African swine fever virus (ASFV) is producing a devastating pandemic that, since 2007, has spread to a contiguous geographical area from central Europe to Asia. In July 2021, ASFV was detected in the Dominican Republic, the first report of the disease in the Americas in more than 40 years. ASFV is a large, highly complex virus harboring a large dsDNA genome that encodes for more than 150 genes. The majority of these genes have not been functionally characterized. Bioinformatics analysis predicts that ASFV gene A859L encodes for an RNA helicase, although its function has not yet been experimentally assessed. Here, we evaluated the role of the A859L gene during virus replication in cell cultures and during infection in swine. For that purpose, a recombinant virus (ASFV-G-∆A859L) harboring a deletion of the A859L gene was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. Recombinant ASFV-G-∆A859L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, demonstrating that the A859L gene is non-essential for ASFV replication. Experimental infection of domestic pigs demonstrated that ASFV-G-∆A859L replicates as efficiently and induces a clinical disease indistinguishable from that caused by the parental ASFV-G. These studies conclude that the predicted RNA helicase gene A859L is not involved in the processes of virus replication or disease production in swine. African swine fever (ASF) is currently affecting the swine production industry in a large geographical area stretching across Europe and into East and Southeast Asia. In July 2021, the Dominican Republic reported its first outbreak of ASF, which had been absent from North America for 40 years. As a result, global swine industries are threatened by significant economic losses and human populations face food insecurity [The etiological agent, African swine fever virus (ASFV), is a large, structurally complex virus with a large (more than 180 kb pairs) double-stranded DNA genome . ASFV enA small number of ASFV genes have been successfully deleted from the ASFV genome  ,16,17,18Six ASFV genes with potential RNA helicase activity have been identified using functional genomics: QP509L, Q706L, D1133L, B962L, F105L and A859L . The gen6 cells per ml. ASFV Georgia (ASFV-G) was a field isolate kindly provided by Dr. Nino Vepkhvadze from the Laboratory of the Ministry of Agriculture (LMA) in Tbilisi, Republic of Georgia [50 . After adsorption for 1 h at 37 °C under 5% CO2, the inoculum was removed, cells rinsed with PBS twice, and further incubated with macrophage media for 2, 24, 48, 72 and 96 h at 37 °C under 5% CO2. At described times post-infection, the cells were frozen at ≤−70 °C and thawed, and the lysates were titrated by HAD50/mL in primary swine macrophage cell cultures in 96-well plates. All samples were run simultaneously to avoid inter-assay variability. The presence of the virus was assessed by hemadsorption (HA), and virus titers were calculated as previously described [Primary cultures of blood-derived swine macrophages were performed as previously described . Ficoll- Georgia . Growth escribed .® RNA Cleanup Kit . One μg of RNA was used to produce cDNA using qScript cDNA SuperMix  that was used for the qPCR. As previously described , we usedPrimers and probes for the detection of the A859L gene were designed using the ASFV Georgia 2007/1 strain (GenBank Accession #NC_044959.2). Primer forward: 5′-GTGTGATCTCCCGCCTATG-3′, reverse: 5′-CTTCCACAGGAGTTATCACCAG-3′ and probe: 5′-FAM/AGCCATCTTTGCCCTCTGATCCG /MGBNFQ-3′. Primers and probes for the detection of p72 gene: forward 5′-CTTCGGCGAGCGCTTTATCAC-3′, reverse: 5′-GGAAATTCATTCACCAAATCCTT-3′ and probe: 5′-FAM-CGATGCAAGCTTTAT -MGB NFQ-3′. Primers and probes for the detection of CP204L (p30) gene: forward 5′-GACGGAATCCTCAGCATCTTC-3′, reverse: 5′-CAGCTTGGAGTCTTTAGGTACC-3′ and probe: 5′-FAM-TGTTTGAGCAAGAGCCCTCATCGG-MGB NFQ-3′. Primers and probes for the detection of the β-actin gene: forward 5′-GACCTGACCGACTACCTCATG-3′, reverse: 5′-TCTCCTTGATGTCCCGCAC-3′ and probe: 5′-FAM-CTACAGCTTCACCACCACGGC-MGB NFQ-3′. All qPCRs were conducted using the TaqMan Universal PCR Master Mix  using the following amplification conditions: One step at 55 °C for 2 min, followed by one denaturation step at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 65 °C for 1 min.ASFV lacking the A859L gene (ASFV-G-∆A859L) was developed by homologous recombination between the genome of the parental ASFV-G and a recombination transfer vector as described elsewhere . The recASFV DNA was extracted from infected cell cultures showing 90–100% CPE, using the nuclear extraction kit (Active Motif cat# 40010), the nucleus and cytoplasmic fractions were separated, the cytoplasmic fraction was used to isolate the viral DNA, following the manufacturer’s protocol. In brief, ASFV infected cells were collected and incubated in the hypotonic buffer for 15 min on ice or until the cell membrane dissolved. The nucleus fraction is separated by centrifugation. The cytoplasmic fraction is collected, and DNA is extracted by adding 10% 3M Na0Ac by volume to the sample  and an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) with a Ph of 6.5–6.9 (Sigma-Aldrich P3803-100ML), then centrifuged for max speed in a tabletop centrifuge. The aqueous layer is then ethanol-precipitated using 2 volumes of 100% ethanol, washed with the same volume of 70% ethanol and dried the resulting DNA pellet is then reconstituted in sterile water. We then used this DNA library for NGS sequencing using Nextera XT kit in the NextSeq  following the manufactures protocol. Sequence analysis was performed using CLC Genomics Workbench software .2 HAD50 of ASFV-G-∆A859L and compared with a group of five pigs inoculated with 102 HAD50 of ASFV-G. Clinical signs  and changes in body temperature were recorded daily throughout the experiment. Blood samples were obtained at 0, 4 and 7 days post-inoculation (pi). Animal experiments were performed under biosafety level 3 conditions in the animal facilities at Plum Island Animal Disease Center, following a strict protocol approved by the Institutional Animal Care and Use Committee (225.01-16-R_090716).The virulence of ASFV-G-∆A859L was evaluated using 35–40 kg commercial breed swine. A group of five pigs was intramuscularly (IM) inoculated with 10https://pfam.xfam.org/ (accessed on: 22 November 2021) showed the existence of a T5orf172 domain (residues 5 to 78), a type III restriction enzyme subunit (residues 166–332) that contains a DEAD/DEAH box helicase domain (residues 170–332), and followed by a helicase conserved C-terminal domain (residues 399–499). Blast analysis of these domains showed an identity around 50 to 30% with the helicase-like protein of different bacteria and parasite species. The A859L gene encodes for a virus protein predicted, through functional genomics, to be an RNA helicase . BesidesInterestingly, blast analysis between A859L protein and other predicted RNA helicases in ASFV, such as QP509L and Q706L, indicates that there is a low percentage of identity (28–26% and 30.16%) in the type III restriction enzyme subunit with these proteins. Furthermore, an identity of 32.61% was depicted just between A859L and QP509L proteins in the DEAD/DEAH box helicase domain. No other significant percentages of identity were found in other functional domains, indicating the potential functional differences between A859L and the other two predicted ASFV RNA helicases.To assess the diversity of the A859L protein across multiple ASFV isolates available through the NCBI GenBank database, we conducted a pairwise comparison analysis. The average A859L protein is 859 residues in length. The % identity at the nucleotide and amino acid levels across multiple ASFV isolates was 90.03–99.96% and 85.56–99.88%, respectively. Overall, 146 variable sites were found in the protein alignment, reflecting the multiple missense mutations accumulated during the evolution of the A859L protein , suggesting that substitutions at these sites may be promoting an adaptative advantage to the Eurasia lineage. Interestingly, position 427 situated at the helicase conserved C-terminal domain appears as a polymorphic site in the alignment (A-V-E), with residue E highly conserved among most of the ASFVs with exception of the Eurasia lineage (A), supporting the potential evolutionary relevance of this site. Conversely, the change in the residue 149 appeared associated just to the isolate Estonia 2014. Furthermore, a deletion at residue 146 appears distinctive of all isolates belonging to the Eurasia lineage  . RemarkaTo determine when the A859L gene is transcribed during the replication cycle, a time-course experiment was performed to analyze the kinetics of RNA transcription in primary swine macrophages infected with ASFV strain Georgia. Swine macrophage cultures were infected at an MOI = 1 with ASFV-G, and cell lysate samples were taken at 4, 6, 8 and 24 hpi. The presence of A859L RNA was detected by two-step RT-PCR, as described in the Material and Methods section. Transcription of A859L was detected at 4 hpi and remained stable until 24 hpi . The patThe relative level of conservation of the A859L gene among different ASFV isolates and its predicted function as an RNA helicase  suggest To evaluate A859L during ASFV replication in cell cultures and in infected animals, a recombinant deletion mutant of the highly virulent ASFV Georgia 2007 isolate (ASFV-G) lacking the A859L gene was developed (ASFV-G-∆A859L). The A859L gene was deleted by swapping 813 amino acid residues in the A859L ORF with a p72mCherry cassette by homologous recombination . A regio® 500, a total of 3,433,439 reads aligned to the reference genome, and the comparative analysis of ASFV-G-∆A859L and ASFV-G genomes confirmed a deletion of 2439 nucleotides, in agreement with the designed genomic modifications. Additionally, the ASFV-G-∆A859L genome possesses an insertion of 1226 nucleotides consistent with the insertion of the p72-mCherry cassette sequence. No additional genomic differences were detected between ASFV-G-∆A859L and ASFV-G, confirming that no undesired changes were introduced during the process of production and purification of ASFV-G-∆A859L. In addition, NGS also indicated the absence of parental ASFV-G genome as a potential contaminant in the ASFV-G-∆A859L stock.To evaluate the accuracy of the genetic modifications applied to the genome of ASFV-G-∆A859L as well as the integrity of the remaining virus genome, the full genome sequence was obtained by NGS using an Illumina NextSeqTo understand the possible function of A859L during virus replication, the in vitro kinetics of ASFV-G-∆A859L replication was evaluated and compared to that of the parental ASFV-G. A multistep growth curve using swine macrophage cultures as a substrate was performed. Macrophages were infected (MOI of 0.01) with either recombinant ASFV-G-∆A859L or parental ASFV-G. Samples to evaluate virus yields were collected at 2, 24, 48, 72 and 96 h post-infection (pi). The results demonstrated that ASFV-G-∆A859L exhibited a very similar growth kinetic to that of the parental ASFV-G without any significant differences in virus yields at any of the evaluated times post-infection . Therefore, removal of the A859L gene from the ASFV-G genome does not affect the capability of ASFV-G-∆A859L to replicate in swine macrophages. This is an interesting result, considering the predicted function of the A859L gene as a helicase, particularly since other ASFV genes, also predicted to function as helicases, have been shown to be critical for virus replication .2 HAD50. A control group was also IM inoculated but with 102 HAD50 of ASFV-G. As expected, all animals inoculated with virulent ASFV-G had a rise in body temperature (>40 °C) on 4–5 days pi. This was rapidly followed by the development of clinical disease   . The cliAll animals inoculated with ASFV-G-∆A859L developed a clinical disease indistinguishable from animals inoculated with ASFV-G. The timeline of the appearance of clinical signs and their severity were similar to those observed in the ASFV-G-inoculated animals. These results indicate that deletion of the A859L gene from the genome of ASFV-G does not affect virus virulence in domestic swine.6.5–107.5 HAD50/mL) on day 4 pi, remaining high until day 7 pi, when all animals were euthanized. All animals inoculated with ASFV-G-∆A859L had viremia values similar to those inoculated with the parental virus by day 4 pi, reaching maximum titers by day 7 pi, when all animals were euthanized (Systemic virus replication in animals was assessed by determining viremia titers throughout the experimental period. Viremias in animals IM infected with parental ASFV-G had expected high titers (10thanized . TherefoThese results suggest that deletion of A859L from the genome of ASFV-G does not significantly affect virus replication or virulence in domestic swine. To further confirm that ASFV-G-∆ A859L was responsible for the virulent phenotype and viremia levels observed, the virus was isolated from the blood of ASFV-G-∆A859L-infected animals and analyzed by NGS. The results obtained by sequencing samples from three animals confirmed the absence of any significant differences with the full-length genome sequence of the ASFV-G-∆A859L stock. In summary, we determined that A859L is a non-essential gene since its deletion from the ASFV-G genome does not significantly alter virus replication either in vitro, in swine macrophage cultures, or during infection in vivo and, importantly, is not critical for ASFV virulence in swine. One potential explanation as to why A859L is non-essential would be the potential replacement of the helicase gene function by other ASFV genes with predicted helicase function ,24. The


Over the past 25 years, the acceleration of achievements in the development of oligonucleotide-based therapeutics has resulted in numerous new drugs making it to the market for the treatment of various diseases. Oligonucleotides with alterations to their scaffold, prepared with modified nucleosides and solid-phase synthesis, have yielded molecules with interesting biophysical properties that bind to their targets and are tolerated by the cellular machinery to elicit a therapeutic outcome. Structural techniques, such as crystallography, have provided insights to rationalize numerous properties including binding affinity, nuclease stability, and trends observed in the gene silencing. In this review, we discuss the chemistry, biophysical, and structural properties of a number of chemically modified oligonucleotides that have been explored for gene silencing. Since themistry  117]. C. CO-MOE-y groups . The proO-MOE soon became the gold standard alkyl modification, owing to its improvement in therapeutically relevant properties. Compared to 2'-OMe RNA, the 2'-O-MOE RNA analogue has similar or even increased RNA binding affinity, as well as a tenfold increase in nuclease resistance , s, sN-guane (GENA)  167], 2, 2N-guane (GENA)  [168–169-Me-ENA)  170], a, aN-guan-Me-ENA)  [171–172O-benzyl-ᴅ-arabinofuranosyl chloride [O-benzoyl-2-deoxy-2-fluoro-α-ᴅ-arabinofuranosyl bromide (Arabino nucleic acids (ANA) are analogs of RNA where the hydroxy group at C2' is inverted . In fluochloride . The 2'- bromide  [176–180Tm relative to the PS-DNA/RNA duplex, however, RNase H-mediated cleavage of RNA was diminished for the duplex formed with PS-FANA relative to PS-DNA [c-MYB mRNA with a persistent silencing effect [Hybridization studies of uniformly modified ANA of mixed nucleobase composition to complementary RNA revealed reduced thermal stability relative to the corresponding DNA/RNA duplex by approximately 1.5 °C per modification –183. A so PS-DNA . Improveo PS-DNA . PS-FANAg effect .-endo (east) conformation that is readily accommodated in a B-form duplex [endo sugar pucker [endo sugar conformation has been reported for the DNA strand in DNA/RNA hybrid duplexes, the natural substrate of RNase H [A 1.55 Å crystal structure of a Dickerson–Drew dodecamer containing fluoroarabinothymine revealed that these modified nucleotides adopt an O4'm duplex   and southern (C2'-endo) configurations for the respective alpha and beta isomers [N-Iodosuccinimide promoted the alkoxylation of the 4'–5'-enol acetates yielded the corresponding 5'-acetoxy-5'-iodo-4'-methoxy intermediates [N,N-dimethylformamide (DMF) followed by a sodium borohydride reduction to give the 4'-alkoxy products [Modifications at the C4' sugar position  have lon isomers . In 2015 isomers . N-Iodosmediates . These iproducts . The 4'-products . In contproducts –204. Rosproducts . Oligomeproducts . These sproducts . In contproducts .endo) sugar pucker, albeit without the need for the bicyclic structures typical for locked nucleic acids [The incorporation of fluorine at the C4' position has long constituted a challenge owing to the instability of the glycosidic bond in the resulting nucleosides. This modification is desirable due to its involvement in the mode of action of the natural antibiotic nucleocidin ,205. Damic acids . The 2',ic acids . Standaric acids . Damha aic acids .endo) sugar pucker despite the 2'-βF, which generally leads to the adoption of a southern or eastern pucker [endo (north) conformation [2',4'-diF-modified siRNA sequences were capable of triggering RNAi with high efficiency, and the incorporation of multiple residues in the guide (antisense) strand yielded more potent siRNAs than those containing LNA or FANA modifications . 2',4'-dn pucker . This mon pucker . In 2018n pucker . This woormation , even inormation . siRNA cormation .19F NMR [endo (north) conformation [O-β-glucosylated nucleocidin, an intermediate in the biosynthetic pathway of nucleocidin, was stable, they may be able to successfully achieve the synthesis of the 4'-F-rU phosphoramidite through a selective protection of the hydroxy groups in stages [3/MeOH to give 5'-iodo-4'-fluorouridine [O-acetyl group with NH3/MeOH, some TBDMS transfer to the C3' position is seen [19F NMR results show that not only does this modification allow for discernment between ssRNA and dsRNA, but it also allows for the identification of mismatches and the binding of RNA-processing proteins with chemical shift dispersions as large as 4 ppm, suggesting that this modification has a wide use for the determination of a variety of RNA structures through NMR spectroscopy [Recently, Zhou provided the first synthesis of a 4'-F-rU  phosphor19F NMR . This moormation , similarormation . Zhou ren stages . Startinouridine . Selectiouridine . Treatmeouridine . The aut is seen . 5'-DMT  is seen . 19F NMRtroscopy .C-aminoalkyl-2'-O-methyl (endo (south) conformation [OMe    has showendo (north) sugar puckering of furanose nucleosides [R and S configurations, respectively [Herdewijn was the first to describe the synthesis as well as the biophysical, structural, and biological characterization of hexitol nucleic acids (HNA), mannitol nucleic acids (MNA), and altritol nucleic acids (AtNA) –220. Theleosides . MNA andectively –220. HNAectively . HNA alsectively . mRNA trectively . AtNA/RNectively .19F NMR structural studies of nucleic acids [In 2011, a work detailing the first synthesis of both isomers of 3'-fluoro-modified hexitol nucleic acid (FHNA and Ara-FHNA)  was public acids . It was ic acids .The published data show that incorporation of a 3'-fluorine atom in the trans-diaxial orientation relative to the base in FHNA  leads toR-6'-Me-FHNA is highly destabilizing, whereas S-6'-Me-FHNA leads to duplex stabilization [R-6'-Me-FHNA [R-6'-Me-FHNA perturbs the structure of water surrounding the O2P atoms which will further reduce the pairing affinity of the R isomer [Methylation at the C6' position further influences the RNA affinity of nucleic acids containing these modifications. lization . This trlization . The 1.2-Me-FHNA . AdditioR isomer .Herdewijn recently published the synthesis of 4'-aminotritylhexitol nucleosides for the eventual synthesis of N4' → P6' phosphoramidates of aminohexitol nucleic acids (AHNA) , as wellTm of the siRNA duplex but is less destabilizing than a mismatch  [Bacillus halodurans RNase H (which was bound to the duplex but did not exert an influence on its structure), revealed distances of 3.09 and 3.12 Å for the F4(dF)···N6(A) atoms of the two dF:A pairs similar to the O4(T)···N6(A) distance (2.96 and 3.11 Å) observed for the native DDD [2) revealed a F4(rF)···N6(A) distance of approximately 4 Å between the rF:A pairs [2'-Deoxydifluorotoluyl (dF) nucleoside derivatives  were firand G/A) . Severaland G/A) ,237–240.:A pairs .OMe, 2'-F, and 2'-O-MOE. Moreover, combinations of these modifications in an oligonucleotide leads to a synergistic effect enhancing their therapeutic properties. Such combinations of nucleotide and backbone modifications with numerous analogs that have been developed will continue as an exciting direction for the next generation of oligonucleotide-based therapeutics. Rational design of future modifications with improved properties may be gleaned from insights from structural techniques. For example, stability, gene silencing and structural studies of chemically modified oligonucleotides containing fluorine at the sugar and nucleobase have provided insights into the role of noncovalent interactions on the properties of these molecules. The partnership between organic synthesis, biophysical chemistry, biochemistry and structural biology continues to guide the design and drive the achievements for oligonucleotide-based therapeutics.Chemically modified oligonucleotides have come of age as a class of therapeutic agents for a number of diseases. Taking inspiration from the structure, properties and biological roles of nucleic acids, scientists have employed chemistry to prepare a diverse collection of modifications to the architecture of this molecule imbuing desirable characteristics for applications as a therapeutic agent. In addition, many nucleic acid analogs have been explored for additional studies including investigation of artificial genetic systems, catalysts, and sensors. Amongst the oligonucleotide-based therapeutics that have been approved as drugs, the dominating modifications are the phosphorothioate backbone and at the C2'-position (of ribose) including 2'-


IL-4 and IL-4R cytokine genes are responsible for immune response in the reproductive system and are related to embryonic implantation and fetal survival during pregnancy in females. However, to date, their effects on litter size traits in pigs have been not elucidated. Therefore, the present study was conducted to verify the porcine IL-4 and IL-4R polymorphisms and assess how they affect litter size traits in commercial pigs. The findings suggested that the porcine IL-4 g.134993898T > C and IL-4R c.1577A > T polymorphisms were associated with the litter size traits. Therefore, the porcine IL-4 and IL-4R genes may be potentially effective genetic markers to improve the litter size traits in pigs.The IL-4 and IL-4R genes and to assess their effects on litter size traits in commercial pigs. Single nucleotide polymorphisms (SNPs) in the porcine IL-4 and IL-4R genes were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. A non-coding SNP of IL-4 g.134993898T > C and a non-synonymous SNP of IL-4R c.1577A > T  were found to be segregating in Landrace sows. The IL-4 g.134993898T > C polymorphism was significantly associated with the number of piglets weaned alive (NWA) trait. The IL-4R c.1577A > T polymorphism was significantly associated with the number born alive (NBA) and NWA traits. Moreover, the accumulation of favorable alleles of these two SNP markers revealed significant associations with the NBA, NWA, and mean weight of piglets at weaning (MWW) traits. These findings indicate that the porcine IL-4 and IL-4R genes may contribute to the reproductive traits of pigs and could be used as candidate genes to improve litter size traits in the pig breeding industry.The interleukin-4 (IL-4) and interleukin-4 receptor (IL-4R) are cytokines that are involved in the immune and reproductive systems. This study aimed to verify the polymorphisms in the porcine  EphA4) [RNF4) [LIF) [IL-6) [OPN) [Litter size traits are among the most important traits for breeding in commercial pig production. Increasing litter size is of great economic interest as a means of enhancing the productivity of the pig industry ,2,3. MajEphA4) , ring fi) [RNF4) , leukemi4) [LIF) , interle) [IL-6) , and ost6) [OPN) . Current6) [OPN) ,14 and h6) [OPN) .IL-4 mRNAs are increased in the uterine endometrium during implantation in pigs and humans [IL-4 gene has been mapped on the long arm of the Sus scrofa chromosome 2 (SSC2q) and is composed of four exons and three introns that are encoded with a peptide of 133 amino acids . Besides, the porcine IL-4 gene is closely located  to the quantitative trait loci (QTL) regions for ovulation rates (128.8–145.1 Mb), total number born (139.9–140.9 Mb), mummified fetuses (133.4–145.1 Mb), numbers of stillborn (128.5–137.1 Mb), the mortality of piglets (138.7–143.7 Mb), gestation length (153.0 Mb), and age at puberty (133.3–157.3 Mb) [IL-4 gene have been characterized and reported in the Ensembl database . Moreover, associations of the IL-4 polymorphisms with the recurrent abortion, pregnancy specific-disorder of pre-eclampsia, allergic diseases, and serum immunoglobulin E (IgE) levels have been reported in humans and mice [IL-4 gene is involved in implantation and pregnancy, thus the IL-4 gene is important to litter size traits.The IL-4 is an anti-inflammatory cytokine that is mainly produced by T-helper (Th) 2 cells, natural killer (NK) cells, mast cells, and basophils . It is id humans ,14,15. Id humans . The IL-57.3 Mb) ,20,21,22and mice ,25,26,27IL-4R is an immune gene that encodes the alpha-chain of the IL-4R molecule, a type-I transmembrane protein that can bind IL-4 and IL-13, and mediates its effect through kinases of the Janus kinase (JAK) family, leading to tyrosine phosphorylation of several substrates in cells [IL-4R mRNAs are expressed in the endometrium and conceptus [IL-4R gene has been mapped on Sus scrofa chromosome 3 (SSC3). It contains ten exons and nine introns that are encoded with a peptide of 824 amino acids . Moreover, the porcine IL-4R gene is closely located  to the QTL regions for total number born (15.3–20.0 Mb), litter birth intervals (11.9–33.7 Mb), and age at puberty (11.1–27.20 Mb) [IL-4R gene have been characterized and reported in the Ensembl database . Moreover, the polymorphisms of IL4R gene are associated with the pregnancy disorders and various cancer types in humans [The in cells ,29. The onceptus ,31. The 7.20 Mb) ,32. The rty 11.1–.20 Mb [Ials 11.9–.7 Mb, ann humans ,35,36.IL-4 and IL-4R genes are responsible for immune response in the reproductive system of mammals. Moreover, their functions are critical for embryonic implantation and fetal survival during pregnancy, as well as their positions, which are closely located to the QTLs for reproductive traits in pigs. Therefore, the IL-4 and its receptor (IL-4R) genes can be regarded as positional and functional candidate genes for the determination of the reproductive traits of pigs. However, information on the association of the IL-4 and IL-4R genes with litter size traits in pigs has been limited. Thus, the porcine IL-4 and IL-4R SNPs were selected based on their segregation in the pig population to elucidate their association with litter size traits. In the present study, we have verified the polymorphisms in porcine IL-4 and IL-4R genes, while their association with litter size traits was assessed in commercial pigs.All these shreds of evidence suggest that the Blood samples were taken from a total of 323 sows of the Landrace pig breed. These Landrace breeding stocks were obtained from a commercial nucleus herd that is established for improved growth performance and reproductive traits. All sows were reared under commercial conditions of the Betagro Hybrid International Company, Thailand. These sows were kept in closed houses with an evaporative cooling system and were fed a corn–soybean-based diet containing 16% crude protein and 3388 kcal/kg digestible energy. The reproductive performance traits of the sows were assessed in 1162 litters (1 to 8 parities) and were recorded in terms of litter size. These traits consisted of total number born (TNB), number born alive (NBA), the number of piglets weaned alive (NWA), mean birth weight of the piglets (MBW), and mean weight of piglets at weaning . Genomic DNA was extracted from blood samples using the Chelex method  and keptIL-4 gene, specific primers were designed based on the available nucleotide sequence information (GenBank accession number: NC_010444.3) to cover four exons and three introns of the porcine IL-4 gene, as shown in IL-4 gene using each primer  to find out the SNPs segregating in this pig population. To verify the SNPs in the porcine IL-4R gene, five non-synonymous SNPs  of the porcine IL-4R gene were selected based on the restriction enzymes available in the Ensembl database . These were used to verify the SNP in the Landrace pig population. The specific primers of the porcine IL-4R gene were designed based on relevant nucleotide sequence information (GenBank accession number: NC_010445.3), as shown in IL-4 and IL-4R genes were used purposely for association analysis in this study. These SNPs were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The PCR amplification was performed in a total volume of 20 μL consisting of 50 ng of a genomic DNA sample, 1× (NH4)2SO4 buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.4 μM each primer . The PCR conditions were as follows: 94 °C for 3 min at the initial denaturing stage; followed by 35 cycles of 94 °C for 30 s, 58–60 °C for 30 s, and 72 °C for 30 s; and then 5 min at 72 °C to complete the reaction. The PCR products were digested with 2.5 U of the restriction enzyme  for each fragment , YSj represents the fixed effect of year-season (j = 1–8), Gk is representative of the fixed effect of the genotypes for IL-4 and IL-4R (k = 1–3) or the accumulated favorable alleles for IL-4 and IL-4R (k = 0–4), and eijkl represents the residual error. Moreover, the additive effect was calculated as the half difference between the two homozygous genotypes and the dominance effect was estimated as the deviation of the heterozygous genotype effect from the mean effect of the two homozygous genotypes [t-test on significant deviations from zero. Furthermore, linear regression between the number of favorable alleles of porcine IL-4 and IL-4R genes and least square mean values of the litter size traits were analyzed.Allelic and genotypic frequencies were calculated. Hardy–Weinberg equilibrium (HWE) was analyzed using the chi-square test. Association analysis of porcine enotypes . The estIL-4 gene, the DNA segments of exons and introns of the porcine IL-4 gene were sequenced. An SNP in intron 3 of the porcine IL-4 gene was found and corresponded to an SNP of porcine IL-4 g.134993898T > C (rs329453960) locus in Ensembl data . It was detected with the restriction enzyme BsuRI. Two specific alleles revealed two fragments of 295 and 38 bp for allele T and three fragments of 180, 115, and 38 bp for allele C  was found in exon 8. It was a non-synonymous mutation leading to a non-conservative amino acid exchange at position 526 from glutamine to leucine (Q526L). This polymorphic site was detected with the restriction enzyme AluI. Two specific alleles revealed a 137 bp fragment for allele A and two fragments of 119 and 18 bp for allele T . The chi-square test revealed that the genotype distributions of the porcine IL-4 g.134993898T > C and IL-4R c.1577A > T loci within Landrace sows were in agreement with the HWE specifications (p > 0.05).The genotypic and allelic frequencies of the porcine IL-4 and IL-4R genotypes on litter size traits were assessed in the Landrace sows. The porcine IL-4 and IL-4R polymorphisms were associated with litter size traits of pigs. Association of the porcine IL-4 g.134993898T > C polymorphism with litter size traits is shown in IL-4 g.134993898T > C polymorphism with any litter size traits was found in the first parity of sows. However, the porcine IL-4 g.134993898T > C polymorphism was significantly associated with the NWA trait in the later parities of sows. The sows with the TT and TC genotypes had higher NWA values than the sows with the CC genotype. The significant additive effect for the NWA trait was detected in the later parities of sows. Thus, the porcine IL-4 g.134993898T allele seems to be a favorable allele for litter size traits in pigs. Association of the porcine IL-4R c.1577A > T polymorphism with litter size traits is shown in IL-4R c.1577A > T polymorphism with any litter size traits in the first parity of sows. However, the porcine IL-4R c.1577A > T polymorphism was significantly associated with the NBA and NWA traits in later parities of sows. Notably, the sows with the TT genotype had higher NBA and NWA values than the sows with the AA and TA genotypes. Additionally, the significant additive effects for the NBA and NWA traits were observed in later parities of sows. Thus, the porcine IL-4R c.1577T allele seems to be a favorable allele for litter size traits in pigs.The effects of porcine IL-4 g.134993898T > C and IL-4R c.1577A > T on litter size traits are shown in IL-4 and IL-4R genotypes seemed to have advantageous effects on litter size traits. On the other hand, the smallest number of favorable alleles (CCAA) of porcine IL-4 and IL-4R genotypes seemed to have disadvantageous effects on these traits.The effects of the accumulated favorable alleles of porcine Increasing litter size is of significant economic interest in the pig industry ,2,3. EmbIL-4 and/or IL-4R genes are associated with allergic diseases [IL-4 and IL-4R genes with respect to reproductive performance traits in commercial pigs. The porcine IL-4 g.134993898T > C and IL-4R c.1577A > T polymorphisms were found to be segregating in Landrace sows. At the porcine IL-4 g.134993898T > C and IL-4R c.1577A > T loci, three possible genotypes were observed in pigs. The IL-4 g.134993898T and IL-4R c.1577A were major alleles in this pig population. The results of the chi-square test revealed that the porcine IL-4 g.134993898T > C and IL-4R c.1577A > T polymorphisms in these sows met the HWE specifications. This result indicates that the porcine IL-4 g.134993898T > C and IL-4R c.1577A > T polymorphisms were under homeostasis when accompanied by the effects of artificial selection.IL-4 is an anti-inflammatory Th2 cytokine that can bind IL-4R to regulate IgE production, immune response, and embryonic implantation ,29,42. Sdiseases ,24, diabdiseases , cancersdiseases , and prediseases ,26,27. IIL-4 is mapped on SSC2 at position 134.9 Mb. Recently, a conducted genome-wide association study (GWAS) revealed that the porcine IL-4 gene is located within QTLs regions for the TNB (139.9–140.9 Mb) and piglet mortality traits (138.7–143.7 Mb) in pigs [IL-4 gene are upregulated in the endometrium during implantation [IL-4 gene plays a crucial role in embryo implantation and may be important for litter size traits.The porcine  in pigs . MoreoveNB 139.9–0.9 Mb anIL-4 gene had a significant association with litter size traits in pigs. A positive effect of the favorable IL-4 g.134993898T allele on litter size traits was found. The IL-4 g.134993898T > C polymorphism was found to be located in the non-coding sequence of the porcine IL-4 gene. We hypothesize that this SNP might be in linkage disequilibrium with other causal polymorphisms that may be located in another region of the porcine IL-4 gene. The association of the porcine IL-4 gene with litter size traits may be attributed to the balance of cytokines in endometrium during implantation period and the regulation of endometrial cytokines by IL-4. The IL-4 polymorphisms may be implicated in the impaired Th1/Th2 cytokine balance and may influence implantation defects and pregnancy disorders. Previous studies have demonstrated that increased anti-inflammatory Th2 cytokines  and decreased pro-inflammatory Th1 cytokines  levels in endometrium are important for successful pregnancy [IL-4 polymorphisms are associated with the recurrent abortion and pregnancy specific disorder of pre-eclampsia in humans [IL-4 polymorphisms may influence the other endometrial cytokines in promoting embryo implantation and placental development during pregnancy. Previous studies have reported that the endometrial cytokines of leukemia inhibitory factor (LIF) and vascular endothelial growth factor (VEGF) are upregulated by IL-4 [VEGF and IL-4 expression levels are associated with recurrent spontaneous miscarriage or recurrent abortion in women [LIF and VEGF genes are associated with litter size traits in pigs [IL-4 gene may contribute to embryonic implantation and be related to litter size traits in pigs.In this study, the polymorphism of the porcine regnancy ,15. Howeregnancy ,45. Moren humans ,27. Furt by IL-4 ,47,48. T by IL-4 . The dec in pigs . This evIL-4R gene is mapped on SSC3 at position 19.5 Mb and is closely located to the QTL regions for total number born (15.3–20.0 Mb) and some reproductive performance traits (11.9–33.7 Mb) in pigs [IL-4R gene is involved in the gene-transcription factor networks for TNB trait in pigs [IL-4R mRNAs are expressed in the endometrium, embryonic disc, and trophectoderm in pigs [IL-4R gene plays an important role in embryo implantation and may be related to litter size traits in pigs.The porcine orn 15.3–.0 Mb and in pigs . It has  in pigs . Moreove in pigs ,31. ThisIL-4R c.1577A > T polymorphism was significantly associated with the NBA and NWA traits. A positive effect of the favorable IL-4R c.1577T allele on litter size traits was found. Remarkably, the porcine IL-4R c.1577A > T polymorphism revealed a non-synonymous mutation and exhibited a changing amino acid residue at position Q526L in the cytoplasmic domain of the IL-4R structural molecule. This domain region is important to the IL-4 signaling transduction response and exhibits several key functions to regulate the tyrosine phosphorylation and intracellular signal transduction pathways. It contains numerous tyrosine (Y) residues  and sequence motifs of the binding sites for the insulin receptor substrate-1 (IRS-1), IRS-2, and signal transducer and activator of transcription 6 (STAT6) in terms of the growth and gene expression of the regulation domains [1419CCCAGAGCC1427, 1448CCCAGAGCC1456, 1448CCCAGAGCT1456, and 1569CCCAGAGCT1577) were observed in the cytoplasmic region of the porcine IL-4R gene. Interestingly, the polymorphism of IL-4R c.1577A > T (Q526L) was also located at the nucleotide position 1577 on the 1569CCCAGAGCT1577 motif of the porcine IL-4R mRNA sequence (Genbank accession no. NM_214340.1). These sequence motifs might have affected its expression or relationships to functional variations. However, the variant Q526L of the porcine IL-4R gene has not yet been functionally identified. It has been hypothesized that the observed amino acid change of Q526L might serve as the functional explanation of these traits, although the possibility of mutations inside or outside the coding region in close linkage disequilibrium with porcine IL-4R c.1577A > T (Q526L) should not be excluded.In this study, the porcine  domains ,50 was oIL-4R gene with litter size traits in pigs. However, several studies have demonstrated that the polymorphisms of IL4R gene are associated with the recurrent abortion, spontaneous preterm birth, and pre-eclampsia in humans [IL-4R variants with the recurrent abortion has been reported in Iranian women [IL-4R gene are associated with LIF mRNAs and protein expression levels [LIF gene are association with the recurrent abortions in women [IL-4R gene may be implicated in litter size traits in pigs.There is limited literature on the association of the n humans ,33,34,35n humans . In contan women . Furthern levels  and variin women  and littin women . This evIn order to establish an elite breeding stock with high prolificacy traits in pigs, there is an essential procedure to accumulate identified genes or combine more affected genes from multiple parents into a superior genotype of the breeding stock, which is called gene pyramiding schema . The genIL-4 and IL-4R genotypes are associated with diabetes, cancer, and pemphigus foliaceus in humans [IL-4 and IL-4R genotypes on litter size traits were observed in this study (data not shown). Therefore, it was explored whether the accumulated favorable alleles of the porcine IL-4 and IL-4R genotypes are associated with litter size traits in pigs. Interestingly, the significant effect of the accumulated favorable alleles (IL-4 g.134993898T and IL-4R c.1577T) of porcine IL-4 and IL-4R genes on litter size traits was exhibited. The increased numbers of favorable alleles were positively correlated with the TNB, NBA, and NWA traits in sows  can be used in marker-assisted selection to select the individuals with higher litter size traits. These findings suggest the gene pyramiding effects of porcine IL-4 and IL-4R genes may provide advantages for the breeding industry. The results in this study indicate that the porcine IL-4 and IL-4R genes could be expected to be involved in the reproductive processes of pigs, especially with regard to litter size traits. Further studies are required to confirm the association of these SNPs with litter size traits in larger population samples and various commercial pig breeds.Several studies have reported that the interactions of the n humans ,56,57. U in sows . Moreove in sows . This evIL-4 and IL-4R genes are associated with the reproductive traits of pigs. The porcine IL-4 g.134993898T > C and IL-4R c.1577A > T polymorphisms had clear effects on NBA, NWA, and MWW traits in commercial pigs. Moreover, the accumulation of favorable alleles of porcine IL-4 and IL-4R genes is positively correlated with the litter size traits. These findings emphasize the importance of the porcine IL-4 and IL-4R genes in the reproductive traits of pigs. Therefore, the porcine IL-4 and IL-4R genes may be potential candidate genes for the genetic improvement of litter size traits in the pig breeding industry.In this study, we found that polymorphisms of porcine


It is shown that the PO-6 can effectively bind to the CD123+ AML cells and the micellar formulation mPO-6 increases the dissolution stability and the specific binding capacity. When injected intravenously, mPO-6 significantly prolongs the survival of the refractory AML mice by interfering CD123/IL-3 axis, evidenced by the down regulation of phosphorylation of STAT5 and PI3K/AKT and the inhibition of activated NF-κB in the nucleus, as well as by the analysis results of next generation RNA-sequencing (RNA-seq) with the bone marrow of the AML mice. The antagonistic effect leads to the significantly reduction of AML cells infiltration in the bone marrow of the AML mice. In conclusion, mPO-6 could provide a potent antagonistic therapeutic approach for targeted treatment of AML.Acute myeloid leukemia (AML) is a common malignant heterogeneous hematopoietic disease with very low average 5-year survival rate due to the refractory feature and high rate of relapse. CD123 is highly expressed on multiple types of AML cells, especially leukemia stem cells, and closely associated with the poor prognosis of AML. Aiming to meet the urgent demand to targeted therapeutics for the refractory AML patients, herein we synthesize a CD123 antagonistic peptide (PO-6) loaded in nanomicelles (The online version contains supplementary material available at 10.1186/s13045-021-01206-y. Acute myeloid leukemia (AML) is a common malignant heterogeneous hematopoietic disease with very low average 5-year survival of 25% due to the refractory feature and high rate of relapse , 2. CD12+ AML cell line MOLM-13 cells in a concentration-dependent manner while weakly bind to the CD123− AML cell line HL-60 and chronic myeloid leukemia cell line K562 cells  with the average diameter of 38 nm  has been obtained based on screening of the cell culture from a group of de novo designed peptides targeting to various segments of the CD123 protein   of the AML mice post three intravenous administrations of ted Fig. A, and thted Fig. B. Furtheted Fig. C. Wester BM Fig. D, which  BM Fig. E observe BM Fig. F detecte BM Fig. G of the In summary, we report a novel and chemically synthesized peptide with antagonistic function towards CD123. The peptide in its micellar formulation displayed significant anti-leukemia activities in the refractory AML mice, providing an effective and safe therapeutics to the refractory AML treatments.Additional file 1. Supplementary material and methods.  Fig. S1. Affinity of PO-6 and mPO-6 to different leukemia cells. Fig.S2. The TEM image of mPO-6. Fig. S3. Confocal microscopy observation of PO-6 binding with MOLM-13 cells. Fig. S4.mPO-6 inhibited the antibodies binding to CD123. Fig. S5. Affinity of mPO-6 to the AML blasts. Fig. S6. CD123 expression on GFP+ cells in BM of AE & CKITD816V mice. Fig. S7. Therapeutic efficacy of Ara-C or HHT in AE & CKITD816V mice. Fig. S8. The fluorescent representative imaging of main organs at designated time points after mPO-6-FITC injection in healthy mice. Fig. S9. Acute toxicity evaluation of mPO-6 in healthy mice. Table S1. Human and mouse CD123 alignment of the extracellular amino acid sequences.Additional file 2. Materials and Methods.


The Apo B/A1 ratio is a major factor that predicts future cardiovascular outcomes. However, it is unclear whether the apolipoprotein B (Apo B)/apolipoprotein A1 (Apo A1) is a better predictor of future outcome than the total cholesterol (TC)/HDL-C ratio or lipoprotein (a) (Lp (a)) after the percutaneous coronary intervention (PCI). Therefore, we performed this study to evaluate the impact of the Apo B/A1 ratio on the patients who achieved LDL-C below 70 mg/dL one year after PCI.We included 448 PCI patients whose LDL-C levels were below 70 mg/dL at follow-up. The Apo B/A1 ratio, TC/HDL-C ratio, and Lp (a) levels were measured at the time of PCI and follow-up, and decreases in these parameters between baseline and follow-up were assessed as potential markers to predict major cardiovascular adverse events (MACEs).During a median follow-up period of 38.0 months, 115 MACEs were recorded. The tertile with the lowest decrease in the Apo B/A1 ratio (≤ 0.146) showed a lower MACE survival rate compared to the other tertiles. There were no differences in MACE survival rates for the TC/HDL-C ratio or Lp (a) levels.The Apo B/A1 ratio had better predictive accuracy for clinical outcomes compared to the TC/HDL-C ratio and Lp (a) level. A lower decrease in the Apo B/A1 ratio may be a residual risk factor for MACEs in patients who have reached LDL-C levels below 70 mg/dL after PCI. The 2018 American Heart Association (AHA)/American College of Cardiology (ACC) guideline strongly suggests that low-density lipoprotein cholesterol (LDL-C) should be maintained below 70 mg/dL in patients with clinical atherosclerotic cardiovascular disease by using statins to achieve 50% or greater reduction  = 0.485, p = 0.620, LDL-C decrease: [AUC] = 0.556, p = 0.072) In multivariate analysis using logistic regression, the lowest tertile of LDL-C (≤ 87.6 mg/dL) and use of non-high-intensity statins at the time of PCI were the independent predictors of the lowest tertile for the decrease in the Apo B/A1 ratio  levels may be associated with a poor prognosis after PCI , 15, 16.The results of a meta-analysis demonstrated that the percentage reduction of LDL-C is more important for secondary prevention than for the achievement of a target LDL-C level . EuropeaCho et al.  demonstrIt has been reported that the decrease in the Apo B/A1 ratio varies depending on the type and intensity of statin or addition of ezetimibe. Patients who used high-intensity statins or added ezetimibe had a more reduced Apo B/A1 ratio than those who used non-high-intensity statins , 24. TanOur study had several limitations. First, because of the retrospective nature of this study, there were differences in duration for lipid and clinical follow-up for each patient. However, this study was conducted with patients who had good treatment compliance and arrived at the target LDL-C 1 to 3 year after statin use, and the clinical follow-up duration (median 38.0 months) was long enough to assess the effects of the decrease in the Apo B/A1 ratio. Second, this study was a single-center study and was conducted with a relatively small number of patients. Third, since the type of statins could be changed during clinical follow-up at the discretion of the physician, it was difficult to evaluate the effects of these statin changes on the lipid profiles and future clinical outcomes.The Apo B/A1 ratio had greater predictive accuracy for clinical outcomes compared with the TC/HDL-C ratio and Lp (a) after PCI. A lower decrease in the Apo B/A1 ratio over time can be a residual risk factor of MACE in patients who have reached LDL-C levels below 70 mg/dL after PCI.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.