Single-cell roadmap of human gonadal development.

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.

Mapping Human Reproduction with Single-Cell Genomics.

The trillions of cells in the human body develop as a result of the fusion of two extremely specialized cells: an oocyte and a sperm. This process is essential for the continuation of our species, as it ensures that parental genetic information is mixed and passed on from generation to generation. In addition to producing oocytes, the female reproductive system must provide the environment for the appropriate development of the fetus until birth. New genomic and computational tools offer unique opportunities to study the tight spatiotemporal regulatory mechanisms that are required for the cycle of human reproduction. This review explores how single-cell technologies have been used to build cellular atlases of the human reproductive system across the life span and how these maps have proven useful to better understand reproductive pathologies and dissect the heterogeneity of in vitro model systems.

Characterizing domain-specific open educational resources by linking ISCB Communities of Special Interest to Wikipedia.

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.

Investigating the mechanism of action of DNA-loaded PEGylated lipid nanoparticles.

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.

Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking.

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.

Bulk tank milk quality data is unlikely to give useful information about dairy cow welfare at herd level.

This research communication explores the value of routinely collected bulk tank milk quality data for estimating dairy cattle welfare at herd level. Selected bulk tank milk quality parameters (somatic cell count, total bacterial count, urea, protein and fat contents) recorded during the years 2014-2016 in 287 Italian dairy farms were compared with the animal welfare data of each farm. The welfare assessment data were extracted from the database of the Italian Reference Centre for Animal Welfare (CReNBA), which includes the outputs of the application of the CReNBA welfare assessment protocol for dairy cows, used at national level for on-farm controls. The statistical analysis was carried out using the correlation coefficient for Kendall's Tau ranks, in order to investigate the presence of a categoric relationship between the selected bulk tank milk quality parameters and the overall animal welfare score or the scores of the single areas A (farm management and staff training), B (housing) and C (animal-based measures). Somatic cell count, total bacterial count, urea and proteins demonstrated only a few statistically significant and very weak correlations with farm animal welfare data, while no significant correlations were obtained for milk fat content. Given the weak correlations found, the selected bulk tank milk parameters seems to be able to provide only limited information about the welfare level of the herd, thus it could be difficult to use them for drawing up a pre-screening model for identifying herds at risk of poor welfare.

Increased soluble urokinase plasminogen activator receptor (suPAR) levels in neovascular age-related macular degeneration: a role for inflammation in the pathogenesis of the disease?

PURPOSE: To evaluate the plasma concentration of the soluble form of the urokinase-type plasminogen activator receptor ((s)uPAR), an established biomarker of chronic inflammation, in patients affected by neovascular age-related macular degeneration. METHODS: Forty consecutive patients affected by age-related macular degeneration and 52 subjects with no history of the disease were included in this case-control study. The two groups of individuals considered for the study were matched for age, sex, and class of medications taken. Plasma concentration of suPAR was measured using a specific ELISA assay (suPARnostic, Birkeroed, Denmark). RESULTS: The case and control groups were similar for age, gender distribution, weight, height, and systolic and diastolic blood pressure, as well as for dyslipidemia and high blood pressure medication (P > 0.28). The plasma concentrations of suPAR were significantly increased in patients with neovascular age-related macular degeneration when compared to controls (6.19 ± 2.2 ng/ml, vs 5.21 ± 1.5, respectively, mean ± SD P = 0.01). CONCLUSIONS: Patients with neovascular age-related macular degeneration display increased plasma levels of suPAR, suggesting that chronic inflammation may be involved in the pathogenesis of the disease.

The interaction between uPAR and vitronectin triggers ligand-independent adhesion signalling by integrins.

The urokinase-type plasminogen activator receptor (uPAR) is a non-integrin vitronectin (VN) cell adhesion receptor linked to the plasma membrane by a glycolipid anchor. Through structure-function analyses of uPAR, VN and integrins, we document that uPAR-mediated cell adhesion to VN triggers a novel type of integrin signalling that is independent of integrin-matrix engagement. The signalling is fully active on VN mutants deficient in integrin binding site and is also efficiently transduced by integrins deficient in ligand binding. Although integrin ligation is dispensable, signalling is crucially dependent upon an active conformation of the integrin and its association with intracellular adaptors such as talin. This non-canonical integrin signalling is not restricted to uPAR as it poses no structural constraints to the receptor mediating cell attachment. In contrast to canonical integrin signalling, where integrins form direct mechanical links between the ECM and the cytoskeleton, the molecular mechanism enabling the crosstalk between non-integrin adhesion receptors and integrins is dependent upon membrane tension. This suggests that for this type of signalling, the membrane represents a critical component of the molecular clutch.

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process.

EmptyDropsMultiome discriminates real cells from background in single-cell multiomics assays.

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%.

Machine-learning-guided recognition of α and ß cells from label-free infrared micrographs of living human islets of Langerhans.

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.

Effects of vaccination timing and target pathogens on performances and antimicrobial use in long-transported Charolais beef cattle from France to Italy - A retrospective study.

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.

Using Expert Elicitation for ranking hazards, promoters and animal-based measures for on-farm welfare assessment of indoor reared beef cattle: an Italian experience.

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.

Unveiling nanoscale optical signatures of cytokine-induced ß-cell dysfunction.

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.

Safety Evaluation and Probabilistic Health Risk Assessment of Cow Milk Produced in Northern Italy According to Dioxins and PCBs Contamination Levels.

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.

Farmed fish welfare during slaughter in Italy: survey on stunning and killing methods and indicators of unconsciousness.

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.

Paratuberculosis, Animal Welfare and Biosecurity: A Survey in 33 Northern Italy Dairy Goat Farms.

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.

DMRT1 regulates human germline commitment.

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.

Brain-Derived Neurotrophic Factor, Kynurenine Pathway, and Lipid-Profiling Alterations as Potential Animal Welfare Indicators in Dairy Cattle.

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.

Yolk sac cell atlas reveals multiorgan functions during human early development.

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.