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RNase2 is the member of the RNaseA family most abundant in macrophages. Here, we knocked out RNase2 in THP-1 cells and analysed the response to Respiratory Syncytial Virus (RSV). RSV induced RNase2 expression, which significantly enhanced cell survival. Next, by cP-RNAseq sequencing, which amplifies the cyclic-phosphate endonuclease products, we analysed the ncRNA population. Among the ncRNAs accumulated in WT vs KO cells, we found mostly tRNA-derived fragments (tRFs) and second miRNAs. Differential sequence coverage identified tRFs from only few parental tRNAs, revealing a predominant cleavage at anticodon and D-loops at U/C (B1) and A (B2) sites. Selective tRNA cleavage was confirmed in vitro using the recombinant protein. Likewise, only few miRNAs were significantly more abundant in WT vs RNase2-KO cells. Complementarily, by screening of a tRF & tiRNA array, we identified an enriched population associated to RNase2 expression and RSV exposure. The results confirm the protein antiviral action and provide the first evidence of its cleavage selectivity on ncRNAs. Funding: Open Access Funding provided by Universitat Autònoma de Barcelona. This research was founded by Research work was supported by the Ministerio de Economía y Competitividad (SAF2015-66007P), co-financed by FEDER funds, by Agencia Estatal de Investigación (PID2019-106123GB-I00/AEI/10.13039/501100011033) and by Fundació La Marató de TV3 (2080310). LL and JL were supported by China Scholarship Council (CSC) predoctoral fellowships

RNase2, also named the Eosinophil derived Neurotoxin (EDN), is one of the main proteins secreted by the eosinophil secondary granules. RNase2 is also expressed in other leukocyte cells and is the member of the human ribonuclease A family most abundant in macrophages. The protein is endowed with a high ribonucleolytic activity and participates in the host antiviral activity. Although RNase2 displays a broad antiviral activity, it is mostly associated to the targeting of single stranded RNA viruses. To explore RNase2 mechanism of action in antiviral host defence we knocked out RNase2 expression in the THP1 monocyte cell line and characterized the cell response to human Respiratory Syncytial Virus (RSV). We observed that RSV infection induced the RNase2 expression and protein secretion in THP1 macrophage-derived cells, whereas the knockout (KO) of RNase2 resulted in higher RSV burden and reduced cell viability. Next, by means of the cP-RNAseq methodology, which uniquely amplifies the RNA 2’3’cyclic-phosphate-end products released by an endonuclease cleavage, we compared the ncRNA population in native and RNase2-KO cell lines. Among the ncRNAs accumulated in WT versus KO cells, we found mostly tRNA-derived fragments and secondly miRNAs. Analysis of the differential sequence coverage of tRNAs molecules in native and KO cells identified fragments derived from only few parental tRNAs, revealing a predominant cleavage at anticodon loops and secondarily at D-loops. Inspection of cleavage region identified U/C and A, at 5’ and 3’ sides of cleavage sites respectively (namely RNase B1 and B2 base binding subsites). Likewise, only few selected miRNAs were significantly more abundant in WT versus RNase2-KO cells, with cleavage sites located at the end of stem regions with predominance for pyrimidines at B1 but following an overall less defined nucleotide specificity.Complementarily, by screening of a tRF&tiRNA PCR array we identified an enriched population of tRNA-derived fragments associated to RNase2 expression and RSV infection. The present results confirm the contribution of the protein in macrophage response against virus infection and provide the first evidence of its cleavage selectivity against ncRNA population. A better understanding of the mechanism of action of RNase2 recognition of cellular RNA during the antiviral host defence should pave the basis for the design of novel antiviral drugs.

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Background The microbiota of the female reproductive tract is increasingly recognized as playing fundamental roles in animal reproduction. To explore the relative contribution of reproductive tract microbiomes to egg production in chickens, we investigated the microbiota in multiple reproductive and digestive tract sites from 128 female layer (egg-producing) chickens in comparable environments. Results We identified substantial differences between the diversity, composition, and predicted function of site-associated microbiota. Differences in reproductive tract microbiota were more strongly associated with egg production than those in the digestive tract. We identified 4 reproductive tract microbial species, Bacteroides fragilis, Bacteroides salanitronis, Bacteroides barnesiae, and Clostridium leptum, that were related to immune function and potentially contribute to enhanced egg production. Conclusions These findings provide insights into the diverse microbiota characteristics of reproductive and digestive tracts and may help in designing strategies for controlling and manipulating chicken reproductive tract microbiota to improve egg production.

Tuberculosis (TB) is still a leading cause of death worldwide. Treatments remain unsatisfactory due to an incomplete understanding of the underlying host&ndash, pathogen interactions during infection. In the present study, weighted gene co-expression network analysis (WGCNA) was conducted to identify key macrophage modules and hub genes associated with mycobacterial infection. WGCNA was performed combining our own transcriptomic results using Mycobacterium aurum-infected human monocytic macrophages (THP1) with publicly accessible datasets obtained from three types of macrophages infected with seven different mycobacterial strains in various one-to-one combinations. A hierarchical clustering tree of 11,533 genes was built from 198 samples, and 47 distinct modules were revealed. We identified a module, consisting of 226 genes, which represented the common response of host macrophages to different mycobacterial infections that showed significant enrichment in innate immune stimulation, bacterial pattern recognition, and leukocyte chemotaxis. Moreover, by network analysis applied to the 74 genes with the best correlation with mycobacteria infection, we identified the top 10 hub-connecting genes: NAMPT, IRAK2, SOCS3, PTGS2, CCL20, IL1B, ZC3H12A, ABTB2, GFPT2, and ELOVL7. Interestingly, apart from the well-known Toll-like receptor and inflammation-associated genes, other genes may serve as novel TB diagnosis markers and potential therapeutic targets.

Background: The microbiota of the digestive and reproductive systems has a prominent role in animal health and performance, but the extent of its contribution is difficult to determine. In chickens, the effect of host genetics on the reproductive and digestive tract microbiota is unclear, and the means by which digestive/reproductive microbiomes help improve egg production in chicken are unknown.Results: To gain insight into this, we examined genomes from 128 chickens reared under identical conditions and described their digestive (crop, gizzard and small intestine) and reproductive tract (vagina, uterus and isthmus) microbiota. Although the diversity, composition and predicted function of the digestive and reproductive tract microbiota exhibited notable microbiota variation substantially between different parts, host genetics had limited effects on the reproductive and digestive tract microbial community. The digestive and reproductive tract microbiota had a significant effect on egg production (accounting for 52.31% - 98.86% of the variance), after correcting for host genetic effects; in particular, the uterus and isthmus microbiota accounted for an average of 93.59% and 98.86%, respectively, of variance in egg production. We further identified four reproductive tract microbial species which were related to immune system, Bacteroides fragilis, Bacteroides salanitronis, Bacteroides barnesiae and Clostridium leptum, that were significantly positively correlated with egg production. Chickens with a lower abundance of these species had produced significantly fewer eggs at 300 days of age (37.13 vs. 113.75) than those with a higher abundance of these microorganisms. These taxa indicate potential roles play in promoting reproductive performance. Especially uterus and isthmus tract microbiota were major factors in regulating the chicken egg production.Conclusions: Host genetics has limited effect on digestive/reproductive microbiome composition. The distinct site-associated chicken microbiome may be determined by the differences of their physical function. These findings may help design strategies for controlling and altering the digestive/reproductive tract microbiota in chickens to improve egg production.

The human RNase3 is a member of the RNaseA superfamily involved in host immunity. RNase3 is expressed by leukocytes and shows broad-spectrum antimicrobial activity. Together with a direct antimicrobial action, RNase3 exhibits immunomodulatory properties. Here, we have analysed the transcriptome of macrophages exposed to the wild-type protein and a catalytic-defective mutant (RNase3-H15A). The analysis of differently expressed genes (DEGs) in treated THP1-derived macrophages highlighted a common pro-inflammatory “core-response” independent of the protein ribonucleolytic activity. Network analysis identified the epidermal growth factor receptor (EGFR) as the main central regulatory protein. Expression of selected DEGs and MAPK phosphorylation were inhibited by an anti-EGFR antibody. Structural analysis suggested that RNase3 activates the EGFR pathway by direct interaction with the receptor. Besides, we identified a subset of DEGs related to the protein ribonucleolytic activity, characteristic of virus infection response. Transcriptome analysis revealed an early pro-inflammatory response, not associated to the protein catalytic activity, followed by a late activation in a ribonucleolytic-dependent manner. Next, we demonstrated that overexpression of macrophage endogenous RNase3 protects the cells against infection by Mycobacterium aurum and the human respiratory syncytial virus. Comparison of cell infection profiles in the presence of Erlotinib, an EGFR inhibitor, revealed that the receptor activation is required for the antibacterial but not for the antiviral protein action. Moreover, the DEGs related and unrelated to the protein catalytic activity are associated to the immune response to bacterial and viral infection, respectively. We conclude that RNase3 modulates the macrophage defence against infection in both catalytic-dependent and independent manners. Electronic supplementary material The online version of this article (10.1007/s00018-020-03695-5) contains supplementary material, which is available to authorized users.

Timeless(Tim) is considered to function as an essential circadian clock gene inDrosophila. Putative homologues of theDrosophila timelessgene have been identified in both mice and humans. WhileDrosophilacontains two paralogs,timelessandtimeout, acting in clock/light entrainment and chromosome integrity/photoreception, respectively, mammals contain only oneTimhomolog. In this paper, we study the phylogeny of thetimeless/timeoutfamily in 48 species [including 1 protozoan (Guillardia theta), 1 nematode (Caenorhabditis elegans), 8 arthropods and 38 chordates], for which whole genome data are available by using MEGA (Molecular Evolutionary Genetics Analysis). Phylogenetic Analysis by Maximum Likelihood (PAML) was used to analyze the selective pressure acting on metazoantimeless/timeoutgenes. Our phylogeny clearly separates insecttimelessandtimeoutlineages and shows that non-insect animalTimgenes are homologs of insecttimeout. In this study, we explored the relatively rapidly evolvingtimelesslineage that was apparently lost from most deuterostomes, including chordates, and fromCaenorhabditis elegans. In contrast, we found that thetimeoutprotein, often confusingly called “timeless” in the vertebrate literature, is present throughout the available animal genomes. Selection results showed thattimeoutis under weaker negative selection thantimeless. Finally, our phylogeny oftimeless/timeoutshowed an evolutionary conservation of the circadian clock genetimeoutin Metazoa. This conservation is in line with its multifunctionality, being essential for embryonic development and maintenance of chromosome integrity, among others.