Caenorhabditis elegans. Specifically, our results point to SKI-exosome decay and pelota-based ribosome removal as key steps facilitating suppression and clearance of prematurely-terminated translation complexes. These results suggest a model in which premature stop codons elicit nucleolytic cleavage, with the nonstop pathway disengaging ribosomes and degrading the resultant RNA fragments to suppress ongoing expression.Nonsense-mediated mRNA decay is the process by which mRNAs bearing premature stop codons are recognized and cleared from the cell. While considerable information has accumulated regarding recognition of the premature stop codon, less is known about the ensuing mRNA suppression. During the characterization of a second, distinct translational surveillance pathway (nonstop mRNA decay), we trapped intermediates in nonsense mRNA degradation. We present data in support of a model wherein nonsense-mediated decay funnels into the nonstop decay pathway in  S. cerevisiae and C. elegans revealed protein factors responsible for NMD (Saccharomyces cerevisiae NMD is thought to occur predominantly through decapping and 5’>3’ exonucleolytic degradation (Nonsense-mediated decay (NMD) (reviewed in ) is a tr for NMD . In the radation , while sDrosophila where transient knockdown of nonstop decay factors stabilized nonsense mRNA fragments . Given ragments .S. cerevisiae  . Among d flies) . ContribC. elegans) where nonstop decay can be genetically manipulated. During our characterization of C. elegans’ nonstop, we uncovered an unexpected link with NMD. Our subsequent results support a nonsense-mediated decay model in which recognition of premature stop codons results in cleavage of mRNAs at stop codons, generating truncated mRNAs which are further repressed by nonstop decay.Here, we report a metazoan system  . Again, we observed a ~6-fold loss of the nonstop mRNA, suggesting the changes in unc-54 mRNA level are not a secondary consequence of the Unc phenotype. At the protein level, we observed a > 100-fold loss of the UNC-54 nonstop protein  , the anip allele . To cont protein , and a lunc-54(cc2865) nonstop allele:There is reason to expect at least three independent mechanisms repressing expression of the The first 28 3’UTR-encoded amino acids are sufficient to elicit ~20-fold repression at the protein level . This isAdditional repression would be expected to elicit nonstop decay via translation of the poly(A) tail at the level of:nonstop protein decay, in which release of the nascent peptide is coupled to repressive mechanisms (e.g. ), andnonstop mRNA decay, in which ribosome stalling leads to degradation of the mRNA .These multiple functionally redundant yet independent repressive mechanisms likely underlaid our failures to identify trans-acting factors through genetic screens or via candidate-based approaches . In lighunc-54::gfp, generating unc-54(cc4092) a ‘T2A-nonstop reporter’. While this strain exhibited little difference in RNA levels compared to the T2A-less version (We reasoned that release of the nascent peptide prior to translation of the 3’UTR and poly(A) tail would allow the UNC-54::GFP reporter protein to escape peptide repression acting from translation of the 3’UTR or poly(A) tail. Reporter protein levels, however, would still be tied to mRNA decay mechanisms. The 15 amino acid ‘stop-and-go’ T2A peptide co-translationally releases the upstream peptide, after which the ribosome resumes translation of downstream sequences. Indeed, a T2A peptide can liberate upstream protein from 3’UTR- or poly(A)-encoded protein repressive mechanisms . We ther version , protein version . These ounc-54(cc4092) T2A-nonstop reporter to screen for mutants that restored reporter protein expression. In ~55,000 mutagenized genomes, we isolated 17 recessive mutants with a similar phenotype: higher reporter expression and a mild but incomplete rescue of animal movement. Some isolates exhibited a weak rescue of egg-laying defects. It is notable that no mutant completely rescued the reporter, suggesting that the alleles recovered only partially restored UNC-54::GFP reporter expression.We used the skih-2(IV) for the 14 mutants mapping to chrIV, and in the gene ttc-37(V) for the three mutations mapping to chrV   . Altogetdom34/pelota ribosome rescue factor. Loss of dom34/pelota modestly increases nonstop mRNA levels in multiple organisms (C. elegans r74.6 as a sequence homolog of dom34/pelota. Three lines of evidence suggest r74.6 (hereafter pelo-1) is the functional ortholog of dom34/pelota:Conspicuously absent from hits in our screen was a homolog of the rganisms . We idendom34/pelota, and has conserved functional residues known to be important for function in other systems While loss of either e at 23C . This sypelo-1 deletion conferred a small, but reproducible increase in the T2A-nonstop reporter protein  allele, in wild type as well as skih-2, pelo-1, and skih-2 pelo-1 mutant backgrounds. To measure the effects of skih-2 and pelo-1 mutations on mRNA levels, we performed RNA-seq . Loss of skih-2 increased RNA levels from the unc-54 allele . A ribosome in the act of translating an mRNA will protect ~28–30 nt of mRNA upon RNase digestion in C. elegans  in each strain mirrored the increases observed by RNA-seq . Howeverpelo-1 . The incotprints , and wasotprints . The inche 3’end . The shahe 3’end . This ache 3’end : endonucrevisiae  and S. pS. pombe , pointinS. cerevisiae and C. elegans: PELO-1 rescues ribosomes stalled on 3’-truncated RNA fragments, and SKI ensures efficient nonstop mRNA clearance.Altogether our data support a model for nonstop decay conserved between C. elegans opens up the possibility of identifying endogenous SKI/PELO substrates. In the above analyses, we observed a population of endogenous mRNAs whose behavior mirrored that of the unc-54(cc4092) reporter: an accumulation of 15-18nt Ribo-seq footprints in pelo-1 animals. We identified a population of 723 of these messages that exhibited reproducible accumulation of 15-18nt Ribo-seq footprints relative to 28-30nt Ribo-seq footprints specifically in a skih-2(cc2854) pelo-1(2849) mutant  mutant , see als) mutant . We reasxbp-1, the homolog of S. cerevisiae hac1  reporter or xbp-1: stop codon-less isoforms with truncated RNA fragments at or just upstream of the mRNA 3’end. This was not the case. Instead, endogenous SKI/PELO targets exhibited an accumulation of 15-18nt Ribo-seq reads at stop codons. For example, in addition to the tRNA endonuclease site in xbp-1, we noted an accumulation of 15-18nt Ribo-seq reads overlapping the stop codon that would terminate the unspliced xbp-1 reading frame  yielded comparatively few endogenous SKI/PELO substrates (S. cerevisiae by their increased accumulation of 15-18nt Ribo-seq fragments relative to longer Ribo-seq fragments (hac1 (xbp-1 homolog) and a handful of other mRNAs. Individual examination of these mRNAs failed to reveal accumulation of 15-18nt Ribo-seq reads over stop codons. We thus conclude that while at least one nonstop substrate is conserved (hac1/xbp-1), there is substantial difference in the number and nature of endogenous SKI/PELO-targeted RNAs between S. cerevisiae and C. elegans. We set out to better understand the link between stop codons and nonstop in C. elegans.Both the number of endogenous SKI/PELO targets, and the location of 15-18nt Ribo-seq reads in bstrates . We thusragments . Our anaC. elegans distribution of 15-18nt Ribo-seq reads genome-wide. In wild type and skih-2 animals, we observed an approximately uniform distribution of 15-18nt Ribo-seq reads across the open-reading frame, and less in untranslated regions  and ~5-fold for pelo-1(cc2849) skih-2(cc2854)). The endogenous SKI/PELO targets exhibited an even greater increase over these same positions (>13-fold for pelo-1(cc2849) skih-2(cc2854)), consistent with our gene-by-gene analyses  in the A-site . In eukaryotes, eRF1 recognizes stop codons in the ribosomal A-site, facilitating translation termination and peptide release. We examined 15-18nt Ribo-seq fragments for clues as to their ond base . It was ond base . Truncatond base . Thus, se A-site . The incswp-1, rsp-7, b0495.8, rsp-5, ubl-1, rsp-6, y57g11c.9, c12d8.1, asd-1, tos-1, hrpf-1, cyl-1  is a premature stop codon (Qln >Stop) mutation known to elicit nonsense-mediated decay (unc-54(e1092) allele into the skih-2(cc2854) pelo-1(cc2849) mutant. We observed substantial accumulation of 15-18nt Ribo-seq footprints at the e1092 site  nonstop reporter. Thus, introduction of a premature stop codon is associated with an accumulation of reads in a manner consistent with nonstop RNA decay of the transcript.First, we tested whether activation of nonsense decay is sufficient to target an mRNA to nonstop. We inserted an early stop codon allele known to be a nonsense decay target to see if it could elicit nonstop decay. ed decay . We intr092 site . As with092 site . The phaskih-2(cc2854) pelo-1(cc2849) unc-54(e1092)) to examine unc-54 RNA levels and species (unc-54: the unc-54 mRNA upstream of e1092 exhibited >10 fold more reads than the unc-54 mRNA downstream of e1092. This observation is consistent with a model where SKIH-2 and PELO-1 are required for clearance of unc-54(e1092) mRNA. In the skih-2/pelo-1 mutant, nonstop degradation of the truncated unc-54(e1092) open-reading frame is inefficient, and stalled ribosomes stabilize the degradation fragments generated by nonsense decay.We also performed RNA-seq on the same sample  pelo-1(cc2849) unc-54(e1092) smg-1(e1228). In the smg-1(e1228) mutant there was a net reduction in 15-18nt Ribo-seq footprints on the unc-54(e1092) transcript (unc-54(e1092) in the smg-1(e1228) mutant (unc-54(e1092) also evened out in the smg-1(e1228) mutant. Thus loss of an intact nonsense-mediated mRNA decay pathway accompanies an apparent loss of targeting of the unc-54(e1092) nonsense mRNA via nonstop decay. We noted that a minority of 15-18nt Ribo-seq fragments overlapping e1092 remained in the smg-1(e1228) mutant. The source of this residual population of fragments is unclear, although possibilities include (1) residual activity of the nonsense-mediated decay pathway in the smg-1(e1228) background, whether from truncated SMG-1 protein or readthrough of e1228, (2) SMG-1-independent nonsense-mediated decay activity, or (3) a low level of nonsense-mediated decay-independent generation of nonstop fragments.Second, we tested whether induction of nonstop at an early stop codon depended on known nonsense factors. se mRNAs . We thusanscript . This de) mutant . The dissmg-1(e1228). As with unc-54(e1092), the endogenous SKI/PELO targets exhibited a loss of 15-18nt Ribo-seq footprints relative to 28-30nt Ribo-seq footprints in the smg-1(e1228) mutant (smg-1(e1228) conferred a loss of 15-18nt Ribo-seq footprints specifically over the stop codon (unc-54(e1092), a residue of stop codon footprints persisted even in the smg-1(e1228) mutant (see preceding paragraph).Third, we tested whether endogenous SKI/PELO targets were relieved from nonstop targeting upon loss of nonsense-mediated decay. We examined the behavior of endogenous SKI/PELO targets with and without ) mutant . Thus, top codon . The losskih-2/pelo-1 with smg-1(+) or smg-1(e1228) revealed that the majority of endogenous SKI/PELO targets lose short footprints in the smg mutant (smg-1(r910) mutant  data. One explanation for this is that the short footprint assay is capable of identifying relatively minor transcript isoforms that are targeted by NMD relative to the background of 15-18nt Ribo-seq reads. If such isoforms are sufficiently rare, even a large fold de-repression in a smg mutant would be masked in RNA-seq by a lack of change in more abundant isoforms produced from the same gene. Consistent with this idea, we manually inspected several SKI/PELO targets and observed 15-18nt Ribo-seq reads accumulated at out-of-frame stop codons, although often without a transcript annotation that would explain such a translation termination event.A gene-by-gene comparison of 15-18nt Ribo-seq read counts in g mutant . Consistset from , Figure smg-1(e1228), among them xbp-1. Examination of xbp-1 revealed that while the bulk of 15-18nt Ribo-seq reads over the early stop codon were lost in the smg-1 mutant, some internal reads remained  and f38e11.9 (...ACACTTCTCCCAAAAA^), where ^ indicates the site of untemplated A’s. Our ability to identify premature polyadenylation sites was likely hampered by an apparent bias in our short read libraries against A/T-rich sequences (Among the remaining ~20 largely equences .smg-1(e1228) allele into the skih-2(cc2854) pelo-1(cc2849) background improved animal health. This was most readily manifest in a partial rescue of fertility defects  holds true for an engineered premature stop codon (e1092), (2) holds true for endogenous nonsense targets, and (3) depends on the known nonsense factor SMG-1.Thus, the activity of the nonsense-mediated decay pathway in C. elegans provides a remarkable tool in understanding the process. Several conclusions stem from this analysis:Several features have hampered the study of nonstop mRNA decay in vivo. By allowing a dissection of partially redundant aspects of nonstop decay and its consequences for target transcripts in the cell, we found that Nonstop mRNA decay is redundant. Because the cell responds to nonstop events through both protein and mRNA decay, two parallel pathways must be compromised to see an effect on a phenotypic reporter. Here we apply a tool (the T2A-based reporter) to study nonstop mRNA decay separate from nonstop protein decay. The T2A-based reporter enables both reverse and forward genetic studies of nonstop mRNA decay, and we expect that similar reporters may be useful in other systems where both mRNA and protein decay occur.C. elegans or S. cerevisiae  yields approximately normal levels of protein expression.Removal of a ribosome from the 3’end of a nonstop mRNA is a critical cellular function. To-date there is no identified mutation or combination of mutations that yield complete derepression of a nonstop mRNA in revisiae . The scaC. elegans. Here, the ability to knockout nonstop decay factors provided an opportunity to define and study endogenous SKI/PELO targets. The nonessentiality of a seemingly core gene expression function, while surprising, has precedent: C. elegans tolerates some alterations to translation termination , and paralogy with other proteins . Nonetheless, it is clear that complexes with similarity to the yeast SKI complex have important roles in a variety of RNA degradation processes in diverse systems  and ttc-37 (ski3 homolog) from a genome-wide screen provides strong evidence that a comparable SKI complex exists in C. elegans and is required for nonstop mRNA decay.This experimental system has facilitated a characterization of roles for the SKI complex in mRNA decay. The in yeast . Subsequop decay . Outsidens, e.g. ), low ses . Converaccepted . Here thC. elegans offers a functional companion to these (and ongoing) structural studies of SKI and the ribosome. For example, here we isolated skih-2(cc4142), a Gly >Glu missense mutation that mutates a conserved glycine packed at the interface of Ski2p and helix 16 of the small subunit rRNA  We would expect a more diffuse accumulation of 15-18nt Ribo-seq reads over premature stop codons. Instead, we observed a discrete and phased accumulation of 15-18nt Ribo-seq reads starting over the premature stop codon. (b) We would expect a population of intermediate size footprints (e.g. 19-28nt) that may arise from ribosomes translating to or colliding with 3’>5’ exonucleases. We did not detect these intermediate fragments, although it is possible they are unstable and/or not detectable with our current Ribo-seq protocols.Discerning between these and additional models will be a subject of future research.S. cerevisiae. This may be because nonsense decay is fundamentally different in S. cerevisiae and C. elegans. For example, S. cerevisiae lacks evident homologs of four smg genes (among them smg-5 and smg-6) required for nonsense decay in metazoans such as C. elegans and mammals. An immediate question for future research is whether other eukaryotes  exhibit nonsense/nonstop coupling as in C. elegans, or not as in S. cerevisiae. Answering this question may be technically challenging as pelota is essential in mammals  yields neurodegenerative phenotypes in mice  of protein production may be degraded by nonstop protein decay. Under this model, the expression and toxicity of a prematurely truncated protein would be mitigated by nonstop decay. This has implications for genetic studies where nonsense alleles are used as loss-of-function alleles for a given protein of interest, as well as the ~11% of human inherited disease that occurs due to premature stop codon mutations .skih-2 and pelo-1 mutants were grown at 16C. CRISPR/Cas9 was used to introduce edits (All strains were derived from ‘N2’ (VC2010) background  unless oce edits , and in skih-2/pelo-1 double mutant larvae from 16C to 23C allowed that animal to produce a handful of progeny prior to becoming sterile. The resultant progeny spent their entire lives at 23C, and we analyzed their brood size as described above. No power analysis was performed to determine the number of animal broods to assay. The experimenter was blinded to genotype while picking animals and counting brood size.Brood size was measured by picking a single larvae to a freshly seeded small NGM plate. Every 24–48 hr, the animal was picked to a fresh plate until it stopped producing progeny. Its offspring were allowed to grow for a few days to make counting progeny easier. Plates exhibiting microbial contamination, or where the adult crawled off the edge, were excluded from the counts shown. For brood size calculations at 23C, we found that shifting Western blotting was performed as described . BrieflyRNA-seq was performed essentially as described in , unc-54(e1092), etc.), we created custom versions of the genome with a modified unc-54 locus and annotations. Prior to mapping, reads were collapsed to remove PCR duplicates, using the unique molecular adaptor (NNNNNN) ligated on the RNA with AF-JA-34. PCR duplicates consisted of no more than a few percent of reads for any given library. Collapsed reads were mapped to the genome with STAR (v2.4.2a) allowing for one mismatch. Uniquely mapping reads were size restricted, then assigned to genes according to C. elegans’ ensembl release 83 annotations.We used ensembl release 83 (WBcel235) of the unc-54 poly(A) site, the number of reads at each position was counted and stored as an array. We then shifted the array by x bases, and calculated pearson’s correlation coefficient with the starting array. We performed this analysis for x = 0,1,...100, and plotted the correlation coefficient . We tabulated gene counts for both 15-18nt and 28-30nt Ribo-seq libraries, restricted to reads with 5’ends mapping between  relative to the start and stop codon, respectively  screen, the mutagen was 50 mM EMS. For the PD4092 (unc-54::gfp::T2A::nonstop) screen, a cocktail of 25 mM EMS and 0.5 mM ENU was used. Animals were washed twice with M9, and allowed to recover for 24 hr on NGM plates with food. Mutagenized P0 animals were dissolved in sodium hypochlorite for ~7’, leaving behind their eggs (mutant F1). Eggs were placed on an unseeded plate and larvae were allowed to starve and arrest as L1.~100 larvae were plated per small NGM, and 2 days later healthy F1 adults were counted to ascertain the number of genomes screened. At the F2/3 generation, animals were screened for increased movement and/or GFP. Only one isolate was kept per plate, ensuring independence of observed mutations.unc-54::mCherry animals were made by CRISPR/Cas9, males isolated, then mated to each suppressor strain. Hawaiian (CB4856) is a wild C. elegans isolate with a SNP every 700–1300 base pairs. The F1 cross progeny were isolated and allowed to self for 2–3 generations, after which 20–50 GFP-positive animals were picked to a new plate. After a few more generations, genomic DNA was isolated via proteinase K digestion and phenol/chloroform extraction.Suppressor loci were mapped similar to , as depiC. elegans genome (Ensembl Release 83) using bowtie2 (ver. 2.2.6  1) Based on knowledge from other organisms, the authors speculate that SMG6 is responsible for the endonucleolytic cleavage at the premature termination codon in NMD substrates in C. elegans. The authors should test if this is true to finalize their model.smg-6/skih-2/pelo-1 triple mutant. The expectation from that experiment is the same whether smg-6 is the causative nuclease or not (see smg-1 experiment). We expect loss of any of smg-1 through smg-7 to yield the same effect on the distribution of short ribosome footprints. In any smg mutant the SMG machinery has lost the ability to detect  premature stop codons.  Definitive testing of SMG-6 as the nuclease will be done by us and others in future studies by other means .We have considered profiling a 2) The authors speculate that NSD may also be involved in the degradation of the upstream fragments produced by NMD in other organisms. Is this likely? There are some papers that have identified endonucleolytic cleavages by SMG6 transcriptome-wide at nucleotide resolution in human cell lines . Often cleavage sites cluster downstream of the premature termination codon, which would mean that the upstream fragment would still contain a stop codon. This does not appear to be the case in C. elegans and should be considered by the authors.Most studies on NMD cleavage sites have been conducted in cells with an intact nonstop decay system. Because we would expect SKI/PELO to clear cleavages within or upstream of the stop codon, one would expect to detect only those cleavages outside of ORFs . We expect that future studies in diverse systems  after SKI and PELO activity have been ablated will clarify whether this is the case.C. elegans. Determining the full extent to which NMD is coupled to nonstop across organisms is beyond the scope of this current study, though we have referenced the above work in our manuscript .Another group (working in flies), detected endonucleolytic cleavage near stop codons, and demonstrated that the upstream RNA fragment is subject to nonstop decay . We note that at least some studies in mammalian cells have detected cleavage within the stop codon  similar to what we observe in Reviewer #3:[…] In light of previous publications, it might be interesting to categorize the annotated endogenous SKI/PELO-1 targets into different groups that might include previously identified NMD targets (i.e. found in a screen for mRNA levels in a UPF1/SMG1 delete), prematurely polyadenylated mRNAs (if they have been documented in C. elegans or by looking for reads possessing iterated As at their 3' ends), or some other group.C. elegans (see first comment for review #1, updated We have included a more in-depth analysis of the SKI/PELO targets in


Valsa mali, were found to colonize the apple bark, thus providing ample opportunity for HGT to occur. In the present study, we identified 32 HGT events in V. mali by combining phyletic distribution-based methods with phylogenetic analyses. Most of these HGTs were from bacteria, whereas several others were from eukaryotes. Three HGTs putatively functioned in competition with actinomycetes, some of which showed a significant inhibitory effect on V. mali. Three HGTs that were probably involved in nitrogen uptake were also identified. Ten HGTs were thought to be involved in pathogenicity because they were related to known virulence factors, including cell wall-degrading enzymes and candidate effector proteins. HGT14, together with HGT32, was shown to contribute to bleomycin resistance of V. mali.These results suggest that HGT drives the adaptive evolution of V. mali. The HGTs identified here provide new clues for unveiling the adaptation mechanisms and virulence determinants of V. mali.Horizontal gene transfer (HGT) often has strong benefits for fungi. In a study of samples from apple canker in Shaanxi Province, China, diverse microbes, along with the necrotrophic pathogen  Horizontal gene transfer (HGT) is the stable transmission of genetic materials between species through any mechanism other than vertical inheritance25Valsa mali, is one of the most destructive diseases affecting apple trees, causing significant yield losses in eastern Asia8V. mali and therefore may contribute genes to V. mali via HGT, because shared habitat is a major factor driving transfers15V. mali may have ample opportunity for HGT to occur. We have previously reported the V. mali genome sequence, which suggests a potential adaptation to colonize woody bark13V. mali. The functions of two HGTs of interest were verified experimentally. The results will provide new clues for unveiling adaptation mechanisms and virulence determinants in V. mali.Valsa canker caused by the ascomycetous Identifying HGT in eukaryotes is difficult because of their highly complex genome content. The gold standard for identifying HGT with confidence is phylogenetic incongruenceMost of these HGTs were derived from bacterial sources 16, probaV. mali during infection of apple bark, reported by us previouslyV. mali-apple interaction. Their specific adaptive values will be discussed in the following section.Functional annotations showed that the HGTs identified mainly affected enzymes (69%) from diverse metabolic pathways and genes 19%) with unknown functions . The rol% with unV. mali provide ample opportunity for other microbes to colonize. HGT14 and HGT32, bleomycin (Bm) resistance proteins, might enable V. mali to be resistant to bleomycin which is a family of antibiotics produced by actinomycetes that cause cell death in eukaryotes and prokaryotesV. mali, Fusarium graminearum and Aspergillus nidulans was examined under three levels of Bm stress . Compared wiht F. graminearum and A. nidulans, V. mali was significantly more resistant   in the Transporter Classification Database. DAL5 transports several substrates and is sensitive to nitrogen catabolite repression24252627V. mali might possibly utilize new sources of nitrogen.The types of nutrients that can be utilized by fungi are determined largely by their membrane transporters, because these microbes feed exclusively by osmotrophy. Thus, acquiring a novel transporter gene might enable fungi to utilize a new source of nutrition or gain a competitive advantage against other microbes in their ecological niches. HGT11, a major facilitator superfamily (MFS) transporter of a basidiomycetous source, was found only in the genus V. pyri) . HGT11 hde novo by the α-aminoadipate (AAA) pathway in higher fungi, and by the diaminopimelate (DAP) pathway in bacteria and plantsDapE gene (HGT1) in the DAP pathway by horizontal gene transfer  during V. mali infection  and a uridine nucleosidase (HGT17), which putatively function in the mannose biosynthetic process and nucleotide metabolism, respectively. A putative fructosyl amino acid oxidase (HGT12), which putatively functions in catabolizing naturally occurring fructosyl amino acidsV. mali infectionValsa and is a mitochondrial enzyme that detoxifies cyanideArabidopsisV. mali infects apple trees mainly through woundsV. mali-apple interaction.After pathogen attack, many plants produce low molecular weight antimicrobial compounds. Likewise, several cytochrome P450 genes potentially involved in secondary metabolite biosynthesis in apples are upregulated after V. mali can degrade woody tissues through secreted cell wall-degrading enzymes (CWDEs)1012V. mali, at least six CWDEs involved in HGT events were identified in the current study . These results suggest that HGT2 is a potentially important virulence factor in V. mali. Beyond their enzymatic functions, CWDEs can also act as a microbe-associated molecular pattern (MAMP) that activates plant defence responses, as reported for endopolygalacturonases (pectinase) of Botrytis cinereaPhytophthora sojaeV. mali-apple interactions is in progress.As a typical necrotrophic pathogen on apples, nt study . Among t. Another protein that probably functions in regulating plant immunity is HGT18, a small secreted protein with unknown function that was strongly activated during infection (Phytophthora spp.)  is an important plant hormone involved in defence responses against biotrophic pathogensnV. mali . Neverthnfection . HGT18 sra spp.) .V. mali. Most of these HGTs were of bacterial origin, and several were of eukaryotic origin. Three HGTs putatively functioned in competition with actinomycetes some of which showed a significant inhibitory effect on V. mali. Three HGTs that are likely to be involved in nitrogen uptake were identified in V. mali, which can effectively utilize nitrogen in apple bark. Ten HGTs were thought to be involved in pathogenicity, as they were related to known virulence factors. HGT14, together with HGT32, was shown to contribute to bleomycin resistance of V. mali. These results suggest that HGT drives the adaptive evolution of V. mali.By combing phyletic distribution-based methods and phylogenetic analyses, we identified 32 HGT events in the apple canker pathogen V. maliR threshold ranging from 0.2 to 0.9, Q value < 0.01)The proteome of http://itol.embl.de/)http://tree.bio.ed.ac.uk/software/figtree/).For phylogenetic analysis, the protein sequences of the top 100 blastp hits for each HGT candidate were retrieved from GenBank. Multiple sequence alignments were performed using MAFFT (v7.245)et al.The putative functions of HGTs were predicted with the PfamValsa mali, Aspergillus nidulans and Fusarium graminearum) was evaluated under three levels of bleomycin stress . Fungal isolates were grown on potato dextrose agar (PDA) media at 25 °C in the dark. Diameters of colonies were measured two days post inoculation. Each assay was repeated ten times. The data were analysed with ANOVA or t-test by using the online tool VassarStats (http://www.vassarstats.net/).The bleomycin resistance of three fungi , and G6PDH gene was used as an internal referenceFor RT-qPCR analysis, fungi were grown in potato dextrose broth at 25 °C in the dark for two days. Total RNA was extracted using a Quick RNA isolation Kit  according to the manufacturer’s instructions. First strand cDNA synthesis and qPCR were performed using a RevertAid First Strand cDNA Synthesis Kit  and RealStar Green Mixture , respectively. The qPCR assays were performed with a CFX96 ConnectHow to cite this article: Yin, Z. et al. Horizontal gene transfer drives adaptive colonization of apple trees by the fungal pathogen Valsa mali. Sci. Rep.6, 33129; doi: 10.1038/srep33129 (2016).


Nasonia vitripennis, a model for hymenopteran reproduction and development. Our work demonstrates that N. vitripennis, like other examined hymenopterans, exhibits characteristics indicative of an abortive meiosis, including slight enlargement of spermatocytes preceding meiotic initiation. However, we saw no evidence of cytoplasmic buds containing centrioles that are produced from the first abortive meiotic division, which occurs in the honeybee. In contrast to other previously studied hymenopterans, N. vitripennis males produce sperm in bundles that vary widely from 16 to over 200, thus reflecting a range of cellular divisions. Our results highlight interesting variations in spermatogenesis among the hymenopteran insects, and together with previous studies, they suggest a pattern of progression from meiosis to a more mitotic state in producing sperm.Males of hymenopteran insects, which include ants, bees and wasps, develop as haploids from unfertilized eggs. In order to accommodate their lack of homologous chromosome pairs, some hymenopterans such as the honeybee have been shown to produce haploid sperm through an abortive meiosis. We employed microscopic approaches to visualize landmark aspects of spermatogenesis in the jewel wasp  Drosophila melanogaster. Initially, a single germ cell produced from a stem cell division, termed a gonialblast, undergoes exactly four rounds of mitosis with incomplete cytokinesis, forming a cyst of 16 interconnected germ cells referred to as spermatogonia 2. Following a round of S-phase and enlargement in volume by ~25-fold3, these cells, now referred to as primary spermatocytes, pass synchronously through the two divisions of meiosis, producing 64 round, haploid spermatids4. The chromatin of these cells becomes remodeled such that the highly basic histone proteins are removed from DNA, and it is repackaged with sperm nuclear basic proteins (SNBPs), including protamines5. Each spermatid, including its nucleus, becomes highly elongated as it forms a flagellated tail and a pair of long, tubular mitochondrial derivatives2. Grouped in bundles of exactly 642, the spermatids undergo individualization through an actin-myosin-dependent process in which they become separated into mature sperm8.Spermatogenesis is one of the most elaborate cell transformational processes in animal development. Much of what is known about spermatogenesis in insects stems from numerous studies in the dipteran fruit fly, 9. Interestingly, in hymenopteran insects, which includes all bees, ants, wasps and sawflies, males arise as haploids from unfertilized eggs (reviewed in10). To be clear, males of these organisms contain only a single set of chromosomes inherited from the maternal parent. This characteristic makes homologous chromatid alignment and recombination in the male germ line impossible, thus begging the question of how haploid males produce sperm.A central question is to what degree deviations from this general paradigm of spermatogenesis occur in other insects. One outstanding example stems from studies performed in several bee species and a handful of other related insects that pertain to meiosis. In diploids, meiosis involves the side-by-side alignment of homologous chromatid pairs and the physical exchange of genetic material between them during the first meiotic divisionApis mellifera, like in D. melanogaster, the primary spermatocytes undergo cellular enlargement before entry into meiosis11. However, interestingly, in A. mellifera the nuclear envelope fails to break down in each spermatocyte during meiosis I even though the chromatin condenses into chromatids12. Each spermatocyte becomes clavate, or tear-dropped, in shape as it appears to undergo division. However, this division fails to occur, resulting in the original primary spermatocyte as the product. In the Indian honey bee Apis cerana indica, the first meiotic division does indeed occur, but the products consist of a large daughter cell containing the undivided nuclear material and a much smaller “cytoplasmic bud” containing no nuclear material12. Additionally, previous TEM studies in A. mellifera and other bees have noted the presence of small cytoplasmic buds containing extra, unused centrioles produced before or during the abortive division13. The second meiotic division in these insects is more conventional, being symmetrical and involving complete nuclear envelope breakdown and sister chromatid separation, resulting in haploid spermatids that subsequently undergo the final stages of spermatogenesis. It is presumed that the abortive division during meiosis I is an alteration of the basic cellular meiotic programming that compensates for the lack of homologous chromosomes in haploid males that is needed to properly produce haploid sperm – i.e., the reductive division step is eliminated because it is neither necessary nor desirable in haploid individuals.Previous work has shown that in the honey bee, Nasonia vitripennis, noted a less pronounced cellular increase of primary spermatocytes than occurs in A. mellifera14. Additionally, the number of sperm per bundle, and, therefore, the number of germ cell divisions producing sperm, varies among different species. For example, a predatory wasp Microstigmus arlei has 32 spermatids per bundle (5 divisions)15, some bee species have 32 spermatids per bundle while others have 64 spermatids per bundle (5 or 6 divisions)13, and the rose sawfly Arge pagana has either 256 or 512 spermatids per bundle (7 or 8 divisions)16. These patterns seem to follow a 2n rule where n is the number of germ cell divisions giving rise to sperm17. An interesting question is what cellular factor(s) determine the number of germ cell divisions for a given species.Although abortive meiosis I may be a common feature of spermatogenesis in hymenopteran insects, there appear to be some notable variations in aspects of spermatogenesis that have been found in a few examined species. For example, early TEM studies in males of the jewel wasp, N. vitripennis using a combination of several different microscopic approaches. N. vitripennis, a parasitoid of different blowfly species19, has gained prominence over the past two decades as a rising model insect for genetic and genomic studies aimed at understanding the evolution and development of axis patterning21, sex determination22, de novo centrosome formation23, host-symbiont interactions26, and venom production28. Additionally, recent work has focused on a paternally transmitted B chromosome known as PSR , which, through an unknown mechanism, causes complete elimination of the paternal (sperm-derived) half of the N. vitripennis genome during the first embryonic mitotic division30. Investigating these biological processes in N. vitripennis, particularly those involving the male germ line, would be greatly facilitated by a better understanding of spermatogenesis in this organism.These noted cellular variations in spermatogenesis, and the fact that this developmental process has been limited primarily to TEM studies in only a few hymenopteran species, led us to further investigate this process in the jewel wasp N. vitripennis, like other previously studied hymenopterans, undergoes an abortive first meiotic division to produce sperm. However, we observed several unique characteristics of spermatogenesis in N. vitripennis, including a relatively small increase in primary spermatocyte size compared to A. mellifera, and a highly variable number of spermatids per bundle. We propose that these and other characteristics may reflect an evolutionary progression from meiosis toward a fully mitotic state of sperm production in N. vitripennis.Our study here reveals certain cellular characteristics supporting the likelihood that N. vitripennis, we examined the male reproductive tract during wasp pupal development, when much of spermatogenesis occurs31. The male reproductive tract of N. vitripennis is bilaterally symmetrical, with each of the two sides beginning with an oval-shaped testis that connects at its base through a duct to a seminal vesicle, and then to an accessory gland, before joining to the ejaculatory duct . The two sides of the male reproductive tract connect at a position that is distal  to the two accessory glands  germ cells organized into cysts, and (ii) individual cells that contain very large nuclei and are interspersed among the cysts of germ cells , the nuclei appeared small, round, and hyper-condensed 11 and D. melanogaster (25-fold)2. Second, taking advantage of previously published transcriptome data for N. vitripennis germ line tissues37, we looked to see if any genes whose functions are restricted to meiosis are expressed in the wasp testis38. Five of eight such genes – DMC1, HOP2, MND1, MSH4, and SPO11 – are expressed at comparable levels in both the testis and ovary  would produce haploid gametes, regardless of how many mitotic divisions occur in any given cyst.Moreover, D. melanogaster, produce large amounts of specific RNAs and proteins that are actively transported through cytoplasmic bridges into the developing oocyte39. If trophocytes function in transporting needed factors to the germ cells, then their transport system would need to be different from that between the oocyte and its nurse cells because the trophocytes are not physically connected to the germ cells. However, although very speculative, we propose that trophocytes may express “diffusible” factors that help to regulate the overall mitotic/developmental progression of the germ cells of each cyst so that they are coordinated more-or-less together across large regions of the testis. Such an in trans means of developmental regulation would be expected to be somewhat imprecise, and is consistent with the variable number of spermatids per bundle within a given testis.Our observations show that the trophocytes interspersed among the cysts of germ cells have unique transcriptional properties. Our current work does not directly address the function(s) of these cells. However, it is intriguing to speculate on their possible roles based on knowledge of the polyploid nurse cells present in the female germ line. Specifically, nurse cells, which undergo 10–12 rounds of endoreplication by the end of oogenesis in The work presented here adds to a broader understanding of spermatogenesis in hymenopteran insects, especially regarding interesting variation in the cellular dynamics of gametogenesis present within this group. Additionally, these findings provide a number of hypotheses regarding the mechanics and evolution of spermatogenesis in a haploid individual that will be important to test in future studies.33. Wasp lines of the AsympC line were propagated by placing together equal numbers of males and females (10 of each sex) in glass vials containing ~10 Sarcophaga bullata blowfly pupae and a few drops of 50% honey in water. PSR crosses were conducted as previously described40. Wasps were maintained in an incubator at 25 °C on 12 hr light/dark cycles.The majority of our analyses were performed on reproductive tissues taken from male wasps of the AsymC genetic background; however, cytological analysis of a repetitive satellite sequence, PSR4317, was performed on the PSR + genetic background, which contains the supernumerary B chromosome PSRSarcophaga bullata host pupae at the appropriate stage based on wasp pupal color pattern  at 1:200; mouse anti-acetylated alpha-Tubulin (Santa Cruz Biotech) at 1:100; mouse anti-RNA Polymerase II phosphorylated at Serine residue 2 (Abcam) at 1:500. Secondary antibodies and their dilutions in the same buffer were: anti-rabbit Alexa488 and anti-mouse Cy5 .Fixed testes were stained with primary antibodies overnight on a platform rocker at 4 °C, washed three times at 15 minutes each with 1 × PBT. Secondary antibodies were then added to testes and incubated at room temperature on a platform rocker and in the dark  for 1 hour. The tissues were then washed three additional times with 1 × PBT (still covered) before mounting on a slide with Vectashield mounting medium with DAPI (Vector Laboratories).in situ hybridization (FISH) was performed as previously described33 using fluorescence labeled DNA oligonucleotide probes synthesized by IDT. PSR22 RNA was detected as in33, using a DIG-labeled LNA probe (5′-AATATCCAATCATAAGTCGAGACTTT-3′) generated by Exiqon. FISH was performed using the procedures and reagents provided by the manufacturer in the miRCURY kit. 28S ribosomal RNA and vasa RNA were detected using antisense DIG-labeled RNA probes synthesized in vitro from PCR-generated templates via conventional techniques41.DNA fluorescence Fluorescence confocal microscopy was conducted with a Leica TCS SPE confocal microscope. Images were collected either as a single image, or as Z-series and subsequently merged to capture for inclusion of details in different focal planes. All images were exported in JPEG format and then processed in Adobe Photoshop CS5 v. 12.The fixative used was Karnovsky’s Cacodylate Buffered Glutaraldehyde-Formaldehyde (1965), which consists of 3% glutaraldehyde and 3% formaldehyde in 0.1 M cacodylate buffer. All the testes were dissected while the pupa were immersed with fixative at room temperature. The testes within each group were placed into a 1.5 mL microfuge tube filled with fixative, left for 1 hour at room temperature, and then placed at 4 °C overnight. The next day, the samples were warmed to room temperature, the excess fixative removed, and a small amount of low melt agarose  added and mixed to disperse the testes. After letting the agarose cool, the agarose blocks were cut out, removed from the microfuge tubes, and placed into fresh fixative. While in new fixative, the agarose blocks were cut in half longitudinally, and the 2 pieces were placed into vials with new fixative. The vials were then kept at 4 °C overnight. The next day, the samples were warmed to room temperature, washed with 0.1 M cacodylate buffer and then post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for one hour. After washing in 0.1 M cacodylate buffer, the samples were dehydrated to 50% ethanol and then en bloc stained in 5% uranyl acetate and 50% ethanol for 60 minutes. The samples were then dehydrated in a graded ethanol series to 100% and then infiltrated with Epon 812 with propylene oxide as the transition fluid. After polymerization at 60 °C for 48 hours, the blocks were ultrathin sectioned, and the sections were stained in uranyl acetate and lead citrate. The sections were viewed in a Hitachi H7000 TEM at 75 kV. Images were captured using an AMT  2k × 2k CCD camera. All chemicals were supplied by EMS  unless otherwise specified.Supplementary Information


The export of organic acids is typically proton or sodium coupled and requires energetic expenditure. Consequently, the cell factories producing organic acids must use part of the carbon feedstock on generating the energy for export, which decreases the overall process yield. Here, we show that organic acids can be exported from yeast cells by voltage-gated anion channels without the use of proton, sodium, or ATP motive force, resulting in more efficient fermentation processes. Xenopus oocytes and in Saccharomyces cerevisiae engineered for dicarboxylic acid production. Among the tested transporters, the Mae1(p) from Schizosaccharomyces pombe had the highest activity toward succinic, malic, and fumaric acids and resulted in 3-, 8-, and 5-fold titer increases, respectively, in S. cerevisiae, while not affecting growth, which was in contrast to the tested transporters from the tellurite-resistance/dicarboxylate transporter (TDT) family or the Na+ coupled divalent anion–sodium symporter family. Similar to SpMae1(p), its homolog in Aspergillus carbonarius, AcDct(p), increased the malate titer 12-fold without affecting the growth. Phylogenetic and protein motif analyses mapped SpMae1(p) and AcDct(p) into the voltage-dependent slow-anion channel transporter (SLAC1) clade of transporters, which also include plant Slac1(p) transporters involved in stomata closure. The conserved phenylalanine residue F329 closing the transport pore of SpMae1(p) is essential for the transporter activity. The voltage-dependent SLAC1 transporters do not use proton or Na+ motive force and are, thus, less energetically expensive than the majority of other dicarboxylic acid transporters. Such transporters present a tremendous advantage for organic acid production via fermentation allowing a higher overall product yield.Biobased C4-dicarboxylic acids are attractive sustainable precursors for polymers and other materials. Commercial scale production of these acids at high titers requires efficient secretion by cell factories. In this study, we characterized 7 dicarboxylic acid transporters in  Microbial cell factories are designed to convert cheap substrates into added-value products, such as fuels, commodity, specialty chemicals, and drugs. It is usually advantageous if the cells efficiently secrete the products into the medium. This minimizes feedback inhibition and toxicity, prevents product degradation, and facilitates downstream processing , 2.Streptomyces griseoviridis  is likely due to the low energy requirement for organic acids export.C4-dicarboxylic acids, such as succinic, malic, and fumaric acid, are widely used in food, pharmaceutical, and polymers industries . These a15 studies , 20. In S. pombe, previously shown to be effective for malate and succinate production in S. cerevisiae . We also selected S. cerevisiae mitochondrial citrate transporter Ctp1(p). From the bacterial transporters, we chose two dicarboxylate transporters EcDcuc(p) and EcDcub(p) from E. coli (EcDcub(p), a putative transporter called AsDct(p) from the succinate producer Actinobacillus succinogenes. The genes encoding EcDcuc(p) and EcDcub(p) in E. coli are colocalized with the fumarase B gene and with genes involved in citrate utilization. Finally, we found a putative transporter SLC13(p) that was colocalized with succinyl-CoA synthetase and oxoglutarate dehydrogenase genes in A. succinogenes. The selected 7 transporter candidates were phylogenetically classified into 5 transporter families  and Ctp1(p) were shown to localize in the plasma membrane of oocytes, while their nontagged variants were expressed in the cytosolic space , which could also be converted into succinate and malate through the TCA cycle within oocytes. After incubating the oocytes in a buffer for 3 h, the concentrations of exported dicarboxylic acid were measured by LC-MS. EcDcuc(p), EcDcub(p), AsSlc13(p), and AsDct(p) were able to export citrate (SpMae1(p), the most closely related transporter to the Dcu(p) transporters did not show citrate export capability (SpMae1(p) was able to export fumarate, succinate, and malate (AsDct(p) and ScCtp1(p) fused to the Orai11(p) peptide (Leader-ScCtp1) showed some detectable export of fumarate and malate.The candidate membrane transporters  were sub citrate B. SpMae1pability B. On thed malate B. AsDct(S. cerevisiae strain engineered for the production of C4-dicarboxylic acids. The strain was created based on the evolved pyruvate decarboxylase-deficient strain (Xenopus oocyte screen: SpMae1(p), ScCtp1(p), AsDct(p), and AsSlc13(p) (SpMae1(p) showing the highest transport rate (SpMae1 (ScCtp1(p), AsDct(p), and AsSlc13(p) affected the growth of yeast, resulting in a lower final OD600 and a slower glucose utilization. On the contrary, the expression of SpMae1(p) did not exhibit a negative effect on the growth of yeast cells, and the glucose consumption was similar to the control strain without a heterologous transporter  B. In agrort rate C. We fou (SpMae1 C. The ti (SpMae1 C. As expnsporter C.SpMae1(p) transporter was initially identified on the basis of a mutant defective in malate uptake (SpMae1(p)’s ability to improve the export of carboxylic acids, including malate from yeast cell factories (SpMae1(p) did not impair the growth of yeast cells, while increasing the secretion of acids several fold. This was an indication that the production of acids was not coupled to growth, e.g., due to improved redox balance. The other transporters that also increased the secretion of acids all inhibited the cellular growth. We, therefore, hypothesized that the SpMae1(p) transporter must have a different transport mechanism with less energetic expenditure. To investigate this, we performed a protein motif search on SpMae1(p) using the Pfam library version 31 with 16,712 models (available at ftp://ftp.ebi.ac.uk/pub/databases/Pfam/releases/) and Gene3d models version 16 including 65,016 models (available at http://download.cathdb.info/gene3d/v16.0.0/gene3d_hmmsearch/). SpMae1(p) was annotated as a voltage-dependent slow activating (S-type) anion channel 1 (SLAC1) with e values below 10−72 (SpMae1(p) in S. cerevisiae is sulfite pump ScSsu1(p), and it contains a SLAC1 Pfam motif. The phylogenic relationship with other annotated Slac1(p) transporters from plants (SpMae1(p) did not show any proton exchange in a previous study (SpMae1(p) is more similar (TM-score 0.704) to the experimentally determined structure of Haemophilus influenza HiTehA(p) .The e uptake , 31, 32.actories , 23. In ow 10−72 . The clom plants , bacterim plants , and witm plants , which am plants , 36. It us study , which iiTehA(p)  as a bacSpMae1[p] in transmembrane domain 3 and F329_SpMae1[p] in transmembrane domain 9). These residues close the transporter tunnel with their phenyl rings (SpMae1(p) is 100% conserved in all of the investigated SLAC1 transporters, the F107 was substituted by similar amino acid tyrosine in AtSlah1(p) and AtSlah4(p) (SpMae1(p)F329A and a double-residue mutant SpMae1(p)F107A,F329A and expressed them individually in S. cerevisiae. These changes removed the phenyl rings, respectively, from the middle or middle and cytosolic faces of the transport channel  on malate secretion  (S. cerevisiae (SpMae1(p) without the C-terminal peptide, which was 40% lower than the native protein. The expression levels were determined using C-terminal fusion with a GFP protein (Alignment of Slac1(p) transporters, including Mae1(p) transporters, uncovered 2 conserved phenylalanine residues located within the transport channel (F107_yl rings B and C. Slah4(p) C. To exa channel B. While  channel D, the siecretion E. We add peptide F. To exapMae1(p) F and fourevisiae E. This d protein E.AtSlac1(p) from plant A. thaliana, HiTehA(p) from bacterium H. influenza, and AcDct(p) from fungus A. carbonarius. We also included an ALMT member, AtAlmt12(p) from A. thaliana. Among the examined transporters, AcDct(p) expression resulted in a 12-fold increase in malate titer, while the rest of the transporters lead to a smaller increase in 10–20% (SpMae1(p) increased malic acid titer 3-fold and improved the growth F329A. There were no significant changes in the activities of AtSlac1(p) or HiTehA(p). Expression of the mutant AcDct(p)F354A resulted in a lower titer of malic acid than in the control strain not expressing a heterologous transporter at low pH (AcDct(p)F354A had a severe growth defect at neutral pH . The GFP signal for SpMae1(p) and AcDct(p) mutants was 40–50% lower than for the native transporters. The decreased expression of SpMae1(p) cannot explain the complete loss of activity by the mutated variant of SpMae1(p), so we can conclude that phenylalanine residue F329 is essential for the transporter activity.For the SLAC1 members, we also examined the effect of mutations corresponding to the t low pH C. Curiouutral pH A, so we S. cerevisiae can grow in an acidic medium with a pH range of 3–6, which reduces the need for neutralization and allows direct recovery of an undissociated form of acids (S. cerevisiae membranes (S. pombe (AcDct(p), the homolog of SpMae1(p), was found to boost C4-dicarboxylic acid production in A. carbonarius (SpMae1(p) was annotated as a member of the TDT family and was believed to use a proton as the motive force (SpMae1(p) is highly active for the export of malate, succinate, and fumarate in oocytes (SpMae1(p) did not affect the cellular growth, in contrast to ScCtp1(p), AsDct(p), and AsSlc13(p) (SpMae1(p) and AcDct(p) as members of the voltage-dependent Slac1(p) transporters (SpMae1(p) and AcDct(p) are most likely equipped with mechanisms used by their evolutionary and structurally closely related transporters of the SLAC1 family . This is in contrast to the TDT family where the activity of transporters is coupled with a proton or Na+ ions. We recently addressed the energetic evolution of transporters, both at the level of cellular transportome and also transporter family levels  C. Our phsporters . Togethesporters , Slac1 has the potential for further improvements via engineering for higher transport efficiencies. There are two highly conserved phenylalanine residues in SpMae1(p) (SpMae1(p) (SpMae1(p), while closing the transport channel in the substrate-free state, are also necessary for the transport of carboxylic acids (Unraveling the transport mechanism of pMae1(p) . One of pMae1(p) B and C. pMae1(p) , which ipMae1(p) D. Howeveic acids A and C. ic acids .S. pombe had a very high activity toward C4-dicarboxylic acids  in both Xenopus oocytes and yeast S. cerevisiae and that SpMae1(p) did not inhibit the growth of yeast cells both at neutral and at low pH. A homolog AcDct(p) from A. carbonarius could also increase the production of malate in yeast without inhibiting the growth, albeit only at neutral pH. We present evidence that SpMae1(p) and AcDct(p) belong to the voltage-gated anion channel family SLAC1 and their expression results in energetically efficient export of dicarboxylic acids. This finding is important for engineering efficient cell factories for the production of biobased organic acids.To summarize, we showed that the Mae1(p) transporter from X. laevis oocytes by RoboInject . Candidates of membrane transporters were expressed in the S. cerevisiae cell factory designed to produce malic acid. A Leica TCS SP5-II confocal microscope was used for localization studies. HMMER version 3.1b2 and Phyre2 were used for protein motif identification and structure predictions, respectively. Chemicals were quantified by HPLC and LC-MS. Detailed experimental procedures can be found in SI Appendix, Supplementary Materials and Methods and Tables S1–S5.All of the DNA constructs were built using the USER fusion technique. In vitro transcribed cRNAs were injected into the Supplementary File


The reported issues with miRNA isolation using traditional RNA reagents necessitates the optimisation of miRNA isolation from challenging samples. In this study we compared six commercial RNA extraction kits to evaluate their ability to isolate miRNA from ovine plasma. We also compared three methods for quantification of small RNA extracted from plasma to determine the most reliable. Using minimal sample inputs of fresh and frozen plasma from five sheep, we compared the six kits (Kit A-F) using quantitative PCR. Operational factors were also assessed for each kit. Kits A and B provided the best detection of the miRNA qPCR reference genes across fresh and frozen samples (p < 0.001) followed by Kit C. The Qubit and microRNA assay provided the least variation , followed by the NanoDrop  and Agilent Bioanalyzer . We identify Kit A to be optimal for isolating miRNA from small volumes of fresh and frozen ovine plasma, and Kit B the top performing kit taking into consideration miRNA detection and operational factors. The Qubit fluorometer using a microRNA assay was the most reliable miRNA quantification method. One such biomarker that is frequently assessed is microRNA (miRNA), a small (approximately 22 nucleotides in length) subset of non-coding RNA which are critical mediators of gene expression. These short transcripts limit protein translation through a process known as “gene silencing”, where miRNA bind to mRNA molecules and either transiently block translation of proteins, or degrade the bound mRNA2.At the forefront of disease research in both human and veterinary medicine, predictive and diagnostic biomarkers have the potential to be highly specific and informative. Biofluids such as plasma, serum, saliva, or urine are common targets for biomarker profiling as they are collected by minimally invasive techniques and contain biologically relevant molecules which may be indicative of disease outcomes6. These molecules are thought to be excellent biomarker candidates as they are stable in blood and biofluids despite high levels of RNase, and are abundant in the circulation associated with extracellular vesicles and protein complexes7. In their role as regulators of translation, miRNA represent a complex interplay between the host immune system and invading pathogens, supporting the need for research into their biological function and control of disease processes.The expression of miRNA is notably altered during both bacterial and viral infection, as well as diseases such as cancer and diabetes9. Sample quality and processing are important factors affecting the quality and quantity of miRNA isolated; the profile of commonly used reference miRNA can be significantly altered in haemolysed plasma samples, while rupturing of cellular components of blood may alter the miRNA profile13. Similarly, the configuration of the miRNA itself may influence its ability to be effectively profiled. Sequences with lower GC content and stable secondary duplex structures have been shown to be less stable and therefore at risk of being lost during the extraction process14. It is evident that a range of factors affect the success of miRNA isolation, and therefore it is important that the chosen extraction protocol can tolerate these sample constraints.Challenges remain with respect to the isolation and accurate quantification of miRNA, especially from complex samples such as plasma. The small size of miRNA, along with high levels of contaminating protein and inhibitors present in plasma, reduces the efficacy of current molecular isolation methods. Commercially available extraction kits are commonly used in place of traditional RNA isolation protocols as they offer a faster and often simpler method of RNA isolation: these kits may be further specialised for miRNA isolation and a range of sample types, including serum and plasma, plant material, and tissue. The association of miRNAs with protein complexes such as lipoproteins and RNA-binding proteins further complicates isolation and quantification, as co-isolation of proteins may confound quantitative measurements and interfere with downstream applicationsOften, utilising a combination of sample lysis and silica-based spin column technology, commercial kits precipitate and bind RNA to the column membrane, allowing for easy elution and reduced sample loss when sequential pelleting of the RNA is performed. While many kits cater for total RNA isolation, several newer options are specialised for miRNA isolation and provide protocols for the isolation of either total RNA including miRNA, or for the selective isolation of miRNA only. The wide range of kits currently on the market provides a surplus of options for RNA and miRNA research; however, as there are many options, it is often necessary to test several kits for their suitability for specific applications and minimisation of extraction bias based on GC content and thermostability.15. Methods relying on fluorometric detection of nucleic acids may be more sensitive alternatives with greater tolerance for the presence of contaminants.The measurement and quantification of isolated miRNA are similarly important aspects. Traditionally, spectrophotometric methods have been used for RNA measurement that rely on the absorbance of UV-visible light at specific wavelengths to measure particle concentration, however at low concentrations this technique can often present inaccurate and inflated results16. Thus, an evaluation of the suitability of isolation protocols for specific sample types was undertaken. Discovery and profiling of biomarkers, as one of the main uses of isolated miRNA, necessitates that the starting volume is kept minimal, while still maintaining the sensitivity to detect low abundance targets. Here we have compared the suitability of six commercial kits, some specialised for miRNA isolation and others more broadly for several RNA populations, for applicability to the isolation of miRNA from low volumes ovine plasma. Several miRNAs, with varying GC content and thermostability , were quantified by qPCR and common RNA quantification methods were compared.To date there is limited knowledge and research into the plasma miRNome of sheep, and there are currently no reports on the optimal methods for ovine miRNA analysis. While miRNA research in human or murine models may provide the groundwork for ruminant studies, the biological and physiological differences in plasma composition and the variability of plasma component levels due to diet and metabolism necessitates investigation of the applicability of these kits and protocols for ruminant samplesA comparison of six different commercial kits for miRNA isolation from fresh or frozen ovine plasma was performed (Kits A-F). Additionally, three methods of RNA quantification were compared. An overview of the experimental design used for the assessment of isolation methods for ovine plasma miRNA is shown in Fig. All three methods of RNA quantification assessed were able to detect RNA in extracts from frozen plasma. The Qubit microRNA assay was able to quantify low abundance targets with the least within-sample variation , while also detecting RNA from each individual sample Fig. . Both thTo assess the success of miRNA extractions using different commercial kits, ten miRNA were detected by qPCR from each of the sample extracts Figs.  and 4. OThe repeatability of extractions from each kit was also assessed, with the coefficient of variation (CV) calculated for both fresh and frozen samples across all miRNA. CV was calculated for each of the five biological replicates to provide an overview of the variability between technical replicates . There was a significant difference between the cDNA input of fresh and frozen plasma in Kit B, with fresh samples having a higher amount of cDNA in the qPCR reaction on average compared to the frozen plasma samples (p < 0.001).A range of operational factors were also considered, consisting of subjective aspects associated with the use and application of each kit for miRNA isolation Table . These fThe average yield from each of the kits was considered as one of the key factors Fig. . Kits A Similar operational factors were also considered for the methods of RNA quantification Table ; the fac19. The successful isolation of miRNA from small volumes of complex biological samples, such as plasma which contain high levels of RNase and contaminating proteins including albumins and globulins, is an added challenge. Commercial column-based total RNA and miRNA extraction kits are often favoured over traditional guanidium thiocyanate/phenol-chloroform RNA isolation methodologies due to reports that the latter can result in isolation of selective miRNA populations, as well as often resulting in lower quantities of impure total RNA from smaller sample volumes21. A similar study comparing isolation kits miRNA recovery from both plasma and cerebrospinal fluid showed that TRIzol did not perform as well as commercial extraction kits, even when combined with on-column clean-up22. TRIzol extracted plasma had consistently higher Cq values across multiple miRNA and lower overall RNA recovery. Several more conflicting studies over the application of the gold standard RNA isolation methods to circulating miRNA recovery have been reported, which display favourable results over kit based protocols, suggesting that there is a high degree of variability and operator influence on its success24. Following these reports, commercial kits optimised for biological samples such as plasma and serum which may be used with small sample volumes, are ideal for miRNA profiling and biomarker discovery in archived or clinical samples26.Optimisation of the nucleic acid extraction step is a critical aspect of study design as this can impact the detection assays and experimental techniques that can be utilised, as well as the quality of the results and robustness of datacfRNA Serum and Plasma) is targeted for cell-free RNA including miRNA and mRNA, while Kit E (Isolate II) fractionates the RNA populations based on size to select for small RNAs. Both Kit E (PureLink RNA) and Kit F  are for total RNA populations, with no enrichment for small RNA, although they provide protocols for capturing the small RNA populations in the total RNA extract.In this study, we compared the use of six extraction kits from a range of manufacturers for their suitability for use with miRNA from either fresh or frozen ovine plasma samples. Due to the apparent variability of traditional methods in efficacy across samples and operators, we chose to examine these kits for their applicability to future miRNA profiling and potential biomarker studies. The chosen kits varied in their specificity for miRNA extraction, however, were included in the study as more broadly applicable RNA isolation kits are often cheaper and more widely used in general laboratory settings. Kits A (miRNeasy Serum/Plasma) and B (miRNeasy Serum/ Plasma Advanced) both extract total RNA, although enrich for small RNA populations <200nt. Kit C , were chosen for the qPCR measurement aspect of the study. While these miRNA may not cover the entire spectrum of GC content and ΔG observed in miRNA, they provide an insight into the performance of the extraction kits in contending with isolation of differing miRNA.The miRNeasy Serum/Plasma kit was optimal across samples, displaying the least variation between fresh and frozen plasma samples as well as requiring a minimal amount of plasma input. The miRNeasy Serum/Plasma Advanced kit was the next best kit, with similar miRNA detection in fresh plasma samples and also requiring low sample input for extraction. While the Serum/Plasma Advanced kit performed worse for frozen samples, this may have been due to a lower cDNA concentration than the fresh plasma samples. The Quick-cfRNA Serum and plasma kit was the third-best kit, however showed significantly worse performance when applied to frozen plasma samples. The added requirement for extra equipment, such as a vacuum and manifold, decrease the rating on ease of use in relation to the operational factors considered as this could be an obstacle for some laboratories. The extended incubation time and sample digestion (between 1–2 hours) required by this kit also increased the time required per sample and decreased the number of samples able to be processed simultaneously.The miRNeasy kits and the Quick-cfRNA were serum and plasma specific, suggesting that the specifically targeted kits performed better than those accommodating several sample types, such as the PureLink and Monarch Total RNA kits, and may necessitate the added cost for miRNA studies.27. While the PureLink RNA and Monarch Total RNA kits involved fast and simple isolation methods, their poorer performance in detecting miRNA and greater variability between samples from fresh and frozen plasma, and across replicates of samples from the same animal, produced poorer and less reliable results. While it is worth noting that the varying elution volumes of each of the kits will impact the concentration of RNA, the reverse transcription reaction showed similar levels of cDNA and no significant difference between cDNA quantity from different extraction kits (data not shown).The PureLink RNA, Isolate, and Monarch Total RNA kits performed similarly for ovine plasma across miRNA detection and the secondary performance parameters. While the Isolate kit required the least amount of sample (100 µl), higher Cq values and lack of detection for certain miRNAs reduced the suitability of this kit for plasma miRNA studies. Issues with column clogging despite centrifugation to remove cryoprecipitates and cellular debris further contributed to the lower efficacy of this extraction kit, with similar issues noted in other challenging biological samples14. While the initial report of selective loss of low GC/high ΔG miRNA populations during the extraction process was using traditional guanidium thiocyanate/phenol-chloroform reagents, it is an issue which must be investigated even in commercial kits. It is therefore of importance to assess the degree to which these kits mitigate the extraction bias of low GC/high ΔG miRNA and take this into consideration when selecting appropriate reagents.One important aspect impacting the performance of extraction kits is the miRNA GC content and the stability of secondary structuresA similar expression pattern was observed between both miRNeasy kits. While the Serum/Plasma and Serum/Plasma Advanced kits showed higher Cq values for miRNA with low GC% and high ΔG such as miR-144, −103, and −345-3p, the effect of these factors was better mitigated than in non-miRNA specific kits. Both miRNeasy kits were able to detect all miRNA and better handle the impact of low GC/high ΔG miRNA in the extraction process.For the Quick-cfRNA kit, there were higher Cq values across all miRNA for frozen samples, however especially for miRNA with low GC% and high ΔG in frozen samples, suggesting that the freeze/thaw process may also be impacting the kits performance.An effect of low GC/high ΔG on detection of miRNA by qPCR was also seen in the remaining kits , however the overall higher Cq values reduced the observable magnitude and impact of these factors on general kit performance.In comparing methods for the quantification of miRNA extracts from small volumes of ovine plasma samples, the Qubit with a microRNA assay was the least variable. The NanoDrop spectrophotometer also displayed an acceptably low level of variation between measurements, however the values provided were markedly higher than those from the Qubit and Bioanalyzer. As all measurements were taken from RNA from the same extraction and tube, we believe the high measurement values and apparent lack of sensitivity of the NanoDrop is due to the detection of contaminants such as phenol and silica carried over from extractions. Due to the dye-based methods of the other assays, these are tolerated and are not confounding measurements of nucleic acids.Conversely, the Bioanalyzer showed high variation between sample measurements compared to the Qubit and NanoDrop, although with greater reliability in measurement values than the NanoDrop. While only two measurements per sample were taken for the Bioanalyzer compared to the 3 for the other methods, likely contributing in part to the higher variation between readings, the raw differences between the measurements can be assumed to make up much of the variation. The high cost of the Bioanalyzer chips and reagents is also disadvantage of the method, while the Qubit machine and reagents provide a more cost-effective and accessible alternative.15. It appears that fluorometric assays like the Qubit may be better suited to miRNA studies, particularly when difficult samples are analysed, with our results, and others, demonstrating consistent measurement and lower variation than the microfluidic methodology of the Bioanalyzer28. However, only the Agilent Bioanalyzer provides a detailed quality assessment of the RNA integrity29. Although the operational factors showed the NanoDrop as the top method for quantification, the variability and the seemingly high measurements provided reduces its applicability to miRNA. The simple methodology of the Qubit fluorometer may also provide less room for operator error than the Bioanalyzer, which requires more technical steps including chip loading and priming and may be more applicable to multiple users and reduce time required for measurements.Thorough investigation into the ideal miRNA quantification methodologies in plasma samples have produced similar results, indicating that spectrophotometric methods may be insufficient for small quantities of plasma miRNAAll downstream steps from reverse transcription to qPCR were undertaken with the Qiagen miScript system with the same volume of RNA used for the generation of cDNA. Starting cDNA quantity did not influence amplification and qPCR performance. As this study was foremost a technical methodology assessment, levels of gene expression were not measured, and miRNA used solely to determine amplification of targets.33, and may be a viable option for enhanced miRNA recovery.We have identified methodologies for the initial extraction of miRNA from plasma samples suitable for downstream use in qPCR, however further work to optimise extraction protocols for other downstream assays is of importance. The addition of carrier RNA or glycogen have been shown to increase yields and improve amplification performance35. Overall, the miRNeasy Serum/Plasma and Serum/Plasma Advanced kit displayed superior qPCR performance, however as the Advanced kit and did not require long incubation and digestion times it was considered to be the most applicable kit. When used with the Qubit microRNA assay, concentrated and pure miRNA can be extracted from small volumes of either fresh or frozen plasma and measured with a high degree of sensitivity.This study has identified suitable kits for the isolation of miRNA from ovine plasma based on a combination of qPCR performance and secondary user-focused parameters. While there was no difference between the top performing kits (miRNeasy Serum/Plasma and Serum/Plasma Advanced kits) in both qPCR performance and operational factors, the Advanced kit offers a non-toxic, phenol-free alternative for miRNA extraction. This allows the protocol to be undertaken on the bench without the need for specialist protective equipment and reduces the time required per sample, by replacing the phase separation step. Studies using human plasma have found similar results regarding these kits and their favourable performance in the isolation of miRNA suitable for qPCR detection assays, supporting the conclusion that column-based extraction methods are optimal for complex biological samplesg for 20 minutes at 4 °C (Beckman Coulter Allegra X-12R). The supernatant was then transferred to a sterile tube leaving approximately 1 cm above the buffy coat to avoid contamination with white blood cells and platelets. Plasma was aliquoted into 200 µl volumes, visually inspected for haemolysis, and either frozen at −80 °C or stored at 4 °C for immediate RNA extraction.Whole blood was collected into EDTA-coated vacutainer tubes  via jugular venepuncture from 5 healthy sheep. All samples were processed on the day of collection and plasma separated by centrifugation at 2,000 × cfRNA Serum & Plasma , Kit D: Isolate II miRNA , Kit E: PureLink RNA Mini kit , Kit F: Monarch Total RNA Miniprep kit . All extractions were performed in duplicate for each animal, except for the PureLink and Monarch kits due to sample and reagent availability. Following extraction and elution of RNA, samples were immediately frozen at −80 °C.RNA was extracted from either fresh or frozen plasma according to the manufacturer’s instructions using 6 commercially available kits; Kit A: miRNeasy Serum/Plasma , Kit B: miRNeasy Serum/Plasma Advanced , Kit C: Quick-Briefly, Total RNA was isolated from 200 µl of plasma as per the manufacturer’s recommendations and eluted in 14 µl of RNase-free water.The miRNeasy Serum/Plasma Advanced kit is a phenol-free alternative to the original miRNeasy Serum/Plasma kit. Total RNA was extracted from 200 µl of plasma according to the manufacturer’s protocol and eluted in 20 µl of RNase-free water.Total RNA was extracted from 200 µl of plasma according to the manufacturer’s instructions using a vacuum pump and manifold for initial sample processing. RNA was eluted in 15 µl of RNase-free water.A 100 µl aliquot of plasma was used for extractions with the Isolate II kit, as per manufacturer’s instructions. Enriched small RNA (<200nt) was eluted in 50 µl of RNA Elution Buffer, the minimum amount required to adequately wet the column.Total RNA was isolated from 200 µl of plasma as per the manufacturer’s recommendations and RNA eluted in 30 µl of RNase-free water.As per the manufacturer’s recommendations, total RNA was extracted from 200 µl of plasma and eluted in the minimum suggested volume of 50 µl of RNase-free water.Three miRNA quantification methods, NanoDrop, Qubit Fluorometer, and Agilent 2100 Bioanalyzer, were compared for their suitability to provide a reasonable measurement of RNA quantity extracted from plasma. RNA was extracted from 200 µl of frozen plasma using the Qiagen miRNeasy Serum/Plasma Advanced kit and frozen at −80 °C. A single kit was used to avoid variability arising from isolation method, and the Serum/Plasma Advanced kit chosen due to the low time input required and the phenol-free formula. All measurements were performed in triplicate, except for the Bioanalyzer, from 5 biological replicates. Measurements were taken from the same RNA eluate and mixed via pipetting prior to sample loading.A NanoDrop One UV-Vis spectrophotometer (ThermoScientific) was used for RNA assessment this utilises the absorbance of light by nucleic acids to determine their concentration. Prior to measurement, the NanoDrop was blanked with 2 µl of the same nuclease-free water that the RNA was eluted in. Briefly, 2 µl of each RNA sample was loaded onto the NanoDrop pedestal and the arm lowered. Absorbance measurements were taken for each sample and the concentration (ng/µl) calculated.The Qubit® microRNA Assay Kit was used alongside the Qubit® 2.0 Fluorometer  to measure the quantity of RNA in each sample. With a stated core detection range of 5–500 ng/mL, the microRNA Assay Kit uses fluorometric detection of a miRNA specific dye to determine concentration, and provides 2 standards, 0 ng/µl and 10 ng/µl, to generate a standard curve prior to sample measurement. Each standard (10 µl) was diluted in 190 µl of working solution (1:200 dilution of microRNA Reagent in microRNA Buffer), while 2 µl of RNA sample was diluted in 198 µl of working solution. The mixtures were vortexed and incubated for 2 minutes in the dark at room temperature before measurements were taken. Qubit values were provided as ng/mL, and sample concentration was calculated using the Qubit® 2.0 Fluorometer’s dilution calculator.x g for 10 minutes. Gel-dye mix (9 µl) was pipetted into the appropriate well and the chip primed, followed by 9 µl of gel-dye mixture into the remaining gel wells. Conditioning solution (9 µl) was added to appropriate well, and 5 µl of the Small RNA marker to each sample and ladder well. The heat denatured ladder (70 °C for 2 minutes) was pipetted into the ladder well, and 1 µl of each denatured sample into each of the sample wells. The chip was then vortexed for 60 seconds and read using the Agilent 2100 Bioanalyzer.Evaluation of RNA quantity was assessed using the Agilent Bioanalyzer and Small RNA chip and reagents. This system employs microfluidic technology to electrophoretically separate and fluorescently label nucleic acids including RNA and miRNA. Small RNA chips and reagents were primed and prepared according to the manufacturer’s instructions. Briefly, 2 µl of dye was mixed with 40 µl of filtered gel mixture and centrifuged at 13,000 Reverse transcription for all RNA extracts was performed using the miScript RT kit  according to the manufacturer’s instructions. A master mix was prepared on ice containing 4 µl of 5x HiSpec Buffer, 2 µl of 10x Nucleics Mix, 2 µl of Reverse Transcriptase, and 2 µl of RNase-free water; per reaction to a total volume of 20 µl. 10 µl of master mix was added to 10 µl of the total RNA extracted from each plasma sample (n = 100) and incubated in a Corbett PalmCycler 96 thermal cycler using the following conditions: 37 °C for 60 minutes, followed by 95 °C for 5 minutes to inactivate the reverse transcriptase. cDNA was kept on ice and diluted with 200 µl of RNase-free water. Diluted cDNA was stored at −80 °C until use.cDNA synthesis efficiency and success were determined using a Qubit® ssDNA assay and the Qubit® 1.0 fluorometer. Two standards provided with the kit (0 ng/µL and 20 ng/µL) were made from the stock solutions and used to generate a standard curve. Briefly, 10 µl of each standard was diluted in 190 µl of working solution (1:200 dilution of ssDNA Reagent in ssDNA buffer), while 2 µl of sample was diluted in 198 µl of working solution. The mixtures were vortexed and incubated for 2 minutes in the dark at room temperature before measurements were taken. Measurements were used to estimate starting concentration of the RNA sample, qPCR cDNA input, and the efficiency of the reverse transcription reaction from a consistent input volume of 10 µL.Quantitative (q)PCRs were carried out using an Mx3000p Real-time PCR system  and the miScript SYBR Green PCR Kit  for 10 miRNA with varying GC content and thermostability  , 2.5 µl of each of the miScript specific primer and the miScript Universal Primer, and 2.5 µl of nuclease-free water. Amplification was under the following conditions: 95 °C for 15 minutes; 40 cycles of 94 °C for 15 seconds, 55 °C for 30 seconds, 70 °C for 30 seconds with fluorescence data acquisition occurring at the end of each cycle, followed by 1 cycle of 95 °C for 1 minute, 65 °C for 30 seconds, and 97 °C for 30 seconds. Cq values represent the cycle at which the detected fluorescence crosses the threshold. A dissociation curve was generated with ovine samples run alongside a bovine control to ensure amplification of the correct PCR product and homology of miRNA sequences across both species. Any samples with no amplification values were re-tested to confirm the result.36. This method accounts for each technical replicate, as well as the efficiency of the reaction to control for differences between qPCR runs and plate to plate variability and transforms raw Cq values to a normal logscale distribution.Raw Cq values were transformed to efficiency-weighted Cq (Cw) values prior to analysis using the common base methodStatistical analyses were performed using Genstat (v18.0). Data were grouped according to treatment (i.e. fresh or frozen plasma), gene, and extraction kit. A REML (restricted maximum likelihood) with Fishers LSD was performed to measure significant differences between the means of different treatments and kits, and accounting for the varying GC content of each miRNA.A two-way ANOVA was used to analyse the cDNA quantity for fresh and frozen samples and assess variation in the cDNA input into the PCR reaction for both fresh and frozen samples. The interaction between kit and treatment was evaluated and significance assessed at the 5% level.For each of the quantification methods, the coefficient of variation (CV) was calculated for intra-measurement variation and expressed as a percentage, as well as the standard error of the mean (SEM).All procedures were carried out under approval from the University of Sydney’s Animal Ethics Committee (AEC No. 2017/2149).Supplementary figures.


Colloidal gold nanoparticles are targeting probes to improve varlitinib delivery into cancer cells. The nanoconjugates were designed by the bioconjugation of pegylated gold nanoparticles with varlitinib via carbodiimide-mediated cross-linking and characterized by infrared and X-ray photoelectron spectroscopy. The drug release response shows an initial delay and a complete drug release after 72 h is detected. In vitro experiments with MIA PaCa-2 cells corroborate that PEGAuNPsVarl conjugates increase the varlitinib toxic effect at very low concentrations (IC50 = 80 nM) if compared with varlitinib alone (IC50 = 259 nM). Our results acknowledge a decrease of drug side effects in normal cells and an enhancement of drug efficacy against to the pancreatic cancer cells reported. Varlitinib is a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) family, controlling cell growth, differentiation, and survival. It selectively and reversibly binds to both EGFR ErbB-1) and Her-2/neu (ErbB-2) and prevents their phosphorylation and activation  and Her-. The tyrN-ethyl-N′-(3-dimetylaminopropyl) carbodiimide (EDC) and sulfo-N-hydroxysuccinimide (NHSS) is used to mediate the formation of linkage between carboxylic and amino-terminated groups [The primary problem in the cancer treatments with chemotherapeutic agents has been the high toxicity and low bioavailability of the anticancer therapy. The tumour heterogeneity and the MDR are the key challenge in anticancer therapy . Trying d groups .In the present study, the aim was to obtain conjugated gold nanoparticles to evaluate its effect into human pancreatic cells: MIA PaCa-2, a pancreatic cancer cell line that express high levels of HER2/neu and EGFR ,29,30, aPegylated gold nanoparticles were synthesized and conjugated with varlitinib via carbodiimide-mediated cross-linking. The nanoconjugate was characterized by using ultraviolet visible (UV-Vis), dynamic light scattering (DLS) and laser Doppler velocimetry, Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) techniques, in vitro drug release, and in vitro drug stability analysis. Our results showed that pegylated gold nanoparticles represent a promising drug delivery nanosystem, enhancing the varlitinib cell toxicity in pancreatic cancer cell lines.4; 99.99% trace metals basis, 30 wt % in dilute HCl—were acquired from Sigma-Aldrich Química . a-thiol-w-carboxyl (polyethylene glycol) (HS-C11-(EG)3-OCH2-COOH; molecular weight 394.57 Da) was obtained from Prochimia .Varlitinib was acquired from Selleck Chemicals LLC . Phosphate buffered saline (PBS) and fetal bovine serum (FBS) was purchased from Invitrogen Co. . Dimethyl sulfoxide (DMSO), trisodium citrate dihydrate and tetrachloroauric (III) acid—HAuCl2 humidified atmosphere at 37 °C.Immortalized human pancreatic duct epithelial cells (hTERT-HPNE) were provided by Professor M. A. Hollingsworth . Human pancreatic carcinoma cells (MIA PaCa-2) were obtained from the LGC Standards . The cells were maintained in DMEM medium, supplemented with 10% FBS under 5% CO4 through a solution of trisodium citrate [Gold nanoparticles (AuNPs) were prepared by the reduction process of HAuCl citrate ,32. Theng) to remove the unbound varlitinib drug.Varlitinib was conjugated to PEGAuNPs using the EDC/NHSS coupling  for 2.5 h. The PEGAuNPVarl was centrifuged , at 25 °C.PEGAuNPs and PEGAuNPsVarl were analysed by UV-Vis spectroscopy , using a 1 cm quartz cuvette, at room temperature.−1, resolution of 4 cm−1, and 64 scans, at 25 °C.The suspensions of PEGAuNPsVarl and PEGAuNPs, and varlitinib solutions were analysed by ATR-FTIR spectroscopy . Spectral scanning was acquired in the 4000–400 cmTEM images were acquired using a Jeol JEM-1400 , JEOL operated at 60 kV. An amount of 5 μL of each sample was placed on carbon formvar-coated grid and let to adsorb for 5 min. After, the grid was washed twice with deionized (DI) water to remove the excess.XPS was performed on a Kratods Axis Ultra HAS instrument  using a monochromator Al X-ray anode source operated at 90 W. Samples—AuNPs, PEGAuNPs, and PEGAuNPsVarl—were prepared by drop on a clean microscope slide and the drops were allowed to air dry before the analysis.g, 15 min) and the supernatant was collected to measure varlitinib concentration by fluorescence measures . The conjugation efficiency was evaluated by:The PEGAuNPsVarl formulations were centrifuged  . Varlitinib concentration of the dialysate buffer was determined with time through fluorescence measures using a microplate reader  .v/v) in PBS solution, at 37 °C. Samples were evaluated at several time points post incubation during 72 h by using DLS, UV-Vis spectroscopy and laser Doppler velocimetry.PEGAuNPs 8 nM and PEGAuNPsVarl 5 nM were incubated in PBS 0.01 M, at 4 °C and in FBS 10% (2 humidified atmosphere at 37 °C) and allowed to adhere for 24 h. Then, the cells were treated for 48 h with varlitinib and PEGAuNPsVarl at the concentrations ranging between 10 and 1000 nM varlitinib. Cells were fixed with 10% (w/v) TCA for 60 min on ice. Next, the cells were washed with water and stained with 50 μL of SRB solution. The unbound dye was removed by washing with 1% (v/v) acetic acid. The dried cells and the protein-bound stain were solubilized with 10 mM Tris solution. The SRB absorbance was measured at 560 nm in a microplate reader . The IC50  and GI50 (50% of growth inhibition) values were determined. The absorbance of the wells containing the NPs or drug and the absorbance of the wells containing untreated cells following a 48-h incubation period were subsequently compared with that of the wells containing the cells that have been fixed at time zero (corresponding to incubation of the nanoparticles and drug).In vitro cytotoxicity of varlitinib and PEGAuNPsVarl against pancreatic cell lines was evaluated by SRB (colorimetric) . Brieflyt-test statistical analysis was used to determine statistical significance ((p < 0.05).Values are reported as mean of three independent experiments. Student’s 4 solution with sodium citrate. They present a hydrodynamic diameter of 20.0 ± 0.2 nm  by using the EDC/NHSS crosslinking of carboxylic acids from PEGAuNPs with secondary amine group of varlitinib a, illustw/w) of varlitinib was conjugated with PEGAuNPs (by subtracting the unbound varlitinib in the supernatant solution). Therefore, the final varlitinib concentration in stock PEGAuNPs solution was determined to be 4.4 ± 0.5 µM.The varlitinib conjugation efficiency was determined through fluorescence analysis. Per the data, 84 ± 1%  and sp2—hybridized carbons (such as C=C and C=O). It corroborates with a covalent interaction between AuNPs and PEG-COOH. The signal of N at 399.76 eV is observed for PEGAuNPsVarl  was evaluated by hydrodynamic diameter, zeta potential measurements and time-dependent absorbance spectra, at 37 °C for 72 h . In the The in vitro drug controlled release experimental of PEGAuNPsVarl was performed in PBS  through a regenerated cellulose dialysis membrane with an initial varlitinib concentration in NPs of 4.2 μM. Cmax corresponds to the total amount of the drug added. Results are shown as mean ± SEM of three independent experiments.The in vitro cytotoxic effects after treatment with varlitinib alone and PEGAuNPsVarl were assessed on MIA PaCa-2 and hTERT-HPNE cell. Treatment with PEGAuNPs at concentrations up to 2 nM, during 48 h of incubation, did not presented effect on the cell growth (data not shown) corroborating non-toxicity of the PEGAuNPs . The effThe same trend is observed when analyzing inhibition of cell growth in response to varlitinib alone and conjugated to PEGAuNPs. For MIA PaCa-2s, the nanoconjugate improves the varlitinib activity resulting lower GI50 values e. In 48 For the hTERT-HPNE cells, the same effect is not observed b,d. Our An evan of inhibition hTERT-HPNE cell growth in response to PEGAuNPsVarl and varlitinib alone is observed on . The in vitro release profiles show a delay on varlitinib release due to the coupling process. The PEGAuNPsVarl shows a significant cancer cell survival inhibition for MIA PaCa-2 cells. In fact, cell survival appeared to decrease by 22–80% after PEGAuNPsVarl treatment with varlitinib concentration in range from 10 to 1000 nM, if compared to varlitinib alone. In contrast, cell inhibition in hTERT-HPNE cells by PEGAuNPsVarl is lower, denoting a drop of the nanoconjugate toxic effects in non tumour cells. The varlitinib therapeutic effect is enhanced by the controlled release of the anticancer drug after conjugation with functionalized gold nanoparticles. Our findings indicate that PEGAuNPs can be used as an effective vehicle for varlitinib inhibitor.In summary, a well-defined varlitinib delivery system PEGAuNPsVarl was successfully designed and prepared through the EDC/NHSS coupling reaction with a conjugation efficiency of 84%The drug delivery system shows potential antineoplastic activity for the treatment of EGFR overexpressed family, decreasing drug doses and the multi-drug resistance effects.