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Abstract Meta-analyses show an overall decrease in soil N2O emissions after biochar (BC) amendment. Nonetheless, N2O mitigation with BC cannot be extrapolated to every BC-soil combination, inasmuch as an increase in soil N2O release has been occasionally reported. We hypothesized that BC characteristics are key, and performed two microcosm experiments to advance in the understanding of the properties associated. We first investigated how 22 well-characterized BCs affect N2O emissions in a calcareous soil under denitrification conditions. Whereas most BCs decreased N2O emissions, some substantially increased N2O emissions. In a second experiment, we selected and further characterized eight of the 22 previous BCs. We applied the 15N-gas-flux method to study how these BCs affect denitrification products (N2O and N2) in the same soil. Results indicate that the interaction between BC and the denitrification process depends on the temperature of pyrolysis. Whereas BCs produced at 400 °C tended to increase total denitrification (N2O+N2) by an average of 28%, BCs produced at 600 °C significantly reduced total denitrification by 53%. Nevertheless, this decline in overall denitrification did not result in a decrease of N2O emissions, as there was a strong shift in the N2O/(N2+N2O) ratio favoring N2O. A redundancy analysis revealed a direct correlation between carboxylic groups on BCs surface and N2O emissions. This research enhances our understanding of the interaction of BC with denitrification, particularly concerning the relevance of the temperature of pyrolysis, and opens up new paths for investigation, crucial for optimizing the application of BCs in different soil environments. Graphical Abstract

Abstract The agronomic use of compost and biochar as soil amendments may exhibit contrasting results in terms of soil fertility and plant nutrition. The effects of the biennial application of biochar, compost and a blend of compost:biochar (90:10; % dw:dw) on the agronomical performance of an organically managed and well established 25-year-old olive orchard was assessed 5 years after the initial application. The agronomical evaluation was based on the assessment of the soil physical, chemical, and biological characteristics, and the assessment of the soil fertility by both crop production and nutritional status of the orchard, and the bioassay with olive plantlets. Biochar mainly benefited the physical properties (bulk density, total porosity, aeration, water retention capacity) of soil, especially in the top 0–5 cm. Compost and its blend with biochar improved microbial activity, soil nutritional status (increasing the content of soluble organic C, N, and P) and favoured the formation of aggregates in soil. The bioassay conducted with young plantlets confirmed the enhanced soil fertility status in the three amended treatments, particularly in the case of biochar and its blend with compost. However, this effect was not significantly observed in the adult plants after 5 years of application, reflecting the slow response of adult olive trees to changes in fertilization. Based on these results, alongside the desirable long-residence time of biochar in soil and the ready availability of compost, the blend of biochar with compost assayed in this study is defined as a valid strategy for preparing high quality soil organic amendments.

ABSTRACT The interaction of biochar with mineral fertilization has attracted attention as a strategy to reduce N losses and enhance nitrogen use efficiency. In this study, we investigated the coapplication of biochar with two optimized fertilization strategies based on split urea and a microbial inoculant (Azospirillum brasilense) in a commercial pointed white cabbage crop. Additionally, we evaluated a third optimized N fertilization alternative, a biochar‐based fertilizer (BBF) enriched in plant‐available N, which was developed from the same biochar. We assessed environmental impacts such as greenhouse gasses (GHG) and NH3 emissions, yield‐scaled N2O emissions, and global warming potential (GWP). Additionally, we evaluated agronomical outcomes such as crop yield, plant N, and chlorophyll concentration. Moreover, we examined the N‐fixing gene's total and relative abundance (nifH and nifH/16S). Biochar and BBF exhibited similar crop yield, GHG, and NH3 emissions compared to split applications of the synthetic fertilizer. The main difference was associated with the higher soil C sequestration in biochar and BBF treatments that reduced the associated GWP of these fertilization strategies. Finally, biochar favored the activity of the N‐fixing bacteria spread, compared to the sole application of bacteria and BBF demonstrated a promoting effect in the soil's total abundance of natural N‐fixing bacteria.

IntroductionLong COVID, or post-acute sequelae of SARS-CoV-2 infection (PASC), manifests as persistent and often debilitating symptoms enduring well beyond the initial COVID-19 infection. This disease is especially worrying in children since it can seriously alter their development. Presently, a specific diagnostic test or definitive biomarker set for confirming long COVID is lacking, relying instead on the protracted presence of symptoms post-acute infection.MethodsWe measured the levels of 13 biomarkers in 105 saliva samples (49 from children with long COVID and 56 controls), and the Pearson correlation coefficient was used to analyse the correlations between the levels of the different salivary biomarkers. Multivariate logistic regression analyses were performed to determine which of the 13 analysed salivary biomarkers were useful to discriminate between children with long COVID and controls, as well as between children with mild and severe long COVID symptoms.ResultsPediatric long COVID exhibited increased oxidant biomarkers and decreased antioxidant, immune response, and stress-related biomarkers. Correlation analyses unveiled distinct patterns between biomarkers in long COVID and controls. Notably, a multivariate logistic regression pinpointed TOS, ADA2, total proteins, and AOPP as pivotal variables, culminating in a remarkably accurate predictive model distinguishing long COVID from controls. Furthermore, total proteins and ADA1 were instrumental in discerning between mild and severe long COVID symptoms.DiscussionThis research sheds light on the potential clinical utility of salivary biomarkers in diagnosing and categorizing the severity of pediatric long COVID. It also lays the groundwork for future investigations aimed at unravelling the prognostic value of these biomarkers in predicting the trajectory of long COVID in affected individuals.

Abstract Telomerase RNA (TERC) has a noncanonical function in myelopoiesis binding to a consensus DNA binding sequence and attracting RNA polymerase II (RNA Pol II), thus facilitating myeloid gene expression. The CR4/CR5 domain of TERC is known to play this role, since a mutation of this domain found in dyskeratosis congenita (DC) patients decreases its affinity for RNA Pol II, impairing its myelopoietic activity as a result. In this study, we report that two aptamers, short single-stranded oligonucleotides, based on the CR4/CR5 domain were able to increase myelopoiesis without affecting erythropoiesis in zebrafish. Mechanistically, the aptamers functioned as full terc; that is, they increased the expression of master myeloid genes, independently of endogenous terc, by interacting with RNA Pol II and with the terc-binding sequences of the regulatory regions of such genes, enforcing their transcription. Importantly, aptamers harboring the CR4/CR5 mutation that was found in DC patients failed to perform all these functions. The therapeutic potential of the aptamers for treating neutropenia was demonstrated in several preclinical models. The findings of this study have identified two potential therapeutic agents for DC and other neutropenic patients.

Type I interferonopathies are a heterogenic group of rare diseases associated with an increase in type I interferon (IFN). The main challenge for the study of Type I interferonopathies is the lack of a well-founded animal model to better characterize the phenotype as well as to perform fast and large drug screenings to offer the best treatment options. In this study, we report the development of a transgenic zebrafish model of Type I interferonopathy overexpressing ifih1 carrying the mutation p.Arg742His (Tg(ifih1_mut)), corresponding to the human mutation p.Arg779His. RNA sequence analysis from Tg(ifih1_mut) larvae revealed a systemic inflammation and IFN signature upon a suboptimal poly I:C induction compared with wild-type larvae, confirming the phenotype observed in patients suffering from Type I interferonopathies. More interestingly, the phenotype was manifested in the zebrafish inflammation and Type I IFN reporters nfkb:eGFP and isg15:eGFP, respectively, making this zebrafish model suitable for future high-throughput chemical screening (HTS). Using the unique advantages of the zebrafish model for gene editing, we have generated Tg(ifih1_mut) knocked down for mavs and ikbke, which completely abrogated the Poly I:C induction and activation of the GFP of the reporters. Finally, we used an FDA-approved drug, Baricitinib (Jak1/Jak2 inhibitor), which was able to reduce the inflammation and the ISG expression. Our results demonstrate the potential of this model to further understand AGS pathological mechanisms and to identify novel therapeutic drugs by HTS.

Telomerase reverse transcriptase (TERT) maintains telomere homeostasis, thus ensuring chromosome stability and cell proliferation. In addition, several telomere‐independent functions of human TERT have been described. In this study, we report that TERT binds directly to the TCF binding elements located upstream of the oncomiR miR500A, and induces its transcription. This function was independent of the telomerase activity, as shown with experiments using catalytically inactive TERT and inhibitors of TERT and the TERT RNA component. miR500A was in turn found to target three key components of the Hedgehog signalling pathway: Patched 1; Gli family zinc finger 3; and Cullin 3, thereby promoting cell invasion. Our results point to the crucial role of the TERT–miR500A–Hedgehog axis in tumour aggressiveness and highlight the therapeutic potential of targeting noncanonical TERT functions in cancer.

Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD+) metabolism, but the precise mechanism involved is unknown. Here, we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD+ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation through parthanatos cell death. A study of chronic skin inflammation reveals that hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation via parthanatos cell death, identifying NAMPT, PARP1 and AIFM1 as novel therapeutic targets for psoriasis.

Abstract Background Skin cutaneous melanoma (SKCM) is the most lethal form of skin cancer and while incidence rates are declining for most cancers, they have been steadily rising for SKCM. Serine protease inhibitor, kunitz-type, 1 (SPINT1) is a type II transmembrane serine protease inhibitor that has been shown to be involved in the development of several types of cancer, such as squamous cell carcinoma and colorectal cancer. Methods We used the unique advantages of the zebrafish to model the impact of Spint1a deficiency in early transformation, progression and metastatic invasion of SKCM together with in silico analysis of the occurrence and relevance of SPINT1 genetic alterations of the SKCM TCGA cohort. Results We report here a high prevalence of SPINT1 genetic alterations in SKCM patients and their association with altered tumor immune microenvironment and poor patient survival. The zebrafish model reveals that Spint1a deficiency facilitates oncogenic transformation, regulates the tumor immune microenvironment crosstalk, accelerates the onset of SKCM and promotes metastatic invasion. Notably, Spint1a deficiency is required at both cell autonomous and non-autonomous levels to enhance invasiveness of SKCM. Conclusions These results reveal a novel therapeutic target for SKCM.

Many proteins contain tandemly repeated modules of several amino acids, which act as the building blocks that form the underlying architecture of a specific protein-binding interface. Among these motifs and one of the most frequently observed is ankyrin repeats (ANK), which consist of 33 amino acid residues that are highly conserved. ANK domains span a wide range of functions, including protein–protein interactions, such as the recruitment of substrate to the catalytic domain of an enzyme, or the assembly of stable multiprotein complexes. Here, we report the identification of an evolutionarily conserved protein, that we term Caiap (from CARD- and ANK-containing Inflammasome Adaptor Protein), which has an N-terminal CARD domain and 16 C-terminal ANK domains and is required for the inflammasome-dependent resistance to Salmonella Typhimurium in zebrafish. Intriguingly, Caiap is highly conserved from cartilaginous fish to marsupials but is absent in placental mammals. Mechanistically, Caiap acts downstream flagellin and interacts with catalytic active Caspa, the functional homolog of mammalian caspase-1, through its ANK domain, while its CARD domain promotes its self-oligomerization. Our results therefore point to ANK domain-containing proteins as key inflammasome adaptors required for the stabilization of active caspase-1 in functionally stable, high molecular weight complexes.

This paper reports the results on the agronomic performance of organic amendments in the EU 7th FP project “FERTIPLUS—reducing mineral fertilizers and agro-chemicals by recycling treated organic waste as compost and bio-char”. Four case studies on field-scale application of biochar, compost and biochar-blended compost were established and studied for three consecutive years in four distinct cropping systems and under different agro-climatic conditions in Europe. These included the following sites: olive groves in Murcia (Spain), greenhouse grown tomatoes in Almeria (Spain), an arable crop rotation in Oost-Vlaanderen (Merelbeke, Belgium), and three vineyards in Friuli Venezia Giulia (Italy). A slow pyrolysis oak biochar was applied, either alone or in combination with organic residues: compost from olive wastes in Murcia (Spain), sheep manure in Almeria (Spain), and compost from biowaste and green waste in Belgium and Italy. The agronomical benefits were evaluated based on different aspects of soil fertility (soil total organic carbon (TOC), pH, nutrient cycling and microbial activity) and crop nutritional status and productivity. All amendments were effective in increasing soil organic C in all the field trials. On average, the increase with respect to the control was about 11% for compost, 20% for biochar-blended compost, and 36% for biochar. The amendments also raised the pH by 0.15−0.50 units in acidic soils. Only biochar had a negligible fertilization effect. On the contrary, compost and biochar-blended compost were effective in enhancing soil fertility by increasing nutrient cycling (25% mean increase in extractable organic C and 44% increase in extractable N), element availability (26% increase in available K), and soil microbial activity (26% increase in soil respiration and 2−4 fold enhancement of denitrifying activity). In general, the tested amendments did not show any negative effect on crop yield and quality. Furthermore, in vineyards and greenhouse grown tomatoes cropping systems, compost and biochar-blended compost were also effective in enhancing key crop quality parameters (9% increase in grape must acidity and 16% increase in weight, 9% increase in diameter and 8% increase in hardness of tomato fruits) important for the quality and marketability of the crops. The overall results of the project suggest that the application of a mixture of biochar and compost can benefit crops. Therefore, biochar-blended compost can support and maintain soil fertility.

SUMMARY The study of telomere biology is crucial to the understanding of aging and cancer. In the pursuit of greater knowledge in the field of human telomere biology, the mouse has been used extensively as a model. However, there are fundamental differences between mouse and human cells. Therefore, additional models are required. In light of this, we have characterized telomerase-deficient zebrafish (Danio rerio) as the second vertebrate model for human telomerase-driven diseases. We found that telomerase-deficient zebrafish show p53-dependent premature aging and reduced lifespan in the first generation, as occurs in humans but not in mice, probably reflecting the similar telomere length in fish and humans. Among these aging symptoms, spinal curvature, liver and retina degeneration, and infertility were the most remarkable. Although the second-generation embryos died in early developmental stages, restoration of telomerase activity rescued telomere length and survival, indicating that telomerase dosage is crucial. Importantly, this model also reproduces the disease anticipation observed in humans with dyskeratosis congenita (DC). Thus, telomerase haploinsufficiency leads to anticipation phenomenon in longevity, which is related to telomere shortening and, specifically, with the proportion of short telomeres. Furthermore, p53 was induced by telomere attrition, leading to growth arrest and apoptosis. Importantly, genetic inhibition of p53 rescued the adverse effects of telomere loss, indicating that the molecular mechanisms induced by telomere shortening are conserved from fish to mammals. The partial rescue of telomere length and longevity by restoration of telomerase activity, together with the feasibility of the zebrafish for high-throughput chemical screening, both point to the usefulness of this model for the discovery of new drugs able to reactivate telomerase in individuals with DC.

SUMMARY Although it is known that tumor necrosis factor receptor (TNFR) signaling plays a crucial role in vascular integrity and homeostasis, the contribution of each receptor to these processes and the signaling pathway involved are still largely unknown. Here, we show that targeted gene knockdown of TNFRSF1B in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of TNFRSF1A or the activation of NF-κB rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. In endothelial cells, TNFRSF1A signals apoptosis through caspase-8, whereas TNFRSF1B signals survival via NF-κB. Similarly, TNFα promotes the apoptosis of human endothelial cells through TNFRSF1A and triggers caspase-2 and P53 activation. We have identified an evolutionarily conserved apoptotic pathway involved in vascular homeostasis that provides new therapeutic targets for the control of inflammation- and tumor-driven angiogenesis.

Abstract Chronic inflammatory diseases are associated with hematopoietic lineage bias, including neutrophilia and anemia. We have recently identified that the canonical inflammasome mediates the cleavage of the master erythroid transcription factor GATA1 in hematopoietic stem and progenitor cells (HSPCs). We report here that genetic inhibition of Nlrp1 resulted in reduced number of neutrophils and increased erythrocyte counts in zebrafish larvae. We also found that the NLRP1 inflammasome in human cells was inhibited by LRRFIP1 and FLII, independently of DPP9, and both inhibitors regulated hematopoiesis. Mechanistically, erythroid differentiation resulted in ribosomal stress‐induced activation of the ZAKα/P38 kinase axis which, in turn, phosphorylated and promoted the assembly of NLRP1 in both zebrafish and human. Finally, inhibition of Zaka with the FDA/EMA‐approved drug Nilotinib alleviated neutrophilia in a zebrafish model of neutrophilic inflammation and promoted erythroid differentiation and GATA1 accumulation in K562 cells. In conclusion, our results reveal that the NLRP1 inflammasome regulates hematopoiesis and pave the way to develop novel therapeutic strategies for the treatment of hematopoietic alterations associated with chronic inflammatory and rare diseases.

Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD+) metabolism, but the precise mechanism involved is unknown. Here, we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD+ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation through parthanatos cell death.

TNFα overexpression has been associated with several chronic inflammatory diseases, including psoriasis, lichen planus, rheumatoid arthritis, and inflammatory bowel disease. Paradoxically, numerous studies have reported new-onset psoriasis and lichen planus following TNFα antagonist therapy. Here, we show that genetic inhibition of Tnfa and Tnfr2 in zebrafish results in the mobilization of neutrophils to the skin. Using combinations of fluorescent reporter transgenes, fluorescence microscopy, and flow cytometry, we identified the local production of dual oxidase 1 (Duox1)-derived H₂O₂ by Tnfa- and Tnfr2-deficient keratinocytes as a trigger for the activation of the master inflammation transcription factor NF-κB, which then promotes the induction of genes encoding pro-inflammatory molecules. In addition, pharmacological inhibition of Duox1 completely abrogated skin inflammation, placing Duox1-derived H₂O₂ upstream of this positive feedback inflammatory loop. Strikingly, DUOX1 was drastically induced in the skin lesions of psoriasis and lichen planus patients. These results reveal a crucial role for TNFα/TNFR2 axis in the protection of the skin against DUOX1-mediated oxidative stress and could establish new therapeutic targets for skin inflammatory disorders.

Telomere length and telomerase activity are important factors in the pathobiology of human diseases. Age-related diseases and premature aging syndromes are characterized by short telomeres, which can compromise cell viability, whereas tumour cells can prevent telomere loss by aberrantly upregulating telomerase. The zebrafish (Danio rerio) offers multiple experimental manipulation advantages over other vertebrate models and, therefore, it has been recently considered as a potential model for aging, cancer, and regeneration studies. However, it has only partially been exploited to shed light on these fundamental biological processes. The aim of this study was, therefore, to investigate telomere length and telomerase expression and activity in different strains of zebrafish obtained from different stock centres to determine whether they undergo any changes during aging and regeneration. We found that although both telomerase expression and telomere length increased from embryo to adulthood stages, they drastically declined in aged fish despite telomerase activity was detected in different tissues of old fish. In addition, we observed a weaker upregulation of telomerase expression in regenerating fins of old fish, which well correlates with their impaired regeneration capacity. Strikingly, telomeres were elongated or maintained during the fin regeneration process at all ages and after repeated amputations, likely to support high cell proliferation rates. We conclude that the expression of telomerase and telomere length are closely related during the entire life cycle of the fish and that these two parameters can be used as biomarkers of aging in zebrafish. Our results also reveal a direct relationship between the expression of telomerase, telomere length and the efficiency of tissue regeneration.