Search Results (26)

DNase II, identified in 1947 and named in 1953, is an acidic DNA endonuclease prevalent across organisms and crucial for normal growth. Despite its expression in nearly all human tissues, as well as its biological significance, DNase II’s detailed functions and corresponding mechanisms remain unclear. Although many groups are trying to figure this out, progress is very limited. It is very hard to connect its indispensability with its DNA cleavage activity. In this study, we find that DNase II secreted to saliva can digest RNA in mildly acidic conditions, prompting us to hypothesize that salivary DNase II might digest RNA in the stomach. This finding is consistent with the interesting discovery reported in 1964 that RNA could inhibit DNase II’s activity, which has been largely overlooked. This RNA digestion activity is further confirmed by using purified DNase II, showing activity to digest both DNA and RNA effectively. Here, we suggest redesignating DNase II as DNase II (RNase). The biological functions of DNase II are suggested to recycle intracellular RNA or digest external nucleic acids (both RNA and DNA) as nutrients. This discovery may untangle the mystery of DNase II and its significant biofunctions. Full article

Circular RNA (circRNA) is a type of single-stranded RNA that forms a covalently closed continuous loop, unlike linear RNA. The expression of circRNAs in mammals is often conserved across species and shows tissue and cell specificity. Some circRNA serve as gene regulators. However, the biological function of most circRNAs is unclear. CircRNA does not have 5′ or 3′ ends. The unique structure of circRNAs provides them with a much longer half-life and more resistance to RNase R than linear RNAs. Inflammatory lung responses occur in the pathogenesis and recovery of many lung diseases. Macrophages form the first line of host defense/innate immune responses and initiate/mediate lung inflammation. For example, in bacterial pneumonia, upon pro-inflammatory activation, they release early response cytokines/chemokines that recruit neutrophils, macrophages, and lymphocytes to sites of infection and clear pathogens. The functional effects and mechanisms by which circRNAs exert physiological or pathological roles in macrophage activation and lung inflammation remain poorly understood. In this article, we will review the current understanding and progress of circRNA biogenesis, regulation, secretion, and degradation. Furthermore, we will review the current reports on the role of circRNAs in macrophage activation and polarization, as well as in the process of inflammatory lung responses. Full article

Heat shock protein 47 (HSP47), also known as SERPINH1, functions as a collagen-specific molecular chaperone protein essential for the formation and stabilization of the collagen triple helix. Here, we delved into the regulatory pathways governed by HSP47, shedding light on collagen homeostasis. Our investigation revealed a significant reduction in HSP47 mRNA levels in the skin tissue of older mice as compared to their younger counterparts. The augmented expression of HSP47 employing lentivirus infection in fibroblasts resulted in an increased secretion of type I collagen. Intriguingly, the elevated expression of HSP47 in fibroblasts correlated with increased protein and mRNA levels of type I collagen. The exposure of fibroblasts to IRE1α RNase inhibitors resulted in the reduced manifestation of HSP47-induced type I collagen secretion and expression. Notably, HSP47-overexpressing fibroblasts exhibited increased XBP1 mRNA splicing. The overexpression of HSP47 or spliced XBP1 facilitated the nuclear translocation of β-catenin and transactivated a reporter harboring TCF binding sites on the promoter. Furthermore, the overexpression of HSP47 or spliced XBP1 or the augmentation of nuclear β-catenin through Wnt3a induced the expression of type I collagen. Our findings substantiate that HSP47 enhances type I collagen expression and secretion in fibroblasts by orchestrating a mechanism that involves an increase in nuclear β-catenin through IRE1α activation and XBP1 splicing. This study therefore presents potential avenues for an anti-skin-aging strategy targeting HSP47-mediated processes. Full article

Tumor necrosis is a recurrent characteristic of head and neck squamous cell carcinomas (HNSCCs). There is a need for more investigations on the influence of biomolecules released by these necrotic foci in the HNSCC tumor microenvironment. It is suspected that a fraction of the biomolecules released by necrotic cells are damage-associated molecular patterns (DAMPs), which are known to be natural endogenous ligands of Toll-like receptors (TLRs), including, among others, proteins and nucleic acids. However, there has been no direct demonstration that biomolecules released by HNSCC necrotic cells can activate TLRs. Our aim was to investigate whether some of these molecules could behave as agonists of the TLR3, either in vitro or in vivo. We chose a functional approach based on reporter cell exhibiting artificial TLR3 expression and downstream release of secreted alkaline phosphatase. The production of biomolecules activating TLR3 was first investigated in vitro using three HNSCC cell lines subjected to various pronecrotic stimuli (external irradiation, serum starvation, hypoxia and oxidative stress). TLR3 agonists were also investigated in necrotic tumor fluids from five oral cancer patients and three mouse tumor grafts. The release of biomolecules activating TLR3 was demonstrated for all three HNSCC cell lines. External irradiation was the most consistently efficient stimulus, and corresponding TLR3 agonists were conveyed in extracellular vesicles. TLR3-stimulating activity was detected in the fluids from all five patients and three mouse tumor grafts. In most cases, this activity was greatly reduced by RNAse pretreatment or TLR3 blocking antibodies. Our data indicate that TLR3 agonists are consistently present in necrotic fluids from HNSCC cells and mainly made of dsRNA fragments. These endogenous agonists may induce TLR3, which might lead to a protumorigenic effect. Regarding methodological aspects, our study demonstrates that direct investigations—including functional testing—can be performed on necrotic fluids from patient tumors. Full article

Bacillus licheniformis produces several classes of antimicrobial substances, including bacteriocins, which are peptides or proteins with different structural composition and molecular mass: ribosomally synthesized by bacteria (1.4–20 kDa), non-ribosomally synthesized peptides and cyclic lipopeptides (0.8–42 kDa) and exopolysaccharides (>1000 kDa). Different bacteriocins act against Gram-positive or Gram-negative bacteria, fungal pathogens and amoeba cells. The main mechanisms of bacteriocin lytic activity include interaction of peptides with membranes of target cells resulting in structural alterations, pore-forming, and inhibition of cell wall biosynthesis. DNase and RNase activity for some bacteriocines are also postulated. Non-ribosomal peptides are synthesized by special non-ribosomal multimodular peptide synthetases and contain unnatural amino acids or fatty acids. Their harmful effect is due to their ability to form pores in biological membranes, destabilize lipid packaging, and disrupt the peptidoglycan layer. Lipopeptides, as biosurfactants, are able to destroy bacterial biofilms. Secreted polysaccharides are high molecular weight compounds, composed of repeated units of sugar moieties attached to a carrier lipid. Their antagonistic action was revealed in relation to bacteria, viruses, and fungi. Exopolysaccharides also inhibit the formation of biofilms by pathogenic bacteria and prevent their colonization on various surfaces. However, mechanism of the harmful effect for many secreted antibacterial substances remains unknown. The antimicrobial activity for most substances has been studied in vitro only, but some substances have been characterized in vivo and they have found practical applications in medicine and veterinary. The cyclic lipopeptides that have surfactant properties are used in some industries. In this review, special attention is paid to the antimycobacterials produced by B. licheniformis as a possible approach to combat multidrug-resistant and latent tuberculosis. In particular, licheniformins and bacitracins have shown strong antimycobacterial activity. However, the medical application of some antibacterials with promising in vitro antimycobacterial activity has been limited by their toxicity to animals and humans. As such, similar to the enhancement in the antimycobacterial activity of natural bacteriocins achieved using genetic engineering, the reduction in toxicity using the same approach appears feasible. The unique capability of B. licheniformis to synthesize and produce a range of different antibacterial compounds means that this organism can act as a natural universal vehicle for antibiotic substances in the form of probiotic cultures and strains to combat various types of pathogens, including mycobacteria. Full article

Astrocytes are central nervous system (CNS)-restricted glial cells involved in synaptic function and CNS blood flow regulation. Astrocyte extracellular vesicles (EVs) participate in neuronal regulation. EVs carry RNAs, either surface-bound or luminal, which can be transferred to recipient cells. We characterized the secreted EVs and RNA cargo of human astrocytes derived from an adult brain. EVs were isolated by serial centrifugation and characterized with nanoparticle tracking analysis (NTA), Exoview, and immuno-transmission electron microscopy (TEM). RNA from cells, EVs, and proteinase K/RNase-treated EVs was analyzed by miRNA-seq. Human adult astrocyte EVs ranged in sizes from 50 to 200 nm, with CD81 as the main tetraspanin marker and larger EVs positive for integrin β1. Comparison of the RNA between the cells and EVs identified RNA preferentially secreted in the EVs. In the case of miRNAs, enrichment analysis of their mRNA targets indicates that they are good candidates for mediating EV effects on recipient cells. The most abundant cellular miRNAs were also abundant in EVs, and the majority of their mRNA targets were found to be downregulated in mRNA-seq data, but the enrichment analysis lacked neuronal specificity. Proteinase K/RNase treatment of EV-enriched preparations identified RNAs secreted independently of EVs. Comparing the distribution of cellular and secreted RNA identifies the RNAs involved in intercellular communication via EVs. Full article

The Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that affects the world’s popula-tion with chikungunya disease. Adaptation of the viral life cycle to their host cells’ environment is a key step for establishing their infection and pathogenesis. Recently, the accumulating evidence advocates a principal role of extracellular vesicles (EVs), including exosomes, in both the infection and pathogenesis of infectious diseases. However, the participation of exosomes in CHIKV infec-tion and transmission is not well clarified. Here, we demonstrated that the CHIKV RNA and pro-teins were captured in exosomes, which were released by viral-infected epithelial cells. A viral genomic element in the isolated exosomes was infectious to naïve mammalian epithelial cells. The assay of particle size distribution and transmission electron microscopy (TEM) revealed CHIKV-derived exosomes with a size range from 50 to 250 nm. Treatments with RNase A, Triton X-100, and immunoglobulin G antibodies from CHIKV-positive patient plasma indicated that in-fectious viral elements are encompassed inside the exosomes. Interestingly, our viral plaque for-mation also exhibited that infectious viral elements might be securely transmitted to neighboring cells by a secreted exosomal pathway. Taken together, our recent findings emphasize the evidence for a complementary means of CHIKV infection and suggest the role of exosome-mediated CHIKV transmission. Full article

Extracellular vesicles (EVs) are membranous particles released by all cell types. Their role as functional carrier of bioactive molecules is boosted by cells that actively secrete them in biological fluids or in the intercellular space (interstitial EVs, iEVs). Here we have optimised a method for the isolation and characterization of zebrafish iEVs from whole melanoma tissues. Zebrafish melanoma iEVs are around 140 nm in diameter, as determined by nanoparticle tracking and transmission electron microscopy (TEM) analysis. Western blot analysis shows enrichment for CD63 and Alix in the iEV fraction, but not in melanoma cell lysates. Super resolution and confocal microscopy reveal that purified zebrafish iEVs are green fluorescent protein positive (GFP+), indicating that they integrate the oncogene GFP-HRAS^V12G used to induce melanoma in this model within their vesicular membrane or luminal content. Analysis of RNA-Seq data found 118 non-coding (nc)RNAs differentially distributed between zebrafish melanoma and their iEVs, with only 17 of them being selectively enriched in iEVs. Among these, the RNA components of RNAses P and MRP, which process ribosomal RNA precursors, mitochondrial RNAs, and some mRNAs, were enriched in zebrafish and human melanoma EVs, but not in iEVs extracted from brain tumours. We found that melanoma iEVs induce an inflammatory response when injected in larvae, with increased expression of interferon responsive genes, and this effect is reproduced by MRP- or P-RNAs injected into circulation. This suggests that zebrafish melanoma iEVs are a source of MRP- and P-RNAs that can trigger inflammation in cells of the innate immune system. Full article

Antimicrobial peptides (AMPs), which are natural antibiotics, protect against pathogens invading the urinary tract. RNase 7 with antimicrobial properties has rapid and powerful suppressive effects against Gram-positive and Gram-negative bacterial infections. However, its detailed antibacterial mechanisms have not been fully determined. Here, we investigate whether RNase 7 had an impact on bladder cells under uropathogenic Escherichia coli (UPEC) infection in a high-glucose environment using in vitro GFP-UPEC-infected bladder cell and PE-labeled TLR4, STAT1, and STAT3 models. We provide evidence of the suppressive effects of RNase 7 on UPEC infection and UPEC-induced inflammatory responses by regulating the JAK/STAT signaling pathway using JAK inhibitor and STAT inhibitor blocking experiments. Pretreatment with different concentrations of RNase 7 for 24 h concentration-dependently suppressed UPEC invasion in bladder cells (5 μg/mL reducing 45%; 25 μg/mL reducing 60%). The expressions of TLR4, STAT1, and STAT3 were also downregulated in a concentration-dependent manner after RNase 7 pretreatment (5 μg/mL reducing 35%, 54% and 35%; 25 μg/mL reducing 60%, 75% and 64%, respectively). RNase 7-induced decrease in UPEC infection in a high-glucose environment not only downregulated the expression of TLR4 protein and the JAK/STAT signaling pathway but also decreased UPEC-induced secretion of exogenous inflammatory IL-6 and IL-8 cytokines, although IL-8 levels increased in the 25 μg/mL RNase 7-treated group. Thus, inhibition of STAT affected pSTAT1, pSTAT3, and TLR4 expression, as well as proinflammatory IL-6 and IFN-γ expression. Notably, blocking JAK resulted in the rebound expression of related proteins, especially pSTAT1, TLR4, and IL-6. The present study showed the suppressive effects of RNase 7 on UPEC infection and induced inflammation in bladder epithelial cells in a high-glucose environment. RNase 7 may be an anti-inflammatory and anti-infective mediator in bladder cells by downregulating the JAK/STAT signaling pathway and may be beneficial in treating cystitis in DM patients. These results will help clarify the correlation between AMP production and UTI, identify the relationship between urinary tract infection and diabetes in UTI patients, and develop novel diagnostics or possible treatments targeting RNase 7. Full article

Ovarian granulosa cells (GC) play an essential role in the development and atresia of follicles. Emerging studies suggest that non-coding RNAs are involved in the regulation of GC apoptosis. Here, we aimed to analyze the function of ssc-circINHA-001, coded by the first exon of the inhibin subunit α gene (INHA), in resisting GC apoptosis and follicular atresia by enhancing the expression of the inhibin subunit β A (INHBA) through a cluster of miRNAs. A higher expression of ssc-circINHA-001 in healthy follicles compared to early atretic follicles was detected by qRT-PCR. Its circular structure was confirmed by RNase R treatment and reversed PCR. The function of ssc-circINHA-001 in GC resistance to apoptosis was detected by in vitro transfection of its si-RNA. Furthermore, the dual-luciferase reporter assay suggested that ssc-circINHA-001 adsorbed three miRNAs, termed miR-214-5p, miR-7144-3p, and miR-9830-5p, which share the common target INHBA. A low expression of ssc-circINHA-001 increased the levels of the free miRNAs, inhibited INHBA expression, and thus raised GCs apoptosis through a shift from the secretion of activin to that of inhibin. Our study demonstrated the existence of a circRNA–microRNAs–INHBA regulatory axis in follicular GC apoptosis and provides insight into the relationship between circRNA function and its coding gene in inhibin/activin balance and ovarian physiological functions. Full article

Extracellular vesicles (EVs) can transfer diverse RNA cargo for intercellular communication. EV-associated RNAs have been found in diverse fungi and were proposed to be relevant for pathogenesis in animal hosts. In plant-pathogen interactions, small RNAs are exchanged in a cross-kingdom RNAi warfare and EVs were considered to be a delivery mechanism. To extend the search for EV-associated molecules involved in plant-pathogen communication, we have characterised the repertoire of EV-associated mRNAs secreted by the maize smut pathogen, Ustilago maydis. For this initial survey, we examined EV-enriched fractions from axenic filamentous cultures that mimic infectious hyphae. EV-associated RNAs were resistant to degradation by RNases and the presence of intact mRNAs was evident. The set of mRNAs enriched inside EVs relative to the fungal cells are functionally distinct from those that are depleted from EVs. mRNAs encoding metabolic enzymes are particularly enriched. Intriguingly, mRNAs of some known effectors and other proteins linked to virulence were also found in EVs. Furthermore, several mRNAs enriched in EVs are also upregulated during infection, suggesting that EV-associated mRNAs may participate in plant-pathogen interactions. Full article

Pestiviruses express the unique essential envelope protein E^rns, which exhibits RNase activity, is attached to membranes by a long amphipathic helix, and is partially secreted from infected cells. The RNase activity of E^rns is directly connected with pestivirus virulence. Formation of homodimers and secretion of the protein are hypothesized to be important for its role as a virulence factor, which impairs the host’s innate immune response to pestivirus infection. The unusual membrane anchor of E^rns raises questions with regard to proteolytic processing of the viral polyprotein at the E^rns carboxy-terminus. Moreover, the membrane anchor is crucial for establishing the critical equilibrium between retention and secretion and ensures intracellular accumulation of the protein at the site of virus budding so that it is available to serve both as structural component of the virion and factor controlling host immune reactions. In the present manuscript, we summarize published as well as new data on the molecular features of E^rns including aspects of its interplay with the other two envelope proteins with a special focus on the biochemistry of the E^rns membrane anchor. Full article

New anti-inflammatory treatments are needed for CF airway disease. Studies have implicated the endoplasmic reticulum stress transducer inositol requiring enzyme 1α (IRE1α) in CF airway inflammation. The activation of IRE1α promotes activation of its cytoplasmic kinase and RNase, resulting in mRNA splicing of X-box binding protein-1 (XBP-1s), a transcription factor required for cytokine production. We tested whether IRE1α kinase and RNase inhibition decreases cytokine production induced by the exposure of primary cultures of homozygous F508del CF human bronchial epithelia (HBE) to supernatant of mucopurulent material (SMM) from CF airways. We evaluated whether IRE1α expression is increased in freshly isolated and native CF HBE, and couples with increased XBP-1s levels. A FRET assay confirmed binding of the IRE1α kinase and RNase inhibitor, KIRA6, to the IRE1α kinase. F508del HBE cultures were exposed to SMM with or without KIRA6, and we evaluated the mRNA levels of XBP-1s, IL-6, and IL-8, and the secretion of IL-6 and IL-8. IRE1α mRNA levels were up-regulated in freshly isolated CF vs. normal HBE and coupled to increased XBP-1s mRNA levels. SMM increased XBP-1s, IL-6, and IL-8 mRNA levels and up-regulated IL-6 and IL-8 secretion, and KIRA6 blunted these responses in a dose-dependent manner. Moreover, a triple combination of CFTR modulators currently used in the clinic had no effect on SMM-increased XBP-1s levels coupled with increased cytokine production in presence or absence of KIRA6. These findings indicate that IRE1α mediates cytokine production in CF airways. Small molecule IRE1α kinase inhibitors that allosterically reduce RNase-dependent XBP-1s may represent a new therapeutic strategy for CF airway inflammation. Full article

The pestivirus envelope protein E^rns is anchored in membranes via a long amphipathic helix. Despite the unusual membrane topology of the E^rns membrane anchor, it is cleaved from the following glycoprotein E1 by cellular signal peptidase. This was proposed to be enabled by a salt bridge-stabilized hairpin structure (so-called charge zipper) formed by conserved charged residues in the membrane anchor. We show here that the exchange of one or several of these charged residues reduces processing at the E^rns carboxy-terminus to a variable extend, but reciprocal mutations restoring the possibility to form salt bridges did not necessarily restore processing efficiency. When introduced into an E^rns-only expression construct, these mutations enhanced the naturally occurring E^rns secretion significantly, but again to varying extents that did not correlate with the number of possible salt bridges. Equivalent effects on both processing and secretion were also observed when the proteins were expressed in avian cells, which points at phylogenetic conservation of the underlying principles. In the viral genome, some of the mutations prevented recovery of infectious viruses or immediately (pseudo)reverted, while others were stable and neutral with regard to virus growth. Full article

CircRNAs have been identified to be expressed differently and stably in numerous species and tissues, but their functions in growth hormone (GH) secretion are still largely unknown. In summary, we have revealed a circRNA-miRNA-mRNA network that may play a biological role in the rat pituitary gland. First, we verified the chromosome location information of circAgtpbp1 according to sequencing analysis. The circAgtpbp1 characteristics were authenticated through PCR, qRT–PCR, treating with RNase and fluorescent in situ hybridization (FISH). Second, we detected the expression pattern of circAgtpbp1 in the rat anterior pituitary by qRT–PCR. We also designed circAgtpbp1 siRNA and constructed overexpression plasmid to evaluate the effect of circAgtpbp1 function on GH secretion by qRT–PCR, ELISA and Western blot. CircAgtpbp1 is a stable, truly circular molecule. We found that circAgtpbp1 interacted with miR-543-5p and can regulate GH secretion in pituitary cells through a circAgtpbp1-miR-543-5p-GH axis. Overall, the evidence generated by our study suggests that circAgtpbp1 can act as a sponge of miR-543-5p to reduce the inhibitory effect of miR-543-5p on Gh1 and further promote GH secretion. These findings expand our existing knowledge on the mechanisms of hormone regulation in the pituitary gland. Full article