Upon oxidative stress, mammalian cells rapidly reprogram their translation. This is accompanied by the formation of stress granules (SGs), cytoplasmic ribonucleoprotein condensates containing untranslated mRNA molecules, RNA-binding proteins, 40S ribosomal subunits, and a set of translation initiation factors. Here we show that arsenite-induced stress causes a dramatic increase in the stop-codon readthrough rate and significantly elevates translation reinitiation levels on uORF-containing and bicistronic mRNAs. We also report the recruitment of translation termination factors eRF1 and eRF3, as well as ribosome recycling and translation reinitiation factors ABCE1, eIF2D, MCT-1, and DENR to SGs upon arsenite treatment. Localization of these factors to SGs may contribute to a rapid resumption of mRNA translation after stress relief and SG disassembly. It may also suggest the presence of post-termination, recycling, or reinitiation complexes in SGs. This new layer of translational control under stress conditions, relying on the altered spatial distribution of translation factors between cellular compartments, is discussed. Cell survival under stress requires the cell to mount a precise and rapid response to environmental changes ,2,3. As The response of cells to changing environments under stress also includes the formation of specialized ribonucleoprotein (RNP) biocondensates or microscopically visible RNA granules . Two majInterestingly, while SGs and PBs are physical entities, which are microscopically visible under stress, their protein and RNA components shuttle in and out of them, and SGs themselves are highly dynamic—rapidly assembling and disassembling with the onset and release of stress (reviewed in ). It is The number of proteins identified as SG constituents has been greatly expanded in the past few years. Many of them are related to translational control, mRNA turnover, cell signaling, and other cellular processes. Some of these proteins are directly involved in the regulation of SG formation, some are recruited to SGs, and some interact with SG components ,26,27,28It is well established that SGs contain untranslated mRNAs, 40S ribosomal subunits, and a number of translation initiation factors, which together could form 48S preinitiation complexes (PICs) under normal conditions. Among the factors, the multi-subunit protein eIF3, the cap-binding factor eIF4E, RNA helicase eIF4A, and scaffold protein eIF4G were reported as SG components ,29,30 T. HoweverTranslation termination in eukaryotes is mediated by two release factors, eRF1/Sup45p and eRF3/Sup35p ,35. OnceThe subsequent step of the translation cycle is ribosome recycling, initiated by ABCE1/Rli1p-catalyzed dissociation of the 80S ribosome into mRNA-bound 40S and free 60S subunits ,53,54. ITMA20 and TMA64 or TMA22 and TMA64, produce a synthetic slow-growth phenotype in yeast [In in vitro systems, eIF2D can release tRNA and mRNA from the post-termination complex ,59, whilin yeast . In the in yeast . The absin yeast . The impin yeast ,68. ThusUpon severe stress , the translatome of mammalian cells changes dramatically  for 1 h and then immediately fixed. For puromycin/emetine assays, U2OS cells were treated with 250 µM sodium arsenite for 90 min with the addition of either puromycin (10 µg/mL) (Sigma-Aldrich) to promote SGs or emetine (10 µg/mL) (Sigma-Aldrich) to disassemble SGs after 30 min of arsenite treatment.HEK293T, U2OS, and HeLa cell lines  were grown in DMEM  supplemented with 10% FBS  in the presence of penicillin and streptomycin  in a humidified 5% COThe following primary antibodies were used: rabbit polyclonal anti-eIF2D, anti-eRF1, and anti-eRF3 , anti-ABCE1 , anti-MCT-1 . Mouse anti-G3BP1 (H-10) , and goat anti-eIF3b (N-20)  were used to detect SGs, and mouse anti-p70 S6 kinase, which exhibits cross reactivity with Hedls , was useRabbit IgG-specific secondary antibodies conjugated to Alexa Fluor 555 or mouse IgG-specific secondary antibodies conjugated to Alexa Fluor 488 for ICC in HeLa were obtained from Invitrogen, USA ; anti-goat-Cy5, anti-mouse-Cy2, and anti-rabbit-Cy3 secondary antibodies from Jackson ImmunoResearch Laboratories, USA, were used for ICC in U2OS cells. Cells were grown on glass coverslips for 12 to 24 h, fixed with absolute methanol for 5 min at −20 °C, and post-fixed with 3% paraformaldehyde (Sigma-Aldrich) for 15 min at +4 °C. Cells were permeabilized with 0.5% Triton X-100 for 5 min at RT prior to blocking (10% FBS with 0.1% glycine (Sigma-Aldrich) in PBS) for 1 h at RT. Cells were then incubated in primary antibodies for 1 h at RT or overnight at +4 °C, followed by washing with PBS (3 × 5 min), secondary antibody incubation (1 h at RT), and washing with PBS (3 × 5 min). After immunostaining, the coverslips were mounted using polyvinyl mounting media or Aqua-Poly/Mount with DAPI .Microscopy for HeLa cells was performed using an Olympus IX51 microscope supplied with a 14-bit Olympus XM10 camera and UniBlitz D122 shutter driver  controlled by Micromanager software (v.1.4.22), or with the Axiovert 2000 M microscope  using 63× and 100× Plan-NEOFLUAR objectives. Images were acquired with AxioVision 3.1 software (Carl Zeiss) using the AxioCam HRc digital video camera (Carl Zeiss). Microscopy for U2OS ICC was performed using a Nikon Eclipse E800 microscope  with a 63× Plan Apo objective lens (NA 1.4) and illuminated with a mercury lamp and standard filters for Cy2 , Cy3 , and Cy5 . Images were taken with a Pursuit Monochrome 1.4 MP Digital Camera  using the manufacturer’s software . Raw TIF files were processed with ImageJ 1.52p  or Adobe Photoshop CS5 software . 7G-capped and polyadenylated mRNAs encoding firefly (Fluc), Renilla (Rluc), and NanoLuc (Nluc) luciferases were prepared as previously described [no_uORFluc, uORF1luc, RFluc2, and RF1luc1fus constructs from [Fluc, REI uORF-Fluc, REI Rluc-Fluc, and control Rluc-Fluc, respectively. Plasmids encoding NanoLuc variants, having the Nluc coding region (CDS) extended at the 5′ end by 39 nt derived from the human HBB gene [The mescribed ,70,71. Fcts from  hereafteShort-term mRNA transfection of HEK293T, HeLa, or U2OS cells was performed as described previously . Briefly2AsO3, sodium dihydroarsenite), a commonly used inducer of oxidative stress [While oxidative stress-induced changes in translation initiation and elongation are well studied ,2, the ee stress ,73,74,75e stress  was usedcontrol Fluc) or the same 5′ UTR with a 5-codon long uORF joined out-of-frame with the Fluc CDS (coding region) by the AUGA start-stop sequence (REI uORF-Fluc). Renilla luciferase mRNA (Rluc) with a simple 5′ UTR leader was co-transfected with the Fluc reporters for normalization. Two hours after transfection, cells were harvested and luciferase activities were measured. In our experiments, we used a range of arsenite concentrations to induce moderate to severe oxidative stress [The first system  was usede stress ,76, nameREI uORF-Fluc mRNA, as compared to the control Fluc reporter  [This suggests that the ribosome’s ability to initiate translation at the main the uORF ,78, or dt codon) .Rluc coding region instead of the short uORF, joined by the UGAUG sequence to the Fluc CDS .To exclude a contribution of leaky scanning and 43S sliding, we used a bicistronic reporter mRNA with the full-length op codon . As a coREI Rluc-Fluc, as compared to monocistronic control Rluc-Fluc after normalization to Rluc activity), which reflects the reinitiation rate, changed even more dramatically, >40-fold, rising from ~1:325 in unstressed cells  to ~1:8RLI1, TMA64, TMA20, or TMA22, the orthologs of human genes encoding ribosome recycling factors ABCE1, eIF2D, MCT-1, and DENR, respectively [These results clearly indicate that the proportion of ribosomes reinitiating after the first ORF translation robustly increased under conditions of arsenite-induced oxidative stress. Similar effects were observed earlier in mutant yeast strains with the deletion of ectively ,57.We further investigated whether a rate of stop-codon readthrough is affected by arsenite-induced oxidative stress. We used two reporter mRNAs  encodingwt Nluc mRNA produced 2.5-fold to ~400-fold less luciferase activity in cells treated with the various concentrations of arsenite we tested, the PTC(UGA)-containing mRNA showed only a 1.5-fold to ~20-fold decrease in translation under the same conditions . We found that eRF1 and eRF3 were diffusely distributed in the cytoplasm under normal conditions but recruited into G3BP1-positive foci under arsenite stress  staining. In mammals, eRF1 is encoded by a single e stress . In the e stress .As we found that arsenite-induced SGs contain translation termination factors, we then explored whether the ribosome recycling factors are also present in SGs. First, we examined the localization of ABCE1, an ATPase participating in peptide release, 60S dissociation, and reinitiation ,53,54, bWe then analyzed the subcellular localization of other ribosome recycling factors, i.e., eIF2D, MCT-1, and DENR, which promote tRNA and mRNA release from the post-termination 48S complexes ,57. In aTo further exclude cell type or cell line specificity of the observed phenomena and explore whether the termination and recycling factors may be recruited to PBs, we performed similar IF (immunofluorescence) experiments with human osteosarcoma U2OS cells (a common model to study SGs and PBs), using SG- and PB-specific markers . We confbona fide SGs, we treated U2OS cells with arsenite in the presence of small-molecule drugs, which are known to affect SG dynamics. Puromycin and emetine, two translation inhibitors with different mechanisms of action [SGs are dynamic and translationally modulated foci that are in the equilibrium with polysomes. To further confirm that the termination/recycling factor-positive foci are f action , promotef action . In agref action .The depletion of eRF1 and eRF3 in mammalian cells causes effects similar to those we observed in our study ,41, whilG3BP1 and G3BP2 genes knocked-out [To determine whether such sequestration of translation factors into SGs contributes to the changes in translation fidelity, we assayed readthrough and reinitiation rates in U2OS ddG3BP1/2 cells, which have cked-out . These ccked-out ,87.Unlike the prediction of the sequestration model, we determined an approximately 2-fold increase in the readthrough and reinitiation rates under stress conditions in the mutant cells, as compared to the wt U2OS cells A,B, suggIn arsenite-treated ddG3BP1/2 cells, PBs form but SGs do not . ConsistWe cannot exclude the possibility that despite the lack of microscopically visible SGs in the ddG3BP1/2 cells, SG seeds  or some It is well established that SGs contain a number of translation initiation factors, 40S ribosomal subunits, untranslated mRNAs recruited from disassembled polysomes, many RNA-binding proteins, and additional components affecting mRNA metabolism ,26,28,88In this study, we found that proteins involved in the distinct steps of the translation cycle, i.e., the peptide chain release factors eRF1 and eRF3, as well as recycling and reinitiation factors ABCE1, eIF2D, MCT-1, and DENR, are also present in SGs formed under conditions of arsenite-induced oxidative stress in mammalian cells. A search of the RNA granule database  reveals eRF1 and eRF3 bind to a pre-termination 80S ribosome complex at a stop codon in the A-site, in contact with primarily the 60S subunit . As Intriguingly, we found that, under the conditions we used, the eRF1 and eRF3 relocalization to SGs coincides with a dramatic acceleration of stop-codon readthrough. In general, readthrough events are likely associated with slowed termination dynamics at the stop codon ,98. In tThe modulation of termination fidelity observed in our study may contribute to translational reprogramming of the mammalian cells under stress. It is well established that C-terminally extended isoforms of many proteins diversify their functionality by the alteration of their subcellular localization or activity ,101. RecREI Rluc-Fluc mRNA  activity has been reported for arsenite-treated cells in some cases ,106. Howluc mRNA B also coWe further showed that the ribosome recycling and reinitiation factors ABCE1, eIF2D, MCT-1, and DENR also reside in SGs in arsenite-treated mammalian cells. ABCE1 participates in three consecutive stages of the translation cycle, bringing together peptide release, ribosome recycling, and translation initiation steps ,108,109.Rluc coding region, preceding the reporter Fluc CDS, under stress conditions. This rendered the translation of uORF-containing mRNAs partially resistant to stress, as compared to the control Fluc mRNA without uORF, although the absolute values of Fluc activity were nevertheless lower in the arsenite-treated cells due to a dramatic reduction of the overall ribosome recruitment onto the mRNA 5′ end. However, in the case of REI Rluc-Fluc (where the contribution of leaky scanning and 43S sliding was excluded), we even observed an increase in the absolute values of Fluc activity, providing a basis for physiologically relevant effects. In this case, Fluc activity can also be considered as a proxy of an aberrant 3′ UTR translation, which may contribute to the stress response.DENR, MCT-1, and eIF2D have been reported to regulate the translation of uORF-containing mRNAs in yeast, mammals, and flies ,111,112.The phenomenon of translational resistance of some uORF-containing mRNA to eIF2-inactivating stresses has long been established . ThuORF-Fluc and REI-Fluc translation was observed in yeast lacking eIF2D, MCT-1, or DENR orthologs [As in the case of translation termination, the observed changes in reinitiation rates may be attributed to either a decrease in cytosolic concentration or post-translational modifications of key components involved in the process. Previously, a similar up-regulation in rthologs . Thus, wrthologs  to validIn summary, our study presents evidence that relocalization of translation termination and ribosome recycling/reinitiation factors to SGs and their partial depletion from the cytoplasm during stress coincides with elevated stop-codon readthrough and reinitiation rates. These spatial and functional changes may contribute significantly to reshaping the mammalian translatome under stress conditions.


SAP genes in maize (Zea mays L.). In the present study, the SAP genes were identified from the maize genome. Subsequently, the protein properties, gene structure and duplication, chromosomal location, and cis-acting elements were analyzed by bioinformatic methods. Finally, their expression profiles under osmotic stresses, including drought and salinity, as well as ABA, and overexpression in Saccharomyces cerevisiae W303a cells, were performed to uncover the potential function. The results showed that a total of 10 SAP genes were identified and named ZmSAP1 to ZmSAP10 in maize, which was unevenly distributed on six of the ten maize chromosomes. The ZmSAP1, ZmSAP4, ZmSAP5, ZmSAP6, ZmSAP7, ZmSAP8 and ZmSAP10 had an A20 domain at N terminus and AN1 domain at C terminus, respectively. Only ZmSAP2 possessed a single AN1 domain at the N terminus. ZmSAP3 and ZmSAP9 both contained two AN1 domains without an A20 domain. Most ZmSAP genes lost introns and had abundant stress- and hormone-responsive cis-elements in their promoter region. The results of quantitative real-time PCR showed that all ZmSAP genes were regulated by drought and saline stresses, as well as ABA induction. Moreover, heterologous expression of ZmSAP2 and ZmSAP7 significantly improved the saline tolerance of yeast cells. The study provides insights into further underlying the function of ZmSAPs in regulating stress response in maize.Stress-associated proteins (SAPs) are a kind of zinc finger protein with an A20/AN1 domain and contribute to plants’ adaption to various abiotic and biological stimuli. However, little is known about the  Xenopus laevis and played key roles in innate immunity and cell death [Plants constantly encounter biotic and abiotic stresses from their surroundings. Consequently, plant growth, development and production are restricted by these stimuli, such as drought and salt stress ,2. To avll death ,6,7. Mosll death . Subsequll death ,10,11.OSISAP1 gene showed enhancements in the tolerance of cold, dehydration and salt [OsSAP1 sequence as a reference, 18 and 14 SAP genes (named OsSAP and AtSAP) were identified from the genome of rice and Arabidopsis, respectively, through a sequence similarity approach blast [OsSAPs and AtSAPs have been confirmed to regulate abiotic stress responses such as drought, salt, and temperature [SAPs from wheat (TaSAP5), banana (MusaSAP1), Populus trichocarpa (PtSAP13), Aeluropus littoralis (AlSAP) and Leymus chinensis (LcSAP) positively regulate drought and salt tolerance [In plants, OSISAP1 was first identified as A20/AN1 zinc-finger protein from rice and induced by multiple stresses, including cold, desiccation, salt, submergence and heavy metals, as well as injury . Meanwhiand salt . Thereafch blast . Most Osperature ,16,17,18olerance ,22,23.Prunus persica control flowering time, branching of lateral shoots, and cell expansion [OsiSAP7 negatively regulates ABA signaling to impart sensitivity to water-deficit stress [In addition to abiotic stress response, plant SAPs are found to serve as an important hub to mediate disease resistance and development. For instance, Pha13 containing A20/AN1 zinc finger domains and its homologs AtSAP5, AtSAP9 and OsSAP1 are also involved in virus resistance, basal resistance against pathogen infection and compromising innate immunity ,25,26. Txpansion ,28. The xpansion ,30. Abscxpansion ,32. Avait stress . HoweverSAPs have emerged as promising candidates for improving stress tolerance and growth during unfavorable conditions in plants. As one of the most important crops, maize is a key factor in developing the national economy and maintaining food security [SAPs of maize remain poorly understood. In this study, the ZmSAPs were first identified in the maize genome and comprehensively characterized for protein properties, gene structure and duplication, chromosomal locations, cis-acting regulatory elements and tissue expression profiles. Additionally, the expression profiles of ZmSAPs under different abiotic stress and hormone induction were investigated by RT-qPCR. The function of ZmSAPs was validated in yeast. The study provides insights into the further underlying function of ZmSAPs and helps in understanding the known modes of SAPs action in plants.Previous studies indicate that security . HoweverArabidopsis and rice were used as queries in local BLAST searches, respectively. Totally, 10 SAP genes were identified from the maize genome and named ZmSAP1~ZmSAP10 , with a molecular weight of 16.00 to 32.04 kDa, respectively. All ZmSAP proteins were predicted to be basic proteins with high theoretical isoelectric points ranging from 8.28 to 9.53. The grand average of hydropathicity (GRAVY) and instability indices of all ZmSAPs was <0 and >40, respectively, suggesting they were hydrophilic and instable proteins. Meanwhile, ZmSAPs showed no signal peptides and transmembrane region but were predicted to be nuclear localization.To identify ZmSAPs in maize, the amino acid sequences of 14 and 18 SAPs of ~ZmSAP10 . The encThe CDD analysis and sequence alignments showed that seven ZmSAPs, including ZmSAP1, ZmSAP4, ZmSAP5, ZmSAP6, ZmSAP7, ZmSAP8 and ZmSAP10, had an A20 domain at the N terminus and an AN1 domain at the C terminus, respectively. Only ZmSAP2 possessed a single AN1 domain at the N terminus. ZmSAP3 and ZmSAP9 both contained two AN1 domains without an A20 domain. Moreover, ZmSAP9 had two C2H2 domains . MeanwhiArabidopsis and rice, the amino acid sequences of 10 ZmSAPs, 14 AtSAPs and 18 OsSAPs were muti-aligned and used for phylogenetic tree construction. As shown in To analyze the phylogenetic relationship between ZmSAPs and SAPs of ZmSAPs were located on six chromosomes. There were two ZmSAP genes on chromosome 1 (ZmSAP1 and ZmSAP2), chromosome 2 (ZmSAP3 and ZmSAP4), chromosome 4 (ZmSAP5 and ZmSAP6) and chromosome 7 (ZmSAP8 and ZmSAP9), respectively. The ZmSAP7 and ZmSAP10 were located on chromosome 5 and chromosome 9, respectively , while others possessed a 5’-UTR and 3’-UTR, respectively  sequence of ectively .cis-acting elements analysis exhibited that seven kinds of cis-elements associated with stress response were identified in ZmSAP promoters  was the most abundant cis-element. There were six and five AREs in the ZmSAP3 and ZmSAP6 gene promoters. Meanwhile, except ZmSAP7 and ZmSAP9, the other eight ZmSAP genes contained at least one MBS (Myb binding site) element. In addition, eight kinds of hormone-responsive cis-elements were observed in ZmSAP promoters and associated with different hormones, including ABA (ABRE), ethylene (ERE), MeJA (CGTCA-Motif), salicylic acid (TCA element), auxin (TGA element) and gibberellin  response elements. This suggests that the ZmSAP genes may play different roles in stress and hormone response.The romoters . Among tZmSAPs in different development stages of maize was analyzed using RNA-seq data. We found that ZmSAPs showed no tissue specificity in the transcription level in maize, and ZmSAP6 exhibited a high expression level in all tissues.  were studied by RT-qPCR. Under drought stress mimicked by PEG treatment, the transcription levels of ZmSAP2, ZmSAP3, ZmSAP5 and ZmSAP8 were significantly up-regulated at 6, 12, 12 and 12 h of treatment, respectively. While ZmSAP1, ZmSAP4, ZmSAP6, ZmSAP7 and ZmSAP9 were significantly down-regulated by drought stress  plasmid on the plates with mannitol , Lobularia maritima (LmSAP), Leymus chinensis (LcSAP) also enhanced cell tolerance to salt, ionic and osmotic stresses in yeast [SAPs have exhibited functional diversity in osmotic stress, including drought and salt. For instance, OSISAP1, OsiSAP1, OsiSAP8 and AtSAP5, as positive regulators, improved drought, salt, and osmotic tolerance in transgenic tobacco, rice and Arabidopsis, respectively [SAP genes play a negative role in stress tolerance. For example, rice OsiSAP7 and ZFP185 negatively regulate ABA stress signaling and impart sensitivity to drought and salt stress in transgenic Arabidopsis and rice [PagSAP1 in poplar significantly enhances tolerance to salt stress, increases the K+/Na+ ratio in roots, and alters gene expression related to cellular ion homeostasis [Moreover, it has been proven that heterologous expression of the in yeast ,52,53. Mectively ,16,17,54and rice ,33. Likeeostasis .ZmSAP genes were identified in the maize genome. All ZmSAPs belong to the family of zinc-finger proteins with the A20/AN1 domain. The expression of ZmSAP genes was regulated by osmotic stresses, including drought and salt, as well as ABA. Furthermore, heterologous expression of ZmSAP2 and ZmSAP7 significantly improved the saline tolerance in yeast cells. The study suggests that ZmSAPs may play important roles in response to abiotic stresses and provides insights into further underlying the regulatory mechanisms of ZmSAPs in regulating stress response in maize.In conclusion, a total of 10 https://download.maizegdb.org/Zm-B73-REFERENCE-NAM-5.0/, accessed on 15 October 2021) and used for ZmSAPs search. Subsequently, the 14 and 18 SAP protein sequences of Arabidopsis and rice were downloaded from the Arabidopsis Information Resource  and the Rice Genome Annotation Project  and used as queries to perform local BLASTp search against above maize database by using the BLAST + suite with an E-value 1e−10, respectively [https://pfam-legacy.xfam.org/, accessed on 15 October 2021) and used to further identify ZmSAP candidates using HMMER3.0. Then, the redundant sequences were removed manually to acquire ZmSAPs.The genome and protein sequence data (reference 5.0) of maize B73 were retrieved from the MaizeGDB database , GOR IV , TMHMM Server v. 2.0  and SignalP 4.1 , respectively. The subcellular location was predicted by using WoLF PSORT . The conserved domains and motifs of ZmSAPs were further analyzed via searching the Conserved Domain Search Database  and using Multiple Em for Motif Elicitation (MEME) online program  [According to the method described by Yu et al. , the amier 2021) , respecthttp://www.megasoftware.net, accessed on 28 October 2021) with 1000 bootstrap replications. The phylogenetic tree among ZmSAP members was also constructed using the same method.The amino acid sequence of AtSAPs and OsSAPs were used for phylogenetic analysis against with ZmSAPs of maize. The phylogenetic tree was constructed using MEGA7.0 with the neighbor-joining (NJ) method  with the default parameters [SAP genes of maize, Arabidopsis and rice was analyzed using Dual Synteny Plotter software  [ZmSAPs were obtained from MaizeGDB and then used to analyze the exon–intron organizations and intron type by using Gene Structure Display Server (GSDS) .The physical location of g Circos . The genrameters . The syner 2021) . The cDNZmSAPs were downloaded from MaizeGDB and used for cis-elements analysis by using PlantCARE online software .The 2000 bp upstream of translation start site (TSS) of ZmSAPs in maize development, the expression feature of ZmSAPs in different development stages was analyzed. The RNA-seq data of ZmSAPs in different tissues were obtained from MaizeGDB qTeller center , displayed as fragments per kilobase of the exon model per million mapped fragments (FPKM) values and visualized as a heatmap using TBtools [To examine the potential role of  TBtools .The seeds of the maize inbred line B73 were surface-sterilized and germinated in filter paper for 48 h. The seedlings were transplanted into a Hoagland’s solution for a hydroponic culture under 16 h light at 28 °C/8 h dark at 25 °C periods. At the three-leaf stage, the seedlings with the same size were divided into four groups. Each of the three groups of seedlings was treated with 16% PEG-6000, 250 mM NaCl and 100 μM ABA solution, respectively. One group of seedlings was used as a control without treatment. At 0, 3, 6, 9, 12, and 24 h of treatment, the leaves were sampled, frozen and ground in liquid nitrogen, and stored at −80 °C for an RNA extraction, with three replicates.TM OneC . Subsequently, the 100 ng RNA of every sample was reverse transcribed into cDNA using the PrimeScriptTM reagent kit . The cDNA samples were stored at −20 °C and used for quantitative real-time PCR (qRT-PCR).The total RNA of every sample was extracted by using RNAiso plus kit  according to the manufacturer’s instruction, then treated with RNase-free DNase, and quantified using NanoDropZmSAPs and ZmEF-1a for internal control were designed by using the Primer-BLAST  and synthesized at TSINGKE (China)  using SYBR PremixEx TaqTM II  with three technical replicates. The expression of ZmSAP genes was analyzed and normalized by using the 2−ΔΔCT method of the CFX MangerTM software version 2.0  [t-test.The specific primer pairs of  (China) . As descCA, USA) . The datZmSAPs from cDNA of maize B73 inbred line by PCR amplification. The purified PCR product of every ZmSAP was inserted into the BamHI/XhoI site of the pYES2 vector . The recombinant plasmid and empty vector pYES2 were transformed into Saccharomyces cerevisiae W303a (MATa ade2 ura3 leu2 his3 trp1) by standard PEG lithium acetate method, respectively [ZmSAP was identified by PCR and incubated overnight in liquid YNB-Ura medium to OD600 to 1.0. The specific primer pairs of ZmSAPs were designed using Primer 5, synthesized at TSINGKE (China) , and useectively . The tra−1, 10−2, 10−3, 10−4) using liquid YNB-Ura medium. Then, 8 μL of each diluent was placed on YNB-Ura-Gal 2% solid medium supplemented with 0.5, 1.0, 1.5 M NaCl or 2.0, 2.5, 3.0 M Mannitol, respectively, and cultured at 28 °C in an incubator for 2–3 days for phenotyping. Subsequently, the candidates showing preferential growth vigor on solid medium were selected for dissecting the growth curve. The OD600 of yeast line with candidate genes was adjusted to 0.2, then 1 mL of them was added into 20 mL YNB-Ura-Gal 2% liquid medium, and cultured at 28 °C. At 0, 12, 24, 36, 48 and 72 h, the OD600 of every culture was monitored with three replicates. The yeast transformed by the pYES2 vector was used as a control. The 2% galactose  was added into the YNB-Ura medium to induce the expression of ZmSAPs under the control of the Gal promoter.According to the methods of Ben et al. ,53, with


Coronavirus disease (COVID-19) was first observed in Wuhan, Hubei Province (China) in December 2019, resulting in an acute respiratory syndrome. Only later was COVID-19 considered a public health emergency of international concern and, on 11 March 2020, the WHO classified it as pandemic. Despite being a respiratory virus, the clinical manifestations are also characterized by cardiological involvement, especially in patients suffering from previous comorbidities such as hypertension and diabetes mellitus, its complications being potentially serious or fatal. Despite the efforts made by the scientific community to identify pathophysiological mechanisms, they still remain unclear. A fundamental role is played by the angiotensin 2 converting enzyme, known for its effects at the cardiovascular level and for its involvement in COVID-19 pathogenesis. The goal of this paper was to highlight the mechanisms and knowledge related to cardiovascular involvement during the first pandemic phase, as well as to emphasize the main cardiological complications in infected patients. Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats ,3,4. PreThe aim of this study was to analyze CV involvement/complications following SARS-CoV-2 infection during the first pandemic phase.This review was carried out in line with the PRISMA statement .SCOPUS and Medline (using PubMed as the search engine) databases were searched in order to recognize relevant research available until 23 of June to examine CV complications associated with SARS-COV-2 infection.MeSH entry terms used were “SARS-CoV-2” AND “cardiovascular disease”; “SARS-CoV-2”; “COVID-19”.A search of the research articles that were suitable for inclusion in this review was also done, and the research-significant papers were therein collected and reviewed.The following inclusion criteria were adopted: studies that assessed the SARS-CoV-2 in association with CVD. The following exclusion criteria were applied: scientific articles that were not published in English and/or conference abstracts, and/or reviews or letters.For duplicate studies, only the article with further detailed information was included.Two reviewers (A.C. and E.V.) evaluated articles separately. The title, abstract, and full text of each potentially relevant study were reviewed. Any divergence on the eligibility of the studies was ascertained through debate or by consulting an additional reviewer (C.L.). The following information was extracted from all qualified papers: authors, year of publication, nationality of subjects, and study characteristics.After a free search for scientific literature by reviewers, a total of 1136 (100%) documents were collected. Of these, 230 (20%) were ruled out because of their reviews, 746 (65%) were only abstracts, 34 (3%) were disqualified after subsequent analysis of the title, and 77 (7%) were ruled out because they were not in English. In conclusion, 49 (4%) studies satisfied the inclusion criteria and were encompassed in the systematic review. Before June 2020 there were few studies defining the pathophysiological nature of COVID-19 and there were substantial uncertainties about CV-involvement complications.From the analysis of the 49 studies, the involvement of the CV apparatus in COVID-19 infection was described through the following points: (a) COVID-19 and myocardial injury, (b) acute myocardial infarction (AMI), (c) cardiac arrhythmia and cardiac arrest, (d) heart failure (HF) and cardiogenic and mixed shock, (e) venous thromboembolism event (VTE), (f) heart transplantation, and (g) long-term sequelae of SARS-CoV-2 infection.n = 7386) were males and the mean age was 63.1 ± 8.8 years. The average number of hospitalization days was 28.4 ± 17.3. The percentage of patients with hypertension (Hp) was 66% and with diabetes mellitus (DM) it was 32.5%. The percentage of patients needing ICU was 1.2%; 47.6% of the whole sample deceased, while 52.4% survived.n = 767) [n = 220) [n = 41) [n = 159) [n = 70) [n = 45) [n = 32) [n = 27) [n = 21) [n = 12) [n = 8) [The most frequent complications observed were acute cardiac injury (ACI) n = 767) ,68,70,74n = 220) ,58,64,68[n = 41) ,45, HF (n = 159) ,42,67,74 ,68,70,7[n = 70) ,34,52, a[n = 45) ,31,55,67[n = 32) ,45,60,61[n = 27) ,74, card[n = 21) ,47,54, v [n = 8) ,53,60,67Severe myocardial damage was the most commonly described CV complication in COVID-19.Any of the mechanisms described above can bring to acute cardiac injury (ACI) and rise in cardiac troponins in COVID-19 patients. The relative role of these diverse mechanisms has not been described but direct  myocardial injury due to viral myocarditis or the effect of systemic inflammation seems to be the most common mechanism. These observations were sustained by a previous autopsy study of patients who had died due to SARS during the Toronto SARS outbreak . Oudit eA report from China’s National Health Commission showed that almost 12% of patients without known CVD had elevated troponin levels or cardiac arrest during hospitalization .Early reports indicate that there are two patterns of COVID-19 myocardial damage . One stuA murine model proved pulmonary infection with SARS-CoV also precipitated an ACE2-dependent myocardial infection . Other sCollateral tissue injury and the inflammatory processes that follow vasodilation, endothelial permeability, and leukocyte recruitment lead to further pulmonary damage, hypoxemia, and CV stress. In a subset of patients, the host inflammatory response continues to amplify  and results in systemic inflammation ,76.n = 7/20) of patients who died from SARS [This systemic toxicity, in turn, has the potential to injure distant organs . Reportsrom SARS . Of noterom SARS ,86. It rrom SARS . In Junerom SARS ,87,88,89rom SARS . ECO evarom SARS ,21. Elecrom SARS ,38,51.Acute systemic inflammation enhances the risk of atherosclerotic plaque disruption and AMI ,74,91,92Analysis by Kwong et al.  proved tCardiac arrhythmias are another typical CV phenomenon described in patients infected with COVID-19. Though nonspecific, heart palpitations were part of the incoming symptomology in 7.3% of patients in a cohort of 137 patients admitted for COVID-19 .In hospitalized COVID-19 subjects, cardiac arrhythmia was detected in 16.7% in a Chinese cohort of 138 patients and was more common in ICU patients than in non-ICU ones (44.4% vs. 6.9%) . HoweverIn June 2020 it was unclear whether HF was more commonly due to exacerbation of pre-existing left ventricular dysfunction or to new cardiomyopathy (either due to myocarditis or stress cardiomyopathy) . Right HCOVID-19 patients also have increased risks of VTE ,104. SysApart from the mechanisms by which COVID-19 can affect patients with CVD risk factors, it is also important to consider COVID-19 in the context of especially fragile patients, such as those individuals awaiting transplantation or those who have already undergone transplantation . There aCV complications are likely to occur even after recovery from illness. COVID-19 is a nascent pandemic and, therefore, long-term sequelae are unknown, but there are reports of complications occurring soon after major severe symptoms have disappeared. COVID-19, caused by SARS-CoV-2, is a global pandemic that has affected hundreds of thousands of people and is evolving in real time. The ACE2 enzyme plays a crucial role in CV and immune systems involved in cardiac function, and in the development of hypertension and diabetes mellitus it has been identified as a functional receptor for coronaviruses, including SARS-CoV and the new SARS-CoV-2 . SARS-CoThe SARS-CoV-2 mainly invades alveolar epithelial cells, causing respiratory symptoms that are more acute in CVD patients. CV comorbidities are common in COVID-19 patients who turn out to be at higher risk of morbidity and mortality. It is not known whether the presence of CV comorbidity conditions poses independent risks or if these are conveyed by other factors . Myocardial injury is present in >25% of critical cases and appears in two patterns—severe myocardial injury and dysfunction on presentation and myocardial damage that develops as illness severity intensifies. High prevalence of arrhythmia might be, in part, attributable to metabolic disarray, hypoxia, or neurohormonal or inflammatory stress in the setting of viral infection in patients with or without existing CVD. Right-heart failure and related pulmonary hypertension ought to be also taken into account, in particular when severe parenchymal lung disease and ARDS are at play.Furthermore, many recent studies have demonstrated a large increase in Takotsubo Syndrome incidence associated to Covid-19 . HoweverThe results of this review show that the cardiac muscle, after the lung, is a critical organ in SARS-CoV-2 infection. This agent can affect it with direct infection or cause heart damage through a cytokine-mediated inflammatory process. In the latter, various symptoms may occur, ranging from cardiac arrhythmia to acute myocardial infarction up to cardiac arrest. The main weakness of this article was that this is a new pathogen and little is known about the mechanisms underlying COVID-19 pathogenesis, clinical manifestations, or complications.Furthermore, not all long-term sequelae and how they can affect the health of individuals were known; for these reasons, cardiac examination is extremely important while assessing patients’ health. Indeed, the heart check must be of vital importance in re-admission after COVID-19 disease.The strengths of the study were the limited period observed and the remarkable specificity and sensitivity of the diagnostic processes aimed at distinguishing between symptomatic and asymptomatic subjects, which were useful to reduce the infection.Therefore, further studies will be needed to better understand the pathophysiological mechanisms underlying the heart involvement following COVID-19 infection.


Pleural mesothelioma (PM) is an aggressive cancer with poor prognosis and no effective therapies, mainly caused by exposure to asbestos. Antagonists of growth hormone-releasing hormone (GHRH) display strong antitumor effects in many experimental cancers, including lung cancer and mesothelioma. Here, we aimed to determine whether GHRH antagonist MIA-690 potentiates the antitumor effect of cisplatin and pemetrexed in PM. In vitro, MIA-690, in combination with cisplatin and pemetrexed, synergistically reduced cell viability, restrained cell proliferation and enhanced apoptosis, compared with drugs alone. In vivo, the same combination resulted in a strong growth inhibition of MSTO-211H xenografts, decreased tumor cell proliferation and increased apoptosis. Mechanistically, MIA-690, particularly with chemotherapeutic drugs, inhibited proliferative and oncogenic pathways, such as MAPK ERK1/2 and cMyc, and downregulated cyclin D1 and B1 mRNAs. Inflammatory pathways such as NF-kB and STAT3 were also reduced, as well as oxidative, angiogenic and tumorigenic markers  and growth factors (VEGF and IGF-1). Overall, these findings strongly suggest that GHRH antagonists of MIA class, such as MIA-690, could increase the efficacy of standard therapy in PM. Pleural mesothelioma (PM) is a rare and aggressive cancer, generally diagnosed at an advanced stage and characterized by a poor prognosis, with median overall survival between 6 and 12 months . Of noteGrowth hormone-releasing hormone (GHRH) is a hypothalamic neuropeptide that regulates the release of growth hormone (GH) from the pituitary gland . In addiTo select the best inhibitory concentrations of the different compounds, dose-response experiments were initially performed at 48 h in MSTO-211H human biphasic cell lines, representative of the epithelioid/sarcomatoid PM histological subtype, already employed in our previous study . CisplatBased on cell viability experiments, where different concentrations were tested in combination (data not shown), 1 μM MIA-690, 2 μM cisplatin and 50 nM pemetrexed were selected as best concentrations for the subsequent experiments. Treatment for 48 h with MIA-690 reduced both cell viability and proliferation, and increased apoptosis, an effect observed also with cisplatin/pemetrexed alone. The addition of MIA-690 to cisplatin/pemetrexed produced a further inhibition of both cell viability and proliferation, as well as an elevation in apoptosis, compared with cisplatin/pemetrexed alone D–F. The −/− mice xenografted with MSTO-211H cells, that received 4-week treatment with the different compounds / severe combined immune deficient (SCID)/gamma chainompounds A. Nine aompounds B.per se, enhances the in vivo inhibitory effect of chemotherapy in PM. Similarly, tumor weight was decreased at the end of the experiment after treatment with MIA-690 as single agent or cisplatin/pemetrexed, by 18% and 26%, respectively, and was further reduced by the combination of MIA-690 with the drugs (58% reduction versus vehicle and 22% versus cisplatin/pemetrexed) C. No varTo examine the proliferative pathways involved in the antitumor effects of MIA-690 in combination with cisplatin/pemetrexed, Western blot and real-time PCR analysis were performed in tumor tissues. The activation of mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase 1/2 (ERK1/2), a critical regulator of cell proliferation and survival in many tumors, including PM , was redper se and in combination with chemotherapy drugs, reduces PM tumor growth in vivo via the inhibition of proliferative/oncogenic pathways and activation of apoptosis. Many cancers, including PM, are closely related to cell cycle regulators such as cyclins, which are downregulated by p53/p21 and are promising targets for treatment-refractory cancers ,34. RealThe transcription factors NF-kB and STAT3 play a crucial role in inflammation, tumorigenesis, immunosuppression and chemoresistance and are highly expressed in many cancers, including PM ,36. WestWe next investigated whether the combination of MIA-690 with cisplatin and pemetrexed would further induce the decrease in VEGF and IGF-1 expression in PM xenografts. As expected, MIA-690 as single agent reduced the levels of both VEGF mRNA and IGF-1 protein in tumors, compared with vehicle. A similar effect was observed in mice treated with cisplatin/pemetrexed. Surprisingly, VEGF mRNA levels were potently inhibited by MIA-690/cisplatin/pemetrexed compared with chemotherapeutic agents alone A. IGF-1 Pleural mesothelioma is a rare cancer with a very poor prognosis, for which curative treatment modalities are still lacking. Here, we demonstrate that the GHRH antagonist MIA-690 potentiates the antitumor effects of the chemotherapeutic drugs cisplatin and pemetrexed, in vitro, in mesothelioma cells, and particularly in vivo, in mice PM xenografts. Of note, the in vivo effects of MIA-690 in combination with cisplatin and pemetrexed included the reduction in tumor volume and weight, inhibition of mitotic markers and increase in proapoptotic molecules. Furthermore, the activation of survival, proliferative and inflammatory pathways, the expression of oncogenic markers, cell cycle regulators and angiogenic factors, were blunted in PM tumors of mice treated with MIA-690/cisplatin/pemetrexed, compared with chemotherapy alone.The standard treatment for unresectable PM consists of the combination of cisplatin and pemetrexed, which, however, has not proved to be resolutive and only partially improves the patients’ quality of life ,6; conseThe MAPK ERK1/2 pathway is involved in different cellular processes, e.g., survival and apoptosis, proliferation, differentiation and inflammation, and is often activated in many cancers, including mesothelioma, where it plays a key role in asbestos-induced carcinogenesis ,41. PrevThe p53 tumor suppressor protein has a prominent role in blocking cancer development by inducing apoptosis and cell cycle arrest, and promoting DNA repair and cell senescence. p53 also acts as a transcription factor to activate apoptotic proteins, such as Bax, and inhibits cell cycle regulators such as cyclin B1 and B2 . ImportaAberrant cell proliferation is one of the key hallmarks of cancer and a consequence of dysregulated cell division ; thus, tDysregulation of cMyc expression has been demonstrated in many human cancers, including mesothelioma . MoreoveMetalloproteinases are strongly implicated in most of the dysregulated processes in cancer, such as tumor growth, metastasis and angiogenesis and are highly expressed in mesothelioma, particularly MMP2 and MMP9 . In agreWe also observed a reduction in VEGF mRNA levels in tumors of mice treated with either MIA-690 or cisplatin/pemetrexed alone, in line with our previous findings in PM xenografts . This efInterestingly, the GHRH antagonist MZ-5-516 in prostate cancer cells , as wellOverall, this study provides compelling evidence demonstrating that MIA-690 enhances the antitumor effects of cisplatin/pemetrexed in PM, both in vitro and particularly in vivo, where it acts by inhibiting key oncogenic, proliferative, inflammatory and angiogenic pathways, while increasing proapoptotic molecules. Thus, these novel results suggest that GHRH antagonists could improve the anticancer effectiveness of standard therapy in pleural mesothelioma.0 -Tyr1, D-Arg2, Cpa6, Ala8, Har9, Fpa510, His11, Orn12, Abu15, His20, Orn21, Nle27, D-Arg28, Har29] hGH-RH(1-29)NH2 was synthesized and purified in the laboratory of one of the authors  at the Veterans Affairs Medical Center, University of Miami, Miami, FL, USA, as described previously [The GHRH-R antagonist MIA-690 [PhAc-Adaeviously . For in 2 humidified atmosphere in RPMI-1640 with 10% FBS, 2 mM L-glutamine, penicillin (100 U/mL), streptomycin (100 μg/mL) and 250 ng/mL amphotericin B.The human biphasic MPM cell line MSTO-211H was purchased from American Type Culture Collection (ATCC) . Cells were maintained at 37 °C in a 5% CO3 cells/well. After 48 h, cells were serum-starved for 12 h and incubated with the different stimuli for further 48 h. Cell survival was assessed by MTT assay. Cells were incubated with 1 mg/mL of MTT for approximately 2 h, then the medium was removed, and formazan products solubilized with 100 μL DMSO (MilliporeSigma). Cell viability was assessed by spectrophotometry at 570 nm absorbance using the LT-4000 microplate reader . Cell proliferation was assessed using the 5-bromo-2-deoxyuridine (BrdU) incorporation ELISA kit . Briefly, the cells were incubated with BrdU labeling solution for 2 h at 37 °C. After the removal of the labeling solution, cells were fixed, denatured, and incubated for 90 min with anti-BrdU antibody conjugate, which was subsequently removed by rinsing three times. Finally, cells were incubated in substrate solution at room temperature and proliferation assessed by colorimetric analysis at 450 nm absorbance using the LT-4000 microplate reader . Cells were seeded in 96-well plates at the concentration of 2 × 104 cells/well. Caspase-3 activity was assessed by Caspase-3 Colorimetric Assay Kit  in cell lysates, according to the manufacturer’s instruction. Briefly, cells were resuspended in Cell Lysis Buffer, incubated 10 min at 4 °C, centrifuged and cytosolic extract was used for protein quantification. Next, samples were incubated for 2 h with DEVD-pNA substrate. Caspase-3 activity was assessed by colorimetric detection at 405 nm absorbance using the LT-4000 microplate reader (Euroclone).Cells were seeded into a 6-well plate at a concentration of 3 × 10Drug synergistic effect was performed according to the Chou-Talalay method of synergy quantitation using CompuSyn software . Using a-/- (NSG) male mice were s.c. injected in the right flank with 1 × 106 MSTO-211H cells resuspended in 100 μL PBS 1X/matrigel  solution (ratio 1:1). Tumor growth was measured daily by caliper, according to the equation (L × W2)/2, where L = tumor length and W = tumor width. When tumors became palpable, (volume > 40 mm3), animals were randomized into four groups (n = 9 mice/group) and treated for 28 consecutive days as follows: (1) control group, mice treated with 0.1% DMSO and 10% aqueous propylene glycol solution (vehicle solution for MIA) s.c. and/or physiological solution i.p. (vehicle solution for cisplatin and pemetrexed); (2) MIA-690 (MIA) group, mice treated with 5 μg MIA s.c. daily; (3) cisplatin and pemetrexed (C/P) group, mice treated with 5 mg/kg cisplatin i.p. at days 9 and 23, and 100 mg/kg pemetrexed i.p. at days 3, 5, 9, 18, 23 and 25; (4) MIA-690, cisplatin and pemetrexed (MIA/C/P) group, treated with all three drugs as above. Tumor volumes were monitored, and animals were euthanized 28 days after randomization. Tumors were resected, weighed, and divided for RNA and protein isolation or fixed in 10% buffered formalin. All animal procedures were performed according to institutional guidelines in compliance with national  and international law and policies (new directive 2010/63/EU). The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Italian Ministry of Health (protocol #136/2020-PR 26-01-2020) and by the University of Turin Ethical Committee.All mice were bred at the Animal Facility of the Molecular Biotechnology Center  and housed under 12 h light/dark cycle, with food and drinking provided ad libitum. Six/eight-weeks-old NOD/SCID/gamma chainSections (3 μm-thick) were cut from formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks and stained with H&E or processed for immunohistochemistry (IHC) analysis. The latter was performed using an automated platform  with the following primary antibodies: anti-Ki-67 (30-9) rabbit monoclonal primary antibody ; cleaved Caspase-3 (Asp175) polyclonal rabbit antibody . Antigen retrieval was performed using ULTRA Cell Conditioning Solution (ULTRA CC1) antigen retrieval buffer  for all sections and the Ultraview detection system  was used for all analysis. Appropriate positive and negative controls were included for each immunohistochemical analysis. Image acquisition was performed with Hamamatsu NanoZoomer S210 Digital Slide Scanner . The number of mitotic cells was assessed by counting number of mitoses per 10 high power fields (mitosis/10HPF) on standard Hematoxylin and Eosin (H&E) stained slides (40 × magnification). Only viable cells were considered in each HPF examined within the section.Protein extraction and Western blot analysis were performed as described previously ,44. Prothttp://www.primer3.org/). The endogenous control used was 18S rRNA. Relative quantification was performed using the comparative Ct (2−ΔΔCt) method.Total RNA extraction and reverse transcription to cDNA (1 μg RNA) from PM tumors were performed as described previously . cDNAs wTumor xenograft samples were homogenized in RIPA buffer (MilliporeSigma), sonicated and centrifuged at 14,000 rpm (4 °C for 15 min). Total protein lysates were quantified with Bicinchoninic Acid kit (BCA) from (MilliporeSigma). IGF-1 levels were measured following the manufacturer’s protocol using mouse IGF-I ELISA Kit , as previously described .Results are presented as mean ± SEM. Significance was calculated by one-way, or two-way ANOVA followed by Dunnet’s or Tukey’s multiple comparison test for post hoc analysis. Analysis was performed using GraphPad Prism 8.0 .


Mortierellaceae are important for their remarkable capability to synthesize large amounts of polyunsaturated fatty acids, especially arachidonic acid (ARA). Although many genomes have been published, the quality of these data is not satisfactory, resulting in an incomplete understanding of the lipid pathway in Mortierellaceae. We provide herein two novel and high-quality genomes with 55.32% of syntenic gene pairs for Mortierella alpina CGMCC 20262 and M. schmuckeri CGMCC 20261, spanning 28 scaffolds of 40.22 Mb and 25 scaffolds of 49.24 Mb, respectively. The relative smaller genome for the former is due to fewer protein-coding gene models . The former yields 45.57% of ARA in total fatty acids, while the latter 6.95%. The accumulation of ARA is speculated to be associated with delta-5 desaturase (Delta5) and elongation of very long chain fatty acids protein 3 (ELOVL3). A further genomic comparison of 19 strains in 10 species in three genera in the Mortierellaceae reveals three types of fatty acid synthase (FAS), two of which are new to science. The most common type I exists in 16 strains of eight species of three genera, and was discovered previously and consists of a single unit with eight active sites. The newly revealed type II exists only in M. antarctica KOD 1030 where the unit is separated into two subunits α and β comprised of three and five active sites, respectively. Another newly revealed type III exists in M. alpina AD071 and Dissophora globulifera REB-010B, similar to type II but different in having one more acyl carrier protein domain in the α subunit. This study provides novel insights into the enzymes related to the lipid metabolism, especially the ARA-related Delta5, ELOVL3, and FAS, laying a foundation for genetic engineering of Mortierellaceae to modulate yield in polyunsaturated fatty acids.Fungal species in the family  Aspergillus flavus Link [=A. oryzae (Ahlb.) Cohn], Mortierella alpina Peyronel, M. wolfii B.S. Mehrotra & Baijal, Mucor racemosus Fresen., Lichtheimia corymbifera (Cohn) Vuill., Syncephalastrum racemosum Cohn ex J. Schröt., Geotrichum candidum Link, Umbelopsis isabellina (Oudem.) W. Gams (=Mortierella isabellina Oudem.), and Yarrowia lipolytica  Van der Walt & Arx have recently gained attention and played an important role in the studies of biodiesel and polyunsaturated fatty acids (PUFAs) Mortierel2), Pfam , CAZymes2), Pfam ,42, and 2), Pfam  database2), Pfam  with an  version  was used version . RNAmmer version  and tRNA version  were useA total of 40 strains of early diverging fungi, including 38 strains downloaded from online databases and two strains sequenced herein, were used for phylogenomic analyses . A totalMortierella alpina CGMCC 20262 and M. schmuckeri CGMCC 20261 were aligned using MCScanX for all protein-coding gene models, and then genomic collinearity was analyzed using a dual synteny plotter package [Mortierellaceae were reannotated using the KEGG database [The genome sequences of database .Mortierella alpina CGMCC 20262 is much lower than M. schmuckeri CGMCC 20261 in dry cell weight , total lipid weight , and total lipid content  in total fatty acids . Obviously, these two strains are significantly different, C20:4 (45.57%) being the top one in M. alpina CGMCC 20262, and C18:1 (27.54%) and C16:0 (24.68%) being the top two in M. schmuckeri CGMCC 20261 , and M. schmuckeri CGMCC 20261 is a sister to M. gamsii (MLBV = 100%) and closely related to M. elongata (MLBV = 100%).The Maximum Likelihood phylogenomic tree  suggestsMortierella alpina CGMCC 20262 and M. schmuckeri CGMCC 20261, accounting for 55.32% of all the 24,812 gene models in both strains. A total of 13,726 gene models located in all scaffolds are collinear between Mortierellaceae, genomes of 19 strains in 10 species are de novo annotated or reannotated. We focus on the 13 genes associated with NADPH, precursors, desaturation, and elongation (Delta5 (encoding delta-5 desaturase) is not found in M. gamsii NVP60, and neither is Delta6 (encoding delta-6 desaturase) in M. alpina LL118. With these two exceptions, each gene possesses between one and six copies. The genes HK  and ME  are remarkable as both have between three and six copies , and unites together eight active sites  family consists of four members in d ELOVL6 . ELOVL2 Mortierella alpina is now attracting more and more attention because of its ability to accumulate large amounts of arachidonic acid (ARA) [M. alpina synthesized lipids up to 50% of dry cell weight (DCW) and ARA 30–70% of total lipid content (TLC) [M. alpina CGMCC 20262 falls in these ranges, yielding lipids 17% of DCW and ARA 45.57% of TCL .Among numerous oleaginous fungi, id (ARA) ,16,22. Pnt (TLC) . Herein,% of TCL . In addi% of DCW , much moM. alpina was published in 2011, and consequently lipid synthesis pathway was predicted [Mortierellaceae have shed light on their lipid synthesis mechanism. For example, Mortierella sp. BCC40632 has an n-6 series fatty acid synthesis pathway due to a lack of delta-15 or omega-3 desaturase [M. alpina CGMCC 20262 and M. schmuckeri CGMCC 20261, are sequenced to provide more insights into lipid metabolism , up to 43.3% of total fatty acids [M. alpina is significantly different from other species in the family Mortierellaceae based on the phylogenetic analyses (ELOVL2, ELOVL3, ELOVL4, and ELOVL6, are identified herein in 19 strains of Mortierellaceae (PUFA biosynthesis requires an activity of elongation of very long chain fatty acids proteins, as well as fatty acid desaturases such as delta-5, delta-6, delta-12, and omega-3 desaturase . For exaty acids . In the analyses , which iellaceae .Aspergillus oryzae, Mortierella alpina, Saccharomyces cerevisiae, and Yarrowia lipolytica [Mortierellaceae (Mortierella alpina ATCC 32222, a single subunit consisting of eight catalytic domains/active sites [M. antarctica KOD 1030 only, and type III in Dissophora globulifera REB-010B and Mortierella alpina AD071 (The fatty acid synthase plays an important role in lipid synthesis, catalyzing the synthesis of saturated fatty acids from acetyl-CoA and malonyl-CoA. A variety of fatty acid synthases were found in fungi, such as polytica ,12,73,74ellaceae . Type I ve sites . Types Ina AD071 .Overall, the rapid increase in genomic data is providing more materials for tackling fatty acid biosynthesis, discovering more potential genes, and finalizing the understanding of the genetic foundation of lipid metabolism.Mortierella alpina CGMCC 20262 and M. schmuckeri CGMCC 20261, were sequenced using the PacBio Sequel and Illumina NovaSeq 6000 platform. To explore the lipid metabolism in Mortierellaceae, a total of 19 genomes were reannotated. The results suggest that delta-5 desaturase and elongation of very long chain fatty acids protein 3 probably promoted the accumulation of polyunsaturated fatty acids, especially arachidonic acid. Besides, three types of fatty acid synthase, including two novel ones, were identified. Consequently, with the increase in public genomic data, a comprehensive analysis on lipid metabolism will discover more genes or protein-encoding models, providing more information for subsequent genetic engineering of oleaginous fungi.In this paper, two new genomes,


Prolonged plasma treatment of aqueous solutions of vitamin C, 1,4-hydroquinone, and 1,4-benzoquinone respectively, leads to their decomposition. Studies of the mechanisms in plasma-induced processes can help to prevent side effects in medicine, agriculture, and food disinfection.Cold atmospheric pressure plasma (CAPP) is widely used in medicine for the treatment of diseases and disinfection of bio-tissues due to its antibacterial, antiviral, and antifungal properties. In agriculture, CAPP accelerates the imbibition and germination of seeds and significantly increases plant productivity. Plasma is also used to fix molecular nitrogen. CAPP can produce reactive oxygen and nitrogen species (RONS). Plasma treatment of bio-tissue can lead to numerous side effects such as lipid peroxidation, genotoxic problems, and DNA damage. The mechanisms of occurring side effects when treating various organisms with cold plasma are unknown since RONS, UV-Vis light, and multicomponent biological tissues are simultaneously involved in a heterogeneous environment. Here, we found that CAPP can induce in vitro oxidation of the most common water-soluble redox compounds in living cells such as NADH, NADPH, and vitamin C at interfaces between air, CAPP, and water. CAPP is not capable of reducing NAD CAPPs p, and O3 . These C3 and HNO2 which are useful for the production of nitrogen compounds and fertilizers [Cold plasma can behave as a catalyst for the oxidation of nitrogen gas, occurring at the plasma/water and plasma/air interfaces ,21,22,23RONS are known to participate in developmental processes by acting as signaling molecules for cell proliferation and differentiation, programmed cell death, seed germination, gravitropism, root hair growth, pollen tube development, and senescence. The use of cold plasma in food has proven to be an emerging technology. Cold plasma technology has been used in the food industry to reduce microbial count, degrade mycotoxin, inactivate enzymes, increase the concentration of bioactive compounds, and reduce allergens in food products.Cold plasma has many biomedical applications ,18,24,25+, NADPH, NADP+, vitamin C (ascorbic acid), as well as hydrophilic and hydrophobic quinones in reduced or oxidized forms [How does cold atmospheric pressure radio frequency plasma interact with water-soluble redox components of living cells? The most common examples in nature are water-soluble redox components such as NADH, NADed forms ,30,31,32There are many publications about the side effects in biological tissues of various organisms in vivo when treated with cold plasma ,33,34,353, HNO2, H2O2, O3, and NOx compounds. The majority of RONS, He+ cations, and radicals are strong oxidants. 3. Electrochemical mechanisms and redox potentials of nitrogen and water oxidation at the cold atmospheric plasma/water interface were analyzed earlier [3 and small impurities of less stable compounds HNO2 and H2O2 are also produced. The process of aqueous phase acidification during nitrogen fixation starts at the interface between cold plasma and water [It was demonstrated recently that cold plasma/water and plasma/air interfaces can have catalytic properties for charge-transfer reactions . The maj+ is a coenzyme in single cells and multicellular organisms. NAD+ and NADH strongly absorb ultraviolet light because of adenine. The oxidized form of NAD+ absorbs light at a lower wavelength than the reduced form NADH  does not reduce NAD+ to NADH during one hour of treatment  is a water-soluble antioxidant. Ascorbic acid in aqueous solutions is reversibly oxidized to dehydroascorbic acid (DHA)  and thenAscorbic acid can transfer a single electron, because of the resonance-stabilized nature of its radical ion, called semidehydroascorbate (AR). Ascorbic acid can also transfer two electrons to acceptors synchronously without formation of the intermediate semidehydroascorbate radical ion ,38,39. AHydroquinone and semiquinone can participate in disproportionation, autoxidation, and cross-oxidation reactions. Hydroquinone participates in vivo in metabolic reactions of methylation, sulfation, or formation of glucuronides, turning them into biologically inactive compounds that are eliminated from the body.Absorption spectra of 1,4-hydroquinone and 1,4-benzoquinone before and after treatment with cold He-plasma are shown in 2O2-dependent oxidation of quinone’s double bonds, or in reactions of quinones with O2•−.Cold plasma does not reduce 1,4-benzoquinone but can oxidize it B. It is + cations, and UV radiation at the plasma/air and plasma/water interfaces -ascorbic acid sodium salt (vitamin C) at interfaces between air, cold atmospheric pressure plasma, and water . CAPPJ ioquinone  despite oquinone , 1,4-hydoquinone  leads toed water . Among tAnother possible process of the acidification of an aqueous phase could be the oxidation of water at the plasma/water interface:Redox reactions  produce acidification of an aqueous phase. Cold atmospheric pressure He-plasma jet produces acidification of an aqueous phase, oxidation of water-soluble compounds in the reduced form, such as NADH, NADPH, and L(+)-ascorbic acid. CAPPJ can induce decomposition of water-soluble redox compounds such as L(+)-ascorbic acid B, 1,4-hyThere are many beneficial effects of short low dose CAPPJ applications on bio-tissues . These strips were used for the detection of H2O2, NO2−, and NO3− in water after the treatment with cold atmospheric pressure He-plasma jet. Time of the aqueous solution treatment was shown in β-Nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH), β-nicotinamide adenine dinucleotide sodium salt hydrate (NAD+), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), 1,4-hydroquinone, 1,4-benzoquinone, and L(+)-ascorbic acid sodium salt  were purchased from Sigma-Aldrich (USA). HThe plasma was powered with a high voltage pulsed DC system consisting of a Matsusada AU-10P60 10 kV DC power supply , an IXYS PVX-4110 pulse generator , and a DG-1022Z digital function generator . The system was operated with an 8 kV pulse amplitude, 6 kHz pulse frequency, 1 μs pulse width, and ~70 ns pulse rise and fall time. The experimental setup was described earlier . Helium Each sample of aqueous solutions was thoroughly stirred with a magnetic stirrer during treatment with cold atmospheric pressure He-plasma jet to ensure a homogeneous composition of chemicals in the 20 mL clear borosilicate glass vial . The diaThe digital laser temperature gun, Etekcity lasergrip 800 , was used for the measurement of temperature in the plasma jet, water, and air. The temperature of the aqueous phase during 60 min of plasma treatment was 20 °C.Absorption spectra were recorded using 1 cm quartz cuvettes with the Shimadzu UV-Vis spectrophotometer ISR-2600 Plus .The software SigmaPlot 12  was used for statistical analysis of experimental data. All experimental results were reproduced at least 14 times.+ and 1,4-benzoquinone despite the presence of free electrons in CAPP. Plasma is used in biomedical research directly on multicomponent biological tissues [Cold atmospheric pressure radio frequency plasma (CAPP) is widely used in medicine, agriculture, food processing for the treatment of various diseases, and disinfection of biological tissues and food due to its antibacterial, antiviral, and antifungal properties. The mechanisms of these multicomponent processes in biological tissues remain partially elucidated. CAPP in air can produce reactive oxygen and nitrogen species (RONS) including radicals, ions, and relatively stable molecules. Cold atmospheric pressure plasma can induce oxidation of the most common water-soluble redox compounds in multicellular organisms, such as NADH, NADPH, L(+)-ascorbic acid sodium salt (vitamin C), and quinones at interfaces between three fluid phases: air, cold atmospheric pressure plasma, and water. CAPPJ is unable to reduce NAD tissues ,47. To c tissues . Studies


Tubers and tuberous root crops are essential carbohydrate sources and staple foods for humans, second only to cereals. The developmental phase transition, including floral initiation and underground storage organ formation, is controlled by complex signaling processes involving the integration of environmental and endogenous cues. FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1/CENTRORADIALIS (TFL1/CEN), members of the phosphatidylethanolamine-binding protein (PEBP) gene family, play a central role in this developmental phase transition process. FT and FT-like proteins have a function to promote developmental phase transition, while TFL1/CEN act oppositely. The balance between FT and TFL1/CEN is critical to ensure a successful plant life cycle. Here, we present a summarized review of the role and signaling network of PEBP in floral initiation and underground storage organ formation, specifically in tubers and tuberous root crops. Lastly, we point out several questions that need to be answered in order to have a more complete understanding of the PEBP signaling network, which is crucial for the agronomical improvement of tubers and tuberous crops. Solanum tuberosum) and yam (Dioscorea spp.), and tuberous roots or starchy root crops, such as cassava (Manihot esculenta) and sweet potato (Ipomea batatas), are important carbohydrate sources and staple foods for humans, second only to cereals [Billions of people around the world consume tubers and tuberous root crops. Tuber crops, such as potato , such as the high-affinity sucrose/Hd StSUT4 ,11,12. Fd StSUT4 ,14, suggPhosphatidylethanolamine-Binding Protein (PEBP) gene family [The floral initiation and underground storage organ development are controlled by overlapping regulatory processes involving the e family . In thisPEBPs were first purified from the bovine brain and characterized as having affinities with several organic anions and phospholipids, e.g., phosphatidylethanolamine (PE) . The oveMOTHER OF FT AND TFL1 (MFT)-like genes, FLOWERING LOCUS T (FT)-like genes, and TERMINAL FLOWER 1 (TFL1)/CENTRORADIALIS (CEN)-like genes [MFT-like genes, whereas further duplication and divergence of FT/TFL1-like genes have occurred in vascular plants [FT, TWIS SISTER OF FT (TSF), and MFT are known as floral activators, while BROTHER OF FT AND TFL1 (BFT), TFL1, and ARABIDOPSIS THALIANA CENTRORADIALIS (ATC) act as floral repressors [Arabidopsis  [On the basis of the genome sequence, PEBPs can be classified into three main groups, ke genes . Algae ar plants . In angir plants ,21. In gr plants ,22. In Apressors . Intereshaliana) ,25.FT-, TFL1/CEN-, BFT-, and MFT-like genes were identified in the selected tubers and tuberous root crops, including potato, yam, cassava, and sweet potato  has eight FT-like genes, three TFL1/CEN-like genes, one BFT-like gene, and one MFT-like gene. Cassava has two FT-like genes, five TFL1/CEN-like genes, one BFT-like gene, and two MFT-like genes. Lastly, sweet potato has 10 FT-like genes, 11 TFL1/CEN-like genes, and seven MFT-like genes. Among the identified FT-like genes, until now, only PGSC0003DMG400016179 (StSP3D) and Manes_12G001600 (MeFT1) have been characterized as floral activators [PGSC0003DMG400023365 (StSP6A) was found to be responsible for promoting potato tuberization [StSP5G, the repressor of StSP6A [PGSC0003DMG400014322 (StCEN), which has been identified as a negative regulator of floral development and tuber formation [BFT than TFL1/CEN , BvFT1, acts as a floral repressor due to mutations that correspond to Y134N, G137Q, and W138Q in Arabidopsis FT [Hordeum vulgare), HvFT4, acts as a floral repressor likely due to W138A mutation [The divergence of FT-like genes involves a positive selection of the fourth exon after the duplication event . Among topsis FT , while bmutation ,34.Arabidopsis FT (StSP5G-like) has two mutations corresponding to R83K and W138N in Arabidopsis FT which are crucial for FT movement [Arabidopsis FT, DRNTG_29881 has a Y85H mutation, DRNTG_11086 has a W138M mutation, DRNTG_11087 has W138M and Q140H mutations, and DRNTG_20845 has a Q140P mutation. Furthermore, DRNTG_16474 has a V70I mutation, which is crucial for FT movement [Arabidopsis FT. Nevertheless, functional studies of the remaining FT-, BFT-, and TFL1/CEN-like genes in potato, yam, cassava, and sweet potato are essential to understand the genetic redundancy and the signaling network of PEBPs in the regulation of flowering time and underground storage organ formation.The majority of FT-like proteins from tubers and tuberous root crops have conserved critical residues in comparison with opsis FT B. Interemovement , respectPEBP [Arabidopsis, flower in response to increasing day length, whereas other plants, including rice and sorghum, flower under short-day (SD) conditions. Photoperiodic flowering is controlled by the integration of light perception and internal circadian rhythm [Arabidopsis, LD conditions induce high levels of FT and TSF expression, which eventually promote flowering [FT and TSF expression at dusk under LD conditions [ATC is activated by SD conditions, especially at night time, likely to prevent flowering under noninductive SD conditions [Internal and external cues strictly control the timing of flowering by regulating PEBP   [FT and TSF expression via phytohormone abscisic acid (ABA) [BFT expression, which likely serves as a buffering mechanism to counteract FT and TSF activation [FT-dependent and -independent pathways [FT-dependent and -independent pathways [Temperatures also control the flowering time of Arabidopsis by flowering earlier under warm temperatures and later under cool temperatures  Figure A. Warm t4 (PIF4) . Moreove4 (PIF4) . Droughtid (ABA) ,48. Intetivation . The phypathways . Lastly,pathways  , LEAFY (LFY), FRUITFULL (FUL), and APETALA1 (AP1) [Arabidopsis BFT, TSF, and ATC were also reported to interact with FD [Adiantum capillus-veneris is also capable of interacting with FD [Arabidopsis PEBPs contain the 14-3-3-binding motif, this suggests a dynamic complex formation to fine-tune floral formation  . Further with FD ,58,59, w with FD . The int with FD . Consideormation B. FurtheArabidopsis, the molecular mechanism of flowering time regulation in tubers and tuberous root crops is still elusive [Unlike in the model plant  elusive . Most tu elusive ,64,65,66 elusive . Further elusive ,69,70. TS. tuberosum ssp. andigena), cultivated in the Andean highlands, and the modern potato (S. tuberosum ssp. tuberosum), originating from the lowlands of southern Chile [Arabidopsis, StSP3D is expressed in the middle of the day instead of at dusk [Arabidopsis CO results in downregulation of StSP3D and late flowering in potato plants [StCO is encoded by three tandem genes, CONSTANS-like 1 (StCOL1), StCOL2, and StCOL3 [StCOL1 using RNA interference accelerates potato flowering time [Arabidopsis [StSP5G through the TGTGGT motif in the StSP5G promoter [StSP3D and, thus, delays flowering [Potato can be classified into two subspecies, the wild Andean varieties  acts as a negative regulator of the flowering time through the regulation of StSP3D (BBX24 in Arabidopsis flowering [CYCLING DOF FACTOR 1 (stCDF1) results in a late flowering phenotype [CDF1 as a floral repressor in Arabidopsis [Arabidopsis CDF1 prevents flowering through repressing CO and FT transcription [StSP6A [StSP6A was found to prevent potato flowering, as StSP6A knockdown plants flowered early, while plants with high StSP6A levels flowered late [StSP6A seems unrelated to the regulation of StSP3D expression [StSP6A, using constitutive 35S promoter, accelerated the flowering of Arabidopsis and potato [StSP6A and StSP3D are expressed in the leaves and stem, it is interesting to speculate whether StSP6A competes with StSP3D to interact with protein transporters such as the MCTPs [The potato f StSP3D A, in conlowering ,76. Howehenotype , consistbidopsis ,79. Intecription ,81, StCD [StSP6A   [StSP3D-independent manner [Arabidopsis, high light prevents the transcription of the floral repressor, FLOWERING LOCUS C (FLC), through nuclear accumulation of the PHD type transcription factor [Arabidopsis and GA biosynthesis and signaling [In addition to the photoperiod, temperature is one of the seasonal cues that control the flowering time of many plant species . A cold demissum . In Araberatures . Cold niersicum) . Furthert manner . In Arabn factor ,92. Consignaling ,94, it iArabidopsis, which flower early in warm temperatures [Arabidopsis, the expression levels of MeFT1 and MeFT2 are mostly unperturbed by temperature, while PIF4 expression increases in warm temperatures (similar to Arabidopsis) [MeFT1 and MeFT2 [Arabidopsis and rice [Cassava’s flowering time is induced by LD conditions and cool temperature (early flowering at 22 °C and late flowering at 34 °C) ,95 FiguB. The teeratures . Howeveridopsis) ,86. As sidopsis) . The drond MeFT2   to promote flowering . Howevererminacy . Furthererminacy  , while LD conditions inhibit tuber formation and stimulate vine and leaf growth [Underground storage organ formation, including tubers and tuberous roots, is essential for plant survival under adverse environmental conditions. Day length regulates potato tuber formation in a genotype-dependent manner. For instance, the wild Andean varieties strictly require SD conditions to promote tuber formation, while the modern potato cultivar is more adapted to LD conditions . SD condf growth ,100. Intf growth , suggestStPhyB induces tuber formation in noninductive conditions (LD and SD + night break) [Arabidopsis, where PhyB induces CO degradation [Arabidopsis CONSTANS-like 7 is stabilized by PhyB [StSP5G transcription, a negative regulator of StSP6A [D. alata) is also likely connected with FT-like genes, DaFT1 and DaFT2 [Similar to flowering time, photoperiodic tuber formation is controlled by integrating light signaling and the circadian clock . Light st break) . Furthert break) ,105  [Arabidopsis. Furthermore, the BELLRINGER-1-like transcription factor (StBEL5) and its interacting partner, the StKNOX transcription factor, have been identified as activators of StCDF1 and StSP6A [The natural variation of nditions . StCDF1 ormation ,109. Thed StSP6A ,111. StBEL5 are regulated by photoperiod through the PhyB/microRNA miR172 cascade [Arabidopsis, miR172 and miR156 have been shown to promote and inhibit tuber formation, respectively, partly through the regulation of StSP6A [Arabidopsis [The expression levels of  cascade . Similarf StSP6A ,113. Botf StSP6A ,113,114.bidopsis   tuberous root formation [Suppressing Expression of SP6A (SES), an upstream regulator of StSP6A [SES reduces the accumulation of StSP6A transcripts to reduce the sink strength and delay tuber formation [SES is likely a potato-specific small RNA, as no homolog of -pri-miRNASES was found in the Solanaceae genome [StSP6A, especially in warm temperatures [Arabidopsis, the interaction between TOC1 and PIF4 mediates the circadian gating of thermomorphogenesis [EARLY FLOWERING 3, and florigen movement, could potentially elucidate the molecular mechanism of temperature-responsive underground storage organ formation [Temperature is another seasonal cue that controls underground storage organ formation. Warm temperatures inhibit potato tuber formation and sweet potato and Mexican turnip  tubers [FT reduces the tuberous root growth and accelerated flowering of cassava, suggesting a relationship between both developmental processes [A previous report suggested that the tuberigen signal, StSP6A, prevents floral formation in an StSP3D-independent manner . However) tubers ,122,123,rocesses   to activate tuber formation . Moreove germin3 . StSP6A  germin3  , a plant-specific TCP transcription factor, are crucial for tuber formation in potato species . Anothermeristem , suggestmeristem . StGA2ox1, a GA-catabolizing enzyme [StSP6A also forms an alternative complex with St14-3-3 and StABL1 to promote tuber formation . StABL1 g enzyme . The facg enzyme ,131. Intg enzyme , suggestPEBP gene family [Arabidopsis [Arabidopsis PEBPs are involved in flowering time regulation [PEBP in flowering time and underground storage organ formation is essential to elucidate the PEBP signaling network in each tuber and tuberous root crop;All gulation ,23, whilStSP3D and StSP6A [Arabidopsis could accelerate the characterization of the regulatory module of the PEBP-gene family in tubers and tuberous root crops  section of the Solanum genus, suggests that the non-tuber-bearing phenotype of Etuberosum is caused by the deletion of the fourth exon of SP6A [Some plants develop underground storage organs to survive adverse environmental conditions . Whole-g of SP6A . HoweverFloral initiation and underground storage organ formation are complex processes that integrate environmental cues and endogenous signals through the regulation of master regulators, i.e., the e family ,22. FT ae family . Subtle e family ,35,36. Tbidopsis ,22,39,71


Epithelial ovarian cancers are age-associated diseases, usually diagnosed at an advanced stage. lncRNA has been discovered to interplay with N6-methyladenosine (m6A), working in tandem to promote cancer progression and worsening patient outcomes. This study is aimed at investigating the roles and mechanism of m6A-related lncRNA signature on ovarian cancers. 50 of chemotherapeutic drugs were calculated, and CMap was run to identify the potential compounds and their corresponding mechanisms.  We retrieved TCGA and CGGA sequencing data to identify m6A-related lncRNA signature and constructed an m6A score (MS) using the LASSO algorithm. A clinical nomogram was then established to predict the overall survival of patients. Subsequently, GSEA analyses were conducted to obtain pathways involved. Expression of HLA genes, 28 tumor-infiltrating lymphocyte infiltration, and anticancer cycle were analyzed the immunological differences between high-MS and low-MS groups. Finally, immune checkpoint gene expressions and ICNRP1, TNFSF9, and VSIR was observed in the high-MS group. Finally, the high-MS group also predicted low IC50 of vinorelbine and vorinostat.  We identified 16 m6A-related lncRNAs and constructed an MS model. The high-MS group showed a poor prognosis. A clinical nomogram consists of MS, and age was constructed and predicted the 1-, 3-, and 5-year survival with high accuracy. GSEA analyses presented downregulated antigen processing and presentation pathways. Immunocyte infiltrating analyses demonstrated that high-MS was associated with high infiltration of Treg cells, macrophages, and low Th1/Th2 rate. Also, high expression of immune checkpoint genes  This study constructed a robust prediction model for prognostic management and revealed the cross-talk between m6A and immunosuppression. Besides, the m6A lncRNA signature can predict the chemotherapeutic drug response. These will shed light on the development of novel therapeutic strategies and render survival benefits for ovarian patients. Ovarian cancer is one of the most predominant gynecological diseases in the world, containing a highly heterogeneous group of malignant tumors in etiology and genetic features. The largest proportion of ovarian cancer is epithelial, presenting an advanced stage at the time of first diagnosis  and highN6-methyladenosine (m6A) has emerged as the most common modification of RNA; its reversible functions were mediated by three types of functional components: “writers,” “readers,” and “erasers.” The “writers” were complexes of methyltransferases, such as METTL3 and METTL14, adding m6A on RNAs, while the “erasers,” like the demethylase FTO, removes the m6A away. Some specific RNA binding proteins (“readers”) can recognize the m6A and affect the fate of RNAs, including YTHDC1 and IGF2BPs . Long nom6A-related lncRNA was engaged in multiple biological processes affecting cancer progress . RecentlHere, we identified m6A-related lncRNA to construct a LASSO-based prognostic model called m6A score (MS), for predicting the survival of ovarian cancer patients. The roles of MS signature in cancer immune and drug sensitivities were also explored in multiple levels. This study offers a robust prediction tool in patient prognostic management and sheds light on the cross-talk between m6A and immune-related cancer properties. This will trigger novel therapeutic strategies targeting the critical m6A-related lncRNAs and improve the prognosis of ovarian cancer patients.n = 377 were retrieved via the R package “TCGAbiolinks” from https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga. The Gene Expression Omnibus (GEO) sample datasets GSE26193 (n = 107) were obtained using the R package “GEOquery” from https://www.ncbi.nlm.nih.gov/geo/. All expression data were transferred to transcripts per million (TPM) for further analyses. TCGA dataset was assigned as the training set, and the GEO dataset was used for independent validation. A total of 4183 lncRNAs were extracted from TCGA RNA matrix.The clinical features, RNA expression, copy number variation (CNV), and single nucleotide polymorphism (SNP) data of The Cancer Genome Atlas (TCGA) samples with CBLL1, VIRMA, METTL14, METTL3, RBM15, RBM15B, WTAP, and ZC3H13 as writers; ALKBH5 and FTO as erasers; and ELAVL1, FMR1, HNRNPA2B1, HNRNPC, IGF2BP1, IGF2BP2, IGF2BP3, LRPPRC, YTHDC1, YTHDC2, YTHDF1, YTHDF2, and YTHDF3 as readers. The lncRNAs were filtered according to their correlations with the 23 m6A regulators; those lncRNAs with absolute correlation coefficients less than 0.3 were removed. Subsequently, the lncRNAs passing the filtration were input to the univariate Cox hazard analysis for identifying the survival-associated lncRNAs, and the least absolute shrinkage and selection operator (LASSO) algorithm [li refers to the expression level of the lncRNA i and βi means the calculated coefficients of lncRNA i from the LASSO algorithm. Their corresponding coefficients were visualized in a lollipop chart.We first selected the 23 m6A-related regulators as symbols of m6A modification , includilgorithm  was perfThe training set and validating set were divided into high-MS and low-MS groups according to the median value of the m6A score. Kaplan-Meier curves were plotted to verify whether the MS can separate the overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS) rate of the two groups. Further, receiver operating characteristic curve (ROC) was performed to estimate the accuracy of the MS for 1-, 3-, and 5-year OS prediction.The downloaded SNP and CNV data were organized to compare the differences between high-MS and low-MS groups. CNV includes amplifications and deletions among the genomic region; the two CNV events were measured by GSITIC 2.0 . BesidesThe MS as well as the clinical factors  was analyzed by univariate Cox regression and multivariate Cox regression successively to obtain the independent parameters for establishing a quantitative prediction nomogram . The nomhttp://software.broadinstitute.org/gsea/msigdb). We then conducted GSEA [To discover the biological mechanisms of the lncRNA signature functions, we seek the pathways on which the lncRNA signature was enriched. Gene set variation analysis (GSVA)  was perfted GSEA  to calcuted GSEA  from MSiHLA-E, HLA-DPB2, HLA-C, HLA-J, HLA-DQB1, HLA-DQB2, HLA-DQA2, HLA-DQA1, HLA-A, HLA-DMA, HLA-DOB, HLA-DRB1, HLA-H, HLA-B, HLA-DRB5, HLA-DOA, HLA-DPB1, HLA-DRA, HLA-DRB6, HLA-L, HLA-F, HLA-G, HLA-DMB, HLA-DPA1).The tumor microenvironment contains various stromal cells and immunocytes. We estimated the stromal infiltrating via calculation of stromal score and tumor purity by Estimation of STromal and Immune cells in MAlignant Tumours using Expression data (ESTIMATE) . Besideshttps://tcia.at/). The coefficients of the correlations between the MS and 28 TILs were calculated, and their correlations were visualized in the correlation plot. Subsequently, the correlation between MS and the antitumor cell cycle [To clarify the immune landscape, we explore the immunocyte infiltration via analysis of 28 tumor-infiltrating lymphocytes (TILs)  on The C50) of commonly applied chemotherapy drugs for obtaining the drug sensitivities of ovarian cancer patients, including cisplatin, doxorubicin, gemcitabine, paclitaxel, vinorelbine, and vorinostat. Finally, we screened candidate chemical compounds targeting the m6A features based on the 202 differentially expressed genes identified between high-MS and low-MS patients; this analysis was performed by the mechanism of actions (MoA) among those compounds using CMap tools (https://clue.io/) [The immune checkpoint mediates the suppression of lymphocyte activation and escapes cancers from immune supervision. To investigate whether immune checkpoints play roles in the procancer activities, we compared the expression level of PD-1, CTLA4, and 14 immune checkpoint genes between high-MS and low-MS groups. We also estimated the predicted half-maximal inhibitory concentration (IClue.io/) .p value < 0.05 was regarded as statistically significant. Log-rank test was used for the Kaplan-Meier curves of TCGA and GEO dataset patient survival analyses. For normally distributed variables, we used Student's t-test to conduct the pairwise comparisons, and for nonnormally distributed variables, the Wilcoxon test was performed. Euclidean distance adopted in hierarchical cluster analysis was applied to the heatmap graphing. Finally, Spearman correlation analysis was used to compute the significance of correlations between variables; absolute correlation coefficients > 0.3 were considered as correlated. For the symbols, ∗∗∗, ∗∗, ∗, and NS refer to p < 0.001, <0.01, <0.05, and not significant, respectively [The statistical analyses were conducted in the R software (version 3.6.3). A two-sided ectively .A total of 4183 lncRNAs were adopted to seek the m6A-associated factors by correlation analysis. Sixty-three lncRNAs with absolute correlation coefficients of more than 0.3 were screened out and subsequently applied to the univariate Cox hazard analysis. The univariate Cox analysis produced 19 lncRNAs related to survival, and the 19 lncRNAs were input to LASSO regression analysis for selecting the candidate variables used for model construction. Finally, 16 lncRNAs were obtained with their corresponding coefficients presented Figures . The 16 To validate the prognostic value of the MS, we conducted survival analyses by plotting Kaplan-Meier curves. As a result, the survival rate can be separated between the high-MS and low-MS groups, and the high-MS group showed low survival probabilities both in training and validating sets Figures . In addiThe constructed MS was capable of distinguishing the patient risks; we further explored whether patients in the two groups bore dissimilar CNV and SNP rates. The CNV consists of two forms: copy number amplifications and deletions. As exhibited in Figures To testify whether the MS, patient age, tumor stage, and grade can function as independent predictors for establishing a clinical nomogram, we conducted univariate and multivariate Cox regression analyses for these features. The MS and age were retained to be integrated as predictive factors . SubsequThe GSEA was performed on the 16 m6A-associated lncRNA signature, and the top 3 positively and top 4 negatively enriched KEGG pathways were presented. The positively enriched pathways include calcium, extracellular matrix (ECM) receptor, and focal adhesion signaling pathway , and theThe functional analyses have suggested the immune diversity between high-MS and low-MS groups. On top of that, we compared the tumor environment infiltrating levels between the two groups. The calculation of stromal score and tumor purity showed the high infiltration of stromal cells in the high-MS group Figures . We alsoTo explicit the immunocyte infiltration map, we further performed ssGSEA analyses on the MS based on 28 tumor-infiltrating lymphocytes (28 TILs) retrieved from The Cancer Immunome Atlas. As presented in NPR1, TNFSF9, and VSIR genes were significantly highly expressed in the high-MS group (50) of commonly used chemotherapy drugs, including cisplatin, vinorelbine, and vorinostat. As result, high-MS cancers presented lower estimated IC50 of vinorelbine and vorinostat (Figures Since the high-MS group exhibited immunosuppressive features, we then analyzed the immune checkpoint marker levels between the two groups to seek whether immune checkpoints played roles in their immunosuppression. We noticed that the MS group ; the heaMS group , implyin Figures , suggest Figures . These rIn this review, we constructed a robust m6A score to predict the prognosis of ovarian cancer patients using the m6A lncRNA signature. Among various cancers, m6A lncRNA prognostic models have presented high accuracy in predicting the patients' direct prognosis or therapeutic response and in revealing the mechanism of tumor malignancy –16. For Establishment of the MS identified 12 novel m6A-related lncRNA with prognostic value, including WAC-AS1, TRAM2-AS1, SH3RF3-AS1, PCOLCE-AS1, MYCNOS, LINC01270, LINC00592, LAMTOR5-AS1, FOXN3-AS1, DLGAP1-AS2, DICER1-AS1, and ARHGAP26-AS1. WAC-AS1 was identified as a protective lncRNA in glioma , and LAMm6A has been discovered to correlate with antitumor immunity and immune evasion , 37 in cNRP1, TNSF9, and VSIR. NRP1 targeted inhibitors were found to enhance the proliferation and antitumor effect of CD8+ T cells [TNSF9 was a conserved pan-cancer marker of Treg cells that affect CD8+ T cell activity [VSIR was also found to regulate Treg cells in combination with CTLA-4 [Since Treg cells and Th1/Th2 balance can suppress anticancer immunity via immune checkpoint-related mechanisms , 43, the T cells ; it was  T cells , 46. TNSactivity . VSIR wah CTLA-4 , as wellh CTLA-4 . The dis50 of vinorelbine and vorinostat in the high-MS group has suggested their promising efficacy. Since no clinical trial of these two drugs' effects on ovarian cancer patients has been conducted, the future exploration of benefits from them depending on the MS status is required.However, there are numerous limitations to our study that should be considered. Our research was only based on TCGA database, and we do not conduct vitro assays for hub lncRNAs. Beyond the effects on the immune, MS can predict the sensitivities to chemotherapeutic drugs. The low estimated ICComprehensively, this study established a robust tool for prognostic management of ovarian cancer patients and providing candidate adjuvant chemotherapy, as well as novel therapeutic strategies targeting the cross-talk between m6A and immunosuppression.