Salmonella enterica, is a deaminase that quenches the reactive metabolite 2-aminoacrylate generated by pyridoxal 5’-phosphate-dependent enzymes and ultimately spares certain enzymes from damage. The accumulation of 2-aminoacrylate can damage enzymes and lead to growth defects in bacteria, plants, and yeast. While all subfamily members have been annotated as imine deaminases based on the RidA characterization, experimental evidence to support this annotation exists for a single protein outside the RidA subfamily. Here we report that six proteins, spanning Rid subfamilies 1–3, deaminate a variety of imine/enamine substrates with differing specific activities. Proteins from the Rid2 and Rid3 subfamilies, but not from the RidA and Rid1 subfamilies deaminated iminoarginine, generated in situ by the Pseudomonas aeruginosa D-arginine dehydrogenase DauA. These data biochemically distinguished the subfamilies and showed Rid proteins have activity on a metabolite that is physiologically relevant in Pseudomonas and other bacteria.The Rid (YjgF/YER057c/UK114) protein family is a group of small, sequence diverse proteins that consists of eight subfamilies. The archetypal RidA subfamily is found in all domains, while the Rid1-7 subfamilies are present only in prokaryotes. Bacterial genomes often encode multiple members of the Rid superfamily. The best characterized member of this protein family, RidA from  The te (2AA) , 19–21.S. enterica protein when tested in vivo and in vitro [The biochemical role of RidA has been characterized primarily in the context of specific PLP-dependent enzymes that proceed through 2-aminoacrylate (2AA), which spontaneously tautomerizes in solution to 2-iminopropionate prior to hydrolysis to a stable ketoacid. In the context of PLP-dependent enzymes, RidA increases the rate of 2AA conversion to pyruvate by facilitating hydrolysis, which is otherwise mediated by solvent water  9, 22, , 22, 23.in vitro . Signifiin vitro , 25–30, S. enterica genome encodes RidA, in addition to YoaB and STM1549, which are members of subfamily Rid2 and Rid7, respectively. The genome of Escherichia coli encodes five putative Rid proteins, two of the RidA subfamily, one of the Rid2 subfamily, a Rid7 subfamily protein and RutC [Pseudomonas aeruginosa, with nine Rid proteins, Streptomyces coelicolor, with eleven, and various Bradyrhizobia that encode up to sixteen putative Rid proteins [Unlike the RidA subfamily, which spans the three domains, subfamilies Rid1-Rid7 are found exclusively in prokaryotes . BacteriS. enterica, respectively) [Members of the Rid family are universally annotated as imine deaminases, yet beyond the RidA subfamily, there is a single report of activity for a Rid2 subfamily member . Based oin vitro assays and in vivo complementation of a ridA mutant were used to assess deaminase activity and substrate specificity of six proteins spanning subfamilies Rid1, Rid2 and Rid3. Iminoarginine deaminase was identified as an activity unique to proteins of the Rid2 and Rid3 subfamilies. In total, these studies bolstered the annotation of the Rid superfamily and suggested a role for Rid family members in facilitating metabolic steps that involve unstable intermediates.This study was initiated to validate the broad functional annotation of the Rid superfamily and extend our understanding of the Rid subfamilies beyond the RidA subfamily. Both Salmonella enterica were derivatives of serovar Typhimurium LT2 and are described, with relevant plasmids, in Escherichia coli BL21AI was used for protein overexpression. Primers used to generate plasmids are listed in Escherichia coli. Other plasmids were constructed by amplifying the appropriate gene from genomic DNA, and ligating into a modified pBAD24 vector (pCV1) [Strains of r (pCV1)  or into r (pCV1) . PlasmidS. enterica and Lysogeny Broth (LB) was used for E. coli. Cells were grown in super broth  for protein overexpression. Difco BiTek agar was added (15 g/ liter) for solid medium. No-carbon E medium (NCE) supplemented with MgSO4 (1 mM) [Crotalus adamanteus), D-amino acid oxidase from porcine kidney (A5222) and catalase from bovine liver (C9322) were hydrated and stored at 4°C. Tris base was purchased from Amresco .Difco Nutrient Broth (8 g/ liter) with NaCl (5 g/ liter) was used as rich medium for 4 (1 mM) , trace m4 (1 mM)  and glycS. enterica strains grown overnight in rich medium (2 mL) were used to inoculate the indicated minimal medium (1% inoculum). Growth was monitored as optical density (OD) at 650 nm for strains grown in biological triplicate at 37°C with shaking. Growth curves were plotted using GraphPad Prism (version 6.0g).Liquid growth analyses were done in a 96-well microtiter plate using a BioTek Elx808 plate reader with continuous shaking (set to slow on the machine). E. coli strain BL21AI harboring the appropriate pET construct. RidA protein was purified as described previously [650 between 0.7 and 1.0. Arabinose  was added to induce T7 RNA polymerase production and cultures were shifted to 22°C (PA5083), 28°C (RidA), 30°C  or 37°C (PSPTO_3006) for 18 hours. Cells were harvested by centrifugation at 7,000 x g and the pellets were stored at -80°C until use. Binding buffer  was added to thaw cells (2 mL per gram wet cell weight) along with lysozyme (1 mg/ mL) and DNase (20 Units/ mL), and the cells were lysed with a French pressure cell. The lysate was clarified by centrifugation  and passed through a 0.45 μm syringe filter (Argos Technologies) prior to being loaded onto a 5 mL Ni-NTA FPLC column of HisPur resin (ThermoFisher Scientific) on an ÄKTA FPLC equipped with a UV lamp and Frac920 fraction collector. Protein was eluted with a 0–100% gradient of elution buffer  per the column manufacturer’s instructions . Elution of polyhistidine-tagged proteins was monitored by absorbance at 280 nm over the gradient and occurred between 20–80% elution buffer. Fractions containing desired protein  were pooled and concentrated with a 7,000 molecular weight cut-off protein concentrator (Millipore). The preparations were dialyzed into storage buffer  using a PD-10 desalting column . Proteins (0.125–2 μg) were subjected to SDS/PAGE and purity was assessed using a Foto/Analyst FX (Fotodyne) imager and TotalLab Quant v11 densitometry software. Purified PFL_1835 was enriched to greater than 90% purity, while all other Rid proteins used in this study were enriched to greater than 98% purity. Protein concentration was quantified using BCA Protein Assay (Thermo Scientific), and the samples were frozen in liquid nitrogen and stored at -80°C.Rid proteins were purified from eviously . ProteindauA. An overnight culture (10 mL) grown in LB containing kanamycin was used to inoculate 1.5 liters of super broth containing kanamycin. A total culture volume of 3 liters was grown at 37°C until the cultures reached an OD650 of 0.6. L-arabinose (3 grams) was added to each flask, and the temperature was shifted to 18°C for 16 additional hours. A sample (5 mL) was pelleted, resuspended in buffer , sonicated, and activity confirmed using a dye-based crude extract dehydrogenase assay [DauA was partially purified from DM15625, a BL21AI strain harboring pET28-All assays were performed at room temperature (~25°C).-1 cm-1).A coupled assay using cysteine desulfhydrase-dependent pyruvate formation and the oxidation of NADH by lactate dehydrogenase was used to assess Rid activity as previously described . The ass-1 cm-1) to calculate the rate of semicarbazone formation.The LOX-based assay for Rid activity was adapted from a previously described continuous assay . Simply,Reaction mixtures contained potassium pyrophosphate , neutralized semicarbazide (10 mM), bovine liver catalase (24 Units), FAD (30 μM), and ~ 1 μM DauA. The reaction was initiated with the addition of D-arginine (1 mM), and the absorbance at 248 nm was monitored for ten minutes. Similar to assays with LOX, the path length was determined for each well, and the molar extinction coefficient for semicarbazone was used to calculate the rate of semicarbazone formation.Pseudomonas aeruginosa, and Acinetobacter baylyi. Members of Rid2 came from S. enterica, Pseudomonas aeruginosa, and Pseudomonas syringae, and members from Rid3 came from Pseudomonas syringae and Pseudomonas fluorescens. Source organism, locus tag and sequence alignment of each protein are shown in Representative members of Rid1, Rid2, and Rid3 subfamilies, predicted to have deaminase activity due to the presence of the relevant arginine residue, were chosen for study. Rid1 representatives were from ridA mutant have been attributed to the accumulation of 2-aminoacrylate in vivo [in vitro assay for RidA activity uses a PLP-dependent dehydratase to generate 2-aminoacrylate in situ. In situ generation of the 2AA substrate is necessary due to its short (~1.5 s) half-life in aqueous solution [in vitro using cysteine desulfhydrase  as the in situ generator. The data in Growth defects of a  in vivo –21, 41. solution . In thissolution . Six RidS. enterica strains lacking ridA have a growth defect in minimal medium containing 5 mM serine or 250 μM cysteine due to the accumulation of 2AA [in vivo function of representative members of the subfamilies Rid1-3. Rid-protein encoding genes were cloned into pBAD24 vectors, putting their expression under the control of arabinose. Each of the plasmids, in addition to control plasmids pDM1439 and pBAD24, was transformed into strain DM12920 (ridA). The resulting strains were grown in minimal medium containing glycerol (30 mM) as the sole carbon source with the addition of serine . This result indicated that expression of the ridA gene from the pBAD plasmid without induction (presumed to be lower than with the other genes with induction), was sufficient to complement the loss of the chromosomal locus. Second, four of the six remaining plasmids supported growth of the ridA mutant when the respective genes were induced by arabinose . Finally, protein ACIAD3089 from A. baylyi supported growth of the ridA mutant, without induction, when cysteine was present (panel F), but required induction to allow growth in the presence of serine (panel B). Significantly, the pattern of complementation did not correlate with subfamily assignment of the proteins. It was formally possible that some  of the negative results in vivo were due to instability of the gene products, which was not specifically tested here. However, the ability to purify the relevant proteins in active forms appeared to minimize this possibility.n of 2AA , 23, 43.S. enterica [in situ from a range of L-amino acids using an FAD-dependent mechanism. In an assay mixture with Rid proteins included, the resulting imines have three fates that are depicted in Imine deaminase activity of the six Rid proteins was quantified using L-amino acid oxidase (LOX) in an assay described in a previous study, which used proteins RidA, YoaB Rid2), and STM1549 (Rid7) from , and STMA. baylyi ACIAD3089 protein had a level of activity similar to RidA across substrates. With a few outliers, the remaining proteins, all from Pseudomonads, displayed activity on all substrates similar to or higher than, the RidA protein. A notable observation was that the non-RidA proteins had significantly more activity than RidA with the imine derived from L-Phe and L-His. The significance of this substrate specificity is unclear, but it may hint at a physiological role of Rid proteins.In total, the data in Pseudomonas, the source of the relevant enzymes.When arginine was used as a LOX substrate, representatives from the Rid1, Rid2 and Rid3 subfamilies appeared to have more deaminase activity than RidA (data not shown). This activity distribution piqued our interest due to the presence of iminoarginine in the metabolism of P. aeruginosa, L-arginine is generated in a racemization pathway that includes D-Arg dehydrogenase (a.k.a. D-Arg:acceptor oxidoreductase (deaminating), EC 1.4.99.6, DauA), to convert D-Arg to 5-guanidino-2-ketopentanoate via an iminoarginine (5-carbamimidamido-2-iminopentanoate) intermediate [in vivo. Such a scenario would require the release of the iminoarginine from the DauA active site to make it available for the Rid proteins. This possibility was addressed in vitro with purified DauA, monitoring semicarbazone formation with a strategy similar to that of the LOX assay. In the absence of a Rid protein, control reactions were initiated with D-Arg (1 mM) and showed the rate of semicarbazone product formation was linear over a DauA protein concentration between 0.3–6 μM (data not shown). The effect of Rid proteins on the rate of semicarbazone formation was determined and the data are reported as a change in absorbance at 248 nm as a function of time  participates in the pathway of D-arginine racemization in vivo  had detrimental effects. We hypothesize that the general role of Rid proteins is to facilitate reactions that are limited by the rate of hydrolysis allowed by water, and thus make a variety of metabolic pathways more efficient. In total, efficiency provided by Rid proteins, and other potential metabolic modulators, would increase overall organismal fitness enough to explain the selective pressure that maintained this superfamily as reflected in its broad conservation.Activity in  in vivo . While iP. aeruginosa.Despite their role in overall fitness, identifying specific pathways that produce substrates for Rid proteins is hampered by the expectation that any growth defects would be subtle. Defining the physiological roles of Rid proteins will require multiple approaches including creative phenotypic analysis, targeted biochemical approaches and a fair amount of serendipity. The results here provide a framework to explore the role of Rid proteins, specifically in the racemization of arginine in


L. rhamnosus isolated from cheese. The proposed TA system comprises two convergently transcribed RNAs: a toxin RNA encoding a 29 amino acid peptide named Lpt and an antitoxin non-coding RNA. Both toxin and antitoxin RNAs resulted upregulated under conditions mimicking cheese ripening. The toxicity of the Lpt peptide was demonstrated in E. coli by cloning the Lpt ORF under the control of an inducible promoter. Bioinformatics screening of the bacterial nucleotide database, shows that regions homologous to the Lpt TA locus are widely distributed in the Lactobacillus genus, particularly within the L. casei group, suggesting a relevant role of TA systems in plasmid maintenance of cheese microbiota.Plasmids carry genes that give bacteria beneficial traits and allow them to survive in competitive environments. In many cases, they also harbor toxin-antitoxin (TA) systems necessary for plasmid maintenance. TA systems are generally characterized by a stable “toxin”, a protein or peptide capable of killing the cell upon plasmid loss and by an unstable “antitoxin”, a protein or a non-coding RNA that inhibits toxin activity. Here we report data toward the identification of a RNA-regulated TA system in the plasmid DNA of  Lactobacillus rhamnosus is a non-starter lactic acid bacterium that plays a significant role during cheese ripening, contributing to the formation of flavor. In long-ripened cheeses it persists throughout the whole ripening time, due to its ability to adapt to changing environmental conditions, however the metabolic response of L. rhamnosus to different ecosystems is still poorly understood.1. Several studies have been carried out to better comprehend the bacterial metabolic response under these adverse conditions, particularly with the aim to identify biochemical pathways that promote bacterial survival and growth within the cheese habitat2. Recently we identified a set of genes related to the growth of L. rhamnosus in a cheese-like medium by cDNA-amplified fragment length polymorphism (cDNA-AFLP), a sensitive transcriptomic technologies for gene expression analyses3. In this study, the upregulation of a transcript related to a Lactobacillus casei plasmid was also reported. To date, more than 20 species of Lactobacillus containing plasmids have been identified and over 400 lactic acid bacteria (LAB) plasmids have been isolated and characterized4. Among these, only four plasmids have been isolated from L. rhamnosus and their sequence is available in GenBank . Plasmids often carry genes that are essential for survival under adverse conditions5. In particular, LAB plasmids encode traits related to phages or antibiotics resistance, lactose catabolism and production of proteolytic enzymes or bacteriocins4. LAB plasmids may also harbor genes encoding toxin-antitoxin (TA) systems necessary for plasmid maintenance11, a phenomenon known as plasmid addiction or post-segregational killing.Fermented foods like cheese represent challenging environments for bacteria because the nutrients are often exhausted and waste products are abundant. In addition, other factors such as moisture content, salt concentration, pH and oxygen concentration can affect survival, growth and metabolism of the cheese microbiota12. When a plasmid-free variant is produced, owing to a replication error or to defects in plasmid maintenance, a rapid depletion of the antitoxin occurs in newborn plasmid-free cells. Under these conditions, the stable toxin inherited from the mother cell no longer neutralized by the antitoxin, causes cell death13. Homologous systems have also been found in bacteria chromosomes as part of integrated mobile elements15. However, newly identified TA loci of the bacterial chromosomes do not show homology with mobile genetic elements14, suggesting that TA systems might have different biological functions.Plasmid TA loci are two-component systems made of a stable “toxin”, capable of killing the cell, and an unstable “antitoxin” that inhibits the toxin activity17. Among these, type I TA system is characterized by a toxin peptide and an antitoxin RNA capable of interfering with the toxin mRNA translation either by preventing ribosome binding or by promoting mRNA degradation. Studies focused on type I TA systems are limited, probably because the small size of the toxin peptide and the complex prediction of small RNAs make their recognition difficult18.To date, at least six types of TA systems with different genetic architectures and regulatory activities have been identifiedL. rhamnosus isolated from Parmigiano-Reggiano cheese. The proposed TA system includes a 29 amino acid hydrophobic toxin named Lpt (Lactobacillus plasmid toxin) and a 74 nucleotides antitoxin RNA. The antitoxin RNA harbours a 24 nt sequence complementary to a region encompassing the start codon of the toxin mRNA. Both toxin and antitoxin RNA are upregulated in a cheese-like medium, suggesting a possible role under stress conditions. Moreover, bioinformatics analysis shows that regions homologous to the proposed TA locus are widely distributed in plasmids harbored by the L. casei group  and by L. brevis, suggesting a relevant function of TA systems in the Lactobacillus genus.In this work we report data in support of the presence of a type I TA system in the plasmid DNA of two different strains of. L. rhamnosus PR1473 grown in MRS and Cheese Broth (CB) media evaluated by using cDNA-AFLP with different primer sets. By comparing these results with those previously observed for the strain PR10193 . These homologous sequences are annotated in the database as non-coding DNA regions and no significant similarity was found by searching the protein database with the Blastx program19. However, a search in the Conserved Domain (CD) database20 revealed a marginally significant similarity (E = 8 × 10−3) between an open reading frame (ORF) encoding a 29 amino acids peptide and the faecalis plasmid stabilization toxin (Fst) domain. A sensitive homology search with the HHPRED program21, using the peptide sequence of the hypothetical ORF as a query, resulted in the identification of several bacterial toxins. The highest similarity (E = 2 × 10−17) was found with Fst, a type I TA system toxin, which was first identified in the pAD1 plasmid of Enterococcus faecalis23. Based on these hints we analyzed a larger region of the pLBPC-2 plasmid in order to assess the existence of a complete regulatory region characterizing the toxin-antitoxin mechanism. By means of bioinformatics tools, two small convergently transcribed RNAs (RNA I and RNA II), typical of type I TA systems10, were identified . Interestingly enough, the plasmid sequences overexpressed in the two L. rhamnosus strains in CB medium  located at the 5′ end of toxin RNA is essential to ensure RNA stability24, while the flanking stem loop region (SL), including the ribosomal binding site (RBS) sequence, is implicated in an intramolecular mechanism of translation repression25. In analogy with the Fst TA system, in the structural model of the Lpt-encoding RNA  was carried out to evaluate the expression level of the toxin mRNA I in ium Fig. .Figure 4L. rhamnosus represents the predominant species26. The relative expression of Lpt mRNA was confirmed also in situ, with slight variations between the two samples  shows a small delay, probably due to basal transcription of the lac promoter. In the presence of lactose (black triangles), a condition that induces the expression of the Lpt peptide, growth was strongly inhibited. This result clearly demonstrates that the predicted Lpt peptide has a toxic effect on E. coli.To verify the toxicity of the predicted Lpt peptide, the corresponding cDNA was cloned in the lactose-inducible expression vector pSRKKmE. coli σ70-RNAP, deposited onto freshly-cleaved mica and imaged in air by AFM as described in the Methods. Representative full-scan AFM images of the complexes are reported in Fig. To verify that the predicted promoter sequences of RNA I and RNA II are actual promoters, we employed Atomic Force Microscopy (AFM) to map the position of RNA polymerase (RNAP) bound along a DNA template of known sequence. First, the 431 bp DNA sequence carrying the entire TA system was cloned into pGEM T-easy to obtain plasmid pGEM-TA (see Methods). Using this construct and primers mapping on the vector DNA, a linear DNA fragment of 1065 bp was obtained by PCR. Within this fragment, the RNA I promoter is located 498 bp from the upstream DNA end, whereas the RNA II promoter is located at 276 bp from the upstream DNA end Fig. . Promote29 and their effect on the position distribution of RNAP-DNA complexes was analyzed. Figure wt RNA II promoter of this template remains unaffected. Likewise, the inactivation of the RNA II promoter drastically reduces the number of RNAP bound in the template region corresponding to this promoter  and RNA II (antitoxin) promoters, we transformed E. coli with plasmids containing the entire TA sequence either in the wild type form or with the RNA I and RNA II promoters alternatively inactivated. In these constructs, transcription of RNA I and RNA II is under the control of their wt or mutated natural promoters.To evaluate the 600 of about 1.7–1.8 in 6 hours. The OD value remains stable in the next 5 hours for E. coli transformed with plasmid containing the inactivated RNA I promoter (white circles). Conversely, the OD600 values of E. coli transformed with plasmid containing the wild type sequence (black circles) or the inactivated RNA II promoter (grey circles) decrease slowly but constantly in the next 5 hours, with a more consistent drop for cells with inactivated RNA II promoter. These in vivo results show specific E. coli phenotypes associated with RNA I or RNA II promoter inactivation and further validate the role of the predicted promoter sequences.Figure L. rhamnosus PR1473 as a query. Interestingly enough, all the sequences with significant similarity are located on plasmids carried by bacteria belonging to the Lactobacillus genus. In particular, we were able to identify 6 plasmids isolated from L. paracasei, 3 isolated from L. casei, 2 from L. brevis and 1 from L. rhamnosus . To evaluate the association between the TA locus and RepB, a synteny analysis30 of the different plasmids containing sequences homologous to Lpt was performed  systems are characterized by a small toxin peptide and an antitoxin RNA capable of targeting the toxin mRNA. In this work we describe how the detection of an overexpressed RNA by transcriptomic analysis, lead us to the identification of a putative type I TA system in the plasmid DNA of two L. rhamnosus strains. Despite the overexpressed RNA shares significant sequence similarity with plasmid DNA of the L. casei group, detection of the TA system was complex. Firstly, the nucleotide regions with the highest identities, corresponding to plasmid pLBPC-2 of L. paracasei and plasmid pNCD0151 of L. casei, are annotated in the databases as non-coding sequences. Secondly, no homologs were found by searching the protein database with the Blastx program. Thirdly, although in the literature there are many reports describing toxin-antitoxin systems, only a few regard type I TA systems. This lack of information is probably due to the small size of the toxin peptide and to the challenging bioinformatics prediction of small RNAs. Known and new type I TA systems were recently identified in different bacterial lineages by using a computational approach based on PSI-Blast and tBlastn18, however, none of these referred to plasmids of the Lactobacillus genus. It was only thanks to a more sensitive tool for protein structure prediction (HHpred)21, that we could find a correlation between the overexpressed RNA in L. rhamnosus and the Fst protein family. Fst is a type I TA system, first identified on plasmid pAD1 from E faecalis, comprising two convergently transcribed small RNAs: RNA I coding for a toxin peptide and RNA II acting as an antitoxin22. Both RNA I and RNA II contain complementary DR sequences that are involved in the regulation of Fst expression. Because of similarities in the structural organization of the Lpt TA system with the Fst TA system, we hypothesize a common mechanism of action. Experiments conducted in E. coli have shown a strong inhibition of cell growth when the predicted Lpt toxin was expressed under the control of an inducible promoter. Furthermore, RACE experiments lead us to the identification of an RNA I transcript that was shorter than that predicted in-silico, suggesting that RNA I might be post-transcriptionally processed. Processing of RNA I has also been proposed for the Fst TA system as a mechanism of translation activation14, even though the processed transcript has never been isolated. This finding supports the idea that the 5′ UTR of Lpt-encoding RNA might have a role in the regulation of translation process during toxin synthesis. For the Fst system, it has been indeed demonstrated that an helix (UH) and a stem-loop (SL) in the 5′ UTR control the stability of the RNA and its translation25. In addition, it has also been proposed that a region of RNA I (DR), including the Fst toxin start codon, is sequestered by a complementary region (DR) of the antitoxin RNA II preventing toxin translation23.The putative role of TA systems is generally referred to plasmid stability and maintenance but also other biological functions have been proposed. In particular, TA systems, defined one of the most versatile global regulatory system in bacteria70-RNAP, we determined that the predicted promoters are actual promoters and no other strong promoters are present in the TA system DNA region. Furthermore, analysis of the promoter occupancy revealed that the RNA II promoter has a higher affinity than the RNA I promoter, suggesting that the unstable antitoxin is under the control of a stronger promoter. Such a different promoter strength could be due to the particular sequence of the −10 and −35 hexamers but also to the different length of the intervening sequence between the two promoter elements . When E. coli was transformed with a vector carrying the Lpt TA system under the control of these natural promoters, growth was not hampered, even when the antitoxin promoter was inactivated, suggesting that the TA system may be regulated by Lactobacillus specific factors. However, we systematically observed that after the exponential phase the OD600 of cells transformed with an inactivated antitoxin promoter dropped more rapidly than the OD600 of cells transformed either with an inactivated toxin promoter or with a wt promoter. This behavior may indicate that a toxin concentration threshold must be reached before Lpt can exert its toxic effect.By mapping the position of DNA bound σLactobacillus genus and in particular in L. casei, L. paracasei and L. rhamnosus. Notably, the distribution of this putative type I TA system is restricted to plasmid DNA because when the entire TA locus was used as a query to search in the nucleotide database, no significant homology was found with bacterial chromosomes. Surprisingly, in a recent multiple approach study18, highlighting the wide distribution of type I TA modules in plasmid and chromosome DNA, the Lpt toxin and its homologous peptides were not described.In this study, we also found that Lpt and its homologous peptides are widespread among L. rhamnosus have been proposed as a marker for strains diversity34. The synteny analysis of plasmids containing sequences homologous to Lpt reveals that they share a limited region of similarity encompassing Lpt and two neighboring genes potentially involved in plasmid replication. A physical association between genes encoding toxic peptides and genes involved in plasmid replication has been reported also for other TA systems31.Lpt homologous peptides encoded by different plasmids show a distribution which seems to be strain-dependent instead of specie-dependent, probably reflecting a possible horizontal gene transfer. In previous studies, type II TA systems encoded by the chromosome of L. casei group are generally used in commercial and traditional fermented foods. In dairy products their presence is due to the natural occurrence in milk and to the ability of growing in curd and cheese, becoming dominant species in ripening cheese26. Ripening represents a crucial step for bacterial growth and survival because different factors, such as salt concentration, pH, moisture, temperature and nutrient starvation, can cause environmental changes leading to stress-induced reactions. Recently, it has been shown that species of L. casei group can survive in a hostile environment by means of different mechanisms of adaptation37. Plasmids can contribute to this stress tolerance by encoding traits that confer adaptive advantages4.Closely genetically related species of the 38, TA system regulation has never been reported for microorganisms grown in food or in food-like environments. In this work, we demonstrate the upregulation of putative toxin and antitoxin RNAs in L. rhamnosus grown in cheese mimicking conditions. In addition, toxin-encoding RNA was also detected directly in Parmigiano Reggiano cheese ripened either 6 or 12 months. It should be noted that despite in vitro RNAP promoter recruitment experiments show that the antitoxin promoter is stronger than the toxin promoter, we find that the ratio between the absolute amount of toxin and antitoxin RNA is in the range 1.6–2.9, in all the experimental conditions analyzed. Although these results do not allow definitive conclusions about the expression regulation of the Lpt peptide, the widespread distribution in the Lactobacillus genus of this putative TA system, underlines the importance of plasmid maintenance which, in turn, can affect the composition of the viable bacterial population strictly correlated to technological performance. In vivo and in vitro experiments aimed to unravel the expression regulation and the mechanism of action of the Lpt toxic peptide are in progress.Despite the presence in the literature of many studies concerning the physiological role of TA systems, particularly in pathogenic microorganismsL. rhamnosus PR1019 and PR1473 were isolated from Parmigiano Reggiano cheese (PR) at 4 and 20 months of ripening, respectively39. Strains were identified by 16 S rDNA gene sequencing40 and species-specific PCR41. Both strains were cultivated in MRS broth (Oxoid) or Cheese Broth (CB) at 30 °C, under anaerobiosis, for 24 or 48 h, respectively. CB is a culture medium that mimics raw-milk long-ripened cheese, prepared according to42.L. rhamnosus PR1473 and PR1019 were grown in MRS or CB medium and the total RNA was extracted at the end of logarithmic phase by using the RNeasy Protect Bacteria Mini Kit (QIAGEN). cDNA synthesis and cDNA-AFLP analysis were carried out as described in42 and in Supplementary Methods.43 and re-amplified using unlabeled selective primers (Table Transcript-derived fragments overexpressed in CB medium were eluted from the gel as described inrs Table . Amplifi19 by using the overexpressed cDNA sequence identified by AFLP or the complete Lpt TA system region as a query. Coding region search was conducted with BlastX (nr database) and CD-search (Ver. 3.12) programs, without the filter for low-complexity regions, using the cDNA sequence as a query. Protein homology determination was performed with the HHPRED program21 (http://toolkit.tuebingen.mpg.de/hhpred) on the Pfam and PDB databases. The identification of putative promoter sequences was carried out with the BPROM service (http://www.softberry.com), while the prediction of terminator sequences was carried out with the “Arnold finding terminators” web service (http://rna.igmors.u-psud.fr). RNA secondary structures were predicted by using the RNAfold web service (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). Sequence alignments were constructed by Clustal Omega44 and rendered with the GeneDoc or ESPript345 (http://espript.ibcp.fr) programs.NCBI Nucleotide database was searched with the Blastn programhttp://www.dveltri.com/simplesynteny/) using the protein sequences identified in the pLBPC-2 plasmid as a gene query file and the DNA sequences of 10 Lpt-encoding plasmids as a genome contig file. The Lpt consensus sequence obtained from peptide alignment  was performed with the SimpleSinteny web server (L. rhamnosus PR1019 e PR1473 grown in MRS or CB medium by using Plasmid DNA Extraction Mini Kit (Fisher Molecular Biology). The predicted complete sequence, including all the regulatory regions, was amplified by standard PCR with primers TA-plus and TA-minus  and random hexamer primers according to the manufacturer’s protocol. qRT PCR was carried out as described in Supplementary Methods.Quantitative reverse transcription PCR (qRT PCR) was carried out on cDNA obtained from RNA of L. rhamnosus PR1473 plasmid DNA as template and two sequence specific primers: the NdeI-tailed Lpt-plus and the BamHI-tailed Lpt-minus . Mutant RNA I and mutant RNA II were obtained by introducing a three-point mutation in the −10 hexamer . Mutation fidelity was verified by sequencing.pGEM-TA plasmid harboring the entire TA locus was obtained by cloning the 431 bp DNA amplified from wt RNA I and wt RNA II promoters or mutant RNA I and wt RNA II promoters or wt RNA I and mutant RNA II promoters, were obtained by standard PCR from the corresponding pGEM-TA plasmid with oligonucleotides pGEM-AFM-plus and pGEM-AFM-minus (Table 260 nm. Transcription complexes were assembled and imaged by AFM as described in Supplementary Methods. DNA contour length measurements were performed as described in46 and in Supplementary Methods.The 1065 bp DNA templates harboring us Table . All DNAE. coli XL1 Blue were transformed with the recombinant plasmid pGEM-TA harbouring the entire Lpt TA locus under the control of wt or alternatively inactivated natural promoters. Growth assays were carried out in LB medium and cell growth was monitored by absorbance at 600 nm for twelve hours.Supplementary Information


RNA editing, or post-transcriptional changes in the sequences of RNAs, is being increasingly recognized as an important player in the regulation of gene expression in vertebrates and invertebrates. Different types of RNA editing have been reported. This review discuss the type of RNA editing caused by cellular enzymes known as adenosine deaminases that act on RNAs (ADARs), and it's significance in the lifecycle of an RNA virus, hepatitis delta virus.


For the first time in the literature, our group has managed to demonstrate the existence of plant RNAs in honey samples. In particular, in our work, different RNA extraction procedures were performed in order to identify a purification method for nucleic acids from honey. Purity, stability and integrity of the RNA samples were evaluated by spectrophotometric, PCR and electrophoretic analyses. Among all honey RNAs, we specifically revealed the presence of both plastidial and nuclear plant transcripts: RuBisCO large subunit mRNA, maturase K messenger and 18S ribosomal RNA. Surprisingly, nine plant microRNAs  were also detected and quantified by qPCR. In this context, a comparison between microRNA content in plant samples  and their derivative honeys was carried out. In addition, peculiar microRNA profiles were also identified in six different monofloral honeys. Finally, the same plant microRNAs were investigated in other plant food products: tea, cocoa and coffee. Since plant microRNAs introduced by diet have been recently recognized as being able to modulate the consumer’s gene expression, our research suggests that honey’s benefits for human health may be strongly correlated to the bioactivity of plant microRNAs contained in this matrix. MicroRNAs (miRNAs), together with short interfering RNAs (siRNAs) and other small groups of small RNA sequences, are included in the large class of small RNAs (sRNAs). MiRNAs are non-coding RNAs, highly abundant in living cells and whose length ranges between 19 and 24 nucleotides [lin-4) was identified, by Lee et al. [Caenorhabditis elegans Maupas. Since then, miRNAs have been found both in simpler organisms, such as Chlamydomonas reinhardtii P.A.Dang., and in superior ones, included Homo sapiens L., suggesting that their function was not merely an accessory but was indispensable during evolution [The first miRNA  Heynh., C. elegans, Mus musculus L. and H. sapiens) remains scarcely documented [MiRNAs are produced from endogenous genes (MIR genes) which are phylogenetically conserved and which may be evolved independently within each life kingdoms . Indeed,cumented .MiRNAs have been demonstrated to be fundamental for the modulation of gene expression in the cells where they are synthesized. For example, in humans, miRNAs are able to regulate about 30% of the whole gene set . In partVice versa, if the pairing between miRNA and gene messenger is imperfect, mRNA is subjected to translation repression, possibly altering ribosome sliding or stability on the transcript or inhibiting the coupling of eIF4E factor with mRNA cap [In general, each miRNA determines the gene silencing of one, or more, specific RNA messengers (mRNAs). For miRNAs, two action mechanisms have been widely illustrated, although some minor aspects still remain ambiguous. When miRNA is perfectly  complementary with a portion of nucleotide sequence of its mRNA target , the transcript is rapidly degraded, probably favoring the removal of the poly-A tail, thus making it less stable. mRNA cap , 10.MiRNA biogenesis has been widely described in the literature and follows a very similar pathway in both animal and plants, although some steps are carried out by orthologue enzymes. In briefly, from the MIR genes, miRNAs are transcribed by RNA polymerase II and then finely processed in the nucleus before being transported into the cytoplasm. Here, miRNAs can perform their regulatory activity on mRNA targets by being associated to the RISC complex (RNA induced silencing complex) , 11, 12.phenomenon [Oryza sativa L. miRNA186a could be detected in both the blood and tissues of mammals on a rice diet. Moreover, this plant miRNA, surprisingly still in active form, showing sequence homology with the human mRNA codifying for Low Density Lipoprotein Receptor Adapter Protein 1 (LDLRAP1), was also able to reduce the translational process of LDLRAP1 transcript in animal cells, thus determining an increase of LDL level in plasma. These data clearly confirmed that plant miRNAs, introduced by diet, could strongly influence consumers’ gene expression, acting as communication molecules between different kingdoms. However, this potential role of plant miRNAs is strongly dependent on the fact that, with respect to animal ones, they are characterized by a 3’-terminal nucleotide whose sugar is methylated in position 2’: this feature endows the miRNAs with extreme stability, resistance to degradation (i.e. low pH), preservation to temperature variation (i.e. boiling) and absorption processes by intestinal mucosae [The discovery of the existence of cross-kingdom interactions mediated by miRNAs has been one of the most significant breakthroughs of the last decade. However, only a few studies have reported this enomenon –16. Amonenomenon , where t mucosae –21. All Malus domestica Borkh., Robinia pseudoacacia L., Castanea sativa Mill., Eucalyptus sp., Tilia cordata Mill., Rosmarinus officinalis L.). In fact, since honey derives from the re-elaboration of floral secretions by honeybees (Apis mellifera ligustica Spinola) [Therefore, the main objective of this research has been to demonstrate, for the first time in literature, the existence of plant RNAs and miRNAs in different samples of monofloral honeys (Spinola) , we had Spinola) , 24; it Camellia sinensis L.) and cocoa (Theobroma cacao L.) and coffee seed powders (Coffea arabica L.), have been similarly analyzed for analogous purposes.Finally, other plant derived-commercial products, such as desiccated tea leaves (M. domestica flowers were sampled from 4 adult apple trees (ten flowers per tree) grown and cultivated in the Botanical Gardens of Rome “Tor Vergata”. R. pseudoacacia nectar (about 4 mL) was collected, by a Hamilton syringe (max vol. 50 μL), from Black Locust flowers harvested, in the Botanical Gardens of Rome “Tor Vergata”, from 4 different bloomed trees. Apple flowers and R. pseudoacacia nectar were instantaneously processed after sampling. Honeys were kindly provided by the Honey Research Center of the University of Rome “Tor Vergata”, after certification by melissopalynological analysis [M. domestica, R. pseudoacacia, C. sativa, Eucalyptus sp., T. cordata, R. officinalis) were studied. Desiccated tea leaves (C. sinensis) and cocoa (T. cacao) and coffee seed powders (C. arabica) were obtained each from 3 respective commercial sachets; as such, they were industrially processed and ready to be used by consumers.Forty analysis . In parttotal RNA extraction kit), samples were lysed in the presence of guanidinium isothiocyanate, a chaotropic salt able to protect RNA from endogenous RNases; the samples were then processed through a Spin Cartridge containing a clear silica-based membrane that bound RNA. Impurities were removed by subsequent washing, and purified total RNA (long and short sequences) was eluted in RNase-free water. By using mirPremier microRNA Isolation Kit , samples were treated by a 2-mercaptoethanol enriched-lysis buffer that specifically solubilized small/micro RNA, inactivated ribonucleases and neutralized interfering plant secondary metabolites. Large RNA and genomic DNA, which both remained insoluble, were removed by centrifugation from the lysate, along with other cellular debris. Therefore, only small/micro RNA was captured onto a silica binding column, as specified in the manufacturer’s guidelines. Residual impurities were removed by repeated washings, and small/micro RNA was eluted in RNase-free water. The third extraction method (named home-made RNA extraction) was carried out according to a Sambrook [pasteur pipette. The whole solution was conserved at -70°C for 1h and, then, centrifuged for 30 minutes at 4°C at 13,000 RPM. Finally, the pellet was resuspended in RNase-free water and treated, according to manufacturer’s guidelines, with DNAse I , in order to destroy residual contaminating DNA molecules. All RNA samples were stored at -70°C until their analysis. RNA concentration and purity were determined with a Nanodrop ND1000 spectrophotometer (NanoDrop Technologies). RNA separation and visualization was performed by agarose-formaldehyde gel electrophoresis according to the Sambrook [For RNA extraction, plant samples  were completely pulverised using mortar and pestle, in the presence of liquid nitrogen, while honeys and nectars were used in their original form. Three different extraction procedures were employed. According to the first method, Pure Link RNA Kit  were added for each retrotranscription reaction, in order to control the absence of nucleases during the procedures and the efficiency of cDNA synthesis and qPCR assay. PCR amplifications of plant genes  were performed by a Biorad (IQ5) thermocycler. PCR procedure and conditions, reagent concentrations and amplicon detection through gel electrophoresis were all identical to those widely reported in Gismondi et al. [5’-UCUUUCCUAUCCCUCCCAUUCC-3’), miR156a (5’-UGACAGAAGAGAGUGAGCAC-3’), miR396c (5’-UUCCACAGCUUUCUUGAACUU-3’), miR171a (5’-UGAUUGAGCCGCGCCAAUAUC-3’), miR858 (5’-UUCGUUGUCUGUUCGACCUGA-3’), miR162a (5’-UCGAUAAACCUCUGCAUCCAG-3’), miR159c (5’-GAAUUCCUUCUCCUCUCCUUU-3’), miR395a (5’-CUGAAGUGUUUGGGGGAACUC-3’) and miR2118a (5’-CUACCGAUGCCACUAAGUCCCA-3’). The 5S rRNA  was used as internal loading control to normalize of qPCR results (relative quantization). In fact, the amount of each miRNA was determined using the 2-ΔΔCt formula, where the threshold cycle (Ct) of the target miRNA measured in one sample is normalized with respect to the internal reference gene  and to the respective value obtained in the second sample (ΔΔCt). qPCR negative controls  were performed throughout to confirm adequacy and accuracy of the detection system. Results were expressed as mean ± standard deviation (s.d.) and all the experiments were repeated at least three times starting from different and independent RNA extractions. The significance of the results was measured by one-way ANOVA test using PAST software .Total cDNA was synthesized by joining, in a final volume of 25 μL for 90 min at 37°C, the following reagents: 0.4 mM each dNTP , 20 U ribolock RNase inhibitor , 0.5 μg random hexamer primers , 200 U moloney murine leukemia virus reverse transcriptase , 1X enzyme buffer, 10 mM dithiothreitol and 0.5 μg RNA, previously heated at 65°C for 2 minutes. By contrast, the synthesis of microRNA cDNAs was carried out by using a reverse transcription kit specific for miRNAs , according to manufacturer’s instructions. In particular, 10i et al. . The choi et al. , for rbci et al. , for 18Si et al. , 30: miRThe beneficial effects of a plant food-based diet on human health have been widely documented in literature ,32. In gApis mellifera ligustica Spinola) [For these reasons, the present work aimed to investigate if plant miRNAs could be detectable even in the honey, a sugary matrix which derives from the re-elaboration of plant products  by honeybees (Spinola) , in ordeSpinola) , 24.total RNA extraction kit, home-made RNA extraction and miRNA extraction kit, as widely reported in “Materials and Methods” section.To determine the best procedure for miRNA extraction, three different methods were carried out, named respectively M. domestica miRNome and which we used as our main reference for the choice of miRNAs to detect in the current study [M. domestica flowers were also subjected to miRNA extraction.The first sample we decided to process was an apple honey because, given the recent publication of a paper describing nt study . To moni260/280 and A260/230 are usually employed to measure the purity level of nucleic acids since nucleotides, RNA and DNA all absorb at 260 nm; A260/280 and A260/230 ratios of, respectively, ~2.00 and ~2.00–2.20 generally indicate a “pure” RNA sample [As shown in A sample .Home-made RNA extraction recovered the highest concentrations of RNA, in all samples, compared to the other two methods. It was unexpected to notice that total RNA extraction method was not able to purify an amount of RNA similar to that obtained by home-made RNA extraction, while the same result was quite predictable for miRNA extraction kit which is specifically designed to extract only small and micro RNAs .RNA collected by and 1.60 , did not, 230 nm . These dHome-made extraction, as in the case of the previous method, has allowed us to obtain an impure RNA: maybe, the presence of proteins, derived from the samples, or phenol residues, used during the extraction procedure, negatively affected the A260/280 ratio [260/230 ratios reported in 260/230 value still revealed the predominant sugary nature of this matrix.80 ratio . After a260/280 and A260/230 ratios were measured for the small/micro RNAs purified by miRNA extraction kit. In both samples, no peculiar contamination was detected, while even elevated purity values reached  and a long trait located on the nuclear one (18S rDNA of 844 bp). These regions were chosen for two principal reasons: i) They represented some of the best plant DNA barcodes, nucleotide successions highly conserved in plant kingdom and were very easy to amplify and detect , 37–39; total RNA extraction method and home-made RNA extraction produced amplicons. These fragments were detected, analyzed and verified in length, through a molecular weight (M.W.), by agarose gel electrophoresis  and then analyzed by real-time PCR. The presence of nine different plant miRNAs , chosen for their abundant expression, wide distribution in various plant districts and elevated nucleotidic conservation in plant kingdom [All these  kingdom , 30, 40, kingdom , 42. In M. domestica flower samples. Apple honey miRNAs purified by total RNA extraction method (−2 and 10−6 folds) than in apple flowers.As described in n method , panel Ahome-made RNA extraction  analyses to fully characterize the honey miRNome. At this point, it is necessary to underline how our study demonstrated that honey’s botanical and geographical origin might influence, both the quality and the quantity, of honey’s miRNA content, so suggesting the existence of a specific miRNA-based fingerprint for each honey. In addition, we cannot exclude that honey can also contain some C. arabica, C. sinensis and T. cacao commercial products (Finally, as supplementary study, we investigated the presence of plant miRNAs in products , panel F


Multiple categories of markers are targeted including CK, human epidermal growth factor receptor family markers, Hedgehog pathway markers, human papillomavirus markers, and protein arginine methyltransferase 5. In addition, an integrated method of RNA ISH and fluorescent ICC staining was developed to visualize CTCs on both mRNA and protein levels. The robustness of the integrated co-ICC and RNA ISH staining was demonstrated by a series of tests on representative tumor markers of different categories. The integrated staining can incorporate the advantages of both RNA ISH and fluorescent ICC staining and provide more intense signals and more specific bindings. With this integrated staining methodology, distinct staining patterns were applied in this report to facilitate the searching and characterization of rare subgroups of CTCs. These results support the existence of diverse groups of CTCs at both protein and mRNA transcript levels and provide an analytical tool for the research on CTCs of rare subgroups.Circulating tumor cells (CTCs) are routinely identified as cytokeratin (CK)-positive, epithelial cell adhesion molecule (EpCAM)-positive, and CD45-negative and are enriched based on EpCAM. However, there are a number of methodological challenges regarding both isolation and characterization of these rare CTCs including downregulation or absence of EpCAM in a variety of solid tumors leading to the omission of subpopulations of CTCs, difficulties in analyzing RNA and protein targets in CTCs due to the rarity of these cells, and low levels of targets and technological limitations of visualizing the targets of interest on each individual cell. Building on our previous CTC research on CD45-based negative magnetic separation and four-color fluorescent immunocytochemical (ICC) staining, RNA  Circulating tumor cells (CTCs) are tumor cells or tumor-related cells that are shed into human circulatory systems and areas associated with cancer metastasis. They have the potential to serve as liquid biopsy material that can provide a wealth of real-time information on patient disease state, treatment efficacy, prognosis, and potential drug targets. This is particularly helpful due to the challenges in obtaining serial metastasis biopsies and to distinguish the differences between metastasis and primary tumor biology. Although this idea is promising, the field has faced multiple challenges in the isolation and characterization of CTCs that have impacted the broad acceptance of the utility of these circulating cells , 2.The most common method to identify and enumerate CTCs is through a positive selection by targeting the epithelial cell adhesion molecule (EpCAM). However, the EpCAM-based positive selection brings significant challenges in using CTCs as diagnostic and prognostic tumor markers, or as predictive markers for developing therapeutic drug targets, since more than one-third of patients with metastatic diseases do not have detectable CTCs by EpCAM-based technologies . In factFluorescent immunocytochemical (ICC) staining is typically used to analyze CTCs with multiple tumor-related markers, including cytokeratin (CK), human epidermal growth factor receptor (HER) family markers, stem cell markers, etc. , 12. AdmThe prevailing way to perform mRNA-level analysis on CTCs is to use RT-PCR or microarray analysis on the extracted mRNA from the CTCs that are isolated by oncomarker-based positive selection –16. Mostin situ hybridization (ISH) on mRNA is a viable alternative to RT-PCR or microarray analysis. RNA ISH is a visualized technique to fluorescently label a specific nucleic acid sequence on mRNA with short anti-sense nucleic acid sequences. Dynamic mRNA transcription and movement research shows that the quantification and timeliness of RNA ISH are adequate to track the appearance and the location of single mRNA molecules  viral mRNA with the integrated RNA ISH and ICC staining of tumor markers in CTCs. In this study, a comprehensive analytical tool for heterogeneous rare CTCs was established to enrich CTCs and visualize them on both mRNA and protein levels with a variety of tested markers.2 atmosphere. Cell lines were harvested by washing the adherent cells with phosphate-buffered saline (PBS) and subsequently incubating with Accutase  for 10 min at 37°C to detach cells from the culture flasks. Accutase was then neutralized with the culture medium before pelleting the cells by centrifuging at 350 × g for 5 min. Cells were finally resuspended in culture medium for downstream experiments.Breast cancer cell lines, MCF-7 (HTB-22) and BT474 (HTB-20), and tongue cancer cell line, SCC-4 (CRL-1624), were procured from ATCC. HPV-positive cell lines, SCC-47 (UM-SCC-47) and SCC-90 (SCC-090), were provided by Dr. Quintin Pan’s lab, Department of Otolaryngology, The Ohio State University. SCC-47 was originally acquired from EMD Millipore, whereas SCC-90 cell line was originally created by Dr. Gollin’s lab with previously reported source and protocol . HedgehoPatients with various metastatic solid tumor malignancies, older than 18 years of age, were enrolled in Institutional Review Board (IRB)-approved protocols that permit CTC characterization of blood specimens. All patients gave their informed consent to participate in the study. After several standard blood tubes were drawn for routine chemotherapy labs, either prior to initiation of a new line of systemic therapy or at progression, peripheral blood (7.8–17.7 mL) was collected in BD Vacutainer Heparin tubes (BD Biosciences) for CTC enumeration and processed within 4 h of blood collection. The clinical characteristics of patients are summarized in Table S1 in Supplementary Material.Healthy donor blood was collected from volunteer donors after obtaining informed consent using an IRB-approved protocol at The Ohio State University Medical Center and was processed in the same manner as tumor patient blood samples. The protocol was approved by the Cancer Institutional Review. Source leukocytes (buffy coat), also as a control, were purchased from American Red Cross, Central-Southeast Ohio region.10 of the ratio between the total nucleated cell number before NME and that after NME. Figure Blood samples were kept at ambient temperature and subjected to a CTC enrichment procedure within 4 h after blood collection using our NME approach, as described previously , 25. BriNucleated cell counts were determined prior to RBC lysis, after RBC lysis, and after NME. Lysis efficiency, nucleated cell log depletion, and total cell log depletion were used to evaluate the performance of the NME process. The calculation for these three parameters was introduced in our earlier publication . As prev4 NME cells from the blood of a patient or healthy donor were used for each RNA ISH slide. Approximately 2 × 104 source leukocytes spiked with 5 × 103 cells from a cancer cell line were applied on each control slide. Cell suspensions were spun onto superfrost plus slides (Fisher Scientific) and fixed by 4% paraformaldehyde for 10 min. Subsequently, the cells were treated with QuantiGene ViewRNA ISH Cell Assay Kit (Affymetrix), following the manufacturer’s protocol. Different nucleic acid probes with type 1, type 4, and type 6 fluorescent labeling conjugations were used to detect the corresponding mRNA sequences. Here, type 1, type 4, and type 6 are the fluorescent probes provided by Affymetrix Inc., fitting the orange, green, and far red filter sets, respectively. The slides were then mounted with ProLong Gold Anti-fade Reagent with DAPI (Invitrogen) and sealed with nail polish.Approximately 2 × 104 NME cells from the blood of a patient or healthy donor were used for each RNA ISH slide. Approximately 2 × 104 source leukocytes spiked with 5 × 103 cells from a cancer cell line were applied on each control slide. After following the same protocol for RNA ISH analysis as described above without the protease-incubation and DAPI-mounting step in the manufacturer’s protocol, cells were then blocked with normal serum blocking solution (NSBS)  for 30 min and washed with PBS for 5 min. Subsequently, one or two primary antibodies from different species of hosts  were diluted to 5 μg each antibody/ml with Normal Antibody Diluent  and applied to the slides. The slides were then incubated for 30 min in dark at room temperature and washed three times with 1× PBS containing 0.5% Tween-20 (PBST). Each washing was approximately 5 min. The secondary antibody  was diluted to 5 μg each antibody/ml with PBST and applied onto the slides. The slides were incubated for 30 min in the dark at room temperature and washed three times for 5 min with PBST. The slides were then mounted with ProLong Gold Anti-fade Reagent with DAPI (Invitrogen) and sealed with nail polish.Approximately 2 × 10Nikon Eclipse 80i Advance Research Microscopy system was employed in this research. The Nikon Eclipse 80i Advance Research Microscope was equipped with Nikon IntensiLight C-HGFi and DS-Qi1Mc digital microscope camera. Four filter sets were used to detect and filter the signals, as shown in Table S3 in Supplementary Material. The microscopy automatic stage-moving system was designed and programed in our lab to linearly scan and capture pictures of all cells on slides. The motors were from Zaber Tech (T-LSM025A) and were controlled and automatized by the LabVIEW  program developed in our lab.The number of nucleated cells was counted before and after NME. The cell suspension was diluted at 1:25 for the cells before enrichment and at 1:10 for the cells after enrichment with 3% acetic acid. The cells in acetic acid were let stand for 10 min before counted using Fuchs-Rosenthal hemocytometer.The enriched and stained cells on the slides were automatically counted by NIS-elements Br (Nikon Instruments Inc.) on DAPI channel. Gates for the cell circularity, size, and brightness on the nucleic acid staining were set by the NME cells from the buffy coat processed and stained in the same manner with the same protocol. The tumor-related cells that were positive on each tumor marker or combination of tumor markers were counted manually from the pictures taken by the microscopy with automatic stage-moving system.10 depletion, the average total cell log10 depletion, and the average lysis efficiency were 2.65, 5.64, and 80.0%, respectively. That is, an average of more than 99.78% of nucleated blood cells was removed by our CD45-targeted NME. For the blood specimens from healthy donors, the average nucleated cell log10 depletion, the average total cell log10 depletion, and the average lysis efficiency were 2.52, 5.76, and 70.8%, respectively.For the blood specimens from patients with solid tumors, the average nucleated cell logInitially, RNA ISH was tested with the probes targeting β-actin (ACTB), HER2, and EGFR on BT474 and SCC-4 cell lines [Figure A measure of the specificity of RNA ISH probes was performed with customized sense probes. The sense probes were designed with the same amplification and fluorescent reporting structures as these used for the specific targeting probes , but with complementary and opposite binding sequences. Theoretically, this sense probe should have zero affinity for the targeted mRNA. Figure After the establishment of the protocol of RNA ISH test and the verification of specificity, the integrated RNA ISH and ICC staining procedure was developed to analyze different markers in the same cell at mRNA and protein levels. ICC staining was attempted on intracellular markers, which is usually more challenging as it requires additional permeabilization of the cell membrane that creates higher potential for non-specific bindings, comparing with ICC staining on surface markers. RNA ISH was assigned to multiple tumor markers in HER family and Hedgehog pathway to verify the robustness of RNA ISH. HER signaling is one of the most established and clinically studied pathways. And often, the status of ErbB receptor tyrosine kinase family, such as EGFR and HER2, dictates treatments for breast cancer patients. While the targeted therapies are very effective, patients may develop resistance, which results in poor outcome. Hedgehog signaling is a very valuable drug target in such case because the hedgehog molecules, such as Gli1 and Smo, are upregulated in endocrine-resistant tumors . Figure To further verify the robustness of the integrated RNA ISH and ICC staining, the correlation of positive signals from RNA ISH and fluorescent ICC staining was tested. Specifically, both cell surface proteins and the corresponding mRNAs were targeted and stained on the same cell with integrated RNA ISH and ICC staining. Figure Having verified the robustness of the incorporated RNA ISH and ICC staining on some prevailing tumor markers, we extended the application to tumor markers of different categories, including viral targets (this section) and enzymatic targets (next section). HPV16 infection is associated with the basal cells of the stratified epithelium and found in some specific types of cervical and head and neck cancers –32. AmonLabeling non-structural or membrane bound proteins, such as enzymes, in CTCs, is challenging for ICC staining, for a number of reasons, including the mobility of the proteins after cell membrane permeabilization, low protein expression level, and low specificity of antibodies. Protein arginine methyltransferase 5 (PRMT5), a Type II arginine methyltransferase, which has been reported to be involved in the regulation of multiple tumorigenesis pathways, usually can only be stained by immunohistostaining on tumor biopsy , 36. WitThe integrated RNA ISH and fluorescent ICC staining enables a multimodality confirmation of the positive expression of specifically targeted markers at different levels, as opposed to relying on a single antibody clone with a reported reactivity to specific isoforms of the targeted proteins. As we demonstrated in the previous study, in the prevailing ICC staining, even targeting the very common hematopoietic surface marker, CD45, can produce significantly different results depending on the selected clone of antibody and even the manufacturer where the clone is procured . Considevia these approaches, the heterogeneity of CTCs prevents the conclusive determination of various marker combinations on a cell-by-cell basis. To eliminate the errors from CTC heterogeneity in parallel RNA ISH and fluorescent ICC staining, the integrated RNA ISH and fluorescent ICC staining provides a solution to get information from both RNA ISH and ICC staining on the same cells.Exploiting the advantages of RNA ISH, several recently published studies on CTCs demonstrated the ability of RNA ISH to visualize and quantify epithelial markers, Wnt pathway markers, and CD45 , 38. In To further investigate the specificity and selectivity of our RNA ISH technology, various combinations of cell types, probes, antibodies, and targeting markers were tested in this research. Unlike antibodies that frequently have low level of non-specific binding, especially on blood samples, we did not observe any positive signals in the cell types that were assumed to be negative for the specially targeted RNA ISH probes. The sense controls presented in Figure The RNA ISH technology in this study creates a signal amplification tree using branched single-stranded DNA. This amplification tree can provide 400 binding sites for each labeling probe binding to single target sequence on mRNA. Several fluorescent molecules can be bound to each binding site and thus achieve a theoretical 8000-fold amplification for each mRNA target sequence bound. It has been reported that the high selectivity and high amplification levels in this RNA ISH probe design enable the tracking of single transcript of target mRNA or microRNA, as well as the quantitative analysis of the transcription level of the targeting gene. This quantitative analysis is precise enough to create a dynamic profile on the synthesis, transportation, and degradation of the nascent mRNAs after a chemical induction on the transcription of the corresponding targeted gene . Some laDespite the merits and superiority of RNA ISH over ICC staining in specificity, reactivity, and quantitation as discussed above, ICC staining, which targets proteins, may provide more direct information of CTCs since protein is the functional unit in most signal pathways. In the common practice of tumor cell line analysis, RNA ISH and ICC staining are applied to support each other on parallel slides from the same batch of cells of certain tumor cell lines. However, we suggest that unlike the relatively homogeneous tumor cell lines, the highly heterogeneous CTCs require all staining markers in all staining methods on the same cells. Our integrated RNA ISH and ICC staining can fulfill this challenging requirement and provide an analytical method for comprehensive visualized labeling on different markers in tumor-related pathways.A challenge with this RNA ISH technology combined with ICC is exposure time. Since for a specific marker, the amount of mRNA is usually lower than the amount of the corresponding protein, the signal from RNA ISH is usually weaker  than that from ICC staining. Correspondingly, the camera exposure time for RNA ISH staining under the fluorescent microscopy scanning is about 5–20 times longer than that for ICC staining. Hence, in a visualized fluorescent characterization of the enriched CTCs which inevitably have some healthy, irrelevant cells, the automated fluorescent microscopy scanning takes 5–20 times longer to detect and analyze the target cells on the slides with RNA ISH staining than those with ICC staining. One solution in using an integrated RNA ISH and ICC staining protocol might consist of an automated fluorescent microscopy scanning system to quickly search the tumor-related target cell populations by ICC staining. Once identified, these targeted cells could then be analyzed for RNA ISH staining. This is part of the reason why the selection of the ICC markers in our integrated RNA ISH and ICC staining are usually prevailing tumor markers with high expression, such as CK and HER family markers.For the ease of the access and better resolution in our current presentation, instead of confocal imaging which typically uses laser light, we chose to use a traditional epi-fluorescence microscope with excitation and emission filters, dichroic mirrors, and an intensified mercury bulb. This approach requires longer exposure time, accompanied with the increased potential of the signal “leaking” from the other ICC dyes into the RNA ISH channel. For example, it is suggested that weak and diffuse orange color in the EGFR-type 1 images in Figures via RT-PCR and RNA microarrays (In some images from RNA ISH slides (i.e., Figure roarrays , 41. FurThe research aims to provide a robust separation and characterization method for heterogeneous CTCs. The CTCs were initially enriched by our NME process, in which all blood cells were concentrated and labeled with CD45 antibody that is conjugated with magnetic beads. The labeled cells then flowed through our customized QMS system where the non-hematopoietic cells without CD45-magnetic beads labeling were enriched. To compliment prevailing ICC staining, we introduced RNA ISH into our CTC characterization method and evaluated its specificity and sensitivity. Furthermore, the integrated RNA ISH and ICC staining was developed to allow visualized multiparameter analysis at both RNA and protein levels.The integrated RNA ISH and fluorescent ICC staining provides an approach to study the heterogeneity of CTCs by incorporating the strengths of antibody labeling and RNA ISH. To verify the practicability and robustness of this integrated staining, tumor markers of different categories, including epithelial markers, HER family markers, Hedgehog pathway markers, viral markers, enzymatic markers, etc., were tested on both cell line and NME cells from healthy donors and patients.Currently, in our lab, the high expressing or well-studied tumor markers are stained with ICC staining, which has higher signal intensity, to shorten the duration of automated detection of CTCs. Meanwhile, RNA ISH is applied on novel markers to perform visualized, specific, and precise detection. The ongoing work in our laboratory is focused on the use of spectral deconvolution technology not only to sharpen the images for each fluorescent signal but also to extend the number of targets stained with both ICC and RNA ISH technology up to seven distinct markers as well as the detailed quantitative evaluation of the staining results.YW, K-JJP, CD, PA, and BM conducted the experiments. QP, MZ, ML, and JC designed the experiments. YW, K-JJP, and JC wrote the text.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.


Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity  and 100% sensitivity . This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification. Escherichia coli  in that it amplifies a gene target in the presence of a fluorescent reporter dye. Following amplification, the product is exposed to an increasing temperature gradient to denature and reduce the helicity of the double-stranded oligonucleotide, releasing the fluorescent dye. Once released, the dye undergoes a conformational change that reduces the amount of fluorescence produced. An HRM-capable thermocycler will record the fluctuations in fluorescence and produce a melt curve unique to the sequence of the amplicon analyzed. This technique is capable of comparing the similarity of two amplicons because the melt curve of each oligonucleotide is determined by its nucleotide sequence, oligonucleotide length, and primary structure , 12. TheEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. . A direct comparison of the derivative melt curves between strains showed that all ST131 isolates shared similar profiles that were distinct from the profiles of non-ST131 isolates. With this multiplexed reaction, ST131 identification requires the peaks to be defined by temperature, as well as by the temperature difference between the peaks. The temperatures of the comparator ST131 melt profile peaks were evaluated across all validation runs. The profile peak temperatures for ST131 controls in the adk, gyrB, mdh, and recA master mixture were 84.63 ± 0.16°C, 85.60 ± 0.13°C, 87.24 ± 0.33°C, and 87.54 ± 0.09°C  of HRM data using ScreenClust HRM software  were selected to develop this methodology  and those for adk, gyrB, mdh, and recA (primer set 2). The primer concentrations remained the same as in the singleplex reaction mixtures, and the primer combinations were selected to minimize melt curve overlap between individual amplicons. Following amplification of these primer sets against both ST131 and non-ST131 templates under the cycling conditions described above, the MLST amplicons were subjected to HRM analysis in the optimized range of 80 to 95°C. The derivative melt curves generated were compared between sequence types to evaluate the discriminatory power of this methodology. Both melt curves from E. coli isolates of unknown sequence type were compared to ST131 melt profiles .The sequences of the MLST amplicons produced in this study were determined offsite at Functional Biosciences, Inc. , by Sanger sequencing . Samples were prepared to company submission standards, and the nucleotide calling at each position was determined by a Phred score greater than 20. The resulting sequences were then identified using the As an alternative to manually comparing the derivative melt curves of each sample, Qiagen ScreenClust HRM software was employed to differentiate between sequence types using clustering analysis. After selecting samples by master mixture, the software automatically processes the data by principal component analysis (PCA) and clusters the samples into groups based on the variance in fluorescence between clinical isolates. Briefly, a residual melt curve was automatically constructed from the mean values of normalized data for each isolate. The differentials of the residual melt curves were plotted and analyzed by PCA to construct a feature that reflected the greatest variance of each isolate from the group. The first three principal components in PCA reflect the greatest degree of variance, and these values were graphed onto a scatterplot. The principal components were further processed by the ScreenClust HRM software to group isolates with similar variances into clusters. With the exception of selecting the isolate samples for analysis, the ScreenClust HRM software automates the analysis. While ScreenClust HRM software analysis is currently only available for the Qiagen platform, open-source software may be employed to perform PCA and clustering analysis as described in the supplemental material.rgl and mclust packages were used for three-dimensional (3-D) visualization and clustering, respectively (–The ABI 7500 system does not include PCA software, but HRM data from this platform can be exported and analyzed with third-party software. One free, open-source option is the R software environment, available from the R Project for Statistical Computing. PCA is part of the functionality of the R 3.2.2 base package, and the ectively –34. Perfctively – and a rectively –.