The physiology of SARS-CoV-2 virus/host interactions is not well understood. To better understand host/virus interactions, we performed a CRISPR activation screen to identify host genes that confer resistance to authentic SARS-CoV-2. This highlighted 34 new candidate genes that may alter the course of infection. We validated that 7 of these genes can suppress authentic SARS-CoV-2 infection, including the innate immune receptor P selectin, which increases SARS-CoV-2 spike-dependent binding to cells, while protecting from infection. P selectin also promotes binding to SARS-CoV-2 variants, SARS-CoV-1, and Middle East respiratory syndrome spike proteins, suggesting a general role for P selectin in highly pathogenic coronavirus infections. Importantly, P selectin protein expression driven by synthetic mRNA can block SARS-CoV-2 infection. Naturally, P selectin is expressed on platelets, and we show that it promotes spike-mediated platelet aggregation. P selectin is also expressed on the endothelium, where SARS-CoV-2 spike interactions are also P selectin dependent. In vivo, SARS-CoV-2 uses P selectin to home to capillary beds where the virus interacts with platelets and endothelium, and blocking this interaction can clear vascular-associated pulmonary SARS-CoV-2.

Per- and polyfluoroalkyl substances (PFAS) threaten ecosystems worldwide due to their persistence, bioaccumulation, and toxicity. Through a global-scale meta-analysis of 119 aquatic and terrestrial food webs from 64 studies, we analyse 1009 trophic magnification factors (TMFs) for 72 PFAS and identify key variability drivers. On average, PFAS concentrations double with each trophic level increase (mean TMF = 2.00, 95% CI:1.64-2.45), though magnification varies considerably by compound. Notably, the industrial alternative F-53B exhibits the highest magnification (TMF = 3.07, 95% CI:2.41-3.92), a critical finding given its expanding use and minimal regulatory scrutiny. Methodological disparities across studies emerge as the dominant source of TMF variability. Our models explain 85% of the variation in TMFs, underscoring predictive capacity. This synthesis establishes PFAS as persistent trophic multipliers and provides a framework to prioritise high-risk compounds and harmonise biomagnification assessments. Our results call for consideration of stricter PFAS regulation to curb cascading ecological and health impacts.

Per- and polyfluoroalkyl substances (PFAS) threaten ecosystems worldwide due to their persistence, bioaccumulation, and toxicity. Through a global-scale meta-analysis of 122 aquatic and terrestrial food webs from 64 studies, we analyse 1,009 trophic magnification factors (TMFs) for 72 PFAS and identify key variability drivers. PFAS concentrations systematically doubled with each trophic level increase (mean TMF=2.00, 95% CI:1.64-2.45), confirming widespread biomagnification across ecosystems. Methodological disparities across studies emerged as the dominant source of TMF variability. Our models explained 84% of the variation in TMFs, underscoring predictive capacity. Notably, the industrial alternative F-53B exhibited the highest magnification (TMF=3.07, 95% CI:2.41-3.92), a critical finding given its expanding use and minimal regulatory scrutiny. This synthesis establishes PFAS as persistent trophic multipliers and provides a framework to prioritise high-risk compounds and harmonise biomagnification assessments. Our results call for consideration of stricter PFAS regulation to curb cascading ecological and health impacts.

Directed evolution is a process of mutation and artificial selection to breed biomolecules with new or improved activity. Directed evolution platforms are primarily prokaryotic or yeast-based, and stable mammalian systems have been challenging to establish and apply. To this end, we develop PROTein Evolution Using Selection (PROTEUS), a platform that uses chimeric virus-like vesicles to enable extended mammalian directed evolution campaigns without loss of system integrity. This platform is stable and can generate sufficient diversity for directed evolution in mammalian systems. Using PROTEUS, we alter the doxycycline responsiveness of tetracycline-controlled transactivators, generating a more sensitive TetON-4G tool for gene regulation with mammalian-specific adaptations. PROTEUS is also compatible with intracellular nanobody evolution, and we use it to evolve a DNA damage-responsive anti-p53 nanobody. Overall, PROTEUS is an efficient and stable platform to direct evolution of biomolecules within mammalian cells.

Background: Per- and polyfluoroalkyl substances (PFAS) are a large group of manufactured chemicals. Since the beginning of their commercial manufacturing in the 1950s, PFAS haven’t only found their way into numerous industrial and commercial applications, but also into the bloodstream of the majority of the human population, the natural environment and its wildlife. Exposure to high levels of PFAS can create health risks for humans and animals which may exacerbate the effects of other anthropogenic impacts faced by wildlife species. To gain a comprehensive overview of the abundance and distribution of PFAS in wildlife species, and to better understand the risk of PFAS exposure on threatened species and PFAS transfer into human food chains, we will collate the available literature into a systematic evidence map and bibliometric analysis.Methods: We will conduct a comprehensive systematic literature search on Scopus, Web of Science and the ‘grey literature’. For screening purposes, we will use decision trees, scanning title, abstract and keywords first. The next step includes full-text screening performed by two reviewers. We will only consider publications in English, peer-reviewed articles, pre-prints and theses. We will limit our search to 31 PFAS types (based on a previous study). A pilot search on Scopus resulted in ~250 potentially relevant publications. We will scan all publications included in the systematic map for predetermined indicators of quality and potential study-level biases. In addition, we will extract bibliometric records from Scopus and perform network analysis. We will present the results using a narrative summary, tables (database), bar plots and colour-coded maps. Results will be available on a dedicated freely accessible website. Discussion: This study will provide critical insight into the gaps and clusters of the literature with regards to the PFAS concentration in wildlife. Therefore, our study will inform and direct future research efforts to fill the gaps revealed.Systematic review registration: osf.io osf.io/gnt2y

Background: Per- and polyfluoroalkyl substances (PFAS) are a class of widely used anthropogenic chemicals. Concerns regarding their persistence and potential adverse effects have led to multiple secondary research publications. Here, we aim to assess the resulting evidence base in the systematic secondary literature by examining research gaps, evaluating the quality of reviews, and exploring interdisciplinary connections. Methods: This study employed a systematic evidence-mapping approach to assess the secondary literature on the biological, environmental, and medical aspects of exposure to 35 fluorinated compounds. The inclusion criteria encompassed systematic reviews published in peer-reviewed journals, pre-prints, and theses. Comprehensive searches across electronic databases and grey literature identified relevant reviews. Data extraction and synthesis involved mapping literature content and narrative descriptions. We employed a modified version of the AMSTAR2 checklist to evaluate the methodological rigour of the reviews. A bibliometric data analysis uncovered patterns and trends in the academic literature. A research protocol for this study was previously pre-registered (osf.io/2tpn8) and published (Vendl et al., Environment International 158 (2022) 106973). The database is freely accessible through the interactive and user-friendly web application of this systematic evidence map at https://hi-this-is-lorenzo.shinyapps.io/PFAS_SEM_Shiny_App/. Results: Our map includes a total of 175 systematic reviews. Over the years, there has been a steady increase in the annual number of publications, with a notable surge in 2021. Most reviews focused on human exposure, whereas environmental and animal-related reviews were fewer and often lacked a rigorous systematic approach to literature search and screening. Review outcomes were predominantly associated with human health, particularly with reproductive and children’s developmental health. Animal reviews primarily focused on studies conducted in controlled laboratory settings, and wildlife reviews were characterised by an over-representation of birds and fish species. Recent reviews increasingly incorporated quantitative synthesis methodologies. The methodological strengths of the reviews included detailed descriptions of study selection processes and disclosure of potential conflicts of interest. However, weaknesses were observed in the critical lack of detail in reporting methods. A bibliometric analysis revealed that the most productive authors collaborate within their own country, leading to limited and clustered international collaborations. Conclusions: In this overview of the available systematic secondary literature, we map literature content, assess reviews’ methodological quality, highlight data gaps, and draw research network clusters. We aim to facilitate literature reviews, guide future research initiatives, and enhance opportunities for cross-country collaboration. Furthermore, we discuss how this systematic evidence map and its publicly available database benefit scientists, regulatory agencies, and other stakeholders by providing access to current systematic secondary literature on PFAS exposure.

Legumain is a lysosomal cysteine protease that has been implicated in an increasing amount of physiological and pathophysiological processes. However, the upstream mechanisms regulating the expression and function of legumain are not well understood. Here, we provide in vitro and in vivo data showing that vitamin D3 (VD3) enhances legumain expression and function. In turn, legumain alters VD3 bioavailability, possibly through proteolytic cleavage of vitamin D binding protein (VDBP). Active VD3 (1,25(OH)2D3) increased legumain expression, activity, and secretion in osteogenic cultures of human bone marrow stromal cells. Upregulation of legumain was also observed in vivo, evidenced by increased legumain mRNA in the liver and spleen, as well as increased legumain activity in kidneys from wild-type mice treated with 25(OH)D3 (50 µg/kg, subcutaneously) for 8 days compared to a control. In addition, the serum level of legumain was also increased. We further showed that active legumain cleaved purified VDBP (55 kDa) in vitro, forming a 45 kDa fragment. In vivo, no VDBP cleavage was found in kidneys or liver from legumain-deficient mice (Lgmn−/−), whereas VDBP was cleaved in wild-type control mice (Lgmn+/+). Finally, legumain deficiency resulted in increased plasma levels of 25(OH)D3 and total VD3 and altered expression of key renal enzymes involved in VD3 metabolism (CYP24A1 and CYP27B1). In conclusion, a regulatory interplay between VD3 and legumain is suggested.

For over two decades pluripotent stem cells have promised a renewable source of β cells to treat patients with type 1 diabetes. Major efforts to optimize the differentiation, survival, and function of transplanted stem cell-derived tissue have recently delivered clinically meaningful metabolic benefits using a perforated encapsulation device that promotes integration with recipient vasculature under the protection of systemic immunosuppression. Despite this success, the journey is not over as a universal cure will require a larger β cell mass. Here, we summarize recent interdisciplinary advances that could maximize the functional β cell mass within transplanted devices and provide an immune privileged niche that could eliminate the need for systemic immunosuppression.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the degeneration of motor neurons. Mutations in the cyclin F (CCNF) and fused in sarcoma (FUS) genes have been associated with ALS pathology. In this study, we aimed to investigate the functional role of CCNF and FUS in ALS by using genome editing techniques to generate zebrafish models with genetic disruptions in these genes. Sequence comparisons showed significant homology between human and zebrafish CCNF and FUS proteins. We used CRISPR/Cas9 and TALEN-mediated genome editing to generate targeted disruptions in the zebrafish ccnf and fus genes. Ccnf-deficient zebrafish exhibited abnormal motor neuron development and axonal outgrowth, whereas Fus-deficient zebrafish did not exhibit developmental abnormalities or axonopathies in primary motor neurons. However, Fus-deficient zebrafish displayed motor impairments in response to oxidative and endoplasmic reticulum stress. The Ccnf-deficient zebrafish were only sensitized to endoplasmic reticulum stress, indicating that ALS genes have overlapping as well as unique cellular functions. These zebrafish models provide valuable platforms for studying the functional consequences of CCNF and FUS mutations in ALS pathogenesis. Furthermore, these zebrafish models expand the drug screening toolkit used to evaluate possible ALS treatments.

COVID-19 causes a clinical spectrum of acute and chronic illness and host / virus interactions are not completely understood 1,2 . To identify host factors that can influence SARS-CoV-2 infection, we screened the human genome for genes that, when upregulated, alter the outcome of authentic SARS-CoV-2 infection. From this, we identify 34 new genes that can alter the course of infection, including the innate immune receptor P-selectin, which we show is a novel SARS-CoV-2 spike receptor. At the cellular level expression of P-selectin does not confer tropism for SARS-CoV-2, instead it acts to suppress infection. More broadly, P-selectin can also promote binding to SARS-CoV-2 variants, SARS-CoV-1 and MERS, acting as a general spike receptor for highly pathogenic coronaviruses. P-selectin is expressed on platelets and endothelium 3 , and we confirm SARS-CoV-2 spike interactions with these cells are P-selectin-dependent and can occur under shear flow conditions. In vivo , authentic SARS-CoV-2 uses P-selectin to home to airway capillary beds where the virus interacts with the endothelium and platelets, and blocking this interaction can clear vascular-associated SARS-CoV-2 from the lung. Together we show for the first time that coronaviruses can use the leukocyte recruitment system to control tissue localization, and this fundamental insight may help us understand and control highly pathogenic coronavirus disease progression.

Respiratory diseases are the 2nd leading cause of death globally. The current treatments for chronic lung diseases are only supportive. Very few new classes of therapeutics have been introduced for lung diseases in the last 40 years, due to the lack of reliable lung models that enable rapid, cost-effective, and high-throughput testing. To accelerate the development of new therapeutics for lung diseases, we established two classes of lung-mimicking models: (i) healthy, and (ii) diseased lungs – COPD. To establish models that mimic the lung complexity to different extents, we used five design components: (i) cell type, (ii) membrane structure/constitution, (iii) environmental conditions, (iv) cellular arrangement, (v) substrate, matrix structure and composition. To determine whether the lung models are reproducible and reliable, we developed a quality control (QC) strategy, which integrated the real-time and end-point quantitative and qualitative measurements of cellular barrier function, permeability, tight junctions, tissue structure, tissue composition, and cytokine secretion. The healthy model is characterised by (i) continuous tight junctions, (ii) physiological cellular barrier function, (iii) a full thickness epithelium composed of multiple cell layers, and (iv) the presence of ciliated cells and goblet cells. Meanwhile, the disease model emulates human COPD disease: (i) dysfunctional cellular barrier function, (ii) depletion of ciliated cells, and (ii) overproduction of goblet cells. The models developed here have multiple competitive advantages when compared with existing in vitro lung models: (i) the macroscale enables multimodal and correlative characterisation of the same model system, (ii) the use of cells derived from patients that enables the creation of individual models for each patient for personalised medicine, (iii) the use of an extracellular matrix proteins interface, which promotes physiological cell adhesion and differentiation, (iv) media microcirculation that mimics the dynamic conditions in human lungs. Our model can be utilised to test safety, efficacy, and superiority of new therapeutics as well as to test toxicity and injury induced by inhaled pollution or pathogens. It is envisaged that these models can also be used to test the protective function of new therapeutics for high-risk patients or workers exposed to occupational hazards.

Abstract The obesity epidemic, largely driven by the accessibility of ultra‐processed high‐energy foods, is one of the most pressing public health challenges of the 21st century. Consequently, there is increasing concern about the impacts of diet‐induced obesity on behavior and cognition. While research on this matter continues, to date, no study has explicitly investigated the effect of obesogenic diet on variance and covariance (correlation) in behavioral traits. Here, we examined how an obesogenic versus control diet impacts means and (co‐)variances of traits associated with body condition, behavior, and cognition in a laboratory population of ~160 adult zebrafish (Danio rerio). Overall, an obesogenic diet increased variation in several zebrafish traits. Zebrafish on an obesogenic diet were significantly heavier and displayed higher body weight variability; fasting blood glucose levels were similar between control and treatment zebrafish. During behavioral assays, zebrafish on the obesogenic diet displayed more exploratory behavior and were less reactive to video stimuli with conspecifics during a personality test, but these significant differences were sex‐specific. Zebrafish on an obesogenic diet also displayed repeatable responses in aversive learning tests whereas control zebrafish did not, suggesting an obesogenic diet resulted in more consistent, yet impaired, behavioral responses. Where behavioral syndromes existed (inter‐class correlations between personality traits), they did not differ between obesogenic and control zebrafish groups. By integrating a multifaceted, holistic approach that incorporates components of (co‐)variances, future studies will greatly benefit by quantifying neglected dimensions of obesogenic diets on behavioral changes.

COVID-19 patients display a wide range of disease severity, ranging from asymptomatic to critical symptoms with high mortality risk. Our ability to understand the interaction of SARS-CoV-2 infected cells within the lung, and of protective or dysfunctional immune responses to the virus, is critical to effectively treat these patients. Currently, our understanding of cell-cell interactions across different disease states, and how such interactions may drive pathogenic outcomes, is incomplete. Here, we developed a generalizable and scalable workflow for identifying cells that are differentially interacting across COVID-19 patients with distinct disease outcomes and use this to examine eight public single-cell RNA-seq datasets (six from peripheral blood mononuclear cells, one from bronchoalveolar lavage and one from nasopharyngeal), with a total of 211 individual samples. By characterizing the cell-cell interaction patterns across epithelial and immune cells in lung tissues for patients with varying disease severity, we illustrate diverse communication patterns across individuals, and discover heterogeneous communication patterns among moderate and severe patients. We further illustrate patterns derived from cell-cell interactions are potential signatures for discriminating between moderate and severe patients. Overall, this workflow can be generalized and scaled to combine multiple scRNA-seq datasets to uncover cell-cell interactions.

The obesity epidemic is among the most serious and rapidly growing public health challenges of the 21st century. This rapid increase is concerning as obesity appears to negatively impact cognition and behaviour. Furthermore, some studies suggest that this negative effect could be carried across generations from both mothers and fathers although evidence is not consistent. Here, we attempt to address how obesogenic diets in the parental generation (F0) can impact offspring’s cognition and anxiety intergenerationally (F1) in a zebrafish model. We compare both mean trait values and their variances. Using a multifactorial design, we created a total of four groups: F1T (treatment mothers x treatment fathers); F1M (treatment mothers x control fathers); F1P (treatment fathers x control mothers); and F1C (control mothers x control fathers, F1C); and subjected them to anxiety tank tests and aversive learning assays. When both parents were exposed, offspring (F1T) displayed the poorest aversive learning, while offspring that only had one parent exposed (F1P and F1M) learnt the aversive learning task the best. Zebrafish in all groups displayed no statistically significant differences in anxiety-associated behaviours. Males and females also performed similarly in both anxiety and aversive learning assays. While all F1 groups had similar levels of fasting blood glucose, variance in glucose levels were reduced in F1P and F1T indicating the importance of investigating heteroskedasticity between groups. Furthermore, anxiety behaviours of these two groups appeared to be less repeatable. To our knowledge, this is the first study to test the intergenerational effects of an obesogenic diet on zebrafish cognition. Our multifactorial design as well as repeated tests also allowed us to disentangle maternal and paternal effects (as well as combined effects) and accurately detect subtle information such as between-individual variation. However, our work is restricted to aversive learning. It would be interesting to see whether appetitive and other types of learning produce similar results in zebrafish offspring.

Directed evolution uses cycles of gene diversification and selection to generate proteins with novel properties. While traditionally directed evolution is performed in prokaryotic systems, recently a mammalian directed evolution system (viral evolution of genetically actuating sequences, or "VEGAS") has been described. Here we report that the VEGAS system has major limitations that preclude its use for directed evolution. The deconstructed Sindbis virus (SINV) genome that comprises the VEGAS system could no longer promote Sindbis structural gene (SSG)-dependent viral replication. Moreover, viral particles generated using the VEGAS system rapidly lost the target directed evolution transgene, and instead, "cheater" particles, primarily containing RNA encoding SINV structural components, arose. By sequencing, we found that this contamination came from RNA provided during initial SINV packaging, not RNA derived from the VEGAS system. Of note, both the structural RNA and target transgenes used in the VEGAS system contain viral packaging sequences. The impact of SINV "cheater" particles could be potentially overcome in the context of a robust VEGAS circuit, but since SSG complementation is also defective in the VEGAS system, selection for authentic evolution products is not currently possible. Similar results have been obtained in independent laboratories. Taken together, these results show that the VEGAS system does not work as described and, without significant redesign, cannot be used for mammalian directed evolution campaigns.

Abstract The obesity epidemic is concerning as obesity appears to negatively impact cognition and behavior. Furthermore, some studies suggest that this negative effect could be carried across generations from both mothers and fathers although evidence is not consistent. Here, we attempt to address how obesogenic diets in the parental generation (F0) can impact offspring's cognition and anxiety intergenerationally (F1) in a zebrafish model. We compare both mean trait values and their variances. Using a multifactorial design, we created a total of four groups: F1T (treatment mothers × treatment fathers); F1M (treatment mothers × control fathers); F1P (treatment fathers × control mothers); and F1C (control mothers × control fathers, F1C); and subjected them to anxiety tank tests and aversive learning assays. When both parents were exposed, offspring (F1T) displayed the poorest aversive learning, while offspring that only had one parent exposed (F1P and F1M) learnt the aversive learning task the best. Zebrafish in all groups displayed no statistically significant differences in anxiety‐associated behaviors. Males and females also performed similarly in both anxiety and aversive learning assays. While all F1 groups had similar levels of fasting blood glucose, variance in glucose levels were reduced in F1P and F1T indicating the importance of investigating heteroskedasticity between groups. Furthermore, anxiety behaviors of these two groups appeared to be less repeatable. To our knowledge, this is the first study to test the intergenerational effects of an obesogenic diet on zebrafish cognition. Our multifactorial design as well as repeated tests also allowed us to disentangle maternal and paternal effects (as well as combined effects) and accurately detect subtle information such as between‐individual variation.

Although ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of factors controlling SARS-CoV-2 host interactions has not been described. Here we used whole genome CRISPR activation to identify host factors controlling SARS-CoV-2 Spike binding. The top hit was a Toll-like receptor-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 Spike binding where it forms a cell surface complex with LRRC15 but does not support infection. Instead, LRRC15 functioned as a negative receptor suppressing both pseudotyped and live SARS-CoV-2 infection. LRRC15 is expressed in collagen-producing lung myofibroblasts where it can sequester virus and reduce infection in trans. Mechanistically LRRC15 is regulated by TGF-β, where moderate LRRC15 expression drives collagen production but high levels suppress it, revealing a novel lung fibrosis feedback circuit. Overall, LRRC15 is a master regulator of SARS-CoV-2, suppressing infection and controlling collagen production associated with “long-haul” COVID-19.

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous in the environment and often ingested with food. PFAS exposure in people can have detrimental health consequences. Therefore, reducing PFAS burdens in food items is of great importance to public health. Here, we investigated whether cooking reduces PFAS concentrations in animal-derived food products by synthesizing experimental studies. Further, we examined the moderating effects of the following five variables: cooking time, liquid/animal tissue ratio, cooking temperature, carbon chain length of PFAS and the cooking category (oil-based, water-based & no-liquid cooking). In our systematic review searches, we obtained 512 effect sizes (relative differences in PFAS concentration between raw and cooked samples) from 10 relevant studies. These studies exclusively explored changes in PFAS concentrations in cooked seafood and freshwater fish. Our multilevel-meta-analysis has revealed that, on average, cooking reduced PFAS concentrations by 29%, although heterogeneity among effect sizes was very high (I2 = 94.65%). Our five moderators cumulatively explained 49% of the observed heterogeneity. Specifically, an increase in cooking time and liquid/animal tissue ratio, as well as shorter carbon chain length of PFAS (when cooked with oil) were associated with significant reductions in PFAS concentrations. The effects of different ways of cooking depended on the other moderators, while the effect of cooking temperature itself was not significant. Overall, cooking can reduce PFAS concentrations in blue food (seafood and freshwater fish). However, it is important to note that complete PFAS elimination requires unrealistically long cooking times and large liquid/animal tissue ratios. Currently, literature on the impact of cooking of terrestrial animal produce on PFAS concentrations is lacking, which limits the inference and generalisation of our meta-analysis. However, our work represents the first step towards developing guidelines to reduce PFAS in food via cooking exclusively with common kitchen items and techniques.