Sleeping Beauty kit sets provide rapid and accessible generation of artificial antigen-presenting cells for natural killer cell expansion.

Artificial antigen-presenting cells (aAPCs) offer a cost effective and convenient tool for the expansion of chimeric antigen receptor (CAR)-bearing T cells and NK cells. aAPCs are particularly useful because of their ability to efficiently expand low-frequency antigen-reactive lymphocytes in bulk cultures. Commonly derived from the leukemic cell line K562, these aAPCs lack most major histocompatibility complex expression and are therefore useful for NK cell expansion without triggering allogeneic T-cell proliferation. To combat difficulties in accessing existing aAPC lines, while circumventing the iterative lentiviral gene transfers with antibody-mediated sorting required for the isolation of stable aAPC clones, we developed a single-step technique using Sleeping Beauty (SB)-based vectors with antibiotic selection options. Our SB vectors contain options of two to three genes encoding costimulatory molecules, membrane-bound cytokines as well as the presence of antibiotic-resistance genes that allow for stable transposition-based transfection of feeder cells. Transfection of K562 with SB vectors described in this study allows for the surface expression of CD86, 4-1BBL, membrane-bound (mb) interleukin (IL)-15 and mbIL-21 after simultaneous transposition and antibiotic selection using only two antibiotics. aAPCs successfully expanded NK cells to high purity (80-95%). Expanded NK cells could be further engineered by lentiviral CAR transduction. The multivector kit set is publicly available and will allow convenient and reproducible in-house production of effective aAPCs for the in vitro expansion of primary cells.

Active Nematic Flows over Curved Surfaces.

Cell monolayers are a central model system in the study of tissue biophysics. In vivo, epithelial tissues are curved on the scale of microns, and the curvature's role in the onset of spontaneous tissue flows is still not well understood. Here, we present a hydrodynamic theory for an apical-basal asymmetric active nematic gel on a curved strip. We show that surface curvature qualitatively changes monolayer motion compared with flat space: the resulting flows can be thresholdless, and the transition to motion may change from continuous to discontinuous. Surface curvature, friction, and active tractions are all shown to control the flow pattern selected, from simple shear to vortex chains.

A retrospective observational study of critically unwell patients retrieved from Thames Hospital between April 2018 and December 2020.

INTRODUCTION In New Zealand, critically ill patients who present to rural hospitals are typically treated, stabilised and transferred to facilities where more appropriate resources are available. AIM The aim of this study was to describe patients who presented critically unwell and required retrieval from Thames Hospital in the Waikato region. METHODS Notes were reviewed retrospectively for patients who were retrieved from Thames Hospital between 1 April 2018 and 31 December 2020. Patients were excluded if they were retrieved from the offsite birthing centre or their notes were not available to the authors. RESULTS During the study period, 56 patients were retrieved by intensive care teams based at Waikato, Starship or Auckland Hospitals. Patients had a median age of 57 years and most were female (60.7%). Maori patients were over-represented in the retrieval cohort compared with the population presenting to the emergency department (30.4% vs. 20.1%, P < 0.001). We found that 41% of patients presented after-hours when there was only one senior medical officer available on site and 70 procedures were performed, including rapid sequence induction, which was required by 19.6% of patients. DISCUSSION This study describes a population of critically unwell patients who were retrieved from a rural hospital. The key finding is that nearly half of these patients presented after-hours when there was only one senior medical officer available on site. This doctor also has sole responsibility for all other patients in the hospital. We recommend that referral centres streamline the retrieval processes for rural hospitals.

Metabolic and Mitochondrial Functioning in Chimeric Antigen Receptor (CAR)-T Cells.

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized adoptive cell therapy with impressive therapeutic outcomes of >80% complete remission (CR) rates in some haematological malignancies. Despite this, CAR T cell therapy for the treatment of solid tumours has invariably been unsuccessful in the clinic. Immunosuppressive factors and metabolic stresses in the tumour microenvironment (TME) result in the dysfunction and exhaustion of CAR T cells. A growing body of evidence demonstrates the importance of the mitochondrial and metabolic state of CAR T cells prior to infusion into patients. The different T cell subtypes utilise distinct metabolic pathways to fulfil their energy demands associated with their function. The reprogramming of CAR T cell metabolism is a viable approach to manufacture CAR T cells with superior antitumour functions and increased longevity, whilst also facilitating their adaptation to the nutrient restricted TME. This review discusses the mitochondrial and metabolic state of T cells, and describes the potential of the latest metabolic interventions to maximise CAR T cell efficacy for solid tumours.

Fine-scale computations for adaptive processing in the human brain.

Adapting to the environment statistics by reducing brain responses to repetitive sensory information is key for efficient information processing. Yet, the fine-scale computations that support this adaptive processing in the human brain remain largely unknown. Here, we capitalise on the sub-millimetre resolution of ultra-high field imaging to examine functional magnetic resonance imaging signals across cortical depth and discern competing hypotheses about the brain mechanisms (feedforward vs. feedback) that mediate adaptive processing. We demonstrate layer-specific suppressive processing within visual cortex, as indicated by stronger BOLD decrease in superficial and middle than deeper layers for gratings that were repeatedly presented at the same orientation. Further, we show altered functional connectivity for adaptation: enhanced feedforward connectivity from V1 to higher visual areas, short-range feedback connectivity between V1 and V2, and long-range feedback occipito-parietal connectivity. Our findings provide evidence for a circuit of local recurrent and feedback interactions that mediate rapid brain plasticity for adaptive information processing.

Automated data extraction from historical city directories: The rise and fall of mid-century gas stations in Providence, RI.

The location of defunct environmentally hazardous businesses like gas stations has many implications for modern American cities. To track down these locations, we present the directoreadr code (github.com/brown-ccv/directoreadr). Using scans of Polk city directories from Providence, RI, directoreadr extracts and parses business location data with a high degree of accuracy. The image processing pipeline ran without any human input for 94.4% of the pages we examined. For the remaining 5.6%, we processed them with some human input. Through hand-checking a sample of three years, we estimate that ~94.6% of historical gas stations are correctly identified and located, with historical street changes and non-standard address formats being the main drivers of errors. As an example use, we look at gas stations, finding that gas stations were most common early in the study period in 1936, beginning a sharp and steady decline around 1950. We are making the dataset produced by directoreadr publicly available. We hope it will be used to explore a range of important questions about socioeconomic patterns in Providence and cities like it during the transformations of the mid-1900s.

Automated identification of multinucleated germ cells with U-Net.

Phthalic acid esters (phthalates) are male reproductive toxicants, which exert their most potent toxicity during fetal development. In the fetal rat, exposure to phthalates reduces testosterone biosynthesis, alters the development of seminiferous cords and other male reproductive tissues, and induces the formation of abnormal multinucleated germ cells (MNGs). Identification of MNGs is a time-intensive process, and it requires specialized training to identify MNGs in histological sections. As a result, MNGs are not routinely quantified in phthalate toxicity experiments. In order to speed up and standardize this process, we have developed an improved method for automated detection of MNGs. Using hand-labeled histological section images with human-identified MNGs, we trained a convolutional neural network with a U-Net architecture to identify MNGs on unlabeled images. With unseen hand-labeled images not used in model training, we assessed the performance of the model, using five different configurations of the data. On average, the model reached near human accuracy, and in the best model, it exceeded it. The use of automated image analysis will allow data on this histopathological endpoint to be more readily collected for analysis of phthalate toxicity. Our trained model application code is available for download at github.com/brown-ccv/mngcount.

Sex Differences in Problem Alcohol Use in High School as a Function of Recent Sexual Violence Victimization or Perpetration.

To investigate sex differences in associations between sexual violence victimization (SVV), sexual violence perpetration (SVP), and binge drinking and/or alcohol problems among high school students. While SVV has been linked to problem alcohol use among young women, little research has addressed the unique associations of SVV and SVP on alcohol use/problems within both sexes. A cross-sectional analysis of 16,992 high school students' self-reports of past-year SVP and SVV was used where SVV/SVP was defined by three tactics (sexual coercion, drug/alcohol-facilitated or incapacitated sex, and physically forced sex). Alcohol measures included past-month binge drinking and past-year alcohol problems. Rates of SVV were twice as high in females (21.2% vs. 13.3%), and SVP rates were twice as high in males (10.9% vs. 5.2%). SVV and SVP were each associated with an increased rate of current binge drinking and problem alcohol use for both sexes, across increasing numbers of SV tactics and within each of three tactics. After controlling for demographic and other risk factors including SVP, drug/alcohol-facilitated or incapacitated SVV was more strongly linked to binge drinking and alcohol problems among females. SVP was more strongly linked to binge drinking and alcohol problems among males (adjusting for SVV and other covariates). No sex differences emerged in associations between coerced or physically forced SVV/SVP and alcohol-related outcomes. Both SVV and SVP are associated with an increased likelihood of binge drinking and alcohol problems for males and females. Important sex differences emerged when SV tactics are considered.

Unfolding of polymers tethered to viscoelastic substrates.

The problem of globular polymer unfolding under applied force is a widely-studied fundamental topic in biological and chemical physics, with important applications in cell biology. Much of the existing theoretical and experimental literature focuses on the case where force is applied while fixing the opposite end of the polymer chain in space. However, in a realistic biological microenvironment, forces will be applied against viscoelastic references, and the deformation of the folded polymer chain will be combined with the deformation of viscoelastic substrate. In this paper, we consider several simple viscoelastic models for the substrate, and show that its relaxation properties determine the unfolding kinetics. In particular, for low pulling forces, substrates with longer relaxation times cause lower unfolding rates for the pulled polymer chain, whereas for high forces, those substrates with longer relaxation times instead produce higher unfolding rates.

Diffusion of active particles in a complex environment: Role of surface scattering.

Experiments have shown that self-propelled particles can slide along the surface of a circular obstacle without becoming trapped over long times. Using simulations and theory, we study the impact of boundary conditions on the diffusive transport of active particles in an obstacle lattice. We find that particle dynamics with sliding boundary conditions result in large diffusivities even at high obstacle density, unlike classical specular reflection. These dynamics are very well described by a model based on run-and-tumble particles with microscopically derived reorientation functions arising from obstacle-induced tumbles. This model, however, fails to describe fine structure in the diffusivity at high obstacle density predicted by simulations for pusherlike collisions. Using a simple deterministic model, we show that this structure results from particles being guided by the lattice. Our results thus show how nonclassical surface scattering introduces a dependence on the lattice geometry at high densities. We discuss implications for the study of bacteria in complex environments.

CAMKII as a therapeutic target for growth factor-induced retinal and choroidal neovascularization.

While anti-VEGF drugs are commonly used to inhibit pathological retinal and choroidal neovascularization, not all patients respond in an optimal manner. Mechanisms underpinning resistance to anti­VEGF therapy include the upregulation of other proangiogenic factors. Therefore, therapeutic strategies that simultaneously target multiple growth factor signaling pathways would have significant value. Here, we show that Ca2+/calmodulin-dependent kinase II (CAMKII) mediates the angiogenic actions of a range of growth factors in human retinal endothelial cells and that this kinase acts as a key nodal point for the activation of several signal transduction cascades that are known to play a critical role in growth factor-induced angiogenesis. We also demonstrate that endothelial CAMKIIγ and -δ isoforms differentially regulate the angiogenic effects of different growth factors and that genetic deletion of these isoforms suppresses pathological retinal and choroidal neovascularization in vivo. Our studies suggest that CAMKII could provide a novel and efficacious target to inhibit multiple angiogenic signaling pathways for the treatment of vasoproliferative diseases of the eye. CAMKIIγ represents a particularly promising target, as deletion of this isoform inhibited pathological neovascularization, while enhancing reparative angiogenesis in the ischemic retina.

Universal Kinetics of the Onset of Cell Spreading on Substrates of Different Stiffness.

When plated onto substrates, cell morphology and even stem-cell differentiation are influenced by the stiffness of their environment. Stiffer substrates give strongly spread (eventually polarized) cells with strong focal adhesions and stress fibers; very soft substrates give a less developed cytoskeleton and much lower cell spreading. The kinetics of this process of cell spreading is studied extensively, and important universal relationships are established on how the cell area grows with time. Here, we study the population dynamics of spreading cells, investigating the characteristic processes involved in the cell response to the substrate. We show that unlike the individual cell morphology, this population dynamics does not depend on the substrate stiffness. Instead, a strong activation temperature dependence is observed. Different cell lines on different substrates all have long-time statistics controlled by the thermal activation over a single energy barrier ΔG ≈ 18 kcal/mol, whereas the early-time kinetics follows a power law ∼t5. This implies that the rate of spreading depends on an internal process of adhesion complex assembly and activation; the operational complex must have five component proteins, and the last process in the sequence (which we believe is the activation of focal adhesion kinase) is controlled by the binding energy ΔG.

Measurement of bystander actions in violence intervention evaluation: Opportunities and Challenges.

PURPOSE OF REVIEW: This review discusses design and methodological challenges specific to measuring bystander actions in the evaluation of bystander-based violence prevention programming. "Bystanders" are defined as people who are present immediately before, during and/or after a violent event, but are not a perpetrator nor the intended victim. Bystander-based violence prevention programs seek to prevent or mitigate violent events by empowering bystanders to intervene on acts of violence and social norms that promulgate violence. RECENT FINDINGS: Effective bystander-based violence prevention programs demonstrate increased bystander intentions, actions, and attitudes [Bringing in the Bystander;12 iSCREAM;33; The Men's Project;20 and Green Dot,3] lowered violence acceptance scores19,21,22,23,36 and reduced sexual violence perpetration and victimization.3••,6,20 However, bystander-based violence prevention programs are methodologically challenging to evaluate, due to the wide diversity of programs being implemented and the multifactorial and contextual nature of acts of violence. SUMMARY: Measures of bystander actions temporally-connected to specific, high-risk opportunities are recommended approaches to capture bystander experiences and address the methodological challenges in measuring bystander actions and evaluating violence prevention programming.

Specific binding of a polymer chain to a sequence of surface receptors.

This paper considers a biologically relevant question of a Gaussian chain (such as an unfolded protein) binding to a sequence of receptors with matching multiple ligands distributed along the chain. Using the characteristic time for a tethered ligand to bind to a surface receptor, we study the case of multiple binding to a linear sequence of receptors on the surface. The tethered binding time is determined by the entropic barrier for the chain to be stretched sufficiently to reach the distant receptor target, and a restriction on chain conformations near the substrate. Adsorption (multiple-site binding) is shown to be dominated by a simple zipper sequence, only occasionally accelerated by loop formation. However, when the number of receptors increases, a competing rate-limiting process takes over: the center of mass of the remaining free chain has to drift down the line of receptors, which takes longer when the receptors are close and the entropic pulling force is low. As a result, the time for the complete chain adsorption is minimised by a certain optimal number of receptors, depending on the distance to be traversed by the free end, and the chain length.

Focal Adhesion Kinase: The Reversible Molecular Mechanosensor.

Sensors are the first element of the pathways that control the response of cells to their environment. Protein complexes that produce or enable a chemical signal in response to a mechanical stimulus are called "mechanosensors". In this work, we develop a theoretical model describing the physical mechanism of a reversible single-molecule stiffness sensor. Although this has the potential for general application, here we apply the model to focal adhesion kinase, which initiates the chemical signal in its active phosphorylated conformation, but can spontaneously return to its closed folded conformation. We find how the rates of conformation changes depend on the substrate stiffness and the pulling force applied from the cell cytoskeleton. We find the sensor is homeostatic, spontaneously self-adjusting to reach a state where its range of maximum sensitivity matches the substrate stiffness. The results compare well with the phenotype observations of cells on different substrates.

Non-exponential kinetics of unfolding under a constant force.

We examine the population dynamics of naturally folded globular polymers, with a super-hydrophobic "core" inserted at a prescribed point in the polymer chain, unfolding under an application of external force, as in AFM force-clamp spectroscopy. This acts as a crude model for a large class of folded biomolecules with hydrophobic or hydrogen-bonded cores. We find that the introduction of super-hydrophobic units leads to a stochastic variation in the unfolding rate, even when the positions of the added monomers are fixed. This leads to the average non-exponential population dynamics, which is consistent with a variety of experimental data and does not require any intrinsic quenched disorder that was traditionally thought to be at the origin of non-exponential relaxation laws.

Unfolding of globular polymers by external force.

We examine the problem of a polymer chain, folded into a globule in poor solvent, subjected to a constant tensile force. Such a situation represents a Gibbs thermodynamic ensemble and is useful for analysing force-clamp atomic force microscopy measurements, now very common in molecular biophysics. Using a basic Flory mean-field theory, we account for surface interactions of monomers with solvent. Under an increasing tensile force, a first-order phase transition occurs from a compact globule to a fully extended chain, in an "all-or-nothing" unfolding event. This contrasts with the regime of imposed extension, first studied by Halperin and Zhulina [Europhys. Lett. 15, 417 (1991)], where there is a regime of coexistence of a partial globule with an extended chain segment. We relate the transition forces in this problem to the solvent quality and degree of polymerisation, and also find analytical expressions for the energy barriers present in the problem. Using these expressions, we analyse the kinetic problem of a force-ramp experiment and show that the force at which a globule ruptures depends on the rate of loading.

Antenatal imaging of anomalies of the corpus callosum: a decade of experience.

PURPOSE: To assess the diagnostic accuracy of antenatal ultrasound and foetal magnetic resonance imaging (FMRI) over the past decade in the diagnosis of anomalies of the corpus callosum in a tertiary referral centre. METHODS: A single tertiary referral centre ultrasound database was searched for cases of suspected callosal anomalies between 2003 and 2012. All subsequent ultrasound scans, MRIs, neonatal imaging, postmortem investigations and birth records were reviewed. Callosal anomalies were classified into isolated or complex based on the presence or absence of accompanying congenital anomalies. RESULTS: Forty-three cases of callosal anomaly were detected; 60 % were investigated by FMRI revealing additional diagnoses in 23 %; half of which were anomalies of cortical development. Of those considered isolated who underwent FMRI, 21 % were diagnosed with additional anomalies, changing the classification to a complex callosal anomaly. CONCLUSION: In cases of callosal anomaly suspected on ultrasound, FMRI provides greater certainty and the potential to identify significant additional anomalies. The additional information may alter or clarify prognosis and help parents to better understand the pathology, allowing for informed decisions about the pregnancy to be made. However, some cases may still be diagnosed with additional anomalies after delivery and parents should be aware of such limitations of antenatal imaging.