Impact of nutrient excess on physiology and metabolism of Sulfolobus acidocaldarius.

Overflow metabolism is a well-known phenomenon that describes the seemingly wasteful and incomplete substrate oxidation by aerobic cells, such as yeasts, bacteria, and mammalian cells, even when conditions allow for total combustion via respiration. This cellular response, triggered by an excess of C-source, has not yet been investigated in archaea. In this study, we conducted chemostat cultivations to compare the metabolic and physiological states of the thermoacidophilic archaeon Sulfolobus acidocaldarius under three conditions, each with gradually increasing nutrient stress. Our results show that S. acidocaldarius has different capacities for the uptake of the two C-sources, monosodium glutamate and glucose. A saturated tricarboxylic acid cycle at elevated nutrient concentrations affects the cell's ability to deplete its intermediates. This includes deploying additional cataplerotic pathways and the secretion of amino acids, notably valine, glycine, and alanine, while glucose is increasingly metabolized via glycogenesis. We did not observe the secretion of common fermentation products, like organic acids. Transcriptomic analysis indicated an upregulation of genes involved in fatty acid metabolism, suggesting the intracellular conservation of energy. Adapting respiratory enzymes under nutrient stress indicated high metabolic flexibility and robust regulatory mechanisms in this archaeon. This study enhances our fundamental understanding of the metabolism of S. acidocaldarius.

A proteogenomic atlas of the human neural retina.

The human neural retina is a complex tissue with abundant alternative splicing and more than 10% of genetic variants linked to inherited retinal diseases (IRDs) alter splicing. Traditional short-read RNA-sequencing methods have been used for understanding retina-specific splicing but have limitations in detailing transcript isoforms. To address this, we generated a proteogenomic atlas that combines PacBio long-read RNA-sequencing data with mass spectrometry and whole genome sequencing data of three healthy human neural retina samples. We identified nearly 60,000 transcript isoforms, of which approximately one-third are novel. Additionally, ten novel peptides confirmed novel transcript isoforms. For instance, we identified a novel IMPDH1 isoform with a novel combination of known exons that is supported by peptide evidence. Our research underscores the potential of in-depth tissue-specific transcriptomic analysis to enhance our grasp of tissue-specific alternative splicing. The data underlying the proteogenomic atlas are available via EGA with identifier EGAD50000000101, via ProteomeXchange with identifier PXD045187, and accessible through the UCSC genome browser.

High-throughput single-cell screening of viable hybridomas and patient-derived antibody-secreting cells using punchable microwells.

Monoclonal antibodies (mAbs) hold significant potential as therapeutic agents and are invaluable tools in biomedical research. However, the lack of efficient high-throughput screening methods for single antibody-secreting cells (ASCs) has limited the diversity of available antibodies. Here, we introduce a novel, integrated workflow employing self-seeding microwells and an automated microscope-puncher system for the swift, high-throughput screening and isolation of single ASCs. The system allows for the individual screening and isolation of up to 6,400 cells within approximately one day, with the opportunity for parallelization and efficient upscaling. We successfully applied this workflow to both hybridomas and human patient-derived B cells, enabling subsequent clonal expansion or antibody sequence analysis through an optimized, single-cell nested reverse transcription-polymerase chain reaction (RT-PCR) procedure. By providing a time-efficient and more streamlined single ASC screening and isolation process, our workflow holds promise for driving forward progress in mAb development.

From follicle to blastocyst: microRNA-34c from follicular fluid-derived extracellular vesicles modulates blastocyst quality.

BACKGROUND: Within the follicular fluid, extracellular vesicles (EVs) guide oocyte growth through their cargo microRNAs (miRNAs). Here, we investigated the role of EVs and their cargo miRNAs by linking the miRNAs found in EVs, derived from the fluid of an individual follicle, to the ability of its oocyte to become a blastocyst (competent) or not (non-competent). METHODS: Bovine antral follicles were dissected, categorized as small (2-4 mm) or large (5-8 mm) and the corresponding oocytes were subjected to individual maturation, fertilization and embryo culture to the blastocyst stage. Follicular fluid was pooled in 4 groups (4 replicates) based on follicle size and competence of the corresponding oocyte to produce a blastocyst. Follicular fluid-derived EVs were isolated, characterized, and subjected to miRNA-sequencing (Illumina Miseq) to assess differential expression (DE) in the 4 groups. Functional validation of the effect of miR-34c on embryo development was performed by supplementation of mimics and inhibitors during in vitro maturation (IVM). RESULTS: We identified 16 DE miRNAs linked to oocyte competence when follicular size was not considered. Within the large and small follicles, 46 DE miRNAs were driving blastocyst formation in each group. Comparison of EVs from competent small and large follicles revealed 90 DE miRNAs. Cell regulation, cell differentiation, cell cycle, and metabolic process regulation were the most enriched pathways targeted by the DE miRNAs from competent oocytes. We identified bta-miR-34c as the most abundant in follicular fluid containing competent oocytes. Supplementation of miR-34c mimic and inhibitor during IVM did not affect embryo development. However, blastocyst quality, as evidenced by higher cell numbers, was significantly improved following oocyte IVM in the presence of miR-34c mimics, while miR-34c inhibitors resulted in the opposite effect. CONCLUSION: This study demonstrates the regulatory effect of miRNAs from follicular fluid-derived EVs on oocyte competence acquisition, providing a further basis for understanding the significance of miRNAs in oocyte maturation and embryonic development. Up-regulation of miR-34c in EVs from follicular fluid containing competent oocytes and the positive impact of miR-34c mimics added during IVM on the resulting blastocysts indicate its pivotal role in oocyte competence.

Average Nucleotide Identity and Digital DNA-DNA Hybridization Analysis Following PromethION Nanopore-Based Whole Genome Sequencing Allows for Accurate Prokaryotic Typing.

Whole-genome sequencing (WGS) is revolutionizing clinical bacteriology. However, bacterial typing remains investigated by reference techniques with inherent limitations. This stresses the need for alternative methods providing robust and accurate sequence type (ST) classification. This study optimized and evaluated a GridION nanopore sequencing protocol, adapted for the PromethION platform. Forty-eight Escherichia coli clinical isolates with diverse STs were sequenced to assess two alternative typing methods and resistance profiling applications. Multi-locus sequence typing (MLST) was used as the reference typing method. Genomic relatedness was assessed using Average Nucleotide Identity (ANI) and digital DNA-DNA Hybridization (DDH), and cut-offs for discriminative strain resolution were evaluated. WGS-based antibiotic resistance prediction was compared to reference Minimum Inhibitory Concentration (MIC) assays. We found ANI and DDH cut-offs of 99.3% and 94.1%, respectively, which correlated well with MLST classifications and demonstrated potentially higher discriminative resolution than MLST. WGS-based antibiotic resistance prediction showed categorical agreements of ≥ 93% with MIC assays for amoxicillin, ceftazidime, amikacin, tobramycin, and trimethoprim-sulfamethoxazole. Performance was suboptimal (68.8-81.3%) for amoxicillin-clavulanic acid, cefepime, aztreonam, and ciprofloxacin. A minimal sequencing coverage of 12× was required to maintain essential genomic features and typing accuracy. Our protocol allows the integration of PromethION technology in clinical laboratories, with ANI and DDH proving to be accurate and robust alternative typing methods, potentially offering superior resolution. WGS-based antibiotic resistance prediction holds promise for specific antibiotic classes.

Proteomics data in vitiligo: a scoping review.

An unbiased screening of which proteins are deregulated in vitiligo using proteomics can offer an enormous value. It could not only reveal robust biomarkers for detecting disease activity but can also identify which patients are most likely to respond to treatments. We performed a scoping review searching for all articles using proteomics in vitiligo. Eight manuscripts could be identified. Unfortunately, very limited overlap was found in the differentially expressed proteins between studies (15 out of 272; 5,51%) with variable degrees of the type of proteins and a substantial variety in the prevalence of acute phase proteins (range: 6-65%). Proteomics research has therefore brought little corroborating evidence on which proteins are differentially regulated between vitiligo patients and healthy controls or between active and stable vitiligo patients. While a limited patient size is an obvious weakness for several studies, an incomplete description of patient characteristics is an unfortunate and avoidable shortcoming. Additionally, the variations in the used methodology and analyses may further contribute to the overall observed variability. Nonetheless, more recent studies investigating the response to treatment seem to be more robust, as more differentially expressed proteins that have previously been confirmed to be involved in vitiligo were found. The further inclusion of proteomics analyses in clinical trials is recommended to increase insights into the pathogenic mechanisms in vitiligo and identify reliable biomarkers or promising drug targets. A harmonization in the study design, reporting and proteomics methodology could vastly improve the value of vitiligo proteomics research.

How temperature modulates the expression of pathogenesis-related molecules of the cross-kingdom pathogen Lasiodiplodia hormozganensis.

Lasiodiplodia hormozganensis, initially recognized as a fungal plant pathogen, is recognized now acknowledged as a potential threat to humans. However, our understanding of the pathogenesis mechanisms of Lasiodiplodia species remains limited, and the impact of temperature on its pathogenicity is unclear. This study aims to elucidate the effects of temperature on the biology of L. hormozganensis, focusing on the expression of pathogenesis-related molecules and its ability to function as a cross-kingdom pathogen. We conducted experiments at two different temperatures, 25 and 37 °C, analyzing the proteome and transcriptome of L. hormozganensis. Using strain CBS339.90, initially identified as L. theobromae but confirmed through ITS and tef1-α sequence analysis to be L. hormozganensis, we aimed to understand the fungus's protein expression under varying temperature conditions. Results from the functional analysis of the secretome at 25 °C showed a noteworthy presence of proteins related to carbohydrate metabolism, catabolism, plant cell wall degradation, and pathogenesis. However, when grown at 37 °C, the fungus exhibited an increased production of stress response and pathogenesis-related proteins. Our findings identified various pathways crucial for pathogenesis in both plants and humans, suggesting that L. hormozganensis possesses the genetic foundation to infect both hosts. Specific pathogenesis-related proteins, including the phytotoxin snodprot1, aspartic protease aspergillopepsin, and virulence protein SSD1, were also identified. Concluding, we propose a possible mechanism of how L. hormozganensis adapts to different temperatures. The shift in temperature results in the expression of genes that favor human related pathogenesis molecules.

Myeloid A20 is critical for alternative macrophage polarization and type-2 immune-mediated helminth resistance.

Background: Protective immunity against intestinal helminths requires induction of robust type-2 immunity orchestrated by various cellular and soluble effectors which promote goblet cell hyperplasia, mucus production, epithelial proliferation, and smooth muscle contractions to expel worms and re-establish immune homeostasis. Conversely, defects in type-2 immunity result in ineffective helminth clearance, persistent infection, and inflammation. Macrophages are highly plastic cells that acquire an alternatively activated state during helminth infection, but they were previously shown to be dispensable for resistance to Trichuris muris infection. Methods: We use the in vivo mouse model A20myel-KO, characterized by the deletion of the potent anti-inflammatory factor A20 (TNFAIP3) specifically in the myeloid cells, the excessive type-1 cytokine production, and the development of spontaneous arthritis. We infect A20myel-KO mice with the gastrointestinal helminth Trichuris muris and we analyzed the innate and adaptive responses. We performed RNA sequencing on sorted myeloid cells to investigate the role of A20 on macrophage polarization and type-2 immunity. Moreover, we assess in A20myel-KO mice the pharmacological inhibition of type-1 cytokine pathways on helminth clearance and the infection with Salmonella typhimurium. Results: We show that proper macrophage polarization is essential for helminth clearance, and we identify A20 as an essential myeloid factor for the induction of type-2 immune responses against Trichuris muris. A20myel-KO mice are characterized by persistent Trichuris muris infection and intestinal inflammation. Myeloid A20 deficiency induces strong classical macrophage polarization which impedes anti-helminth type-2 immune activation; however, it promotes detrimental Th1/Th17 responses. Antibody-mediated neutralization of the type-1 cytokines IFN-γ, IL-18, and IL-12 prevents myeloid-orchestrated Th1 polarization and re-establishes type-2-mediated protective immunity against T. muris in A20myel-KO mice. In contrast, the strong Th1-biased immunity in A20myel-KO mice offers protection against Salmonella typhimurium infection. Conclusions: We hereby identify A20 as a critical myeloid factor for correct macrophage polarization and appropriate adaptive mucosal immunity in response to helminth and enteric bacterial infection.

HES6 knockdown in human hematopoietic precursor cells reduces their in vivo engraftment potential and their capacity to differentiate into erythroid cells, B cells, T cells and plasmacytoid dendritic cells.

Hematopoiesis is driven by molecular mechanisms that induce differentiation and proliferation of hematopoietic stem cells and their progeny. This involves the activity of various transcription factors, such as members of the Hairy/Enhancer of Split (HES) family, and important roles for both HES1 and HES4 have been shown in normal and malignant hematopoiesis. Here, we investigated the role of HES6 in human hematopoiesis using in vitro and in vivo models. Using bulk and scRNA-seq data, we show that HES6 is expressed during erythroid/megakaryocyte and pDC development, as well as in multipotent precursors and at specific stages of T- and B-cell development following preBCR and preTCR signalling, respectively. Consistently, knockdown of HES6 in cord blood-derived hematopoietic precursors in well-defined in vitro differentiation assays resulted in reduced differentiation of human hematopoietic precursors towards megakaryocytes, erythrocytes, pDCs, Band T-cells. In addition, HES6 knockdown HSPCs displayed reduced colony forming unit capacity in vitro and impaired potential to reconstitute hematopoiesis in vivo in a competitive transplantation assay. We demonstrate that loss of HES6 expression impacts cell cycle progression during erythroid differentiation and provide evidence for potential downstream target genes that impact these perturbations. Thus, our study uncovers new insights for a role of HES6 in human hematopoiesis.

Myb overexpression synergizes with the loss of Pten and is a dependency factor and therapeutic target in T-cell lymphoblastic leukemia.

T-lineage acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 10%-15% of pediatric and 25% of adult ALL cases. Although the prognosis of T-ALL has improved over time, the outcome of T-ALL patients with primary resistant or relapsed leukemia remains poor. Therefore, further progress in the treatment of T-ALL requires a better understanding of its biology and the development of more effective precision oncologic therapies. The proto-oncogene MYB is highly expressed in diverse hematologic malignancies, including T-ALLs with genomic aberrations that further potentiate its expression and activity. Previous studies have associated MYB with a malignant role in the pathogenesis of several cancers. However, its role in the induction and maintenance of T-ALL remains relatively poorly understood. In this study, we found that an increased copy number of MYB is associated with higher MYB expression levels, and might be associated with inferior event-free survival of pediatric T-ALL patients. Using our previously described conditional Myb overexpression mice, we generated two distinct MYB-driven T-ALL mouse models. We demonstrated that the overexpression of Myb synergizes with Pten deletion but not with the overexpression of Lmo2 to accelerate the development of T-cell lymphoblastic leukemias. We also showed that MYB is a dependency factor in T-ALL since RNA interference of Myb blocked cell cycle progression and induced apoptosis in both human and murine T-ALL cell lines. Finally, we provide preclinical evidence that targeting the transcriptional activity of MYB can be a useful therapeutic strategy for the treatment of T-ALL.

Contribution of combined stressors on density and gene expression dynamics of the copepod Temora longicornis in the North Sea.

The impact of multiple environmental and anthropogenic stressors on the marine environment remains poorly understood. Therefore, we studied the contribution of environmental variables to the densities and gene expression of the dominant zooplankton species in the Belgian part of the North Sea, the calanoid copepod Temora longicornis. We observed a reduced density of copepods, which were also smaller in size, in samples taken from nearshore locations when compared to those obtained from offshore stations. To assess the factors influencing the population dynamics of this species, we applied generalised additive models. These models allowed us to quantify the relative contribution of temperature, nutrient levels, salinity, turbidity, concentrations of photosynthetic pigments, as well as chemical pollutants such as polychlorinated biphenyls and polycyclic aromatic hydrocarbons (PAHs), on copepod density. Temperature and Secchi depth, a proxy for turbidity, were the most important environmental variables predicting the densities of T. longicornis, followed by summed PAH and chlorophyll concentrations. Analysing gene expression in field-collected adults, we observed significant variation in metabolic and stress-response genes. Temperature correlated significantly with genes involved in proteolytic activities, and encoding heat shock proteins. Yet, concentrations of anthropogenic chemicals did not induce significant differences in the gene expression of genes involved in the copepod's fatty acid metabolism or well-known stress-related genes, such as glutathione transferases or cytochrome P450. Our study highlights the potential of gene expression biomonitoring and underscores the significance of a changing environment in future studies.

Genome-wide methylome stability and parental effects in the worldwide distributed Lombardy poplar.

BACKGROUND: Despite the increasing number of epigenomic studies in plants, little is known about the forces that shape the methylome in long-lived woody perennials. The Lombardy poplar offers an ideal opportunity to investigate the impact of the individual environmental history of trees on the methylome. RESULTS: We present the results of three interconnected experiments on Lombardy poplar. In the first experiment, we investigated methylome variability during a growing season and across vegetatively reproduced generations. We found that ramets collected over Europe and raised in common conditions have stable methylomes in symmetrical CG-contexts. In contrast, seasonal dynamics occurred in methylation patterns in CHH context. In the second experiment, we investigated whether methylome patterns of plants grown in a non-parental environment correlate with the parental climate. We did not observe a biological relevant pattern that significantly correlates with the parental climate. Finally, we investigated whether the parental environment has persistent carry-over effects on the vegetative offspring's phenotype. We combined new bud set observations of three consecutive growing seasons with former published bud set data. Using a linear mixed effects analysis, we found a statistically significant but weak short-term, parental carry-over effect on the timing of bud set. However, this effect was negligible compared to the direct effects of the offspring environment. CONCLUSIONS: Genome-wide cytosine methylation patterns in symmetrical CG-context are stable in Lombardy poplar and appear to be mainly the result of random processes. In this widespread poplar clone, methylation patterns in CG-context can be used as biomarkers to infer a common ancestor and thus to investigate the recent environmental history of a specific Lombardy poplar. The Lombardy poplar shows high phenotypic plasticity in a novel environment which enabled this clonal tree to adapt and survive all over the temperate regions of the world.

Baselining physiological parameters in three muscles across three equine breeds. What can we learn from the horse?

Mapping-out baseline physiological muscle parameters with their metabolic blueprint across multiple archetype equine breeds, will contribute to better understanding their functionality, even across species. Aims: 1) to map out and compare the baseline fiber type composition, fiber type and mean fiber cross-sectional area (fCSA, mfCSA) and metabolic blueprint of three muscles in 3 different breeds 2) to study possible associations between differences in histomorphological parameters and baseline metabolism. Methods: Muscle biopsies [m. pectoralis (PM), m. vastus lateralis (VL) and m. semitendinosus (ST)] were harvested of 7 untrained Friesians, 12 Standardbred and 4 Warmblood mares. Untargeted metabolomics was performed on the VL and PM of Friesian and Warmblood horses and the VL of Standardbreds using UHPLC/MS/MS and GC/MS. Breed effect on fiber type percentage and fCSA and mfCSA was tested with Kruskal-Wallis. Breeds were compared with Wilcoxon rank-sum test, with Bonferroni correction. Spearman correlation explored the association between the metabolic blueprint and morphometric parameters. Results: The ST was least and the VL most discriminative across breeds. In Standardbreds, a significantly higher proportion of type IIA fibers was represented in PM and VL. Friesians showed a significantly higher representation of type IIX fibers in the PM. No significant differences in fCSA were present across breeds. A significantly larger mfCSA was seen in the VL of Standardbreds. Lipid and nucleotide super pathways were significantly more upregulated in Friesians, with increased activity of short and medium-chain acylcarnitines together with increased abundance of long chain and polyunsaturated fatty acids. Standardbreds showed highly active xenobiotic pathways and high activity of long and very long chain acylcarnitines. Amino acid metabolism was similar across breeds, with branched and aromatic amino acid sub-pathways being highly active in Friesians. Carbohydrate, amino acid and nucleotide super pathways and carnitine metabolism showed higher activity in Warmbloods compared to Standardbreds. Conclusion: Results show important metabolic differences between equine breeds for lipid, amino acid, nucleotide and carbohydrate metabolism and in that order. Mapping the metabolic profile together with morphometric parameters provides trainers, owners and researchers with crucial information to develop future strategies with respect to customized training and dietary regimens to reach full potential in optimal welfare.

Osteopontin characterizes bile duct-associated macrophages and correlates with liver fibrosis severity in primary sclerosing cholangitis.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is an immune-mediated cholestatic liver disease for which pharmacological treatment options are currently unavailable. PSC is strongly associated with colitis and a disruption of the gut-liver axis, and macrophages are involved in the pathogenesis of PSC. However, how gut-liver interactions and specific macrophage populations contribute to PSC is incompletely understood. APPROACH AND RESULTS: We investigated the impact of cholestasis and colitis on the hepatic and colonic microenvironment, and performed an in-depth characterization of hepatic macrophage dynamics and function in models of concomitant cholangitis and colitis. Cholestasis-induced fibrosis was characterized by depletion of resident KCs, and enrichment of monocytes and monocyte-derived macrophages (MoMFs) in the liver. These MoMFs highly express triggering-receptor-expressed-on-myeloid-cells-2 ( Trem2 ) and osteopontin ( Spp1 ), markers assigned to hepatic bile duct-associated macrophages, and were enriched around the portal triad, which was confirmed in human PSC. Colitis induced monocyte/macrophage infiltration in the gut and liver, and enhanced cholestasis-induced MoMF- Trem2 and Spp1 upregulation, yet did not exacerbate liver fibrosis. Bone marrow chimeras showed that knockout of Spp1 in infiltrated MoMFs exacerbates inflammation in vivo and in vitro , while monoclonal antibody-mediated neutralization of SPP1 conferred protection in experimental PSC. In human PSC patients, serum osteopontin levels are elevated compared to control, and significantly increased in advanced stage PSC and might serve as a prognostic biomarker for liver transplant-free survival. CONCLUSIONS: Our data shed light on gut-liver axis perturbations and macrophage dynamics and function in PSC and highlight SPP1/OPN as a prognostic marker and future therapeutic target in PSC.

MicroRNA-146b negatively affects bovine embryo development and quality.

MicroRNAs (miRNAs), which can be carried inside extracellular vesicles (EVs), play a crucial role in regulating embryo development up to the blastocyst stage. Yet, the molecular mechanisms underlying blastocyst development and quality are largely unknown. Recently, our group identified 69 differentially expressed miRNAs in extracellular vesicles (EVs) isolated from culture medium conditioned by bovine embryos that either developed to the blastocyst stage or did not (non-blastocysts). We found miR-146b to be more abundant in the EVs derived from media conditioned by non-blastocyst embryos. Using RT-qPCR, we here confirmed the upregulation of miR-146b in non-blastocyst (arrested at 2-4 cell and morula stage) embryos compared to blastocysts (p<0.005), which coincides with the upregulation of miR-146b in EVs derived from the medium of these non-blastocysts. To evaluate a functional effect, bovine embryo culture media were supplemented with miR-146b mimics, resulting in significantly decreased embryo quality, with lower blastocyst rates at day 7 and lower total cell numbers, while the opposite was found after supplementation with miR-146b inhibitors, which resulted in reduced apoptosis rates (P < 0.01). Transcriptomic analysis of embryos treated with miR-146b mimics or inhibitors showed differential expression (P < 0.01) of genes associated with apoptosis, cell differentiation, and the RNA Pol II transcription complex, including WDR36, MBNL2, ERCC6l2, PYGO1, and SNIP1. Overall, miR-146b is overexpressed in non-blastocyst embryos and in EVs secreted by these embryos, and it regulates genes involved in embryo development and apoptosis, resulting in decreased embryo quality.

Correction: Sallam et al. DNA Methylation Alterations in Fractionally Irradiated Rats and Breast Cancer Patients Receiving Radiotherapy. Int. J. Mol. Sci. 2022, 23, 16214.

Radia Tamarat and Susana Constantino Rosa Santos were not included as authors in the original publication [...].

Myeloid OTULIN deficiency couples RIPK3-dependent cell death to Nlrp3 inflammasome activation and IL-1ß secretion.

Loss-of-function mutations in the deubiquitinase OTULIN result in an inflammatory pathology termed "OTULIN-related autoinflammatory syndrome" (ORAS). Genetic mouse models revealed essential roles for OTULIN in inflammatory and cell death signaling, but the mechanisms by which OTULIN deficiency connects cell death to inflammation remain unclear. Here, we identify OTULIN deficiency as a cellular condition that licenses RIPK3-mediated cell death in murine macrophages, leading to Nlrp3 inflammasome activation and subsequent IL-1ß secretion. OTULIN deficiency uncoupled Nlrp3 inflammasome activation from gasdermin D-mediated pyroptosis, instead allowing RIPK3-dependent cell death to act as an Nlrp3 inflammasome activator and mechanism for IL-1ß release. Accordingly, elevated serum IL-1ß levels in myeloid-specific OTULIN-deficient mice were diminished by deleting either Ripk3 or Nlrp3. These findings identify OTULIN as an inhibitor of RIPK3-mediated IL-1ß release in mice.

Targeted haplotyping in pharmacogenomics using Oxford Nanopore Technologies' adaptive sampling.

Pharmacogenomics (PGx) studies the impact of interindividual genomic variation on drug response, allowing the opportunity to tailor the dosing regimen for each patient. Current targeted PGx testing platforms are mainly based on microarray, polymerase chain reaction, or short-read sequencing. Despite demonstrating great value for the identification of single nucleotide variants (SNVs) and insertion/deletions (INDELs), these assays do not permit identification of large structural variants, nor do they allow unambiguous haplotype phasing for star-allele assignment. Here, we used Oxford Nanopore Technologies' adaptive sampling to enrich a panel of 1,036 genes with well-documented PGx relevance extracted from the Pharmacogenomics Knowledge Base (PharmGKB). By evaluating concordance with existing truth sets, we demonstrate accurate variant and star-allele calling for five Genome in a Bottle reference samples. We show that up to three samples can be multiplexed on one PromethION flow cell without a significant drop in variant calling performance, resulting in 99.35% and 99.84% recall and precision for the targeted variants, respectively. This work advances the use of nanopore sequencing in clinical PGx settings.

A spatial human thymus cell atlas mapped to a continuous tissue axis.

T cells develop from circulating precursors, which enter the thymus and migrate throughout specialised sub-compartments to support maturation and selection. This process starts already in early fetal development and is highly active until the involution of the thymus in adolescence. To map the micro-anatomical underpinnings of this process in pre- vs. post-natal states, we undertook a spatially resolved analysis and established a new quantitative morphological framework for the thymus, the Cortico-Medullary Axis. Using this axis in conjunction with the curation of a multimodal single-cell, spatial transcriptomics and high-resolution multiplex imaging atlas, we show that canonical thymocyte trajectories and thymic epithelial cells are highly organised and fully established by post-conception week 12, pinpoint TEC progenitor states, find that TEC subsets and peripheral tissue genes are associated with Hassall's Corpuscles and uncover divergence in the pace and drivers of medullary entry between CD4 vs. CD8 T cell lineages. These findings are complemented with a holistic toolkit for spatial analysis and annotation, providing a basis for a detailed understanding of T lymphocyte development.

HIV-PULSE: a long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization.

A deep understanding of the composition of the HIV-1 reservoir is necessary for the development of targeted therapies and the evaluation of curative efforts. However, current near full-length (NFL) HIV-1 proviral genome sequencing assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. To address this, we developed a high-throughput, long-read sequencing assay called HIV-PULSE (HIV Proviral UMI-mediated Long-read Sequencing). This assay uses unique molecular identifiers (UMIs) to tag individual HIV-1 genomes, allowing for the omission of the limiting dilution step and enabling long-range PCR amplification of many NFL genomes in a single PCR reaction, while simultaneously overcoming poor single-read accuracy. We optimized the assay using HIV-infected cell lines and then applied it to blood samples from 18 individuals living with HIV on antiretroviral therapy, yielding a total of 1308 distinct HIV-1 genomes. Benchmarking against the widely applied Full-Length Individual Proviral Sequencing assay revealed similar sensitivity (11 vs 18%) and overall good concordance, although at a significantly higher throughput. In conclusion, HIV-PULSE is a cost-efficient and scalable assay that allows for the characterization of the HIV-1 proviral landscape, making it an attractive method to study the HIV-1 reservoir composition and dynamics.