Tissue and cellular spatiotemporal dynamics in colon aging.

Tissue structure and molecular circuitry in the colon can be profoundly impacted by systemic age-related effects, but many of the underlying molecular cues remain unclear. Here, we built a cellular and spatial atlas of the colon across three anatomical regions and 11 age groups, encompassing ~1,500 mouse gut tissues profiled by spatial transcriptomics and ~400,000 single nucleus RNA-seq profiles. We developed a new computational framework, cSplotch, which learns a hierarchical Bayesian model of spatially resolved cellular expression associated with age, tissue region, and sex, by leveraging histological features to share information across tissue samples and data modalities. Using this model, we identified cellular and molecular gradients along the adult colonic tract and across the main crypt axis, and multicellular programs associated with aging in the large intestine. Our multi-modal framework for the investigation of cell and tissue organization can aid in the understanding of cellular roles in tissue-level pathology.

Phylogenomics and the rise of the angiosperms.

Angiosperms are the cornerstone of most terrestrial ecosystems and human livelihoods1,2. A robust understanding of angiosperm evolution is required to explain their rise to ecological dominance. So far, the angiosperm tree of life has been determined primarily by means of analyses of the plastid genome3,4. Many studies have drawn on this foundational work, such as classification and first insights into angiosperm diversification since their Mesozoic origins5-7. However, the limited and biased sampling of both taxa and genomes undermines confidence in the tree and its implications. Here, we build the tree of life for almost 8,000 (about 60%) angiosperm genera using a standardized set of 353 nuclear genes8. This 15-fold increase in genus-level sampling relative to comparable nuclear studies9 provides a critical test of earlier results and brings notable change to key groups, especially in rosids, while substantiating many previously predicted relationships. Scaling this tree to time using 200 fossils, we discovered that early angiosperm evolution was characterized by high gene tree conflict and explosive diversification, giving rise to more than 80% of extant angiosperm orders. Steady diversification ensued through the remaining Mesozoic Era until rates resurged in the Cenozoic Era, concurrent with decreasing global temperatures and tightly linked with gene tree conflict. Taken together, our extensive sampling combined with advanced phylogenomic methods shows the deep history and full complexity in the evolution of a megadiverse clade.

Critical Assessment of Novel Developments in HPGR Technology Using DEM.

Advances in high-pressure grinding roll (HGPR) technology since its first commercial application in the cement industry include new roll wear protection techniques and new confinement systems. The latter contribute to reductions in the edge effects in an attempt to reach a more homogenous product size along the rolls. Additional advances in this technology have been made in recent years, while modeling and simulation tools are also reaching maturity and can now be used to subject such novel developments to detailed scrutiny. This work applies a hybrid approach combining advanced simulations using the discrete element method, the particle replacement model and multibody dynamics to a phenomenological population balance model to critically assess two recent advances in HPGR technology: spring-loaded cheek plates and the offset roller press. Force and torque controllers, included in the EDEM 2022.1 software, were used to describe the responses of the geometries in contact with the granular material processed. Simulations showed that while the former successfully reduced the lateral bypass of the material by as much as 65% when cheek plates became severely worn, the latter demonstrated lower throughput and higher potential wear but an ability to generate a finer product than the traditional design.

Altered microbial bile acid metabolism exacerbates T cell-driven inflammation during graft-versus-host disease.

Microbial transformation of bile acids affects intestinal immune homoeostasis but its impact on inflammatory pathologies remains largely unknown. Using a mouse model of graft-versus-host disease (GVHD), we found that T cell-driven inflammation decreased the abundance of microbiome-encoded bile salt hydrolase (BSH) genes and reduced the levels of unconjugated and microbe-derived bile acids. Several microbe-derived bile acids attenuated farnesoid X receptor (FXR) activation, suggesting that loss of these metabolites during inflammation may increase FXR activity and exacerbate the course of disease. Indeed, mortality increased with pharmacological activation of FXR and decreased with its genetic ablation in donor T cells during mouse GVHD. Furthermore, patients with GVHD after allogeneic hematopoietic cell transplantation showed similar loss of BSH and the associated reduction in unconjugated and microbe-derived bile acids. In addition, the FXR antagonist ursodeoxycholic acid reduced the proliferation of human T cells and was associated with a lower risk of GVHD-related mortality in patients. We propose that dysbiosis and loss of microbe-derived bile acids during inflammation may be an important mechanism to amplify T cell-mediated diseases.

Repeated immunization with ATRA-containing liposomal adjuvant transdifferentiates Th17 cells to a Tr1-like phenotype.

In many autoimmune diseases, autoantigen-specific Th17 cells play a pivotal role in disease pathogenesis. Th17 cells can transdifferentiate into other T cell subsets in inflammatory conditions, however, there have been no attempts to target Th17 cell plasticity using vaccines. We investigated if autoantigen-specific Th17 cells could be specifically targeted using a therapeutic vaccine approach, where antigen was formulated in all-trans retinoic acid (ATRA)-containing liposomes, permitting co-delivery of antigen and ATRA to the same target cell. Whilst ATRA was previously found to broadly reduce Th17 responses, we found that antigen formulated in ATRA-containing cationic liposomes only inhibited Th17 cells in an antigen-specific manner and not when combined with an irrelevant antigen. Furthermore, this approach shifted existing Th17 cells away from IL-17A expression and transcriptomic analysis of sorted Th17 lineage cells from IL-17 fate reporter mice revealed a shift of antigen-specific Th17 cells to exTh17 cells, expressing functional markers associated with T cell regulation and tolerance. In the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, vaccination with myelin-specific (MOG) antigen in ATRA-containing liposomes reduced Th17 responses and alleviated disease. This highlights the potential of therapeutic vaccination for changing the phenotype of existing Th17 cells in the context of immune mediated diseases.

AutonoMS: Automated Ion Mobility Metabolomic Fingerprinting.

Automation is dramatically changing the nature of laboratory life science. Robotic lab hardware that can perform manual operations with greater speed, endurance, and reproducibility opens an avenue for faster scientific discovery with less time spent on laborious repetitive tasks. A major bottleneck remains in integrating cutting-edge laboratory equipment into automated workflows, notably specialized analytical equipment, which is designed for human usage. Here we present AutonoMS, a platform for automatically running, processing, and analyzing high-throughput mass spectrometry experiments. AutonoMS is currently written around an ion mobility mass spectrometry (IM-MS) platform and can be adapted to additional analytical instruments and data processing flows. AutonoMS enables automated software agent-controlled end-to-end measurement and analysis runs from experimental specification files that can be produced by human users or upstream software processes. We demonstrate the use and abilities of AutonoMS in a high-throughput flow-injection ion mobility configuration with 5 s sample analysis time, processing robotically prepared chemical standards and cultured yeast samples in targeted and untargeted metabolomics applications. The platform exhibited consistency, reliability, and ease of use while eliminating the need for human intervention in the process of sample injection, data processing, and analysis. The platform paves the way toward a more fully automated mass spectrometry analysis and ultimately closed-loop laboratory workflows involving automated experimentation and analysis coupled to AI-driven experimentation utilizing cutting-edge analytical instrumentation. AutonoMS documentation is available at https://autonoms.readthedocs.io.

Assessing the difference in contamination of retail meat with multidrug-resistant bacteria using for-consumer package label claims that indicate on-farm antibiotic use practices- United States, 2016-2019.

BACKGROUND: Antibiotic use in food-producing animals can select for antibiotic resistance in bacteria that can be transmitted to people through contamination of food products during meat processing. Contamination resulting in foodborne illness contributes to adverse health outcomes. Some livestock producers have implemented antibiotic use reduction strategies marketed to consumers on regulated retail meat packaging labels ("label claims"). OBJECTIVE: We investigated whether retail meat label claims were associated with isolation of multidrug-resistant organisms (MDROs, resistant to ≥3 classes of antibiotics) from U.S. meat samples. METHODS: We utilized retail meat data from the U.S. Food and Drug Administration National Antimicrobial Resistance Monitoring System (NARMS) collected during 2016-2019 for bacterial contamination of chicken breast, ground turkey, ground beef, and pork chops. We used modified Poisson regression models to compare the prevalence of MDRO contamination among meat samples with any antibiotic restriction label claims versus those without such claims (i.e., conventionally produced). RESULTS: In NARMS, 62,338 meat samples were evaluated for bacterial growth from 2016-2019. Of these, 24,446 (39%) samples had label claims that indicated antibiotic use was restricted during animal production. MDROs were isolated from 2252 (4%) meat samples, of which 71% (n = 1591) were conventionally produced, and 29% (n = 661) had antibiotic restriction label claims. Compared with conventional samples, meat with antibiotic restriction label claims had a statistically lower prevalence of MDROs (adjusted prevalence ratio: 0.66; 95% CI: 0.61, 0.73). This relationship was consistent for the outcome of any bacterial growth. IMPACT: This repeated cross-sectional analysis of a nationally representative retail meat surveillance database in the United States supports that retail meats labeled with antibiotic restriction claims were less likely to be contaminated with MDROs compared with retail meat without such claims during 2016-2019. These findings indicate the potential for the public to become exposed to bacterial pathogens via retail meat and emphasizes a possibility that consumers could reduce their exposure to environmental reservoirs of foodborne pathogens that are resistant to antibiotics.

Throwing IL-1ß for a loop.

The lncRNA AMANZI exerts cis-regulatory control of IL-1ß-mediated inflammation.

Animal drug shortages limit veterinary therapeutic options and introduce artifacts in antimicrobial sales reporting.

Supply chain issues disrupt veterinary care and cause downstream consequences that alter the practice of veterinary medicine. Antimicrobials are just 1 class of pharmaceuticals that have been impacted by supply chain issues over the last couple of years. Since February 2021, 2 sponsors/manufacturers of penicillin products have reported shortages in the active pharmaceutical ingredient. With the release of the 2021 Summary Report on Antimicrobials Sold or Distributed for Use in Food-Producing Animals by the FDA, a key finding was a 19% decrease in penicillin sales and distribution from 2020 to 2021. Herein, we provide our clinicians' professional perspective regarding how drug shortages, specifically that of penicillin, might contribute to misconstrued patterns in antimicrobial use and what can be done by veterinarians and the FDA to minimize the impact of an antimicrobial drug shortage on animal health and well-being.

Human In vitro Modeling Identifies Adjuvant Combinations that Unlock Antigen Cross-presentation and Promote T-helper 1 Development in Newborns, Adults and Elders.

Adjuvants are vaccine components that can boost the type, magnitude, breadth, and durability of an immune response. We have previously demonstrated that certain adjuvant combinations can act synergistically to enhance and shape immunogenicity including promotion of Th1 and cytotoxic T-cell development. These combinations also promoted protective immunity in vulnerable populations such as newborns. In this study, we employed combined antigen-specific human in vitro models to identify adjuvant combinations that could synergistically promote the expansion of vaccine-specific CD4+ cells, induce cross-presentation on MHC class I, resulting in antigen-specific activation of CD8+ cells, and direct the balance of immune response to favor the production of Th1-promoting cytokines. A screen of 78 adjuvant combinations identified several T cell-potentiating adjuvant combinations. Remarkably, a combination of TLR9 and STING agonists (CpG + 2,3-cGAMP) promoted influenza-specific CD4+ and CD8+ T cell activation and selectively favored production of Th1-polarizing cytokines TNF and IL-12p70 over co-regulated cytokines IL-6 and IL-12p40, respectively. Phenotypic reprogramming towards cDC1-type dendritic cells by CpG + 2,3-cGAMP was also observed. Finally, we characterized the molecular mechanism of this adjuvant combination including the ability of 2,3-cGAMP to enhance DC expression of TLR9 and the dependency of antigen-presenting cell activation on the Sec22b protein important to endoplasmic reticulum-Golgi vesicle trafficking. The identification of the adjuvant combination CpG + 2,3-cGAMP may therefore prove key to the future development of vaccines against respiratory viral infections tailored for the functionally distinct immune systems of vulnerable populations such as older adults and newborns.

The interplay between trehalose and dextran as spray drying precursors for cationic liposomes.

Successful oral delivery of liposomes requires formulations designed to withstand harsh gastrointestinal conditions, e.g., by converting to solid-state followed by loading into gastro-resistant delivery devices. The hypothesis was that the use of dextran-trehalose mixtures for spray drying would improve the rehydration kinetics of dried liposomes. The objectives were to determine the protective capacity of trehalose-dextran dehydration precursors and to increase the concentration of liposomes in the dry formulation volume. The study successfully demonstrated that 8.5% dextran combined with 76.5% trehalose protected CAF®04 liposomes during drying, with the liposome content maintained at 15% of the dry powder. Accordingly, the rehydration kinetics were slightly improved in formulations containing up to 8.5% dextran in the dry powder volume. Additionally, a 2.4-fold increase in lipid concentration (3 mM vs 7.245 mM) was achieved for spray dried CAF®04 liposomes. Ultimately, this study demonstrates the significance of trehalose as a primary carrier during spray drying of CAF®04 liposomes and highlights the advantage of incorporating small amounts of dextran to tune rehydration kinetics of spray-dried liposomes.

Sensitivity to targeted UBA1 inhibition in a myeloid cell line model of VEXAS syndrome.

Somatic UBA1 mutations in hematopoietic cells are a hallmark of Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic (VEXAS) syndrome, which is a late-onset inflammatory disease associated with bone marrow failure and high mortality. The majority of UBA1 mutations in VEXAS syndrome comprise hemizygous mutations affecting methionine-41 (M41), leading to the expression of UBA1M41T, UBA1M41V, or UBA1M41L and globally reduced protein polyubiquitination. Here, we used CRISPR-Cas9 to engineer isogenic 32D mouse myeloid cell lines expressing hemizygous Uba1WT or Uba1M41L from the endogenous locus. Consistent with prior analyses of patients with VEXAS syndrome samples, hemizygous Uba1M41L expression was associated with loss of the UBA1b protein isoform, gain of the UBA1c protein isoform, reduced polyubiquitination, abnormal cytoplasmic vacuoles, and increased production of interleukin-1ß and inflammatory chemokines. Vacuoles in Uba1M41L cells contained a variety of endolysosomal membranes, including small vesicles, multivesicular bodies, and multilamellar lysosomes. Uba1M41L cells were more sensitive to the UBA1 inhibitor TAK243. TAK243 treatment promoted apoptosis in Uba1M41L cells and led to preferential loss of Uba1M41L cells in competition assays with Uba1WT cells. Knock-in of a TAK243-binding mutation, Uba1A580S, conferred TAK243 resistance. In addition, overexpression of catalytically active UBA1b in Uba1M41L cells restored polyubiquitination and increased TAK243 resistance. Altogether, these data indicate that loss of UBA1b underlies a key biochemical phenotype associated with VEXAS syndrome and renders cells with reduced UBA1 activity vulnerable to targeted UBA1 inhibition. Our Uba1M41L knock-in cell line is a useful model of VEXAS syndrome that will aid in the study of disease pathogenesis and the development of effective therapies.

Co-adjuvanting DDA/TDB liposomes with a TLR7 agonist allows for IgG2a/c class-switching in the absence of Th1 cells.

Class-switching to IgG2a/c in mice is a hallmark response to intracellular pathogens. T cells can promote class-switching and the predominant pathway for induction of IgG2a/c antibody responses has been suggested to be via stimulation from Th1 cells. We previously formulated CAF®01 (cationic liposomes containing dimethyldioctadecylammonium bromide (DDA) and Trehalose-6,6-dibehenate (TDB)) with the lipidated TLR7/8 agonist 3M-052 (DDA/TDB/3M-052), which promoted robust Th1 immunity in newborn mice. When testing this adjuvant in adult mice using the recombinant Chlamydia trachomatis (C.t.) vaccine antigen CTH522, it similarly enhanced IgG2a/c responses compared to DDA/TDB, but surprisingly reduced the magnitude of the IFN-γ+Th1 response in a TLR7 agonist dose-dependent manner. Single-cell RNA-sequencing revealed that DDA/TDB/3M-052 liposomes initiated early transcription of class-switch regulating genes directly in pre-germinal center B cells. Mixed bone marrow chimeras further demonstrated that this adjuvant did not require Th1 cells for IgG2a/c switching, but rather facilitated TLR7-dependent T-bet programming directly in B cells. This study underlines that adjuvant-directed IgG2a/c class-switching in vivo can occur in the absence of T-cell help, via direct activation of TLR7 on B cells and positions DDA/TDB/3M-052 as a powerful adjuvant capable of eliciting type I-like immunity in B cells without strong induction of Th1 responses.

Distance and destination of retail meat alter multidrug resistant contamination in the United States food system.

Antibiotic-resistant infections are a global concern, especially those caused by multidrug-resistant (MDR) bacteria, defined as those resistant to more than three drug classes. The animal agriculture industry contributes to the antimicrobial resistant foodborne illness burden via contaminated retail meat. In the United States, retail meat is shipped across the country. Therefore, understanding geospatial factors that influence MDR bacterial contamination is vital to protect consumers and inform interventions. Using data available from the United States Food and Drug Administration's National Antimicrobial Resistance Monitoring System (NARMS), we describe retail meat shipping distances using processor and retailer locations and investigated this distance as a risk factor for MDR bacteria meat contamination using log-binomial regression. Meat samples collected during 2012-2014 totaled 11,243, of which 4791 (42.61%) were contaminated with bacteria and 835 (17.43%) of those bacteria were MDR. All examined geospatial factors were associated with MDR bacteria meat contamination. After adjustment for year and meat type, we found higher prevalence of MDR contamination among meat processed in the south (relative adjusted prevalence ratio [aPR] 1.35; 95% CI 1.06-1.73 when compared to the next-highest region), sold in Maryland (aPR 1.12; 95% CI 0.95-1.32 when compared to the next-highest state), and shipped from 194 to 469 miles (aPR 1.59; 95% CI 1.31-1.94 when compared to meats that traveled < 194 miles). However, sensitivity analyses revealed that New York sold the meat with the highest prevalence of MDR Salmonella contamination (4.84%). In this secondary analysis of NARMS data, both geographic location where products were sold and the shipping distance were associated with microbial contamination on retail meat.

The PTPN2/PTPN1 inhibitor ABBV-CLS-484 unleashes potent anti-tumour immunity.

Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance1,2. The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity3-6. However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8+ T cell function by enhancing JAK-STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes.

The HLA advantage: Why COVID-19 benched you but not your co-worker.

An HLA variant confers protective immunity against COVID-19 through cross-reactive T cells induced by seasonal coronaviruses.

Genesis-DB: a database for autonomous laboratory systems.

Summary: Artificial intelligence (AI)-driven laboratory automation-combining robotic labware and autonomous software agents-is a powerful trend in modern biology. We developed Genesis-DB, a database system designed to support AI-driven autonomous laboratories by providing software agents access to large quantities of structured domain information. In addition, we present a new ontology for modeling data and metadata from autonomously performed yeast microchemostat cultivations in the framework of the Genesis robot scientist system. We show an example of how Genesis-DB enables the research life cycle by modeling yeast gene regulation, guiding future hypotheses generation and design of experiments. Genesis-DB supports AI-driven discovery through automated reasoning and its design is portable, generic, and easily extensible to other AI-driven molecular biology laboratory data and beyond. Availability and implementation: Genesis-DB code and installation instructions are available at the GitHub repository https://github.com/TW-Genesis/genesis-database-system.git. The database use case demo code and data are also available through GitHub (https://github.com/TW-Genesis/genesis-database-demo.git). The ontology can be downloaded here: https://github.com/TW-Genesis/genesis-ontology/releases/download/v0.0.23/genesis.owl. The ontology term descriptions (including mappings to existing ontologies) and maintenance standard operating procedures can be found at: https://github.com/TW-Genesis/genesis-ontology.

Keratin-positive giant cell-rich tumors of soft tissue with HMGA2::NCOR2 fusions.

BACKGROUND: Giant cell tumor of soft tissue (GCT-ST) is a rare soft tissue neoplasm that is morphologically similar to but genetically distinct from giant cell tumor of bone. A novel keratin-positive GCT-ST (KPGCT-ST) harboring HMGA2::NCOR2 fusions was recently discovered. Fewer than 30 cases have been described; herein is reported an additional seven. METHODS: Cases diagnosed as GCT-ST were retrieved from institutional archives and consultation files. The histopathologic characteristics were assessed, and the electronic medical record was reviewed. RESULTS: Seven tumors were identified in six women and one man with a median age of 23 years. All patients underwent excision; no recurrences or metastases were noted during a median follow-up period of 7 months. Histopathologically, the tumors were characterized by a multinodular proliferation of keratin-positive mononuclear cells with evenly admixed osteoclast-like giant cells and absent neoplastic bone. A fibrous capsule with lymphoid cuffing was frequently seen. Foamy macrophages, inflammation, hemorrhage, and hemosiderin were variably present. The HMGA2::NCOR2 fusion was detected in all cases. CONCLUSIONS: Our findings support previously reported hypotheses that KPGCT-ST is a spectrum of the same entity as the recently described xanthogranulomatous epithelial tumor. Although follow-up data are limited, to date, KPGCT-ST appears to follow an indolent course.

Genetic modification of inflammation- and clonal hematopoiesis-associated cardiovascular risk.

Clonal hematopoiesis of indeterminate potential (CHIP) is associated with an increased risk of cardiovascular diseases (CVDs), putatively via inflammasome activation. We pursued an inflammatory gene modifier scan for CHIP-associated CVD risk among 424,651 UK Biobank participants. We identified CHIP using whole-exome sequencing data of blood DNA and modeled as a composite, considering all driver genes together, as well as separately for common drivers (DNMT3A, TET2, ASXL1, and JAK2). We developed predicted gene expression scores for 26 inflammasome-related genes and assessed how they modify CHIP-associated CVD risk. We identified IL1RAP as a potential key molecule for CHIP-associated CVD risk across genes and increased AIM2 gene expression leading to heightened JAK2- and ASXL1-associated CVD risk. We show that CRISPR-induced Asxl1-mutated murine macrophages had a particularly heightened inflammatory response to AIM2 agonism, associated with an increased DNA damage response, as well as increased IL-10 secretion, mirroring a CVD-protective effect of IL10 expression in ASXL1 CHIP. Our study supports the role of inflammasomes in CHIP-associated CVD and provides evidence to support gene-specific strategies to address CHIP-associated CVD risk.