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Anti-platelet factor 4 (PF4)/heparin immune complexes that cause heparin-induced thrombocytopenia (HIT) activate complement via the classical pathway. Previous studies have shown that the alternative pathway of complement substantially amplifies the classical pathway of complement activation through the C3b feedback cycle.These studies sought to examine the contributions of the alternative pathway to complement activation by HIT antibodies.Using IgG monoclonal (KKO) and/or patient-derived HIT antibodies, we compared the effects of classical pathway (BBK32 and C1-esterase inhibitor [C1-INH]), alternative pathway (anti-factor B [fB] or factor D [fD] inhibitor) or combined classical and alternative pathway inhibition (soluble complement receptor 1 [sCR1]) in whole blood or plasma.Classical pathway inhibitors BBK32 and C1-INH and the combined classical/alternative pathway inhibitor sCR1 prevented KKO/HIT immune complex-induced complement activation, including release of C3 and C5 activation products, binding of immune complexes to B cells, and neutrophil activation. The alternative pathway inhibitors fB and fD, however, did not affect complement activation by KKO/HIT immune complexes. Similarly, alternative pathway inhibition had no effect on complement activation by unrelated immune complexes consisting of anti-dinitrophenyl (DNP) antibody and the multivalent DNP--keyhole limpet hemocyanin antigen.Collectively, these findings suggest the alternative pathway contributes little in support of complement activation by HIT immune complexes. Additional in vitro and in vivo studies are required to examine if this property is shared by most IgG-containing immune complexes or if predominance of the classic pathway is limited to immune complexes composed of multivalent antigens.

The earliest events during human tumour initiation, although poorly characterized, may hold clues to malignancy detection and prevention1. Here we model occult preneoplasia by biallelic inactivation of TP53, a common early event in gastric cancer, in human gastric organoids. Causal relationships between this initiating genetic lesion and resulting phenotypes were established using experimental evolution in multiple clonally derived cultures over 2 years. TP53 loss elicited progressive aneuploidy, including copy number alterations and structural variants prevalent in gastric cancers, with evident preferred orders. Longitudinal single-cell sequencing of TP53-deficient gastric organoids similarly indicates progression towards malignant transcriptional programmes. Moreover, high-throughput lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programmes repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference and a marked degree of phenotypic convergence in premalignant epithelial organoids. These data imply predictability in the earliest stages of tumorigenesis and show evolutionary constraints and barriers to malignant transformation, with implications for earlier detection and interception of aggressive, genome-instable tumours.

Recent advances in synthetic biology are enabling exciting technologies, including the next generation of biosensors, the rational design of cell memory, modulated synthetic cell differentiation and generic multi-functional bio-circuits. These novel applications require the design of gene circuits leading to sophisticated behaviours and functionalities. At the same time, designs need to be kept minimal to avoid compromising cell viability. Bifurcation theory of dynamical systems provides powerful tools to address complex nonlinear dynamics and multifunctionality, linking model topology and kinetic parameters with circuit behaviour. However, the challenge of incorporating bifurcation analysis to automated design has not been accomplished so far. In this work we present an optimisation-based method for the automated forward design of synthetic gene circuits with specified bifurcation diagrams, allowing us to find minimal topologies optimizing the required functionalities and taking into account additional requirements and/or context specifications. We apply the method to design of gene circuits exhibiting the so called mushroom bifurcation, a relatively unexplored multi-functional behaviour of particular relevance for developmental biology. Using the results of the optimisation analysis we explore the capabilities of the resulting circuits for possible applications in advanced biosensors, memory devices, and synthetic cell differentiation.

SUMMARYTelomeres are prone to formation of the common oxidative lesion 8-oxoguanine (8oxoG), and the acute production of 8oxoG damage at telomeres is sufficient to drive rapid cellular senescence. OGG1 and MUTYH glycosylases initiate base excision repair (BER) at 8oxoG sites to remove the lesion or prevent mutation. Here, we show OGG1 loss or inhibition, or MUTYH loss, partially rescues telomeric 8oxoG-induced senescence, and loss of both glycosylases results in a near complete rescue. Loss of these glycosylases also suppresses 8oxoG-induced telomere fragility and dysfunction, indicating that single-stranded break (SSB) intermediates arising downstream of glycosylase activity impair telomere replication. The failure to initiate BER in glycosylase-deficient cells suppresses PARylation at SSB intermediates and confers resistance to the synergistic effects of PARP inhibitors on damage-induced senescence. Our studies reveal that inefficient completion of 8oxoG BER at telomeres triggers cellular senescence via SSB intermediates which impair telomere replication and stability.

Purpose:Advanced-stage gastrointestinal cancers represent a high unmet need requiring new effective therapies. We investigated the antitumor activity of a novel T cell–engaging antibody (B7-H6/CD3 ITE) targeting B7-H6, a tumor-associated antigen that is expressed in gastrointestinal tumors.Experimental Design:Membrane proteomics and IHC analysis identified B7-H6 as a tumor-associated antigen in gastrointestinal tumor tissues with no to very little expression in normal tissues. The antitumor activity and mode of action of B7-H6/CD3 ITE was evaluated in in vitro coculture assays, in humanized mouse tumor models, and in colorectal cancer precision cut tumor slice cultures.Results:B7-H6 expression was detected in 98% of colorectal cancer, 77% of gastric cancer, and 63% of pancreatic cancer tissue samples. B7-H6/CD3 ITE-mediated redirection of T cells toward B7-H6–positive tumor cells resulted in B7-H6–dependent lysis of tumor cells, activation and proliferation of T cells, and cytokine secretion in in vitro coculture assays, and infiltration of T cells into tumor tissues associated with tumor regression in in vivo colorectal cancer models. In primary patient-derived colorectal cancer precision-cut tumor slice cultures, treatment with B7-H6/CD3 ITE elicited cytokine secretion by endogenous tumor-infiltrating immune cells. Combination with anti-PD-1 further enhanced the activity of the B7-H6/CD3 ITE.Conclusion:These data highlight the potential of the B7-H6/CD3 ITE to induce T cell–redirected lysis of tumor cells and recruitment of T cells into noninflamed tumor tissues, leading to antitumor activity in in vitro, in vivo, and human tumor slice cultures, which supports further evaluation in a clinical study.

Supplementary Figure 1 (A) OGAP® Screen of tumor and non-tumorous (normal, non-diseased tissues). (B) B7-H6 mRNA expression in gastrointestinal cancer tissues (TCGA). Supplementary Figure 2 B7-H6 expression on cell lines Supplementary Figure 3 Influence of B7-H6/CD3 ITE on the B7-H6-induced activation of NK-92® MI cells. Supplementary Figure 4 Pharmacokinetic profile in NOG mice.

Oxidative stress is a primary cause of cellular senescence and contributes to the etiology of numerous human diseases. Oxidative damage to telomeric DNA has been proposed to cause premature senescence by accelerating telomere shortening. Here, we tested this model directly using a precision chemoptogenetic tool to produce the common lesion 8-oxo-guanine (8oxoG) exclusively at telomeres in human fibroblasts and epithelial cells. A single induction of telomeric 8oxoG is sufficient to trigger multiple hallmarks of p53-dependent senescence. Telomeric 8oxoG activates ATM and ATR signaling, and enriches for markers of telomere dysfunction in replicating, but not quiescent cells. Acute 8oxoG production fails to shorten telomeres, but rather generates fragile sites and mitotic DNA synthesis at telomeres, indicative of impaired replication. Based on our results, we propose that oxidative stress promotes rapid senescence by producing oxidative base lesions that drive replication-dependent telomere fragility and dysfunction in the absence of shortening and shelterin loss.

Molecular clocks that record cell ancestry mutate too slowly to measure the short-timescale dynamics of cell renewal in adult tissues. Here, we show that fluctuating DNA methylation marks can be used as clocks in cells where ongoing methylation and demethylation cause repeated ‘flip–flops’ between methylated and unmethylated states. We identify endogenous fluctuating CpG (fCpG) sites using standard methylation arrays and develop a mathematical model to quantitatively measure human adult stem cell dynamics from these data. Small intestinal crypts were inferred to contain slightly more stem cells than the colon, with slower stem cell replacement in the small intestine. Germline APC mutation increased the number of replacements per crypt. In blood, we measured rapid expansion of acute leukemia and slower growth of chronic disease. Thus, the patterns of human somatic cell birth and death are measurable with fluctuating methylation clocks (FMCs).

Neoplastic transformation and genome instability are enhanced by replication stress, conditions that slow or stall DNA replication forks. Consequently, cancer cells require multiple enzymes and checkpoint signaling pathways to mitigate replication stress for their viability and proliferation. Targeting proteins that enhance cancer cell survival during replication stress is a recent approach in clinical strategies, especially when targets produce synthetic lethality. DNA polymerase eta (Pol η) has many key functions in genome stability, particularly for translesion synthesis. Here we demonstrate that endogenous Pol η displays significant protein induction and forms intense foci throughout the nucleus in response to replication stress induced by drugs that do not directly form DNA adducts. During replication stress, Pol η-deficient cells displayed hyperactivation of the ATR replication checkpoint and arrested late in the cell cycle. During recovery from replication stress, Pol η-deficient cells continue to display aberrant phenotypes, including delayed cell-cycle progression, apoptosis, and cell survival. Depletion or inhibition of ATR was synthetically lethal with Pol η deficiency, particularly when tumor cells were treated with replication stress-inducing drugs. Together our data expand knowledge of the cellular environments that increase endogenous Pol η expression beyond DNA damaging agents and demonstrate that Pol η regulation is central to the replication stress response. Because Pol η is aberrantly expressed in several tumor types, our results are critical for developing more effective chemotherapy approaches and identify coinhibition of Pol η and ATR as a potential therapeutic strategy.Significance:This study demonstrates that replication stress upregulates Pol η (POLH) in tumor cells and reveals a role for Pol η in tumor cell recovery following replication stress.

Supplementary Table 1 and Figures S1-S7

Punishing and rewarding experiences can change the valence of sensory stimuli and guide animal behaviour in opposite directions, resulting in avoidance or approach. Often, however, a stimulus is encountered with both positive and negative experiences. How is such conflicting information represented in the brain and resolved into a behavioural decision? We address this question by dissecting a circuit for sexual conditioning inC. elegans. In this learning paradigm, an odour is conditioned with both a punishment (starvation) and a reward (mates) resulting in odour approach. We find that negative and positive experiences are both encoded by the neuropeptide PDF-1 being released from and acting on different neurons. Each experience creates a separate, parallel memory in the circuit for odour processing. This results in the sensorimotor representation of odour being different in naïve and sexually conditioned animals despite both displaying approach. Our results reveal that the positive valence of a stimulus is not represented in the activity of any single neuron class but flexibly represented within the circuit according to the experiences and predictions associated with the stimulus.

SynBioBrain: building biological computers from bacterial Using synthetic biology, we can now engineer bacteria into whole-cell biosensors where sensing, transduction and output occur within the living cell. Applications include the detection of harmful environmental agents, bioprocess monitoring, and detecting medically relevant biomarkers. Engineering microbial consortia allows biosensor information to be integrated and processed in a distributed fashion. In this project, we construct biological computers formed from engineered bacterial populations that communicate using intercellular signalling molecules. Our approach opens up new ways to perform biological computation.

BACKGROUND Challenges exist with heterotaxy due to the complexity of heart disease, abnormal venous connections, and infection risks. This study aims to understand heart transplant outcomes for children with heterotaxy. METHODS All children with congenital heart disease listed for transplant from 1993 to 2018 were included. Those with and without heterotaxy were compared. Waitlist outcomes and survival post-listing and transplant were analyzed. Post-transplant risk factors were identified using multiphase parametric hazard modeling. RESULTS There were 4814 children listed, of whom 196 (4%) had heterotaxy. Heterotaxy candidates were older (5.8 ± 5.7 vs 4.2 ± 5.5 years, p < 0.01), listed at a lower urgency status (29.8% vs 18.4%, p < 0.01), more commonly single ventricle physiology (71.3% vs 59.2%, p < 0.01), and less often supported by mechanical ventilation (22% vs 29.1%, p < 0.05) or extracorporeal membrane oxygenation (3.6% vs 7.5%, p < 0.05). There were no differences in waitlist outcomes of transplant, death, or removal. Overall, post-transplant survival was worse for children with heterotaxy: one-year survival 77.2% vs 85.1%, with and without heterotaxy, respectively. Heterotaxy was an independent predictor for early mortality in the earliest era (1993-2004), HR 2.09, CI 1.16-3.75, p = 0.014. When stratified by era, survival improved with time. Heterotaxy patients had a lower freedom from infection and from severe rejection, but no difference in vasculopathy or malignancy. CONCLUSIONS Mortality risk associated with heterotaxy is mitigated in the recent transplant era. Early referral may improve waitlist outcomes for heterotaxy patients who otherwise have a lower status at listing. Lower freedom from both infection and severe rejection after transplant in heterotaxy highlights the challenges of balancing immune suppression.

For many years, researchers studied EFs in the laboratory with a focus on understanding an individual child's development and brain processes in a controlled environment. Building on this foundational research, there is a growing interest in EFs in the context of a child's dynamic, social world and the contextual and compositional factors influencing EF development. This paper provides a descriptive view of EFs in 1,112 K-3 children from six schools in Phoenix, AZ. The study's goals were to examine (1) variation in EF scores between and within schools and classrooms, (2) predictors of variation in children's spring EF scores, and (3) individual and compositional predictors of children's spring EF scores. Our findings indicate greater variation in children's EF within schools than between, with very little or no variation arising from differences between schools. Though we observed greater variation within classrooms than between them, a notable amount of variance in children's spring EF scores appears to arise from differences between classrooms. Classroom-level variables, including a fall leave-out classroom mean (without the students' own score) and the number of children in the top or bottom grade-level quartiles in each classroom, were significant predictors of variation in spring EF scores as well as in fall to spring changes in EF. In some cases, the classroom variables were stronger predictors than individual fall scores. Findings suggest that understanding variation and cultivating growth in EF skills requires intervention, measurement, and analytic approaches that extend beyond the individual to include compositional features of the classroom environment. This article is protected by copyright. All rights reserved.

A novel method of producing fluorine-18 for Positron emission tomography (PET) scan applications is evaluated theoretically. The method is based upon the 3He(d,p)4He nuclear reaction from which the protons produced are used to produce fluorine-18 via the 18O(p,n)18F reaction. The potential advantage of such a system over cyclotron-based production is of lower input beam energy, which may lower the cost of the system and potentially allow for onsite production. Two theoretical designs were investigated. The first utilizes a helium-3 beam incident on a deuterated plastic target such as Mylar which is backed with an oxygen-18 heavy-water (H2O18) target. The second design utilizes a super-heavy-water (D2O18) target effectively combining both targets into one. Theoretical yield calculations were performed for both designs and the practicalities, primarily those of thermal and target degradation effects were assessed. To produce sufficient fluorine-18 yield a 1 MeV helium-3 beam at 100 mA was simulated. For this beam it was calculated that 310 MBq of fluorine-18 activity, as required to scan a 74 kg patient would be generated in a 69 min or an 18 min production run for the Mylar and super-heavy-water systems respectively. The simulated beam is at 100 kW power and without significant cooling would vaporize the target materials with the melting point of Mylar and boiling point of water calculated to be breached within 0.41 μs and 0.45 μs from beam-on. To achieve sufficient fluorine-18 yield the helium-3 beam power had to be increased to impractical levels making the systems technically infeasible.

G-protein coupled receptors (GPCRs) sense a wide variety of stimuli, including lipids, and transduce signals to the intracellular environment to exert various physiological responses. However, the structural features of GPCRs responsible for detecting and triggering responses to distinct lipid ligands have only recently begun to be revealed. 14,15-epoxyeicosatrienoic acid (14,15-EET) is one such lipid mediator that plays an essential role in the vascular system, displaying both vasodilatory and anti-inflammatory properties. We recently reported multiple low-affinity 14,15-EET-binding GPCRs, but the mechanism by which these receptors sense 14,15-EET remains unclear. Here, we have taken a combined computational and experimental approach to identify and confirm critical residues and properties within the lipid-binding pocket. Furthermore, we generated mutants to engineer selected GPCR-predicted binding sites to either confer or abolish 14,15-EET-induced signaling. Our structure-function analyses indicate that hydrophobic and positively charged residues of the receptor-binding pocket are prerequisites for recognizing lipid ligands such as 14,15-EET and possibly other eicosanoids.

Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness., Analysis of live-cell imaging and single-cell genome sequencing data of colorectal cancer organoids identifies temporal dynamics of sub-chromosomal copy-number amplifications.

No current treatments target microvascular reperfusion after stroke, which can contribute to poor outcomes even after successful clot retrieval. The G protein-coupled receptor GPR39 is expressed in brain peri-capillary pericytes, and has been implicated in microvascular regulation, but its role in stroke is unknown. We tested the hypothesis that GPR39 plays a protective role after stroke, in part due to preservation of microvascular perfusion. We generated GPR39 knockout (KO) mice and tested whether GPR39 gene deletion worsens capillary blood flow and exacerbates brain injury and functional deficit after focal cerebral ischemia. Stroke was induced in male and female GPR39 KO and WT littermates by 60-min middle cerebral artery occlusion (MCAO). Microvascular perfusion was assessed via capillary red blood cell (RBC) flux in deep cortical layers in vivo using optical microangiography (OMAG). Brain injury was assessed by measuring infarct size by 2,3,5-triphenyltetrazolium chloride staining at 24 h or brain atrophy at 3 weeks after ischemia. Pole and cylinder behavior tests were conducted to assess neurological function deficit at 1 and 3 weeks post-stroke. Male but not female GPR39 KO mice exhibited larger infarcts and lower capillary RBC flux than WT controls after stroke. Male GPR39 KO mice also exhibited worse neurologic deficit at 1 week post-stroke, though functional deficit disappeared in both groups by 3 weeks. GPR39 deletion worsens brain injury, microvascular perfusion, and neurological function after experimental stroke. Results indicate that GPR39 plays a sex-dependent role in re-establishing microvascular flow and limiting ischemic brain damage after stroke.

Purpose: Advanced-stage gastrointestinal cancers represent a high unmet need requiring new effective therapies. We investigated the antitumor activity of a novel T cell–engaging antibody (B7-H6/CD3 ITE) targeting B7-H6, a tumor-associated antigen that is expressed in gastrointestinal tumors. Experimental Design: Membrane proteomics and IHC analysis identified B7-H6 as a tumor-associated antigen in gastrointestinal tumor tissues with no to very little expression in normal tissues. The antitumor activity and mode of action of B7-H6/CD3 ITE was evaluated in in vitro coculture assays, in humanized mouse tumor models, and in colorectal cancer precision cut tumor slice cultures. Results: B7-H6 expression was detected in 98% of colorectal cancer, 77% of gastric cancer, and 63% of pancreatic cancer tissue samples. B7-H6/CD3 ITE-mediated redirection of T cells toward B7-H6–positive tumor cells resulted in B7-H6–dependent lysis of tumor cells, activation and proliferation of T cells, and cytokine secretion in in vitro coculture assays, and infiltration of T cells into tumor tissues associated with tumor regression in in vivo colorectal cancer models. In primary patient-derived colorectal cancer precision-cut tumor slice cultures, treatment with B7-H6/CD3 ITE elicited cytokine secretion by endogenous tumor-infiltrating immune cells. Combination with anti-PD-1 further enhanced the activity of the B7-H6/CD3 ITE. Conclusion: These data highlight the potential of the B7-H6/CD3 ITE to induce T cell–redirected lysis of tumor cells and recruitment of T cells into noninflamed tumor tissues, leading to antitumor activity in in vitro, in vivo, and human tumor slice cultures, which supports further evaluation in a clinical study.

BACKGROUND The Berlin Heart EXCOR Pediatric (EXCOR) ventricular assist device (VAD) was introduced in North America nearly 2 decades ago. The EXCOR was approved under Humanitarian Device Exemption status in 2011 and received post-market approval (PMA) in 2017 from Food and Drug Administration. Since the initial approval, the field of pediatric mechanical circulatory support has changed, specifically with regard to available devices, anticoagulation strategies, and the types of patients supported. This report summarizes the outcomes of patients supported with EXCOR from the Advanced Cardiac Therapies Improving Outcomes Network (ACTION) registry. These data were part of the PMA surveillance study (PSS) required by the Food and Drug Administration. METHODS ACTION is a learning collaborative of over 40 pediatric heart failure programs worldwide, which collects data for all VAD implantations as one of its initiatives. All patients in North America with EXCOR implants reported to ACTION from 2018 to 2020 ( n = 72) who had met an outcome were included in the EXCOR PSS group. This was compared with a historical, previously reported Berlin Heart EXCOR study group (Berlin Heart study [BHS] group, n = 320, 2007‒2014). RESULTS Patients in the PSS group were younger, were smaller in weight/body surface area, were more likely to have congenital heart disease , and were less likely to receive a bi-VAD than those in the BHS group. Patients in the PSS group were less likely to be in Interagency Registry for Mechanically Assisted Circulatory Support Profile 1 and were supported for a longer duration. The primary anticoagulation therapy for 92% of patients in the PSS group was bivalirudin . Success, defined as being transplanted, being weaned for recovery, or being alive on a device at 180 days after implantation, was 86% in the PSS group compared with 76% in the BHS group. Incidence of stroke was reduced by 44% and the frequency of pump exchange by 40% in the PSS group compared with those in the BHS group. Similarly, all other adverse events , including major bleeding, were reduced in the PSS group. CONCLUSIONS The PSS data, collected through ACTION, highlight the improvement in outcomes for patients supported with EXCOR compared with the outcomes in a historical cohort. These findings may be the result of changes in patient care practices over time and collaborative learning.

The use of biologics in positron emission tomography (PET) imaging is an important area of radiopharmaceutical development and new automated methods are required to facilitate their production. We report an automated radiosynthesis method to produce a radiolabelled biologic via facile inverse electron demand Diels–Alder (IEDDA) “click” chemistry on a single GE FASTLab™ cassette. We exemplified the method by producing a fluorine-18 radiolabelled interleukin-2 (IL2) radioconjugate from a trans-cyclooctene (TCO) modified IL2 precursor. The radioconjugate was produced using a fully automated radiosynthesis on a single FASTLab™ cassette in a decay-corrected radiochemical yield (RCY, d.c.) of 19.8 ± 2.6% in 110 min (from start of synthesis); the molar activity was 132.3 ± 14.6 GBq μmol−1. The in vitro uptake of [18F]TTCO-IL2 correlated with the differential receptor expression (CD25, CD122, CD132) in PC3, NK-92 and activated human PBMCs. The automated method may be adapted for the radiosynthesis of any TCO-modified protein via IEDDA chemistry., A fully automated and efficient radiosynthesis of a novel interleukin-2 radioconjugate from a single FASTLab™ cassette.

We have exemplified a pretargeted approach to interrogate hypoxia in live cells using radioactive bioorthogonal inverse electron demand Diels–Alder (IEDDA) “click” chemistry. Our novel 18F-tetrazine probe ([18F]FB-Tz) and 2-nitroimidazole-based TCO targeting molecule (8) showed statistically significant (P < 0.0001) uptake in hypoxic cells (ca. 90 %ID per mg) vs. normoxic cells (, Bioorthogonal IEDDA “click” can interrogate intracellular hypoxia using a radioactive reporter molecule.

A suboptimal response to the 2-dose COVID-19 vaccine series in the immunocompromised population prompted recommendations for a 3rd primary dose. We aimed to determine the humoral and cellular immune response to the 3rd COVID-19 vaccine in immunocompromised children.Prospective cohort study of immunocompromised participants, 5-21 years old, who received 2 prior doses of an mRNA COVID-19 vaccine. Humoral and CD4/CD8 T-cell responses were measured to SARS-CoV-2 spike antigens prior to receiving the 3rd vaccine dose and 3-4 weeks after the 3rd dose was given.Of the 37 participants, approximately half were solid organ transplant recipients. The majority (86.5%) had a detectable humoral response after the 2nd and 3rd vaccine doses, with a significant increase in antibody levels after the 3rd dose. Positive T-cell responses increased from being present in 86.5% to 100% of the cohort after the 3rd dose.Most immunocompromised children mount a humoral and cellular immune response to the 2-dose COVID-19 vaccine series, which is significantly augmented after receiving the 3rd vaccine dose. This supports the utility of the 3rd vaccine dose and the rationale for ongoing emphasis for vaccination against COVID-19 in this population.Most immunocompromised children mount a humoral and cellular immune response to the 2-dose COVID-19 vaccine series, which is significantly augmented after receiving the 3rd vaccine dose. This is the first prospective cohort study to analyze both the humoral and T-cell immune response to the 3rd COVID-19 primary vaccine dose in children who are immunocompromised. The results of this study support the utility of the 3rd vaccine dose and the rationale for ongoing emphasis for vaccination against COVID-19 in the immunosuppressed pediatric population.

The field of optimal experimental design uses mathematical techniques to determine experiments that are maximally informative from a given experimental setup. Here we apply a technique from artificial intelligence—reinforcement learning—to the optimal experimental design task of maximizing confidence in estimates of model parameter values. We show that a reinforcement learning approach performs favourably in comparison with a one-step ahead optimisation algorithm and a model predictive controller for the inference of bacterial growth parameters in a simulated chemostat. Further, we demonstrate the ability of reinforcement learning to train over a distribution of parameters, indicating that this approach is robust to parametric uncertainty.

This chapter explores the development of university-wide research data management conversations and collaborations involving key stakeholders at a Research 1 (R1) higher education institution in the southeastern United States of America. Research data management conversations led to collaborations of several key stakeholders across campus (i.e., university, office of research, research compliance office, information technology [IT], researchers, academic units, library) resulting in the development of an inaugural Supporting Data Management at University of Florida (UF) Proposal to the Office of Research draft document in 2019. Funding agencies, research foundations, and research associations increasingly request plans outlining how scientific data from funded research will be annotated, managed, shared, and stored. The National Institutes of Health (NIH) new Policy on Data Management and Sharing becomes effective January 25, 2023. The authors employed a participatory action research method to explore evolving aspects of the data science ecosystem, including research data management.

The earliest events during human tumor initiation, while poorly characterized, may hold clues to malignancy detection and prevention1. Here we model occult pre-neoplasia by bi-allelically inactivatingTP53, a common early event in gastric cancer, in human gastric organoids. Causal relationships between this initiating genetic lesion and resulting phenotypes were established using experimental evolution in multiple clonally derived cultures over two years.TP53loss elicited progressive aneuploidy, including copy number alterations and structural variants prevalent in gastric cancers, with evident preferred orders. Longitudinal single cell sequencing ofTP53deficient gastric organoids similarly indicates progression towards malignant transcriptional programs. Moreover, high-throughput lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programs repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference, and a striking degree of phenotypic convergence in pre-malignant epithelial organoids. These data imply predictability in the earliest stages of tumorigenesis and reveal evolutionary constraints and barriers to malignant transformation with implications for earlier detection and interception of aggressive, genome instable tumors.

The majority of cancer genomic data are generated from bulk samples composed of mixtures of cancer subpopulations, as well as normal cells. Subclonal reconstruction approaches based on machine learning aim to separate those subpopulations in a sample and reconstruct their evolutionary history. However, current approaches are entirely data-driven and agnostic to evolutionary theory. We demonstrate that systematic errors occur in the analysis if evolution is not accounted for, and this is exacerbated by multi-sampling of the same tumor. We present a novel approach for model-based tumor subclonal reconstruction (MOBSTER) that combines machine learning with theoretical population genetics. Using public whole-genome sequencing data from 2,606 samples from different cohorts, new data and synthetic validation, we show this method is more robust and accurate than current techniques in single sample, multi-region and longitudinal data. This approach minimizes the confounding factors of non-evolutionary methods, leading to more accurate recovery of the evolutionary history of human cancers.

The functions of secreted animal mucuses are remarkably diverse and include lubricants, wet adhesives, protective barriers, and mineralizing agents. Although present in all animals, many open questions related to the hierarchical architectures, material properties, and genetics of mucus remain. Here, we summarize what is known about secreted mucus structure, describe the work of research groups throughout the world who are investigating various animal mucuses, and relate how these studies are revealing new mucus properties and the relationships between mucus hierarchical structure and hydrogel function. Finally, we call for a more systematic approach to studying animal mucuses so that data sets can be compared, omics-style, to address unanswered questions in the emerging field of mucomics. One major result that we anticipate from these efforts is design rules for creating new materials that are inspired by the structures and functions of animal mucuses. acceptedVersion

Accurate quantification of absorbed radiation dose is important for safe and effective delivery of radiation therapy. An important aspect to this is reference dosimetry, which is performed under reference conditions specified by international codes of practice. Such measurements are usually performed in a water phantom. In the Sun Nuclear Corporation (SNC) three‐dimensional (3D) scanner water tank system the detector holder is fixed to a horizontal metallic drive rail (MDR) which is in close proximity to the active volume of the detector. In this project, the dosimetric effects of the MDR on reference dosimetry were investigated for MV photons, MeV electrons, and kV photons by comparing reference dosimetry measurements in the SNC 3D scanner against similar measurements in a Standard Imaging (SI) one‐dimensional (1D) tank and against measurements in the SNC 3D scanner with an additional, custom‐made spacer placed beneath the chamber holder to increase the chamber ‐ MDR separation. A second experiment investigated the difference in chamber reading dependent on chamber to MDR separation by fixing the chamber in the tank independently of the MDR and successively moving the MDR vertically to alter the separation. The results showed that measurements in the SNC 3D scanner agree with both SI 1D tank and SNC 3D scanner with spacer to within ±0.3% for MV photons, ±0.1% for electrons and ±1.2% for kV photons within the calculated setup uncertainty. The second experiment showed that the contribution of backscatter from the MDR was significant if the distance between MDR and chamber was reduced below the chamber's designed position in the SNC 3D scanner. The exception was for kV photons where the contribution of backscatter from the MDR was measured to be 0.5% at the designed distance. Further investigation would be useful for kV photons, where the experiment showed relatively large measurement uncertainties.

Mammalian telomeres are guanine-rich sequences which cap the ends of linear chromosomes. While recognized as sites sensitive to oxidative stress, studies on the consequences of oxidative damage to telomeres have been primarily limited to experimental conditions which cause oxidative damage throughout the whole genome and cell. We developed a chemoptogenetic tool (FAP-mCER-TRF1) to specifically induce singlet oxygen at telomeres, resulting in the formation of the common oxidative lesion 8-oxo-guanine. Here, we describe this tool and detail how to generate cell lines which express FAP-mCER-TRF1 at telomeres and verify the formation of 8-oxo-guanine.

Bovine tuberculosis (bTB) is a globally distributed zoonotic disease with significant economic impacts. Control measures in Great Britain include testing for and culling diseased animals. Farmers receive compensation for the value of culled animals, but not for the consequential costs of having to comply with testing and associated control measures. Such uncompensated costs can be significant. We present results of a survey of 1,600 dairy and beef farm holdings conducted in England and Wales to update and improve estimates of these consequential costs.Estimated costs are positively skewed and show considerable variance, which is in agreement with previous, smaller scale surveys of bTB: most farms experiencing bTB incur modest costs but some suffer significant costs. Testing, movement restrictions and output losses account for over three quarters of total uncompensated costs. Total costs rise with herd size and duration of controls. The composition of consequential costs changes as total costs increase, with an increasing proportion of the costs being associated with output losses and movement restrictions, and a decreasing proportion of costs associated with testing costs. Consequential costs tend to be higher for dairy than beef herds but this is likely due to larger herd sizes for dairy.Overall we find the total farm costs of bTB surpass those compensated for by Government in Great Britain. This study contributes to the public-private cost-sharing debate as farmers bear some of the economic burden of a disease breakdown. The methodology and results presented are crucial for informed Government and farmer decision-making. The identification of potential risk factors in this study was challenging but is of relevance outside GB.

Telomeres present inherent difficulties to the DNA replication machinery due to their repetitive sequence content, formation of non-B DNA secondary structures, and the presence of the nucleo-protein t-loop. Especially in cancer cells, telomeres are hot spots for replication stress, which can result in a visible phenotype in metaphase cells termed “telomere fragility”. A mechanism cells employ to mitigate replication stress, including at telomeres, is DNA synthesis in mitosis (MiDAS). While these phenomena are both observed in mitotic cells, the relationship between them is poorly understood; however, a common link is DNA replication stress. In this review, we will summarize what is known to regulate telomere fragility and telomere MiDAS, paying special attention to the proteins which play a role in these telomere phenotypes.

The microcirculation serves crucial functions in adult heart, distinct from those carried out by epicardial vessels. Microvessels are governed by unique regulatory mechanisms, impairment of which leads to microvessel-specific pathology. There are few treatment options for patients with microvascular heart disease, primarily due to limited understanding of underlying pathology. High throughput mRNA sequencing and protein expression profiling in specific cells can improve our understanding of microvessel biology and disease at the molecular level. Understanding responses of individual microvascular cells to the same physiological or pathophysiological stimuli requires the ability to isolate the specific cell types that comprise the functional units of the microcirculation in the heart, preferably from the same heart, to ensure that different cells have been exposed to the same in-vivo conditions. We developed an integrated process for simultaneous isolation and culture of the main cell types comprising the microcirculation in adult mouse heart: endothelial cells, pericytes, and vascular smooth muscle cells. These cell types were characterized with isobaric labeling quantitative proteomics and mRNA sequencing. We defined microvascular cell proteomes, identified novel protein markers, and confirmed established cell-specific markers. Our results allow identification of unique markers and regulatory proteins that govern microvascular physiology and pathology.

The flow shear-driven Kelvin-Helmholtz (KH) instability is ubiquitous in planetary magnetospheres. At Earth these surface waves are important along the dawn and dusk flanks of the magnetopause boundary while at Jupiter and Saturn the entire dayside magnetopause boundary can exhibit KH activity due to corotational flows in the magnetosphere. Kelvin-Helmholtz waves can be a major ingredient in the so-called viscous-like interaction with the solar wind. In this paper, we review the KH instability from the perspective of hybrid (kinetic ions, fluid electrons) simulations. Many of the simulations are based on parameters typically found at Saturn’s magnetopause boundary, but the results can be generally applied to any KH-unstable situation. The focus of the discussion is on the ion kinetic scale and implications for mass, momentum, and energy transport at the magnetopause boundary.

Predictability is a fundamental requirement in biological engineering. As we move to building coordinated multicellular systems, the potential for such systems to display chaotic behaviour becomes a concern. Therefore understanding which systems show chaos is an important design consideration. We developed a methodology to explore the potential for chaotic dynamics in small microbial communities governed by resource competition, intercellular communication and competitive bacteriocin interactions. We show that we can expect to find chaotic states in relatively small synthetic microbial systems, understand the governing dynamics and provide insights into how to control such systems. This work is the first to query the existence of chaotic behaviour in synthetic microbial communities and has important ramifications for the fields of biotechnology, bioprocessing and synthetic biology.

Hypoxia is a complex microenvironmental condition known to regulate choline kinase α (CHKA) activity and choline transport through transcription factor hypoxia-inducible factor-1α (HIF-1α) and, therefore, may confound the uptake of choline radiotracer [18F]fluoromethyl-[1,2-2H4]-choline ([18F]-D4-FCH). The aim of this study was to investigate how hypoxia affects the choline radiotracer dynamics. Three underlying mechanisms by which hypoxia could potentially alter the uptake of the choline radiotracer, [18F]-D4-FCH, were investigated: 18F-D4-FCH import, CHKA phosphorylation activity, and the efflux of [18F]-D4-FCH and its phosphorylated product [18F]-D4-FCHP. The effects of hypoxia on [18F]-D4-FCH uptake were studied in CHKA-overexpressing cell lines of prostate cancer, PC-3, and breast cancer MDA-MB-231 cells. The mechanisms of radiotracer efflux were assessed by the cell uptake and immunofluorescence in vitro and examined in vivo (n = 24). The mathematical modelling methodology was further developed to verify the efflux hypothesis using [18F]-D4-FCH dynamic PET scans from non-small cell lung cancer (NSCLC) patients (n = 17). We report a novel finding involving the export of phosphorylated [18F]-D4-FCH and [18F]-D4-FCHP via HIF-1α-responsive efflux transporters, including ABCB4, when the HIF-1α level is augmented. This is supported by a graphical analysis of human data with a compartmental model (M2T6k + k5) that accounts for the efflux. Hypoxia/HIF-1α increases the efflux of phosphorylated radiolabelled choline species, thus supporting the consideration of efflux in the modelling of radiotracer dynamics.

Cancer genomic medicine relies on targeting driver genes. However, current catalogues of cancer drivers are mostly based on indirect measurements of mutation frequencies, positions or types, rather than their effect on clonal expansionsin vivo. Moreover, non-genetic drivers are largely unknown, as are the epigenetic and transcriptomic effects of genetic drivers. Here we perform spatial computational inference on multiomic data with matched whole-genome sequencing, ATAC-seq and RNA-seq. Using 436 samples, we directly quantify the contribution, or lack thereof, of putative driver genes to subclonal expansionsin vivoin 30 colorectal carcinomas (4-33 samples per patient, median=15). Although subclonal neutral evolution was widespread (13/26 cases with sufficient data), there were cases with clear evidence of subclonal selection (6/26) in which we measured epigenetic and transcriptomic differences between subclonesin vivo. In 7/26 cases we could not distinguish between neutral or selective evolution with the available data. We identified expanding subclones that were not driven by known genetic alterations, and propose candidate epigenetic drivers. We identified the distinguishing patterns of genomic heterogeneity produced in fast, exponentially growing tumours (7/26) versus neoplasms growing only at the periphery (19/26), as well as identifying clonally intermixed (16/28 cases with sufficient data) versus segregated malignancies (10/28). Our model-based approach measures genetic and non-genetic subclonal selection, or lack thereof, in space and time and allowsin vivocomparisons of the emergent phenotypic properties of subclones within human tumours.

Whole-cell biosensors hold potential in a variety of industrial, medical and environmental applications. These biosensors can be constructed through the repurposing of bacterial sensing mechanisms, including the common two-component system. Here we report on the construction of a range of novel biosensors that are sensitive to acetoacetate, a molecule that plays a number of roles in human health and biology. These biosensors are based on the AtoSC two-component system. An ordinary differential equation model to describe the action of the AtoSC two-component system was developed and sensitivity analysis of this model used to help inform biosensor design. The final collection of biosensors constructed displayed a range of switching behaviours, at physiologically relevant acetoacetate concentrations and can operate in several Escherichia coli host strains. It is envisaged that these biosensor strains will offer an alternative to currently available commercial strip tests and, in future, may be adopted for more complex in vivo or industrial monitoring applications.

Pluto energies of a few kiloelectron volts and suprathermal ions with tens of kiloelectron volts and above. We measure this population using the Pluto Energetic Particle Spectrometer Science Investigation (PEPSSI) instrument on board the New Horizons spacecraft that flew by Pluto in 2015. Even though the measured ions have gyroradii larger than the size of Pluto and the cross section of its magnetosphere, we find that the boundary of the magnetosphere is depleting the energetic ion intensities by about an order of magnitude close to Pluto. The intensity is increasing exponentially with distance to Pluto and reaches nominal levels of the interplanetary medium at about 190

With a sample of 7,752 fourth- to seventh-grade students in 25 schools which were randomized at the school level to condition, this article reports experimental impacts of an enhanced version of Wo...

To evaluate the efficacy of neurobehavioural rehabilitation (NbR) programmes, services should employ valid, reliable assessment tools; the ability to detect change on repeated assessment is a particular requirement. The United Kingdom Rehabilitation Outcomes Collaborative (UKROC) requires neurorehabilitation services to collect data using a standardized basket of measures, but the responsiveness and usefulness of using these in the context of NbR remains unknown. Anonymous data collected at two assessments for 123 people were examined using multiple methods to determine responsiveness of four outcome measures routinely used in NbR (HoNOS-ABI, FIM + FAM UK, MPAI-4, SASNOS). Predictive validity of two measures of rehabilitation complexity (RCS-E, SRS) regarding the extent of difference scores on these outcome measures at reassessment was also determined. All four outcome measures demonstrated responsiveness, with higher levels for SASNOS and MPAI-4 when only participants categorized as "most likely to change" at first assessment were analyzed. Predictive validity of the RCS-E and SRS in estimating the extent of change was variable. SRS was only predictive of improvement on the MPAI-4 whilst RCS-E was not predictive at all. Recommendations are made regarding ideal characteristics of NbR outcome measures, along with the need to develop measures of rehabilitation complexity specifically conceptualized for these programmes.