Flow-Xl: a new facility for the analysis of crystallization in flow systems.

Characterization of crystallization processes in situ is of great importance to furthering knowledge of how nucleation and growth processes direct the assembly of organic and inorganic materials in solution and, critically, understanding the influence that these processes have on the final physico-chemical properties of the resulting solid form. With careful specification and design, as demonstrated here, it is now possible to bring combined X-ray diffraction and Raman spectroscopy, coupled to a range of fully integrated segmented and continuous flow platforms, to the laboratory environment for in situ data acquisition for timescales of the order of seconds. The facility used here (Flow-Xl) houses a diffractometer with a micro-focus Cu Kα rotating anode X-ray source and a 2D hybrid photon-counting detector, together with a Raman spectrometer with 532 and 785 nm lasers. An overview of the diffractometer and spectrometer setup is given, and current sample environments for flow crystallization are described. Commissioning experiments highlight the sensitivity of the two instruments for time-resolved in situ data collection of samples in flow. Finally, an example case study to monitor the batch crystallization of sodium sulfate from aqueous solution, by tracking both the solute and solution phase species as a function of time, highlights the applicability of such measurements in determining the kinetics associated with crystallization processes. This work illustrates that the Flow-Xl facility provides high-resolution time-resolved in situ structural phase information through diffraction data together with molecular-scale solution data through spectroscopy, which allows crystallization mechanisms and their associated kinetics to be analysed in a laboratory setting.

Optimization of 15N-13C double-resonance NMR experiments under low temperature magic angle spinning dynamic nuclear polarization conditions.

Dynamic nuclear polarization (DNP) enhanced magic angle spinning (MAS) solid-state NMR carried out at 25 K enables rapid acquisition of multi-dimensional 13C-15N correlation spectra for protein structure studies and resonance assignment. Under commonly used DNP conditions, solvent deuteration reduces 1H-15N cross polarization (CP) efficiencies, necessitates more careful optimization, and requires longer high-power 15N radio-frequency pulses. The sensitivity of 2D heteronuclear correlation experiments is potentially impaired. Here we show that 2D 15N-13C experiments based on 13C-15N transferred echo double resonance (TEDOR) methods outperform 2D experiments based on CP transfers in a fully deuterated solvent, and are competitive with CP-based experiments when the solvent is only partially deuterated. Additionally, we show that optimization of TEDOR-based 2D experiments is simpler than optimization of CP-based experiments under 25 K MAS conditions.

Conformations of a Low-Complexity Protein in Homogeneous and Phase-Separated Frozen Solutions.

Solutions of the intrinsically disordered, low-complexity domain of the FUS protein (FUS-LC) undergo liquid-liquid phase separation (LLPS) below temperatures TLLPS in the 20-40° C range. To investigate whether local conformational distributions are detectably different in the homogeneous and phase-separated states of FUS-LC, we performed solid state nuclear magnetic resonance (ssNMR) measurements on solutions that were frozen on sub-millisecond time scales after equilibration at temperatures well above (50° C) or well below (4° C) TLLPS. Measurements were performed at 25 K with signal enhancements from dynamic nuclear polarization. Crosspeak patterns in two-dimensional (2D) ssNMR spectra of rapidly frozen solutions in which FUS-LC was uniformly 15N,13C-labeled were found to be nearly identical for the two states. Similar results were obtained for solutions in which FUS-LC was labeled only at Thr, Tyr, and Gly residues, as well as solutions of a FUS construct in which five specific residues were labeled by ligation of synthetic and recombinant fragments. These experiments show that local conformational distributions are nearly the same in the homogeneous and phase-separated solutions, despite the much greater protein concentrations and more abundant intermolecular interactions within phase-separated, protein-rich "droplets". Comparison of the experimental results with simulations of the sensitivity of 2D crosspeak patterns to an enhanced population of ß-strand-like conformations suggests that changes in conformational distributions are no larger than 5-10%. Statement of Significance: Liquid-liquid phase separation (LLPS) in solutions of proteins with intrinsically disordered domains has attracted recent attention because of its relevance to multiple biological processes and its inherent interest from the standpoint of protein biophysics. The high protein concentrations and abundant intermolecular interactions within protein-rich, phase-separated "droplets" suggests that conformational distributions of intrinsically disordered proteins may differ in homogeneous and phase-separated solutions. To investigate whether detectable differences exist, we performed experiments on the low-complexity domain of the FUS protein (FUS-LC) in which FUS-LC solutions were first equilibrated at temperatures well above or well below their LLPS transition temperatures, then rapidly frozen and examined at very low temperatures by solid state nuclear magnetic resonance (ssNMR) spectroscopy. The ssNMR data for homogeneous and phase-separated frozen solutions of FUS-LC were found to be nearly identical, showing that LLPS is not accompanied by substantial changes in the local conformational distributions of this intrinsically disordered protein.

The Use of Gonadotropin Releasing Hormone to Manage Intraspecific Agonism in a Coalition of Male Lions (Panthera leo).

While housing nonbreeding all-male social groups of African lion (panthera leo) is a necessary part of managing this polygynous species ex situ, resulting intraspecific agonistic behavior can negatively impact animal welfare and guest experience, undermining two primary objectives of modern zoological gardens. Improvest is a gonadotropin releasing factor analog-diphtheria toxoid conjugate marketed for temporary immunological castration which has proven successful in reducing aggression in other zoo-housed species. To the authors' knowledge, the use of this technique has not been described in male African lions; reticence to use GnRH agonists may stem from concern about phenotypic effects (mane loss) and zoo visitor perception. We describe the use of Improvest in conjunction with other management changes to manage agonism in a coalition of African lions (3.0) housed at the Oakland Zoo. Daily agonism scores were calculated via animal care staff records, fecal testosterone levels were measured, and monthly photos were taken to monitor phenotypic changes. While agonism scores varied seasonally in three pretreatment years, a significant (p < 0.05) reduction in the frequency of agonistic behavior began within 2 months of initial treatment. Two lions showed testosterone suppression within 2 months of receiving the first vaccine, while the third showed suppression beginning in the period 4 months after the first vaccine. Mane loss occurred in all three lions, and time to mane regrowth varied between individuals. Improvest, combined with other management strategies, decreased overall fecal testosterone and intraspecific agonism in these lions, and may be an effective tool in other male coalitions.

A spectroscopic analysis code for spatially resolved x-ray absorption data from the COAX platform.

Sophisticated tools such as computer vision techniques in combination with 1D lineout type analyses have been used in automating the analysis of spectral data for high energy density (HED) plasmas. Standardized automation can solve the problems posed by the complexity of HED spectra and the quantity of data. We present a spectroscopic code written for automated and streamlined analysis of spatially resolved x-ray absorption data from the COAX platform on Omega-60. COAX uses radiographs and spectroscopic diagnostics to provide shock position and density information. We also obtain the more novel spectral-derived spatial profile of the supersonic radiation flow into a low-density foam. Considerable effort has been spent modernizing our previous spectroscopic analysis method, including the development of new tools characterized by a faster runtime and minimal user input to reduce bias and a testing suite for verifying the accuracy of the various functions within the code. The new code analyzes our spectroscopic images in 1-2 min, with added uncertainty and confidence.

Emotion regulation mediates the relation between intolerance of uncertainty and emotion difficulties: A longitudinal investigation.

Intolerance of uncertainty has been proposed as a transdiagnostic factor in emotional disorders. Despite comprehensive empirical evidence demonstrating the association between intolerance of uncertainty and emotional disorders, the underlying mechanism remains elusive. Drawing on theoretical frameworks and empirical studies, the current study proposed that emotion regulation emerges as a potential mechanism. We explored the connections among intolerance of uncertainty, eight emotion regulation strategies (both adaptive and maladaptive), and emotional difficulties (specifically anxiety and depression) using a three-wave longitudinal approach (N = 341). Our findings revealed that heightened intolerance of uncertainty predicted increased anxiety but not depression over time. Greater intolerance of uncertainty significantly predicted elevated levels of maladaptive emotion regulation strategies including experiential avoidance, thought suppression, rumination, and reassurance-seeking. Adaptive strategies (i.e., mindfulness, cognitive reappraisal, problem-solving) predicted lower anxiety and/or depression whereas maladaptive emotion regulation strategy rumination predicted greater levels of anxiety and depression. Surprisingly, thought suppression predicted lower levels of anxiety and depression. More importantly, our analysis showed that both rumination and thought suppression served as significant mediators in the relationship between intolerance of uncertainty and both anxiety and depression. These results hold implications for future interventions, emphasising rumination and thought suppression as potential targets for interventions aimed at alleviating emotional difficulties in individuals with intolerance of uncertainty.

Characterization of diamond and organic scintillation detectors utilizing radiation sources for continuous plasma operation.

This paper provides a comprehensive study of neutron calibration methodologies, specifically highlighting the capabilities for n-γ discrimination in diamond and EJ-309, and stilbene scintillation detectors. The calibration process detailed in this study includes pulse height analysis and pulse shape discrimination, relying on the analysis of charge deposition resulting from both γ and neutron interactions. Utilizing 60Co and 252Cf radiation sources, the energy spectra of these sources are obtained. The characterized detectors were used in ST40 experiments and allowed acquiring neutron signal during a plasma shot with good agreement among diamond and scintillation detectors. Then, the diamond detector was cross-calibrated against indium activation foils placed at the same location in proximity to the ST40 during plasma shots: both detectors measured a neutron flux of ≈106 cm-2 s-1 at ≈1 m distance from the tokamak center, and the discrepancy between the diamond detector and the activation foils is ≈25%.

Ductile-to-brittle transition and yielding in soft amorphous materials: perspectives and open questions.

Soft amorphous materials are viscoelastic solids ubiquitously found around us, from clays and cementitious pastes to emulsions and physical gels encountered in food or biomedical engineering. Under an external deformation, these materials undergo a noteworthy transition from a solid to a liquid state that reshapes the material microstructure. This yielding transition was the main theme of a workshop held from January 9 to 13, 2023 at the Lorentz Center in Leiden. The manuscript presented here offers a critical perspective on the subject, synthesizing insights from the various brainstorming sessions and informal discussions that unfolded during this week of vibrant exchange of ideas. The result of these exchanges takes the form of a series of open questions that represent outstanding experimental, numerical, and theoretical challenges to be tackled in the near future.

Evaluation of a portable, lightweight modular system to deliver high inspired oxygen to trauma casualties without the use of pressurised cylinders.

INTRODUCTION: Administering supplemental oxygen is a standard of care for trauma casualties to minimise the deleterious effects of hypoxaemia. Forward deployment of oxygen using pressurised cylinders is challenging, for example, logistics (weight and finite resource) and environmental risk (fire and explosion). Oxygen concentrators may overcome these challenges. Although previous studies successfully demonstrated fractional inspired oxygen (FiO2) >0.8 using oxygen concentrators and ventilators, the systems did not fulfil the size, weight and power requirements of agile military medical units. This study evaluated whether a modular system of commercially available clinical devices could supply high FiO2 to either ventilated or spontaneously breathing casualties. METHODS: As a proof of principle, we configured an Inogen One G5 oxygen concentrator, Ventway Sparrow ventilator and Wenoll rebreather system to ventilate a simulated lung (tidal volume 500 mL). Casualty oxygen consumption (gas withdrawal inspiratory limb) and carbon dioxide (CO2) production (CO2 added expiratory limb) were simulated (respiratory quotient of 0.7-0.8). Three circuit configurations were evaluated: open (supplementary oxygen introduced into air inlet of ventilator); semiclosed (ventilator replaces rebreather bag of Wenoll, oxygen connected to either ventilator or Wenoll); and semiclosed with reservoir tubing (addition of 'deadspace' tube between ventilator patient circuit and Wenoll). Data presented as mean and 95% reference range. RESULTS: There were modest increases in FiO2 with increasing Inogen settings in 'open' configuration 0.23 (0.23-0.24) and 0.30 (0.28-0.32) (Inogen output 420 and 1260 mL/min, respectively). With the 'semiclosed' configuration and oxygen added directly into rebreather circuit, FiO2 increased to 0.36 (0.36-0.37). The addition of the 'reservoir tubing' elevated FiO2 to 0.78 (0.71-0.85). FiO2 remained stable over a 4-hour evaluation period. Fractional inspired carbon dioxide CO2 increased over time, reaching 0.005 after 170 (157-182) min. CONCLUSION: Combining existing lightweight devices can deliver high (>0.8) FiO2 and offers a potential solution for the forward deployment of oxygen without needing pressurised cylinders.

Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations.

Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting one to three genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein-DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.

From admissions to licensure: education data associations from a multi-centre undergraduate medical education collaboration.

This paper reports the findings of a Canada based multi-institutional study designed to investigate the relationships between admissions criteria, in-program assessments, and performance on licensing exams. The study's objective is to provide valuable insights for improving educational practices across different institutions. Data were gathered from six medical schools: McMaster University, the Northern Ontario School of Medicine University, Queen's University, University of Ottawa, University of Toronto, and Western University. The dataset includes graduates who undertook the Medical Council of Canada Qualifying Examination Part 1 (MCCQE1) between 2015 and 2017. The data were categorized into five distinct sections: demographic information as well as four matrices: admissions, course performance, objective structured clinical examination (OSCE), and clerkship performance. Common and unique variables were identified through an extensive consensus-building process. Hierarchical linear regression and a manual stepwise variable selection approach were used for analysis. Analyses were performed on data set encompassing graduates of all six medical schools as well as on individual data sets from each school. For the combined data set the final model estimated 32% of the variance in performance on licensing exams, highlighting variables such as Age at Admission, Sex, Biomedical Knowledge, the first post-clerkship OSCE, and a clerkship theta score. Individual school analysis explained 41-60% of the variance in MCCQE1 outcomes, with comparable variables to the analysis from of the combined data set identified as significant independent variables. Therefore, strongly emphasising the need for variety of high-quality assessment on the educational continuum. This study underscores the importance of sharing data to enable educational insights. This study also had its challenges when it came to the access and aggregation of data. As such we advocate for the establishment of a common framework for multi-institutional educational research, facilitating studies and evaluations across diverse institutions. This study demonstrates the scientific potential of collaborative data analysis in enhancing educational outcomes. It offers a deeper understanding of the factors influencing performance on licensure exams and emphasizes the need for addressing data gaps to advance multi-institutional research for educational improvements.

Distal colonocytes targeted by C. rodentium recruit T-cell help for barrier defence.

Interleukin 22 (IL-22) has a non-redundant role in immune defence of the intestinal barrier1-3. T cells, but not innate lymphoid cells, have an indispensable role in sustaining the IL-22 signalling that is required for the protection of colonic crypts against invasion during infection by the enteropathogen Citrobacter rodentium4 (Cr). However, the intestinal epithelial cell (IEC) subsets targeted by T cell-derived IL-22, and how T cell-derived IL-22 sustains activation in IECs, remain undefined. Here we identify a subset of absorptive IECs in the mid-distal colon that are specifically targeted by Cr and are differentially responsive to IL-22 signalling. Major histocompatibility complex class II (MHCII) expression by these colonocytes was required to elicit sustained IL-22 signalling from Cr-specific T cells, which was required to restrain Cr invasion. Our findings explain the basis for the regionalization of the host response to Cr and demonstrate that epithelial cells must elicit MHCII-dependent help from IL-22-producing T cells to orchestrate immune protection in the intestine.

Localisation of vibrational modes in high-entropy oxides.

The recently-discovered high-entropy oxides (HEO's) offer a paradoxical combination of crystalline arrangement and high disorder. They differ qualitatively from established paradigms for disordered solids such as glasses and alloys. In these latter systems, it is well known that disorder induces localised vibrational excitations. In this article, we explore the possibility of disorder-induced localisation in Mg0.2Co0.2Ni0.2Cu0.2Zn0.2O, the prototypical HEO with rock-salt structure. To describe phononic excitations, we model the interatomic potentials for the cation-oxygen interactions by fitting to the physical properties of the parent binary oxides. We validate our model against the experimentally determined crystal structure and optical conductivity. The resulting phonon spectrum shows wave-like propagating modes at low energies and localised modes at high energies. Localisation is reflected in signatures such as participation ratio and correlation amplitude. Finally, we argue that mass disorder can be increased to enhance localisation. We consider a hypothetical material, high-entropy telluride-oxide, where tellurium atoms are admixed into the anion sublattice. This shows a larger localised fraction, with additional localised modes appearing in the middle of the spectrum. Our results demonstrate that HEO's are a promising platform to study Anderson localisation of phonons.

Correction: Sizing multimodal suspensions with differential dynamic microscopy.

Correction for 'Sizing multimodal suspensions with differential dynamic microscopy' by Joe J. Bradley et al., Soft Matter, 2023, 19, 8179-8192, https://doi.org/10.1039/D3SM00593C.

Experimental Evidence for Millisecond-Timescale Structural Evolution Following the Microsecond-Timescale Folding of a Small Protein.

Prior work has shown that small proteins can fold (i.e., convert from unstructured to structured states) within 10 µs. Here we use time-resolved solid state nuclear magnetic resonance (ssNMR) methods to show that full folding of the 35-residue villin headpiece subdomain (HP35) requires a slow annealing process that has not been previously detected. ^{13}C ssNMR spectra of frozen HP35 solutions, acquired with a variable time τ_{e} at 30 °C after rapid cooling from 95 °C and before rapid freezing, show changes on the 3-10 ms timescale, attributable to slow rearrangements of protein sidechains during τ_{e}.

Quantitative characterization of run-and-tumble statistics in bulk bacterial suspensions.

We introduce a numerical method to extract the parameters of run-and-tumble dynamics from experimental measurements of the intermediate scattering function. We show that proceeding in Laplace space is unpractical and employ instead renewal processes to work directly in real time. We first validate our approach against data produced using agent-based simulations. This allows us to identify the length and time scales required for an accurate measurement of the motility parameters, including tumbling frequency and swim speed. We compare different models for the run-and-tumble dynamics by accounting for speed variability at the single-cell and population level, respectively. Finally, we apply our approach to experimental data on wild-type Escherichia coli obtained using differential dynamic microscopy.

Characterization and Control of the Run-and-Tumble Dynamics of Escherichia Coli.

We characterize the full spatiotemporal gait of populations of swimming Escherichia coli using renewal processes to analyze the measurements of intermediate scattering functions. This allows us to demonstrate quantitatively how the persistence length of an engineered strain can be controlled by a chemical inducer and to report a controlled transition from perpetual tumbling to smooth swimming. For wild-type E. coli, we measure simultaneously the microscopic motility parameters and the large-scale effective diffusivity, hence quantitatively bridging for the first time small-scale directed swimming and macroscopic diffusion.

Optimizing non-Newtonian fluids for impact protection of laminates.

Non-Newtonian fluids can be used for the protection of flexible laminates. Understanding the coupling between the flow of the protecting fluid and the deformation of the protected solids is necessary in order to optimize this functionality. We present a scaling analysis of the problem based on a single coupling variable, the effective width of a squeeze flow between flat rigid plates, and predict that impact protection for laminates is optimized by using shear-thinning, and not shear-thickening, fluids. The prediction is verified experimentally by measuring the velocity and pressure in impact experiments. Our scaling analysis should be generically applicable for non-Newtonian fluid-solid interactions in diverse applications.

The UK Divide: Does Having a Pembrolizumab-Chemotherapy Option in Head and Neck Cancer Matter? Real-world Experience of First-line Palliative Pembrolizumab Monotherapy and Pembrolizumab-Chemotherapy Combination in Scotland.

AIMS: The Scottish Medical Consortium recently approved first-line pembrolizumab monotherapy or in combination with chemotherapy for head and neck squamous cell carcinoma in the palliative setting, contrasting with the decision made by the National Institute for Health and Care Excellence, who approved monotherapy alone in England and Wales. The aim of this study was to provide real-world performance data for first-line pembrolizumab-containing treatments for head and neck squamous cell carcinoma in the palliative setting in Scotland. MATERIALS AND METHODS: We analysed the electronic records of patients who started pembrolizumab-containing treatment between 1 March 2020 and 30 September 2021. Outcomes included overall survival, progression-free survival (PFS), the duration of response and the disease control rate. Data were compared with the KEYNOTE-048 study and clinical factors were evaluated for association with survival. RESULTS: Our cohort included 91 patients (median follow-up 10.8 months). Patient characteristics were similar to those in the KEYNOTE-048 study, although our cohort had a higher proportion of patients with newly diagnosed, non-metastatic disease. For patients receiving monotherapy (n = 76), 12- and 24-month overall survival were 45% and 27%, respectively. For patients receiving pembrolizumab-chemotherapy (n = 15), 12-month overall survival was 60% (24-month overall survival had not yet been reached). Experiencing one or more immune-related adverse event (irAE; versus no irAEs), of any grade, was associated with favourable overall survival and PFS for patients receiving monotherapy in both univariable Log-rank analysis (median overall survival 17.4 months versus 8.6 months, respectively, P = 0.0033; median PFS 10.9 months versus 3.0 months, respectively, P < 0.0001) and multivariable analysis (Cox proportional hazards regression: overall survival hazard ratio 0.31, P = 0.0009; PFS hazard ratio 0.17, P < 0.0001). CONCLUSION: Our real-world data support the KEYNOTE-048 study findings and the value of combination treatment options. Additionally, our data show that irAEs of any grade, as reported in routine clinical records, are associated with better outcomes in this patient group, adding to the growing body of evidence showing that irAEs are generally a positive marker of programmed death-ligand 1 (PD-L1) inhibitor response.