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Background: Agronomic uses of biochar have been intensely explored in the last 15 years. Recently, a new generation of biochar-based fertilizers has been developed. Raw biochar (BCH), nitrogen-enriched biochar (N + BCH) or urea were added to a coir fiber-based substrate for tomato cultivation, to assess seed germination, growth and fruiting of two cultivars (Cuarenteno and Moneymaker)., Results: BCH stimulated seed germination and early radicle growth, possibly because of the presence of karrikins detected in both BCH and N-BCH (0.039 and 0.044 mg kg -1 , respectively). However, BCH reduced growth in adult plants in both cultivars, probably because of ammonium retention, causing low-N-stress-related symptoms such as accumulation of flavonoids in the leaf. Urea was toxic for seed germination because of the fast release of ammonium, but caused a positive effect on adult plant growth and yield, increasing chlorophyll in both cultivars, quantum yield and ascorbic acid in cv. Cuarenteno, and decreasing flavonoids and peroxide in leaves of both cultivars. Unlike urea, N + BCH showed a positive impact on plant growth and yield, but without releasing high amounts of ammonium or negatively affecting seed germination. Nitrogen-rich amendments reduced phosphorus and increased iron leaf content in both cultivars., Conclusion: BCH can be effectively used as a growth medium constituent in nurseries for seedling production, whereas N + BCH offers a promising alternative to urea or other nitrogen mineral fertilizers for crop cultivation. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

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., (© 2024. The Author(s).)

Introduction: Long 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., Methods: We 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., Results: Pediatric 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., Discussion: This 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Tyrkalska, Pérez-Sanz, Franco-Martínez, Rubio, Tvarijonaviciute, Martínez-Subiela, Méndez-Hernández, González-Aumatell, Carreras-Abad, Domènech-Marçal, Cerón, Cayuela, Mulero and Candel.)

Zebrafish are an emergent animal model to study human diseases due to their significant genetic similarity to humans, swift development, and genetic manipulability. Their utility extends to the exploration of the involvement of inflammation in host defense, immune responses, and tissue regeneration. Additionally, the zebrafish model system facilitates prompt screening of chemical compounds that affect inflammation. This study explored the diverse roles of inflammatory pathways in zebrafish development and aging. Serving as a crucial model, zebrafish provides insights into the intricate interplay of inflammation in both developmental and aging contexts. The evidence presented suggests that the same inflammatory signaling pathways often play instructive or beneficial roles during embryogenesis and are associated with malignancies in adults.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bernal-Bermúdez, Martínez-López, Martínez-Morcillo, Tyrkalska, Martínez-Menchón, Mesa-del-Castillo, Cayuela, Mulero and García-Moreno.)

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., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

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., (© 2023. Springer Nature Limited.)

Fish are the most diverse and successful group of vertebrate animals, with about 30,000 species. The study of fish immunity is of great importance for understanding the evolution of vertebrate immunity, as they are the first animals to show both innate and adaptive immune responses. Although fish immunity is similar to that of mammals, there are obvious differences, such as their dependence of ambient temperature, their poor antibody response, and lack of antibody switching and lymph nodes. In addition, several important differences have also been found between the innate immune responses of fish and mammals. Among these, we will discuss in this review the high resistance of fish to the toxic effects of lipopolysaccharide (LPS) which can be explained by the absence of a Toll-like receptor 4 (Tlr4) ortholog in most fish species or by the inability of the Tlr4/Md2 (Myeloid differentiation 2) complex to recognize LPS, together with the presence of a negative regulator of the LPS signaling complex formed by the TLR-like molecule Rp105 (Radioprotective 105) and Md1. Taken together, these data support the idea that, although TLR4 and RP105 arose from a common ancestor to fish and tetrapods, the TLR4/MD2 receptor complex for LPS recognition arose after their divergence about 450 million years ago., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Atopic dermatitis (AD) is a chronic inflammatory skin disease of very high prevalence, especially in childhood, with no specific treatment or cure. As its pathogenesis is complex, multifactorial and not fully understood, further research is needed to increase knowledge and develop new targeted therapies. We have recently demonstrated the critical role of NAD + and poly (ADP-ribose) (PAR) metabolism in oxidative stress and skin inflammation. Specifically, we found that hyperactivation of PARP1 in response to DNA damage induced by reactive oxygen species, and fueled by NAMPT-derived NAD + , mediated inflammation through parthanatos cell death in zebrafish and human organotypic 3D skin models of psoriasis. Furthermore, the aberrant induction of NAMPT and PARP activity was observed in the lesional skin of psoriasis patients, supporting the role of these signaling pathways in psoriasis and pointing to NAMPT and PARP1 as potential novel therapeutic targets in treating skin inflammatory disorders. In the present work, we report, for the first time, altered NAD + and PAR metabolism in the skin of AD patients and a strong correlation between NAMPT and PARP1 expression and the lesional status of AD. Furthermore, using a human 3D organotypic skin model of AD, we demonstrate that the pharmacological inhibition of NAMPT and PARP reduces pathology-associated biomarkers. These results help to understand the complexity of AD and reveal new potential treatments for AD patients.

One of the most studied defense mechanisms against invading pathogens, including viruses, are Toll-like receptors (TLRs). Among them, TLR3, TLR7, TLR8 and TLR9 detect different forms of viral nucleic acids in endosomal compartments, whereas TLR2 and TLR4 recognize viral structural and nonstructural proteins outside the cell. Although many different TLRs have been shown to be involved in SARS-CoV-2 infection and detection of different structural proteins, most studies have been performed in vitro and the results obtained are rather contradictory. In this study, we report using the unique advantages of the zebrafish model for in vivo imaging and gene editing that the S1 domain of the Spike protein from the Wuhan strain (S1WT) induced hyperinflammation in zebrafish larvae via a Tlr2/Myd88 signaling pathway and independently of interleukin-1β production. In addition, S1WT also triggered emergency myelopoiesis, but in this case through a Tlr2/Myd88-independent signaling pathway. These results shed light on the mechanisms involved in the fish host responses to viral proteins., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Different activation and N-doping treatments were used to produce biochar-based fertilizers (BBFs) with increased N concentration and slow N release. Pristine biochars were produced by pyrolysis of olive tree pruning feedstock at low and high temperatures (400 and 800 °C). These biochars were activated either by ultrasonication, or oxidation with hydrogen peroxide (H 2 O 2 ) or nitric acid (HNO 3 ) to increase their N retention potential. Subsequently biochars were enriched with N with either urea or ammonium sulfate. The activation of low-temperature biochars with HNO 3 was the most effective treatment leading to new surface carboxylic groups that facilitated the later enrichment with N. When treated with urea, BBFs reached 7.0 N%, whereas the H 2 O 2 activation only allowed an increase up to 2.0 N%. The use of urea as the external N source was the most efficient for incorporating N. Urea treated biochars had a water-soluble fraction that represented up to 14.5 % of the total N. The hydrolyzable N fraction, composed by amides and simple N heterocycles originated by the N-doping treatments, and nitro groups generated from HNO 3 activation, represented up to 60 % of the total N. This study relates the N chemical forms in the new BBFs to potential N availability in soil. The presence of water-soluble, hydrolyzable and non-hydrolyzable N implied that these BBFs may supply N that would be progressively available for plants, acting as slow-release fertilizers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Functional or compositional perturbations of the microbiome can occur at different sites, of the body and this dysbiosis has been linked to various diseases. Changes in the nasopharyngeal microbiome are associated to patient's susceptibility to multiple viral infections, supporting the idea that the nasopharynx may be playing an important role in health and disease. Most studies on the nasopharyngeal microbiome have focused on a specific period in the lifespan, such as infancy or the old age, or have other limitations such as low sample size. Therefore, detailed studies analyzing the age- and sex-associated changes in the nasopharyngeal microbiome of healthy people across their whole life are essential to understand the relevance of the nasopharynx in the pathogenesis of multiple diseases, particularly viral infections. One hundred twenty nasopharyngeal samples from healthy subjects of all ages and both sexes were analyzed by 16S rRNA sequencing. Nasopharyngeal bacterial alpha diversity did not vary in any case between age or sex groups. Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were the predominant phyla in all the age groups, with several sex-associated. Acinetobacter , Brevundimonas , Dolosigranulum , Finegoldia , Haemophilus , Leptotrichia , Moraxella , Peptoniphilus , Pseudomonas , Rothia , and Staphylococcus were the only 11 bacterial genera that presented significant age-associated differences. Other bacterial genera such as Anaerococcus , Burkholderia , Campylobacter , Delftia , Prevotella , Neisseria , Propionibacterium , Streptococcus , Ralstonia , Sphingomonas , and Corynebacterium appeared in the population with a very high frequency, suggesting that their presence might be biologically relevant. Therefore, in contrast to other anatomical areas such as the gut, bacterial diversity in the nasopharynx of healthy subjects remains stable and resistant to perturbations throughout the whole life and in both sexes. Age-associated abundance changes were observed at phylum, family, and genus levels, as well as several sex-associated changes probably due to the different levels of sex hormones present in both sexes at certain ages. Our results provide a complete and valuable dataset that will be useful for future research aiming for studying the relationship between changes in the nasopharyngeal microbiome and susceptibility to or severity of multiple diseases.

Although COVID-19 has only recently appeared, research studies have already developed and implemented many animal models for deciphering the secrets of the disease and provided insights into the biology of SARS-CoV-2. However, there are several major factors that complicate the study of this virus in model organisms, such as the poor infectivity of clinical isolates of SARS-CoV-2 in some model species, and the absence of persistent infection, immunopathology, severe acute respiratory distress syndrome, and, in general, all the systemic complications which characterize COVID-19 clinically. Another important limitation is that SARS-CoV-2 mainly causes severe COVID-19 in older people with comorbidities, which represents a serious problem when attempting to use young and immunologically naïve laboratory animals in COVID-19 testing. We review here the main animal models developed so far to study COVID-19 and the unique advantages of the zebrafish model that may help to contribute to understand this disease, in particular to the identification and repurposing of drugs to treat COVID-19, to reveal the mechanism of action and side-effects of Spike-based vaccines, and to decipher the high susceptibility of aged people to COVID-19., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

The coronavirus disease 2019 (COVID-19) pandemic turned the whole world upside down in a short time. One of the main challenges faced has been to understand COVID-19-associated life-threatening hyperinflammation, the so-called cytokine storm syndrome (CSS). We report here the proinflammatory role of Spike (S) proteins from different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern in zebrafish. We found that wild-type/Wuhan variant S1 (S1WT) promoted neutrophil and macrophage recruitment, local and systemic hyperinflammation, emergency myelopoiesis, and hemorrhages. In addition, S1γ was more proinflammatory S1δ was less proinflammatory than S1WT, and, notably, S1β promoted delayed and long-lasting inflammation. Pharmacological inhibition of the canonical inflammasome alleviated S1-induced inflammation and emergency myelopoiesis. In contrast, genetic inhibition of angiotensin-converting enzyme 2 strengthened the proinflammatory activity of S1, and angiotensin (1-7) fully rescued S1-induced hyperinflammation and hemorrhages. These results shed light into the mechanisms orchestrating the COVID-19-associated CSS and the host immune response to different SARS-CoV-2 S protein variants.

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., Competing Interests: I have read the journal policy’s and the authors of this manuscript have the following competing interests: A patent for the use of parthanatos inhibitors to treat psoriasis and atopic dermatitis has been registered by Universidad de Murcia and Instituto Murciano de Investigación Biosanitaria (#PCT/EP2020/083380).

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure and cancer predisposition syndrome caused by mutations in telomerase or telomeric proteins. Here, we report that zebrafish telomerase RNA ( terc ) binds to specific DNA sequences of master myeloid genes and controls their expression by recruiting RNA Polymerase II (Pol II). Zebrafish terc harboring the CR4-CR5 domain mutation found in DC patients hardly interacted with Pol II and failed to regulate myeloid gene expression in vivo and to increase their transcription rates in vitro. Similarly, TERC regulated myeloid gene expression and Pol II promoter occupancy in human myeloid progenitor cells. Strikingly, induced pluripotent stem cells derived from DC patients with a TERC mutation in the CR4-CR5 domain showed impaired myelopoiesis, while those with mutated telomerase catalytic subunit differentiated normally. Our findings show that TERC acts as a transcription factor, revealing a target for therapeutic intervention in DC patients., Competing Interests: Competing interest statement: L.I.Z. is a founder and stockholder of Fate Therapeutics, Inc., Scholar Rock, and Camp4 Therapeutics.

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., (© 2021 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Hematopoiesis is a complex process through which immature bone marrow precursor cells mature into all types of blood cells. Although the association of hematopoietic lineage bias (including anemia and neutrophilia) with chronic inflammatory diseases has long been appreciated, the causes involved are obscure. Recently, cytosolic multiprotein inflammasome complexes were shown to activate inflammatory and immune responses, and directly regulate hematopoiesis in zebrafish models; this was deemed to occur via cleavage and inactivation of the master erythroid transcription factor GATA1. Herein summarized are the zebrafish models that are currently available to study this unappreciated role of inflammasome-mediated regulation of hematopoiesis. Novel putative therapeutic strategies, for the treatment of hematopoietic alterations associated with chronic inflammatory diseases in humans, are also proposed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Biochar (BC) has been shown to influence microbial denitrification and mitigate soil N2O emissions. However, it is unclear if BC is able to directly stimulate the microbial reduction of N2O to N2. We hypothesized that the ability of BC to lower N2O emissions could be related not only to its ability to store electrons, but to donate them to bacteria that enzymatically reduce N2O. Therefore, we carried out anoxic incubations with Paracoccus denitrificans, known amounts of N2O, and nine contrasting BCs, in the absence of any other electron donor or acceptor. We found a strong and direct correlation between the extent and rates of N2O reduction with BC's EDC/EEC (electron donating capacity/electron exchange capacity). Apart from the redox capacity, other BC properties were found to regulate the BC's ability to increase N2O reduction by P. denitrificans. For this specific BC series, we found that a high H/C and ash content, low surface area and poor lignin feedstocks favored N2O reduction. This provides valuable information for producing tailored BCs with the potential to assist and promote the reduction of N2O in the pursuit of reducing this greenhouse gas emissions., (© FEMS 2020.)

Psoriasis is a skin inflammatory disorder that affects 3% of the human population. Although several therapies based on the neutralization of proinflammatory cytokines have been used with relative success, additional treatments are required. The in silico analysis of gene expression data of psoriasis lesional skin and an analysis of vitamin B6 metabolites in the sera of psoriasis patients point to altered vitamin B6 metabolism at both local and systemic levels. Functional studies showed that vitamin B6 vitamers reduced skin neutrophil infiltration, oxidative stress and Nfkb activity in two zebrafish models of skin inflammation. Strikingly, inhibition of glycogen phosphorylase L (Pygl) and glucose-6-phosphate dehydrogenase (G6pd), two vitamin B6-regulated enzymes, alleviated oxidative-stress induced inflammation in zebrafish skin inflammation models. Despite the central role of G6pd in antioxidant defenses, the results of the study demonstrate that glycogen stores and G6pd fuel NADPH oxidase to promote skin inflammation, revealing novel targets for the treatment of skin inflammatory disorders., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The zebrafish has become an excellent model for the study of inflammation and immunity. Its unique advantages for in vivo imaging and gene and drug screening have allowed the visualization of dual oxidase 1 (Duox1)-derived hydrogen peroxide (H 2 O 2 ) tissue gradients and its crosstalk with neutrophil infiltration to inflamed tissue. Thus, it has been shown that H 2 O 2 directly recruits neutrophils via the Src-family tyrosine kinase Lyn and indirectly by the activation of several signaling pathways involved in inflammation, such as nuclear factor κB (NF-κB), mitogen activated kinases and the transcription factor AP1. In addition, this model has also unmasked the unexpected ability of H 2 O 2 to induce the expression of the gene encoding the key neutrophil chemoattractant CXC chemokine ligand 8 by facilitating the accessibility of transcription factors to its promoter through histone covalent modifications. Finally, zebrafish models of psoriasis have shown that a H 2 O 2 /NF-κB/Duox1 positive feedback inflammatory loop operates in this chronic inflammatory disorder and that pharmacological inhibition of Duox1, but not of downstream mediators, inhibits inflammation and restores epithelial homeostasis. Therefore, these results have pointed out DUOX1 and H 2 O 2 as therapeutic targets for the treatment of skin inflammatory disorders, such as psoriasis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy. Although novel emerging drugs are available, the overall prognosis remains poor and new therapeutic approaches are required. PP2A phosphatase is a key regulator of cell homeostasis and is recurrently inactivated in AML. The anticancer activity of several PP2A-activating drugs (e.g., FTY720) depends on their interaction with the SET oncoprotein, an endogenous PP2A inhibitor that is overexpressed in 30% of AML cases. Elucidation of SET regulatory mechanisms may therefore provide novel targeted therapies for SET-overexpressing AMLs. Here, we show that upregulation of protein kinase p38β is a common event in AML. We provide evidence that p38β potentiates SET-mediated PP2A inactivation by two mechanisms: facilitating SET cytoplasmic translocation through CK2 phosphorylation, and directly binding to and stabilizing the SET protein. We demonstrate the importance of this new regulatory mechanism in primary AML cells from patients and in zebrafish xenograft models. Accordingly, combination of the CK2 inhibitor CX-4945, which retains SET in the nucleus, and FTY720, which disrupts the SET-PP2A binding in the cytoplasm, significantly reduces the viability and migration of AML cells. In conclusion, we show that the p38β/CK2/SET axis represents a new potential therapeutic pathway in AML patients with SET-dependent PP2A inactivation.

Acute myeloid leukemia (AML) is an aggressive disease associated with very poor prognosis. Most patients are older than 60 years, and in this group only 5-15% of cases survive over 5 years. Therefore, it is urgent to develop more effective targeted therapies. Inactivation of protein phosphatase 2 A (PP2A) is a recurrent event in AML, and overexpression of its endogenous inhibitor SET is detected in ~30% of patients. The PP2A activating drug FTY720 has potent anti-leukemic effects; nevertheless, FTY720 induces cardiotoxicity at the anti-neoplastic dose. Here, we have developed a series of non-phosphorylable FTY720 analogues as a new therapeutic strategy for AML. Our results show that the lead compound CM-1231 re-activates PP2A by targeting SET-PP2A interaction, inhibiting cell proliferation and promoting apoptosis in AML cell lines and primary patient samples. Notably, CM-1231 did not induce cardiac toxicity, unlike FTY720, in zebrafish models, and reduced the invasion and aggressiveness of AML cells more than FTY720 in zebrafish xenograft models. In conclusion, CM-1231 is safer and more effective than FTY720; therefore, this compound could represent a novel and promising approach for treating AML patients with SET overexpression., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

Chronic inflammatory diseases are associated with altered hematopoiesis that could result in neutrophilia and anemia. Here we report that genetic or chemical manipulation of different inflammasome components altered the differentiation of hematopoietic stem and progenitor cells (HSPC) in zebrafish. Although the inflammasome was dispensable for the emergence of HSPC, it was intrinsically required for their myeloid differentiation. In addition, Gata1 transcript and protein amounts increased in inflammasome-deficient larvae, enforcing erythropoiesis and inhibiting myelopoiesis. This mechanism is evolutionarily conserved, since pharmacological inhibition of the inflammasome altered erythroid differentiation of human erythroleukemic K562 cells. In addition, caspase-1 inhibition rapidly upregulated GATA1 protein in mouse HSPC promoting their erythroid differentiation. Importantly, pharmacological inhibition of the inflammasome rescued zebrafish disease models of neutrophilic inflammation and anemia. These results indicate that the inflammasome plays a major role in the pathogenesis of neutrophilia and anemia of chronic diseases and reveal druggable targets for therapeutic interventions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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 C ARD- and A NK-containing Inflammasome A daptor P rotein), 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.

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., Competing Interests: The authors have declared that no competing interests exist.

Dyskeratosis congenita (DC) is an inherited disorder with mutations affecting telomerase or telomeric proteins. DC patients usually die of bone marrow failure. Here we show that genetic depletion of the telomerase RNA component (TR) in the zebrafish results in impaired myelopoiesis, despite normal development of haematopoietic stem cells (HSCs). The neutropenia caused by TR depletion is independent of telomere length and telomerase activity. Genetic analysis shows that TR modulates the myeloid-erythroid fate decision by controlling the levels of the master myeloid and erythroid transcription factors spi1 and gata1, respectively. The alteration in spi1 and gata1 levels occurs through stimulation of gcsf and mcsf. Our model of TR deficiency in the zebrafish illuminates the non-canonical roles of TR, and could establish therapeutic targets for DC.

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.

Objective: The aim of this study is to identify a set of microRNAs (miRNAs) as prognostic molecular biomarkers for the progression of Barrett esophagus (BE) to esophageal adenocarcinoma (EAC) to rationalize the surveillance programs in patients with BE., Background: Histological dysplasia is currently used as the main biomarker to identify the BE patients at high risk for developing EAC. Although miRNA expression profiles in BE and EAC have been reported, it has not been established which set of miRNAs could constitute a robust diagnostic test to predict the progression of BE to EAC., Methods: miRNAs associated with progression of BE to EAC were identified using miRNA sequencing analysis. Further validation by quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in 2 groups of BE patients who either developed or did not develop adenocarcinoma after at least 5 years of follow-up., Results: Twenty-three miRNAs were identified by miRNA sequencing analysis in the carcinogenesis process associated with BE. qRT-PCR analysis using independent tissue samples confirmed differential expression for 19 of them (miR-let-7c, 7, 146a, 149, 153, 192, 192*, 194, 194*, 196a, 196b, 200a, 203, 205, 215, 424, 625, 625*, and 944). However, only miR-192, 194, 196a, and 196b showed a significantly higher expression in BE samples from patients with progression to EAC compared with those who did not progress to EAC., Conclusions: These findings suggest that the expression pattern of a modest number of miRNAs in metaplasia biopsies could identify the BE patients at high risk for developing EAC. Therefore, it has potential use for the control and treatment of this malignancy.

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.

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.

It has long been established that lower vertebrates, most notably fish and amphibians, are resistant to the toxic effect of LPS. Furthermore, the lack of a TLR4 ortholog in some fish species and the lack of the essential costimulatory molecules for LPS activation via TLR4 (i.e., myeloid differentiation protein 2 (MD-2) and CD14) in all the fish genomes and expressed sequence tag databases available led us to hypothesize that the mechanism of LPS recognition in fish may be different from that of mammals. To shed light on the role of fish TLRs in LPS recognition, a dual-luciferase reporter assay to study NF-kappaB activation in whole zebrafish embryos was developed and three different bony fish models were studied: 1) the gilthead seabream (Sparus aurata, Perciformes), an immunological-tractable teleost model in which the presence of a TLR4 ortholog is unknown; 2) the spotted green pufferfish (Tetraodon nigroviridis, Tetraodontiformes), which lacks a TLR4 ortholog; and 3) the zebrafish (Danio rerio, Cypriniformes), which possesses two TLR4 orthologs. Our results show that LPS signaled via a TLR4- and MyD88-independent manner in fish, and, surprisingly, that the zebrafish TLR4 orthologs negatively regulated the MyD88-dependent signaling pathway. We think that the identification of TLR4 as a negative regulator of TLR signaling in the zebrafish, together with the absence of this receptor in most fish species, explains the resistance of fish to endotoxic shock and supports the idea that the TLR4 receptor complex for LPS recognition arose after the divergence of fish and tetrapods.

Telomerase confers limitless proliferative potential to most human cells through its ability to elongate telomeres, the natural ends of chromosomes, which otherwise would undergo progressive attrition and eventually compromise cell viability. However, the role of telomerase in organismal aging has remained unaddressed, in part because of the cancer-promoting activity of telomerase. To circumvent this problem, we have constitutively expressed telomerase reverse transcriptase (TERT), one of the components of telomerase, in mice engineered to be cancer resistant by means of enhanced expression of the tumor suppressors p53, p16, and p19ARF. In this context, TERT overexpression improves the fitness of epithelial barriers, particularly the skin and the intestine, and produces a systemic delay in aging accompanied by extension of the median life span. These results demonstrate that constitutive expression of Tert provides antiaging activity in the context of a mammalian organism.

Background: The dual-luciferase assay has been widely used in cell lines to determine rapidly but accurately the activity of a given promoter. Although this strategy has proved very useful, it does not allow the promoter and gene function to be analyzed in the context of the whole organism., Results: Here, we present a rapid and sensitive assay based on the classical dual-luciferase reporter technique which can be used as a new tool to characterize the minimum promoter region of a gene as well as the in vivo response of inducible promoters to different stimuli. We illustrate the usefulness of this system for studying both constitutive (telomerase) and inducible (NF-kappaB-dependent) promoters. The flexibility of this assay is demonstrated by induction of the NF-kappaB-dependent promoters using simultaneous microinjection of different pathogen-associated molecular patterns as well as with the use of morpholino-gene mediated knockdown., Conclusion: This assay has several advantages compared with the classical in vitro (cell lines) and in vivo (transgenic mice) approaches. Among others, the assay allows a rapid and quantitative measurement of the effects of particular genes or drugs in a given promoter in the context of a whole organism and it can also be used in high throughput screening experiments.

The overproduction of cytokines, such us interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), contributes to the pathological complications observed in many inflammatory diseases caused by bacterial endotoxins. The synthesis of these cytokines is tightly regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of gene expression depends on specific cis-acting sequences and trans-acting factors. Thus, the presence of adenylate- and uridylate-rich (AU-rich) elements (AREs) has been described in the 3'-untranslated regions (UTRs) of many unstable mammalian mRNAs. Although, it represents the most widespread, phylogenetically conserved and efficient determinant of mRNA stability among those so far characterized in mammalian cells, no studies are available on the functional relevance of this sequence in non-mammalian vertebrates. In this contribution, we study the enzymatic activity of various luciferase reporter constructs, containing or lacking the 3'UTR of IL-1beta and TNFalpha from different fish species, and report the finding that bony fish AREs are able to decrease luciferase activity but are less potent than their mammalian counterparts. Surprisingly, the 3'UTR of the IL-1beta from the cartilaginous fish small spotted catshark had the greatest ability to decrease luciferase activity. Lastly, the functional significance of the above was confirmed by measuring the half-life of IL-1beta and TNFalpha mRNAs in gilthead seabream leukocytes by blocking transcription with actinomycin D. Both cytokine mRNAs were unstable with an estimated half-life of about 45 min in control and activated cells.

A key process in organ homeostasis is the mobilization of stem cells out of their niches. We show through analysis of mouse models that telomere length, as well as the catalytic component of telomerase, Tert, are critical determinants in the mobilization of epidermal stem cells. Telomere shortening inhibited mobilization of stem cells out of their niche, impaired hair growth, and resulted in suppression of stem cell proliferative capacity in vitro. In contrast, Tert overexpression in the absence of changes in telomere length promoted stem cell mobilization, hair growth, and stem cell proliferation in vitro. The effects of telomeres and telomerase on stem cell biology anticipate their role in cancer and aging.

Transcriptional factor CarD is the only reported prokaryotic analog of eukaryotic high-mobility-group A (HMGA) proteins, in that it has contiguous acidic and AT hook DNA-binding segments and multifunctional roles in Myxococcus xanthus carotenogenesis and fruiting body formation. HMGA proteins are small, randomly structured, nonhistone, nuclear architectural factors that remodel DNA and chromatin structure. Here we report on a second AT hook protein, CarD(Sa), that is very similar to CarD and that occurs in the bacterium Stigmatella aurantiaca. CarD(Sa) has a C-terminal HMGA-like domain with three AT hooks and a highly acidic adjacent region with one predicted casein kinase II (CKII) phosphorylation site, compared to the four AT hooks and five CKII sites in CarD. Both proteins have a nearly identical 180-residue N-terminal segment that is absent in HMGA proteins. In vitro, CarD(Sa) exhibits the specific minor-groove binding to appropriately spaced AT-rich DNA that is characteristic of CarD or HMGA proteins, and it is also phosphorylated by CKII. In vivo, CarD(Sa) or a variant without the single CKII phosphorylation site can replace CarD in M. xanthus carotenogenesis and fruiting body formation. These two cellular processes absolutely require that the highly conserved N-terminal domain be present. Thus, three AT hooks are sufficient, the N-terminal domain is essential, and phosphorylation in the acidic region by a CKII-type kinase can be dispensed with for CarD function in M. xanthus carotenogenesis and fruiting body development. Whereas a number of hypothetical proteins homologous to the N-terminal region occur in a diverse array of bacterial species, eukaryotic HMGA-type domains appear to be confined primarily to myxobacteria.