Nicotiana benthamiana). Even more extraordinarily, comparably modified UpA-high mutants displayed no pathology and over a million-fold reduction in replication. Tobacco plants with knockdown of RDP6 displayed similar attenuation of CpG- and UpA-high mutants suggesting that restriction occurred independently of the plant siRNA antiviral responses. Despite the evolutionary gulf between plant and vertebrate genomes and encoded antiviral strategies, these findings point towards the existence of novel virus restriction pathways in plants functionally analogous to innate defence components in vertebrate cells.Frequencies of CpG and UpA dinucleotides in most plant RNA virus genomes show degrees of suppression comparable to those of vertebrate RNA viruses. While pathways that target CpG and UpAs in HIV-1 and echovirus 7 genomes and restrict their replication have been partly characterised, whether an analogous process drives dinucleotide underrepresentation in plant viruses remains undetermined. We examined replication phenotypes of compositionally modified mutants of potato virus Y (PVY) in which CpG or UpA frequencies were maximised in non-structural genes (including helicase and polymerase encoding domains) while retaining protein coding. PYV mutants with increased CpG dinucleotide frequencies showed a dose-dependent reduction in systemic spread and pathogenicity and up to 1000-fold attenuated replication kinetics in distal sites on agroinfiltration of tobacco plants ( Viruses may also display compositional abnormalities, most notably, suppression of the frequencies of CpG and UpA dinucleotides that have, in mammalian RNA viruses and retroviruses, been shown to influence replication6. In part, these biases in RNA viruses may be influenced by the genomic composition of the hosts they infect, potentially mirroring the suppression of CpG and TpA dinucleotides in vertebrate and plant DNA genomes9. In these latter groups, suppression of CpG dinucleotides originates from a mechanism in which the cytosine of the CpG dinucleotides is targeted for methylation rendering it susceptible for mutation to thymine by deamination10. Genomes of methylation-heavy organisms such as vertebrates and higher plants show extensive under-representation of CpG dinucleotides12. It is estimated that 70–80% of cytosines in CpG motifs are methylated in mammalian genomes13 while CpG methylation in plants is much less extensive (24%)14 and largely limited to transposons and other repetitive DNA elements15. Plant genome methylation also occurs in a wider range of contexts, with 1.5% of cytosines methylated in CHH motifs  and around 6.7% in CHG sites14. Methylation plays essential regulatory roles in gene expression with functions ranging from epigenetic control16 and anti-viral defence via transcriptional (transposons) gene silencing. The latter is mediated by the RNAi pathway and requires the biogenesis and amplification of 24 nucleotide (nt)-sized small-interfering (si)-RNAs by DCL3 and RNA-dependent RNA polymerase 2 (RDR2), respectively17. Less is understood about UpA dinucleotides suppression. However, RNA degrading nucleases involved in mRNA turnover may target UpA dinucleotides and U + A rich sequences in mRNA for degradation and UpA frequencies may be regulated for this reason18.There is increasing evidence that many phenotypic properties of a virus and aspects of its interaction with hosts are determined by structural and compositional attributes of its genome. Single stranded RNA virus genomes may be internally base-paired, creating often extensive duplex or higher order structures that interact with replication proteins or translation factors19. However, while it could be conceived that suppression of UpA may occur in response to the same cellular factors that govern the stability of cellular mRNAs18, what mediates or drives the suppression of CpGs in RNA virus genomes is unclear given that CpG-associated methylation and loss of CpGs is a specifically DNA-based mechanism occurring in the nucleus. In mammalian RNA viruses, artificially increasing CpG and UpA frequencies in a range of mammalian RNA virus genomes have a severe attenuating effect on replication of echovirus 7 (E7) that is independent of interferon-mediated cellular responses to virus infection, stress response pathways and apoptosis5. Recently, zinc finger antiviral protein (ZAP) was shown to directly bind high CpG RNA sequences of HIV-1 and restricted its replication4. Our recent investigation of restriction mechanisms found no evidence for translational arrest20 or RNA exonuclease-mediated digestion or cleavage of high CpG mutants of E7. We have recently obtained evidence that, in addition to CpG dinucleotides, UpA dinucleotides may be similarly targeted by ZAP and moreover, by RNAseL, a site-specific RNA nuclease may contribute to the attenuation of high CpG mutants of E721.In general, the genomic composition of eukaryotic RNA viruses often recapitulates the compositional characteristics of host mRNA and, in the case of vertebrate and plant viruses, may reproduce the suppressed frequencies of both CpG and UpA dinucleotides of their hosts22 as a model virus for mutagenesis and phenotypic characterization using the previously developed infectious clone23. PVY is a member of the Potyviridae family and particles consist of a non-enveloped filamentous structure containing a positive-sense single-stranded genomic RNA24, of approximately 9.7 Kb in length. The genomic RNA contains an open reading frame (ORF) encoding viral structural and replication proteins, and a short overlapping ORF encoding pretty interesting potyviral protein24. The PYV coding region is flanked by short 5′ and 3′ untranslated regions (UTRs), and with a viral protein genome-linked (VPg) covalently attached to its 5′ end and a 3′ poly(A) tail26. An Agrobacterium tumefaciens mediated delivery method for the infectious clone of PVYNTN was used to investigate the effect of elevated dinucleotide frequencies on virus replication and systemic spread in Nicotiana benthamiana.In contrast to mammalian viruses, there is currently no functional data on effects of similar manipulation of CpG or UpA frequencies on the replication of plant viruses. Their extensive suppression argues that they may be under similar restrictive pressures to enable efficient replication. To address this, we used the PVY-N605 strain of potato virus Y (PVY)Nicotiana species, N. attenuata and N. tomentosiformis , Arabidopsis thaliana and Zea mays (a monocotyledon), along with corresponding sequence datasets from the human genome  and dsRNA viruses (0.66–0.95). For many families in these latter groups, CpG frequencies were substantially higher than those of the analysed plant species (0.45–0.79). For viruses showing suppression of CpG , frequencies were associated with their G + C content , a relationship that closely recapitulates that observed in vertebrate RNA viruses11. There were however, a number of virus groups, including the dsRNA viruses  and several virus groups of + strand RNA viruses  that did not display this relationship  . UpA frequencies were however comparable both between mono- and cotyledon mRNA sequences  strains were comparable  and HpaI (position 8354) of the N19N21 clone of PVY  representing 18% of the genome  over which time symptoms of necrosis, stunted growth, and leaf curling developed in plants infected with WT PVY and PVX  leaves of  16 Fig. . ComparaN. benthamiana agroinoculated with PVX, WT PVY and mutant viruses by double antibody sandwich ELISA (DAS-ELISA) using antibodies against the structural coat protein of PVY  of RDR6 prevents the mounting of a strong 21/22 nt siRNA mediated-PTGS response, leading to increased susceptibility to RNA virus infection29. To investigate whether this pathway was involved in the CpG or UpA-associated attenuation phenotypes, RDR6 knock down (KD) plants using a TRV-based VIGS vector were inoculated with R123-CpGH and R123-UpAH mutant viruses and replication compared with of the WT PYV. Effective KD of RDR6 to 20–40% of constitutive levels was verified by RT-PCR quantitation of mRNA sequences , additionally of UpA dinucleotides30, as if they were either subject to the same compositional constraints that drove the evolution of cellular sequences or cells have evolved to sense non-self RNA based on RNA composition. Suppression of UpA was universal in all plant RNA viruses analysed , both comparable in degree to mammalian RNA viruses11. The underlying basis for UpA suppression in plant mRNA sequences is unclear. The existence of universal although lesser degree of UpA suppression in host DNA sequences of plants and other eukaryotes suggests that it, at least part, originates from a mutagenic process occurring during DNA replication, since the vast majority of DNA sequences are not expressed and potentially fix mutations in the absence of compositional selection. Our previous modelling of dinucleotide frequencies in mammalian and other eukaryotic genomes indeed showed that UpA under-representation could arise secondarily from methylation-associated CpG depletion, rather than direct targeting of TpA dinucleotides in genomic DNA11. This mechanism potentially extends to plant genomes. However, such mutagenic effects are unlikely to be reproduced in the enzymatically quite different process of RNA-dependent RNA transcription of RNA virus genomes. The observation for universal suppression of UpA in RNA viruses therefore supports the hypothesis that they have adapted their composition for replication in the plant cell cytoplasm, very much as previously discussed for mammalian RNA viruses5.Many groups of RNA viruses infecting eukaryotes recapitulate the suppression of CpG and UpA dinucleotides observed in host cell mRNA sequences Fig. 11,19,30,sed Fig. , with me31. Supporting this relationship, suppression of CHG frequencies was much more restricted (O/E ratio of 0.94), commensurate with methylation frequencies of 6.7%, while the absence of detectable suppression of CHH frequencies matched to its extremely low frequency (1.7%) and reversible nature of its methylation.There was substantial suppression of CpG in plant genomic DNA and mRNA sequences Fig.  althoughTymovirales, luteo/tombus/sobemo and –strand RNA viruses reproduced the relationship between CpG representation and G + C content in +strand and −strand mammalian RNA viruses and small DNA viruses11. However members of several other plant virus ssRNA + families  and dsRNA viruses  showed little or no GpG suppression  such as RIG-I and toll-like receptor 3 (TLR3) in vertebrate cells, along with downstream interferon-stimulated effector pathways such as RNAseL. Virus genomic dsRNA may be similarly targeted by siRNA in plants. Under these circumstances, effective shielding of the genome may make suppression of CpG and UpA frequencies unnecessary although such viruses will still produce compositionally abnormal mRNAs that may be targeted by ZAP and RNAseL5. The lack of CpG suppression in the other virus groups is less readily explained - replication of members of the Beniviridae, Closteroviridae, Virgaviridae and Bromoviridae occurs in compartments separated from the cytoplasm36, but this is a general feature of plant RNA virus replication and occurs similarly in tombusviruses, tymoviruses and potyviruses39 where CpG frequencies are systematically suppressed.CpG frequencies in plant RNA viruses (and host genomes) showed both similarities and differences from the universal suppression of UpA. Firstly, not all RNA viruses showed suppression, and for those groups where it was observed, the degree of under-representation was related to G + C content of the genomes 21, a PRR that to date has been considered to specifically target dsRNA. The unexpected involvement of these pathways and their independence from conventional IFN-mediated antiviral responses3 further exemplify the complexity of virus/host interactions, even at the single cell level. The possibilities that these restriction pathways are shared in plant is explored in the following discussion.How the plant recognizes and responds to these compositionally modified sequences is currently unclear. The attenuation of mammalian RNA viruses has been recently shown to be mediated through the action of a novel restriction pathway, in which direct binding of ZAP to CpG-enriched viral RNA sequences somehow induces a profound curtailment of its replicative ability. We have recently shown that ZAP similarly directly binds to and restricts the replication of UpA-enriched mutants of E7 viruses and replicons40. This conjecture is supported by the almost universal development by plant viruses of one or more anti-siRNA evasion pathways, typically through encoding proteins termed viral suppressors of RNA silencing (VSRs) (reviewed in41). In the case of potyviruses, two different VSRs have been identified, HC-Pro that acts through a variety of translational, RNA binding and exosome inhibition pathways (reviewed in42) and VpG that inhibits expression of suppressor of gene silencing 3 (SGS3) a key component of the siRNA pathway43. The difference in replication levels of WT PYV in RDR6 KD plants and controls  is unlikely to play a role in their attenuation. This conclusion is mechanistically supported by the nature of the dsRNA motifs targeted by siRNA. There is no current indication in other systems that increased frequencies of CpG or UpA dinucleotides would enhance the activity of this pathway.At its broadest, plant virus immunity can be divided into two principal strategies. siRNA-mediated cleavage and decay of viral RNA sequences represents a potent defence mechanism shared across eukaryotes and considered by many to represent the primary defence of plants against RNA virus infectionols Fig.  demonstr44. These dimerize and signal through SERKs and SOBIR1 to activate a large number of anti-microbial responses. Only few cases have been reported on the (extracellular) sensing of plant viruses by PRRs. In contrast, most plant viruses trigger an ETI after being sensed by intracellular sensors of innate immunity, the biggest class represent nucleotide binding-leucine-rich repeat (NLR) proteins encoded by single dominant resistance (R) genes45. Their triggering by viral effectors mostly comes with a hypersensitive response (HR), observed by the formation of necrotic local lesions and resulting from apoptosis, a programmed cell death response. NLRs are also present in animals and structurally similar to those of plants. Despite many structural and functional similarities between these and in the stress response pathway conserved across eukaryotes, PRRs, its downstream signalling mechanisms and antimicrobial and R/NLR protein responses represent a system that may have evolved independently and convergently in each kingdom52. In light of this it remains to be questioned whether there are direct homologues of mammalian ZAP or RNaseL in plant cells that mediate the restriction of PVY mutants in the current study.The second and more extensive component of plant defence involves PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). During PTI an array of predominantly cell surface expressed pathogen recognition receptors (PRRs), consisting of receptor kinases and receptor-like proteins, detect a vast range of different PAMPs, expressed on the surfaces of bacteria and fungi in the apoplastic space of plant cells53. While PARPs typically play roles in DNA and RNA metabolism and repair and show a predominantly nuclear cellular distribution, indirect genetic evidence for positive selection suggests that PARP4, PARP9, PARP14 and PARP15 may also have been co-opted to function in vertebrate innate immunity54. Plants possess three paralogues of PARP; however, all three show predominantly nuclear distributions and lack the typical zinc finger domains associated with RNA binding and cleavage; they consequently appear unlikely candidates for functional homologues of ZAP for CpG and UpA recognition in plants. In the absence of any clear candidate pathways for CpG or UpA-enriched RNA sequences, a more robust strategy to identify recognition proteins is to exploit their potential binding to immobilized RNA targets of different compositions (as used to detect ZAP binding to UpA- and CpG-high E7 sequences21) and to identify and characterize bound proteins by mass spectrometry. A comparative analysis of high UpA- and CpG-high RNAs in mammalian, arthropod  and plant cytoplasmic preparations is currently planned.ZAP is a member of the poly-adenosine diphosphate ribosyl transferase protein (PARP) family that is widely distributed across all eukaryotesNicotiana benthamiana plants were grown in contained room at 23 °C ± 2 °C in a 16/8 h light and dark cycle. The Agrobacterium tumefaciens strain GV3101 was used for agroinfiltration of PVY cDNA constructs; strain LBA4404, was used For TRV based VIGS constructs. Both strains of Agrobacterium were grown at 28 °C shaking at 200 rpm.55 comprising the sequence of PYV isolate N605 cloned into the pCAMBIA binary vector (Marker Gene Technologies). Four introns were introduced to facilitate propagation in Escherichia coli and Agrobacterium tumefaciens. The inserted introns were the modified second intron of the Solanum tuberosum light-inducible tissue-specific gene56 and the second intron of Phaseolus vulgaris nitrite reductase gene57. The full sequence of the clone and regions of native and introduced (intronic) genome sections are provided as a Supplementary File.Clone N19N21 pCAMBIA PVY N605 GFPCP was provided by Elisabeth JohansenBstXI and HpaI sites was sub-cloned from wtPVY-pCambia into pJET (ThermoFisher Scientific) using PCR cloning giving wtPVY-pJET construct. Mutant PVY insert sequences  were first cloned into wtPVY-pJET using sites NcoI and BglII for Regions 3 and sites BstXI and NcoI for Regions 1 and 2 and then introduced into wtPVY-pCambia giving six compositionally modified PVY-pCambia constructs  with the respective PVY.pCambia vector, and a single colony was grown overnight in LB3 medium  supplemented with 100 μg/ml kanamycin and 100 μg/ml rifampicin. From the overnight culture, 600 μL were inoculated into 3 ml of induction medium 2SO4, 0.5 g/L sodium citrate dihydrate, 0.25 g/L MgSO4, 0.2% (w/v) glucose, 0.5% (v/v) glycerol, 50 mM acetosyringone and 10 mM MES) and incubated overnight at 28 °C shaking at 200 rpm. The next day, broth was centrifuged at 3000 rpm for 15 min at 28 °C. Following decanting of supernatant, the pellet was resuspended in infiltration medium  and diluted to an optical density at 600 nm (OD600) of 0.4. Plant leaves were agroinfiltrated at the basal side of three weeks old N. benthamiana leaves59.For agroinfiltration of N. benthamiana was performed by virus induced gene silencing (VIGS) using the tobacco rattle virus (TRV) vector system. To this end, N. benthamiana was agroinfiltrated with a combination of TRV1 and TRV2, containing a sequence of the target gene to be silenced. For RDR6 silencing, A. tumefaciens was transformed independently with the either TRV1 or TRV2-RDR6 plasmids, after which, a single colony of each transformant was grown overnight in LB3 medium as described above. Similarly, overnight broth cultures were introduced into induction medium for overnight incubation, then centrifuged and resuspended in the infiltration medium. Both infiltration medium preparations, one for Agrobacterium harbouring TRV1 and another for Agrobacterium harbouring TRV2-RDR6, were diluted to OD600 of 0.4. Equal volumes from both preparations were then mixed together and agroinfiltrated into the basal side of two weeks old N. benthamiana leaves60. Nine days after VIGS agroinoculation, plants were agroinoculated with the PVY mutants. To verify on the onset of gene silencing, as a positive control, plants were infiltrated with TRV-PDS and monitored on bleaching of chlorophyll.Gene knockdown in N. benthamiana were harvested and placed flat against white background in proximity to a linear measuring ruler. Pictures were taken using Canon SLR camera (Cannon) and leaf area was measured using ImageJ software. Differences in leaf area were statistically evaluated using GraphPad Prism software.The four most top leaves of agroinoculated N. benthamiana were screened for green fluorescence signal using ZEISS Observer. Z1 fluorescence microscope mounted with Filter Set 38 for excitation and emission wavelengths of 470/40 and 525/50 respectively. Pictures were processed using AxioVision Release 4.9.1 software.3 × 3 mm sections of topmost third leaf from agroinoculated N. benthamiana leaf tissue using extraction buffer  Tween 20, 2% polyvinyl pyrrolidone (PVP) (Sigma), pH 7.4) using TissueLyser II (Qiagen). Double antibody sandwich (DAS) ELISA using 96-well flat bottom ELISA plates was designed to quantify virus titre. For PVY detection, wells were coated with capture antibody, anti-PVY coating Ab , diluted in bicarbonate buffer  and washed with 2% PBS-T pH 7.4. Extracted virus samples were further diluted in extraction buffer (20-fold dilution) before being added to the wells and were incubated at 4 °C overnight. Detection was with anti-PVY alkaline phosphate conjugated antibody  diluted in extraction buffer and incubated at 4 °C overnight. For PVX detection, same procedure was followed however anti-PVX coating Ab  and anti-PVX alkaline phosphate conjugated antibody  were used. Following overnight incubation, plates were washed with 2% PBS-T pH 7.4, and p-nitrophenylphosphate (pNPP) (Sigma) was used as enzyme substrate. Colour was allowed to develop for 30 minutes at room temperature before the addition of 3 N NaOH to stop the reaction. Absorbance was read at 405 nm.Virus was extracted from N. benthamiana leaf tissue using TRI Reagent Solution (Invitrogen) homogenized using TissueLyser II (Qiagen). Following homogenization, chlorophorm was added (200 ul/1 ml of TRI reagent) and centrifuged at 12,000 g for 20 minutes at 4 °C, after which, the aqueous phase was transformed into equivolume of isopropanol, and centrifuged at 12,000 g for 20 minutes at 4 °C. Pellet RNA was washed 3x with 75% ethanol before being resuspended in DNase/RNAse-free distilled H2O. Extracted RNA was treated with RQ1 Dnase (Promega) at 37 °C for 1 hour.Total RNA was extracted from 100 mg N. benthamiana agroinoculated with VIGS-silenced on RDR6, or GUS (negative control) and treated with RQ1 Dnase (Promega), was used as a template for reverse transcribing cDNA using SuperScript™ II Reverse Transcriptase (ThermoFisher Scientific) and RDR6_cR primer. Following cDNA synthesis, primers pair RDR6_F and RDR_R were used to PCR amplify the cDNA strand using Taq 2X Master Mix (NEB). PCR products were run on 1.0% agarose gels to confirm absence of RDR6 representing band.Total RNA extracted from 61. The data produced during qPCR was processed using StepOne Plus software (ThermoFisher Scientific). Data was presented as relative quantification (RQ) using phosphatase 2 A gene, PP2A, as endogenous reference62. RT-qPCR conditions were 30 min incubation at 48 °C, followed by 10 min at 95 °C, then 40 cycles of 1 min at 60 °C plus 15 s at 95 °C.The qPCR was done using TaqMan qPCR Master Mix (ThermoFisher Scientific) on a OneStep Plus Real-Time PCR system (ThermoFisher Scientific). The primer pair PVY_ F and PVY_R and the probe PVY_P are listed in Table 63; n = 1193; Amalgaviridae: n = 6; Chrysoviridae: n = 8; Partitiviridae: n = 46; Reoviridae: n = 111; Caulimnoviridae: n = 178, Other Ortervirales : n = 51; Bunya-like viruses : n = 44; Rhabdoviridae: n = 108; Secoviridae: n = 125; Betaflexiviridae: n = 268; Potyviridae: n = 202; Alphaflexiviridae: n = 182; Other tymovirales : n = 11; Tymoviridae: n = 85; Sobemovirus: n = 18; Benyviridae: n = 22; Luteoviridae: n = 157; Virgaviridae: n = 247; Bromoviridae: N = 152; Chlosteroviridae: n = 337). Sequences were split into annotated genes to enable analysis of coding regions only. Families and other taxonomic groups representing Baltimore Groups 3–5 and 7 were analysed.Plant virus sequences datasets was constructed from representative sequences of each plant virus species were obtained from the ICTV Virus Metadata ResourceArabidopsis thaliana (chromosomes 1–5), Zea mays (chromosome 9) and Nicotiana attenuata (chromosome 1) were retrieved from Refseq and divided into 5000 base fragments for dinucleotide composition analysis. Coding region sequences from mRNA sequences of the three plant species and N. tomentosiformis were similarly downloaded from the Refseq database and subsequently filtered to remove redundant sequences by selecting the longest splice variant of each mRNA. From these, sequences longer than 450 bases were analysed for dinucleotide composition.DNA genome sequences of 64. Analyses were made using all (near) complete genome sequences of PVY available on Genbank in November, 2014. Sequences <1% divergent from others were discarded. Accession numbers for the analysed sequences are provided in Table Mono- and dinucleotide composition measurements were performed in SSE version 1.3 . Observed/expected frequencies of CpG and TpA/UpA dinucleotides were calculated by normalisation for G and C mononucleotide content. Normalisation of CHG and CHH frequencies was performed by correction for frequencies of their two component dinucleotides instead of the three mononucleotides as the former may themselves by substantially over- or underrepresented. Normalised frequencies of CHG and CHH were calculated as:Dataset 1


The winemaking industry produces large amount of byproducts, including grape pomace, stalks, and lees. Wine lees are a natural source of phenolic compounds, which have important antioxidant and biological properties. Due to the high quantities produced worldwide, this byproduct can be an ideal raw material for obtaining phenolic compounds that could be of interest in the food and pharmaceutical industries. In this mini review, the main characteristics of wine lees as well as their phenolic composition and antioxidant activity have been summarized from the information in the literature. Wine production is an important activity throughout the world. According to the International Organization of Vine and Wine (OIV) , 246.7 mThe winemaking industry produces large amount of byproducts, including grape pomace, stalks and lees. These winemaking byproducts are considered as an important source of bioactive compounds with antioxidant activity, such as phenolic compounds . PhenoliGrape pomace and stalks have been widely studied in relation to its composition and bioactivity ,4,5,6,7.According to literature and EEC regulation No. 337/79, wine lees can be defined as “a residue that is formed at the bottom of wine containers, after fermentation, during storage or after treatments, as well as the residue obtained after the filtration or centrifugation of this product” . On the The main characteristics of wine lees are acidic pH (between 3 and 6), a chemical oxygen demand above 30,000 mg/L, potassium levels around 2500 mg/L, and phenolic compounds in amounts up to 1000 mg/L .This winemaking byproduct is composed of solid and liquid fractions . The solThe composition of wine lees depends on environmental conditions, regions of origin and their agronomic characteristics, the grape variety, and the time of aging in the wood barrels ,16.Wine lees are used for wine ageing, usually for white and sparkling wines, although sometimes they are applied in red wines . Wine agThe first step for the chemical characterization of the phenolic compounds still present in wine lees is the extraction of these compounds. Extraction is a very important step in the recovery of phenolic compounds, and therefore the extraction parameters must be controlled to obtain extracts rich in phenolics. There is no single extraction method; however the most common technique for extraction of phenolic compounds from wine lees is solid–liquid extraction. The main extraction parameters considered are sample pre-treatment , solvent, solvent–solid ratio, extraction mode , temperature, and time.Some papers on the phenolic extraction from wine lees found in literature have been summarized in Regarding pre-treatment, lees are dried at 40 °C or 50 °C in an oven ,23,24,25Regarding the solvent-to-solid ratio parameter, in a study on determination of appropriate ranges of extraction parameters for phenolic compounds from wine lees, the range from 30:1 to 50:1 was selected as the optimal range to obtain the best extraction yield .Bosiljkov et al. (2017) established a highly efficient and eco-friendly extraction method for the anthocyanins in wine lees using natural deep eutectic solvents (NADES). NADES, that are mixtures of choline chloride with a hydrogen donor, coupled with high-efficiency ultrasound-assisted extraction, were an excellent choice for extraction of phenolic compounds from wine lees .Tao et al. (2014)  comparedAlso, microwave-assisted extraction has been used for phenolic extraction from wine lees ,25. PereThe time and temperature for phenolic extraction from wine lees is highly variable. The time can vary from 2 min to 3 h; and the temperature from room temperature to 40 °C .Phenolic compounds are transferred from grape to wine during the maceration, but a high proportion of these compounds remain in the winemaking byproducts such as lees. The wine lees contain phenolic compounds due to the adsorption capacity of yeast cell wall . The phev/v) ethanol:water showing the highest efficiency [v/v) ethanol:water from wine lees and they reported a total phenolic content of 547 mg GAE/g DM. These differences between results may be due to differences in the types of wine lees (grape variety and vinification process) and mainly the extraction process (solvent and extraction method).The total phenolic content of wine lees has been widely evaluated by the Folin–Ciocalteu assay and values were very different between studies. Tao et al. (2014)  indicateficiency . On the ficiency  performeThe contents of total non-flavonoids and total flavonoids have been reported: 1332 and 984 mg GAE/100 g DM, respectively. The content of total anthocyanins was also reported: 383 mg of cyanidin-3-glucoside equivalents/100 g of DM .O-glucoside, quercetin 3-O-glucuronide, quercetin 3-O-galactoside and quercetin 3-O-rutinoside [Vitis labrusca varieties, laricitrin, isorhamnetin, and syringetin, and their glucosides, as well as myricetin derivatives (myricetin 3-O-glucuronide and myricetin 3-O-glucoside) were found [Individual phenolic compounds belonging to flavonoids , phenolic acids, and stilbenes have been identified and quantified in wine lees. tinoside ,25,32. IIn a paper on characterization of wine lees by liquid chromatography and mass spectrometry, flavanols, namely catechin, epicatechin, and procyanidin B2, were tentatively identified, but these compounds were not quantified . In otheVitis vinifera varieties, delphinidin-3-O-glucoside, petunidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside, malvidin-3-O-galactoside, delphinidin-3-O-(6″-p-acetylglucoside), cyanidin-3-O-(6″-p-acetylglucoside), malvidin-3-O-(6″-p-acetylglucoside), delphinidin-3-O-(6″-p-coumaroyl-glucoside), petunidin-3-O-(6″-p-coumaroyl-glucoside), malvidin-3-O-(6″-p-coumaroyl-glucoside) and pelargonidin-3-(6″-p-coumaryl-glucoside) were found [O-glucoside and malvidin-3-(6″-p-coumarylglucoside) were in higher concentrations in wine lees [Anthocyanins have been identified in wine lees. Up to total of 26 anthocyanins were determined in samples of wine lees: derivates of the anthocyanidins delphinidin, cyanidin, petunidin, peonidin and malvidin. In re found ,25,33. Aine lees .non-vinifera Brazilian grape varieties was dominated by diglucoside derivatives in two main forms: 3,5-diglucosides and their p-coumaroylated derivatives (3-(6″-coumaroyl)-glucoside-5-glucosides) of the anthocyanidins previously mentioned [On the other hand, the anthocyanin profile of such by-products from two entioned .p-coumaric acid and their derivates such as trans-caftaric acid and trans-coutaric acid, respectively, have been found in wine lees samples, being caffeic acid and trans-caftaric acid the predominant compounds in both Vitis vinifera and non-vinifera varieties [Regarding phenolic acids, caffeic acid and arieties ,28. Alsoarieties ,28. All cis- and trans-resveratrol, is a stilbene that has been extensively identified in skin of grapes, however its identification in wine lees is less common. This compound has been reported by some authors but at lower concentrations than other phenolics [Resveratrol, henolics ,32.Several pathologies, health conditions and degenerative processes such as atherosclerosis, diabetes, arterial hypertension, cancer, and aging are related to the oxidative stress. Oxidative stress generates an important cell damage because to a high production of reactive oxygen species (ROS), which attack macromolecules such as proteins, DNA, and lipids. Phenolic compounds in wine lees show different antioxidant activities for different assays and therefore lees are a good source of antioxidant compounds that can delay the oxidation of macromolecules .−) based on a hypoxanthine (HPX)/xanthine oxidase (XOD) system. Also, antioxidant activity of wine lees has been determined by cupric reducing antioxidant capacity (FRAP) assay that measures the capacity of antioxidants to reduce the Fe (III) complex of 2,4,6-tripyridyl-s-trizin (TPTZ) to Fe (II)- TPTZ chelate [The antioxidant activity of wine lees has been determined using different methodologies such as 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and oxygen radical absorbance capacity (ORAC) assays that measure the ability of antioxidants to scavenge a radical ,34,35. T chelate .2O (v/v) mixture) have higher antioxidant capacities than the rest of the extracts .Usually, antioxidant activity from winemaking byproducts is directly related to the total concentration of phenolic compounds, being the highest total phenolic content values, which correspond with the highest antioxidant activity values . RegardiO-glucuronide with ORAC and HORAC, and for myricetin with HOSC and FRAP. Regarding anthocyanins, for ORAC and HORAC the correlation was negative and for HOSC and FRAP was positive [Compounds contributing to antioxidant activity have been studied, and it has been established which compounds or group contribute to each assay: flavanols showed negative correlations with ORAC and positive with FRAP assays; specifically, catechin showed statistically significant positive correlation with FRAP and with hydroxyl radical scavenging capacity (HOSC) assays. Flavonols showed different correlations with ORAC, FRAP, hydroxyl radical averting capacity (HORAC), and HOSC. The correlations were significant and positive for quercetin-3-positive .Landeka et al. (2017) determined the antioxidant capacity of wine lees by the DPPH and FRAP methods and results were 259 and 45 mM TE/100 g DL for DPPH and FRAP, respectively. In addition, in this study, wine lees had hypolipidemic and antioxidant properties in animal model .However, wine lees show scavenging activity lower than that found in grape pomace , which cAlthough phenolic compounds are the main compounds contributing to the antioxidant activity, recently another high-value chemical compound has been recovered from wine lees: squalene, which is a natural antioxidant synthesized during sterol biosynthesis in plants .On the other hand, the antioxidant properties of wine lees are also important because they can be used for the elaboration of sparkling wines. The ageing on lees of wines has a close relationship with their phenolic profile .Wine lees are winemaking byproducts with high content of antioxidant compounds, consisting of mainly phenolic compounds. Flavonols and anthocyanins (in wine lees from red grapes) are the most abundant phenolic compounds in wine lees. Further studies are required to determinate other components present in wine lees which could contribute to these antioxidant effects.The exploitation for the potential reuse of wine lees in the wine industry or other industries could be of great interest due to their chemical composition and antioxidant properties.


RNA-based immunization strategies have emerged as promising alternatives to conventional vaccine approaches. A substantial body of published work demonstrates that RNA vaccines can elicit potent, protective immune responses against various pathogens. Consonant with its huge impact on public health, influenza virus is one of the best studied targets of RNA vaccine research. Currently licensed influenza vaccines show variable levels of protection against seasonal influenza virus strains but are inadequate against drifted and pandemic viruses. In recent years, several types of RNA vaccines demonstrated efficacy against influenza virus infections in preclinical models. Additionally, comparative studies demonstrated the superiority of some RNA vaccines over the currently used inactivated influenza virus vaccines in animal models. Based on these promising preclinical results, clinical trials have been initiated and should provide valuable information about the translatability of the impressive preclinical data to humans. This review briefly describes RNA-based vaccination strategies, summarizes published preclinical and clinical data, highlights the roadblocks that need to be overcome for clinical applications, discusses the landscape of industrial development, and shares the authors’ personal perspectives about the future of RNA-based influenza virus vaccines. Influenza virus causes millions of illnesses and up to 650,000 deaths worldwide every year . ApproveVaccination with in vitro transcribed (IVT) messenger RNA (mRNA) was not widely considered as a viable approach until the early 2000s; however, several early studies using various types of mRNA vaccines demonstrated promising preclinical results ,8,9. SemEarly studies using naked (uncomplexed) unmodified RNA for immunization via the intramuscular route demonstrated some efficacy against various infectious pathogens  but subsVarious formats of RNA vaccines against influenza virus have demonstrated potent immunogenicity in preclinical models . MoreoveInfluenza virus infection is a major public health problem; thus, tremendous effort has been invested in improving current vaccines and creating new, more effective vaccines over the past decades. The need for a broadly protective/universal influenza virus vaccine, combined with the enormous infrastructure and research experience behind influenza virus research, and the relative ease of testing influenza virus vaccine efficacy in small animal models, have made it an optimal system for RNA vaccine development. Thus, influenza virus RNA vaccines (using IVT RNA) are among the most extensively studied infectious disease RNA vaccines, with studies dating back over 25 years . Two majThe majority of current SAM vaccines use a modified alphavirus genome that encodes the genes for the RNA replication machinery and the antigen of interest in the place of the viral structural protein-encoding genes ,36. The In summary, several proof-of-concept studies have been published about the efficacy of SAM influenza virus vaccines in preclinical models , but no A substantial amount of research has been invested into the development of directly injectable non-replicating mRNA vaccines. The first study that demonstrated efficacy after vaccination with mRNA was published in 1993. This seminal work showed that administration of influenza virus NP-encoding mRNA complexed in liposomes induced cytotoxic T cell responses in mice . InteresRecently published, highly efficacious influenza virus mRNA vaccines use unmodified or nucleoside-modified full-length HA-encoding mRNAs encapsulated in a LNP carrier. The LNP component of the vaccine has previously been shown to be efficacious for siRNA delivery . CureVacNucleoside-modified mRNA-LNP vaccines represent a new and highly efficacious category of RNA vaccines that can elicit protective immune responses against various pathogens ,46,47,48Coxiella burnetii [RNA vaccines have recently received significant attention and attracted massive academic and industrial investment. A limited number of medium-size biotechnology companies have successfully raised capital to develop innovative RNA vaccines . Both CureVac and Moderna have invested in internal production capabilities. Expected in 2018, CureVac’s “GMP III” facility will have the capacity to produce 10 million doses of GMP mRNA per year, while the commercial scale “GMP IV” facility is currently under construction and will be able to produce up to 30 million mRNA doses per year (www.curevac.com). The company envisions a “GMP V” facility that will be able to increase the capacity to 400 million doses per year . Other companies have also announced investments in developing RNA-based influenza virus vaccines; however, little is currently known about their research strategies  conducted by Moderna Therapeutics . Early results from this Phase I trial using nucleoside-modified mRNA-LNPs are discussed in rategies .Companies focused on developing RNA-based influenza vaccines aim to create a product that is superior to the currently available split (inactivated) or LAIV vaccines. Requirements for innovative, broadly protective influenza virus vaccines have been extensively discussed in the influenza vaccine scientific community  and the Currently used seasonal influenza vaccines show some level of protection against matched viruses but they offer little protection against drifted seasonal or pandemic viruses. A critical requirement for a new class of vaccine is to greatly improve efficacy and breadth of protection. Prophylactic RNA vaccines demonstrated heterologous protection in preclinical models , but tra5–7 PFU or TCID50) and it is in a liquid suspension or large droplet spray that induce only an upper respiratory tract infection after administration into the nares [Large-scale efficacy trials are expensive, and thus, an early indication of the level of protection and cross-protection against drifted strains could be provided by Controlled Human Influenza Virus Infection Models (CHIVIM). The utility of such a challenge system for predicting vaccine effectiveness depends on the nature of the challenge virus, its infectious dose, and route of administration. Currently available challenge viruses . Non-HA based vaccines or LAIV do not have a known correlate and need to rely on large-scale field efficacy trials. The most potent RNA vaccines induce very high HAI titers in small and large animals and HAI titers in the protective range in humans  has been shown to correlate well with protection in preclinical ,72 and cAs discussed in several recent publications ,57, an oCollectively, RNA-based vaccines represent a new vaccine class that can be used to effectively combat influenza virus. As noted, more data from clinical trials, including improved controlled human influenza virus infection models, and large-scale field efficacy trials will be critical to demonstrate the viability of this vaccine technology.


The literature remains seldom on the topic of self-rated health (SRH) among the national working populations of emerging countries. The objectives of the study were to examine the associations of occupational factors with SRH in a national representative sample of the working population in Brazil.This study relied on a cross-sectional sample of 36,442 workers, 16,992 women and 19,450 men. SRH was the studied health outcome. Sixteen occupational factors related to four topics were studied: employment characteristics, working time/hours, psychosocial work factors and physical and chemical work exposures. The associations between occupational factors and SRH were studied using logistic regression models with adjustment for sociodemographic characteristics . The analyses were performed for each gender separately and using weights.The prevalence of poor SRH was 26.71%, this prevalence being higher among women (29.77%) than among men (24.23%). The following risk factors for poor SRH were found among men and women: working as a self-employed worker, clerk/service worker, manual worker, part-time (≤ 20 h/week), exposure to work stress, exposure to high physical activity and exposure to sun. The risk factors for poor SRH among women only were: working as a domestic worker and exposure to noise, and among men, working in the agriculture sector.Our study suggested that occupational factors related to both physical and psychosocial work environment may be associated with SRH in the working population in Brazil. Improving working conditions may be beneficial for health at work in Brazil. Self-rated health (SRH) is a measure of the general health condition as self-perceived by individuals, and can be considered as a general indicator of morbidity and a marker of future morbidity and mortality in the general population , 2. SRH SRH has a multifactorial etiology and a large number of factors of different nature may play a role. As work and occupational exposures are important determinants of health, it may be crucial to identify occupational risk factors for poor SRH. There have been numerous studies exploring occupational factors in association with SRH in various working populations. Among the studied occupational factors, psychosocial work stressors occupy an important place, as almost all studies explored one or more stressors in this topic. For example, studies found the following psychosocial work factors to be associated with SRH: low control or latitude –10, highAlthough the studies have been numerous on the associations between occupational factors and SRH, gaps remain mainly because most of the studies did not explore the occupational factors related to working time/hours and the physical work environment. Regarding the studied populations, most of the studies focused on specific working populations for example on specific occupations, sectors or areas, making the generalisation of the results difficult to national working populations. Furthermore, only half of the studies were able to examine men and women separately, leading to a lack of information about gender differences in this topic. Finally, the majority of the studies came from the more economically developed countries, in particular Europe, and information may be missing for the rest of the world, especially for Latin America.In order to fill these gaps, our study aimed at exploring the associations between a wide range of occupational factors and SRH in a large national representative sample of the Brazilian working population.This study was based on the cross-sectional data of the Brazilian National Health Survey, called in Portuguese, Pesquisa Nacional de Saúde (PNS), set up in 2013 by the Brazilian Institute of Geography and Statistics (IBGE) and the health ministry . As alreSRH was used as a general health status measure and based on the following item: “In general, how would you rate your health?” with response categories rated on a five-point Likert scale: “very good”, “good”, “neither good nor poor”, “poor” and “very poor”. This measure is well-known as a general perceived health tool . SRH wasEmployment characteristics: work status, occupation and economic activities that were coded using standard classifications (ISCO and ISIC respectively), and multiple job-holder (i.e. worker having more than one job).Working time/hours: night/shift work  and working hours a week (collected as a continuous variable and studied in 3 categories).Psychosocial work factors: workplace violence (2 items on violence at the workplace from known or unknown people) and work stress (1 item on stressful work activities).Physical/chemical exposures (1 item each): high physical activity, chemical agents, radioactive agents, urban waste , biological agents, marble dust, noise, and sun.These factors were already constructed and used in our previous publication . A totalSociodemographic characteristics: age, ethnicity , and marital status.Health behaviours: physical activity (1 item on activity within the past 3 months), smoking (1 item on current status) and binge drinking (5 doses or more for men and 4 doses or more for women on one occasion within the past 30 days).Health-related variables: private health insurance plan (1 item) and disability .Educational level Four groups of adjustment variables included:Firstly, a description of the sample was performed for all studied variables. Differences between men and women were tested using Rao-Scott chi-square test.Unadjusted bivariate models between each occupational factor and SRH,Multivariate models with all the occupational factors together (model 1),Multivariate models with all the occupational factors together plus sociodemographic characteristics (model 2).Secondly, the associations of occupational factors with SRH were studied with weighted logistic regression models, using three types of models:With additional adjustment for health behaviours, private health insurance plan and disability,With additional adjustment for education,With the SRH outcome dichotomized into poor (‘poor/very poor’) versus good (‘fair/good/very good’).The following sensitivity analyses explored the robustness of the results:We performed all the statistical analyses for each gender separately, using weights that took the sampling characteristics, non-response and calibration into account, and using SAS 9.4 software.The prevalence of poor SRH was 26.7% (95% CI: 25.9–27.5%) among the total study sample, with a higher prevalence among women than among men: 29.8% versus 24.2% Table .Table 1Bivariate associations are presented in Table Multivariate associations for women are presented in Table Multivariate associations for men are presented in Table Sensitivity analyses showed no change in the results after additional adjustment for health behaviours, private health insurance plan and disability, except for the exposure to noise, that was no longer significant among women. The results were also unchanged after additional adjustment for education, except noise that was not significant anymore among women. The sensitivity analysis using SRH into poor (‘poor/very poor’) versus good (‘fair/good/very good’) showed that some factors were no longer significant, which was expected given the reduced statistical power  among men and 3.7% (95% CI: 3.3–4.2%) among women).Strong differences were observed between genders. Women had a higher prevalence of poor SRH than men and gender-related differences were also observed for almost all studied variables, occupational factors and covariates. Several occupational factors were associated with poor SRH among men and women: being self-employed workers, clerks/service workers, manual workers, working part time (≤20 h/week), exposure to work stress, high physical activity and sun. Gender-specific associations were also observed, for women, between working as domestic workers and exposure to noise and poor SRH, and for men, between working in the agriculture sector and poor SRH.The prevalence of poor SRH was 26.7% (95% CI: 25.9–27.5%) in our study sample; 29.8% for women and 24.2% for men. Previous studies among working populations also observed gender-related differences in the prevalence of poor SRH, women having a higher prevalence than men , 19, 22.Working as a clerk/service worker or manual worker was associated with poor SRH, in line with previous studies, including studies exploring social or occupational differences in SRH, that showed that low-skilled occupational or social groups were more likely to have poor SRH , 29–31. Part-time work was associated with poor SRH in our study, in agreement with the findings from a North American study . This reIn the present study, the association between work stress and poor SRH was observed for both genders. One item (stressful work activities) was used to measure work stress. Job strain, from the job strain model, is related to the combination of high job demands and low decision latitude, and is a well-known measure of work stress. Previous studies showed significant associations of job strain or its components (high demands and low latitude) with SRH –13; our In the present study, a number of occupational physico-chemical exposures, high physical activity and exposure to sun for both genders and exposure to noise among women, were associated with poor SRH. Exposure to high physical activity was associated with poor SRH in our study. Previous studies showed similar results using various measures related to physical demands or ergonomic exposures , 14, 19.The study included the following strengths. It relied on a very large representative national sample of the working population in Brazil, providing reliable findings on occupational exposures and SRH in this country. Our study is also one of the first studies exploring these associations in working populations of Latin America. The response rate was high (92%) and as the survey was national and weights were available and used, a generalisation of the results may be possible to the target population, i.e. the national working population in Brazil. The statistical analyses were done for each gender separately, following the best practices . DiffereOur study also included a number of limitations. The study was cross-sectional, consequently no conclusion about causality can be made, and reverse causation may be possible. A healthy worker effect is also conceivable, that may lead to select healthy people at the workplace and/or at the most exposed jobs, and may lead to an underestimation of the observed associations. The occupational exposures were measured using few items and without validated scales or instruments, something that may lead to potential imprecision and a bias towards the null hypothesis. For example, for the measurement of night/shift work, no precision was given regarding the definition of exposure in the questionnaire . Another example is the measurement of work stress and workplace violence that were not based on validated questionnaires. As the selection of the studied occupational factors relied on the availability of the items in the survey questionnaire, some factors such as job insecurity, temporary employment or work-life conflict may be missing –21, 35. We found significant associations between various occupational factors and poor SRH, especially factors related to work status, occupation, economic activity, work stress and some physico-chemical exposures. More studies may be needed on these associations, especially among the Brazilian working population. Our study suggests that preventive measures oriented towards the reduction of occupational exposures might be beneficial for SRH among working populations. Finally, our study is an attempt to contribute to the literature by addressing these issues among the working populations of Latin America.


To develop a nomogram and validate its use for the intraoperative evaluation of nodal metastasis using shear-wave elastography (SWE) elasticity values and nodal sizeWe constructed a nomogram to predict metastasis using ex vivo SWE values and ultrasound features of 228 axillary LNs from fifty-five patients. We validated its use in an independent cohort comprising 80 patients. In the validation cohort, a total of 217 sentinel LNs were included.p = 0.005) and elasticity ratio  were significantly higher in the metastatic LNs than those in the non-metastatic LNs. However, the mean size of the metastatic LNs was not significantly larger than that of the non-metastatic LNs . The AUC was 0.791  in the validation cohort, and the calibration plots of the nomogram showed good agreement.We developed the nomogram using the nodal size and elasticity values of the development cohort to predict LN metastasis; the area under the curve (AUC) was 0.856 , 0.783–0.929). In the validation cohort, 15 (7%) LNs were metastatic, and 202 (93%) were non-metastatic. The mean stiffness (23.54 and 10.41 kPa, We developed a well-validated nomogram to predict LN metastasis. This nomogram, mainly based on ex vivo SWE values, can help evaluate nodal metastasis during surgery.• A nomogram was developed based on axillary LN size and ex vivo SWE values such as mean stiffness and elasticity ratio to easily predict axillary LN metastasis during breast cancer surgery.• The constructed nomogram presented high predictive performance of sentinel LN metastasis with an independent cohort.• This nomogram can reduce unnecessary intraoperative frozen section which increases the surgical time and costs in breast cancer patients. Axillary lymph node (LN) metastasis is one of the important prognostic factors in breast cancer , 2. SentShear-wave elastography (SWE) can quantitatively calculate the elasticity parameters of target lesions and provide values, such as mean, minimum, and maximum stiffness; standard deviation of elasticity; and elasticity ratio of breast lesions to the adjacent fat tissue. It is helpful in distinguishing breast cancerous lesions because malignant tissues tend to have increased stiffness compared with benign tissues –12. AlsoBased on these results, we hypothesized that the nomogram constructed using ultrasound features and elasticity values measured via intraoperative ex vivo SWE can accurately predict nodal metastasis. Furthermore, the disadvantages of intraoperative frozen section analysis may be compensated if the nomogram precisely predicts sentinel LN metastasis, since ex vivo SWE can be performed easily, and no additional equipment is required, except for ultrasound. For this reason, we developed and validated a nomogram to predict the sentinel LN metastasis using ultrasound features and ex vivo SWE values in this study.In our previous prospective study, a total of 228 axillary LNs, including sentinel LNs obtained from fifty-five patients who underwent breast cancer surgery in Gangnam Severance Hospital from May 2014 to April 2015, were investigated [Our study was approved by the Institutional Review Board of Gangnam Severance Hospital and conducted in accordance with the Good Clinical Practice guidelines and the Declaration of Helsinki principles.SLNB was performed using 99mTc-labeled tin colloid. Intradermal injection of 0.4 mL 30 MBq (0.8 mCi) 99mTc tin colloid diluted in normal saline solution was performed at four peri-areolar sites. Sentinel LNs were determined by employing a gamma detector . The LNs showing a high radioactivity were dissected, after which the gamma detector was used again to confirm the correct sentinel LNs. All radioactive LNs with an account equal to or greater than 10% of the highest radioactive LN were removed. When suspicious LNs not detected by the gamma probe were found after SLNB, they were removed and examined as non-sentinel LNs. If the result of frozen pathology was positive for malignancy, ALND was performed.TM elastography mode [TM; SuperSonic Imagine) on the stiffest region of the excised LNs. The elasticity ratio was calculated automatically when the first ROI was placed on the stiffest region of excised LNs, and the other ROI was immediately placed on the adjacent fat tissue .The characteristics of the patients were analyzed using the chi-square test and independent two-sample A =  − 4.9898 + 0.1498 ×  + 0.0345 × (mean stiffness) + 0.5321 × (ratio)We previously reported the characteristics and elasticity values of the harvested axillary LNs measured using B-mode ultrasound and intraoperative ex vivo SWE in the development cohort . BrieflyIn the nomogram, the estimated probability of LN metastasis could be obtained by summing the scores of each variable and locating such on the total score scale. For instance, a patient with a 16-mm nodal size (11 points), 70-kPa mean stiffness (13 points), and 5 ratio (14 points) would score 38 points, which can be converted into 92.29% probability of LN metastasis Fig. .p value obtained using the Hosmer-Lemeshow goodness-of-fit test was 0.354, indicating a good fit of the model. When internal validation was performed using the 200 bootstrap samples, the mean absolute error was as low as 0.029. Moreover, the calibration plots of the nomogram showed good agreement between the observed and predicted outcomes  was 0.856 , 0.783–0.929) in the development cohort Fig. . The p vmes Fig. .Fig. 3ROp = 0.045). The other clinicopathologic factors were no significantly different between the patients with and without sentinel LN metastasis  (Table p = 0.005) . Meanwhile, there was no difference in nodal size and elasticity values according to histological types of breast cancer  were metastatic, and 202 93.1%) were non-metastatic, including seven (3.2%) micro-metastatic LNs. The metastatic LNs tended to be larger than non-metastatic LNs, but it was not significant  Fig. . Furtherer Table .Table 3UThe nomogram was validated with the independent set including only 217 sentinel LNs. The discriminative power was good, with an AUC of 0.791  Fig. . The calIn this study, we generated a nomogram to predict metastatic LNs based on nodal size and elasticity values such as mean stiffness and elasticity ratio of harvested LNs on B-mode ultrasound with ex vivo SWE using data from our previous study. Thereafter, we tested the predictive ability of the nomogram in an independent set consisting of sentinel LNs. The performance of constructed nomogram to predict sentinel LNs metastasis was high in terms of good discrimination and calibration.Cancer researchers, clinicians, and the public are becoming increasingly interested in statistical models designed to predict the occurrence or the outcome of cancer, along with the efficacy of treatments –27. AmonThe discriminative power of the nomogram was quantified using the AUC exhibiting the accuracy of the test, with an AUC of 0.5 being defined as non-informative; 0.5–0.7, fair; 0.7–0.9, good; and > 0.9, excellent. Our nomogram revealed good discriminative power with the AUC of 0.856 in the development cohort and 0.791 in the validation cohort. In addition, the calibration plot showed good agreement between the observed and predicted probabilities in both the development and validation cohorts. Therefore, our constructed nomogram was suitable in predicting the probability of metastasis among the harvested sentinel LNs.In the validation cohort, the ultrasound and SWE characteristics of sentinel LNs were not different between histologic types of breast cancer in line with previous report . The meaRecently, the American College of Surgeons Oncology Group Z0011 prospective, randomized clinical trial changed the standard approach to axillary surgery, showing that the omission of ALND was possible in early breast cancer patients who underwent breast conserving surgery and adjuvant systemic therapy even with 1–2 metastatic sentinel LNs , 30. SevHowever, one of the major disadvantages of omitting intraoperative frozen section analysis is secondary surgery for ALND. To date, the exact rate of secondary surgery for ALND after skipping intraoperative frozen section analysis has not been well researched. Nevertheless, concerns of secondary operation for ALND can be reduced if intraoperative pathologic examination was performed for sentinel LNs in suspicion of metastasis, as identified by our nomogram.Our study has some limitations. First, the cutoff value of nomogram to determine the intraoperative pathologic examination was not clarified in this analysis. Further prospective studies are needed to verify this issue. Second, the rate of metastatic LNs in the validation cohort was lower than that in the development cohort. It was probably because only sentinel LNs were included in the validation set. The number of metastatic LNs was 15 in the validation cohort of our study; nevertheless, these events were statistically sufficient to perform calibration and discrimination to warrant the use of our nomogram. Finally, concerns about the reproducibility of SWE still remained because interobserver or intraobserver agreements for the elasticity values were not assessed in this study. However, the other previous study showed reliable intraobserver and interobserver reproducibilities of SWE , 36. In In conclusion, our well-validated nomogram can be applied to predict nodal metastasis during SLNB. The clinical application of nomogram, which is mainly based on ex vivo SWE values, may help in reducing unnecessary intraoperative frozen section analysis as well as secondary operation rate in breast cancer patients who underwent SLNB.


Intraoperative consultations, used to guide tumor resection, can present histopathological findings that are challenging to interpret due to artefacts from tissue cryosectioning and conventional staining. Stimulated Raman histology (SRH), a label-free imaging technique for unprocessed biospecimens, has demonstrated promise in a limited subset of tumors. Here, we target unexplored skull base tumors using a fast simultaneous two-channel stimulated Raman scattering (SRS) imaging technique and a new pseudo-hematoxylin and eosin (H&E) recoloring methodology. To quantitatively evaluate the efficacy of our approach, we use modularized assessment of diagnostic accuracy beyond cancer/non-cancer determination and neuropathologist confidence for SRH images contrasted to H&E-stained frozen and formalin-fixed paraffin-embedded (FFPE) tissue sections. Our results reveal that SRH is effective for establishing a diagnosis using fresh tissue in most cases with 87% accuracy relative to H&E-stained FFPE sections. Further analysis of discrepant case interpretation suggests that pseudo-H&E recoloring underutilizes the rich chemical information offered by SRS imaging, and an improved diagnosis can be achieved if full SRS information is used. In summary, our findings show that pseudo-H&E recolored SRS images in combination with lipid and protein chemical information can maximize the use of SRS during intraoperative pathologic consultation with implications for tissue preservation and augmented diagnostic utility. The presence of these artefacts in microscopic sections can result in misinterpretations and subsequent diagnostic pitfalls2.The overarching purpose of intraoperative consultations for tumor surgeries is to obtain crucial information that informs surgical treatment. The tools currently employed include microscopic assessment of a submitted specimen through cytological preparations or cryosectioning and subsequent staining of the tissue by hematoxylin and eosin (H&E). The challenges of generating a diagnostically adequate H&E slide of cryosectioned tissue are organ-specific. In central nervous system specimens, the main challenge often results from limited sample size and general texture heterogeneity of the submitted tissue. In such cases, texture heterogeneity manifests itself as a freeze artefact - the diffuse or focal splitting of sections or cellular rarefaction resulting in loss of diagnostic information3. Skull base tumors face unique challenges due to their low occurrence, presence in deep locations, proximity to critical neurovascular structures, and extension beyond anatomic boundaries4. Accurate intraoperative tissue diagnosis is essential during skull base tumor surgery to maximize tumor removal, as a non-resected tumor can lead to recurrence, treatment failure, and overall poor outcome5. In particular, a rapid diagnosis during the removal of skull base tumors can help the surgeon to choose how aggressive the resection should be. In addition, in some patients, a rapid diagnosis can help to delineate the margins of tumor resection. Additionally, there is an increasing need to preserve tissue from small pathology specimens for downstream molecular ancillary testing, and an intraoperative technique aimed at preserving tissue would be preferable in these cases.In addition to the inherent challenges of cryosectioning, the location of the tumor can add complexity to management. One group of tumors that poses many challenges is skull base tumors which include meningiomas, pituitary adenomas, schwannomas, hemangiopericytomas, chordomas, various types of sarcomas, carcinomas, and metastases among other entities7, confocal microscopy8, two-photon fluorescence combined with second harmonic generation11, Raman spectroscopy12, and coherent Raman scattering (CRS) microscopy13. Among the previous work, CRS stands out amongst other imaging techniques as it provides both morphological and chemical information at a submicron resolution without any staining or chemical labels14. Two CRS methods have been established: coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS). Both CARS and SRS use two pulse lasers (pump and Stokes) to excite intrinsic vibrational motions of molecules coherently and have been shown to be able to provide molecular contrasts that can aid cancer diagnosis13.Many advanced optical imaging techniques have been developed to detect neoplastic cells and to provide diagnostic information with varying degrees of success, including optical coherence tomography16. Specifically, it has been shown that a two-color SRS imaging approach targeting the C–H transition of lipid and protein  allow direct visualization of cell nuclei. The resulting images can be recolored using linear dependence of the signal on lipid and protein content, allowing for a simple and very close simulation of H&E, i.e., stimulated Raman histology (SRH). Orringer et al. and Hollon et al. have tested the efficacy of SRS microscopy as a potential intraoperative H&E staining alternative for cases including glial and metastatic neoplasms with 92% accuracy of tumor subtype determination as assessed by neuropathologists16. Another recent study using the same SRH approach further validated the visual comparison of SRH images to H&E as applied to gastrointestinal tract17. Other studies used two-color SRS images for cancer/non-cancer identification in laryngeal squamous cell carcinoma18 and glioblastoma infiltration in the brain19. However, these studies did not provide pathologist evaluation of SRH or SRS two-color images for cancer subtype determination and thus are insufficient to validate SRS as an alternative to histology. Similar to SRS, CARS microscopy has also been explored for cancer diagnosis22 based on chemical contrasts. Compared to SRH, CARS is further complicated by the presence of a non-resonant background26, which prevents direct conversion of CARS images to H&E images. Adding additional chemical contrasts from second harmonic generation and two-photon fluorescence, it has been shown that multicolor images can be generated and provides useful diagnostic information27. However, whether pathologists can use these images to provide meaningful cancer diagnosis requires further investigation.In particular, it was recently demonstrated that SRS microscopy could provide H&E equivalent information for pathologists to determine cancer subtypes, thereby offering the potential for replacing frozen sectioning as an intraoperative diagnostic tool16, we emphasize assessment of how SRH performs at every step of the typical diagnostic workflow. Moreover, unlike previous studies, instead of training pathologists with SRH images, diagnosis in this study is performed without prior training and thus provides an unbiased assessment of the diagnostic accuracy achievable in an intraoperative setting.While a few studies have validated SRH in limited settings, it remains unclear whether SRH is generally applicable to a diverse set of tumor types encountered in daily pathology practice. Employing two Raman transitions targeting lipids and proteins to generate an H&E alternative works well for entities with higher cytoplasmic lipid content as can be expected in glial neoplasms. This is because the cytoplasmic lipid signals are a primary source of contrast to visualize cell nuclei. However, skull base tumors are inherently more complex because high protein and low lipid concentrations in the cytoplasm or the stromal matrix are common and can pose a challenge in visualizing nuclei. To explore the utility and limitations of SRH in complex tumors, we have set out to conduct a more dedicated investigation of SRH application to skull base tumors. Additionally, we take a different approach from previous studies by focusing on the user  in addition to SRH validation. With only a few publications testing the accuracy of diagnosing histological subtypes29. Specifically, we performed simultaneous two-channel acquisition by using two 90° phase shifted Stokes pulses for orthogonal lock-in detection. This approach allows us to acquire protein and lipid SRS images rapidly for large pieces of tissue by eliminating wavelength switching and sequential acquisition. We image fresh resected tumor tissue collected prospectively during intraoperative consultation and validate the diagnostic capability of SRH through modularized review by board-certified neuropathologists. Our results reveal that SRH is effective in most cases with 87% accuracy relative to H&E stained formalin-fixed paraffin-embedded (FFPE) sections. Moreover, we find that in cases with limited lipid signal to provide the necessary contrast for nuclei visualization, rendering diagnosis is challenging when using strictly SRH, and it can be significantly improved with SRS chemical information.We used a simultaneous two-channel SRS imaging method we and others have developed to enable rapid SRH assessmentSixteen subjects undergoing operations for skull base tumors were recruited with the approval of the University of Washington Institutional Review Board over the course of 12 months, and all research was performed in accordance with relevant guidelines and regulations. Subject eligibility was determined during preoperative evaluation by the neurosurgeon (L.N.S.) participating in this study, and written informed consent was obtained from each subject before surgery. At the time of standard intraoperative frozen section consultation, a fresh tissue sample measuring 0.3 × 0.2 × 0.1 cm on average was allocated for this study. Each piece of fresh and unlabeled tumor sample was placed on a well depression glass slide, moistened with saline and covered with a coverslip in preparation for SRS imaging. Following SRS imaging, each sample was placed in neutrally buffered formalin, processed, and embedded in paraffin for permanent sections. Samples were then sectioned and stained with H&E for standard histopathological analysis. All SRS images with corresponding FFPE sections stained with H&E of skull base tumors were reviewed by a board-certified neuropathologist (G.J-S.), who selected representative portions of SRS images for histological analysis. Slides of frozen sections or cytological preparations performed at the time of intraoperative consultation from all studied cases were also reviewed in this study in order to compare the quality of SRS images with standard intraoperative techniques and to determine the diagnostic utility of SRS microscopy.29. Briefly, the Stokes and pump pulses provided by the fixed  and tunable  outputs, respectively, of a dual-output oscillator  with a repetition rate of 80 MHz. The Stokes pulse was modulated at 20 MHz using an electro-optic modulator (EOM) and split into two separate arms, one of which was then delayed by 12.5 ns (1/80 MHz) relative to the other. The two arms were recombined using a 50:50 beamsplitter and stretched to 2 ps using a grating-based pulse stretcher31. The tunable source was centered at 798 nm, and the pulses were dispersed using 60 cm of glass rods. The average spectral width is estimated to be 300 cm−1 31. The pump and Stokes beams were then combined with a dichroic mirror and overlapped temporally using a delay line in the tunable arm. The resulting beam was sent into a home-built laser scanning microscope. A 25X Olympus water immersion objective (NA = 1.05) was used to focus the beams onto the tissue sample. At the focus, the Stokes beams had an average power of 30 mW each, and the pump had an average power of 40 mW. After passing through the condenser, the Stokes beam was filtered out, and the pump reached a silicon photodiode. SRS signal was detected with a lock-in amplifier. The two orthogonal output of the lock-in amplifier provided the simultaneous two-channel SRS signals for pseudo H&E rendering28. Figure Figure μm × 285 μm per frame) were field-normalized and stitched to recover a complete tissue image (2–6 mm in size). Subsequently, the lipid channel was subtracted from protein channel, as previously demonstrated, to generate lipid corrected protein image  compared to standard intraoperative pathology, which typically includes the frozen section and cytological preparation of the specimen routinely stained with H&E. The survey was performed by three neuropathologists  who were blind to the diagnosis. They received a short clinical and radiographic summary of each case to mimic the circumstance of an intraoperative consultation but had no knowledge of SRS imaging and no training on SRH images. All sixteen studied cases were enrolled in the survey, which consisted of three phases: 1. Review of SRS generated images (SRH); 2. Review of the H&E-stained FFPE sections of the same specimen, and; 3. Review of the H&E-stained frozen sections or cytological preparations performed at the time of the neoplasm extraction for intraoperative diagnosis. Each phase included a series of multiple-choice questions focused on neoplastic appearance , architectural pattern , and nuclear shape . After the initial assessment, the evaluators participated in the diagnostic interpretation of each case that included differential diagnosis  and final diagnosis. Finally, each participant was required to quantify their level of confidence in their diagnosis. The results of the survey were compared to the assessment by collaborating pathologist G.J.-S.  and the neuropathology report .κC) were calculated while comparing each modality  to the assessment by the collaborating pathologist G. J.-S.  and the neuropathology report  for individual case using R software. Additionally, the relative accuracy as a ratio of SRH and H&E FFPE percent agreement was calculated for comparison of SRH to the golden standard of diagnosis. Fleiss’ kappa (κF) was calculated to assess interpathologist reliability.For each pathologist, percent agreement and Cohen’s kappa  are shown Fig. In the case of meningioma, the texture heterogeneity is caused by psammoma bodies . This is a good example of the challenges faced when calcified material is present during cryosectioning. Psammoma bodies are diagnostically helpful when suspecting meningioma, which mitigates poor tissue preservation in this case specifically. On the other hand, conserving features in case of chordoma and chondrosarcoma is important for accurate identification. Figure In other studied types of tumors, similar morphological features found typically in FFPE sections are sufficiently replicated in SRH. For example, SRH of studied pituitary adenoma reveals sheets of relatively monotonous neuroendocrine cells with a moderate amount of cytoplasm and rounded nuclei with bland and occasionally stippled chromatin, depicted in Fig. Finally, SRH provides information on chromatin appearance, which can be used as evidence of preoperative treatment such as embolization. Preoperative endovascular embolization, adjunctive treatment of meningioma, was performed in 5 out of 9 studied meningiomas, and in one studied case resulted in noticeable cytomorphological changes captured on SRH , is also reported. Interpathologist reliability is assessed using Fleiss’s kappa (κF). To limit the impact of interpathologist variation, we report a relative accuracy defined as a ratio of SRH percent agreement and H&E FFPE to highlight the performance of SRH relative to the gold standard.In order to establish the diagnostic utility of SRH, we administer a survey which assesses the interaction of a given neuropathologist with SRH, H&E stained frozen, and FFPE section slides. Unlike previous studiesOverall, the SRH percent agreement for histopathological features is approximately the same as the percent agreement for FFPE and frozen sections between neuropathologists. However, the interpathologist reliability for SRH is consistently lower within a given histopathological feature. These findings could be explained by the fundamental difference of tissue handling in SRH as measurements are conducted on fresh tissue and the final appearance of cellular features as well as tissue architecture could be slightly different when compared to frozen and FFPE. Additionally, the inherent differences in color scale between SRH and H&E stained sections could affect the assessment of the architectural growth pattern. For example, the myxoid and chondroid architectural growth patterns are not visualized sufficiently, as supported by our survey, on SRH due to the proteinaceous background being recolored darker than what is found typically on H&E. Despite the possible shortcomings of SRH when evaluating individual histopathological features, the differential diagnosis was developed successfully  could be challenging for pseudo-H&E recoloring. In conventional H&E staining, hematoxylin preferentially stains nucleic acids dark purple, highlighting nuclear features, whereas eosin stains protein content various shades of pink and red. Pseudo recoloring used in SRH relies on contrast from lipid and protein to recolor protein signal purple, enabling clear visualization of the nucleus. However, the requirement to pseudo-H&E recolor SRS images in SRH limits how chemical information from SRS is presented, which underutilizes the differences in protein and lipid in stromal or cytoplasmic components. The information on lipids is not available in conventional H&E.Although this study is focused on evaluating the diagnostic utility of SRH, it is important to emphasize that protein and lipid information from SRS is available to augment the use of SRH in intraoperative consultation. This can be exemplified in two different cases.versus 6B) shows a high lipid signal in the case of meningioma, while in chondrosarcoma, the supporting cartilaginous matrix is protein-rich. The SRS-based data matches the general knowledge that chondrosarcoma cells can be found in a matrix that is predominantly cartilage, made almost exclusively of protein. Information provided by SRS can thus facilitate a confident diagnosis of chondrosarcoma.Firstly, the studied chondrosarcoma case was misclassified as meningioma due to perceived similarities on SRH Fig. . HoweverSecondly, the occasional challenge of differentiating meningioma and schwannoma cases can be overcome with SRS lipid and protein information without having to resort to immunohistochemistry staining. In schwannoma, the collagen fibers often are present between neoplastic cells in contrast to meningioma, where the collagen fibers usually surround the group of neoplastic cells that can be found in a lobular pattern Fig. . These hIn summary, we highlight in Figs. 16. However, the gold standard in surgical pathology is the use of H&E stained FFPE sections in combination with ancillary studies, including immunohistochemistry, which requires time-consuming tissue processing as well as pathological interpretation. We address the gaps in testing SRH diagnostic capability using the reported original neuropathological diagnosis established for a given case and compare SRH to conventional modalities while accounting for interpathologist variability. In order to thoroughly assess the diagnostic utility of SRH, it is important to modularize the evaluation process to determine precisely the areas where SRH based technology needs improvement. We achieve that by assessing the efficacy at multiple steps on the way to final diagnosis as well as checking the neuropathologist confidence when using the new modality.SRH has shown great promise in intraoperative diagnosis of a limited subset of neoplastic entities, particularly glial tumors. However, the broad utility of SRH and challenges it may face in more diverse tissue remain largely unknown. Furthermore, the evaluation of the diagnostic accuracy conducted in previous studies has only focused on comparing SRH to H&E frozen sections and clinical diagnosis rendered intraoperativelyTaking our unique evaluation approach, we find that neuropathologists were able to establish a neoplasm description, architectural pattern, and nuclear shape with practically the same percent agreement as conventional modalities. Following the diagnostic process, we find that neuropathologists generate differential diagnosis successfully and final SRH diagnostic capability is very close to conventional modalities. In addition to demonstrating the diagnostic capabilities of SRH on this subset of tumors, we demonstrate supporting findings that SRS chemical information with lipid and protein can further help diagnostic process and possibly reduce the need of immunohistochemistry use in selected cases.37. With nuclear segmentation, a separate recoloring technique can be employed. However, incorporating separate recoloring techniques must be generalized across different entities to reduce user variability and avoid inconsistent simulation of H&E. Such inconsistencies can prompt the pathologist to misclassify the type of neoplasm with a detrimental consequence to patients. Moreover, when designing new recoloring schemes, it is essential to use a consistent method without a priori knowledge of H&E as such knowledge would not be available in an intraoperative setting.At the same time, one of the main challenges that the SRS technique is likely to face in broader organ applications is the reliance on lipid and protein contrast to visualize nuclear features which are important to render a pathological diagnosis. For many neoplastic entities, protein/lipid-based simulation of H&E works well. However, for situations where lipid is limited in the cytoplasm, or otherwise nearby stroma, a map of local protein concentration provided by SRS might not be sufficient for successful visualization of a nucleus and general cellular morphology. New recoloring schemes can be considered to mediate such a problem. Nuclear segmentation has been reported in H&E stained slides, fluorescence images, and SRS imagesIn conclusion, we have studied a diverse set of skull base tumors using fast simultaneous 2-channel SRS imaging and new pseudo-hematoxylin and eosin (H&E) recoloring methodology. Due to label-free non-destructive features of SRS technique, we demonstrated that most challenges with cryosectioning and limited amounts of fragile tissue during intraoperative consultation could quickly be addressed using SRS based approach. Using a diverse set of tumors, we determined the potential drawbacks of pseudo-H&E recoloring in selected cases involving insufficient nucleus visualization. While agreeing with previous work on SRS accuracy in intraoperative setting, we determined that SRS is capable of mostly matching the conventional H&E based technique and more importantly, providing additional diagnostically useful information. By following the diagnostic process that a pathologist uses, we discovered that pseudo-H&E recoloring, lipid/protein chemical information, and additional pathologist training to interpret this new information must be considered in tandem to bring SRS into clinical practice.Supplementary information.