Skin and oral mucosa are continuously exposed to potential metal sensitizers while hosting abundant microbes, which may influence the host response to sensitizers. This host response may also be influenced by the route of exposure that is skin or oral mucosa, due to their different immune properties.Streptococcus mitis influences the host response to nickel sulfate (sensitizer) and titanium(IV) bis(ammonium lactato)dihydroxide (questionable sensitizer) in reconstructed human skin (RHS) and gingiva (RHG).Determine how commensal S. mitis for 24 hours. Histology, cytokine secretion, and Toll‐like receptors (TLRs) expression were assessed.RHS/RHG was exposed to nickel or titanium, in the presence or absence of S. mitis increased interleukin (IL)‐6, CXCL8, CCL2, CCL5, and CCL20 secretion in RHS but not in RHG; co‐application with nickel further increased cytokine secretion. In contrast, titanium suppressed S. mitis–induced cytokine secretion in RHS and had no influence on RHG. S. mitis and metals differentially regulated TLR1 and TLR4 in RHS, and predominantly TLR4 in RHG.S. mitis and nickel resulted in a more potent innate immune response in RHS than in RHG, whereas titanium remained inert. These results indicate the important influence of commensal microbes and the route of exposure on the host's response to metals.Co‐exposure of  S. mitis and metals.Host response to  They are both capable of hosting abundant microbes, responding to environmental assault, and maintaining immune homeostasis.Nickel is classically regarded as a contact sensitizer because nickel allergy on the skin and oral mucosa is frequently reported.Allergic manifestations to titanium‐based implants after orthopedic or dental surgery are also being increasingly reported.Staphylococcus aureus.Because microbes trigger innate immune responses similar to those triggered by metals, it can be expected that microbes will also influence sensitization to metals. Furthermore, emerging evidence suggests that microbes maintain a symbiotic relationship with the host and influence both physiological and pathological host events via a group of host receptors known as the toll‐like receptors (TLRs). TLRs have been repeatedly shown to participate in a broad range of host events: nickel‐induced allergy,Streptococcus mitis, a facultative commensal bacteria found on skin and on oral mucosa.The aim of this study is to determine how commensal bacteria influence the response of skin and oral mucosa to potential metal sensitizers in vitro. We exposed RHS and RHG, consisting of a stratified differentiated epithelium on a fibroblast‐populated dermis/laminal propria (collagen hydrogel), to a mixture of metal and bacteria: nickel (II) sulfate hexahydrate or titanium(IV) bis(ammonium lactato)dihydroxide was co‐applied with 22.1Human neonatal foreskin was obtained after informed consent from patients undergoing routine surgical procedures. Human non‐inflamed gingival tissue was obtained from healthy donors undergoing dental implant surgery or wisdom tooth extraction. Skin and gingiva were used anonymously and in accordance with the “Code for Proper Use of Human Tissue” as formulated by the Dutch Federation of Medical Scientific Organizations. Procedures are approved by the local medical research ethics committee of the Amsterdam UMC.6 cells) were seeded onto fibroblast‐populated collagen hydrogels in a 24 mm diameter transwell  and cultured submerged for 3 days. To induce epithelial differentiation, the cultures were then lifted to an air‐liquid interface and cultured for an additional 10 days. Twenty‐four hours before exposure and also at the time of exposure, the cultures were refreshed with medium without penicillin‐streptomycin or hydrocortisone. Cultures were incubated at 37°C, 7.5% CO2, and culture medium was refreshed twice a week.RHS and RHG were constructed exactly as described previously.2.2S. mitis LMG 14557 was cultured anaerobically at 37°C in a modified semi‐defined medium (pH 7.0) prepared exactly as described previously, in the presence of 1% glucose.S. mitis was cultured overnight for 24 hours, and then the pre‐culture was diluted into 1:1000 for another 16 hours growth until within the exponential phase (OD600 around 0.5). OD600 value was measured using SpectraMax Plus 384 . The number of colony forming units (CFUs) of S. mitis at the time of exposure was determined by viable bacterial cell counting (CFU/mL): A sample was taken from the prepared S. mitis exposure, serial dilutions were made and plated on the brain hart infusion (BHI) agar plates, and colonies were counted after 96 hours of anaerobic incubation at 37°C.2.3Titanium(IV) bis(ammonium lactato)dihydroxide solution  and nickel (II) sulfate hexahydrate  were used at the following concentrations: titanium: 20 mg/mL (68 mM) and 40 mg/mL (136 mM); nickel: 3 mg/mL (20 mM) and 15 mg/mL (97 mM). These concentrations were selected because they showed no more than a 5% decrease in metabolic activity ‐2,5‐diphenyltetrazolium bromide assay) in reconstructed human epidermis, compared to unexposed cultures.S. mitis was performed as follows: sterile gauze filters  were placed on top of the RHS or RHG cultures. Then 25 μL of prepared mixture of the following four combinations were applied onto the center of the filters: (a) Hank's Balanced Salt Solution  as control; (b) 109 CFU/mL of S. mitis in HBSS; (c) nickel or titanium at two different concentrations as indicated above; (d) 109 CFU/mL of S. mitis mixed with nickel or titanium at two concentrations. The impregnated filters were kept on the cultures for 24 hours at 37°C, 7.5% CO2, and 95% humidity. To control the amount of S. mitis in the mixtures, CFU counting was performed as described above (section: S. mitis growth condition).Exposure to chemicals and S. mitis (by CFU counting) and RHS/RHG (by MTT assay) was determined. S. mitis samples were taken from the surface of the RHS or RHG using a sterile microbrush. Together with the filter, the brushed samples were sonicated and plated out on BHI agar plates for CFU counting as described above. The viability of RHS and RHG was measured by MTT assay as described previously.After 24 hours exposure, the viability of the applied 2.4RHS and RHG samples were fixed in 4% paraformaldehyde and embedded in paraffin. As previously described,2.5Culture supernatants were collected at the time of harvesting and secretion of proteins determined by commercially available sandwich enzyme‐linked immunosorbent assays (ELISAs). For IL‐6, IL‐10, CCL2, CCL5, and CCL20, antibodies and recombinant proteins were purchased from R&D Systems . For CXCL8, CXCL12, and IL‐18, ELISA kits were used .2.6RHS or RHG epithelium were carefully removed from the fibroblast‐populated collagen hydrogel and washed with PBS. Next, total RNA and protein were isolated from the epithelium using a AllPrep RNA/Protein Kit  following the manufacturer's instructions. Isolated protein was further precipitated using methanol and resuspended in complete lysis‐M buffer (Sigma‐Aldrich). Thereafter, the amount of protein was measured using the Pierce BCA Protein Assay Kit , and RNA was measured using Nanodrop ND‐1000 Spectrophotometer (Thermo Fisher Scientific). The RNA and protein samples were stored at −80°C before further process.2.72 First Strand Kit (Qiagen). Quantitative Reverse Transcriptase PCR (qRT‐PCR) reactions were performed on a ViiA 7 Real‐Time PCR System  using Fast SYBR Green Master Mix (Applied Biosystems) and paired primers : HP205798, OriGene Technologies, Rockville). Individual gene expression levels were normalized to GAPDH expression levels, and shown in fold‐change compared to the RHS or RHG control.Genomic DNA elimination and complementary DNA (cDNA) synthesis were performed using RT2.8As described previously, the isolated proteins were prepared and separated on 4%‐12% Bis‐Tris Plus Gel (Invitrogen) and transferred to a Polyvinylidene fluoride (PVDF) membrane . The membranes were blocked with 2% Bovine Serum Albumin (BSA) in Phosphate Buffered Saline with Tween 20 (PBST) for 1 hour and incubated with antibodies against TLR1, 2, 3, 5, and 6 ; TLR4 ; and tubulin  overnight at 4°C. Thereafter, membranes were washed three times in PBST and further incubated with infrared dye‐conjugated secondary antibodies against mouse  or against rabbit . After washing, the blots were visualized using Sapphire Biomolecular Imager  according to the manufacturer's instruction.2.9S. mitis unexposed and exposed RHS and RHG were compared using unpaired t‐tests. For the dose‐dependent effect of nickel or titanium, differences were compared using one‐way analysis of variance (ANOVA) followed by Bonferroni's multiple‐comparisons test. Statistics were performed using GraphPad Prism version 7.0. Data represent the mean ± standard error of the mean (SEM) of three independent experiments, each performed in duplicate, and consisted of a different skin or mucosa donor (not patient patched) and a different S. mitis inoculum. Differences were considered significant when the P‐value was < .05; *denoting P < .05, **P < .01, and ***P < .001. Data are represented as mean ± SEM.Differences between 33.1Exposure conditions have no detrimental effect on RHS or RHG histology. Both the RHS and RHG featured a stratified, differentiated epithelium on a fibroblast‐populated collagen hydrogel Figure . SimilarS. mitis rRNA (in red) present in the form of biofilms on top of both RHS and RHG and also sparingly within the epithelium (DAPI‐stained keratinocyte nuclei and S. mitis DNA in blue). As examples, RHS exposed to S. mitis  showed the s Figure , RHS expn Figure . After 2s Figure . No signe Figure , or whenm Figure .3.2S. mitis on RHS and RHG, pro‐inflammatory and antimicrobial cytokine secretion was determined  from RHS. Titanium, in the presence or absence of S. mitis, did not influence cytokine secretion from RHG. IL‐1α, IL‐1β, IL‐10, and CCL28 levels were below the detection limit of the ELISA in both RHS and RHG in all experimental conditions (data not shown). Taken together, these results indicate that nickel induces innate cytokine release, which is further enhanced by S. mitis in RHS but not in RHG. In contrast, titanium is inert and even suppresses S. mitis–mediated cytokine release from RHS.In contrast to nickel, exposure of titanium to RHS or RHG did not result in a dose‐dependent increase in cytokine secretion. Notably, titanium even suppressed the 3.4S. mitis and metal exposure. The mRNA expression of TLR2, 4, and 6 was undetectable in all conditions (data not shown). In RHS, TLR1, 3, and 5 mRNA was not regulated (neither increased nor decreased) under any of the experimental conditions investigated  resulted in increased TLR1 mRNA expression  followed by a decrease at the higher concentration (15 mg/mL)  in the complex organotypic model. TLR2, 4, and 6 mRNA levels were not regulated in RHG under any of the experimental conditions studied (data not shown).Exposure of RHG to n Figure . When S.) Figure . In contS. mitis alone did not influence this TLR1 expression, whereas nickel greatly reduced TLR1 expression. When co‐exposed with S. mitis, high nickel concentrations also reduced TLR1 expression. Titanium, either alone or co‐exposure with S. mitis resulted in pronounced clear decrease in TLR1 expression. In contrast, TLR4 expression was suppressed in RHS after S. mitis exposure, as well as metal exposure (concentration dependent). However, co‐exposure of S. mitis with nickel or titanium resulted in TLR4 protein being detectable again. Taken together, we show that for RHS, TLR1 and TLR4 protein was decreased or unaltered by S. mitis and/or metal exposure compared to unexposed RHS  increased TLR4 expression.In contrast to RHS, TLR1 protein levels were negligible in RHG with only a slight band being visible when RHG were exposed to nickel Figure . HoweverS. mitis and metal exposure are able to differentially regulate TLR1 and 4 expression in RHS and RHG, with TLR1 and 4 being involved in RHS, and predominantly TLR4 being involved in RHG.Taken together, these results indicate that 4S. mitis, influence the host response to metals in reconstructed human skin (or RHS) and gingiva (or RHG). Nickel and titanium were chosen for our study because nickel is a well‐characterized contact sensitizer,S. mitis and nickel resulted in a more potent innate immune response in RHS than in RHG. In comparison, titanium remained inert  to commensal microbes, and none have shown such well‐preserved tissue integrity after 24 hours of co‐culture. For example, a recent study described gene regulation in RHG after Streptococcus oralis biofilm exposure via a titanium implant inserted into the RHG for 24 hours; however, no data on tissue viability and limited data on tissue integrity were shown.Candida albicans for 24 hours. However, this study also showed an increased number of caspase‐3 positive cells, strongly suggesting a significant decrease in tissue viability.S. mitis increased the cytokine release of IL‐6, CXCL8, CCL5, and CCL20 in RHS but not in RHG. Such relative inertness of RHG to S. mitis is supported by findings of others describing the regulatory role of S. mitis as an oral commensal on immune balance, where it inhibits CD4+ T cell proliferation, promotes Th17 responses, and induces the secretion of IL‐10 in human monocytes.S. mitis affects the skin. Taken together, our results and those of others support the characteristic of oral mucosa in balancing a potential overactivated immune response induced by commensals. This is necessary for maintaining oral health because the oral cavity is continuously exposed to food antigens, sensitizers, and abundant microorganisms, for example, viruses and fungiPreviously we have described intrinsic differences between skin and gingiva with regard to innate immunology, wound healing, and the response to contact sensitizers.This article describes in detail the results obtained from foreskin keratinocytes. Our previous study using adult abdominal skin showed secreted protein concentrations falling within the ranges observed for the foreskin donors in all cases.S. mitis strongly enhanced the innate immune response to nickel in RHS, especially the release of CXCL8. In contrast, the S. mitis– and nickel‐mediated cytokine response in RHG was considerably lower than in RHS, in line with a more tolerogenic environment  may2NP is also inert for THP‐1 macrophages because it does not influence genes involved in modulating macrophage maturation, inflammatory responses, chemotaxis, and leukocyte migration.2 cannot induce skin sensitization in LLNA in mice.2NPs were found to act as an adjuvant when co‐applied with the bacterial fragments LPS and peptidoglycan;In contrast to nickel, titanium suppressed t Figure . Indeed S. mitis and metals differentially regulate TLR1 and 4 in RHS, and predominantly TLR4 in RHG  have important roles in sensitization and in host–microbe interactions; however, they are absent in the present study. Furthermore, we used only a single bacteria species for the exposure, whereas in vivo, a broad variety of other microbial species  are also in contact with the skin and the oral mucosa. The influence from these on host tissues, especially the pathogens, may provide more information to interpret conflicting results from in vitro and in vivo studies. In addition, sensitization occurs on host tissues, which are already influenced and primed by local commensal microbes, whereas in this study Lin Shang: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; writing‐original draft; writing‐review and editing. Dongmei Deng: Conceptualization; data curation; formal analysis; methodology; supervision; writing‐review and editing. Sanne Roffel: Data curation; formal analysis; investigation; methodology; writing‐review and editing. Susan Gibbs: Conceptualization , funding acquisition , project administration (lead), resources (lead), supervision (lead), validation , writing‐review & editing .Susan Gibbs is scientific advisor for Geneus BioTech CRO (SME). The other authors have no interests to declare.


Salmonella enterica, 2-aminoacrylate (2AA) is a reactive enamine intermediate generated during a number of biochemical reactions. When the 2-iminobutanoate/2-iminopropanoate deaminase  is eliminated, 2AA accumulates and inhibits the activity of multiple pyridoxal 5’-phosphate(PLP)-dependent enzymes. In this study, untargeted proton nuclear magnetic resonance (1H NMR) metabolomics and transcriptomics data were used to uncover the global metabolic response of S. enterica to the accumulation of 2AA. The data showed that elimination of RidA perturbed folate and branched chain amino acid metabolism. Many of the resulting perturbations were consistent with the known effect of 2AA stress, while other results suggested additional potential enzyme targets of 2AA-dependent damage. The majority of transcriptional and metabolic changes appeared to be the consequence of downstream effects on the metabolic network, since they were not directly attributable to a PLP-dependent enzyme. In total, the results highlighted the complexity of changes stemming from multiple perturbations of the metabolic network, and suggested hypotheses that will be valuable in future studies of the RidA paradigm of endogenous 2AA stress.In  The metabolic network of an organism consists of a complex system of biochemical reactions that together result in the behavioral characteristics of the organism. In general, the reactions that make up the metabolic network, and their integration, are governed by chemical and physical constraints placed on the cell by its environment. Microbes in particular face rapidly changing environments and their metabolic networks must be structured to absorb and/or respond to internal and external insults ,2,3. TecSalmonella enterica defines a metabolic model with clearly defined local effects that result in predictable global consequences. Members of the broadly conserved RidA protein family are responsible for the hydrolysis of enamine/imine species, which are generated as intermediates in a variety of biochemical reactions [An important use of global strategies is to expand the scope of a model that has been based on data from biochemical genetic approaches, which often define detail but can lack validation on a global scale. The RidA paradigm of enamine stress in eactions ,18,19,20eactions ,22,23,24S. enterica has over 40 different PLP-dependent enzymes with functions in amino acid metabolism, cofactor/coenzyme biosynthesis, and the synthesis of cell structure components [ridA, and led to the identification of an uncharacterized role for RidA in motility [Overall, mponents ,27. Whilmotility . A majorS. enterica to 2AA stress. Specifically, untargeted NMR metabolomic data were sought to complement existing transcriptomics data. Our understanding of the RidA system at a physiological, genetic, and biochemical level provided a critical basis to guide the interpretation of the global data sets.This study was initiated to further our understanding of the global metabolic response of 1H NMR analysis was performed on the cellular contents of an isogenic pair of S. enterica LT2 strains that differed only at the ridA locus (DM9404 and DM3480) after growth to stationary phase in minimal glucose medium. These data were viewed through a biological lens, specifically considering the role PLP-enzyme-dependent enzymes have in the ridA paradigm [Comprehensive paradigm ,30, to e1H-NMR spectral data  adjusted p value < 0.05. ridA. The relevant metabolites represented diverse areas of the metabolic network and did not suggest a simple model for specific site(s) of 2AA-dependent perturbation. A majority of the metabolites (10/23) were related to, or the product of, amino acid biosynthetic pathways and a significant number (5/23) were components nucleotide metabolism. The former was expected based on the prevalence of PLP-dependent enzymes (targets of 2AA damage) in amino acid metabolism [S. enterica and thus limited the conclusions that could be made about the global metabolic state of S. enterica ridA mutants.In total, 37 metabolites could be structurally identified from the ral data . Table 1lites 10/ were reltabolism ,25,31, wridA mutant strains [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103146). As reported, the transcription of 186 genes in a S. enterica ridA mutant were significantly (FDR < 0.05) elevated and the transcription of 227 genes was significantly decreased when compared to the wild-type parental strain [ridA and WT. These data emphasized that the cellular response to 2AA stress generated transcriptional changes of modest magnitude. Over-representation analysis was performed on all differentially expressed genes, regardless of fold-change intensity, to determine pathways that showed significant enrichment for the differentially expressed genes in a ridA mutant , biotin metabolism (stm00780), amino acid biosynthesis stm01230), one-carbon metabolism (stm00670), ABC transporters (stm02010), and glycine, serine and threonine metabolism (stm00260) were all significantly over-represented as up-regulated in a A mutant . Since m0, one-caridA mutant are consistent with a perturbation in folate metabolism. Tetrahydrofolate (THF) and its derivatives are essential cofactors used to facilitate one-carbon unit transfers in many important pathways [glyA, gcvTHP, tdh, kbl, pabB, and folE) were increased in a ridA mutant, as compared to wild-type  and 2-amino-3-ketobutyrate CoA ligase , which are involved in the conversion of threonine to glycine, are also induced in a ridA mutant. Induction of these genes could lead to the rerouting of threonine for glycine production, ultimately generating additional 5,10-mTHF via the glycine cleavage complex (GCV) (pabB (1.45-fold) and folE (3.21-fold), is also elevated in a ridA mutant. Expression of these genes, which are involved in 7,8-dihydrofolate production, could increase the biosynthesis of THF for use by GlyA and the GVC complex during 5,10-mTHF production. The putative 5,10-mTHF limitation in a ridA mutant would explain the increased expression of multiple pathways that are dependent upon folate metabolism   . Transcrnthesis) . ridA mutant  [ridA background. In fact, SAM biosynthesis is reliant on both 5-methyltetrahydrofolate (5-mTHF) and purines (ATP), making this conclusion consistent with the data and interpretations above. An expected consequence of SAM limitation is the decreased production of products of pathways that rely upon SAM as a cofactor. Consistently, expression of genes involved in the SAM-dependent pathways of biotin , lipoate (lipA), and thiamine (thiCEFSGH and thiMD) biosynthesis were induced, consistent with a limitation for these critical metabolites in a ridA mutant  and represses expression of a number of genes whose expression is increased in a nd folE) ,38. IncrA mutant B.1H NMR metabolomic analysis showed that a ridA mutant had lower endogenous levels of both formate and acetate  facilitates the conversion of 3-phosphooxypyruvate to O-phospho-L-serine during serine biosynthesis, while SerC and N-succinyldiaminopimelate aminotransferase  catalyze N-succinyldiaminopimelate production from N-succinyl-L-amino-6-ketopimelate during lysine biosynthesis  used in this study contains a MudJ1734 transposon insertion [ridA locus (ridA3::MudJ1734) [ridA locus. Minimal medium was no-carbon E medium (NCE) containing 1 mM MgSO4 [Strains used in this work are derivatives of nsertion  disruptiudJ1734) . Strain mM MgSO4 , trace mmM MgSO4 , and 11 ridA mutant (DM3480) strains were grown aerobically overnight in NB medium at 37 °C and used to inoculate (1% inoculum) 250 mL minimal glucose medium in 500 mL non-baffled flasks. Flasks were randomly arranged in an Innova® 44 incubator and cultures were allowed to grow to stationary phase (16 h) shaking at 180 RPM and 37 °C. Cultures were cooled on ice for 5 min and then harvested by centrifugation at 7000× g for 10 min at 4 °C. The supernatant was decanted, pellets were resuspended in 10 mL ddH2O, and transferred to sterile 15 mL conical tubes in which they were pelleted at 7000× g 10 min at 4 °C. Final supernatant was decanted before pellets were frozen in liquid nitrogen and stored at −80 °C prior to cell extractions.Ten biologically independent cultures each of wild-type (DM9404) and 2O (80/20) solvent was added and samples were agitated 7 times  at 1600 rpm in a FastPrep 96 (MPBIO). Samples were then centrifuged at 416× g and 4 °C for 16 min and supernatant was transferred to a new 15 mL conical tube. Homogenization was carried out a second and third time using 4 mL MeOH/H2O with 4 homogenization cycles (150 s) and 2 mL MeOH/H2O with 3 homogenization cycles (150 s), respectively. Pooled supernatants from each sample were concentrated to dryness using a CentriVap Benchtop Vacuum Concentrator (Labconco). The extracts were reconstituted in 600 μL of deuterated 100 mM sodium phosphate buffer (pH 7.4) containing 1 mM of the internal standard DSS  and vortex mixed 2 min. Each sample was transferred into 5 mm SampleJet NMR tubes for NMR analysis.Each frozen bacterial pellet (~1 g) was thawed on ice and homogenized 3 times. For homogenization, 2.6 g of 0.1 mm zirconium beads and 6 mL 4 °C MeOH/HThe 1D proton spectra were collected using the pulse sequence ‘noesypr1d’ from the Bruker library. A mixing time of 5 ms was used, and during the acquisition, 65,536 complex datapoints were collected for the FID using 128 scans, with four additional dummy scans for equilibration and 4 s between scans. The spectral width was 20 ppm. For processing, 32,768 points were used for the spectrum, using an exponential window function of 0.3 Hz before the Fourier transform (FT). After FT and phase correction, a polynomial baseline correction of order 3 was used, and the frequency calibrated to the DSS peak (0.00 ppm).1 increment, along with four dummy scans for the equilibrium and 2 s between scans. For processing, 32,768 points were used for the direct dimension of the spectrum and 512 points for the indirect dimension. An unshifted sine window function was also used in each dimension, along with backward linear prediction of 64 points in the indirect dimension to increase the sensitivity as well as the resolution [https://github.com/artedison/Edison_Lab_Shared_Metabolomics_UGA) and all raw and processed metabolomics data are available on the Metabolomics workbench (http://www.metabolomicsworkbench.org/).The 2D J-RES spectra were collected using the pulse sequence ‘jresgpprqf’ from the Bruker library. 32,768 points were used for the FID in the direct dimension and 64 points for the indirect dimension, with a spectral width of 20 ppm for the direct dimension and 50 Hz for the indirect dimension. Eight scans were acquired for each tsolution . A polyn1H-13C heteronuclear single quantum correlation (HSQC) and 1H-13C HSQC–TOCSY  experiments were used to aid in annotation of 2D J-RES metabolites and were not used for statistical analysis. A total of 37 metabolites were identified in bacterial extractions using COLMARm [Two-dimensional  COLMARm , compreh COLMARm . In shorridA mutants .Two-dimensional (2D) J-RES spectra were used for quantification and annotation of metabolites. A representative 2D J-RES spectra, with significantly altered metabolites annotated, is provided in S. enterica ridA mutants were determined using the Database for Annotation, Visualization and Integrated Discovery (DAVID) pathway enrichment analysis tool [p values, gene identities for hits, and ORA Benjamini-Hochberg FDR values [Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were induced or repressed in sis tool ,33. InpuR values . SignifiR values .1H NMR metabolomics analysis to uncover global metabolic consequences of removing RidA. Importantly, all S. enterica ridA mutant-associated phenotypes to date are not only eliminated by expression of wild-type ridA in trans, but exist as the consequence enamine species (2-aminoacrylate) accumulation [ridA mutant permeated multiple KEGG pathways, including amino acid, coenzyme, and folate metabolism. Complementing this finding, 1H NMR revealed multiple metabolic changes that were involved in amino acid, mixed acid fermentation, and nucleic acid metabolism. Some of the patterns from the transcriptomics and metabolomics data were explained by extrapolating known consequences from 2AA stress. For instance, induced expression of folate-related genes can be tied to damage of GlyA, and decreased isoleucine and leucine content with damage to IlvE. Indeed, many of the shifts in threonine, serine, lysine, and phenylalanine were consistent with the 2AA-dependent damage of PLP-dependent enzymes involved in the metabolism of these metabolites.The current study used comparative transcriptomics and untargeted mulation ,53,54,55ridA mutant could not easily be attributed to the inhibition of one or more PLP-dependent enzyme(s), the known targets of 2AA. These changes are expected to be the consequence of downstream effects on the metabolic network. Importantly, while they are difficult to tie to a specific 2AA target, these effects often prove important for the physiological state of the organism and cause visible phenotypes. This study took advantage of a mechanistically understood but complex paradigm of metabolic stress. The accumulation of 2AA causes a cellular stress that is multipronged, yet subtle, since the activity of multiple enzymes is reduced by 20–60%. The data herein showed that defining changes to the metabolic network created by multiple small perturbations to the system is not straightforward with the tools currently available. The RidA system can provide a valuable template for the continued refinement of global approaches to understand metabolic network structure, since it is understood at a biochemical level and can be manipulated genetically to test hypotheses that arise.Significantly, numerous other metabolic and transcriptional changes in a


Klebsiella pneumoniae typing has been proposed: it consists of an HRM protocol designed on the capsular wzi gene and an HRM-based algorithm of strains clustering. In this study, we evaluated the repeatability and reproducibility of this method by performing the HRM typing of a set of K. pneumoniae strains, on three different instruments and by two different operators. The results showed that operators do not affect melting temperatures while different instruments can. Despite this, we found that strain clustering analysis, performed using MeltingPlot separately on the data from the three instruments, remains almost perfectly consistent. The HRM method under study resulted highly repeatable and thus reliable for large studies, even when several operators are involved. Furthermore, the HRM clusters obtained from the three different instruments were highly conserved, suggesting that this method could be applied in multicenter studies, even if different instruments are used.High resolution melting (HRM) is a fast closed-tube method for nucleotide variant scanning applicable for bacterial species identification or molecular typing. Recently a novel HRM-based method for  Klebsiella pneumoniae is a Gram-negative opportunistic pathogen often present in the gut of healthy individuals but also able to cause severe infections. Furthermore, the bacterium is one of the most important nosocomial pathogens, causing healthcare-acquired infections worldwide with a mortality rate ranging from 20 to 70%  and the World Health Organization (WHO) -based typing is a promising tool for clinical and epidemiological applications  known as “high risk” , from here “CFX96”.Eco Real-Time PCR System (Illumina), from here “Eco_RT”.QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems), from here “QS_6Flex”.HRM analysis (see below) was performed on three different real-time PCR/HRM instruments:The three instruments were placed in three different laboratories in two cities, for details see Additional file 9 cells have been used as starting material for total DNA extraction using the DNeasy blood and tissue kit (Qiagen) following the manufacturer’s instructions.Bacterial strains were freshly streaked on MacConkey agar plate and incubated overnight at 37 °C; then a single colony was inoculated into 5 mL of LB broth (DifcoTM) and incubated overnight at 37 °C with vigorous shaking. For each strain, 1 × 10wzi-3 and wzi-4). Negative controls were added in every HRM analysis for each primer pair.For each strain, the extracted DNA was subjected to six HRM analyses: two operators (AP and MP) independently performed the HRM analyses on the three real-time PCR/HRM instruments listed above. In each of the six HRM analysis, three technical replicates were performed for each strain, amplified with the two primer pairs in the Perini and colleagues , 0.4 µl of each primer (0.4 µM) and 1 µl of template DNA (25–50 ng/µl). The thermal profile was as follows: 98 °C for 2 min, 40 cycles of , 95 °C for 2 min, followed by HRM ramping from 70 to 95 °C. Fluorescence data were acquired at increments of 0.5 °C for CFX96, 0.3 °C QuantStudio 6 Flex, and 0.1 °C for Eco Real-Time PCR System. Each CFX96 and QuantStudio 6 Flex HRM analysis was performed in a single 96-well optical plate, while for the Eco Real-Time PCR System each HRM analysis required three 48-well optical plates.The HRM reaction mix (10 µl) contained: 5 µl of 2x SsoAdvanced Universal SYBRDNA and reagents aliquots for all the experiments were prepared in advance to reduce the risk of contamination. In each experiment, the two operators independently prepared the HRM mixes in a pre-PCR ‘clean’ room using the same pipettes each day for each individual experiment.wzi-3 and wzi-4 primer sets. A preliminary qualitative comparison of the aTms obtained by the different instruments and operators was performed reporting the median, minimum and maximum temperature differences for wzi-3 and wzi-4 aTms and plotting the aTm distributions by boxplots.For each strain, the average of the melting temperatures (aTm) obtained from the three technical replicates were computed for wzi-3 and wzi-4 aTms were investigated as independent and as combined factors. The statistical analyses were performed on 1,000,000 bootstrapping strain subsets, randomly selected with replacement. For each subset, aTms were analyzed using R v.3.6.1 (https://www.r-project.org/) as follows:Normality distribution of aTm values was tested by the Shapiro–Wilk test.Homoskedasticity variances of aTm values between operators and among the three instruments were compared using F test and Bartlett test, respectively.t test (applying the Welch approximation in case of heteroskedasticity variance), otherwise using Mann–Whitney test.If aTms were normally distributed, the operators were compared using If aTms were normally distributed, the instruments were compared using one-way ANOVA , otherwise Kruskal–Wallis test.Independent factors:The effects of operator and instrument, and their interactions, were evaluated using the non-parametric analysis of variance implemented in the art function of ARTool R 3.6.1 package Kay .Combined factors:Then, the effects of operators or instruments on Then, we evaluated the percentage of subsets for which the effect of operator, instrument or their interaction were significant .https://skynet.unimi.it/index.php/tools/meltingplot/). More in detail, for each strain the tool computes the replicates average melting temperatures (aTms) for each primer set. Using the igraph R library (http://igraph.org/), the tool builds a graph connecting the strains with aTm distance ≤ 0.5 °C for every primer set, and it clusters the strains on the basis of their betweenness , the obtained melting temperatures were subjected to HRM clustering analysis using the MeltingPlot tool  are reported in Additional file Forty-three: Boxplots of the aTms from the six HRM experiments are reported in Fig. For either primer sets, the combinations of different operators on the same machine gave a maximum difference below or equal to 0.5 °C, the threshold set by Perini and colleagues  for the 100% of the 1,000,000 bootstrap replicates, while the instrument resulted significant  for all of them. For wzi-3, the non-parametric analysis of the variance found the operator to be significant for 13.37% of the bootstraps replicates, the instrument for 100% of replicates, and the interaction among the two factors (operator/instrument) was never significant among the bootstrap replicates. For wzi-4, the instrument was found significant for 100% of the bootstrap replicates, while the operator and the operator-instrument interaction were never significant.The results of the statistical analysis are summarized in Additional file wzi-3 and wzi-4 aTms using the MeltingPlot tool , the 43 strains were clustered on the basis of We compared the clusters obtained by the three instruments to the Perini et al. wzi HRM protocol for Klebsiella pneumoniae typing , statistical analyses revealed that the operator does not affect the measured melting temperatures. Furthermore, the operators did not affect the results of clustering analysis see Fig.  is highlConversely, the instruments resulted to significantly affect the measured melting temperatures  and, in particular, on two gene regions rich in HRM detectable SNPs. In this way it is possible to have a wider range of melting temperatures than in HRM typing protocols developed around one or a few specific SNPs.The clustering analysing applied by MeltingPlot is based only on the melting temperatures measured by the real time PCR instrument. For this reason the melting curves are not normalized, smoothed or background adjusted as these procedures change the overall shape of the curve but not the melting temperature, i.e. the peak of the derivative melt curve  for the HRM assay, the protocol is able to discriminate only strains for which the melting temperatures differ more than 0.5 °C for at least one of the targets. For this reason, strains harbouring different wzi alleles can cluster together  and short time required to accomplish the analysis (~ 5 h), it is a typing method suitable for the real time monitoring of the epidemiological scenario in a hospital setting. A day by day monitoring of the K. pneumoniae clones circulating in a hospital can allow the prompt detection of the emergence of a nosocomial outbreak and to follow the spreading of the outbreak clone among the patients/wards.In this work we can conclude that the Additional file 1: Table S1. Strains genomic information and Results of clustering analysis. For each strain included in the study the cluster assigned by MeltingPlot tool on the basis of wzi-3 and wzi-4 melting temperatures obtained by each instrument and operator  are reported. The relative hospital of isolation , Multi Locus Sequence Typing (MLST) profiles, the wzi alleles (wzi) and the clusters described in Perini et al. Additional file 2: Table S2. Instruments information. For each instrument used in this work, the model, the short name , the sensitivity and the location are reported.Additional file 3: Table S3. Strains melting temperatures. The instrument used for the HRM analysis, the operator who performed the HRM analysis, the primer set used, the melting temperature replicates  and their average melting temperature (aTm) are reported for each strain.Additional file 4: Table S4. Melting temperature differences among instruments and operators. The median of the average melting temperatures differences for each strain among instruments and operators are reported. In brackets, minimum and maximum difference values are reported. The comparisons between the two operators on the same instrument are reported in bold.Additional file 5: Table S5. Results of statistical analyses. The results of statistical analyses are reported.Additional file 6: Table S6. Cross table of the clusters found in Perini et al.


RNA-binding protein (RBP) has a highly dynamic spatiotemporal regulation process and important biological functions. They are critical to maintain the transcriptome through post-transcriptionally controlling the processing and transportation of RNA, including regulating RNA splicing, polyadenylation, mRNA stability, mRNA localization, and translation. Alteration of each process will affect the RNA life cycle, produce abnormal protein phenotypes, and thus lead to the occurrence and development of tumors. Here, we summarize RBPs involved in tumor progression and the underlying molecular mechanisms whereby they are regulated and exert their effects. This analysis is an important step towards the comprehensive characterization of post-transcriptional gene regulation involved in tumor progression. It is found that there are approximately 1914 human RNA-binding proteins (RBPs), accounting for 7.5% of protein-coding genes . RBPs arMany RBPs consist of multiple repetitive sequences that contain only a few specific basic domains. Structurally, common RNA-binding domains mainly include RNA-recognition motif (RRM), K homology (KH) domain, double-stranded RBD (dsRBD), cold-shock domain (CSD), arginine-glycine-glycine (RGG) motif, tyrosine-rich domain, and zinc fingers (ZnF) of the CCHC, CCCH, ZZ type etc. . Accordihttp://rbpdb.ccbr.utoronto.ca/) including 1171 known RBPs, in which the users can browse by domain and species. And the Cancer Genome Atlas (TCGA) database can be used to download RNA high-throughput sequencing and clinical pathology data for determining the abnormal expression of RBPs between cancerous and normal samples [At present, the research methods of RBPs mainly include homopolymer binding method, ultraviolet cross-linking, SELEX, EMSA, whole-genome in vivo immunoprecipitation, and protein affinity purification . In addi samples . Subsequ samples . The dis samples .Fig. 3TTumor is a complex heterogeneous disease and its pathogenesis is difficult to determine. Tumor is always accompanied with genetic mutations, which disturb the homeostasis of carcinogenic or tumor suppressor signaling pathways . PerspecNcRNA-mediated post-transcriptional regulation is closely related to the expression of RBPs. For example, LIN28 is an RBP mainly composing of two subunits, LIN 28A and LIN 28B, both of which have a similar structure and function. LIN28 is often dysregulated in tumor cells, and the upregulation of LIN28 is inversely related to the poor prognosis of advanced cancer. LIN28 inhibits the processing of let-7 precursor miRNA into mature miRNA in poorly differentiated cells, increases PD-L1 transcription and translation, and then promotes the growth and immune escape of various cancer cells . ConversThe abnormal function of RBP is also caused by abnormal post-translational modifications (PTM) in tumors, originates from multiple signaling pathways and activates the spatial conformation of enzymes, thereby inducing changes in its functional chemical groups, including acetylation, phosphorylation, methylation, ubiquitination, and N6-methyladenosine methylation (m6A)  tails at the 3′ end of the mRNA. This process corresponds to the addition of a long string of adenylate on the 3′ end of the transcript. The pre-mRNA 3′ end processed complexes are mainly composed of CPSF, CSTF, CFI, CFII, CTD and RNAPII. About 85 proteins engage in this process , 143. ThTumor cells express a large number of aberrant mRNAs. The length of these mRNA 3′UTRs is shorter than that in normal cells. Studies have revealed that this phenomenon is usually caused by APA –148. BecThe transcription, translation, stability and subcellular localization of mRNA are closely related to the highly regulated RNA-binding protein (RBP), thereby exerting a variety of cellular functions. In addition to the poly (A) tail at the 3′ end, the structure that impacts the stability of the eukaryotic mRNA has a cap structure at the 5′ end. There are many ways to degrade mRNA, one of which is to start with the hydrolysis of poly (A) tail, and then process to 5′ end decapping and 5′ → 3′ direction degradation, or after poly (A) tail hydrolysis, degradation in the 3′ → 5′ direction . To dateOn the one hand, RBPs can enhance the stability of mRNA to regulate the protein expression of its target genes. For example, the PTBP family belongs to RBPs, and there are three main members PTBP1, PTBP2, and PTBP3. PTBP1 and PTBP2 regulate mRNA splicing, stability, localization and translation . HoweverSome mRNAs are transcribed in the nucleus, then transported into the cytoplasm and translated to produce proteins. However, other mRNA transcripts directly target specific regions for local translation or distribution, resulting in an asymmetric distribution of cytoplasmic proteins . The subThe translation process is intricate and can consist of three stages: initiation, extension and termination. Translational regulation of most mRNAs occurs at the initial stage. RBPs, such as the 5′cap-binding complex eIF4F and poly (A)-binding protein (PABP), are required for cyclization and translational activation of mRNAs. In cancer, almost all key oncogenic signaling pathways are abnormal and cause translational disorders such as the PI3K/AKT/mTOR, RAS/MAPK and Wnt/β-catenin signaling pathways –197. It RBPs are increasingly appreciated for their participation in the entire life course of RNA . Recent Notably, in recent years, reversible modification of N6-methyladenosine (m6A) on mRNA has been shown to be a common phenomenon . Both mRThis article mainly discusses the structure and abnormal expression of RBPs in tumors and focuses on the molecular mechanism thereby they exert their effects in cancer. Since RBPs are involved in various processes of tumors, suggesting that RBPs participate in a very complex regulatory network and change numerous characteristics of tumors Fig. . RBPs noAs more and more cancer-related RBPs are discovered, identifying their target genes is needed. A key issue is that the binding of many RBPs to RNA does not depend on classical RBDs , so iden


Mass spectrometry (MS) can reliably detect and localize all mass‐altering modifications of ribonucleic acids (RNA), but current MS approaches that allow for simultaneous de novo sequencing and modification analysis generally require specialized instrumentation. Here we report a novel RNA dissociation technique, radical transfer dissociation (RTD), that can be used for the comprehensive de novo characterization of ribonucleic acids and their posttranscriptional or synthetic modifications. We demonstrate full sequence coverage for RNA consisting of up to 39 nucleotides and show that RTD is especially useful for RNA with highly labile modifications such as 5‐hydroxymethylcytidine and 5‐formylcytidine. Ready to dissociate: Cobalt(III) hexammine can transfer hydrogen atoms to RNA anions and generate radical ions that dissociate into specific fragments for de novo characterization of modified RNA by mass spectrometry. This approach is especially useful for RNA with highly labile modifications, such as 5‐hydroxymethylcytidine and 5‐formylcytidine. In RTD, cobalt(III)hexamine ([CoIII(NH3)6]3+) serves as the reagent for the production of RNA radical ions that dissociate into d and w fragments upon collisional activation, along with c and y fragments that form through the well‐established mechanism for phosphodiester backbone bond cleavage.EDD of RNA,1 (Table III(NH3)6]3+  illustrate the efficient formation of (M+CoIII(NH3)6−n H)n−3)−6−9 H)6− ions  at 51 eV produced ions formed by loss of NH3, 2 NH3, 6 NH3, and . Fragments with [CoIII(NH3)6]3+ attached were a minor fraction (ca. 2 %) and of the a, c, and y type but not the d and w type. Of all the fragments from RNA backbone cleavage , about 5 % were a, 6 % c, 37 % d, 9 % y, and 42 % w. In addition to c and y fragments, CAD can also produce complementary a and w fragments (Scheme d and w fragments from CAD of (M+CoIII(NH3)6−n)n−3)−6−7 H)4− ions with Co attached all carried [CoIII(NH3)6]3+, whereas those from CAD of (M+CoIII(NH3)6−10 H)7− all carried CoII. For the (M+CoIII(NH3)6−n H)n−3)−6 attached decreased with increasing energy used for CAD , Scheme 3 molecules. Moreover, the c and y fragments with CoII attached must have formed by a mechanism other than phosphodiester backbone bond cleavage and subsequent loss of 6 NH3, as the latter cannot account for the change in oxidation state from CoIII to CoII.The D Figure  B, whichIII(NH3)6−n H)n−3)− increased substantially n−3)− could be radical (M+CoIII+H.−n H)n−3)−.( ions (formed by H. transfer from NH3 to the RNA) as well as even‐electron (M+CoII−(n−1)H)n−3)−( ions (formed by electron transfer to CoIII and proton transfer to the RNA). As CAD (51 eV) of even‐electron (M+CoII−8 H)6− ions from ESI of solutions of RNA 1 with cobalt(II) acetylacetonate did not produce any d and far fewer w fragments  than CAD of (M+CoIII(NH3)6−9 H)6− ions, we conclude that a substantial fraction of the ions formed by loss of (5 NH3+.NH2) are radical (M+CoIII+H.−9 H)6−. ions that can dissociate into d and w fragments (reaction (2), Scheme c and y fragments that did not carry [CoIII(NH3)6]3+ likely involved nonradical (M+CoII−8 H)6− ions ), as their mass values were consistent with CoII but not CoIII. Moreover, CAD of (M+CoIII(NH3)6−n H)n−3)−, which agrees with the established nonradical mechanism for RNA dissociation into c and y fragments that involves the 2′‐OH group.With increasing energy and net charge of the (M+Co5 NH3+.NH increase5 NH3+.NH increase5 NH3+.NH increaseM+CoII−n−H)(n−3)− 3+.NH2) in CAD of (M+CoIII(NH3)6−9 H)6− ions in the energy range 45–57 eV coincided with a steep increase in the number of d and w fragments. However, above 57 eV, the number of c and y fragments increased, whereas the number of d and w fragments decreased ) than for separate H+ and e− transfer and dissociation into c and y fragments (reaction (3)), which is also reflected in the branching ratio between d+w and c+y fragments for n−3=6 and 7  was predominant 6−. and even‐electron (M+CoII−8 H)6− ions are formed by CAD of (M+CoIII(NH3)6−9 H)6− ions of RNA 1, we used collisional activation in the source region of the instrument, isolated the products resulting from loss of (5 NH3+.NH2) 6− ions), and subjected them to CAD (54 eV) in the collision cell. This experiment produced a, c, d, y, and w fragments, with and without CoII attached, from which we conclude that both radical (M+CoIII+H.−9 H)6−. and even‐electron (M+CoII−8 H)6− ions were produced by dissociation of (5 NH3+.NH2) from (M+CoIII(NH3)6−9 H)6− ions. The branching ratio between d+w and c+y fragments was about 4.1, which is somewhat lower than that from CAD of (M+CoIII(NH3)6−9 H)6− ions at 54 eV without collisional activation in the source region (ca. 5.1) but close to that at 57 eV , along with loss of .NH2 and reduction of Co3+ to Co2+. The latter remains bound by electrostatic interactions to either the d or the w fragment after separation of the fragments. For example, about 62 % of the d4 6−9 H)6− ions of RNA 1 at 51 eV carried Co2+ , which adds up to about 100 %. Our proposed mechanism thus provides a rationale for the types of fragments formed (d and w), the unusual loss of (5 NH3+.NH2) from the (M+CoIII(NH3)6−n H)n−3)−6]3+ instead of [CoIII(NH3)6]3+, namely CAD of (M+RuIII(NH3)6−n H)n−3)−CoIII complexes showed only products corresponding to electron and metal transfer but not H. transfer.Our proposed mechanism for the formation of d and w fragments was not significantly affected by the presence or absence of nucleobases and ribose 2′‐OH groups (Table d and w fragments was affected by the net charge of the (M+CoIII(NH3)6−n H)n−3)−, which we tentatively attribute to different sites of Co(NH3)6 binding in the (M+CoIII(NH3)6−n H)n−3)−.The site‐specific extent of RNA backbone cleavage into ps Table , which i4 (39 nt), we found that an increase in the number of [CoIII(NH3)6]3+ adducts from one to two increased the branching ratio between d+w and c+y fragments from about 0.35 (CAD of (M+CoIII(NH3)6−17 H)14− ions at 119 eV) and 0.39 (CAD of (M+CoIII(NH3)6−18 H)15− ions at 111 eV) to 0.89 (CAD of (M+2 [CoIII(NH3)6]−21 H)15− ions at 105 eV), although all three RTD spectra of RNA 4 provided full sequence coverage , 5‐hydroxymethylcytidine (5Chm), and 5‐formylcytidine (5Cf) at position 10, respectively , 5Chm, and 5Cf indicated that the glycosidic bond of C is more stable than those of 5Cm, 5Chm, and 5Cf by factors of 1.07, 1.25, and 1.74, respectively . CAD of RNAs 5, 6, and 7 with C, 5Chm, and 5Cf at position 10, respectively, showed that losses of A, C, and G nucleobases from (M−n H)n− ions (base loss from U was not observed) were not significantly affected by the presence of 5Chm or 5Cf, but that loss of guanine and adenine are favored at lower and higher net negative charge, respectively . Moreover, the up to 11‐fold higher base loss from 5Cf compared to that from A, C, and G confirmed the low stability of the glycosidic bond of 5Cf . For RNA 6, the loss of H2O from 5Chm was competitive with nucleobase loss and similar in extent to nucleobase loss from 5Cf . Notably, the extent of nucleobase and H2O loss from fragments from RTD of (M+CoIII(NH3)6−10 H)7− ions was generally lower than for CAD of (M−7 H)7− ions of RNAs 5, 6, and 7 (Table S1). For example, extensive 5Cf nucleobase loss from a10 (93 %) and c10 (21 %) formed by cleavage of the backbone next to 5Cf at position 10 was observed in CAD of RNA 7, whereas RTD did not produce any c10 or d10 fragments that showed 5Cf nucleobase loss. We conclude that binding of [CoIII(NH3)6]3+ either increases the stability of the glycosidic bond or lowers the energy required for backbone cleavage below that for nucleobase dissociation.To demonstrate RTD of modified RNA, we studied the 17 nt RNAs 59Co), the isotope distributions of RNA and RNA fragments with and without Co are highly similar, and existing algorithms can be used for automated data analysis. The unique RTD radical reactions made possible by [CoIII(NH3)6]3+ considerably expand the repertoire of dissociation techniques for the characterization of RNA by mass spectrometry.In conclusion, we report a new dissociation technique, RTD, that allows for de novo sequence characterization of modified RNA without the need for laborious sample preparation or specialized MS instrumentation. As naturally occurring, stable Co is monoisotopic  chloride  at a flow rate of 1.5 μl min−1. Data reduction utilized the SNAP2 algorithm .Experiments were performed on a 7 T FT‐ICR instrument  equipped with an ESI source, a linear quadrupole for ion isolation, and a collision cell for CAD. RNA was prepared by solid‐phase synthesis, purified by HPLC, desalted,The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The novel coronavirus disease (COVID-19) outbreak was first declared in China in December 2019, and WHO declared the pandemic on 11 March 2020. A fast-rising number of confirmed cases has been observed in all continents, with Europe at the epicentre of the outbreak at this moment.Sexual and reproductive health (SRH) and rights is a significant public health issue during the epidemics. The novel coronavirus (SARS-CoV-2) is new to humans, and only limited scientific evidence is available to identify the impact of the disease COVID-19 on SRH, including clinical presentation and outcomes of the infection during pregnancy, or for persons with STI/HIV-related immunosuppression. Beyond the clinical scope of SRH, we should not neglect the impacts at the health system level and disruptions or interruptions in regular provision of SRH services, such as pre- and postnatal checks, safe abortion, contraception, HIV/AIDS and sexually transmitted infections. Furthermore, other aspects merit attention such as the potential increase of gender-based violence and domestic abuse, and effects of stigma and discrimination associated with COVID-19 and their effects on SRH clients and health care providers. Therefore, there is an urgent need for the scientific community to generate sound clinical, epidemiological, and psycho-social behavioral links between COVID-19 and SRH and rights outcomes. On 31 December 2019, the World Health Organization (WHO) was informed of a cluster of cases of pneumonia of unknown cause, first of which reported on 9 December, detected in Wuhan City, Hubei Province of China. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the causative virus by Chinese authorities on 7 January, with evidence of human-to-human transmission by 20th January. On 30 January 2020 the outbreak is declared a Public Health Emergency of International Concern (PHEIC) and a pandemic on March 11, 2020.On March 16, 2020, the total number of cases outside China had overtaken the total number of cases in China. As of 16th March 2020, 167,511 corona virus disease (COVID-19) cases had been confirmed globally in 148 countries. Europe has now become the epicenter of the pandemic. A fast-rising number of confirmed cases has also been observed in many countries, including the US, Iran, Italy and currently counting cases on all continents [As early as in February, the United Nations Population Fund (UNFPA) Asia and Pacific Regional Office issued its first guidance document and elucidated that SRH is a significant public health issue during epidemics . WHO andCOVID-19 is new to humans, and only limited scientific evidence is available to identify its impact on sexual and reproductive health (SRH). It is inconclusive as to whether transmission from the pregnant woman to the fetus exists in COVID-19 , 6. UndoIt is also important to recognise that reproductive health issues may not be restricted to women, but that men may also suffer consequences. SARS-CoV-2 infection may increase the risk of damage to testicular tissues , and we For both men and women, questions around whether persons with STI/HIV-related immunosuppression are at greater risk of contracting COVID-19 and experiencing poorer recovery rates may also be worthy of investigation given their public health implications. An early February 2020 online survey of persons living with HIV in China indicated this issue as a major source of anxiety within this community .Beyond the clinical scope of SRH, we should not neglect the second tier of impact from COVID-19 on SRH. At the health system level, to support Hubei Province’s emergency response, 189 medical teams, consisting of 21,569 health professionals were assembled across China and dispatched to Hubei Province . AlthougIt has also been suggested that demands of safe abortion services, including information provision, has increased in the hospitals in nearby Hunan Province of China, which may be related to lack of contraceptive commodities or to fear of unknown consequences of infection during pregnancy in the context of an epidemic . These sGender-based and domestic violence is also a major concern of SRH and rights, and the consequences of enforced self-quarantine or compulsory quarantine policy to contain the outbreak are unknown. Evidence shows that quarantine leads to negative psychological effect, including post traumatic stress symptoms, confusion and anger . QuarantSRH and rights is a significant public health issue during outbreaks and it should be a priority. As this pandemic is still evolving, it is urgent for the scientific community to generate solid clinical, epidemiological, and psycho-social behavioral links between COVID-19 and SRH and rights outcomes. In particular, there is a strong need for timely planning and actions for epidemiological research and surveillance of the key vulnerable groups of women and adolescents, and to assess the immediate, medium and long-term effects on their SRH. Perhaps more importantly, we need to solidify operational strategies and actions to protect SRH and rights of women, young people, and vulnerable populations during the epidemic. This requires not only the scientists and physicians, but also policy-makers, community organizations, and international agencies, to work in coordination, trust, and solidarity.


FMR1) gene. The expansion of CGG repeats in the FMR1 mRNA elicits neuronal cell toxicity through two main pathogenic mechanisms. First, mRNA with CGG expanded repeats sequester specific RNA regulatory proteins resulting in splicing alterations and formation of ribonuclear inclusions. Second, repeat-associated non-canonical translation (RANT) of the CGG expansion produces a toxic homopolymeric protein, FMRpolyG. Very few small molecules are known to modulate these pathogenic events, limiting the therapeutic possibilities for FXTAS. Here, we found that a naturally available biologically active small molecule, Curcumin, selectively binds to CGG RNA repeats. Interestingly, Curcumin improves FXTAS associated alternative splicing defects and decreases the production and accumulation of FMRpolyG protein inclusion. Furthermore, Curcumin decreases cell cytotoxicity promptly by expression of CGG RNA in FXTAS cell models. In conclusion, our data suggest that small molecules like Curcumin and its derivatives may be explored as a potential therapeutic strategy against the debilitating repeats associated neurodegenerative disorders.Fragile X-associated tremor ataxia syndrome is an untreatable neurological and neuromuscular disorder caused by unstable expansion of 55–200 CGG nucleotide repeats in 5′ UTR of Fragile X intellectual disability 1 ( Candidate small molecule Curcumin ameliorates expanded CGG RNA mediated disease pathogenesis. Cn (FMRP) . Of inteexp] causing FXTAS. Ideally, a potential compound would prevent non-canonical translation of the CGG repeats sequence into the toxic FMRpolyG protein, as well as correct the alternative splicing defects caused by sequestration of RNA regulatory proteins by expanded CGG nucleotide  , DiGeorgcleotide . Here, wcleotide . We valiexp RNA are able to reverse the toxic consequences of CGG expansions. We searched for naturally available and biologically potent small molecules that would bind to CGG RNA repeat A repeat .exp, UU, CC, and AA hairpin loop structures are formed in r(CUG)exp, r(CCG)exp, and r(CAG)exp RNAs. Binding studies demonstrated that Curcumin showed high selectivity with CGG RNA over other mismatched RNA. Finally, different G-quadruplexes forming DNA  and duplex calf thymus (CT) DNA controls were also tested  and association constant (Ka) were derived using Origin 7.0 software  of the highest affinity binding sites for r(CGGx6), r(CGGx20), r(CGGx40) and r(CGGx60) RNAs are 1.7 × 106 M–1, 3.8 × 107 M–1, 8.5 × 107 M–1, and 8.6 × 107 M–1, respectively  and red shift in negative peaks in a concentration dependent manner owing to the formation of Curcumin-RNA complex . Of intem) were of 1.49, 3.48, 4.19, 5.25, and 6.28°C for r(CGGx1), r(CGGx6), r(CGGx20), r(CGGx40), and r(CGGx60), respectively exp RNAs exp RNAs . Of inteexp RNAs . In contexp RNAs . In paraexp RNAs . As a coexp RNAs . In addiexp RNAs . These dSMN2) and B-cell lymphoma × (Bcl-x) minigenes.Next, we determined cellular potency of Curcumin to correct FXTAS-associated pre-mRNA alternative splicing defects in cultured cell using survival of motor neuron 2 (SMN2 and Bcl-x pre-mRNA splicing (SMN2 mature mRNA contain exon 7 in absence r(CGG)×99, while in presence of r(CGG)×99 approximately 65% of exon 7 included in SMN2 mRNA  pattern of SMN2 at 100 μM.Previous studies reported that Sam68, a splicing regulator, sequestered by expanded CGG repeat transcripts causes dysregulation of splicing . In thissplicing . ApproxiMN2 mRNA . NotablySMN2 minigenes in the absence of CGGx99 expansion  minigene was used to investigate the alternative splicing, as their splicing pattern is not influenced by the CGG expansion exp that cause ALS/FTD and DM1 disease, respectively  translation  developed cell model. Furthermore, Curcumin increases the survival of toxic CGG repeat containing plasmid transfected cells.It is well known that expansion of CGG repeats in the 5′ UTR of mutant FMR1 transcripts causes FXTAS . Previou defects . CGG repnslation . Previouapproach , 2016b. Initially, fluorescence based screening of Curcumin against different mismatched RNA motif and fully paired RNA showed high affinity of Curcumin with CGG RNA motif. Curcumin portrays varied binding characteristics with different mismatched RNA motifs as it has different binding constant values for different RNA sequences. Lower affinity of Curcumin with different higher repeat mismatched RNAs, yeast t-RNA and G-quadruplex forming DNA suggests the selectively of Curcumin with CGG expansion. In addition, increase in the number of CGG repeat enhances the binding affinity of Curcumin as revealed by dissociation constant difference of r(CGGx6) and r(CGGx60) RNA. Taken together, binding of Curcumin appear to be sequence, topology and stoichiometry dependent , 2019a.Furthermore, ITC data supports the interactions between Curcumin with CGG expansion . In genevia small molecules ameliorate FXTAS associated pathogenic defects  repeat RNAs which further suggested the similar mode of binding. Moreover, constant CD spectrum changes of CGG RNA elucidated the intercalation mode binding of Curcumin within CGG hairpin structure , 2019a.  defects . Recentl defects , b. In cthe same .Similarly, Curcumin mediated stabilization of CGG repeat RNA could improve splicing defect and RAN translation. As we observed, Curcumin treatment improves the splicing defect in developed cell model . Curcumiexp through small molecule correlates with the inhibitory effect of RAN translation exp motifs that is vital for the effective inhibition of RANT, further support the effect of RNA stabilization on RANT. Second model, binding of compound to CGG expansion interrupt ribosomal machinery to start translation from random position. Additionally, Curcumin have been reported for neuroprotective effect in other neurological disorder also such as Alzheimer’s, Parkinson’s, frontotemporal dementia, and Huntington disease etc. Administration of Curcumin improved the cell viability through various mechanism including inhibits amyloid formation and its toxicity in Alzheimer disease, reduce oxidative damage, inflammation, and synuclein protein aggregation in Parkinson disease, decrease aggregation of tau proteins in frontotemporal dementia. Curcumin treatment attenuates ischemia-induced oxidative stress and neuronal cell death in hippocampal tissue. Further, Curcumin inhibits the accumulation and aggregation of prion protein inside the neuroblastoma cell. It also facilitates anti-anxiety activity by decrease level of TNF-α and MMP-9 in Autism spectrum disorder nslation  as stabidisorder .via small molecule ameliorates FXTAS associated pathogenic defects  or small molecule enhancing thermostability of r(CGG)exp correlates with their inhibitory effect on RAN translation (read through to mRNA. A second model is that binding of compounds to CGG expansions can simply occupy the RNA and prevent ribosomal machinery to start non-canonical translation. Similarly, Curcumin binding to CGG repeats can prevent further association and interaction of RNA binding proteins that leads to FXTAS-associated splicing defects. Furthermore, Curcumin is able to cross blood brain barrier due to its lipophilic nature, despite of its poor bioavailability. Henceforth, Curcumin reach brain in biological effective concentration and ameliorate neurodegeneration (There is a strong need of effective and safe therapeutic modalities for FXTAS. Previous studies have reported that the stabilization of CGG repeat RNA  defects . Recentl defects , 2019a. nslation  as RNA hneration . To furtneration . In concAll datasets generated for this study are included in the article/AK conceived and designed the project. AV, EK, and SM performed the experiments. AV and AK analyzed the data and drafted the manuscript. AK, AM, and NC-B performed critical analysis to the manuscript.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.


Long INterspersed Element-1 (LINE-1) is an autonomous retroelement able to “copy-and-paste” itself into new loci of the host genome through a process called retrotransposition. The LINE-1 bicistronic mRNA codes for two proteins, ORF1p, a nucleic acid chaperone, and ORF2p, a protein with endonuclease and reverse transcriptase activity. Both proteins bind LINE-1 mRNA in cis and are necessary for retrotransposition. While LINE-1 transcription is usually repressed in most healthy somatic cells through a plethora of mechanisms, ORF1p expression has been observed in nearly 50% of tumors, and new LINE-1 insertions have been documented in a similar fraction of tumors, including prostate cancer.Here, we utilized RNA ImmunoPrecipitation (RIP) and the L1EM analysis software to identify ORF1p bound RNA in prostate cancer cells. We identified LINE-1 loci that were expressed in parental androgen sensitive and androgen independent clonal derivatives. In all androgen independent cells, we found higher levels of LINE-1 RNA, as well as unique expression patterns of LINE-1 loci. Interestingly, we observed that ORF1p bound many non-LINE-1 mRNA in all prostate cancer cell lines evaluated, and polyA RNA, and RNA localized in p-bodies were especially enriched. Furthermore, the expression levels of RNAs identified in our ORF1p RIP correlated with RNAs expressed in LINE-1 positive tumors from The Cancer Genome Atlas (TCGA).Our results show a significant remodeling of LINE-1 loci expression in androgen independent cell lines when compared to parental androgen dependent cells. Additionally, we found that ORF1p bound a significant amount of non-LINE-1 mRNA, and that the enriched ORF1p bound mRNAs are also amplified in LINE-1 expressing TCGA prostate tumors, indicating the biological relevance of our findings to prostate cancer.The online version contains supplementary material available at 10.1186/s13100-021-00233-3. Many factors have played a role in shaping the evolution of the human genome. In particular, the Long INterspersed Element 1 (LINE-1) retrotransposon, has significantly contributed in shaping the size, structure, and expression of the human genome over millions of years . Since LFull length LINE-1 sequences consist of a 5’UTR/promoter, two open reading frames, coding for ORF1p and ORF2p proteins, and a 3′ UTR with a polyA signal . FollowiStudies have demonstrated that both LINE-1 proteins, ORF1p and ORF2p, may be necessary for LINE-1 retrotransposition . ORF1p iDNA methylation, histone modifications, and RNA interference all limit LINE-1 expression and function in somatic cells –35. HoweThe repetitiveness of LINE-1 sequences poses a challenge to identifying actively transcribed LINE-1 loci , 43. Howp = 0.03). This likely reflects the fact that LNCaP-95 and LNCaP-abl are derived from LNCaP through androgen deprivation, whereas 22Rv1 is clonally independent.We recently showed that our tool, L1EM, is able to quantify RNA expression at specific LINE-1 loci from RNA-seq data . We alsoWhile the enrichment for LINE-1 RNAs with intact ORF1 indicates that we were successful in immunoprecipitating LINE-1 RNPs, we were surprised by the fact that LINE-1 RNA was such a small fraction of the immunoprecipitated transcripts. Others have also noted widespread associations between ORF1p and host mRNAs , 46, andHLTF, PCDHGA9, PCDHGB5, RELN, SMC2, SI, and SP110. The full list of mRNAs enriched in each experiment can be found in Table SReads from younger LINE-1 families  ranged from 0.1% of all reads in LNCaP to 0.15% of all reads in LNCaP-abl. We therefore wondered whether LINE-1 mRNA was dissociating from ORF1p during the IP procedure, freeing ORF1p to interact with other mRNA species present in the cell lysates. To test this possibility, we performed UV crosslinking of LNCaP cells prior to a 1-h or 3-h immunoprecipitation. These IPs yielded slightly less LINE-1 RNA: < 0.1% of reads in all experiments, indicating that ORF1p was unlikely to disassociate from the LINE-1 mRNA during the IP and that most of the identified interactions of ORF1p and LINE-1 mRNAs formed in the cells, before lysis. Analysis of the non-LINE-1 RNA present in the ORF1p immunoprecipitates showed that, in all experiments, the majority of reads represented ribosomal RNA, which was not depleted from the pool of mRNAs in our experimental design. However, we also found a much greater fraction of reads aligning to exonic sequences in ORF1p IP compared to control (input or IgG IP) . We therefore performed Gene Set Enrichment Analysis (GSEA)  using Rep = 5.9 × 10− 24). Mandal et al. hypothesized that ORF1p bound mRNAs are poised for pseudogene retrotransposition. However, we only found a weak correlation between the number of times a gene has been processed into a pseudogene [ρ = 0.11, p = 1.1 × 10−6). Thus, pseudogenes alone are likely an insufficient explanation for the prevalence of mRNAs in the ORF1p IP.A similar enrichment of many host mRNAs was previously reported by Mandal et al. (2013) in the context of ectopic LINE-1 expression . We founeudogene , 53 and ρ = 0.4, p ≈ 0, Fig. p = 2.6 × 10−77, Figure Sρ = 0.15, p = 7.1 × 10−43, Fig. p = 1.1 × 10−5, Figure Sρ =  − 0.08, p = 1.4 × 10−12), indicating that the positive relationship between ORF1p bound and SG localized RNA can be explained by the fact that many RNAs are enriched in both p-bodies and SG. The Mandal et al. data also has stronger overlap with p-body RNA  than with SG RNA  [ρ = 0.19, p = 1.2 × 10−66 under unstressed conditions and ρ = 0.18, p = 2.5 × 10−56 under stress conditions. These pellets include stress granules and other RNAs, but not p-bodies [We reasoned that ORF1p localization to some phase separated RNP granules may explain why the RNAs that immunoprecipitated with ORF1p were particularly enriched for polyA RNAs. If ORF1p localizes to such a granule, we would expect to IP not only mRNAs directly bound ORF1p, but also those that indirectly interact with ORF1p through extended RNA/protein interactions. Because ORF1p is primarily cytoplasmic , two typ× 10− 9) . We alsop = 1.2 × 10−73, Figure Sρ = 0.39, p ≈ 0, Fig. p ≈ 0, Figure SBecause p-bodies are important regulators of mRNA processing, we hypothesized that LINE-1, or at least ORF1p expression, could have an impact on the abundance of certain mRNAs. To investigate this, we conducted an siRNA knockdown of LINE-1 and examined the mRNA levels of 12 genes enriched in our ORF1 IP and in p-bodies. Upon knockdown, we observed a reduction in mRNA levels in eleven out of twelve of the genes we measured Figure S. FurtherThe reactivation of LINE-1 expression in prostate cancer has raised new questions regarding its role in cancer initiation and progression. Sequencing studies have shown active retrotransposition in prostate tumors, and an increased rate of LINE-1 retrotransposition in metastatic prostate cancer compared to primary tumors . HoweverUnexpectedly, we found that among all the RNA bound by ORF1p only a small percentage is represented by LINE-1 mRNA. While our analysis showed that our ORF1p IP was enriched for full length and complete ORF1p LINE-1 mRNA, indicating successful LINE-1 ORF1p immunoprecipitation, the vast majority of RNA enriched in our ORF1 IP consisted of non-LINE-1 transcripts Fig. . InteresWe were particularly surprised to find MT -RNA enrichment in the crosslinked ORF1p IP experiments. MT-mRNAs are polyadenylated, and it is possible that ORF1p could bind them directly. While no study has shown ORF1p localizing in the mitochondria, ORF1p may bind MT-RNA that has been released from dysfunctional mitochondria. However, we did also find that MT-rRNAs are also enriched . We considered whether this enrichment might be the result of MT-DNA contamination as mtDNA contamination can be amplified by the many MT genome copies that exist in each cell. Globally, we find that the vast majority of reads align to rRNA or exons, indicating that DNA contamination is not likely to be a significant factor in our analysis. However, MT specific DNA contamination is difficult to rule as most of MT genome is transcribed as a single transcript. A third possibility is that crosslinking connects mitochondria to nearby ORF1p containing RNPs. Mitochondria are involved in interferon signaling  and RNA Given strong evidence from previous studies indicating that ORF1p locates to stress granules (SG), we expected to find a large overlap between ORF1p bound mRNAs and SG enriched mRNAs. Instead, we found only a small overlap that can be explained by the overlap between p-body and SG RNA. In contrast, we found a much larger overlap between ORF1p bound mRNAs and p-body enriched mRNAs compared to SG RNAs. This may indicate that endogenously expressed ORF1p localizes to p-bodies in LNCaP cells, or it may indicate that ORF1p promotes the mislocalization of p-body RNAs to sites of ORF1p accumulation. Alternatively, the high levels of L1 sequences may lead to the formation of a third type of body/granule with intermediate properties.The discrepancy between our results and the previous studies pointing to SG as sites of LINE-1 proteins/RNA localization, may be explained by the fact that LNCaP cells were “unstressed” in our study. Goodier et al. (2007) did find endogenous ORF1p localizing with SG markers in embryonal carcinoma cell lines . HoweverWhether ORF1p localizes to p-bodies or causes p-body RNA mislocalization, it may be interfering with the normal processing of p-body RNAs. Our results show that ORF1p-bound mRNAs expression correlates with LINE-1 expression in prostate cancer. Additionally, upon LINE-1 knockdown, we observed a decrease in p-body mRNA levels that were enriched in our ORF1IP. Thus, LINE-1 ORF1p expression may be interfering, directly or indirectly, with the processing/degradation of certain mRNAs. In particular, almost all of the genes that are significantly correlated with LINE-1 RNA in prostate cancer were also enriched in p-bodies, indicating that LINE-1 may interfere with the degradation of p-body associated RNAs. In yeast, p-body proteins are involved in the regulation of gene expression related to DNA replication stress resistance , possiblOur study, summarized in Fig. LNCaP (CRL-1740) and 22Rv1 (CRL-2505) cells were purchased from ATCC and maintained in RPMI 1640 with 10% FBS. LNCaP-abl, and LNCaP-95 cell lines were generous gifts from Z. Culig, and J. Isaacs, respectively and maintained in RPMI 1640, phenol red free, and 10% charcoal dextran stripped FBS. Cells were regularly screened for mycoplasma.For crosslinked samples, cells were kept on ice, washed twice with cold PBS, and UV-crosslinked using a Stratalinker 2400 with 150 mJ/cm^2 at 254 nm. Four 15 cm plates of each cell type, ~ 70% confluency, were used for each immunoprecipitation. Cells were washed with cold PBS and centrifuged at 1500 rpm (485 x g) for 5 min at 4 °C. RNA immunoprecipitation was conducted using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore Sigma 17–700) as follows. Cells were resuspended in 400 μl RIP lysis buffer supplemented with protease inhibitor cocktail and RNase inhibitor, and snap frozen to − 80 °C. Magnetic beads were incubated with 10 μg ORF1p (Millipore Sigma MABC1152) or mouse IgG (Santa Cruz sc-2025) antibody per 50 μl of magnetic beads (200uL beads used per condition). Cells were thawed and centrifuged at 14,000 rpm  for 10 mintues at 4 °C. Supernatant was collected and mixed with RIP Immunoprecipitation buffer, supplemented with EDTA and RNase Inhibitor, and antibody bound magnetic beads. Bead/antibody complexes and cell lysates were rotated at 4 °C for either 1 h or 3 h. Beads were then washed 7x with 500uL RIP Wash Buffer, and then incubated with Proteinase K Buffer for 30 min at 55 °C. Supernatant was separated from magnetic beads and RNA was isolated using a phenol chloroform extraction. Lastly, an on-column DNase digestion and RNA cleanup was performed using a RNeasy MinElute Cleanup Kit according to manufacturer’s protocol .Sequencing library was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB E7770S) according to manufacturer’s protocol, each sample prepared with its own unique barcode (NEB E7600S). Prepared libraries were sequenced as paired end 36-cycle reads (20 M) on the NextSeq 500. Reads were demultiplexed with Illumina bcl2fastq v2.20 requiring a perfect match to indexing BC sequences.Reads were aligned to the hg38 human reference genome using the STAR aligner and TCGA mRNA analysis pipeline options . Locus srRNA was quantified using samtools  to countORF1p IP enrichment for each gene was estimated using DEseq2 . Note th5 LNCaP cells were treated twice with siRNA for 48 h using Lipofectamine RNAiMAX . LINE-1 siRNA contained a pool of siRNA  custom made by Thermo Fisher Scientific . Protein was collected by lysing cells in RIPA buffer  for 10 minutes, spun at 14,000 rpm for 5 min, and normalized using a Bradford Assay .7 × 10RNA was collected using the Qiagen RNeasy Plus Mini Kit 74134) followed by a DNase digestion (Thermo Fisher Scientific AM1907) according to manufacturer’s protocol. RNA was reverse transcribed using the Verso cDNA kit (Thermo Scientific- AB1453A). qPCR was conducted using SYBR Green Master Mix  with gene specific primers  LINE-1 RNA in the input for each cell LINE-1. (B) LINE-1 RNA after ORF1p-IP enrichment.Additional file 2: Figure S2. Heatmap of the specific intact loci immunoprecipitated in each cell line. Expansion of Fig. Additional file 3: Figure S3. Overlap between transcripts enriched in ORF1p IP and in cytoplasmic granules. (A) Overlap between ORF1p IP enrichment and p-body enrichment [richment . A 5% FDrichment . (C) OveAdditional file 4: Figure S4. qPCR quantification of p-body and ORF1 IP RNA after siRNA knockdown of LINE-1. (A) qPCR of top ORF1 IP and p-body genes after 48 h siRNA LINE-1 knockdown. Normalized using RPL19. (B) Western blot of 48 h siRNA LINE-1 knockdown used for qPCR in (A). Demonstrates knockdown of ORF1p protein.Additional file 5: Table S1. L1EM estimates of locus specific LINE-1 expression for each of the samples.Additional file 6: Table S2. Enrichment of genes in ORF1p IP vs input estimated by DESeq2.Additional file 7: Table S3. Primers used in qPCR experiments.


Guanosine- and uridine-rich single-stranded RNA (GU-rich RNA) is an agonist of Toll-like receptor (TLR) 7 and TLR8 and induces strong immune responses. A nanostructured GU-rich RNA/DNA assembly prepared using DNA nanotechnology can be used as an adjuvant capable of improving the biological stability of RNA and promoting efficient RNA delivery to target immune cells. To achieve a sustained supply of GU-rich RNA to immune cells, we developed a GU-rich RNA/DNA hydrogel (RDgel) using nanostructured GU-rich RNA/DNA assembly, from which GU-rich RNA can be released in a sustained manner. A hexapod-like GU-rich RNA/DNA nanostructure, or hexapodRD6, was designed using a 20-mer phosphorothioate-stabilized GU-rich RNA and six phosphodiester DNAs. Two sets of hexapodRD6 were mixed to obtain RDgel. Under serum-containing conditions, GU-rich RNA was gradually released from the RDgel. Fluorescently labeled GU-rich RNA was efficiently taken up by DC2.4 murine dendritic cells and induced a high level of tumor necrosis factor-α release from these cells when it was incorporated into RDgel. These results indicate that the RDgel constructed using DNA nanotechnology can be a useful adjuvant in cancer therapy with sustained RNA release and high immunostimulatory activity. Guanosine- and uridine-rich single-stranded RNA (GU-rich RNA) is a typical agonist of Toll-like receptor 7 (TLR7) and TLR8 in the endosomes of most antigen presenting cells ,2,3,4,5.In the development of a GU-rich RNA-based adjuvant, efficient delivery to endosomes of antigen presenting cells expressing TLR7/8 is necessary. However, endocytic RNA uptake by antigen presenting cells is limited and single-stranded RNA is easily hydrolyzed by the RNase present in biological environments ,7,8. MorAs an approach to solve these problems, we developed a tetrapod-like nanostructured GU-rich RNA/DNA assembly, or tetrapodRD3, comprising one phosphorothioate-stabilized RNA and three phosphodiester DNAs . The tetBased on these considerations, this study aimed to develop a new RNA adjuvant capable of sustained RNA release, targeting TLR7/8. To this end, we designed a hexapod-like nanostructured GU-rich RNA/DNA assembly, or hexapodRD6, consisting of a 20-mer phosphorothioate GU-rich RNA and six phosphodiester DNAs. Two sets of hexapodRD6, i.e., hexapodRD6-1 and hexapodRD6-2, both of which had complementary cohesive ends to each other, were prepared and mixed together to obtain the GU-rich RNA/DNA hydrogel, or RDgel. We first evaluated RDgel formation and GU-rich RNA release from the hydrogel. We then examined the cellular uptake of the RDgel and tumor necrosis factor (TNF)-α release after addition to mouse macrophage-like RAW264.7 cells and mouse dendritic DC2.4 cells. Finally, we added the conditioned media of RDgel-treated RAW264.7 cells to mouse colon26 tumor cells and examined their effects on tumor cell proliferation.Formation of hexapodRD6-1, hexapodRD6-2, and RDgel was evaluated by polyacrylamide gel electrophoresis (PAGE) analyses b,c. HexaHydrogel formation was also evaluated by adding PBS onto the PI-stained hexapodRD6-1 or RDgel. HexapodRD6-1 was rapidly mixed with PBS and was diluted d. On theThe release profile of GU-rich RNA from RDgel was evaluated using a Transwell chamber system, in which FAM-ORN-1 released from RDgel could be detected in the lower well. Release of ORN-1 from RDgel can be prolonged by controlling the RDgel preparation conditions. Two parameters, the concentrations of nucleotides and metal ions, were selected and RDgel was prepared under various conditions. RDgel uptake by mouse dendritic DC2.4 cells was evaluated by flow cytometry . The meaThe immunostimulatory activity of RDgel was evaluated by the TNF-α release from DC2.4 cells a. DC2.4 ORN-1, hexapodRD6-1, or RDgel was added to RAW264.7 cells, and the culture supernatant was collected as conditioned medium at 24 h after addition. The conditioned medium was then added to mouse colon26 tumor cells. GU-rich ssRNA is an adjuvant that can efficiently induce immune responses and increase vaccine potency. To enhance the effectiveness of vaccines, sustained stimulation of the innate immune system by an adjuvant is desirable. Although hydrogels can be used for sustained RNA release, conventional hydrogels have various problems such as complicated preparation processes. The newly developed RDgel in the present study was composed only of nucleic acids and was easy to prepare. This study revealed an approach that can solve the problems associated with conventional sustained RNA release systems.The release rate of FAM-ORN-1 from FAM-RDgel was faster than the disintegration rate of FAM-RDgel. There are mainly three types of RNA forms released from RDgel: ORN-1, hexapodRD6, and the connecting structure of multiple hexapodRD6. RDgel is disintegrated and oligonucleotides and/or nanostructured RNA are then released from RDgel. ORN-1 conjugated to the end of hexapodRD6 can be dissociated without hydrogel disintegration. Therefore, it is considered that the RNA release rate calculated based on the fluorescence intensity derived from FAM was higher than the gel disintegration rate. The high immunostimulatory activity induced by RDgel is considered a result of efficient cellular uptake of RDgel. It was expected that the connecting structure of multiple hexapodRD6 released from RDgel results in a high cellular uptake of RNA. It was assumed that RDgel was not taken up by the cell as it was, but that RNA, hexapodRD6, or the connecting structure of multiple hexapodRD6 released from the RDgel was taken up by DC2.4 cells. Polypod-like nanostructured DNA (polypodna) are recognized by the nucleic acid receptors expressed on the surface of immune cells and are delivered to endosomes by clathrin-mediated endocytosis. We previously reported that polypodna are taken up, at least partially, through macrophage scavenger receptor-1 (MSR1) . MSR1 isThe time course of FAM-ORN-1 from RDgel was delayed in proportion to the concentration of RNA/DNA and metal ions . It is pWe previously reported that GU-rich ORN containing the HIV-1 U5 region and tetrapod-like RNA/DNA nanostructure with GU-rich ORN were specifically recognized by human TLR8 . TherefoTLR7/8 activation is reported to induce an anti-tumor effect by the release of cytokines such as TNF-α and interleukin-12 ,18 and bWe previously reported that DNA hydrogel containing an unmethylated cytosine-phosphate- guanine (CpG) DNA induced significant cytokine production and the delay of EG7-OVA tumor growth in vivo ,22. CatiIn conclusion, the RDgel developed in this study showed sustained RNA release and high immunostimulatory activity. RDgel can be formed by mixing two sets of self-assembled hexapodRD6. The results of the present study show that the RDgel can be used as an RNA adjuvant useful for cancer immunotherapy targeting TLR7/8.RPMI 1640 medium was purchased from Nissui Pharmaceutical Co., Ltd. . Opti-modified Eagle’s medium (Opti-MEM) and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific Inc. . Sodium chloride, sodium bicarbonate, potassium chloride, Tris-HCl, ethylenediaminetetraacetic acid (EDTA), ammonium chloride, and glucose were purchased from Wako Pure Chemicals Industries, Ltd. . Monothioglycerol, MEM non-essential amino acids, and penicillin-streptomycin-glutamine mixed solution were purchased from Nacalai Tesque, Inc. . The 20 bp ladder was purchased from Takara Bio . Propidium iodide, R848, and ovalbumin were obtained from Merck KGaA . Alexa Fluor 488 annexin V/ Dead Cell Apoptosis kit with Alexa Fluor 488 annexin V and PI for Flow Cytometry was purchased from Invitrogen . All other chemicals were of the highest grade available and were used without further purification.Phosphorothioate oligoribonucleotide (ORN)-1 and its labeled form with 6-FAM conjugated at the 3′-end (FAM-ORN-1) were purchased from FASMAC Corporation . ORNs were synthesized at the 1 μmol scale and purified using HPLC. Phosphodiester oligodeoxynucleotides (ODNs) were purchased from Integrated DNA Technologies, Inc. . ODNs were synthesized at the 100 nmol scale and desalted. ORN-1, ODN-1, ODN-2, ODN-3, ODN-4, ODN-5, and ODN-6 were designed to form hexapodRD6-1. ORN-1, ODN-7, ODN-8, ODN-9, ODN-10, ODN-11, and ODN-12 were designed to form hexapodRD6-2. The sequences of ORN-1 and the ODNs are listed in l-glutamine at 37 °C in humidified air containing CO2. The murine dendritic cell line, DC2.4, was grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 0.2% sodium bicarbonate, 100 IU/mL penicillin, 100 μg/mL streptomycin, 2 mM l-glutamine, 50 μM monothioglycerol, and MEM non-essential amino acids at 37 °C in humidified air containing CO2.RAW264.7 murine macrophage-like cells and colon26 murine colon carcinoma cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 0.2% sodium bicarbonate, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2 mM Each oligonucleotide was dissolved in Tris-EDTA buffer . Appropriate molar ratios of the oligonucleotides included in 100–300 μM of hexapodRD6 were mixed with 150 mM sodium chloride. They were heated at 95 °C for 5 min to dissociate into single strands, 65 °C for 2 min, and 62 °C for 1 min to form double strand as designed, and were then slowly cooled to 4 °C at the rate of 2 °C per min. RDgel was prepared by mixing hexapodRD6-1 and hexapodRD6-2 at the room temperature. The formulation of the nanostructure and hydrogel was confirmed by PAGE. PAGE analysis was performed with a 10% polyacrylamide gel at 200 V for 60 min. Oligonucleotides were stained with SYBR GOLD and were observed using a LAS3000 imaging system . FAM-labeled oligonucleotides were observed without SYBR GOLD staining. Separately, hexapodRD6 and RDgel were stained by mixing with 1 mg/mL of PI solution. Then, PI-labeled hexapodRD6 or RDgel were overlaid with PBS and their miscibility was evaluated.RNA release from RDgel was measured using a 12-well Transwell chamber system . RDgel was dripped onto the Transwell and PBS was added to the lower well. Transwell was incubated at 37 °C and PBS was collected after 1, 3, 6, 12, and 24 h, and the absorbance from nucleic acid in PBS and fluorescence intensity derived from FAM-ORN-1 were measured. RNA release from RDgel with 50, 100, or 150 μM RNA/DNA was measured using the above method. RNA release from RDgel prepared with sodium ions  or magnesium ions  was also examined.RDgel containing FAM-ORN-1 (FAM-RDgel) was overlaid with a solution containing nonheat-inactivated FBS at a final concentration of 10% and incubated at 37 °C. After 1, 2, 4, 6, 8, 16, or 24 h of incubation the supernatant was sampled and the fluorescence of the supernatant was measured using Wallac 1420 ARVO MX .4 cells/well were incubated with FAM-labeled ORN-1 or tetrapodRD3 diluted in 0.1 mL of Opti-MEM at a final concentration of 0.2 μM for 1, 2, 4, 8, and 16 h at 37 °C. Cells were harvested and washed twice with PBS. The fluorescent intensity of the cells was determined by flow cytometry  using the Kaluza software , and the mean fluorescence intensity (MFI) was calculated based on the histograms of the fluorescence intensity of the cells.DC2.4 cells on a 96-well plate at a density of 5 × 104 cells/well and incubated for 24 h before treatment. Then, ORN-1, hexapodRD6, or RDgel diluted in 0.1 mL of Opti-MEM at a final concentration of 1 μM were added to the cells. The supernatants were collected after 20 or 8 h and stored at –80 °C until use. The level of TNF-α in the supernatants was determined by ELISA using OptEIA sets .DC2.4 cells and RAW264.7 cells were seeded on 96-well plates at a density of 5 × 104 cells/well were incubated with oligonucleotides  diluted in 0.1 mL of Opti-MEM for 8 h at 37 °C. The culture supernatant of RAW264.7 cells (culture medium) was added to colon26 cells seeded at a density of 5 × 104 cells/well. The treated colon26 cells were harvested and the number of live cells were counted. The harvested cells were stained with Alexa Fluor 488-labelled anti-annexin V antibody and propidium iodide (PI) from the Alexa Fluor 488 annexin V/Dead Cell Apoptosis kit for Flow Cytometry. The proportion of the stained cells was determined by flow cytometry.RAW264.7 cells seeded on 96-well plates at a density of 5 × 10t-test for comparisons between two groups. A p value of < 0.05 was considered statistically significant.The data were statistically evaluated by one-way analysis of variance followed by the Tukey–Kramer test for multiple comparisons and Student’s