasi ; Pinharanda, Ana ; Davey, John ; Garza, Sylvia Fernanda ; Abondano Almeida, Diana ; Merrill, Richard M. ; McMillan, W. Owen ; Schulz, Stefan ; Jiggins, Chris D.
Plants and insects often use the same compounds for chemical communication, but not much is known about the genetics of convergent evolution of chemical signals. The terpene (E)-β-ocimene is a common component of floral scent and is also used by the butterfly Heliconius melpomene as an anti-aphrodisiac pheromone. While the biosynthesis of terpenes has been described in plants and microorganisms, few terpene synthases (TPSs) have been identified in insects. Here, we study the recent divergence of 2 species, H. melpomene and Heliconius cydno, which differ in the presence of (E)-β-ocimene; combining linkage mapping, gene expression, and functional analyses, we identify 2 novel TPSs. Furthermore, we demonstrate that one, HmelOS, is able to synthesise (E)-β-ocimene in vitro. We find no evidence for TPS activity in HcydOS (HmelOS ortholog of H. cydno), suggesting that the loss of (E)-β-ocimene in this species is the result of coding, not regulatory, differences. The TPS enzymes we discovered are unrelated to previously described plant and insect TPSs, demonstrating that chemical convergence has independent evolutionary origins.
Behaviorally irrelevant feature matching increases neural and behavioral working memory readout
(2024)
There is an ongoing debate about whether working memory (WM) maintenance relies on persistent activity and/or short-term synaptic plasticity. This is a challenging question, because neuroimaging techniques in cognitive neuroscience measure activity only. Recently, neural perturbation techniques have been developed to tackle this issue, such as visual impulse perturbation or “pinging”, which reveals (un)attended WM content during maintenance. There are contrasting explanations of how pinging reveals WM content, which is central to the debate. Pinging could reveal mnemonic representations by perturbing content-specific networks or by increasing the neural signal-to-noise ratio of active neural states. Here we tested the extent to which the neural impulse response is patterned by the WM network, by presenting two different impulse stimuli. If the impulse interacts with WM networks, the WM-specific impulse response should be enhanced by physical overlap between the initial memory item and the subsequent external perturbation stimulus. This prediction was tested in a delayed orientation match-to-sample task by matching or mismatching task-irrelevant spatial frequencies between memory items and impulse stimuli, as well as probes. Matching probe spatial frequency with memory items resulted in faster behavioral response times and matching impulse spatial frequency with memory items increased the specificity of the neural impulse response as measured from EEG. Matching spatial frequencies did neither result in globally stronger neural responses nor in a larger decrease in trial-to-trial variability compared to mismatching spatial frequencies. The improved neural and behavioural readout of irrelevant feature matching provide evidence that impulse perturbation interacts directly with the memory representations.
The activity-silent framework of working memory (WM) posits that the neural activity during object perception and encoding leaves behind patterned, “activity-silent” neural traces that enable WM maintenance without the need for continuous, memory-specific neural activity. The presence of such traces in the memory network subsequently patterns its responses to external stimulation, which can be used to readout the contents of WM using an impulse perturbation or “pinging” approach. The extent to which the neural impulse response is patterned by the WM network should be modulated by the physical overlap between the initial memory item and the subsequent external perturbation stimulus, with higher overlap increasing WM readout. Here we tested this prediction in a delayed orientation match-to-sample task, by either matching or mismatching task-irrelevant spatial frequencies between memory items and impulse stimuli, and between memory items and probes. Matching frequencies resulted in faster behavioral response times, and increased the WM-specificity of the neural impulse response as measured from the EEG signal. We found no evidence that matching spatial frequencies resulted in globally stronger or different neural responses, but rather in distinct neural activation patterns. The beneficial effects of feature matching in our task support the tenets of the activity-silent framework of WM, and confirm that impulse perturbation interacts directly with the representations that are held in memory.