Here we disclose crystal structures of RNA hybridizing with SNA and with L-aTNA. The heteroduplexes show unwound right-handed helical structures. Unlike canonical A-type duplexes, the base pairs in the heteroduplexes align perpendicularly to the helical axes, and consequently helical pitches are large. The unwound helical structures originate from interactions between nucleobases and neighbouring backbones of L-aTNA and SNA through CH–O bonds. In addition, SNA and L-aTNA form a triplex structure via C:G*G parallel Hoogsteen interactions with RNA. The unique structural features of the RNA-recognizing mode of L-aTNA and SNA should prove useful in nanotechnology, biotechnology, and basic research into prebiotic chemistry.Xeno nucleic acids, which are synthetic analogues of natural nucleic acids, have potential for use in nucleic acid drugs and as orthogonal genetic biopolymers and prebiotic precursors. Although few acyclic nucleic acids can stably bind to RNA and DNA, serinol nucleic acid (SNA) and L-threoninol nucleic acid (L- aTNA/RNA and SNA/RNA heteroduplexes to further our structural understanding of how synthetic nucleic acids hybridize with natural nucleic acids.Serinol nucleic acid and L-threoninol nucleic acid can bind to RNA and DNA, endowing them with potential as nucleic acid-based drugs. Here the authors prepare single crystals of L- In the past decades, many nucleic acid analogues have been developed. Artificial analogues with 2′-ribose modifications such as 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and locked nucleic acids (LNAs) are used in the nucleic acid-based drugs and drug candidates14. The increased binding affinities of these modified analogues for RNA result from the C3′-endo conformation of pentose ring16. Not only RNA analogues alternate sugar-based XNAs have been developed. Six-membered ring-based hexitol nucleic acid (HNA) has restricted ring conformation to form an A-type structure in the duplex with RNA18. Five-membered ring group α-L-threofuranosyl-(3′→2′) nucleic acid (TNA), which has been used as a model of pre-RNA polymer possesses the phosphodiester group connecting at a different position from that in natural nucleic acids21. Although the backbone unit of TNA is one atom shorter than that of natural nucleic acids that have six-atom backbone repeat, TNA is capable of forming stable duplex with complementary RNA19. In addition to cyclic scaffolds, ribose-inspired acyclic XNAs have been synthesized and characterized because the acyclic XNAs provide extremely resistant against enzymatic degradation22. However, acyclic ribose modified analogues decrease the stabilities of the heteroduplex with RNA25. Also, development of acyclic nucleic acids composing simple structure such as glycerol nucleic acid (GNA), which is most simplified acyclic backbone of propylene glycol28, and zip nucleic acid, which is connecting through six-bonds system in analogy to natural nucleic acid by phosphonomethylglycerol unit29 have been attempted. Although their homoduplexes are highly stable, the stabilities of heteroduplexes with natural nucleic acids are much lower than that of unmodified DNA or RNA duplexes. Peptide nucleic acid (PNA), non-charged type acyclic nucleic acid, binds with high affinity to RNA and DNA32, however, synthesis of long oligomer and purine-rich sequence is technically very difficult due to poor solubility.Xeno nucleic acids (XNAs) are synthetic analogues that retain natural nucleobases but are replaced with backbone structures different from DNA and RNA. They have potential for use in nucleic acid-based drugs, in development of artificial genetic polymers, and in the prebiotic fieldaTNA) can form stable duplexes with RNA in a sequence-specific manner35. Since they are structurally simple, readily synthesized, excellent water solubility, and high nucleases resistance, various applications have been realized based on hybridization with RNA such as a high-sensitive molecular beacon and nucleic acid-based drug candidates, including siRNAs, anti-miRNA oligonucleotides, and exon-skipping type antisense oligonucleotides43. However, how SNA and L-aTNA hybridize with natural nucleic acid have remained unknown. We initially assumed that helicity of duplex of D-aTNA, which is enantiomer of L-aTNA, is right handed based on the similarity of CD spectrum to that of DNA and structural modelling44, and that L-aTNA homoduplex had left-handed helicity. However, based on CD studies of PNA of known structure45, the CD signal of L-aTNA is indicative of right-handed helicity35. Interestingly, the handedness of an SNA homoduplex depends on the oligomer sequence33. Despite the importance of the acyclic nucleic acid, only limited structural information is available. For development of acyclic nucleic acids, for applications to tools or materials, and to facilitate de novo design of artificial polymerases for these acyclic nucleic acids, structural information is helpful. In the present study, we successfully prepared single crystals of L-aTNA/RNA and SNA/RNA heteroduplexes and solved the first crystal structures at 1.70–1.75 Å resolution. L-aTNA/RNA and SNA/RNA form right-handed helical structures with large helical pitch involving Watson–Crick base pairs and parallel-type Hoogsteen base pairs.We recently discovered acyclic nucleic acids serinol nucleic acid (SNA) and L-threoninol nucleic acid (L-aTNA strand LT8a (3′-GCAGCAGC-1′) with an RNA strand R8Br (5′-GCUGC-BrU-GC-3′) and an SNA strand S8a ((S)-GCAGCAGC-(R)) with the R8Br were prepared. Melting analyses and CD spectroscopy analyses confirmed that complexes were formed  stabilized through canonical Watson–Crick base pairing :G7(R8Br-1)*G1(LT8a-2) and C2(LT8a-2):G7(R8Br-2)*G1(LT8a-1) (the colons indicate the Watson–Crick pairs and the asterisks indicate Hoogsteen interactions)  Figs.  and 2b. lex Fig. . The twolex Fig. .Fig. 1DiaTNA/RNA duplex, a single SNA/RNA duplex is an antiparallel right-handed helix stabilized by canonical Watson–Crick base pairing, and two duplexes are stabilized by formation of two parallel triplex through Hoogsteen base pairs of C(SNA):G(RNA)*G(SNA), result in formation of dimer of duplex structure  are observed in the SNA/RNA crystal  and four RNA strands R8Br-3 to 6)  and SNA/RNA (average 1.2°) are much smaller than that of A-type RNA/RNA duplex  and other XNA/RNA heteroduplexes , in which base pairs are stacked in perpendicular to the axis52. In contrast, values of X-displacement of L-aTNA/RNA and SNA/RNA are much higher than that of dsDNA which show no displacement from the helical axis . The lower inclination and larger displacement cause lower helical twist of the L-aTNA/RNA and SNA/RNA structures  compared to 33.0° for dsRNA and 30.0° for LNA/RNA   Fig. . HoweveraTNA/RNA and SNA/RNA adapt unwound helical structures, we next compared torsion angles of RNAs in dsRNA, L-aTNA/RNA, and SNA/RNA duplexes  of the RNA in the heteroduplex with L-aTNA and SNA are between 70° and 100°, the range observed for typical A-type helix3, with the exception of those for the terminal residues of each strand. In contrast, the average values for the backbone torsion angle α associated with the phosphodiester bond (O3′-P-O5′-C5′) are relatively smaller for the RNA strand in the heteroduplexes than those observed in the dsRNA duplex. The average values for the glycosidic torsion angle χ between sugar and base  are also smaller than those observed in the dsRNA. Differences of these angles, although which are very little, make P–P distances of the L-aTNA/RNA and SNA/RNA larger. Consequently, helical pitches of the heteroduplexes larger relative to that of the dsRNA  are different from that observed in typical C:G*G triplex. This allows them to form parallel type Hoogsteen interaction. The unique triplex forming abilities of L-aTNA and SNA will be an advantage for applying triplex-based theranostics.We found that two duplexes interact through triplex formations to form dimer of duplex structures. Interestingly the triplexes were formed by parallel-type Hoogsteen base pairing between Gs and a Watson–Crick G:C base pairs. Conventionally G:C Watson–Crick base pair in DNA and RNA forms parallel Hoogsteen base pair with C, but this type of interaction is disfavoured at physiological pH because protonation of C in third strand is required for formation of the Hoogsteen interaction21. On the other hand, acyclic type XNA studies showed that acyclic nucleic acids can form homoduplex and heteroduplex with RNA or DNA, even if amino acid derivatives are used as backbones35, indicating that ribose is not necessary for formation of stable duplex structure. These findings raise up the fundamental question why nature selected ribose as backbone of genetic materials and amino acid as backbone of proteins, products of gene. It is also possible that acyclic nucleic acid, capable of hybridizing with RNA, derived from amino acid derivatives similar to our SNA and L-aTNA served as evolutionary intermediates or competitors of genetic material. To answer this, it might be important to consider the optimal helical structure for compact packing of large genetic polymer.Nucleic acids play important roles as the blueprints for construction of all living organisms. It is hypothesized that RNA served as the precursor in a prebiotic worldThus, the structural data reported here will expand the scope of application of acyclic nucleic acid analogues in prebiotic studies as well as in nucleic acid-based drug and nanotechnology.S)-GCAGCAGC-(R), S7b: (S)-GCTGCTG-(R), LT8a: 3′-GCAGCAGC -1′, LT7b: 3′- GCTGCTG-1′, R8Br:5′ GCUGCBrUGC-3′, R8a: 5′-GCAGCAGC-3′, and R7b:5′-GCUGCUG-3′, were obtained from HSS Co., Ltd.. Purified oligomers were dissolved in 10 mM Tris-HCl (pH 7.0) and then duplexes were prepared at a final concentration of 1 mM. S8a/R8Br and LT8a/R8Br were annealed by heating for 10 min at 95 °C and then gradually cooling to 4 °C. Crystallization conditions were screened with commercially available sparse matrix screening kits. Crystals of S8a/R8Br were grown using the sitting drop method in 0.1 M HEPES (pH 6.5), 75 mM CaCl2, and 28% PEG400, and crystals of LT8a/R8Br were grown using the sitting drop method in 0.1 M HEPES (pH 7.5), 200 mM CaCl2, and 28% PEG400 at 20 °C.The oligomers used in this study, S8a:  were dissolved in 10 mM HEPES buffer (pH 7.4) with 100 mM CaCl2. The melting curves were obtained with a Shimazu UV-1800 by measuring the change in absorbance at 260 nm versus temperature. Melting curves were measured with a temperature ramp of 0.5 °C min−1. Tm values were determined from the maximum in the first derivative of the melting curve. For CD, L-aTNA, SNA, and/or RNA (4.0 μM) were dissolved in 10 mM HEPES buffer (pH 7.4) with 100 mM CaCl2. Spectra were recorded at 5 °C using a JASCO model J-820 instrument.L-The oligonucleotides mixtures were buffer-exchanged into 100 mM triethylammonium acetate, pH 7.0, by passing the oligonucleotides through Bio-Rad Micro Bio-Spin 6 columns. After buffer exchange, samples were immediately analysed by nanoflow electrospray ionization mass spectrometry using gold-coated glass capillaries made in house . Spectra were recorded on a Waters SYNAPT G2-Si HDMS mass spectrometer in negative ionization mode at 1.13 kV with 150 V sampling cone voltage and source offset voltage, 4 V trap and 2 V transfer collision energies, and 5 mL/min trap gas flow. The spectra were calibrated using 2 mg/mL cesium iodide dissolved in 50% 2-propanol and analysed using MassLynx software (Waters).LT8a, 2650.7; R8Br, 2578.3; LT8a/R8Br duplex, 5228.9; dimer of LT8a/R8Br duplex, 10457.9; S8a, 2538.5; R8Br, 2578.3; S8a/S8a duplex, 5077.1; S8a/R8Br duplex, 5116.8; dimer of S8a/R8Br duplex, 10233.6; LT7b, 2314.6; LT8a/LT7b duplex, 4965.2; dimer of LT8a/LT7b duplex, 9930.5; S7b, 2216.5; S8a/S7b duplex, 4755.0; dimer of S8a/S7b duplex, 9510.0; S7b/S7b duplex, 4432.9.Calculated masses (singly charged states) are as follows: Supplementary Information


N. benthamiana infected by tomato spotted wilt virus (TSWV) or capsicum chlorosis virus (CaCV) demonstrated the presence of abundant vsiRNAs, with host-specific differences evident for each pathosystem. Despite the biogenesis of vsiRNAs in capsicum and N. benthamiana, TSWV and CaCV viral loads were readily detectable. In response to tospovirus infection, the solanaceous host species also generated highly abundant virus-activated small interfering RNAs (vasiRNAs) against many endogenous transcripts, except for an N. benthamiana accession lacking a functional RDR1 gene. Strong enrichment for ribosomal protein-encoding genes and for many genes involved in protein processing in the endoplasmic reticulum suggested co-localisation of viral and endogenous transcripts as a basis for initiating vasiRNA biogenesis. RNA-seq and RT-qPCR-based analyses of target transcript expression revealed an inconsistent role for vasiRNAs in modulating gene expression in N. benthamiana, which may be characteristic of this tospovirus-host pathosystem. Tospoviruses infect numerous crop species worldwide, causing significant losses throughout the supply chain. As a defence mechanism, plants use RNA interference (RNAi) to generate virus-derived small-interfering RNAs (vsiRNAs), which target viral transcripts for degradation. Small RNA sequencing and in silico analysis of capsicum and  Orthotospovirus, family Tospoviridae, order Bunyavirales [Frankliniella and Thrips species) are the primary transmission vectors for these tospoviruses and are also replication hosts, though without associated symptoms [Tospoviruses are taxonomically classified in the genus avirales . In targavirales . Capsicuavirales . Thrips symptoms .Both CaCV and TSWV have single-stranded RNA genomes that comprise five open reading frames across three segments, with one negative-sense and two ambisense. The small (S) RNA encodes a non-structural RNA-silencing suppressor protein (NSs) and the nucleocapsid protein (N). The medium (M) RNA encodes a cell-to-cell movement protein (NSm) and the precursor of the two envelope glycoproteins (GP), with the Large (L) RNA coding for an RNA-directed RNA polymerase (RdRp), also called L protein ,6. Arachis hypogaea; Fabaceae), tomato , and Nicotiana benthamiana (Solanaceae) [Viral RNAs act as both a substrate for and target of the host plant’s RNA interference (RNAi) defence pathways, leading to viral lifecycle disruption through specific transcript degradation. Double-stranded RNAs (dsRNAs) arising from processes such as viral replication are cleaved by Dicer-like enzymes into virus-derived small-interfering RNA (vsiRNA) duplexes of primarily 21 and 22 nucleotides (nt) . VsiRNAsanaceae) ,9,10. Arabidopsis thaliana [Brassica napus leaves, with demonstrated roles in cleavage of host as well as viral transcripts [Brassica rapa, and Brassica napus by cauliflower mosaic virus (CaMV), a double-stranded DNA virus, also produced vasiRNAs [As well as targeting viral transcripts, the plant’s RNAi machinery has several roles in endogenous gene regulation, acting both transcriptionally and post-transcriptionally. Recently, multiple studies have revealed that virus infection triggers the biogenesis of siRNAs targeting host transcripts , in addition to vsiRNAs in thaliana ,12,13. Vthaliana . Though nscripts . InteresvasiRNAs . Target vasiRNAs ,16. N. benthamiana was reported [N. benthamiana accession used or differences between CMV 2b and TSWV NSs vasiRNA sequestration might be responsible. Using a transgenic approach, the addition of a functional RDR1 to the N. benthamiana accession used resulted in the generation of vasiRNAs following tomato leaf curl Gujarat virus infection, and as with the Brassicaceae species, differential regulation of target genes [Following their identification and characterisation in the Brassicaceae, the search for vasiRNAs was extended to solanaceous species. Initially, a lack of detectable vasiRNAs in TSWV-infected reported . The autet genes . N. benthamiana and capsicum (Capsicum annum). N. benthamiana varieties with and without the ability to generate vasiRNAs were used to shed light on the role of vasiRNAs in disease progression as well as endogenous host gene regulation. For each of the species examined, abundant vsiRNA biogenesis against both TSWV and CaCV was evident. In hosts with a functional RDR1, vasiRNA generation was also robust, with localisation of substrate host transcripts proposed as a selective mechanism. Interestingly, the generation of vasiRNAs in N. benthamiana did not result in a general downward trend in target host gene expression attenuation, suggesting many competing factors impact the expression of these genes during infection. In this study, vsiRNA and vasiRNA responses to TSWV and CaCV infection were examined using next-generation sequencing of solanaceous species including Capsicum annuum) cv. Yolo Wonder, with the genome of this isolate previously sequenced [C. annuum cv. Yolo Wonder, Nicotiana benthamiana LAB, and N. benthamiana Western Australia  were used as host plants. All plant material described here was used for TSWV-infection and/or CaCV-infection assays, small RNA sequencing, and RNA-seq.CaCV isolate QLD-3432, provided by the Queensland Department of Agriculture and Fisheries (DAF), was maintained in capsicum  and resistant (Red Defender) tomato varieties following TSWV infection were also reanalysed. Red Defender’s resistance is associated with hypersensitivity induced by the w-5 gene . The genw-5 gene . N. benthamiana at the five-six leaf stage were mechanically inoculated with homogenates from TSWV (QLD-1) or CaCV (QLD-3432) infected plant tissue of the same species. For mechanical inoculation, one or two apical leaves from a systemically infected symptomatic plant were homogenised with chilled mortar and pestle in 10 mM sodium phosphate buffer (pH 7.0) containing 0.4% 2-mercaptoethanol and rub-inoculated to carborundum-dusted leaves of host/test plants.Capsicum at the four-leaf stage and N. benthamiana plants were virus-inoculated. As a negative control, sap from non-infected plants was used for mechanical inoculation (mock treatment). At least three plants were used for each treatment. Virus infection of plants was confirmed by the progression of symptoms, qPCR or DAS-ELISA using TSWV or CaCV kits from Agdia, Inc.  and following the manufacturer’s instructions. Plant images showing symptoms were processed using Adobe Lightroom Classic  to ensure the evenness of exposure and white balance. The youngest two leaves of capsicum plants and leaves two and three of N. benthamiana treatments, apical leaf tissue was collected ten days after mechanical inoculation, snap-frozen in liquid nitrogen, and stored at −80 °C. Tissue was subsequently ground with a chilled mortar and pestle, and total RNA was extracted using TRIsure™ reagent , following the manufacturer’s instructions. RNA samples were suspended in nuclease-free water, and the quantity and purity were determined using a NanoDrop™ spectrophotometer . The quality of the RNA was assessed by the Australian Genome Research Facility  using a Bioanalyzer . For all capsicum and N. benthamiana RNA samples were submitted to AGRF for small RNA sequencing. Small RNA libraries were prepared using the New England Biolabs  kit protocol and selecting for RNA of 18–33 nucleotides (nt). Illumina HiSeq HT Chemistry was used with 50 bp single reads. Samples were distributed across three flow cells. Small RNA sequencing of tomato samples has previously been described [Capsicum and escribed .N. benthamiana LAB and WA accessions 10 days after TSWV or mock inoculation. RNA-seq of N. benthamiana samples was conducted by Genewiz, with non-strand specific library preparation carried out prior to generation of approximately 20 M paired-end reads per sample on a NovaSeq platform. Raw sequence data is available at the NCBI Sequence Read Archive under BioProject ID PRJNA784690.To examine differential expression of vasiRNA targets, RNA-seq was performed on total RNA from apex tissues of http://hannonlab.cshl.edu/fastx_toolkit/, accessed on 1 December 2021) to remove 5′ adapters and collapse reads to unique sequence:count couplets. Reads were aligned to TSWV and CaCV genome segments and endogenous transcript sequences using the SCRAM aligner [https://github.com/FelixKrueger/TrimGalore, accessed on 1 December 2021), and then vasiRNA-target transcript abundance was quantified with Kallisto [Small RNA deep sequencing read files were processed with the FASTX-Toolkit  for TSWV-infected and mock-inoculated capsicum, tomato (Marglobe), and N. benthamiana (WA genotype) samples. For each differentially expressed (DE) transcript, the longest open reading frame (ORF) was identified and translated to a peptide using a custom Python script. Peptides were assigned KEGG identifiers using BLASTKoala [KEGG pathway analysis involved identifying transcripts that had significant differences in complementary vasiRNAs . Changes in transcript abundance were calculated for mock vs. infected samples as −ΔΔCt to meet the assumptions of the statistical tests used. As an RT- control to rule out genomic DNA contamination, RNA samples underwent PCR with MyTaq™ DNA polymerase , with no amplification evident. Primers used for qPCR are shown in RNA samples were first treated with TURBO DNase I™  and reactions terminated with 15 mM EDTA. cDNA was synthesised using the sensiFAST™ cDNA synthesis kit with 1.88 mM MgClN. benthamiana. Of the two N. benthamiana genotypes, N. benthamiana WA had previously unknown TSWV susceptibility, and N.benthamiana LAB with a non-functional RDR1 is considered susceptible to many plant viruses, including TSWV [Responses to CaCV or TSWV infection were investigated in two host plant species of the Solanaceae family: capsicum and ing TSWV ,28. Following TSWV or CaCV inoculation in capsicum, mild leaf mottling and yellowing symptoms were evident compared to mock-inoculated plants at 10 dpi . CaCV anN. benthamiana LAB showed mild symptoms with CaCV or TSWV infection compared to the mock treatment  enzymes process double-stranded RNA into 21 and 22 nt duplexes . Due to  RT-qPCR . The genb and WA . N. benthamiana by TSWV  and resistant (Red Defender) tomato varieties following TSWV infection. Like capsicum and e tomato , while tve to WA , indicatN. benthamiana WA samples and very low alignments in their mock-inoculated counterparts (Heat Shock Cognate 70-2 (HSC70-2), Calnexin 1 (CNX1), and Next to BRCA 1 (NBR1) in TSWV- and CaCV-infected capsicum and N. benthamiana WA displayed the distribution of 21 and 22 nt hotspots in both orientations  and Calnexin transcripts in Marglobe but not in Red Defender where vasiRNAs were absent  were more variable . Expression levels of individual targets were also analysed via RT-qPCR for N. benthamiana WA and LAB, and additionally, capsicum  enzyme processing, with DCL1 and DCL4 principally generating 21 nt siRNAs, DCL2 generating 22 nt siRNAs, and DCL3 generating 24 nt siRNAs in plants [Arabidopsis have shown that DCL4-mediated 21 nt siRNAs generated from dsRNA hairpins are significantly more abundant than their DCL2-mediated 22 nt counterparts [N. benthamiana. If the enhanced potency of systemic silencing induced by 22 nt siRNAs in Arabidopsis also occurs in the Solanaceae, it does not appear to result in reduced viral load, particularly in the N. benthamiana WA/TSWV pathosystem. Differences in relative TSWV and CaCV vsiRNA abundance for tospovirus RNA segments and genes were evident between hosts. Abundant vsiRNAs were, however, still generated in the functional RDR1 lacking n plants . Though terparts ,33, simiN. benthamiana LAB and WA accessions 10 days post-inoculation, TSWV and CaCV infection subsequently proved lethal to the LAB plants, whereas the WA plants survived, albeit in a weakened state. It has previously been demonstrated that the introduction of transgenic RDR1 into N. benthamiana LAB prevents lethality caused by tobacco mosaic virus (TMV) but does not prevent the systemic spread of the virus [Solanum tuberosum) RDR1 transcripts does not affect susceptibility to potato virus X or TMV, as measured by the level of viral coat protein [N. benthamiana WA has a functional RDR1, this supports the notion that its presence is the determinant of enhanced survival or at least a significant contributor. Despite similar symptoms, viral loads and accumulation of vsiRNAs in he virus . Further protein . Given tN. benthamiana WA generate large numbers of vasiRNAs against a specific subset of endogenous transcripts, while N. benthamiana LAB does not, providing further support for RDR1’s role in vasiRNA biogenesis. Profiles of vasiRNA alignments to target transcripts show the presence of hotspots in both orientations, similar to vsiRNA alignments to viral RNAs. Many endogenous vasiRNA targets identified in earlier reports for Brassicaceae and Solanaceae [Apart from acting together with RDR6 in the production of vsiRNAs, RDR1’s role in generating vasiRNAs has been proposed to contribute to enhanced plant longevity under viral challenge ,15,16,18lanaceae ,15,16,18NBR1 are associated with virus defence, though others act as important components for virus replication or systemic movement through the plant  with flippase activity is essential for RDR6, RDR1, and vasiRNA production during CMV infection [To shed light on the conserved selection of vasiRNA-target transcripts, we examined the expression of these transcripts as well as their putative functions and cellular localisation. The vast range of expression of these transcripts suggests that high expression itself in response to tospovirus infection is not how this subset of genes is selected. KEGG analysis revealed that many vasiRNA-associated transcripts were either ribosomal protein-encoding transcripts or encoded proteins located in the ER that are involved in protein processing and folding. For instance, both -folding . It is pnfection ,13. Manynfection . Indeed,nfection . WhetherN. benthamiana LAB, which lacks vasiRNAs and a functional RDR1. The interplay between plant viruses and their hosts remains an active area of investigation, with this work showing that plant responses involving RNAi and the generation of vsiRNAs and vasiRNAs are conserved, with the functional differences apparent between different pathosystems a possible focus for enhancing crop protection. When taken together, these findings demonstrate the evolutionary persistence of the host gene targeting vasiRNA biogenesis pathway, though its function in tospovirus-Solanaceae interactions remains unclear. Abundant virus-derived vsiRNAs targeting TSWV and CaCV transcripts are apparent in multiple Solanaceous species, including


Stylophora pistillata, a coral species which dominates reef flats in the central Red Sea. Phototransects were conducted at each reef flat zone, and the density of S. pistillata, the planar area of each colony, and the occurrence of partial mortality were calculated using the program ImageJ. Each colony was also assigned a color morph . Density and mean size were extremely variable, both among reef flat zones and reefs, but overall, both metrics were lower on the midshelf reefs. The yellow color morph accounted for nearly 90% of colonies surveyed and dominated most reef flats assessed, with the exception of one site where 81% of colonies were purple morphs. There were no spatial trends in the percentage of colonies suffering partial mortality, but overall there is a positive correlation with size class and proportion of colonies with partial mortality. Despite few trends emerging from assessing individual parameters, a PERMANOVA analysis revealed differences among reef flat zones in most of the reefs, highlighting the importance of multivariate analysis. The data presented here serve as a baseline for monitoring and may identify possible future demographic changes to this important coral species in a region increasingly affected by bleaching events.Demographic analyses offer insight into the state of a population. Here, we surveyed different reef flat zones  of six reefs over a cross-shelf gradient to characterize the population structure of  At a fundamental level the growth and maintenance of the reef environment are dependent on the demographic rates of individual coral species 2. In recent decades, coral species have been severely affected by anthropogenic disturbances, compromising the stability of their populations and the resilience of coral reefs worldwide.Coral reef ecosystems are underpinned by hermatypic coral species, which construct the reef structure providing habitat for approximately one quarter of marine life3. Historically, coral percent cover has been used as the standard monitoring variable in benthic reef communities, and is a useful metric that encapsulates very general information about the state of a coral reef. However, this traditional approach obscures details that can be useful in understanding population dynamics. This method ignores the actual abundance and sizes of individual corals, since “percent coral cover” only measures the total space occupied by a coral category. Assessing size structure  is a better tool for understanding the population trajectories of a species because it captures demographic information not contained in other methods like percent cover6. Therefore, simple metrics such as size structure can offer a more in-depth “snapshot” of the state of a population. Often, size structure is used to infer the state of coral populations under anthropogenic stressors, such as pollution7, climate change11, and fishing pressure13. Therefore, in an era of intense degradation as a result of local and global disturbances, a more comprehensive evaluation of the status of coral reefs, potential causes of decline, and projected trajectories is essential to guide mitigation and conservation measures.Long-term monitoring is ideal for gathering information on trends of population growth and decline, which can shed light on how species respond to changes in environmental conditions or disturbances. However, long-term data often are not available for many regions, especially remote coral reefs or historically understudied systems such as the Red Sea14. In corals, abundance is the product of several demographic processes, including asexual reproduction, settlement and mortality rates, while the size of individual coral colonies is dependent on growth rates and partial mortality17. The resulting size frequency distribution provides a way to compare demographic information among different species, populations or sites14. For example, a size-frequency distribution that is negatively skewed indicates an overabundance of large individuals in a population, while positive skew denotes a population dominated by small individuals. Previous studies have suggested that negatively skewed size-frequency distributions are potentially indicative of a degraded reef18. The implication is that there are few young individuals to replenish older individuals in a population, and may be due to a lack of (or unsuccessful) settlement/recruitment or high mortality rates for smaller size classes. While skewness alone cannot indicate the nature of degradation, it can be relevant for future investigations to understand the cause of demographic shifts.Size structure comprises two key metrics: abundance and the size of all individuals20, temperature21 and water quality22 can influence coral size structure. Environmental conditions can vary drastically at the scale of the reef, particularly as a function of wave exposure and water depth; sites near the exposed side of a reef usually experience less variability than sheltered sites in a variety of parameters 23. Perhaps the most extreme gradient that exists over this small scale is over the shallow reef flat from exposed to sheltered sites. The reef flat is typically the shallowest part of the reef and its height is limited by sea level24. The reef flat habitat is subjected to frequent disturbances like low tides, high temperature variability, and extreme wave action25. Though the benthic community on the reef flat is less diverse than the reef slope, the reef flat habitat is important for trophodynamics of the entire reef system26.Coral size structure varies naturally in time and space. Abiotic factors such as depth24. In the central Red Sea, the branching coral Stylophora pistillata is common in shallow areas and can be found across all reef flat zones29. Stylophora pistillata has been called an r-strategist because it quickly colonizes unstable environments, reproduces early in its lifetime, grows quickly, and has high population turnover30. These characteristics allow S. pistillata to be dominant in the disturbance-prone reef flat habitat, while in more stable environments it tends to be outcompeted by other coral species30. The reef flat habitat is therefore an ideal system to study S. pistillata size structure, as this coral’s high population turnover is likely to reflect recent natural or anthropogenic disturbance history.Typically reef flats can be defined as either coral- or rubble-dominated, and within each reef flat clear zonation in the biological community can be observed from the high wave energy windward side of the reef to the low wave energy leeward side31 and common coral species over cross-shelf32 and large-scale gradients28. However, despite being the most abundant species on reef flats in the region27, no studies assessing size-class structure have been conducted on S. pistillata populations, and intra-reef comparisons are lacking. Such information is critical for understanding population dynamics in a region that is increasingly affected by coral bleaching events34. Here, we conduct a population structure assessment of S. pistillata by evaluating size structure, color morph distribution, and incidence of partial mortality to investigate large and small-scale patterns of variability across the shelf and within each reef across the reef flat.Previous studies in the Red Sea have examined the abundance and size class structure of juvenile pocilliporid coralsS. pistillata . Temperature was logged for some reefs starting in June 2019 and near-continuous profiles were obtained until the time of the study. Unfortunately, many sites are missing data for long periods of time due to logistical constraints. We therefore chose to focus on the two reefs with the most complete datasets: the offshore reef Shib Nazar and the nearshore reef Abu Shousha. We highlighted a six-week period of 2020 when temperatures were highest and calculated the degree-heating weeks for each reef zone. Degree-heating weeks is a measure of accumulated heat stress commonly used in coral biology36. We used a baseline of 31.8 °C, one degree above the mean maximum for this region, which has been used in previous studies for this area37.As water flows across the reef flat, it warms during the day and can result in a temperature gradient from the exposed to sheltered sidesS. pistillata colonies38. Size was measured by calibrating each image to a known length (referencing the transect tape in the photo) and using the “area” measurement tool in ImageJ to trace the contours of each colony, resulting in the planar area39 . Since the water depth on reef flats varied slightly between reefs and with tidal oscillations, the distance between the camera from the substrate was not equal for all surveys, resulting in differences in the area covered for each photo. To standardize abundance counts between reefs, we measured the area of each photo to obtain the total area surveyed for each site, and expressed abundance in terms of density . For all colonies considered in the analysis (> 50% living tissue), we also quantified partial mortality between sites and reefs. For each colony we also assigned a color morph category: yellow, purple, or mixed colors, if the colony was partially both colors. We also calculated the proportion of juvenile colonies based on previous studies that consider colonies of S. pistillata and related species less than 10 cm22 planar area likely to be juveniles40. Finally, summing the total area of all colonies allowed us to calculate the percent cover of S. pistillata for each reef flat zone to compare with colony density.At each of the reef sites, we conducted three replicate phototransect surveys of 10 m length. Transect locations were haphazardly selected to begin near the temperature logger deployed at each of the sites, and were laid parallel to the reef edge with 3–5 m separating the start and end of each replicate. Photos were taken along the entire length of the transect via snorkel using a Canon G7 X camera . Photos were later processed in the software ImageJ to count the number and size of a39 Fig. b. All coS. pistillata in our study system. Colony size was first log transformed to normalize the distributions. We separated size classes into bins of 0.5 on the log scale for visualizing size structure . The descriptive statistics  were calculated for each site individually, as well as for each reef and the total population sampled; these analyses were performed in R (R Development Core Team 2018) using the package “moments”42. We compared log-transformed size-frequency distributions using two-sided Kolmogorov–Smirnov tests for reef zones within each reef. Due to extreme variability among reefs, we did not perform these tests for reef flat zones between different reefs. For each of the assessed parameters , we performed a non-parametric Kruskal–Wallis analysis to test for differences among reef flat zones, reefs, and shelf position. When significant differences were found, pairwise Mann–Whitney U tests were performed.We plotted the size class structure of each reef site, the total for each reef, and the entire sampled population of 43). The model included three factors: shelf ; reef ; and reef flat zone . The analysis was conducted on the distance matrix generated using the Euclidean distance and considering mean colony size, colony density, percentage of purple colonies, and percentage of colonies with partial mortality as variables. Percent juveniles were not included in the analysis as this parameter correlated strongly with mean colony size . Data was normalized and transformed (square root) before the analysis.A Permutational Multivariate Analysis of Variance (PERMANOVA) was carried out in Primer v7  compared to the offshore reefs ( 60.27 ± 3.87 cm2) and nearshore reefs (50.28 ± 4.99 cm2) . Offshore and nearshore reefs did not have significantly different colony sizes .Overall, an area of 279.16 mS. pistillata was similarly variable over multiple spatial scales, and ranged from 0.44 colonies m−2 at the exposed reef flat zone of Tahala North to 11.93 colonies m−2 at the midreef reef flat zone of Cement Wreck , which is higher than the midshelf reefs .Density of 1) ranged from − 0.66 at the Shark Reef exposed zone to 1.46 at the Tahala North exposed zone (Table 2), which is a measure of the effect of outliers on a distribution, was variable among sites but mostly platykurtic  or mesokurtic . The only cases where sites’ kurtosis was leptokurtic  were the exposed reef flat zones of Tahala North and Shark Reef   . However, the colony density of the exposed zone is nearly three times lower than the midreef zone , and the mean colony sizes differ significantly . In addition, their size-frequency distributions are significantly different .Summing the planar area of 05) Fig. . Since pS. pistillata found in the largest size class.Incidence of partial mortality ranged from 0 to 47% of colonies between reef flat zones Table . There wThe proportion of purple colonies per site varied widely, from 0% at several sites to 81% in the exposed reef flat zone of Al Fahal. Mixed color morphs are uncommon, only accounting for 0.7% of colonies from three reefs. Overall, yellow colonies dominated reef flats in this study, accounting for nearly 90% of all colonies surveyed  and midshelf  reefs are different from each other within groups. Considering reef flat zones within each reef, in Abu Shousha the reef flat zones were not significantly different from each other, while Tahala North, Al Fahal and Cement Wreck showed differences between all reef flat zones. Shark Reef only had significant differences between the exposed and sheltered reef flat zones, while Shib Nazar had significant differences between sheltered and both midreef and exposed reef flat zones Table .Table 2P6. Using size frequency distributions instead of percent coral cover is more informative for understanding demographic trajectories. In this study, we found large variability of the size class structure of S. pistillata both within and among reefs. We did not observe a clear pattern for either cross-shelf or within-reef scales for any of the parameters analyzed separately . However, multivariate analysis conducted on these parameters showed that most reefs had significant differences between some or all of their reef flat zones. Our results for two reefs with differing thermal profiles suggest that environmental factors such as temperature may be partly responsible for driving spatial differences in S. pistillata population structure. In the Red Sea, few historical datasets are available, especially outside of the northern Gulf of Aqaba region. Although S. pistillata from this region tend to be resilient to high temperatures, further warming due to climate change may result in temperatures that exceed their thermal tolerance, especially when coupled with local stressors like pollutants44. This demographic “snapshot” will therefore serve as a baseline assessment of this common species, which is essential for future biomonitoring. Such baselines have revealed changes in size structure of Stylophora corals in other regions. For example, in the Great Barrier Reef, the genus Stylophora has experienced major declines in overall abundance over the past two decades, with concomitant shifts in size class distribution10. In Kenya, Stylophora underwent a significant reduction in median colony size following the mass bleaching event of 199812.Assessments of size class structure can yield insights into coral population statusThe within-reef patterns of temperature were broadly similar between the two reefs assessed. The midreef and sheltered reef flat zones tended to have larger daily ranges and higher maximum temperatures than the exposed reef flat zones of both reefs Fig. b,c. In aS. pistillata populations on several reef flats in the central Red Sea. The information contained in the size frequency distributions reveal extreme variability on both inter-and intra-reef scales. We chose to use belt transects as a survey method, which may have a small bias towards counting smaller colonies. However, based on our personal observations, we are confident our results accurately reflect the true size structure. Previous studies of coral populations have also found variable size class structure across sites. Adjeroud et al. (2007) note that although they found significant variation in the size structure of two coral species in Moorea, they found no clear patterns in depth or location that could explain the differences, and suggest that other stochastic processes may be at play.The results presented here offer a snapshot of 20. Considering shelf position, the offshore reefs have the highest density and largest colonies of S. pistillata, perhaps indicating these are more suitable habitats for sustaining larger populations of S. pistillata than the reefs closer to shore  of the size class distribution can reveal important details about population structure. Considering the sites individually, half are negatively skewed, indicating a lack of smaller colonies and over-abundance of large colonies Table . HoweverS. pistillata is still common across much of the reef, the sandy substrate may influence growth and juvenile mortality, which affects population density. In addition, the exposed zone of Tahala North experiences seasonal Sargassum blooms which can completely cover any corals growing there for several months per year. We speculate the extremely low density of S. pistillata colonies there is due to periodic Sargassum blooms which may negatively affect S. pistillata growth50 or recruitment51. The second reef which deserves more attention is Shark Reef, which also had low overall density of S. pistillata. During the late summer of 2020, a bleaching event affected the reef system in the study region, but Shark Reef was the most impacted reef (pers. obvs.). During an October 2020 research expedition, we noted parts of the reef crest on Shark Reef experienced 100% bleaching , whereas the other reefs in this study were minimally impacted. While we were not able to assess the reef flat on this expedition, it’s possible that habitat was also heavily impacted and the population of S. pistillata could have experienced high mortality rates. Interestingly, during a mass bleaching event in the central Red Sea in 2015/2016, Shark Reef (refered to as Qita Al-Kirsh) was also heavily impacted by bleaching, moreso than other midshelf and offshore reefs assessed34. This may be an indication that Shark Reef is more susceptible to disturbance, although it is not clear what factors may be responsible.Two of the reefs in this study are of note and deserve further discussion. Tahala North exhibits different substrate characteristics than the other reefs, with a layer of sand covering the pavement across most of the reef flat. Although 52, and the percentage of colonies with partial mortality tends to increase with increasing colony size55. Our results support this pattern, with the smallest size classes containing no colonies suffering partial mortality and an increasing proportion of partial mortality with increasing size class  reached the highest incidence of partial mortality of 50%  and produce both male and female gametes at larger sizes (diameter > 4 cm)41. Approximating the colony’s planar area as a circle, this equates to a cutoff of roughly 12.57 cm2, meaning only the smallest adults will be producing male-only gametes. Given that our results show the frequency of partial mortality is very rare in colonies of small size classes, partial mortality is unlikely to drastically affect the ratio of male/female gametes produced by S. pistillata colonies.As modular organisms, corals frequently suffer partial mortalityass Fig. . The larS. pistillata colonies over the entire study are. The dominance of yellow colonies on these reef flats is notable, as previous work has shown that purple morphs of S. pistillata typically outcompete the yellow morphs57. However, a related species that also exhibits yellow and purple color morphs (Pocillipora damnicornis) shows a similar pattern of yellow morphs dominating shallow areas and purple colonies more common below 3 m depth58. The spatial distribution of color morphs of other coral species are often related to the light environment59, and can influence disease or bleaching susceptibility61. However, the biological significance of color morphs in S. pistillata is not clear. Studies on the photochemistry of the pocilliporin pigments, which give the purple morphs their characteristic color, showed no evidence of photoprotection, function as an accessory pigment, or UV protection62. It may be that the yellow morphs of S. pistillata are better adapted to warmer temperatures, higher irradiances, or some other factor unique to reef flat environments, and the purple morphs are competitively dominant only in deeper areas. Color morphs may also harbor different symbiont communities, which may have different tolerances to temperature and irradiance59. Although S. pistillata from the Red Sea tend to exhibit a depth zonation in their Symbiodiniaceae communities , a recent study conducted in the same area as our study found that both genera can be present in shallow-water colonies63. Regardless, the high percentage of purple morphs on Al Fahal is notable. The exposed reef flat zone is dominated by juvenile purple S. pistillata, which could indicate a recent recruitment pulse for that site. However, there is also a high proportion of adult purple colonies across all reef flat zones of Al Fahal. The higher abundance of purple morphs here may be the result of other stochastic processes.Purple colonies accounted for just 10% of S. pistillata at these sites. The multivariate analysis revealed that Abu Shousha was the only reef assessed which did not have significant differences between its reef flat zones. While other factors are likely also responsible, the similarity in temperature profiles between Abu Shousha’s reef flat zones may indicate that the environmental conditions are similar across the entire reef flat, which is reflected in the similarity of populations of S. pistillata. On the other hand, the sheltered reef flat zone of Shib Nazar was different from both the midreef and exposed reef flat zones  populations of S. pistillata, future monitoring is needed to track population changes and relate them to potential disturbances . We hope the data presented here will foster future studies addressing which other abiotic processes and variables may play a role in this small-scale variability of size structure. Such information may help explain why some reefs experience mass bleaching of corals while other nearby reefs are largely spared. Finally, evaluating the role of biotic interactions  at different spatial scales may also shed light on differences in population structure of S. pistillata in the region.As the dominant branching coral species inhabitating reef flats in the region, Supplementary Information.


P = 1.5 atm, t = 800 ms, and τ = 1500 ms) ensured formation of a lap joint with the required mechanical and structural properties .The aim of this study is to substantiate the use machine learning methods to optimize a combination of ultrasonic welding (USW) parameters for manufacturing of multilayer lap joints consisting of two outer PEEK layers, a middle prepreg of unidirectional carbon fibers (CFs), and two energy directors (EDs) between them. As a result, a mathematical problem associated with determining the optimal combination of technological parameters was formulated for the formation of USW joints possessing improved functional properties. In addition, a methodology was proposed to analyze the mechanical properties of USW joints based on neural network simulation (NNS). Experiments were performed, and threshold values of the optimality conditions for the USW parameters were chosen. Accordingly, NNS was carried out to determine the parameter ranges, showing that the developed optimality condition was insufficient and required correction, taking into account other significant structural characteristics of the formed USW joints. The NNS study enabled specification of an extra area of USW parameters that were not previously considered optimal when designing the experiment. The NNS-predicted USW mode ( Ultrasonic welding (USW) is a common method for joining thermoplastic materials  and their composites, primarily for structural applications. This technology and equipment have been well-developed and widely used in various industries. The USW process includes three main stages:- Initially, the clamping force is gradually increased to squeeze the parts being welded. At this stage, ultrasonic (US) vibrations are not applied.- Then, US vibrations of a given frequency and amplitude are initiated at the same clamping force. In this case, frictional heating and melting of the mated surface layers of the welded parts occurs due to their mutual movement relative to each other. As a result, processes of mixing, mass transfer, and mutual penetration of polymer chains develop.- The third stage involves molten material solidification at the same clamping force. US vibrations are no longer applied.The above-mentioned USW parameters (clamping force and durations of both application of US vibrations and holding under pressure after the end of the US application) can exert opposite (competing) effects on the structure formation. Additionally, it is necessary to take into account some properties of the joined polymers, such as the glass transition and melting temperatures, their molecular structures (molecular weight and polarity), etc. It is known that the diffusion rheological interaction of molecules typically occurs at the USW joint interface with the formation of a structural transition zone under pressure. Such mutual penetration develops when the polymers are in a viscous state and their molecules possess the maximum mobility and the minimum packing density ,2,3,4,5.The USW method has been applied in high-tech industries to join continuously reinforced polymer composites based on high-performance polymers (HPP). In ,7,8,9, iMany authors have discussed and used various methods to optimize technological parameters, including contradicting parameters . MultifaLap shear strength (LSS)  is a conIn addition to continuous fiber or fabric reinforced polymer laminates, US welding is applied to join particulate polymer composites, the strength properties of which are much lower than those of reinforced polymer laminates ,30,31,32The aim of this study was to substantiate the use of machine learning methods to optimize a combination of USW parameters for manufacturing of multilayer lap joints consisting of two outer PEEK layers, a middle prepreg of unidirectional carbon fibers (CFs), and two EDs between them.ω), (b) the sonotrode vibration amplitude (θ), (c) duration of US vibrations (t), (d) clamping duration (of the sonotrode) after the end of US vibrations (τ), clamping pressure (P) (of the sonotrode); (f) porosity of the ED  within preset ranges. The other parameters , providing required mechanical and structural properties of multilayer lap joints based on the results of a limited number of experimental investigations. In the general case, the USW parameters are: (a) frequency of US vibrations (ε), (c) work of strain (A) (area under the stress–strain curve of the corresponding diagram); and the following structural parameter was applied: (d) USW joint thinning (during USW) (Δh). These properties were designated through a vector, P) and duration of US vibrations (t)). In addition to the influence of an arbitrary combination of USW parameters in the experiment, the pattern of this randomness also applies to “measuring”  factors: the error of measuring instruments, the locality of measurements , etc.The following USW joint properties were used as the key mechanical characteristics: (a) ultimate tensile strength , the problem of determining the optimal USW parameters is reduced to interpolation of ns, etc. ,34,35.N measured values in a multidimensional space of the process parameters, i.e., defining such If the  applied ,37. For  applied ,17,38. R applied ,40,41.Taking into account that some properties of the same object are determined, which are characterized by their correlation, the mathematical description of the dependence of the mechanical properties on the USW parameters should be represented as a nonlinear system with multiple inputs and outputs:N vector measurements in a multidimensional space of the USW parameters, i.e., determining an The problem of determining the optimal USW conditions with both known  methods ,42,43.The standard research methodology for analysis of experimental data includes several main stages: (i) design of the experiment, (ii) testing, (iii) preprocessing of the experimental data, (iv) constructing a model or a set of object models, and (v) verification of the model ,44,45.Designing an experiment can significantly reduce the research complexity compared to a full-factor experiment, especially when the number of input parameters exceeds two. The existing approaches to solving this problem are summarized in . In thisAnalysis of the reliability of the results of each experiment and, if necessary, rejection of incorrect data;Analysis of the completeness of the summarized information or, with a small sample, determination of limitations on the simulation results; andAnalysis of the data dimensions.At the stage of preprocessing the experimental results, the following problems were solved:The results were statistically processed according to four test coupons with the confidence interval calculation. Then, according to the accepted confidence interval, one value was discarded. Therefore, the values for three test coupons were used for analysis.z and Another problem associated with almost all data processing and analysis methods is their varying dimensions. It is necessary to choose metrics among known or newly developed metric in both parameter and property spaces. The simplest solution to this problem is to convert both spaces to dimensionless spaces. Such transformations essentially constitute normalization of the original data of the form:According to , it is sIt is generally accepted that a neural network needs to be trained on sufficiently large and representative sample that covers all classes and represents the variability within each class for classification problems. For approximation problems, the meaning of the training sample remains the same, but the sample is represented by single values for individual points in the parameter space. Therefore, the training sample should be formed for the neural network. Accordingly, an assumption is made about the distribution law for the In the final stage, the numerical models are verified. With a sufficiently large (redundant) training sample, a testing sample is selected and cross-checked ,41,43. BThe following components were used to manufacture USW lap joints: 50 × 20 × 2 mm PEEK plates made from “PEEK 770PF” powder  by plunge casting, a PEEK-based prepreg in the form of a tape of unidirectional CFs , and a PEEK film   as an ED.The USW lap joints were formed using a “UZPS-7” ultrasonic welding machine . The plates to be welded were placed in a fixing clamp, which prevented their mutual movement. The dimensions of the sonotrode that was used to apply US vibrations to the overlapped adherends were 20 × 20 mm. The test coupons included five layers, namely two outer PEEK layers, a central prepreg layer, and two intermediate EDs .Tensile tests of the USW joints were performed according to ASTM D5868. The tests were carried out with an “Instron 5582” electromechanical tensile testing machine . The cross-head speed was 1 mm/min. To minimize misalignment during the tests, gaskets made of identical-thickness PEEK plates were installed in wedge grips at the test coupon gripping. The USW joint thinning was measured with a micrometer.The cross-sectional structure of the USW joints was analyzed using a “Neophot 2” optical microscope .t) range was set between 800 and 1200 ms because it was not possible to join the PEEK plates at lower values; however, the prepreg could be damaged at higher levels, resulting in faulty USW joints;The USW duration (P) and its (holding) duration after applying US vibrations (τ) were determined based on the technical characteristics of the USW machine, as well as by visual control of the USW joints.Ranges of the clamping pressure  and their possible changing ranges were preset:ε (mm), (3) A (N·m), and (4) Δh (mm).The data on the mechanical characteristics were ranked according to the Taguchi method in the form of a table of combining all factors and their levels . Each leThe Taguchi method  was implτ) from 500 to 1500 ms was accompanied by a minimal change in ultimate tensile strength. In this case, the clamping pressure (P) and duration of US vibrations (t) were the most significant factors. The next analyzed parameter was elongation at break (P) and duration of US vibrations (t) were the most influential factors, whereas the clamping duration after US vibrations (τ) exerted a much lower influence. Dependences of the work of strain (A) values from the USW parameters showed the same trend as elongation at break (τ) was characterized by a more negligible effect (t) exerted the maximum influence.According to at break b. The clat break c. In tere effect d. Duratit parameter was found to have made the greatest contribution to the increase in mechanical properties . The P parameter had slightly less effect . The τ parameter was characterized by a minimal impact; therefore, its maximum value of 1500 ms was taken as “applied”.Based on the obtained results, the USW parameters were ranked. In this stage, we attempted to obtain the maximum mechanical properties. The scoring system is summarized in P = 3.0 atm, τ = 1200 ms). However, as shown below in the Discussion section, mode #9 gave rise to melting of the prepreg and partially damaged CFs. Therefore, an alternative method was required to determine the optimal combination of USW parameters more objectively, primarily by taking into account the structural characteristics.According to the results obtained using the Taguchi method, the maximum mechanical characteristics (both ultimate tensile strength and elongation at break) were provided by mode #9  and the R determination coefficient were estimated, and correlation schemes of the simulated and real data were drawn. The networks with the best learning outcomes are presented in The following methods were tested as training methods: (i) Levenberg–Marquard, (ii) gradient descent, (iii) random increments, (iv) successive increments, (v) Fletcher–Powell coupled gradients, and (vi) Bayesian regularization ,43,47. Bτ = 1000 ms. The clamping pressure was calculated for each USW duration through the linear dependence of the following type: P(t) = (iP − (t − it)/(jt − it)), where iP, ti, and tj are the values of parameters of the i-th and j-th experiments, respectively.Then, the synthesized artificial neural networks (ANNs) were used to calculate the USW joint properties in the three-dimensional space of the USW parameters. The simulation range was chosen according to the previously accepted normalization limits . ReliabiBased on the obtained results, it was found that after approximation, models 6 and 7 showed a number of excessive extrema, as shown in t and high P values, as well as for both high t and low P levels, the behavior trends of models 1–3 were opposite to those for models 4–7. This did not reflect that these results contradicted the physical meaning but that they required experimental verification. Note the excessive extremes for model 1 in On the other hand, Considering the results of the analysis of the structure and the fracture patterns of the USW joints, as well as based on general considerations about the relationship between the structure and mechanical properties, the choice of the threshold values was substantiated below . For a bThe USW modes that provided low ultimate tensile strength levels of the USW joints were considered unacceptable. In particular, the lowest threshold level of 60 MPa was assigned by the authors. On the other hand, failure of the USW joints showed ultimate tensile strength values slightly higher than that for neat PEEK (~93 MPa), occurred over the base material but not in the fusion zone due to its over-strengthening. The most likely reason for of this phenomenon was damage and partial melting of the prepreg. Therefore, the maximum threshold of 93 MPa was assigned.ε < 2 mm) were excluded. On the other hand, fusion-zone over-strengthening caused strains with the formation of a “neck” in the base material at ε > 7 mm.Based on the same considerations, the modes providing brittle fracture at low elongation at break levels  over the entire volume under consideration. ANN simulation results were combined in a summary (collecting) isoline graph over the 3D space section. The optimal values of technological parameters were highlighted as blue-filled regions. Model 2. The extended optimality region was oriented horizontally at P = 3.0 atm and t from 600 to 1000 ms, which was a fairly wide range of the values. The combination of P ~ 3.0 atm/t ~ 800 ms corresponding to USW mode #2 and resulting in improved mechanical properties should be considered the middle of this region.Model 3. There were two optimality areas. The center of the first optimality area corresponded to P ~ 3.5 atm/t ~ 1000 ms, whereas the second one was observed at P ~ 1.5 atm/t ~ 750 ms. The USW parameters of the first region were close to mode #4 (P = 3.0 atm/t = 1000 ms), when sufficiently high mechanical properties of the USW joint were provided. The parameters of the second region were not experimentally verified in this research. Then, the prediction correctness of this ANN model was verified.Model 4. In this case, there was only one optimality region, with the center at P ~ 3.0 atm/t ~ 1000 ms, which approximately reflected the prediction of the previous model (2) .Model 5. Similar to model 3, this model revealed two optimality areas. Their centers also roughly corresponded to those for model 3: (1) P ~ 3.2 atm/t ~ 1070 ms (close to mode #4); (2) P ~ 1.4 atm/t ~ 800 ms .P = 3.0 atm/t = 800 ms (mode #2), (ii) P = 3.0 atm/t = 1000 ms (mode #4), and (iii) P = 1.5 atm/t = 800 ms (hereafter referred to as mode #10). According to the first two modes , the USW joints were fabricated and tested. According to the third combination (mode #10), new USW joints were manufactured. The results of their tests are analyzed in the Discussion section.The following combinations were determined as optimal: (i) τ parameter showed the least effect according to the applied Taguchi method, it was not analyzed in detail when discussing the results. Consequently, τ value of 1500 ms.Because the According to the stress–strain diagrams , the higt = 1200 ms (modes #7–#9) became apparent from the analysis of the micrographs of their fusion zone. The reasons for the exclusion of the USW joints fabricated at P = 2.0 atm/t = 800 ms; h = 0.42 mm). Therefore, the test coupon failed, with a slight elongation at break value of 3.4 mm an ultimate tensile strength of 76.2 MPa. These levels were within the accepted thresholds of the optimality condition. However, the fusion zone structure was heterogeneous. P). To this end, the same experiments were carried out using mode #10.We also considered it necessary to provide the micrographs of the cross section of the test coupon fabricated according to mode #1  of 800 ms due to the low clamping pressure (P) of 1.5 atm. Furthermore, the clamping pressure (P) minimized possible damage to the prepreg and CFs. Such a mode was predicted by both neural network models 3 and 5. Additionally, this mode was outside of the initial range in the design of the experiment.A USW joint formed according to the mode #10 was characterized as follow: crograph d showed h)) characteristics fell within the specified ranges, all structural changes were not taken into account, i.e., the degree and pattern of both ED and prepreg melting, possible damage of CFs, the formation and degree of porosity, etc. In particular, the optimality condition for the USW parameters could be accompanied by a local violation of the prepreg integrity (mode #4). However, structural characterization was a longer process than the assessment of the USW joint properties, requiring a much greater amount of the experimental data. Therefore, it was necessary to explicitly add parameters characterizing the integrity of the structural elements (the prepreg) to determine the optimal combination of USW parameters. They should be easily and quickly measured without a significant spread over different sections of the USW joints. We will conduct further research in this direction in the future.The following result is of particular importance. Even if both mechanical and structural  of the known data, as well as prognosis of a single property of the joint. It should be stressed that many statistical methods are available to model a single parameter, offering competitive results with those of machine learning methods in terms of accuracy and robustness. We demonstrated that when modeling complex systems with a variety of output parameters, provided they are statistically correlated, an NNS can be adequately employed. The cited papers lack information on the optimality criteria of the USW parameters. The relevant papers on the application of ANNs to solve USW problems ,61,62,63In the current paper, the condition was formalized on achieving the optimality of the USW parameters by attaining the specified mechanical and structural properties in the form of an inequality system (1). The problem of selecting optimal USW parameters based on a small sample was solved in two steps: through continuous approximation of the material property vector with the use of (i) NSS and (ii) determination of the optimal parameters’ range limits. We demonstrated that special attention should be paid to the correctness of the simulation within the approximation and extrapolation ranges of experimental data rather than minimizing the approximation errors of known data (as investigated in numerous papers). As expected, computer simulation with a small training sample was limited due to the use of the simplest ANNs based on a single-layer perceptron, resulting in the need to synthesize several models ,65. TherThe definition of “the optimality combination of the USW parameters” was introduced as a condition for satisfaction of the inequality system (1). The mathematical problem of determining the optimal combination of USW parameters was formulated for the formation of USW lap joint with improved mechanical and structural properties.A methodology for studying the mechanical properties of USW lap joints was proposed based on the design of an experiment via the Taguchi method and experimental data approximation with the use of neural network simulation (NNS).Experiments were performed, and the threshold values of the optimality conditions for the USW parameters were chosen. NNS was accordingly carried out to determine their ranges.P = 3.0 atm/t = 1200 ms combination. This evidence was based on consideration of the mechanical characteristics of the USW joints. However, intense prepreg melting and fracture of CFs occurred during the USW process. Therefore, this mode could not be considered optimal.We demonstrated that according to the Taguchi method, the rational USW parameters corresponded to the We proved that verification of the computer simulation results obtained with a small sample can be carried out only by experimental analysis of their reliability. To obtain formal criteria to assess such facts, it is necessary to conduct additional studies.P = 1.5 atm, t = 800 ms and τ = 1500 ms) ensured formation of lap joints with the required mechanical and structural properties .We analyzed the optimal combination of USW parameters proposed on the basis of NNS. Based on the example of two modes, we demonstrated that the developed optimality condition was insufficient and required correction, taking into account other significant structural characteristics of the formed USW joints. However, the NNS enabled specification of an extra area of USW parameters, which were not previously considered as optimal when designing the experiment. The NNS-predicted optimal US welding mode (The results of this study are related to the use of additional valuable structural characteristics, as well as attracting more complex neural network models. In terms the PEEK-CF layered composite fabrication with the use of US-welding, CF fabric prepregs are suitable for the design of “CF prepreg—PEEK composite” laminates.The following results were obtained in the present study:


Trypanosoma brucei, the parasite that causes sleeping sickness, cycles between an insect and a mammalian host. However, the effect of RNA modifications such as pseudouridinylation on its ability to survive in these two different host environments is unclear. Here, two genome-wide approaches were applied for mapping pseudouridinylation sites (Ψs) on small nucleolar RNA (snoRNA), 7SL RNA, vault RNA, and tRNAs from T. brucei. We show using HydraPsiSeq and RiboMeth-seq that the Ψ on C/D snoRNA guiding 2′-O-methylation increased the efficiency of the guided modification on its target, rRNA. We found differential levels of Ψs on these noncoding RNAs in the two life stages  of the parasite. Furthermore, tRNA isoform abundance and Ψ modifications were characterized in these two life stages demonstrating stage-specific regulation. We conclude that the differential Ψ modifications identified here may contribute to modulating the function of noncoding RNAs involved in rRNA processing, rRNA modification, protein synthesis, and protein translocation during cycling of the parasite between its two hosts. O-methylation (Nm) and pseudouridinylation (Ψ) are the most abundant RNA modifications in eukaryotes  propagates in the midgut and undergoes transformation to a metacyclic stage; these parasites are transferred to the mammalian host upon feeding by the infected fly. In the mammalian host, trypanosomes replicate in the blood as the bloodstream form (BSF)  donates a 5′ exon to all mRNAs (7G and four hypermodified nt (rm (BSF) . Trypanosplicing  and RNA splicing , but lacsplicing . In tranll mRNAs , 14. Thell mRNAs  and undeified nt . The SL ified nt . The maiified nt .T. brucei U1, U2, U4, U5, and U6 snRNAs possess ∼61 Ψs that are mostly guided by snoRNAs   . mtap sind snRNA . Ψ modifnd snRNA . Over 25get mRNA .trans-splicing and mRNA production, suggesting its role in mRNA metabolism  have unique properties compared to other eukaryotes. As an example, Trypanosomes possess a homolog of 7SL RNA, but in contrast to all eukaryotes, the signal recognition particle is composed of two RNA molecules: the 7SL RNA and a tRNA-like molecule, sRNA-76 , 26. Thetabolism .cbf5 silenced cells, the Nm guided by C/D snoRNA was significantly reduced. tRNA levels and Ψ modification were also shown to differ between the BSF and PCF parasites. This study highlights the potential role of Ψ modifications on ncRNAs for gene regulation at different developmental stages.Very little is known regarding the Ψ modification of ncRNAs other than that of the abundant snRNAs . It was Previously we used more than 14 independent biological replicates of small RNA Ψ-seq libraries to detect Ψs mostly on snRNAs involved in splicing . Here, wT. brucei parasites, depleted of ribosomes by ultracentrifugation, and the resulting postribosomal supernatant (PRS) was utilized to extract fractions enriched in small ncRNA (A) (B). To enable quality assessment of PCF and BSF small RNA HydraPsiSeq libraries, the fragmentation profile of the Ψ on SL RNA was determined. SL RNA has a single Ψ28 guided by SLA1 RNA (Ci) suggested that all uridine residues along the SL RNA were fragmented  B. To enaSLA1 RNA . The datSLA1 RNA Ci suggesnt score ) except Cii) indicated that Ψ28 is fully modified in both life stages of the parasite. To examine the ability of the method to detect variations in Ψ level, we also mapped Ψ on RNA derived from cells silenced for mtap and cbf5. MTAP is a protein that was shown to bind to SLA1  verified the dependance of Ψ28 on these two proteins, as previously described  and HydraPsiSeq , and the Ψ was plotted on a scheme showing the experimentally verified secondary structure of vtRNA . This position is modified to a similar degree on both life stages  and is guided by dual functioning snoRNA, as based on small RNA Ψ-seq , the stoichiometry of Ψ100 is reduced upon cbf5 and mtap silencing.Next, we analyzed the Ψs on vtRNA . A singlFig. 1NA Ψ-seq  and HydrAi) with nine sites verified by HydraPsiSeq (Aii). Note that our HydraPsiSeq analysis could not detect Ψ on uridine residues carrying both Ψ and 2′-O-methylation sites in T. brucei rRNA, for an unknown reason. Next, we investigated the localization of these Ψs on the secondary structure depicting the conserved domains of the T. brucei 7SL RNA (Aiii)   of >3 and Ψ-ratio of > 0.01) and based on 14 libraries, 11 putative Ψ sites were detected on 7SL RNA Ai with nraPsiSeq Aii. Note 7SL RNA Aiii 26)Ai) with  complex , 26, 27.n snRNAs Aii 23)..Ai) withAs (Aii) , we obsen snRNAs . Interesn snRNAs . To idenn snRNAs  and S1 . Note that because of the adjacent uridine residues on the snRNA, smaller fragments are generated during hydrazine-aniline cleavage, which are not suitable for sequencing in our experimental setup, and thus, HydraPsiSeq is not suitable for mapping Ψ on such U-rich small RNA molecules. Using small RNA Ψ-seq, we found that U3 snoRNA possess a rich repertoire of 22 Ψs. This is a surprising result since only four Ψs were mapped on human U3 (Biii). Ψs were also located in loop domains such as Box A, A′, B, C, and C′, suggesting that as in U2, U4, and U6 snRNAs, Ψs in U3 are concentrated within RNA–protein interaction domains, as well as in RNA–RNA interaction domains that were shown to be crosslinked to pre-rRNA in the 5′ external transcribed spacer (cbf5 and mtap silencing using Ψ-seq (Biv and v). Indeed all the detected Ψ sites were partially reduced in these silenced cell lines. Twelve snoRNAs were identified to potentially guide Ψ positions on U3 snoRNA by psoralen UV-induced crosslinking  by RiboMeth-seq  (B). This result indicates that the Nm modification guided by these four snoRNAs is strengthened by the Ψ, leading to more efficient methylation by the snoRNA. Note, in all these four snoRNAs, the Ψ is not located in region of base pairing with rRNA target, suggesting that these Ψ might affect the binding of C/D core RNPs as was previously suggested by us, demonstrating that Ψ strengthens the interaction of U2 snRNA with it binding proteins U2A′ and U2B”  B. This rand U2B” .Table 2SAi) with seven Ψ sites verified by small RNA HydraPsiSeq (Aii). The level of the Ψs on H/ACA snoRNA was examined by HydraPsiSeq in the two life stages, and we found cases of both hypermodification and hypomodification of these sites in BSF  and the ARAGORN tRNA detection program (A). The TGIRT enzyme introduces a mutation when encountering a modified nucleotide (B). Our analysis identified all the 66 tRNAs that were previously described in T. brucei (C). Two tRNAs were more abundantly expressed in PCF, and four tRNAs  were more abundantly expressed in BSF (C).It was previously reported that mapping of modifications using conventional reverse transcriptase on tRNAs is not feasible, because the RT terminates readily on the many modifications present on tRNAs. Since the population of expressed tRNAs is not well documented in  library A and the program . To this program , 42 . The tAI was calculated based on total number of reads obtained for each tRNA molecule and the codon usage in each life stage  D. The tAfe stage , 45. Thefe stage , 45.A). Our results (A and B) suggest that the rate of mutation introduced at the Ψ site within the TΨC is higher in RNA treated with CMC (+CMC) compared to untreated RNA (-CMC). Using this method, we detected Ψ sites in 18 tRNAs (B–D). Interestingly on four tRNAs, we detected more than a single Ψ, as was previously reported in humans . The Ψ sites on tRNAs were not reduced upon cbf5 silencing (B and D), suggesting that these Ψ sites are guided by PUS enzymes. Indeed, our recent study showed that PUS1, 3, and 7 guide Ψ on tRNAs, as in yeast and mammals . The Ψ sites in Ψ47 in tRNALeu(TAG) and Ψ54 in tRNAAsp(GTC) were hypermodified in the BSF, and Ψ32 in tRNAArg(CCT) was detected only in BSF , suggesting that some Ψ sites on tRNA are developmentally regulated.The small RNA-Seq using TGIRT was also used to identify Ψs in tRNAs. Recently, it was demonstrated that TGIRT introduces mutations in Ψ sites when treated with CMC . As a poFig. 6n humans   and may miss fragments coming from the termini of small RNAs.T. brucei 7SL RNA . In addition, Ψs are present within the interaction domain between U3 and 5′ external transcribed spacer, which was suggested based on psoralen UV crosslinking between the pre-rRNA and U3 . Th. ThT. brA and U3 . HoweverA and U3 .mtap silencing, suggesting that these modifications are likely guided by PUS enzymes  seem to be more closely related to class-I tRNAs , while the BSF preferentially expressed tRNAs  are more similar to class-II . Th. ThT. brectively C seem toclass-II  (43). ClMost striking was the observation of high adaptability of the tRNA pool to the codon usage on the mRNA of BSF compared to the PCF, suggesting that translation in BSF might be more efficient because of the high tRNA adaptability. Indeed, BSF parasites grow faster than PCF  and adapT. brucei, strain 29 to 13 (T. brucei 427 (cell line 1313-514) was aerobically cultivated at 37 °C under 5% CO2 in HMI-9 medium supplemented with 10% fetal calf serum, 2 μg/ml G418, and 2.4 μg/ml phleomycin  (A). RNA extracted from the PRS was used for library preparation, essentially as described (Whole-cell extracts were prepared from 10150000g) A. RNA exescribed .PRS RNA (5 μg) was treated with 50% hydrazine (Sigma) for 45 min  on ice, and ethanol was precipitated . The RNATo perform HydraPsiSeq, the fragmented RNA (∼800 ng) was dephosphorylated with FastAP thermosensitive alkaline phosphatase (Thermo Scientific), cleaned by Agencourt RNA clean XP beads (Beckman Coulter), and ligated to a 3′ linker using high concentration T4 RNA Ligase 1 (NEB) in a buffer containing dimethyl sulfoxide, ATP, PEG 8000, and RNase inhibitor (NEB). The ligated RNA was cleaned from excess linker using Dynabeads MyOne SILANE beads (Thermo Scientific), and first strand complementary DNA (cDNA) was prepared using the AffinityScript Reverse Transcriptase (Agilent). The RNA was subsequently degraded using 2 μl of 1 M NaOH, and the cDNA was cleaned using Dynabeads MyOne SILANE beads. The cDNA was further ligated to a 3′ adapter using a high concentration T4 RNA Ligase 1 (NEB) and cleaned of excess adapter using Dynabeads MyOne SILANE beads (Thermo Scientific). The adapter-ligated cDNA was PCR enriched using NEBNext high-fidelity (NEB) polymerase (9 PCR cycles), separated on an E-Gel EX agarose gel (Invitrogen), and size selected at the range of 150 to 300 bp (containing ∼30–180 nt corresponding to RNA). The amplicons were gel purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) and sequenced in a Nextseq system (Illumina) in paired end mode (20 million reads for each sample).T. brucei small RNA using Smalt v_0.7.5 (http://www.sanger.ac.uk/resources/software/SMALT/) with default parameters. For each sample, the resulting bam file was sorted and filtered for proper pairs using Samtools v1.9 (https://github.com/FlorianPichot/HydraPsiSeqPipeline), as previously described  was added, and RNA samples were incubated at 90 °C for 20 min  was denatured at 90 °C for 2 min in a thermocycler. Then, an equal volume of buffer  with the following parameters:--stringency 4 --length 30 --paired --retain_unpaired. The alignment to the reference rRNA sequence was done by STAR (ver 2.0.6)   , mapped,eleases) . The 5′-eleases) .tAI was calculated for each mRNA in the two life stages, similarly to the tAI measure of translation efficiency . Here, thttps://eur02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fdataview.ncbi.nlm.nih.gov%2Fobject%2FPRJNA797695%3Freviewer%3Dpbla7s5ift4qbl8i31f0uj1vm8&data=04%7C01%7Ctirza.doniger%40biu.ac.il%7C67e6e9b0f3324592855b08d9d99dd61f%7C61234e145b874b67ac198feaa8ba8f12%7C1%7C0%7C637780094192200189%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=FiPh%2Bb3y1nefbTQbECYgLO7DnOm7B76xKT3EOSamTag%3D&reserved=0). The RiboMeth-seq data is available under the accession number PRJNA836748, PRJNA526606, and PRJNA776556. The small RNA Ψ-seq (The small RNA HydraPsiSeq sequencing data related to this study have been deposited in the NCBI BioProject database under the accession number PRJNA797695 (https://github.com/michaelilab/TB_Pseudo_small_ncRNA.All bioinformatics scripts used in this study are available from the corresponding author upon request. Scripts used to analyze HydraPsiSeq and RiboMeth-seq are available at This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Immune checkpoint inhibitors (ICIs) are a revolutionary breakthrough in the field of cancer by modulating patient’s own immune system to exert anti-tumor effects. The clinical application of ICIs is still in its infancy, and their dosing regimens need to be continuously adjusted. Pharmacokinetic/pharmacodynamic studies showed a significant plateau in the exposure-response curve, with high receptor occupancy and plasma concentrations achieved at low dose levels. Coupled with concerns about drug toxicity and heavy economic costs, there has been an ongoing quest to reevaluate the current ICI dosing regimens while preserving maximum clinical efficacy. Many clinical data showed remarkable anticancer effects with ICIs at the doses far below the approved regimens, indicating the possibility of dose reduction. Our review attempts to summarize the clinical evidence for ICIs regimens with lower-dose, less-frequency, shorter-course, and provide clues for further ICIs regimen optimization. Immune checkpoint inhibitors (ICIs) are revolutionary breakthroughs in the field of cancer in recent years, which have changed the traditional treatment paradigm. At present, the relatively proven ICIs in clinical application include cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors and programmed cell death protein-1 (PD-1) receptor inhibitors/programmed cell death ligand 1 (PD-L1) inhibitors. CTLA-4 is a transmembrane receptor on T cells, which can compete with CD28 to prevent co-stimulation and induce T cell cycle arrest. CTLA-4 inhibitors block the above process and restore the function of T cells to eradicate tumor cells. The U.S. Food and Drug Administration (FDA) approved CTLA-4 inhibitors include: ipilimumab, tremelimumab. PD-1 is expressed on tumor-infiltrating lymphocytes (mainly CD4+ T cells), B cells, natural killer cells, monocytes and dendritic cells, while PD-L1 is highly expressed on tumor cells. The binding of PD-1 to PD-L1 mediates a co-inhibitory signal of T cell activation, thus leading to tumor immune escape. PD-1/PD-L1 inhibitors block the PD-1 signaling pathway, partially restoring T-cells recognition of tumors and inducing immune normalization. Currently FDA-approved PD-1 inhibitors include: nivolumab, pembrolizumab, cemiplimab, dostarlimab, and PD-L1 inhibitors include: atezolizumab, avelumab, durvalumab. In fact, most research on dose intensity reduction of ICI focused on nivolumab, pembrolizumab and ipilimumab, our review also mainly focused on these three ICIs.ICIs belong to monoclonal antibody and their pharmacokinetic/pharmacodynamic properties are distinctly different from those of traditional cytotoxic and small molecule drugs; therefore, determining the optimal dose of ICIs using traditional drug models may face many difficulties. The recommended dosing regimens for ICIs have evolved as experience accumulates. ICIs were initially administered based on body weight, and as population pharmacokinetic data accumulated, fixed-dose regimen was found to improve convenience and reduce waste while preserving efficacy, thus FDA approved nivolumab 240 mg Q2W equivalent to 3 mg/kg Q2W and pembrolizumab 200 mg Q3W equivalent to 2 mg/kg Q3W. Subsequently, high-dose, extended-interval dosing regimens (e.g. nivolumab 480 mg Q4W and pembrolizumab 400 mg Q6W) were added to all approved adult indications based on silico simulations –4, and vConsidering the economic and convenience reasons, many scholars suggest the need to reevaluate the current ICI dosing regimens. They proposed the concept of interventional pharmacoeconomics, hoping to reduce health care costs and perhaps also adverse effects while maintaining treatment efficacy through the development of new dosing regimens , 12. TheThe incidence of irAEs is 70-90% for any grade, 10-40% for grade 3/4, and 0.3-1.2% for fatal (grade 5) irAEs –22. An iDose optimization studies on ICIs are limited, focusing on nivolumab, pembrolizumab, and ipilimumab. An extensive literature search was conducted for the three most widely used ICIs to collect clinical data of each drug. Retrieval method: Firstly, in the PubMed database, the literature was searched by “Clinical Trial”, “Prospective Studies”, “Retrospective Studies” and “nivolumab”, “pembrolizumab”, “ipilimumab”. Secondly, search clinical trials of each drug in Clinicaltrials.gov. Inclusion criteria: retrospective and prospective studies of three ICIs that include off-label dosing regimens, whether monotherapy or combined therapy, whether directly comparing different dosing regimens or simply including cohorts with off-label dosing regimens. Although most attempts at off-labelled dosing regimens are pharmacokinetic/pharmacodynamic simulations, subgroup data from early clinical trials or retrospective studies with small samples, they can still provide clues to the optimization of ICIs (For most ICIs (except ipilimumab), there is no clear relationship between dose and efficacy or safety. The dose-response and exposure-response curves showed an obvious plateau, implying that increasing doses do not contribute to tumor control and that lower-dose ICIs may produce the same effect –63. AgraIn terms of dosing intervals, prolonging the interval still maintains the pharmacodynamic parameters at effective levels. Simulated administration of nivolumab at 240mg Q4W/480mg Q8W regimen and pembrolizumab at 200mg Q6W regimen revealed that serum drug concentrations remained above the minimum effective concentration in more than 95% of patients . The CanPharmacokinetic studies have shown that many variables can influence the clearance of ICIs, such as: gender, race, weight, performance status, tumor volume, drug response, and albumin levels , 75, 76.In the phase I CA209-003 trial , melanomBased on the results of the CheckMate 205 study , The FDAA retrospective study of advanced NSCLC found no significant differences in PFS, OS, or high grade irAEs between pembrolizumab 100mg and 200mg groups, either alone or in combination with chemotherapy . SimilarRationalized medication of ipilimumab is focused on combination therapy with PD-1 inhibitors. Various attempts have been made to reduce the dose in the combination, but the choice of which drug to reduce and by how much to preserve maximum efficacy while reducing toxicity is inconclusive. Given the dose-dependent toxicity of ipilimumab, we prefer to reduce the dose of ipilimumab in combination therapy.Although the combination of 3mg/kg nivolumab with 3mg/kg ipilimumab provides a potential survival benefit, severe toxicity limits its use . Common Many studies have further extended the interval of ipilimumab to 6 weeks , 94–97 oA much lower-dose therapy of nivolumab (0.5 mg/kg) plus ipilimumab (0.3 mg/kg) combined with interleukin 2 and hyperthermia treating 131 cases of multiple advanced cancers showed an ORR of 31.3%, with median PFS reached 10 months, and the incidence of grade 3-4 irAEs was only 8.4% . In a BeThere are relatively few studies of the combination of pembrolizumab and ipilimumab. In the KEYNOTE-029 study, melanoma patients received pembrolizumab 2 mg/kg Q3W in combination with ipilimumab 1 mg/kg Q3W followed by pembrolizumab monotherapy had an ORR of 62% and a 3-year OS of 73% . Cohort In the KEYNOTE-021 study, receiving pembrolizumab plus low-dose ipilimumab in later-line treatment for NSCLC resulted in an ORR of 30% and grade 3-5 irAEs rate of 29% . HoweverGiven these published findings, there is immense potential to reduce the dose intensity of ICIs by applying available pharmacology or clinical data, aided by prospective interventional pharmacoeconomic trials. Given the fact that ICIs are packaged in fixed single vials and drug sharing is not agreed upon in most hospitals, single-dose reduction may not be cost effective. Alternatively, extend the frequency of administrations (with the minimum effective plasma concentration) could reduce costs, adverse events, and patient inconvenience, and is in line with the context of Covid-19 pandemic. In the opinion of Goldstein et.al., the simplest approach to reduce the dose intensity of Atezolizumab or Nivolumab was using a standard dose with an extended interval far greater than the labeled regimens , 14. TheThe treatment efficacy with different durations have been compared across several published clinical trials. Patients in the CA209-003 study were treated with nivolumab for up to 2 years with a 5-year OS of 16% , the CheMelanoma is the most effective and well-studied tumor in immunotherapy, and a series of published studies on selectively discontinuation of ICIs have focused on melanoma. In the revised protocol of the KEYNOTE-001, 006 studies, patients who received pembrolizumab for more than 6 months, whose tumors reached CR and followed by at least two cycles of pembrolizumab could choose to discontinue treatment early. A total of 67 and 23 CR patients discontinued treatment, and their 2-year PFS were 89.9% and 86.4%, which were similar to the outcomes of other enrolled patients , 113. A The best treatment response for most NSCLC patients is PR/SD rather than CR, and the optimal treatment duration may vary from melanoma. The CheckMate 153 study is the first phase III randomized controlled study of ICIs duration to explore whether continuation treatment provides a survival benefit for advanced NSCLC who are progression-free after 1-year nivolumab treatment. The results showed that patients who discontinued nivolumab had an increased risk of relapse and that continued nivolumab could provide a survival benefit. Further stratified analysis showed that CR/PR patients is benefit from continuous treatment, while for SD patients, the median PFS of continuous treatment and 1-year fixed treatment was similar, suggesting that continued treatment was more meaningful for CR/PR patients . A multiA multicenter observational study in India found that a short course of ICIs (0.5-13 months) was comparable in clinical benefit rate to standard ICIs therapy from literatures . Oulu UnA randomized clinical trial initiated by the University of Chicago sought to compare the standard interval and extended interval dosing of nivolumab (240mg Q2W/480mg Q4W vs 240mg Q4W/480mg Q8W) or pembrolizumab (200mg Q3W/400mg Q6W vs 200mg Q6W) in locally advanced or metastatic cancers . RoswellA number of clinical trials focusing on ICIs discontinuation are carrying out. Similar to CheckMate 153, the Japanese phase III SAVE study recruits in silico modelling and simulation, as well as incorporated TDM strategy to monitor trough concentrations within the therapeutic range, enable the provision of precision dosing of ICIs. Various novel phase I/II clinical trial designs have been proposed in the literature to select the optimal biological dose, such as Bayesian adaptive design  has much less instructive for ICIs recommended dose. If the MTD is not reached during dose escalation, the recommended phase 2 dose could be evaluated based on safety profile, pharmacokinetics/pharmacodynamics modelling simulations, early efficacy biomarkers, the variation of immunological composition reflecting immunomodulatory effect, target engagement receptor occupancy model and other new parameters –143. Bese design –148. In e design . Besidese design .Cancer patients are in an immunosuppressed state and are at high risk for COVID-19 infection, with a high incidence of severe cases and mortality , 150. DuThe safety and/or efficacy of the COVID-19 vaccine, as well as the interaction between the vaccine and the ICI, are inconclusive for patients treated with ICI whose immune systems are activated. The limited evidence available support COVID-19 vaccination in patients treated with ICIs. Chen YW  and WaisRational dose selection and optimization of dosing regimens are of clinical importance and are prerequisites for enhancing patients’ medication compliance and obtaining maximum clinical benefits. The management of ICIs is still on research phase, and the approved dosing regimen may not be the best. The previously established approach of early-phase dose-finding clinical trials is not appropriate for current immunotherapy. Various phase I/II clinical trial designs have been proposed to select the optimal biological dose of ICIs, pending test of reasonableness in practice. There is an emergent need to explore the efficacy and safety of modified ICIs treatment strategies  to promote the use of ICIs and reduce drug toxicity and economic wastage. Pharmacokinetic/pharmacodynamic studies, early clinical trials, and small sample attempts suggest that lower-dose and less-frequency administration of ICIs may have durable effects, similar to those of standard dosing regimens. Compared to reduce single-dose, the better way to reduce dose intensity is probably to extend the dosing frequency, which is more economical and convenient, especially in the context of COVID-19 pandemic. The search for the optimal duration of ICIs is also progressing, from a fixed course of treatment to determining the duration of treatment based on treatment response, and further searching for imaging and biological biomarkers to help determine the timing of drug discontinuation. It is important to note that the optimal dosing regimen of ICIs is related to the immunogenicity of the tumor, disease stage, and physical status of patients, and extrapolation of results requires caution. In the era of precision medicine, we pursue individualized treatment rather than using the same schedule for all patients. More pharmacokinetic/pharmacodynamic studies, interventional pharmacoeconomics clinical trials and real-world data, as well as in-depth studies on the mechanisms of ICIs are very essential. These off-labelled dose de-escalation of ICIs in clinical practice would be under the guidance and collaboration of pharmaceutical manufacturers. The correlated research potentially of interest to insurers, government payers, academic institutions, as well as professional/patient associations. Although the study funding and the dissemination of the concepts may be difficult, it is of great interest and urgently needed to reduce the medical stress on both individuals and society.MJ contributed to the study conception and design, data acquisition, data analysis and interpretation, manuscript writing and revision. MJ, YH and GL contributed to the study data acquisition, data analysis and interpretation, original draft preparation. MJ and CC contributed to review and revise the manuscript. All authors agree to be accountable for the content of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.