Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis.

Oxylipins and metabolites from pyroptotic cells act as promoters of tissue repair.

Pyroptosis is a lytic cell death mode that helps limit the spread of infections and is also linked to pathology in sterile inflammatory diseases and autoimmune diseases1-4. During pyroptosis, inflammasome activation and the engagement of caspase-1 lead to cell death, along with the maturation and secretion of the inflammatory cytokine interleukin-1ß (IL-1ß). The dominant effect of IL-1ß in promoting tissue inflammation has clouded the potential influence of other factors released from pyroptotic cells. Here, using a system in which macrophages are induced to undergo pyroptosis without IL-1ß or IL-1α release (denoted Pyro-1), we identify unexpected beneficial effects of the Pyro-1 secretome. First, we noted that the Pyro-1 supernatants upregulated gene signatures linked to migration, cellular proliferation and wound healing. Consistent with this gene signature, Pyro-1 supernatants boosted migration of primary fibroblasts and macrophages, and promoted faster wound closure in vitro and improved tissue repair in vivo. In mechanistic studies, lipidomics and metabolomics of the Pyro-1 supernatants identified the presence of both oxylipins and metabolites, linking them to pro-wound-healing effects. Focusing specifically on the oxylipin prostaglandin E2 (PGE2), we find that its synthesis is induced de novo during pyroptosis, downstream of caspase-1 activation and cyclooxygenase-2 activity; further, PGE2 synthesis occurs late in pyroptosis, with its release dependent on gasdermin D pores opened during pyroptosis. As for the pyroptotic metabolites, they link to immune cell infiltration into the wounds, and polarization to CD301+ macrophages. Collectively, these data advance the concept that the pyroptotic secretome possesses oxylipins and metabolites with tissue repair properties that may be harnessed therapeutically.

Microbes exploit death-induced nutrient release by gut epithelial cells.

Regulated cell death is an integral part of life, and has broad effects on organism development and homeostasis1. Malfunctions within the regulated cell death process, including the clearance of dying cells, can manifest in diverse pathologies throughout various tissues including the gastrointestinal tract2. A long appreciated, yet elusively defined relationship exists between cell death and gastrointestinal pathologies with an underlying microbial component3-6, but the direct effect of dying mammalian cells on bacterial growth is unclear. Here we advance a concept that several Enterobacteriaceae, including patient-derived clinical isolates, have an efficient growth strategy to exploit soluble factors that are released from dying gut epithelial cells. Mammalian nutrients released after caspase-3/7-dependent apoptosis boosts the growth of multiple Enterobacteriaceae and is observed using primary mouse colonic tissue, mouse and human cell lines, several apoptotic triggers, and in conventional as well as germ-free mice in vivo. The mammalian cell death nutrients induce a core transcriptional response in pathogenic Salmonella, and we identify the pyruvate formate-lyase-encoding pflB gene as a key driver of bacterial colonization in three contexts: a foodborne infection model, a TNF- and A20-dependent cell death model, and a chemotherapy-induced mucositis model. These findings introduce a new layer to the complex host-pathogen interaction, in which death-induced nutrient release acts as a source of fuel for intestinal bacteria, with implications for gut inflammation and cytotoxic chemotherapy treatment.

Phase separation-based visualization of protein-protein interactions and kinase activities in plants.

Protein activities depend heavily on protein complex formation and dynamic posttranslational modifications, such as phosphorylation. The dynamic nature of protein complex formation and posttranslational modifications is notoriously difficult to monitor in planta at cellular resolution, often requiring extensive optimization. Here, we generated and exploited the SYnthetic Multivalency in PLants (SYMPL)-vector set to assay protein-protein interactions (PPIs) (separation of phases-based protein interaction reporter) and kinase activities (separation of phases-based activity reporter of kinase) in planta, based on phase separation. This technology enabled easy detection of inducible, binary and ternary PPIs among cytoplasmic and nuclear proteins in plant cells via a robust image-based readout. Moreover, we applied the SYMPL toolbox to develop an in vivo reporter for SNF1-related kinase 1 activity, allowing us to visualize tissue-specific, dynamic SnRK1 activity in stable transgenic Arabidopsis (Arabidopsis thaliana) plants. The SYMPL cloning toolbox provides a means to explore PPIs, phosphorylation, and other posttranslational modifications with unprecedented ease and sensitivity.

GSDMD drives canonical inflammasome-induced neutrophil pyroptosis and is dispensable for NETosis.

Neutrophils are the most prevalent immune cells in circulation, but the repertoire of canonical inflammasomes in neutrophils and their respective involvement in neutrophil IL-1ß secretion and neutrophil cell death remain unclear. Here, we show that neutrophil-targeted expression of the disease-associated gain-of-function Nlrp3A350V mutant suffices for systemic autoinflammatory disease and tissue pathology in vivo. We confirm the activity of the canonical NLRP3 and NLRC4 inflammasomes in neutrophils, and further show that the NLRP1b, Pyrin and AIM2 inflammasomes also promote maturation and secretion of interleukin (IL)-1ß in cultured bone marrow neutrophils. Notably, all tested canonical inflammasomes promote GSDMD cleavage in neutrophils, and canonical inflammasome-induced pyroptosis and secretion of mature IL-1ß are blunted in GSDMD-knockout neutrophils. In contrast, GSDMD is dispensable for PMA-induced NETosis. We also show that Salmonella Typhimurium-induced pyroptosis is markedly increased in Nox2/Gp91Phox -deficient neutrophils that lack NADPH oxidase activity and are defective in PMA-induced NETosis. In conclusion, we establish the canonical inflammasome repertoire in neutrophils and identify differential roles for GSDMD and the NADPH complex in canonical inflammasome-induced neutrophil pyroptosis and mitogen-induced NETosis, respectively.

Factors associated with diet quality among Brazilian individuals with cardiovascular diseases.

BACKGROUND: An individual's dietary pattern contributes in different ways to the prevention and control of recurrent cardiovascular events. However, the quality of the diet is influenced by several factors. The present study aimed to evaluate the quality of the diet of individuals with cardiovascular diseases and determine whether there is an association between sociodemographic and lifestyle factors. METHODS: This is a cross-sectional study carried out with individuals with atherosclerosis (coronary artery disease, cerebrovascular disease or peripheral arterial disease) recruited from 35 reference centres for the treatment of cardiovascular disease in Brazil. Diet quality was assessed according to the Modified Alternative Healthy Eating Index (mAHEI) and stratified into tertiles. For comparing two groups, the Mann-Whitney or Pearson's chi-squared tests were used. However, for comparing three or more groups, analysis of variance or Kruskal-Wallis was used. For the confounding analysis, a multinomial regression model was used. p < 0.05 was considered statistically significant. RESULTS: In total, 2360 individuals were evaluated: 58.5% male and 64.2% elderly. The median (interquartile range [IQR]) of the mAHEI was 24.0 (20.0-30.0), ranging from 0.4 to 56.0 points. When comparing the odds ratios (ORs) for the low (first tertile) and medium (second tertile) diet quality groups with the high-quality group (third tertile), it was observed that there was an association between diet quality with a family income of 1.885 (95% confidence intervals [CI] = 1.302-2.729) and 1.566 (95% CI = 1.097-2.235), as well as physical activity of 1.391 (95% CI = 1.107-1.749) and 1.346 (95% CI = 1.086-1.667), respectively. In addition, associations were observed between diet quality and region of residence. CONCLUSIONS: A low-quality diet was associated with family income, sedentarism and geographical area. These data are extremely relevant to assist in coping with cardiovascular disease because they enable an assessment of the distribution of these factors in different regions of the country.

Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations.

BACKGROUND: Severe acute respiratory syndrome coronavirus (SARS-CoV-2) omicron variant was first detected in South Africa in November 2021. Since then, the number of cases due to this variant increases enormously every day in different parts of the world. Mutations within omicron genome may impair the molecular detection resulting in false negative results during Coronavirus disease 19 (COVID-19) diagnosis. OBJECTIVES: To verify if colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting N and E genes would work efficiently to detect omicron SARS-CoV-2 variant and its sub-lineages. METHODS: SARS-CoV-2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) positive samples were sequenced by next generation DNA sequencing. The consensus sequences generated were submitted to Pangolin tool for SARS-CoV-2 lineage identification. RT-LAMP reactions were performed at 65ºC/30 min targeting N and E. FINDINGS: SARS-CoV-2 omicron can be detected by RT-LAMP targeting N and E genes despite the genomic mutation of this more transmissible lineage. Omicron SARS-CoV-2 sub-lineages were tested and efficiently detected by RT-LAMP. We demonstrated that this test is very sensitive in detecting omicron variant, with LoD as low as 0.4 copies/µL. MAIN CONCLUSIONS: Molecular detection of omicron SARS-CoV-2 variant and its sub-lineages can be achieved by RT-LAMP despite the genomic mutations as a very sensitive surveillance tool for COVID-19 molecular diagnosis.

Perceptions, feelings, and the routine of older adults during the isolation period caused by the COVID-19 pandemic: a qualitative study in four countries.

OBJECTIVE: To evaluate the knowledge, routine, and perception of older adults from four countries about dealing with COVID-19 in the social isolation period. METHODS: Qualitative study with semistructured interviews. Older persons (≥60 years old), who lived in the urban centers of four different countries (Brazil, United States, Italy, and Portugal), were asked about the changes experienced during the pandemic, new habits or restrictions during isolation, sources of social and emotional support, and their knowledge about COVID-19 pandemic. Data was transcribed, codified, and submitted to content analysis. RESULTS: Twenty-five older persons (10 from Brazil, 5 from Italy, 5 from Portugal, and 5 from the United States) were interviewed. Participants reported feeling restricted in their daily life activities and emotional instability. Described adaptations in habits, coping strategies, and greater understanding of the diseased based on information available in the media. There was homogeneity in the statements of the elderly, showing that the pandemic affected them in a similar way, even though they lived in different cultures and contexts. CONCLUSION: The social isolation caused by the COVID-19 pandemic changed the structure for the performance of many occupations, having an impact in the perception social participation and wellbeing of elders. This data can aid health professionals to outline strategies to deal with the impact of the social isolation in older persons.

Frequency and experience in the use of menstrual cycle monitoring applications by Brazilian women.

OBJECTIVES: This study aimed to evaluate frequency and experience in the use of menstrual cycle monitoring applications (apps) by Brazilian women. METHODS: A cross-sectional study was performed through an online survey, announced in social media women's groups, among menstruating Brazilian women aged ≥18 years. The instrument collected sociodemographic, sexual, menstrual and technology usage data of all the women who agreed to participate. RESULTS: Of the 1160 participants, 71.2% used a menstrual cycle monitoring app. The principal motivation for using menstrual cycle apps was to track the menstrual cycle (94.3%), followed by pregnancy avoidance (49.5%). Users rated the apps with a mean 4.4 (standard deviation 0.65) stars out of five. There was a greater likelihood of using an app among women who used behavioural contraceptive methods (odds ratio [OR] 1.8; 95% confidence interval [CI] 1.2, 2.7; p = 0.01), barrier methods (OR 1.6; 95% CI 1.1, 2.5; p = 0.02) and copper- or silver/copper-bearing intrauterine devices (IUDs) (OR 1.9; 95% CI 1.1, 3.5; p = 0.04) and a lower likelihood among women who used hormonal contraception (OR 0.5; 95% CI 0.3, 0.8; p = 0.00) and permanent contraception (OR 0.1; 95% CI 0.0, 0.4; p = 0.00). CONCLUSION: The use of menstrual cycle monitoring apps was quite widespread in the studied group. Satisfaction with app use was considered adequate. The use of menstrual cycle apps was associated with the use of behavioural contraceptive and barrier methods as well as IUDs.

RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis.

Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.