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Human islets of Langerhans are composed mostly of glucagon-secreting α cells and insulin-secreting ß cells closely intermingled one another. Current methods for identifying α and ß cells involve either fixing islets and using immunostaining or disaggregating islets and employing flow cytometry for classifying α and ß cells based on their size and autofluorescence. Neither approach, however, allows investigating the dynamic behavior of α and ß cells in a living and intact islet. To tackle this issue, we present a machine-learning-based strategy for identification α and ß cells in label-free infrared micrographs of living human islets without immunostaining. Intrinsic autofluorescence is stimulated by infrared light and collected both in intensity and lifetime in the visible range, dominated by NAD(P)H and lipofuscin signals. Descriptive parameters are derived from micrographs for ~ 103 cells. These parameters are used as input for a boosted decision-tree model (XGBoost) pre-trained with immunofluorescence-derived cell-type information. The model displays an optimized-metrics performance of 0.86 (i.e. area under a ROC curve), with an associated precision of 0.94 for the recognition of ß cells and 0.75 for α cells. This tool promises to enable longitudinal studies on the dynamic behavior of individual cell types at single-cell resolution within the intact tissue.
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Células Secretoras de Insulina , Aprendizaje Automático , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Glucagón/metabolismo , Islotes Pancreáticos/metabolismo , Rayos InfrarrojosRESUMEN
Multiomic droplet-based technologies allow different molecular modalities, such as chromatin accessibility and gene expression (scATAC-seq and scRNA-seq), to be probed in the same nucleus. We develop EmptyDropsMultiome, an approach that distinguishes true nuclei-containing droplets from background. Using simulations, we show that EmptyDropsMultiome has higher statistical power and accuracy than existing approaches, including CellRanger-arc and EmptyDrops. On real datasets, we observe that CellRanger-arc misses more than half of the nuclei identified by EmptyDropsMultiome and, moreover, is biased against certain cell types, some of which have a retrieval rate lower than 20%.
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Análisis de la Célula Individual , Análisis de la Célula Individual/métodos , Humanos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cromatina/genética , MultiómicaRESUMEN
Antimicrobial use (AMU) in the livestock sector is a major driver of antimicrobial resistance. Italian beef industry strongly relies on the import of young cattle from France, which are commingled in sorting facilities before transportation to Italy. Both commingling and transportation are stressors for animals and lead to higher risk of bovine respiratory disease (BRD), which in turn increases the risk of AMU. This study aimed to investigate how the timing of first BRD vaccination and the different vaccination target pathogens affect AMU and performance of young Charolais beef cattle imported from France to Italy. Information on animal performance, antimicrobial treatments, and vaccinations was available for 60,726 Charolais cattle belonging to 1449 batches in 33 Italian specialised fattening farms between January 2016 and December 2021. Antimicrobial use was estimated using the treatment incidence 100 adapted for Italy (TI100it). A mixed linear model was used to quantify the effects of the vaccination and the time of first administration on slaughter age, carcase weight, and average daily carcase gain. Similarly, a generalised linear mixed model was used to analyse the TI100it. The vaccination programme was usually applied the first day after the animals' arrival to the Italian fattening farms. Most animals were vaccinated with a polyvalent vaccine against infectious bovine rhinotracheitis (IBR), bovine parainfluenza type 3 virus (PI-3), bovine viral diarrhoea virus type 1 and 2 (BVDV), and bovine respiratory syncytial virus (BRSV). The most used class of antimicrobials to treat BRD were the macrolides, followed by aminoglycosides, amphenicols, tetracyclines, aminopenicillins, and fluoroquinolones. Animals that got vaccinated against any of the considered BRD pathogens upon arrival had significantly lower TI100it, greater average daily carcase gain, and reached slaughter age earlier than animals that got vaccinated later. Animals that received the vaccination against BVDV had lower TI100it and greater average daily carcase gain, and animals that received the vaccination against BRSV were younger at slaughter than unvaccinated animals. The vaccination against Mannheimia haemolytica significantly decreased the slaughter age and increased the carcase weight and average daily carcase gain, and the vaccination against PI-3 and Histophilus somni significantly increased the slaughter age. Thus, even if the vaccination programme is essential to tackle BRD, this practice is questionable if applied at arrival to the Italian fattening farms and it is advisable that the vaccination programme is planned before the commingling procedure in France.
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Enfermedades de los Bovinos , Enfermedades Respiratorias , Animales , Bovinos , Estudios Retrospectivos , Vacunación/veterinaria , Enfermedades de los Bovinos/epidemiología , Enfermedades Respiratorias/veterinaria , Antibacterianos , FranciaRESUMEN
Information on slaughter procedures for farmed fish in aquaculture is limited, both in Europe and in Italy, due to a general lack of field data. The aim of this study was to gather information on the procedures used to slaughter fish in Italy and to discuss them considering the WOAH and EFSA recommendations on fish welfare. Using a questionnaire survey, data were collected by official veterinarians in 64 slaughtering facilities where 20 different species of fish were slaughtered. The main species slaughtered were rainbow trout (Oncorhynchus mykiss; 29/64), followed by European sea bass (Dicentrarchus labrax; 21/64), sea bream (Sparus aurata; 21/64), Arctic char (Salvelinus alpinus; 14/64), European eel (Anguilla anguilla; 11/64), sturgeon (Acipenser spp; 11/64), common carp (Cyprinus carpio; 6/64), and brown trout (Salmo trutta fario L.; 5/64). The most applied stunning/killing methods were "asphyxia in ice/thermal shock" and "electric in water bath," followed by "percussion," "asphyxia in air," and "electric dry system." After the application of the method, the assessment of the fish level of unconsciousness was practiced in 72% of the facilities using more than one indicator, with "breathing" and "coordinated movements" the most practiced. The collected data showed a discrepancy between the available recommendations about the welfare of fish at slaughter and what is practiced in many production sites, but for many species precise recommendations are still not available.
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Germline commitment following primordial germ cell (PGC) specification during early human development establishes an epigenetic programme and competence for gametogenesis. Here we follow the progression of nascent PGC-like cells derived from human embryonic stem cells in vitro. We show that switching from BMP signalling for PGC specification to Activin A and retinoic acid resulted in DMRT1 and CDH5 expression, the indicators of migratory PGCs in vivo. Moreover, the induction of DMRT1 and SOX17 in PGC-like cells promoted epigenetic resetting with striking global enrichment of 5-hydroxymethylcytosine and locus-specific loss of 5-methylcytosine at DMRT1 binding sites and the expression of DAZL representing DNA methylation-sensitive genes, a hallmark of the germline commitment programme. We provide insight into the unique role of DMRT1 in germline development for advances in human germ cell biology and in vitro gametogenesis.
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Metilación de ADN , Células Madre Embrionarias Humanas , Humanos , Diferenciación Celular/genética , Células Germinativas/metabolismo , Transducción de SeñalRESUMEN
The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.
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Desarrollo Embrionario , Saco Vitelino , Femenino , Humanos , Embarazo , Coagulación Sanguínea/genética , Macrófagos , Saco Vitelino/citología , Saco Vitelino/metabolismo , Desarrollo Embrionario/genética , Atlas como Asunto , Expresión Génica , Perfilación de la Expresión Génica , Hematopoyesis/genética , Hígado/embriologíaRESUMEN
Pro-inflammatory cytokines contribute to ß-cell failure in both Type-1 and Type-2 Diabetes. Data collected so far allowed to dissect the genomic, transcriptomic, proteomic and biochemical landscape underlying cytokine-induced ß-cell progression through dysfunction. Yet, no report thus far complemented such molecular information with the direct optical nanoscopy of the ß-cell subcellular environment. Here we tackle this issue in Insulinoma 1E (INS-1E) ß-cells by label-free fluorescence lifetime imaging microscopy (FLIM) and fluorescence-based super resolution imaging by expansion microscopy (ExM). It is found that 24-h exposure to IL-1ß and IFN-γ is associated with a neat modification of the FLIM signature of cell autofluorescence due to the increase of either enzyme-bound NAD(P)H molecules and of oxidized lipid species. At the same time, ExM-based direct imaging unveils neat alteration of mitochondrial morphology (i.e. ~ 80% increase of mitochondrial circularity), marked degranulation (i.e. ~ 40% loss of insulin granules, with mis-localization of the surviving pool), appearance of F-actin-positive membrane blebs and an hitherto unknown extensive fragmentation of the microtubules network (e.g. ~ 37% reduction in the number of branches). Reported observations provide an optical-microscopy framework to interpret the amount of molecular information collected so far on ß-cell dysfunction and pave the way to future ex-vivo and in-vivo investigations.
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Neoplasias Pancreáticas , Proteómica , Humanos , Citoesqueleto de Actina , Citocinas , Microscopía FluorescenteRESUMEN
PEGylated lipid nanoparticles (LNPs) are commonly used to deliver bioactive molecules, but the role of PEGylation in DNA-loaded LNP interactions at the cellular and subcellular levels remains poorly understood. In this study, we investigated the mechanism of action of DNA-loaded PEGylated LNPs using gene reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), and fluorescence confocal microscopy (FCS). We found that PEG has no significant impact on the size or nanostructure of DNA LNPs but reduces their zeta potential and interaction with anionic cell membranes. PEGylation increases the structural stability of LNPs and results in lower DNA unloading. FCS experiments revealed that PEGylated LNPs are internalized intact inside cells and largely shuttled to lysosomes, while unPEGylated LNPs undergo massive destabilization on the plasma membrane. These findings can inform the design, optimization, and validation of DNA-loaded LNPs for gene delivery and vaccine development.
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Lípidos , Nanopartículas , Lípidos/química , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Nanopartículas/química , ADN , Polietilenglicoles/química , ARN Interferente PequeñoRESUMEN
Paratuberculosis is a notable infectious disease of ruminants. Goats appear to be particularly susceptible. The survey aimed to investigate the spread of paratuberculosis in Italian goat farming and evaluate whether the presence of the disease could be influenced by welfare and biosecurity deficiencies. A serological survey for paratuberculosis in 33 dairy farms in northern Italy was conducted. Contextually, animal welfare and biosecurity were assessed, using a standardized protocol of 36 welfare indicators and 15 biosecurity indicators which assigns to each farm a welfare and biosecurity score from 0 (any application) to 100% (full application). An overall result of less than 60% was considered insufficient. Nineteen farms (58%) tested positive for paratuberculosis, with a mean intra-herd seroprevalence of 7.4%. Total welfare ranged from 39.56 to 90.7% (mean 68.64%). Biosecurity scores ranged from 10.04 to 90.01% (mean 57.57%). Eight farms (24%) showed poor welfare conditions (welfare score < 60%) and 19 (58%) an unsatisfactory biosecurity condition (biosecurity score < 60%). With respect to the explorative character of the study, an indicative association between seven welfare and biosecurity indicators and paratuberculosis seropositivity was identified. The presence of paratuberculosis in northern Italy dairy goat farms was confirmed. The welfare and biosecurity assessment protocol proved to be an accurate tool, capable of identifying critical points for managing health, welfare and productivity.
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Contamination levels of dioxins and polychlorinated biphenyls (PCBs) were monitored over 2018-2021 in 214 bovine milk samples from farms located in two regions in northern Italy (Lombardy and Emilia-Romagna). The average concentrations of the sum of dioxins and dioxin-like PCBs (0.78 ± 0.55 pg TEQ/g fat) and six non-dioxin-like PCBs (6.55 ± 2.24 ng/g fat) were largely below the maximum, and action limits established at European level, confirming a decreasing trend observed both locally and across Europe in recent years. The impact of contamination levels on chronic dietary exposure of the Italian population to dioxins and PCBs was found to be highly variable based on the type of cow milk (skimmed, semi-skimmed, or whole-fat milk) and the population age group considered. Indeed, a first-tier screening of the potential exposure via determinist methods allowed for the identification of the youngest population as the group with the worst risk profile. The refinement of exposure assessment via Monte Carlo probabilistic methods suggested that, at the less pessimistic middle-bound simulation scenario, infants, toddlers, and children consuming whole cow milk may be exposed to dioxins and PCBs levels above the toxicological reference values with a probability of 76, 56, and 22%, respectively.
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Complete animal welfare evaluation in intensive farming is challenging. With this study, we investigate new biomarkers for animal physical and mental health by comparing plasma expression of biochemical indicators in dairy cows reared in three different systems: (A) semi-intensive free-stall, (B) non-intensive tie-stall, and (C) intensive free-stall. Additionally, protein levels of mature brain-derived neurotrophic factor (mBDNF) and its precursor form (proBDNF) and indoleamine 2,3-dioxygenase (IDO1) specific activity were evaluated in brain samples collected from 12 cattle culled between 73 and 138 months of age. Alterations in plasma lipid composition and in the kynurenine pathway of tryptophan metabolism were observed in the tie-stall-reared animals. The total plasma BDNF concentration was higher in tie-stall group compared to the two free-housing groups. Brain analysis of the tie-stall animals revealed a different mBDNF/proBDNF ratio, with a higher level of proBDNF (p < 0.001). Our data are similar to previous studies on animal models of depression, which reported that inhibition of the conversion of proBDNF in its mature form and/or elevated peripheral kynurenine pathway activation may underlie cerebral biochemical changes and induce depressive-like state behavior in animals.
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On-farm welfare assessment gives the opportunity to monitor and improve the quality of the animal life on the farm. In order to build the first Italian public standard for the welfare of indoor reared beef cattle, a list of 25 potential hazards and 22 potential promoters of beef cattle welfare was judged by a group of national experts by taking into account their negative or positive impacts on the welfare-state of the target population. In addition, the experts were asked to rank a list of 11 animal-based measures for identifying the most appropriate and important for measuring negative welfare outcomes in beef cattle. Based on experts' ratings, an "impact score" (ISoverall) was calculated for the proposed measures. Management hazards and promoters were ranked to have a greater impact on beef cattle welfare than housing factors. Keeping cattle in large (≥ 40 animals) and heterogeneous groups obtained the highest ISoverall among the proposed hazards (ISoverall = 5.54), followed by the presence of animals without free access to drinking water (ISoverall = 4.39) and the use of high-concentrate corn silage diets (concentrate > 80% and fiber < 6%) (ISoverall = 4.39). On the other hand, housing animals in small (≤ 20 animals) and homogeneous groups (ISoverall = 5.41), checking them at least twice a day (ISoverall = 4.62) and rearing cattle in loose housing systems with access to an outdoor area/pasture (ISoverall = 4.27) were ranked among the top 3 promoters. Concerning animal-based measures, experts scored lameness, severe respiratory diseases, body condition scoring and mortality rate to be measures most important for assessing serious welfare impairment.
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Enfermedades de los Bovinos , Industria Lechera , Animales , Bovinos , Granjas , Bienestar del Animal , Italia , Vivienda para Animales , Enfermedades de los Bovinos/epidemiologíaRESUMEN
Lipid nanoparticles (LNPs) are currently having an increasing impact on nanomedicines as delivery agents, among others, of RNA molecules (e.g., short interfering RNA for the treatment of hereditary diseases or messenger RNA for the development of COVID-19 vaccines). Despite this, the delivery of plasmid DNA (pDNA) by LNPs in preclinical studies is still unsatisfactory, mainly due to the lack of systematic structural and functional studies on DNA-loaded LNPs. To tackle this issue, we developed, characterized, and tested a library of 16 multicomponent DNA-loaded LNPs which were prepared by microfluidics and differed in lipid composition, surface functionalization, and manufacturing factors. 8 out of 16 formulations exhibited proper size and zeta potential and passed to the validation step, that is, the simultaneous quantification of transfection efficiency and cell viability in human embryonic kidney cells (HEK-293). The most efficient formulation (LNP15) was then successfully validated both in vitro, in an immortalized adult keratinocyte cell line (HaCaT) and in an epidermoid cervical cancer cell line (CaSki), and in vivo as a nanocarrier to deliver a cancer vaccine against the benchmark target tyrosine-kinase receptor HER2 in C57BL/6 mice. Finally, by a combination of confocal microscopy, transmission electron microscopy and synchrotron small-angle X-ray scattering, we were able to show that the superior efficiency of LNP15 can be linked to its disordered nanostructure consisting of small-size unoriented layers of pDNA sandwiched between closely apposed lipid membranes that undergo massive destabilization upon interaction with cellular lipids. Our results provide new insights into the structure-activity relationship of pDNA-loaded LNPs and pave the way to the clinical translation of this gene delivery technology.
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COVID-19 , Nanopartículas , Animales , Ratones , Humanos , Vacunas contra la COVID-19 , Células HEK293 , Lípidos/química , Ratones Endogámicos C57BL , ADN/química , Nanopartículas/química , ARN Interferente PequeñoRESUMEN
Here we use a combination of two-photon Fluorescence Lifetime Imaging Microscopy (FLIM) of NAD(P)H free/bound ratio in living HIs with post-fixation, immunofluorescence-based, cell-type identification. FLIM allowed to measure variations in the NAD(P)H free/bound ratio induced by glucose; immunofluorescence data allowed to identify single α and ß cells; finally, matching of the two datasets allowed to assign metabolic shifts to cell identity. 312 α and 654 ß cells from a cohort of 4 healthy donors, 15 total islets, were measured. Both α and ß cells display a wide spectrum of responses, towards either an increase or a decrease in NAD(P)H free/bound ratio. Yet, if single-cell data are averaged according to the respective donor and correlated to donor insulin secretion power, a non-random distribution of metabolic shifts emerges: robust average responses of both α and ß cells towards an increase of enzyme-bound NAD(P)H belong to the donor with the lowest insulin-secretion power; by contrast, discordant responses, with α cells shifting towards an increase of free NAD(P)H and ß cells towards an increase of enzyme-bound NAD(P)H, correspond to the donor with the highest insulin-secretion power. Overall, data reveal neat anti-correlation of tissue metabolic responses with respect to tissue insulin secretion power.
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Glucosa , Islotes Pancreáticos , Humanos , Glucosa/metabolismo , NAD/metabolismo , NADP/metabolismo , Islotes Pancreáticos/metabolismo , Insulina/metabolismoRESUMEN
Here we provide demonstration that fast fluorescence fluctuation spectroscopy is a fast and robust approach to extract information on the dynamics of molecules enclosed within subcellular nanostructures (e.g., organelles or vesicles) which are also moving in the complex cellular environment. In more detail, Raster Image Correlation Spectroscopy (RICS) performed at fast timescales (i.e., microseconds) reveals the fast motion of fluorescently labeled molecules within two exemplary dynamic subcellular nanostructures of biomedical interest, the lysosome and the insulin secretory granule (ISG). The measurement of molecular diffusion is then used to extract information on the average properties of subcellular nanostructures, such as macromolecular crowding or molecular aggregation. Concerning the lysosome, fast RICS on a fluorescent tracer allowed us to quantitatively assess the increase in organelle viscosity in the pathological condition of Krabbe disease. In the case of ISGs, fast RICS on two ISG-specific secreting peptides unveiled their differential aggregation propensity depending on intragranular concentration. Finally, a combination of fast RICS and feedback-based 3D orbital tracking was used to subtract the slow movement of subcellular nanostructures from the fast diffusion of molecules contained within them and independently validate the results. Results presented here not only demonstrate the acquired ability to address the dynamic behavior of molecules in moving, nanoscopic reference systems, but prove the relevance of this approach to advance our knowledge on cell function at the subcellular scale.
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Nanoestructuras , Transporte Biológico , Difusión , Movimiento (Física) , Espectrometría de Fluorescencia/métodosRESUMEN
Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.
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Linaje de la Célula , Células Germinativas , Ovario , Diferenciación Sexual , Análisis de la Célula Individual , Testículo , Animales , Cromatina/genética , Cromatina/metabolismo , Femenino , Células Germinativas/citología , Células Germinativas/metabolismo , Células de la Granulosa/citología , Células de la Granulosa/metabolismo , Humanos , Inmunoglobulinas , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana , Proteínas de la Membrana , Ratones , Microscopía Fluorescente , Ovario/citología , Ovario/embriología , Factor de Transcripción PAX8 , Embarazo , Primer Trimestre del Embarazo , Segundo Trimestre del Embarazo , Receptores Inmunológicos , Diferenciación Sexual/genética , Testículo/citología , Testículo/embriología , TranscriptomaRESUMEN
MOTIVATION: Wikipedia is one of the most important channels for the public communication of science and is frequently accessed as an educational resource in computational biology. Joint efforts between the International Society for Computational Biology (ISCB) and the Computational Biology taskforce of WikiProject Molecular Biology (a group of expert Wikipedia editors) have considerably improved computational biology representation on Wikipedia in recent years. However, there is still an urgent need for further improvement in quality, especially when compared to related scientific fields such as genetics and medicine. Facilitating involvement of members from ISCB Communities of Special Interest (COSIs) would improve a vital open education resource in computational biology, additionally allowing COSIs to provide a quality educational resource highly specific to their subfield. RESULTS: We generate a list of around 1500 English Wikipedia articles relating to computational biology and describe the development of a binary COSI-Article matrix, linking COSIs to relevant articles and thereby defining domain-specific open educational resources. Our analysis of the COSI-Article matrix data provides a quantitative assessment of computational biology representation on Wikipedia against other fields and at a COSI-specific level. Furthermore, we conducted similarity analysis and subsequent clustering of COSI-Article data to provide insight into potential relationships between COSIs. Finally, based on our analysis, we suggest courses of action to improve the quality of computational biology representation on Wikipedia.
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Biología Computacional , Análisis por ConglomeradosRESUMEN
Myeloid cells are central to homeostasis and immunity. Characterising in vitro myelopoiesis protocols is imperative for their use in research, immunotherapies, and understanding human myelopoiesis. Here, we generate a >470K cells molecular map of human induced pluripotent stem cells (iPSC) differentiation into macrophages. Integration with in vivo single-cell atlases shows in vitro differentiation recapitulates features of yolk sac hematopoiesis, before definitive hematopoietic stem cells (HSC) emerge. The diversity of myeloid cells generated, including mast cells and monocytes, suggests that HSC-independent hematopoiesis can produce multiple myeloid lineages. We uncover poorly described myeloid progenitors and conservation between in vivo and in vitro regulatory programs. Additionally, we develop a protocol to produce iPSC-derived dendritic cells (DC) resembling cDC2. Using CRISPR/Cas9 knock-outs, we validate the effects of key transcription factors in macrophage and DC ontogeny. This roadmap of myeloid differentiation is an important resource for investigating human fetal hematopoiesis and new therapeutic opportunities.