cGMP via PKG activates 26S proteasomes and enhances degradation of proteins, including ones that cause neurodegenerative diseases.

Because raising cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, we investigated whether agents that raise cGMP may also regulate protein degradation. Treating various cell lines with inhibitors of phosphodiesterase 5 or stimulators of soluble guanylyl cyclase rapidly enhanced multiple proteasome activities and cellular levels of ubiquitinated proteins by activating protein kinase G (PKG). PKG stimulated purified 26S proteasomes by phosphorylating a different 26S component than is modified by protein kinase A. In cells and cell extracts, raising cGMP also enhanced within minutes ubiquitin conjugation to cell proteins. Raising cGMP, like raising cAMP, stimulated the degradation of short-lived cell proteins, but unlike cAMP, also markedly increased proteasomal degradation of long-lived proteins (the bulk of cell proteins) without affecting lysosomal proteolysis. We also tested if raising cGMP, like cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases. Treating zebrafish models of tauopathies or Huntington's disease with a PDE5 inhibitor reduced the levels of the mutant huntingtin and tau proteins, cell death, and the resulting morphological abnormalities. Thus, PKG rapidly activates cytosolic proteasomes, protein ubiquitination, and overall protein degradation, and agents that raise cGMP may help combat the progression of neurodegenerative diseases

A zebrafish model of Ifih1-driven Aicardi–Goutières syndrome reproduces the interferon signature and the exacerbated inflammation of 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

TNFα Impairs Rhabdoviral Clearance by Inhibiting the Host Autophagic Antiviral Response.

TNFα is a pleiotropic pro-inflammatory cytokine with a key role in the activation of the immune system to fight viral infections. Despite its antiviral role, a few viruses might utilize the host produced TNFα to their benefit. Some recent reports have shown that anti-TNFα therapies could be utilized to treat certain viral infections. However, the underlying mechanisms by which TNFα can favor virus replication have not been identified. Here, a rhabdoviral infection model in zebrafish allowed us to identify the mechanism of action by which Tnfa has a deleterious role for the host to combat certain viral infections. Our results demonstrate that Tnfa signals through its receptor Tnfr2 to enhance viral replication. Mechanistically, Tnfa does not affect viral adhesion and delivery from endosomes to the cytosol. In addition, the host interferon response was also unaffected by Tnfa levels. However, Tnfa blocks the host autophagic response, which is required for viral clearance. This mechanism of action provides new therapeutic targets for the treatment of SVCV-infected fish, and advances our understanding of the previously enigmatic deleterious role of TNFα in certain viral infections

The nasopharyngeal microbiome in COVID-19

The development of novel culture-independent techniques of microbial identification has allowed a rapid progress in the knowledge of the nasopharyngeal microbiota and its role in health and disease. Thus, it has been demonstrated that the nasopharyngeal microbiota defends the host from invading pathogens that enter the body through the upper airways by participating in the modulation of innate and adaptive immune responses. The current COVID-19 pandemic has created an urgent need for fast-track research, especially to identify and characterize biomarkers to predict the disease severity and outcome. Since the nasopharyngeal microbiota diversity and composition could potentially be used as a prognosis biomarker for COVID-19 patients, which would pave the way for strategies aiming to reduce the disease severity by modifying such microbiota, dozens of research articles have already explored the possible associations between changes in the nasopharyngeal microbiota and the severity or outcome of COVID-19 patients. Unfortunately, results are controversial, as many studies with apparently similar experimental designs have reported contradictory data. Herein we put together, compare, and discuss all the relevant results on this issue reported to date. Even more interesting, we discuss in detail which are the limitations of these studies, that probably are the main sources of the high variability observed. Therefore, this work is useful not only for people interested in current knowledge about the relationship between the nasopharyngeal microbiota and COVID-19, but also for researchers who want to go further in this field while avoiding the limitations and variability of previous works

Analysis of 16S rRNA Gene Sequence of Nasopharyngeal Exudate Reveals Changes in Key Microbial Communities Associated with Aging

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

Identification of an Evolutionarily Conserved Ankyrin Domain-Containing Protein, Caiap, Which Regulates Inflammasome-Dependent Resistance to Bacterial Infection

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

Differential proinflammatory activities of Spike proteins of SARS-CoV-2 variants of concern

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 51 (S1WT) promoted neutrophil and macrophage recruitment, local and systemic hyperinflammation, emergency myelopoiesis, and hemorrhages. In addition, S1 gamma was more proinflammatory while S1 delta was less proinflammatory than S1WT, and, notably, S1 beta 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

Image_4_A zebrafish model of Ifih1-driven Aicardi–Goutières syndrome reproduces the interferon signature and the exacerbated inflammation of patients.jpeg

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.</p