Stylonychia somatic macronuclei differentiate from germline micronuclei. Thereby, programmed sequence reduction takes place, leading to the elimination of > 95% of germline sequences, which priorly adopt heterochromatin structure via H3K27me3. Simultaneously, 27nt-ncRNAs become synthesized from parental transcripts and are bound by the Argonaute protein PIWI1.During sexual reproduction in the unicellular ciliate These 27nt-ncRNAs cover sequences destined to the developing macronucleus and are thought to protect them from degradation. We provide evidence and propose that RNA/DNA base-pairing guides PIWI1/27nt-RNA complexes to complementary macronucleus-destined DNA target sequences, hence transiently causing locally stalled replication during polytene chromosome formation. This spatiotemporal delay enables the selective deposition of temporarily available histone H3.4K27me3 nucleosomes at all other sequences being continuously replicated, thus dictating their prospective heterochromatin structure before becoming developmentally eliminated. Concomitantly, 27nt-RNA-covered sites remain protected.We introduce the concept of ‘RNA-induced DNA replication interference’ and explain how the parental functional genome partition could become transmitted to the progeny.The online version of this article (10.1186/s13072-018-0201-5) contains supplementary material, which is available to authorized users. Oxytricha (class: Spirotrichea) small non-coding RNAs (sncRNAs) of 27nt in size are synthesized from the parental macronucleus. These target macronucleus-specific sequences in developing macronuclei in association with the PIWI homolog OTIWI-1. These authors proposed that the 27nt-RNAs protect specific sequences from being degraded  for nascent RNA and 5-iodo-2′-deoxyuridine [IdU] or 5-chloro-2′-deoxyuridine [CldU] for nascent DNA), in situ antibody stainings, poly[A]-RNA FISH and subsequent confocal microscopy were done as reported , 21. A lTotal RNA was isolated using Trizol (Sigma-Aldrich) and isopropanol precipitation and further purification on columns. Next, RNA integrity was assayed using the Agilent Bioanalyzer 2000. Only samples with non-fragmented RNA were included.Genomic DNA was isolated from purified nuclei using phenol/chloroform/isoamylic alcohol extraction followed by ethanol precipitation.www.imagif.cnrs.fr). The initial data analysis pipeline was as follows: CASAVA-1.8.2 was used for demultiplexing, Fastqc 0.10.1 for read quality assessment and Cutadapt-1.3 for adaptor trimming, resulting in sequence number for each developmental time point sample between 29.9 and 34.6 Mbp. File conversions, filtering and sorting as well as mapping (Bowtie2), were done using ‘Galaxy’  purification using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs), followed by transcriptomic library preparation (NEBNext Ultra RNA Library Prep Kit for Illumina). Seven libraries were multiplexed per lane and sequenced on a Illumina HiSeq 2000 platform . This work has benefited from the facilities and expertise of the high throughput sequencing core facility of IMAGIF Gif-sur-Yvette . The initial data analysis pipeline was as follows: CASAVA-1.8.2 was used for demultiplexing, Fastqc 0.10.1 for read quality assessment and Cutadapt-1.3 for adaptor trimming, resulting in an average sequence number for each developmental time point sample of 7.58 Mbp. File conversions, filtering and sorting as well as mapping (Bowtie2), were done using ‘Galaxy’  mouse monoclonal antibody and 25 µL magnetic protein A beads (Diagenode). Following the purification of immunocomplexes and several washes with PBS, the samples were heated to 95 °C for 15 min, and then RNAs were purified using Trizol. Small RNA libraries for massive parallel sequencing were made as described above.Immunodetection of PIWI1 and H3K27me3 was done as described , 14.Stylonychia were treated as outlined , 72 °C for 10 min; alternatively, for the 856 bp PCNA amplicon the conditions were: 95 °C for 15 min, 30 cycles of , 72 °C for 10 min.End-point PCR was carried out on a VWR Doppio PCR device using Maxima Hot Start Taq polymerase Master Mix (Qiagen) or Pfu DNA polymerase (ThermoFisher) and SYBR green (Sigma-Aldrich). Standard PCR conditions were as follows: 95 °C for 15 min, 40 cycles of ; alternatively, for the 856 bp PCNA amplicon the conditions were: 95 °C for 15 min, 40 cycles of . Melting of PCR product was performed using a temperature gradient from 55 °C to 95 °C, rising in 0.5 °C increments.Quantitative PCR was performed on a Rotor Gene 6000 (Corbett Life Science) using QuantiTect SYBR green Klenow reaction using the large fragment of DNA polymerase (ThermoFisher) and pGEM-T-easy-PCNA as template upon manufacturers’ recommendations. For priming a modified T7 sequencing primer was used  and after consecutive intervals 10–60 h PC). With emphasis on the probable important roles of histone variants in the course of macronuclear development, Fig. Oxytricha trifallax, disproportionate numbers of mRNAs putatively encoding proteins involved in RNA (and DNA) functions were described recently ) exhibited a less condensed state when compared with micronuclei. Thereafter, we observed a massive 27nt-RNA gain . Subsequently, 27nt-RNAs were still enriched until 60 h PC. At this time point, macronuclear anlagen entered a DNA-poor stage, which followed bulk DNA elimination. After rapidly reaching their maximum at ~ 20 h PC, the concentration of the 27nt-RNAs decreased continuously. Notably, the occurrence of other small-sized RNA (< 21nt) fragments overlapped with the synthesis of 27nt-RNAs, possibly representing debris of the sncRNA biogenesis.Oxytricha [We mapped both sncRNA fractions to the macronuclear genome and to all known micronuclear sequences (complete model genes and thousands of preliminary micronuclear contigs). Clearly and reminiscent of xytricha , 4, the Chlorogonium algae (food source for Stylonychia), plastids or bacteria   . These samples were allocated to different experiments.To gain deeper insight into the putative 27nt-RNA-mediated protection mechanism for MDS retention, we studied the interactions of 27nt-RNAs with PIWI1 and with their target sequences, respectively. For this purpose, we purified macronuclear anlagen from 32P followed by denaturing SDS-PAGE. X-ray film detection of the radioactive tracer revealed a prominent signal being associated with the PIWI1 protein band . Enriched PIWI1-RNPs were end-labelled using polynucleotide kinase and R2 was as high as 0.99634 -gene in PIWI1-minus samples was almost indistinguishable from controls—most IESs were removed between 24  and 30 h PC . A first time observed feature was that PIWI1 appeared to travel physically within extranuclear spheres from parental macronuclei to the young anlage, which at this time point morphologically could not yet be distinguished from micronuclei (m) . In particular, extranuclear PIWI1 overlapped with faint To-Pro-3-positive spheres, indicating that they might have contained significant RNA concentrations. Improved resolution elucidated that PIWI1 subsequently swamped the young anlage, and only in later anlagen stages subtle PIWI1 foci shaped out . Subsequently, these foci became gradually fewer in number. These patterns suggested that PIWI1/27nt-RNA complexes were specifically targeted to discrete sites. Strikingly, this suggestion became obvious when higher magnifications of polytene chromosomes were examined  Fig. c2. In paComplementary, H3K27me3 was selected as a marker for condensed chromatin  reminiscent of canonical H3 that was recognized by anti-H3K27me3 antibodies, if lysine 27 in H3.4 or lysine 32 in H3.8 were trimethylated , which most probably influence its availability as a KTM substrate or readout by putative effector proteins. The expression of other H3 variants seemed to be insignificant with respect to macronuclear development.H3.4 is differentially expressed during chromosome polytenization. Although the K27me3 epitope is also present in H3.8, this 20 kDa H3 variant is restricted to micronuclei . Other HStylonychia and until 20 h PC. Then its expression increased massively during early polytenization from 20 to 30 h PC, before dropping again after 50 h PC —can modify DNA replication in a qPCR  and a heat stable Argonaute protein—Stylonychia PIWI1 can modify linear DNA amplification in a Klenow reaction . When mixes of serially diluted 27nt-RNAs (25–12.8 pM) matching PCNA1 were added to the PCR reactions  and N-terminal His-tag. Subsequently, we cloned His-PIWI1 into pCMV-TNT (Promega) for in vitro expression. Prior to assaying the inhibitory potency on replication, we tested whether in vitro produced His-PIWI1 can bind 27nt-RNAs. We therefore incubated His-PIWI1 with 4TU-RNA mixture obtained after purification of RNA from 4TU-labelled exconjugants, followed by UV cross-linking 365 nm) and PIWI1-RNPs pull-down with PIWI1-specific antibodies . Using t nm and PS. pombe, C. elegans, A. thaliana), it is not assumed that Argonaute/sncRNA complexes bind directly to a DNA target. Rather, they apparently bind via base-pairing to tethered nascent transcripts, which origin from the target regions. Binding of such transcripts through Argonaute/scnRNAs leads to the localized recruitment of chromatin modifiers [Stylonychia  for several model eukaryotes   were injected, at three successive time points. No conspicuous differences or restrictions in growth between mock and bait RNA-treated cells were observed: Following microinjection, cells were grown individually in microtiter plates and inspected in daily routine. The observed growth rates were 2.31-fold/d for bait RNA-treated cells and 2.18-fold/d for mock RNA-treated cells. Intriguingly, we observed a significant reduction in the copy numbers of targeted nanochromosomes between the 1st and 2nd replication cycles after microinjection  Fig. c. When wStylonychia [Our data provide a line of evidence that PIWI1-bound 27nt-RNAs target MDSs via RNA/DNA base-pairing during polytenization, which encompasses a series of 5–6 DNA replication cycles in The termination of MDS module reordering in scrambled micronuclear genes and the removal of interrupting IESs.Targeting/protection of MDSs by PIWI1-bound 27nt-RNA complexes, which leads to locally stalled DNA replication during chromosome polytenization. This sequence protection might lead to the omission/attenuation of H3K27me3 enrichment at MDSs.Subsequent enrichment of H3K27me3 at bulk DNA sequences leading to a unique chromatin structure, which is required for DNA elimination.Thereafter, RNA-induced DNA replication interference would require a series of synchronized events, i.e. briefly:With respect to scrambled gene reordering and IES removal, there is growing consent that a ‘template’ non-coding RNA species that acts on micronuclear genes might occur within a narrow timeframe prior to bulk DNA elimination. It is unknown whether this timeframe overlaps with early chromosome polytenization. The ‘template’ RNA is not yet fully characterized, but it is presumably involved in MDS reordering, IES excision, nanochromosome copy number determination and telomere addition to immature nanochromosomes –19. It iWhereas it is currently technically hardly possible to achieve a better resolution of the exact timing, we assume that 27nt-RNAs act subsequently in the course of serial polytenization on the micronuclear gene loci, which harbour MDSs, thereby protecting them from being entangled in subsequent elimination of bulk DNA. The most plausible scenario is that 27nt-RNAs protect genes as soon as IES excision and MDS reordering have been completed, safeguarding that these immature macronuclear genes remain protected from heterochromatin formation. In theory, RNA/DNA base-pairing of 27nt-RNAs within reordered MDSs would require single-stranded DNA which would be given in a replication bubble, i.e. during polytenization Fig. c3. We obStylonychia. These include the persistence of the parental (old) macronuclei, which could indicate that the translocation of 27nt-RNAs therefrom is blocked, and the developmental arrest of macronuclear anlagen [Stylonychia within developing macronuclei comprise the observation that the micronucleus-specific 20 kDa histone variant H3.8 (formerly ‘protein X’) that usually becomes replaced after synkaryon/zygote formation persists in developing macronuclei, whereas 15 kDa H3 is detectable in Western blots, but does not become methylated at lysine 27 [Whereas the knock-down of PIWI1 by RNAi does not touch the excision of IESs Fig. a, the el anlagen . Furtherysine 27 . We obseysine 27 . It is vEventually, it is imperative that all MDSs must be marked for preservation and bulk DNA must be assembled into a condensed chromatin structure at the time point, when DNA replication/chromosome polytenization is terminated. This expectation is obviously fulfilled: Microscopy of polytene chromosomes demonstrates that PIWI1 and H3K27me3 exhibit mutually exclusive staining patterns. In particular, PIWI1 occurred in non-condensed polytene chromosome regions, whereas H3K27me3 was associated with condensed chromatin bands. We assume that this pattern could be achieved, if MDSs replication became locally and transiently stalled (protected), whereas the continuous replication of unprotected bulk DNA regions would be coupled to the incorporation of specific, temporally available histones, which shape a discrete chromatin structure that determines associated sequences for elimination. Due to technical restrictions, there is currently no close reasoning for all parts of the proposed concept. But in agreement with the requirements of our proposed concept we showed that during polytenization not all sequences become synchronously replicated. In particular, sequences being slated for elimination become earlier polytenized than MDSs during macronuclear development, e.g. the maximum of copy number of the bulk DNA model sequences MaA81 and Stad5 is reached approx. 10–20 h earlier than for the micronuclear gene models TEBPalpha and ACT1 K27me3 could be available (or other chromatin assembly factors that influence the deposition of H3K27me3). Other conceivable scenarios are provided as Supplementary data . While being speculative, these scenarios leave open whether differential efficacy of MDS protection, possibly through differences in 27nt-RNA coverage (which correlate with mRNA levels), could lead to differences in mature MDS preservation. This could contribute to a rough predetermination of nanochromosome copy numbers. In this case MDS protection could be scalable through the mRNA copy number in the parental macronucleus. Notably, the possibility that the parental mRNAs could be the substrates for the synthesis of 27nt-RNAs is supported by the observed tight correlation of 27nt-RNA/mRNA copy numbers and by the observation that 27nt-RNAs cover the transcribed region of nanochromosomes only.Apart from these speculations we summarize that IES excision and MDS reordering, which do not depend on PIWI1, must take place within a restricted time window prior to bulk DNA elimination. This time window possibly opens shortly before the onset of polytene chromosome formation. Subsequently, MDSs become protected and this depends on PIWI1. MDS protection must be achieved during polytene chromosome replication before bulk DNA adopts a heterochromatin structure through H3K27me3 and sequence elimination can start. Thus, the onset of DNA diminution possibly requires an S-phase termination checkpoint. We conclude that a series of precisely timed events is crucial for the tight coordination of these processes and that ‘RNA-induced DNA replication interference’ can in theory provide a mechanistic explanation. Below, in our further discussion we consider additional results to provide stronger support to the plausibility of the RIRI hypothesis.Stylonychia is mainly connected with 27nt-RNA trafficking between parental macronuclei and anlagen, our data suggest this Argonaute homolog could strengthen the DNA replication interference effect during chromosome polytenization in developing macronuclei as it appeared to be the case at least in a Klenow reaction assay. This was reminiscent of reports on non-slicing Argonaute/guide RNA complexes, which function as ‘road blocks’ to inhibit the translation of mRNAs in absence of other auxiliary proteins: In almost all groups of organisms, RNase H degrades selectively the RNA strand in RNA/DNA hybrids. The PIWI-domain in slicing Argonaute proteins in eukaryotes and archaea includes an RNase H-like active site capable for guide strand binding and slicing of RNA/RNA hybrids [E. coli. Interestingly, it was shown that the Tus-Ter only transiently blocks the replisome in the non-permissive direction [Stylonychia. We thus believe that a transient DNA replication ‘road block’ is the most plausible explanation for 27nt-RNA/PIWI1 complexes.We showed in vitro that 27nt-RNAs have the potency to block the DNA replication activity of several DNA polymerases in the vicinity of base-paired homologous sequences. Our assays using 27nt-RNAs alone, 21nt-phDNA or both oligonucleotides in combination with Argonaute homologs  and a putative glutamate finger (including E590 or E594) the PIWI/RNase H-like domain cleavage . HoweverStylonychia, in particular the replication of nanochromosomes in the macronucleus, as a model system, where DNA sequences become replicated in the natural context of chromatin and nuclear architecture in vivo. We observed that microinjection of 27nt-RNAs was sufficient to impair the proper replication of targeted nanochromosomes, which transiently led to their reduced copy numbers in the progeny. We believe that this experimental system provides a practicable approach to provide first evidence that RIRI could exist in vivo. However, since nanochromosome replication in vegetative macronuclei is a process different from polytenization in developing macronuclei, the results should be interpreted with some care. Notably, in contrast to PIWI1, whose expression during macronuclear development exceeds by far all other PIWI-domain proteins in Stylonychia, at least one other PIWI (PIWI2) seems to be constitutively expressed over the life cycle. PIWI2 is the most abundant PIWI-domain protein during vegetative growth  and seven supplementary figures


Cajanus cajan (L.) Millspaugh) is a complex disease due to various factors including the presence of a mixed infection. Comparison of dsRNA profile and small RNA (sRNA) deep sequencing analysis of samples from three locations revealed the presence of Pigeonpea sterility mosaic virus-I and II (PPSMV-I and II) from Chevella and only PPSMV-II from Bengaluru and Coimbatore. PPSMV-I genome consisted of four while PPSMV-II encompassed six RNAs. The two viruses have modest sequence homology between their corresponding RNA 1–4 encoding RdRp, glycoprotein precursor, nucleocapsid and movement proteins and the corresponding orthologs of other emaraviruses. However, PPSMV-II is more related to Fig mosaic virus (FMV) than to PPSMV-I. ELISA based detection methodology was standardized to identify these two viruses, uniquely. Mite inoculation of sub-isolate Chevella sometimes resulted in few- to- many pigeonpea plants containing PPSMV-I alone. The study shows that (i) the N-terminal region of RdRp (SRD-1) of both the viruses contain “cap-snatching” endonuclease domain and a 13 AA cap binding site at the C-terminal, essential for viral cap-dependent transcription similar to the members of Bunyaviridae family and (ii) P4 is the movement protein and may belong to ‘30 K superfamily’ of MPs.Sterility Mosaic Disease (SMD) of pigeonpea ( Cajanus cajan (L.) Millspaugh] in the Indian subcontinent. The susceptible genotypes show proliferation of branches, varied degrees of mosaic and mottling on the leaves2 with a partial or complete sterility of the infected plants 4 in a semi-persistent manner6. PPSMV Patancheru isolate as well as sub-isolate Chevella (contains PPSMV-I and PPSMV-II) infected ICP2376 cultivar produces ring spot symptoms whereas other isolates previously studied3 developed severe mosaic symptoms in the infected plants. This suggests mixed infection of PPSMVs may be the cause of ring spot symptoms coupled with genotype variation in ICP2376, as symptoms caused by individual viruses are not known.Pigeonpea sterility mosaic is a major disease of pigeonpea [nts Fig. . Whereasnts Fig.  infectio7, efforts made recently gave us a clue about the nature of the pathogen10, associated with the diseased plants. Purification of sterility mosaic virus as enveloped spherical particles from the infected plants was not successful. However, an association of virus particles was noticed in infected cells identified as double membrane bodies (DMBs)11. Similarly, plants infected by different emaraviruses containing the DMBs were not recognized earlier as the disease causing agents. However, European mountain ash ringspot-associated virus (EMARaV) and Wheat mosaic virus (WMoV) were purified from the infected plants as enveloped spherical particles14. Purification of sterility mosaic virus from pigeonpea is difficult because of its low titer, besides the SMD infected pigeonpea plants contain interfering polyphenols and secondary metabolites in elevated levels15. Obtaining purified intact virus particles facilitate reliable estimates of genome composition especially in a situation where two emaraviruses exist (PPSMV-I and PPSMV-II) in a mixed infection.Although this disease was first described 85 years backRose rosette virus RRV and PPSMV-I in this study), six  and eight  segmented negative sense RNAs encapsulated in nucleocapsid and transmitted by eriophyid mites belonging to different genera17. However, Redbud yellow ringspot virus18 (RYRSaV), and the newly identified Actinidia chlorotic ringspot-associated virus19 (AcCRaV) have five RNA segments. Woolly burdock yellow vein virus20 (WBYVV) has not been fully characterized and assumed to be a member of genus Emaravirus.Morphologically, all emaraviruses reported till date have virus particles as quasi to spherical with double lipid membrane-bound particles containing four  which encode nucleocapsid proteins (NCP) to facilitate genome encapsidation, with over all limited sequence homology and eriophyid mite transmission. Plant viruses with these traits have been grouped to an unassigned genus The results of the recent studies of Patancheru isolate revealed the association of PPSMV-1 and PPSMV-2 containing a diverse number of RNA genomic segments, in particular PPSMV-1, which is remarkably differerent from our findings. In this study, we described a workable protocol for analysis of virus-associated dsRNA from pigeonpea host. The analysis of total small RNA populations was performed to characterize the genomic RNA segments and variations in PPSMVs. High-throughput sequencing of small RNA (sRNA) was employed to sequence multiple RNA segments that resulted in establishing the complete genome of PPSMV-I containing four genomic RNAs and PPSMV-II containing six genomic RNAs. Furthermore, their evolutionary relationships were studied with other related viruses by performing phylogenetic analysis. This paper will provide an improved understanding of the molecular biology of PPSMVs, their relationship to each other and with pigeonpea sterility mosaic disease.10. A few times that we got plants with PPSMV-I only did not survive to maturity.PPSMV-P sub-isolate Chevella was inoculated routinely and used to obtain 100% infection in the two susceptible cultivars ICP8863 and ICP2376. Eriophyid mite inoculated Fig.  pigeonpeFusarium wilt diseases. Typical SMD symptoms . The RT-PCR predicted product (321 bp) was amplified in PPSMV-P sub-isolate Chevella infected samples  in the susceptible plants infected by PPSMV-P sub-isolate Chevella. SMV-1 and SMV-2 primersles Fig.  but not Tobacco streak virus (8) and Bean common mosaic virus (5) were also obtained in Bng-1 sample. Contigs with a mean length of 404 nucleotides and maximum contig length of 5822 nucleotides were obtained. Homology search revealed that generated contigs showed a relationship with two different members of genus Emaravirus. One group of contigs showed homology with RRV and were identified as genomic segments of PPSMV-I and the second group having homology with FMV were recognized as genomic segments of PPSMV-II. Small RNA sequence analysis revealed that the contigs generated from Mg-1 and Mg-2 samples belonged to PPSMV-I and PPSMV-II, while contigs assembled from Bng-1 sample (PPSMV-B isolate) are related to PPSMV-II. Emaraviruses are known to contain conserved complementary nucleotides (13–14 nt) at 5′ and 3′ regions, resulting in the formation of a panhandle structure, and this region was used as a tag for identification of terminals or complete sequence of RNA segment. Based on these results, primers were designed for PPSMV-I and PPSMV-II, and their genomic segments were authenticated by full-length amplification of each RNA segment, cloned and sequenced.The enriched virus-derived small RNA (vd-sRNA) reads obtained from SMD affected samples of Mg-1, Mg-2, and Bng-1, were assembled to form contigs further used for viral genome assembly. A total of 101 contigs were generated, of which 75 contigs were found specific to viruses by BlastX homology search. For emaraviruses, 62 contigs (23 contigs each of Mg-1 and Mg-2 samples) of PPSMV-P sub-isolate Chevella and 16 contigs from Bng-1 sample of PPSMV-B isolate were generated  RNA viruses. The signals directing RNA synthesis are localized within the 5′ and 3′ end non-translated terminal regions32 and also were attributed to the region responsible for BUNV RNA segment multiplication31. The evidence provided thus suggests that these specific sequences in the termini could be involved in the initiation of transcription and also regulating genome replication34.Following paragraphs describe the nature of the functional four RNAs 1–4) of both the PPSMVs by sequence analysis. The analysis provides evidence, the role they may play in virus multiplication, spreading infection and the variations of RNA-5 among the PPSMV-II isolates. The sequence of each RNA segment was compared with orthologs of known emaraviruses and reasoned that the PPSMVs belong to genus ome Figs  and 4 li of both 38 and use them in viral RNA replication. Similar studies were reported in the case of FMV25. We have identified conserved residues of cap-snatching and cap binding sites at two different domains of the polymerase of PPSMVs  and cleaved 10–13 nucleotides (13–18 in TSWV) downstream of the cap by the endonuclease domain  while interacting with second active cap binding site at the C-terminal endEmaravirus (Table The RNA-1 segment being the largest 7023 nt) Fig. ; Table 13 nt Fig.Bunyaviridae polymerase, and probable cap binding site in these domains in the same order  present in the SRD-1 domain between 66 to 242 aa  motif is present in different viruses belonging to Emaravirus and Tospovirus genera while the later and orthobunyaviruses contain additional DXK motif. KXTDL158–162 motif , are known to play a role in endonuclease activity43. These active residues are highly conserved amongst the genera of Emaravirus, Tospovirus, and Orthobunyavirus.For convenience, the RdRp is described as containing N-terminus SRD-1, a central region as SRD-2 and C-terminus SRD-3 domains Fig.  and iden aa Figs  and 5 anied Fig. , and theOrthobunya- and Tospovirus genera (H….D…PD….DxK…..T), the pattern with the PPSMVs, FMV-JS1, and Emaravirus appear to be H…PD…ExK as of now till the confirmations are made , motif B (NWLQGNLNMISSFVH1211–1225), motif C (SDD1255–1257), motif D (ITLNEKKTY1298–1306), E (EFL1313–1315) and F (KDQRTAXDREIYTGNAQ1049–1065)  Fig. . These aily Fig. .1947–1959 residing in the SRD-3 domain at the C-terminus of PPSMV-I polymerase was identified for the first time as a probable cap binding site (CBS)  and RRV (DHRVYNHWLIPTN1930 to 1942) except that RRV is containing the second residue as H193126. Conserved sites of the endonuclease and probable cap binding sub domains were identified in SRD-1 and SRD-3 domains of PPSMV-I polymerase, primarily by the sequence alignment with members of the Bunyaviridae super family.A stretch of thirteen amino acid with sequence DPLTYNYWVMPTNBS) Fig. . The CBSBunyamwera virus. The N-terminal sequence with amino acid position 66 to 242 (185 aa) was used to develop the 3D structures of the endonuclease. As shown in Fig. 47. Cartoon representation of the endonuclease model of the above viruses showing overall topology and architecture of the protein folding resembles the endonuclease of Bunyamwera virus48 resolved at 1.3 Å , not far away from the center. The endonuclease of PPSMVs, beside their close structural similarity with Bunyamwera virus, also showed similarity with analogs found in the PDB library; La Crosse Orthobunyavirus , Influenza virus A (2W69), B (4WRT) and C (5D98), Pichinde virus (4I1T) and Lassa virus (4MIW) of genus Arenavirus.The tertiary structure of the endonuclease was developed to examine the topology resemblances and the distribution of highly conserved motifs and individual conserved residues mentioned earlier, between PPSMVs, FMV and 3 Å Fig. , compris3 Å Fig. . The con3 Å Fig. ; SI 5A. Bunyaviridae. The N-terminal region of the glycoprotein precursor of PPSMV-I contains a signal peptide cleavage site, located at amino acid position 22 and 23 (AMA↓S) 49. The glycoprotein precursor yields a dimer having a putative cleavage site at FS↓DD, (Ser201 and Asp202), and the precursor may be processed into two Gn (22.4 kDa) and Gc (51.6 kDa) smaller glycoproteins. All emaraviruses have been shown to have the processing site except in RLBV glycoprotein, in which case the strategy of cleavage process remains to be identified27. The glycoprotein contain three transmembrane helices (TMH) at amino acid positions 103–125, 178–200 and 593–615 (TMHMM v.2.0 server50) (http://www.cbs.dtu.dk/cgi-bin). Six potential glycosylation sites were identified at amino acid positions 27, 33, 69, 100, 197 and 44751. The analysis predicted a potential fifth glycosylation site close to the putative processing site amino acid position 197 having a sequence NPS  protein of 648 amino acids Fig. , Table 19. Description of peptide cleavage or signal peptide site information of PPSMV-1 was different from our findings, and the authors reported four out of six glycosylation sites. Furthermore, there was the substitution of 10 amino acids. This precursor protein has an amino acid similarity of 98.15% with PPSMV-I P sub-isolate Chevella (present study).PPSMV (now PPSMV-1) Patancheru isolate P2 sequence published from the previous investigation was partial with 623 amino acids (HF568802) making it thus shorter by missing N-terminal 25 amino acids which contain signal peptide cleavage site. However, the characteristics of precursor described, reflects of a full-length glycoprotein precursor47. The 3D structure was solved at the 12.1 Å resolution and the structures developed in sufficient quality to conclude that various structural elements are similarly arranged to those of PPSMV-II, with an average resolution of 12.7 Å. The precursor proteins of both the viruses showed similar topology with no significant differences in the architecture. PPSMV-I glycoprotein precursor contains 21 α-helices and 17 β-sheets and is shaped like a croissant with panhandle where the croissant and panhandle correspond to the Gn and Gc domains, respectively. There is a clear boundary between the two domains. The N-terminal domain (croissant) is packed compact containing the two transmembrane helices (TMH-1 and TMH-2), the specific glycosylation sites and the panhandle containing the third TMH and the conserved eight residues Phlebo-like motif close to the α-helix 13   Table . PPSMV-I134–140, NRLA183–186 and GYEF204–207 with the NCP of PPSMV-II and members of the genus Emaravirus26 predicted to be involved in RNA-binding27 , distributed in N- and C-terminus, predictably play a role in RNA binding52.RNA-3 is 1,441 nt. long and encodes a single ORF between nt 934 and 102 with 101- and 406-nt NTRs at the 5′ and 3′ends, of a 34.6 kDa (P3) protein of 308 amino acids Fig. , Table 154. In the present study, the P4 sequences of PPSMVs were analyzed and aligned with other emaraviruses, revealing characteristics that are common to emaraviruses. The P4s contain different elements in sequential order; the N-terminal signal peptide , the TMV 30 kDa movement protein domain similar in its sequence and secondary structure. Presence of likely DnaK domain in the C-terminal half and sequences that have been predicted to be RNA binding elements (http://www.imtech.res.in/raghava/pprint/) were identified.The RNA-4 of both the PPSMVs encodes respective P4 proteins assumed to be involved in the cell-to-cell movement, a crucial step in plant virus infections. To support this hypothesis, conclusive evidence was provided from the study of related P4 proteins; FMV and RLBV which have been implicated in cell-to-cell movement57 starting with LP-I68–70 to SWKT195–199. The 30 K domain containing sequences coding for the highly conserved motifs aligned in all emaraviruses   of a 40.8 kDa protein (P4) of 361 amino acids. RNA-4 contains 76 nt and 401 nt as the 5′ and 3′  NTRs, respectively Fig. , Table 1ses Fig.  includin to 77 of8-A9 which are conserved in rest of five RNAs were replaced by T8-T9. Furthermore, the 5′- and 3′-termini complementation was found to be complete to form the putative panhandle with the exception of RNA-5 where the complementation was interrupted by TT replacement at the 5′-end. The four functional RNA segments of both PPSMVs share several common characteristics, while PPSMV-II showed a close relationship to FMV24 as they both uniquely contain six RNA segments, confirming the recent observation10.Six negative-sense single strand genomic RNAs were identified forming the genome of PPSMV-II Figs  and 4B, 1948–1960) as cap binding site could be identified in SRD-1 and SRD-3 domains of polymerase, respectively. The “cap binding” residues are fully conserved with the FMV while moderately conserved with PPSMV-I. Conserved residues of four motifs  in the SRD-1 domain representing the putative endonuclease, between nucleotide position 66 to 242  motif was also identified along with KXTDL157–161 motif  of PPSMV-II is 7009 nt long and slightly smaller to RNA-1 of PPSMV-I (7022 nt) and encodes for a single open reading frame (ORF1) between nucleotides 6930–46 with 45 and 79 nt long NTRs at 5′ and 3′ ends , B (SNWLQGNLNMISSFVH 1210–1225), C (SDD1255–1257), D (ITLNEKKTY1298–1306), E (EFL1313–1315) and F (KDQRTAXDREIYTGNAQ1049–1065) were identified , as closed right hand resembling the 3D pol of typical picornaviruses. The 3D pol contains the conventional sub-domains as fingers, palm, and thumb containing the above, referred as A to F sequence motifs , at which the precursor may be cleaved into two single glycoproteins Gn (22.1 kDa) and Gc (52.3 kDa). In the N-terminus region, a potential signal peptide cleavage site between Gly19 and Thr20 was detected. Additionally, the GP precursor contains three transmembrane helices50 (TMH) at amino acid positions 109–130, 175–192, and 583–599. Five potential N-glycosylation sites at amino acid positions 25, 65, 204, 393 and 438 were predicted, unlike in PPSMV-I, these sites were evenly distributed  of 649 amino acids Fig. , Table 1ted Fig. . Like in130–136, NRLA178–181 and GYEF199–202 . The three motifs were identified around the cleft of the protein. The α-4 helix containing NVVSFNK motif, GYEF motif with α-7 helix and NRLA in a coil between α-5 and α-6 helices (data not shown). The bindN analysis predicted stretches of amino acid sequences at N- and C-terminal regions as potential RNA binding sites (Table RNA-3 is 1362 nt long and encodes a 35 kDa nucleocapsid protein (NCP) of 314 amino acids. RNA-3 encodes a single ORF (P3) between nucleotides 1046 to 102 and contains a 101 and 316 nt NTRs at 5′ and 3′, respectively Fig. , Table 167–69, ASVA-90–93, WTPT-96–99, DxR-113–115 and SWKT-196–199) including at least, parts of TAV segment (T144 to M183) of this domain is mostly homologous to all the emaraviruses except for RLBV and WMoV  of a 40.7 kDa protein (P4) of 361 amino acids. RNA-4 contains 82 and 325 nt at the 5′ and 3′ NTRs, respectively Fig. , Table 1MoV Fig. . The TAV69-SWKT199) was identified  seems almost universal in members of the 30 K family of MPs, including seven emaraviruses. The conserved aliphatic residue I106 of the conserved β-1 sheet region  has been shown in RLBV to mediate membrane association53. It was demonstrated that the conserved residue D127,  is critical for both the movement activity and localization to plasmodesmata of RLBV P4. Examination of the secondary structure of 30 K region of PPSMVs P4 revealed a similar structural pattern of α-helices and β-sheets connected by the coils as predicted earlier57. We used the same nomenclature to describe the secondary structure, from N- to C-terminus. The PPSMVs P4 structural patterns are almost identical with RLBV and FMV. RLBV P4 protein has a secondary structure identified in the 30 K domain as a αA, β-1, β-2, αB, β-3, β-4, β-5, β-6, and β-753. However, minor differences from the mentioned pattern do exist in PPSMVs. The absence of α-helix ‘αB′ between β-sheet 2 and 3 in PPSMV-I and presence of one additional β-sheet-8 in PPSMV-II was noticed.The 131-residue region  and 40.8% similarity with PPSMV-I P4. The P4 possesses 74.7% with FMV and percent similarity of 61.0 to 17.4 with the P4 of other emaraviruses  to eight (PPSMV-II) amino acid motifs were detected as RNA binding elementses Table . Three mes Table . Taken tRNA-5 is 1840 nt long, and contains a single ORF between nt 1534 and 110 with 109 and 306 nt long 5′ and 3′ NTRs, respectively Fig. , Table 1RNA-6 is 1194 nt long, encodes a 27.1 kDa protein (P6) of 238 amino acids with an ORF between nt 785 and 69 Fig. , Table 1Erra kandulu cultivar (PPSMV-P sub-isolate Chevella) and Bng-1 sample (PPSMV-B isolate) generated contigs that revealed close identity with RNA-5. Sequence analysis confirmed a considerable variability among these contigs , WMoV (19.02), AcCRaV (26.53) and a mere 32.77% similarity with closely related FMV, suggesting a high level of variability among RNA-5. However, we noticed that PPSMV-II RNA-5 had an exceptionally high similarity (86.47%) with PPSMV-1 RNA-59. This anomaly can best be explained that PPSMV-1 so called RNA-5 might not relate to its genome. The degree of homology is too high to be expected (for RNA-5) between two different emaraviruses. Furthermore, PPSMV-I genome with four genomic RNA segments could be confirmed by the analysis of pigeonpea cv. ICP8863 plants infected by PPSMV-I alone  from both the viruses were synthesized and used to detect PPSMV-I and PPSMV-II employing RT-PCR, slot blot hybridization, and western blotting methods. The results of these experiments also confirmed the association of different PPSMVs with different isolates studied  infected with PPSMV-P sub-isolate Chevella revealed the presence of PPSMV-I by detecting specific amplicons (927 bp) of PPSMV-I NCP gene and PPSMV-II, by detecting specific amplicons (1078 bp) of PPSMV-II NCP gene. While PPSMV-II alone was detected in Bng-1, Bng-2, and Bng-3 from unknown pigeonpea cultivars infected with PPSMV-B and pigeonpea cvs. CO-5, CO-6, and ICP8863 infected with PPSMV-C isolates  of the PPSMVs  to ensure specific hybridization and used in the slot blot hybridization analysis. Each sample contained the equal amount of total nucleic acids (35 µg) applied to the respective slot on the blot. In this study, positive signals for detection of both PPSMVs NCP was detected only in plants infected by sub-isolate Chevella Fig. . InteresOrthobunyavirus, and Tospovirus Nairovirus, Tenuivirus and Phlebovirus from a common ancestor , LN887952 (RNA-2), LN887953 (RNA-3) and LN887954 (RNA-4). PPSMV-II: LN651310 (RNA-1), LN651182 (RNA-2), LM652701 (RNA-3), LM652703 (RNA-4), LN898418 (RNA-5), LN898419 (RNA-6).Genome sequence of PPSMV-I and PPSMV-II have been submitted to GenBank under the following accession numbers. PPSMV-I: Aceria cajani) transmitted PPSMV-I containing four genomic RNAs and PPSMV-II containing six genomic RNAs. A single ORF is encoded by each genomic segment, which exists as a mixed infection in PPSMV-P sub-isolate Chevella, not found in any known plant diseases caused by emaraviruses and could be transmitted independently by eriophyid mite. However, plants infected by PPSMV-B and PPSMV-C isolates showed PPSMV-II alone. Pigeonpea cv. ICP2376, which produces severe mosaic with sterility symptoms when infected with PPSMV-B and PPSMV-C isolates3, produces chlorotic ring spots on the leaves with no sterility when inoculated with PPSMV-P sub isolate-Chevella  with the infected pigeonpea in three locations studied and co- existence of an additional emaravirus (PPSMV-I) with a smaller genome at one location. Initial analysis revealed that susceptible pigeonpea cv. ICP8863 showing typical SMD symptoms (infected by PPSMV-P sub-isolate Chevella) contain two non-homogeneous RNA-3 segments that encode nucleocapsid proteins and a single homogeneous RNA-3 component with PPSMV-B isolate infection. Interestingly we found doublets of the first four RNA segments subsequently with sub-isolate Chevella. The high-throughput small RNA sequencing facilitated to determine that the eriophyid mite , glycoprotein precursor (P2), nucleocapsid (P3) and movement proteins (P4), respectively. PPSMV-II RNA-5 and RNA-6 encoding P5 and P6 proteins, respectively are not related, with unknown functions. Interestingly RNA-1 to 4 of PPSMV-II has high sequence homology with FMV orthologs, and RNA-5 and RNA-6 have modest to weak homology with WoMV and RLBV orthologs. It is possible that the P5 to P6 proteins of PPSMV-II may be involved in the virus life cycle through different roles including transmission by eriophyid miteset al.9 described the PPSMV-1 genome containing an additional RNA (RNA-5). Conversely, we did not find hits specific to RNA-5 in the sequence analysis that could belong to PPSMV-I. Hits recognized in deep sequencing analysis, corresponding to RNA-5 were matched to PPSMV-II, could be confirmed by RT-PCR analysis using RNA-5 specific primers  and probable thirteen conserved residues of cap-binding site in the central region of SRD-3 domains were identified in the RdRp of PPSMV-I and PPSMV-II  generally employ a ‘cap-snatching′ mechanism for viral mRNA transcription-II Fig. ; Table 3ity Figs  and S6.Emaravirus, Tospovirus, and Bunyavirus. The sequence alignment is primarily representing a conserved region of the palm sub-domain, the most highly conserved feature of all known polymerases61. Interestingly, 3D pol of PPSMVs (PPSMV-II shown here) resembles picornaviruses polymerase as closed right hand organization containing the conventional sub-domains, signifying the probable phylogenetic lineage though emaraviruses have negative strand RNA genome. 3D pol showing typical conserved sequence motifs A to E and motif F of the fingers region  analyzed contains the catalytic center represented by sequence SDD (Ser1255-Asp1256-Asp1257 in PPSMV-II). The glycine residue of GDD motif, which is almost universally conserved in RdRps, has been found substituted by serine in the emaraviruses.The amino acid sequence of central polymerase domain SRD-2 Fig.  of PPSMVion Figs  and S5. Cytorhabdovirus, Nucleorhabdovirus, Tenuivirus, Tospovirus and Emaravirus contain enveloped virus particles are known for the presence of glycoprotein-encoding ORFs22. The presence of the membrane glycoproteins plays a role in the viral entry into a host cell and engage in differential roles in protein folding and intracellular trafficking63. RNA-2 of PPSMVs encodes the glycoprotein precursor that is proteolytically cleaved to yield two viral structural glycoproteins; Gn and Gc, are known to be processed post-translationally. There is a modest amino acid sequence similarity (about 40%) of the precursor protein between PPSMVs and with other emaraviruses. RLBV and WMoV are the least comparable ones. The three-dimensional structure of the precursor protein with unique folding showing the characteristic features described previously. The precursor is shaped like a croissant with a panhandle . 3D structures of the glycoprotein of FMV-JS1 and RRV were found identical with the PPSMVs (data not shown). The structural similarities suggest a common mode of activation for the above emaraviruses despite low sequence homology.Plant viruses belonging to genera dle Fig.  sharing 134–140, NRLA183–186 and GYEF204–207 with the other viruses of the genus Emaravirus as reported16, predicted to be associated with RNA-binding27  specific ‘32-kDa’ protein coding RNA to identify SMD. We noticed that these primers explicitly detect the PPSMV-I and not PPSMV-II in the PPSMV-P sub-isolate Chevella (Supplementary Table The putative nucleocapsid proteins (NCP) encoded by RNA-3 of the PPSMVs with a predicted size of 34.6 and 35.0 kDa, and the 3′ terminal ends of RNAs contain unusually long NTRs. The amino acid sequence of PPSMV-I NCP (34.6 kDa) has modest homology with PPSMV-II (35.0 kDa) and FMV, and weaker homology with the P3 proteins of other emaraviruses. Whereas PPSMV-II showed higher homology with the P3 of FMV (80.0%) and RRV (62.0%). The NCP share a set of conserved amino acid sequence motifs, NVLSFNKet al.27 provided evidence for localization of RLBV P4-GFP fusion protein in the cell wall and the plasmodesmata. Furthermore, emaraviral P4s are capable of rescuing the cell-to-cell movement of a defective Potato virus X by RLBV53 and FMV54 P4s, suggesting that P4 of these viruses are movement proteins and are interchangeable. The multiple sequence alignment of P4 of PPSMVs with other emaraviruses excluding the EMARaV, revealed their overall limited similarity apart from considerable conservation comprising hydrophobic amino acids in the central domain57  are identical to 30 K movement protein . However, the authenticity of this possible similarity has not been determined53. Secondary structures of PPSMVs in this region contain two long α-helices interconnected with long coils.The causal agent requires a genomic entity for facilitating the spread of infection. The RNA-4 of both the PPSMVs encodes respective P4 proteins assumed to be involved in the cell-to-cell movement of the virus. McGavin, n57 Fig. . The sec57 Table  forms thein Fig. 53,55–57.tic Fig.  and sequ8, Koch’s postulates are yet to be fulfilled to recognize the sterility mosaic virus as causal organism of SMD. Clear association of viral RNAs especially PPSMV-II and its consistent presence in all locations studied, could help to rationalize that PPSMV-II could be the actual causal organism although this will be confirmed from the reaming locations. To hypothesize the co-evolution of PPSMVs in one host is still a question that needs to be addressed along with relationship between them in terms of pathogenesis with more experimentation. The identification of these diverse viruses provides a new perspective on the evolutionary origins of multipartite viruses with negative-sense RNAs. In particular, PPSMV-I that is different from PPSMV-II, occupying a phylogenetic position with RRV, RYSaV and EMARaV, containing genomes with four RNA components.Although the first indication of the viral nature of SMD had emerged in 20033. The same nomenclature was used to describe PPSMV isolate, at a given location66. These variants are referred in general terms as isolates; PPSMV-P , PPSMV-B  and PPSMV-C . In addition to Bengaluru and Coimbatore locations, we collected SMD affected leaf samples from Chevella village area (Telangana state), 45 to 50 kilometers away from Patancheru. For sample collection, no specific permission was required. Our study revealed that plants from Chevella were infected with PPSMV-P isolate that contains PPSMV-I and PPSMV-II, named as PPSMV-P sub-isolate Chevella  with typical SMD symptoms were identified, and young leaves were collected. The infected samples were labelled as Mg-1, Mg-2, and Alr representing the three pigeonpea fields. Similarly, second set of samples was collected from Karnataka state around University of agriculture sciences, GKVK campus. Infected leaf samples (PPSMV-B isolate) from Bng-1 (Rajankunte location), Bng-2  and Bng-3 (UAS) were collected as unknown pigeonpea local cultivars. The third set of infected samples was collected from Tamil Nadu Agriculture University (TNAU) fields located at Coimbatore  has been reported affecting pigeonpea plants in the fields69.We surveyed the three above mentioned locations and collected infected leaf samples. Pigeonpea plants  containing ethidium bromide (10 mg/ml) and visualized in gel doc system . Cucumber mosaic virus (CMV) dsRNA, extracted from N. tabacum cv Xanthi was used as dsRNA size marker. Total nucleic acid from infected leaves collected from the three locations and mite inoculated  were isolated by CTAB method72. The quantity of RNA obtained was estimated by spectrophotometer . The protocol resulted in the isolation of 10–20 µg RNA/150 mg of leaf sample.Pigeonpea leaves with mosaic mottling symptoms and non-symptomatic leaf samples as controls of each location, collected from Chevella , Bengaluru  and Coimbatore  as well as mite inoculated pigeonpea cvs. ICP8863 and ICP2376 (35 dpi) were used to isolate dsRNA as described8 to determine the specificity of these primers to PPSMV-I and PPSMV-II. About 1 µg of total RNA from SMD affected Mg-1, Mg-2, Bng-1, Bng-2, Bng-3, CO-5 CO-6 and pigeonpea cv. ICP8863 and mite inoculated ICP8863 (PPSMV-P sub-isolate Chevella) leaf tissues were used as template for the assay. The protocols for RT reaction, first strand cDNA synthesis and polymerase chain reaction of the primers were followed as described8. Total RNA from the non-symptomatic pigeonpea cv. ICP8863 and unknown cultivar  from the three locations were used as control. RT-PCR amplicons were fractionated on 1% agarose gel containing ethidium bromide and visualized in a gel doc .SMV-1 and SMV-2 oligonucleotide primers (known for the amplification of a 321-bp product) were developed from partial nucleocapsid sequence  of PPSMV-P72. Total RNA (7 µg) was fractionated on 12% polyacrylamide gel electrophoresis (PAGE), and the RNA molecules with size ranging 18–30 nts were eluted from the gel. This small RNA population (50 ng) was used for library preparation  by using NEXTflex small RNA library preparation kit V3  at Genotypic Technology genomics facility, Bengaluru. The procedure (in brief) for library preparation was as follows: adapters were ligated at 3′ and 5′ ends of the purified small RNAs. Ligated RNAs were reverse transcribed, and the resulting cDNA library was enriched by PCR (12 cycles). Further resolution, size selection (140–160 bp) of enriched cDNA library was done on 8% PAGE and the fragments were eluted from the gel. The prepared library was quantified by using Qubit fluorometer  and the quality was determined by using high sensitivity Bioanalyzer chip . sRNA (10 nM) enriched fraction was used for single end read sequencing on Illumina GAIIx platform.Small RNA population was separated from total RNA extracted from SMD affected leaves of Mg-1, Mg-2 (from Chevella) and Bng-1  by CTAB methodhttp://genotypic.co. in/SeqQC.html). Adapters were trimmed from the filtered reads used for subsequence assembly. To enrich virus derived small interfering RNA (vd-siRNA) pool, the obtained reads were mapped on pigeonpea genome to remove host specific sRNAs by using Bowtie 2.1.0 program73. Filtered unaligned reads were used for generation of contigs. De novo assembly of short reads was performed by using Velvet -1.2.10 based on de Bruijn graph74; with k-mer17 (often we noticed large k-mer resulting high specificity in contigs). For generation of contigs corresponding to viral genome siRNA reads 20–22 bp length were used for viral genome assembly. The identity of these contigs was established by Blast and BlastX search in NCBI database  La Crosse virus (LACV); genus Tospovirus: represented by Tomato spotted wilt virus (TSWV) and Groundnut bud necrosis virus (GBNV); genus Hantavirus: Hantaan virus (HTNV), Puumala virus (PUUV); genus Tenuvirus: Rice streak virus (RSV), Rice grassy stunt virus (RGSV) genus: Nairovirus: Dugbe virus (DUGV), Crimean-Congo hemorrhagic fever virus (CCHFV). Multiple alignments of protein sequences were performed using ClustalW75. Phylogenetic trees were constructed by MEGA6.076; using neighbour-joining method77 employing maximum likelihood (ML) criterion with 1000 bootstrap replicates, to examine the evolutionary history and evolutionary distance78.To examine the phylogenetic relationship of PPSMVs with related viruses, amino acid sequences of RdRp (P1), NCP (P3) and MP (P4) of PPSMV-I and additional RNA-5 (P5) of PPSMV-II and the orthologs of known emaraviruses were aligned with viral proteins of representative members of family 5–20 MPSKTPFSNMPAASKK*) and PPSMV-II (PPII299–314 *STFLPALEADRLASLP) were synthesized. Peptides were synthesized from GL Biochem limited . These peptides were conjugated to keyhole limpet hemocyanin (KLH) at cysteine residue i.e. “*” position which acts as a carrier. The antisera (PPIAb and PPIIAb) were used to detect the specific viruses.The putative nucleocapsid proteins (NCP) of PPSMV-I and PPSMV-II have a close similarity in amino acid sequence. However, we identified specific unique peptide sequences of both the nucleocapsid proteins to generate antisera. Peptide sequences specific to nucleocapsid of PPSMV-I  were injected at two-week intervals. After ten days of the third booster injection, the animal was bled, and antiserum was collected. IgG fraction from the crude antibody sera was purified by IgG purification kit  following manufacturer instructions. The purified antibodies (PPIAb and PPIIAb) were used for the detection of specific PPSMVs by western blotting. For raising antisera, rabbits were used after approval from the Institutional Animal Ethics Committee under Committee for the purpose of control and supervision on experiment on animals (CPCSEA). All the experiments were conducted as per guidelines and regulations of the committee.PPI and PPII synthetic peptides thus developed of PPSMV-I and PPSMV-II were used as an antigen to generate respective antisera using New Zealander white albino rabbits. Pre-immune bleed of 2 ml per rabbit as control serum was collected on day 0, followed by the primary injection. Two hundred microgram (200 µg) of peptide-KLH conjugates emulsified with Complete Freund’s Adjuvant (CFA)  was injected as a primary injection, to eight-week old rabbits by intramuscular injectionshttp://zhanglab.ccmb.med.umich.edu/I-TASSER), on line server was used to study the proteins81 in their tertiary form. The secondary structures of P4 ‘movement protein’ were predicted using PSIPRED server82 (http://bioinf.cs.ucl.ac.uk/psipred/). The outputs of the I-TASSER data of possible predicted models and the molecular graphics are of high quality. These contained full-length secondary and tertiary structure predictions and was subjected to simulations by an open-source viewer, the PyMol (https://www.pymol.org) and Jmol (http://jmol.sourceforge.net), an interactive graphics program84, for illustrating the three-dimensional chemical structures. These programs facilitated to alter the scheme of the images and to characterize these proteins by identifying conserved residues and the motifs in the protein folding.The three-dimensional structures of polymerase  and glycoprotein precursor (P2) proteins of PPSMV-I and PPSMV-II were studied. The conserved amino acid sequence of viral polymerase at N-terminal SRD-1 domain of P1 (amino acid position 66 to 242) and glycoprotein precursor P2 (PPSMV-I 648 amino acids and PPSMV-II 649 amino acids) proteins were used to develop 3D structures with their characteristic folding. I-TASSER, .Supplementary information


The high resolution crystal structure of bacterial ribosome was determined morethan 10 years ago; however, it contains no information on the structure of thelargest ribosomal protein, S1. This unusual protein comprises six flexiblylinked domains; therefore, it lacks a fixed structure and this prevents theformation of crystals. Besides being a component of the ribosome, protein S1also serves as one of the four subunits of Qβ replicase, the RNA-directedRNA polymerase of bacteriophage Qβ. In each case, the role of thisRNA-binding protein has been thought to consist in holding the template closeto the active site of the enzyme. In recent years, a breakthrough was made instudies of protein S1 within Qβ replicase. This includes the discovery ofits paradoxical ability to displace RNA from the replicase complex anddetermining the crystal structure of its fragment capable of performing thisfunction. The new findings call for a re-examination of the contribution ofprotein S1 to the structure and function of the ribosome. Escherichia coli ribosome, but it also has anunusual structure [Located on a small 30S) subparticle, protein S1 is not just the largestprotein of the tructure , 3, prot0S subpartructure , 5 almostructure  called Otructure ).rpsA gene are lethal [in,which lacks a fixed conformation.Protein S1 is vital to the cell, since deletions of or amber mutations in itse lethal ,9. Howee lethal .Establie lethal . ApparenUndoubtedly, protein S1, with its strong RNA-binding capacity, is important forthe initiation of translation . HoweverIn addition to protein synthesis, protein S1 contributes to other processesthat occur in the cell, both on the ribosome and outside of it . One of Recent results of studies of protein S1 within Qβ replicase havesignificantly advanced our understanding of the structure and function of thisprotein. These results are the subject of this review.Figure).Subsequent structural studies have confirmed this conclusion with further refinement: the N-terminalOB domain (OB1) contains 4 rather than 5 β-strands[The “classic” OB domain consists of ≈70 amino acid residuesand comprises a Greek key barrel of five β strands usually covered by an α-helix.OB do m-strands, 16-18.The name OB domain suggests that it would display affinity for polynucleotides.Indeed, RNA-binding properties were demonstrated for domains OB3 to OB6, 16,19.A special role belongs to the N-terminal segment preceding domain OB1 andconsisting of 20 amino acid residues. In the unbound state, this segment isunstructured  or theQQβ replicase is famous for its unique ability to rapidly amplify RNA.Similar to PCR, the reaction follows an exponential kinetics, since both theoriginal template and its complementary copy serve as templates in the nextamplification round. Therefore, the number of templates increases two-fold ineach round, as long as the replicase remains in molar excess over RNA. However,unlike PCR, the reaction is isothermal: there is no need to increase thetemperature to melt the duplex, since the immediate reaction product comprisesa single-stranded RNA. How does Qβ replicase manage to copy RNA accordingto the principle of complementarity, yet preserve the single strandedness ofthe template and the nascent strand, remains one of the unsolved mysteries ofQβ phage replication .In 1972, Weissmann and colleagues published a paper arguing Weissmann and his colleagues obtained their results when the Qβ replicaseand template concentrations were similar and no exponential synthesis of RNAwas possible. We found that an entirely different result was obtained when thereplicase was in large excess. In that case, protein S1 dramatically stimulatedthe replication of both the Qβ RNA and RQ RNAs. In the presence of proteinS1, most of the product was found to consist of single-stranded RNA, whereas inits absence the product was double stranded . It seemTo verify this assumption, we examined the proportion between thesingle-stranded and double stranded forms of RNA during the elongation in thepresence and absence of protein S1. As a template, we used the 4217 nt-longQβ RNA plus strand, whose copying takes about 4 min at 30°C. To avoidan overestimation of the amount of double-stranded RNA due to its formationupon denaturation of the replicative complex , we testThis result showed us that protein S1 catalyzes the release of asingle-stranded product from the active site of Qβ replicase: in otherwords, it acts as a termination factor. This function seems to be performed byprotein S1 during the replication of any legitimate template  of Qβ reThus, protein S1 performs two distinct functions during the replication ofQβ RNA: the function of a termination factor common to all legitimatetemplates, and a special function performed during initiation on the plusstrand, previously thought to be its only function.E. coli cells, the anti-saltfunction is as important as the termination function because the cytoplasmicconcentration of monovalent cations is even higher than 150 mM [Our direct measurements of the rate of initiation on the plus strand of QβRNA showed that the requirement for protein S1 is not absolute. In a low saltbuffer (50 mM NaCl), initiation in the absence of protein S1 occurred almost asrapidly as in its presence. However, the addition of 50 mM ammonium sulfateresulted in an almost complete inhibition of initiation in the absence ofprotein S1 and only in a two-fold inhibition in its presence (initiation onother legitimate templates was inhibited approximately two-fold regardless ofthe S1 presence) . The addn 150 mM .Figure).It turned out that fragment OB1-2 can replace protein S1 at the terminationstep, while fragment OB1-3 can replace it in the protection of theinitiation step against salt [In order to determine whether all domains of protein S1 are necessary for it toperform its functions, we cloned and purified a series of N-terminal S1fragments containing an increasing number of OB domains fragments can replace protein S1 in all its functions. Previou1-3  while th1-3 [1-2 , the sam1-3 [1-2 . In addi1 and OB2 produces almost noeffect on the structure of the Qβ replicase core. These domains interactwith the β-subunit in the region of the “fingers” domain,which participates in the formation of the active site of replicase, thebinding of RNA, and the unwinding of the complementary strands of thereplicative complex [1 and OB2 and forms a numberof contacts with the β-subunit and EF-Tu [The binding of domains OB complex , 31. Thend EF-Tu . These cnd EF-Tu .1 and OB2 do nothave basic and aromatic amino acid residues capable of forming bonds with thephosphates and nitrogenous bases of RNA, and therefore cannot interact with RNA[2 and presented NMR data demonstrating the ability of this domainto bind RNA [Although the two research groups reported nearly identical structural data, 18, thewith RNA. On the 1-3, rather than OB1-2, to replaceprotein S1 in the anti-salt function means that the third OB domain plays amajor role in the interaction with the M site, whereas domains OB1and OB2 are needed as far as they form a link between domainOB3 and the replicase core.This function is similar to the one commonly assigned to protein S1 whenconsidering its role in translation. The difference is that, while protein S1stimulates initiation of the translation of the vast majority of mRNAs ,it prom3 plays a key role in both the initiation andtermination steps, by interacting with the M site of the Qβ RNA plusstrand during initiation, and with the newly synthesized RNA strand duringtermination [1-2, in which domain OB3 was absentand, therefore, could not participate. Incidentally, neither did they noticethe fact that the same paper demonstrated the key role of domain OB3at the initiation step one and a half year prior to their publication.The Japanese group proposed a mechanism for protein S1 action in which themobile domain OBmination , 34. Altmination , they ap2 adjoins a similarly charged region on the surface of the βsubunit and forms a continuous, positively charged tract leading from theopening through which the synthesized strand is thought to be released from theactive site [The Danish-Russian paper reports that the positively charged region of domainOBive site . Probabl1-2 complex represents the openconformation. In this regard, we would note that protein S1 catalyzes thetermination step even if it is added during the elongation step, but before itscompletion [However, it would be premature to propose a detailed mechanism of termination,since the product is terminated from the closed conformation of Qβreplicase , whereasmpletion . In otheAs a result of the initiation on a legitimate template in the presence of GTP,Qβ replicase acquires a closed conformation from which neither thetemplate nor its complementary copy can dissociate until the elongation iscomplete . This enIn conclusion, we would like to note that the discovery of the ability ofprotein S1 to displace RNA from a complex changes the basic paradigm accordingto which the only purpose of this protein is to hold RNA near the active siteof an enzyme, whether it is a replicase or a ribosome[


Multidrug transporters are membrane proteins that catalyze efflux of antibioticsand other toxic compounds from cells, thereby conferring drug resistance onvarious organisms. Unlike most solute transporters that transport a single typeof compound or similar analogues, multidrug transporters are extremelypromiscuous. They transport a broad spectrum of dissimilar drugs and represent aserious obstacle to antimicrobial or anticancer chemotherapy. Many challengingaspects of multidrug transporters, which are unique, have been studied indetail, including their ability to interact with chemically unrelated drugs, andhow they utilize energy to drive efflux of compounds that are not onlystructurally but electrically different. A new and surprising dimension of thepromiscuous nature of multidrug transporters has been described recently: theycan move long molecules through the membrane in a processive manner. H+, interior negative and/or alkaline). By this means, the freeenergy released by the inward downhill flux of protons with ∆µ̃H+ is utilizedby the multidrug transporter to drive efflux of the drug . One critical featureof these so-called antiporters is that they never allow formation of a continuouschannel across the membrane; the transport pathway is always blocked from either theintracellular or extracellular side of the membrane . This tight seal iscrucial for preventing non-specific leakage of ions through the antiporter, which maycollapse ∆µ̃H+. However, the architecture of these proteins restricts thesize of the transported substrate: only those that can be accommodated structurally inthe occluded state can be transported. Therefore, current wisdom suggests thatprocessive transport of long substrate molecules in several cycles should beimpossible.Except for their promiscuous nature, multidrug transporters probably work like any othertransporter. Phylogenetically, they are members of large solute transporter familiesthat comprise mostly substrate-specific members. For most transporters, including thosethat belong to the MFS superfamily of secondary transporters, a mechanism is suggestedin which binding of substrate occurs on one side of the membrane, followed by transitioninto a substrate-occluded state and its release on the other side. Substrate binding andrelease are achieved by alternating access of binding sites to either side of themembrane by reciprocal opening and closing of the occluded conformer on the opposingsides of the membrane . In addition, many multidrug transporters can also bindhydrophobic drugs from within the membrane bilayer and export them to the medium.Secondary efflux in bacteria is often driven by the proton electrochemical gradient. This unique architecture is a vital clue for understanding theunderlying transport mechanism.The current study by Fluman + stoichiometry, the divalent drugs exhibit a 1:2stoichiometry. Since MdfA functions as a monomer, the finding suggests that divalentdrugs are likely transported in a processive-like mechanism involving two consecutivetransport cycles in which each cationic moiety is transported as if it were a separatesubstrate . Further studies lend strong support to this model by showing thatother divalent substrates with a similar architecture are transported with adrug:H+ stoichiometry greater than unity not only by MdfA, but also byanother multidrug antiporter. Collectively, the study indicates that multidrugantiporters are inherently capable of processivity.Studies of the transport mechanism establish that unlike the usual MdfA substrates, whichhave a 1:1 drug:HAs discussed above, it has been generally believed that processive solute transport isunlikely, since all transporters are tightly sealed either on the extracellular orintracellular side of the membrane. It now seems that at least narrow carbon chains thatbridge positively charged moieties can be threaded through the pathway withoutcompromising the seal . Such carbon chains are particularly suitable for thistask, because they are narrow and may be accommodated inside the protein with relativelyminimal structural perturbation. Another important aspect is the hydrophobic nature ofthe carbon chain, which may allow favorable hydrophobic interactions with the interiorof the antiporter, as happens with lipids. Theoretically, the carbon chain of thesesubstrates mimics the carbon chains of lipids, which are known structural, chemical, andeven functional counterparts of integral membrane proteins, including solutetransporters.In summary: (i) Multidrug antiporters appear to have an even broader substrate-profilethan previously recognized, and the architecture of the substrate may alter thestoichiometry of the transport mechanism. (ii) Antiporters are capable of processivetransport, and they may even be able to transport much larger molecules or polymers,utilizing the same transport mechanism as they use for small solutes.


The growing knowledge on the mechanisms of gene silencing and gene regulation by non-coding RNAs (ncRNA), mainly small interfering RNA (siRNA) and microRNA (miRNA), is providing a significant boost to the development of new therapeutic strategies for the treatment of cancer. However, the design of RNA-based therapeutics is hampered by biopharmaceutical issues, thus requiring the use of suitable delivery strategies. In this regards, lipid nanovectors have been successfully investigated to deliver RNA in different forms of cancer. Compared to other biomaterials, lipids offer advantages such as biocompatibility, biodegradability, easy production, low cost, limited toxicity and immunogenicity. The possibility to formulate these materials in the form of nanovectors allows overcoming biopharmaceutical issues associated to the therapeutic use of RNA, with the possibility to target tumors. This review takes stock of the main lipid nanovectors proposed to deliver ncRNA. For each considered delivery strategy, the rational design and the most meaningful in vitro and in vivo results are reported and discussed. RNA interference (RNAi) is a widespread natural phenomenon conserved across different species  induced by exogenous RNA oligonucleotides  and endogenous small RNA species, such as micro RNA (miRNA) and piwi-interacting RNAs . From thCancer is certainly one of the main targets for RNAi-based therapies. Indeed, oncogenes, mutated tumor suppressor genes, and several other genes involved in tumor progression, are potential targets for gene silencing . CompareThe rapid progresses in the field of RNAi mechanisms and the comprehension of the pivotal role of non-coding RNA (ncRNA) in cancer development and progression has giving rise to positive expectations concerning the discovery of new therapies based on ncRNA molecules, for different forms of cancer. However, the development of RNA-based therapies is hampered by serious drawbacks . NucleicO-methyl, etc.), the number of modified nucleotides, and the position of modified nucleotides on the strand , have to be taken into account to optimize the interaction with the intracellular target and the consequent knockdown efficiency -N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dioleoyl-3-trimethylammonium-propane  DOTAP are largely the most used to prepare CLs -carbamoyl] cholesterol (DC-chol) and 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate, (DOSPA). Among the above-mentioned cationic lipids, DOSPA and DOGS are multivalent cationic lipids characterized by the presence of different ammonium groups, which contribute to a more efficient packing and binding of nucleic acids, compared to monovalent cationic lipids  (DSPE-PEG) lipid. The transfection of let-7a-miR in lung cancers cells was higher compared to untargeted lipoplexes . Th. ThN,N-doperties . Thus, ioperties . Among doperties ,69. The operties ,71,72 wioperties . In anotoperties .Altogether, these results demonstrate that SNALPs can be considered an evolution of cationic lipids, overcoming some issues associated to the latter. Indeed, differently than lipoplex, physical characteristics of SNALPs are quite independent on the preparation conditions, while maintaining the transfection efficiency of the CLs.Core/shell lipid-based nanovectors have been proposed to deliver siRNA and miRNA. The research group of Huang gave a significant contribution to the development of this strategy by developing nanoparticles obtained by RNA complexation with polycation, then covered by a lipid shell. More in detail, in the first studies, RNA  was condensed into the NP core by mixing with protamine, a highly positively-charged peptide already used as transfection reagent for nucleic acids. The high molecular weight DNA was reported to improve the core compaction, while the use of calf thymus DNA, compared to plasmid DNA, was preferred for the limited amounts of immunostimulating CpG motifs . The resw/w) solid fat dispersed in an aqueous phase with the help of surfactants, at a concentration of about 0.5%–5% [Kim et al. proposed to use solid lipid NPs (SLNs) for the delivery of RNA. SLNs are NPs composed of approximately 0.1–30 , involves as first step the direct conjugation of siRNA to PEG  or lipidIn a third approach, the direct conjugation of siRNA with lipids followed by its incorporation in PEG-PE micelles was proposed to completely avoid the use of cationic polymers . By a siEngineered micelles able to combine in one delivery system a number of distinct functional components have also been proposed. In the so-called multifunctional NPs, each single compound plays its role to achieve the maximum therapeutic performance. Such micelles can contain, at the same time, ligands and/or cell penetrating peptides on their surface for selective targeting and enhanced intracellular delivery of a combination of drugs and nucleic acids. In addition, a number of internal and external stimuli for triggered release of the drugs at disease sites have been added. Using this concept, Zhu et al. proposed multifunctional enzyme-sensitive micelles for the co-delivery of siRNA and paclitaxel . In thisDespite, for many years, lipid nanocarriers being confined to laboratory use, at the moment different clinical trials have demonstrated the possibility to use these systems to deliver ncRNA in humans. Clinical trials based on RNAi and involving the use of formulations based on lipid vesicles are summarized in ® for the delivery of miR-34a in cancer. SMARTICLES® are liposomes with amphoteric lipids that offered the possibility to be positively charged at acid pH and anionic at higher pH. This peculiarity allowed a high encapsulation of miRNAs, while the anionic charge present on the surface of the vectors at physiological pH prevents vesicle aggregation. Many studies have already reported promising results with the treatment of vectors encapsulating miR-34a in an orthotopic model of hepatocellular carcinoma [®, namely MRX34, are under clinical evaluation (phase I). Another clinical study ,  involvesarcinoma ,102,103.NCT02110563 is a phase 1 study to assess the safety and tolerability of the anticancer drug DCR-MYC. This formulation is based on a lipid nanoparticle suspension containing a novel synthetic double-stranded RNA (siRNA) against the activation of oncogene MYC involved in the growth of many hematologic and solid tumor malignancies, such as solid tumors, multiple myeloma, non-Hodgkins lymphoma, and pancreatic neuroendocrine tumors. In this study, the nanocarrier is reported as “stable lipid particles”, presumably a technology close to the SNALPs. This study is currently recruiting participants. The same company organized a phase I clinical trial study (NCT02314052) focused on the investigation of the same anticancer drug (DCR-MYC) on patients with hepatocellular carcinoma. Final data collection is scheduled for October 2016. Although any product is, at the moment, approved for clinical use, there is a growing number of clinical studies, suggesting that lipid vesicles could soon enter into clinical practice, paving the way for the therapeutic use of ncRNA for cancer.The use of lipid nanovectors to deliver nucleic acids is not recent and their potential as transfection agents is well established. However, the instability of lipid nanovectors in the biological environment, and the variability of the delivery efficiency, have limited for many years their in vivo application. However, the large number of research studies in the field have been focused on optimizing RNA transfection efficiency, as well as to limit instability in vivo and rapid clearance. In recent years, more clinical trials focused on the use of siRNA and miRNA as new therapeutics, and involving lipid-based nanovector as delivery systems, started. Although further optimization of the lipid nanovectors is still possible, we are certainly very close to the use of these formulations in therapy, thus opening new perspectives for cancer therapy. Moreover, due to the possibility to easily modify the lipid particle with targeting moieties, and taking into account the promising results obtained so far, more selective RNA-based therapy will be possible in the not-too-distant future.


The aims of the present study were to evaluate the predictive value of the platelet-to-lymphocyte ratio for peritoneal metastasis in patients with gastric cancer and to construct an available preoperative prediction system for peritoneal metastasis. A total of 1080 patients with gastric cancer were enrolled in our study. The preoperative platelet-to-lymphocyte ratio and other serum markers and objective clinical tumor characteristics were evaluated by receiver operating characteristic curves. A logistic analysis was performed to determine the independent predictive indicators of peritoneal metastasis. A prediction system that included the independent predictive indicators was constructed and evaluated by receiver operating characteristic curves. Based on the receiver operating characteristic curves, the ideal platelet-to-lymphocyte ratio cutoff value to predict peritoneal metastasis was 131.00. The logistic analysis showed that the platelet-to-lymphocyte ratio was an independent indicator to predict peritoneal metastasis. The area under the receiver operating characteristic curve was 0.599. When integrating all independent indicators , the prediction system more reliably predicted peritoneal metastasis with a higher area under the receiver operating characteristic curve (0.769). The preoperative platelet-to-lymphocyte ratio was an indicator that could be used to predict peritoneal metastasis. Our prediction system could be a reliable instrument to discriminate between patients with gastric cancer with and those without peritoneal metastasis. Gastric cancer (GC) is one of the most common malignant tumors and is the main cause of cancer-related mortality globally, particularly in Asian countries. Owing tGastrectomy and regional lymphadenectomy are the only curative treatment for GC, but these treatments are insufficient for GC with peritoneal metastasis. Furthermore, a preoperative diagnosis of peritoneal metastasis is necessary for the proper selection of neoadjuvant therapy and convComputed tomography (CT), magnetic resonance imaging, and positron-emission tomography integrated with computed tomography (PET-CT) are widely used to predict peritoneal metastasis; however, all have limited success because of their low sensitivity and specificity. Other rIn addition, the use of systemic inflammatory response (SIR) markers such as platelets, neutrophils, lymphocytes, and monocytes has also been widely reported. FurthermTherefore, the aims of this study were to determine the relationship between peritoneal metastasis and preoperative inflammatory markers, and to construct a more useful score system to help improve preoperative diagnostic accuracy by combining the independent related factors. We found that the preoperative platelet-to-lymphocyte ratio was useful for predicting peritoneal metastasis and that our prediction system could be a reliable instrument to discriminate between patients with GC with and without peritoneal metastasis. An individualized multimodality treatment could thus be provided to patients with GC.In this retrospective analysis, the data of 1199 patients with GC who underwent GC surgery at the First Affiliated Hospital of Wenzhou Medical University  from January 2009 to May 2013 were reviewed. All the patients have received Preoperative CT scan and were CT negative for peritoneal metastasis. The following information was collected and recorded: patient’s personal information , tumor characteristics , and blood routine index . The histopathological types were defined as “well differentiated” , “moderately differentiated” , “poorly differentiated” , or “undifferentiated” . The pathological type was divided into the ulcerative group and the nonulcerative group. The diagnoses were confirmed in all patients by histological examination. The exclusion criteria included (1) history of gastric resection (8 patients), (2) liver disease such as cirrhosis (14 patients), (3) history of other malignancies (15 patients), (4) severe bleeding and autoimmune disease (22 patients), (5) preoperative chemoradiotherapy (2 patients), (6) severe inflammation or hematological system diseases (34 patients), and (7) distant metastasis excluding abdominal metastases (6 patients). Finally, 1080 patients were eventually enrolled in this study. This study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University. All patients were aware of the research and signed the informed consent form.According to the Japanese Gastric Cancer Treatment Guidelines (15th edition), the diagnostic criteria for peritoneal metastases were as follows: metastases limited to the greater omentum, lesser omentum, anterior lobe of the transverse mesocolon, pancreatic capsule, and spleen; and metastasis in the upper abdominal peritoneum . These patients with peritoneal metastases were diagnosed by intraoperative frozen section and postoperative pathological diagnosis.Blood specimens, which were obtained within 7 days before surgery, were preserved in tubes containing ethylenediaminetetraacetic acid. A hemocounter  was used to calculate the differential leukocyte, neutrophil, platelet, and monocyte counts. We plotted the receiver operating characteristic (ROC) curves. The values with the maximal Youden index were chosen as the cutoff points of the preoperative PLR and NLR. Thus, the patients were divided into two groups, based on the cutoff point.U test was used to compare the non-normal distributed variables between the peritoneal metastasis group and the non-peritoneal metastasis group. ROC analysis was conducted to determine the performance of the variables. The relationship between clinicopathologic characteristics and the NLR or PLR were analyzed using the χ2-test. In addition, the χ2 test was used for univariate analysis of peritoneal metastasis. Based on the univariate analysis results, a multivariate logistic regression analysis was used to calculate the odds ratio (OR) and 95% confidence interval (CI) of the confirmed independent variables. The area under the ROC curve (AUC) was used to compare the scoring system to other clinicopathologic characteristics. A value of P < 0.05 was statistically significant. Statistical analyses were performed using SPSS software .We performed the Kolmogorov–Smirnov test to determine the normality of continuous parameters such as the neutrophil count, lymphocyte count, platelet count, monocyte count, PLR, NLR, and MLR. The mean and standard deviation values were used for the normal distributed data, whereas the median and interquartile range values were used for the non-normal distributed data. The Mann–Whitney As displayed in P = 0.035). By contrast, the lymphocyte count was higher in patients with GC without peritoneal metastasis (P = 0.034). As is shown in P < 0.05). The ROC curves were then used to further analyze the variables that had a significant difference. As As displayed in The cutoff values of the PLR and NLR for peritoneal metastasis, which were calculated based on the ROC curve, were set to 131.00 and 1.95, respectively . The dia2 = 5.142, P = 0.0162), tumor size , invasion depth , pathological type , lymphatic invasion , NLR , and PLR  were significantly different between patients with GC with and without peritoneal metastasis. By contrast, no significant differences were detected between the two groups with regard to the body mass index, American Society of Anesthesiologists (ASA) score, sex, CEA level, and CA-199 level. Furthermore, these variables were potential independent risk factors. Logistic regression analyses were performed. As P = 0.018), invasion depth , lymphatic invasion , and pathological type . Thus, these factors were the independent predictive indicators of peritoneal metastasis.The chi-square test was used to examine the relationship between clinicopathologic characteristics and peritoneal metastasis. As The independent preoperative predictors were selected, according to the multivariate logistic regression analysis. As is shown in Peritoneal metastasis is usually associated with a poor prognosis in patients with GC. It may At present, imaging techniques such as ultrasonography and CT are the most commonly used means to predict peritoneal metastasis. However, they are not reliable in accurately predicting peritoneal metastasis because peritoneal metastases are not always detectable. The sensitivity of ultrasonography is only 0.09 , whereas that of CT is 0.33 . By contBlood tumor markers such as CEA and CA-199 are commonly used clinically today to help diagnose gastrointestinal tumors. However, the sensitivity of these indicators for diagnosing peritoneal metastasis is insufficient . Thus, iTherefore, the relationship between a tumor and the body inflammatory status has received increasing attention,21,22. TBased on the aforementioned information, we focused on the significance of systemic inflammatory response markers that would be convenient and inexpensive to detect preoperatively. Neutrophils can suppress the activity of immunologic effector cells such as natural killer cells and lymphocytes to inhibit the immune system,27. HoweIn the present study, we first tried to determine whether preoperative circulating monocytes, the PLR, and the MLR could be used to predict peritoneal metastasis. The neutrophil, platelet, and monocyte counts were higher, whereas the lymphocyte count was lower, in the peritoneal metastasis group than in the non-peritoneal metastasis group; however, the differences in the neutrophil and monocyte counts were not statistically significance. For this reason, we speculated that peritoneal metastasis may not be sufficiently sensitive to reflect these differences and the number patients with peritoneal metastasis who were enrolled in the study was insufficient. The PLR and NLR, which amplify single distribution differences, were fortunately significantly different between the two groups, as was expected. The ROC curve further indicated that the PLR and NLR were better predictive markers for peritoneal metastasis with a higher sensitivity and specificity.No previous study has evaluated the relationship between the PLR and peritoneal metastasis; therefore, we determined the cutoff value by using ROC curves. Thus, patients were divided into the high PLR/NLR group and the low PLR/NLR group. Further study found that the NLR and PLR were closely associated with age and several tumor characteristics. This finding was consistent with a previous study showing that older age was an independent risk factor for high NLR. HoweverTo make a more accurate preoperative diagnosis for peritoneal metastasis, we also investigated the significance of tumor characteristics such as tumor size, pathological type, histopathological differentiation, which could be easily obtained by preoperative endoscopy. Our ROC analysis demonstrated that significant predictors such as tumor size and pathological type did have predictive value for peritoneal metastasis. However, similar to the aforementioned preoperative serum markers, the diagnostic accuracy of each single parameter was insufficient. Therefore, we performed a multivariable logistic regression analysis. Through this, we identified the PLR, invasion depth, lymphatic invasion, and pathological type as the preoperative peritoneal metastatic predictors.A combined preoperative score system that included the aforementioned tumor characteristics and inflammatory index was ultimately constructed. A combined ROC analysis was later performed to demonstrate whether the scoring system had improved diagnostic accuracy for peritoneal metastasis. As expected, the scoring system, which consisted of independent preoperative predictors, improved the accuracy (AUC = 0.769) with higher sensitivity (84.09%) and specificity (82.63%). Thus, we concluded that the preoperative score system has a potential diagnostic value for peritoneal metastasis.Before this study, several other predictive systems had been constructed to predict peritoneal metastasis. For example, a predictive equation, which included the variables of tumor size, tumor stage, lymph node invasion, and histological differentiation, had a specificity and sensitivity of 78.3% and 88.5%. HoweverCompared to these studies, our score system, which combined convenient preoperative values such as the depth of invasion, pathological type, lymphatic invasion, and PLR values, provided a credible predictive power .Therefore, taking clinical practicability and accuracy together, our system could serve as a credible tool to predict peritoneal metastasis preoperatively, especially in imaging-negative patients.This present study still has some limitations that should not be neglected. This was a retrospective study; therefore, tumor characteristic parameters such as invasion depth, lymphatic invasion, and pathological type were obtained postoperatively. These parameters could be obtained preoperatively by endoscopy; however, the results may be inaccurate. Futhermore, because of the incomplete data and the partial loss of our follow-up data, we did not add the analysis of prognosis in our study. Moreover, only 101 patients with peritoneal metastasis were enrolled in our study and all of these patients were from a single hospital. Therefore, our findings still require validation by a large prospective multicenter studies. Most importantly, the sensitivity of our score system was not sufficiently high to diagnose peritoneal metastasis. Therefore, it can only be used as an auxiliary tool.This is the first study that has attempted to investigate the relationship between the PLR and peritoneal metastasis. We found that the PLR, which was independently associated with peritoneal metastasis, could be used to predict the condition of peritoneal metastasis and thereby guide individualized treatment. Furthermore, the PLR combined with tumor characteristics such as invasion depth, lymphatic invasion, and pathological type could obviously improve the diagnostic accuracy. Moreover, this new scoring system is an economical and convenient tool that could be used to predict peritoneal metastasis, which will have an important role in imaging-negative patients.S1 File(XLSX)Click here for additional data file.