We show for the first-time that resveratrol downregulates expression of mediators involved in several key signaling pathways IFN-γ, IL-4, PSMB8 (LMP7), and a subset of LPS-induced genes, while lectins induced IFN-γ, IL-4, PSMB8, and many of the same genes as LPS that are important for innate and adaptive immunity. These findings suggest that inflammation may be influenced by common dietary components and this knowledge may be used to prevent or reverse inflammation-based diseases.Inflammation is linked to several human diseases like microbial infections, cancer, heart disease, asthma, diabetes, and neurological disorders. We have shown that the prototype inflammatory agonist LPS modulates the activity of Ubiquitin-Proteasome System (UPS) and regulates transcription factors such as NF-κB, leading to inflammation, tolerance, hypoxia, autophagy, and apoptosis of cells. We hypothesized that proteasome modulators resveratrol and soybean lectin would alter the gene expression of mediators involved in inflammation-induced signaling pathways, when administered ex vivo to human peripheral blood mononuclear blood cells (PBMCs) obtained from normal healthy controls. To test this hypothesis, analysis of RNA derived from LPS-treated human PBMCs, with or without resveratrol and soybean lectin, was carried out using Next Generation Sequencing (NGS). Collectively, the findings described herein suggest that proteasome modulators, resveratrol (proteasome inhibitor) and lectins (proteasome activator), have a profound capacity to modulate  Inflammation is a major contributing factor in the outcomes of diseases like infections, cancer, heart disease, asthma, diabetes, and neurological disorders. Bacterial components such as toxic LPS, peptidoglycan and CpG DNA elicit a cytokine storm; inflammation leading to hypoxia, oxygen deprivation in the tissues and cells, and, finally, cell death. This may lead to septic shock, particularly in individuals with underlying diseases ,2. COVIDOur laboratory focuses on defining the basic underlying molecular and cellular mechanisms by which microbial proinflammatory mediators such as LPS function to regulate inflammation ,15,16,17The cell’s proteasome subunit composition and potential changes to that composition control its enzymatic activities lead to regulation of degradation of crucial Toll-like receptors (TLR)-activated signaling proteins and key metabolic proteins. For example, the 20S proteasome contains at least three major protease activities- specifically chymotrypsin-like , post-acidic , and the trypsin-like activities Z, LMP10) . After a . After Cells of the hematopoietic system (more differentiated cells) are known to contain more immunoproteasome subunits (LMP7. LMP2), as compared to tissue cells or some mouse cells which contain primarily (XYZ) constitutive type subunits . LPS forCollectively, our published and preliminary data and those of others suggest that a differential expression of macrophage (innate immunity) IL-12, IL-1β, IL-6, IL-12 and TNF-α, and T cell cytokines (acquired immunity), such as IFN-γ, and IL-4, which are modulated during development of inflammation and tolerance ,15. IFN-Our overall hypothesis is that the changes in level of gene expression triggered by LPS and/or other proinflammatory stimuli in PBMCs from healthy individuals will be differentially affected by plant constituents such as resveratrol  and soybean lectins  found in human diets ,35, whicResveratrol downregulates some of the inflammation-linked genes in absence of LPS: Resveratrol is present as a dietary component in several fruits and has been shown to be very important for heart health. To analyze the RES-modulated genes, we have included genes that were either downregulated or upregulated by RES, as compared to control PBMCs in EGR1 and EGR2), colony stimulating factor 2 (CSF2), and chemokines involved in inflammation: Gene expression modulated by RES and LPS in human PBMCs. A total of 680 genes were identified as being significantly modulated by various agonists such as LPS , the retinoid receptor (LXR/XRX), and antioxidant activity of vitamin C signaling pathways, which were robustly downregulated.Resveratrol downregulates certain crucial LPS-induced genes such as IFN-γ linked, early growth response 1 and 2 , interferon-γ (IFNG), interleukin 10 (IL-10), interferon-induced protein 44L (IFI44L), and other interferon-induced proteins, IL-27, IL-36G, chemokines, such as CCL2, CCL8, CCL7, and CXCL 9, 10 and 11; enzymes , gamma-glutaminyl transferase 5 (GGT5), sialidase, (NEU4), helicase with zinc finger 2 transcription coactivator (HELZ2), ubiquitin specific peptidase 18 (USP18), maltase-glucoamylase (MGAM); and growth factors such as colony stimulating factor 2 (CSF2), early growth response 2 (EGR2), and platelet-derived growth factor beta polypeptide, (PDGF).Genes that were modulated by RES are listed in Gene expression of proteins modulated (upregulated/downregulated) by LPS and LEC  in human PBMCs: To investigate the modulation of gene expression by LEC10, we compared it with LPS, a potent bacterial agonist . SurprisIFNG, ubiquitin specific peptidase 18 (USP18), chemokine (C-X-C) ligand 11 (CXCL11), radical S-adenosyl methionine domain containing 2 (RSAD2), interferon alpha-inducible protein 6 (IFI6), calcium channel voltage dependent P/Q type alpha 1A (CACNA1A), family with sequence similarity 19 (FAM19A2), lysosomal-associated membrane protein 3 (LAMP3), BCL-like 14 , Sialic acid binding Ig-like lectin (SIGLEC1), ubiquitin-like modifier (ISG15), and Cyclin A1 (CCNA1)  B. Genes IFN-γ, interferon λ (IFN-λ), IL-12A, CD80, CTLA, checkpoint inhibitor in cancer cells, STAT1, 2, HLA-DQB, MHC II, IL-27, Nuclear receptor coactivator 7 NCOA7, enhances the transcriptional activities of several nuclear receptors; PTGER3, IP10, IL-8, and others as compared to control PBMCs. Some of the upregulated genes included TLR9 (receptor for CpG DNA), and CLEC6A (receptor for lectins). The healthy control PBMC, robustly induced gene expression of IFN-γ, TNF-α, LMP7, nitric oxide synthase , and signaling via transcription factor STAT1, and several other mediators in the presence of LPS and LEC. No upregulation of LMP10 was observed in PBMC. However, Insulin receptor was downregulated with LPS and LEC in normal controls. . RES and LPS + RES downregulated gene expression of several LPS induced transcription factors, and LEC50 alone upregulated gene expression of several transcription factors, such as, NFKB1A, TRIM5, MAFF, HDAC9, growth arrest and DNA damage-inducible protein 45 A (GADD45A), IRF7, etc. but downregulates NOTCH3 and OLIGO2, like LPS. A total of 698 genes were upregulated when a combination of LPS + LEC50 was used, as shown in The transcription factors or proteins that affect gene expression were modulated with RES, LEC50 and LPS are listed in E. coli, normally present in the gut of some individuals) and LEC (plant toxin) showed a robust and synergistic reprograming of cells (XYZ/LMP to LMP) by upregulation of PSMB8  and PSMB9 . A similar upregulation of inflammatory genes of mediators involved in signaling pathways in PBMCs from non-diabetic humans in ex vivo experiments was observed and LPS upregulated the IFN-γ and IL-4 induced genes. Conversely, LPS + RES, , downregulated expression of IFNγ- and IL-4 induced genes. These RNAseq data provide support for the hypothesis that RES and LEC can differentially affect the LPS-induced gene expression of proteasome subunits in ex vivo experiments and may also possess a potent immunomodulatory epigenetic effect on the host PBMCs. This novel study provides strong evidence to support the conclusion that LPS , a potent vasoconstrictor, was upregulated and the insulin receptor (INSR) was downregulated by LPS or LEC. These may lead to other disorders such type 2 Diabetes Mellitus and arthritis. However, lectins may be beneficial to people with infections, such as tuberculosis or in certain cancers. These results are significant, and they warrant further clinical research on reprograming the host’s immune system, depending on whether the person has a balanced immune system, is immuno-compromised, or is suffering from an autoimmune disorder. In addition, beneficial properties of RES in sepsis, heart disease and other inflammatory diseases must be studied in detail in humans ,37. In cThe LPS-signaling pathway is well-established ,40,41. Tp50  was upregulated. Moreover, gene expression of TLR4, TLR2, TLR3 and TLR1 was not upregulated by LPS or LEC, because mRNA of these toll receptors was already present in the cell. In contrast, gene expression of TLR7, and TLR9 (present in endosomes) was upregulated by LPS. Thus, LPS upregulated gene expression of TRIF, MyD88, TLR7, TLR9, IRF7, TNF-α, IL-6, IL-12, and IL-2; while LEC10 upregulated all these genes except for MyD88  is a plant-based dietary compound found in grapes, berries, and red wine. Its beneficial effects have been attributed to its anti-inflammatory, antioxidant, anti-aging (due to increase in SIRT1), and anti-tumor properties ,50,51,52PSMB8 (LMP7), and most inflammatory cytokines like LPS  as described below (Lectins (LEC) are used as natural plant insecticides. When consumed by people, they trigger immune responses in people depending on the dose ,59,60,61ed below A–D.IFN-γ (Th1), IL-4 (Th2), IL-2 (Th1), IL-17 A (Th17), Treg , and PSMB8 (LMP7) was upregulated by LPS/LEC in a 3 h ex vivo treatment. Several transcription factor genes activated by IFN-γ (an amplifier of immune response), including STAT2 and STAT1 , were also upregulated by LPS  and LEC (inflammatory), since T cell signaling is also dependent on the LMP subunits of the proteasome . LPS is d by LPS A–D.of IFN-γ, IFN-λ, but not IFN-β. RES inhibited LPS-induced STAT1 more robustly than STAT2, thus inhibiting some of the IFN-γ induced genes . We have also shown for the first time that LEC (with or without LPS) is an activator of the proteasome subunit LMP7, IFN-λ, IFN-β, IFN-γ and several other genes. Notably, LEC 10 and LEC 50 μg/mL upregulated expression of mostly the same genes as LPS 10 ng/mL. This study demonstrates the role of LEC and RES in upregulating/downregulating expression of IL-4 and IFN-γ (Th2 and Th1 cytokines) and of mediators involved in signaling pathways in PBMC obtained from blood from non-diabetic controls.Interestingly, RES downregulated gene expression OAS1), OAS2 and OAS3 (USP18 gene encodes for an important enzyme that removes ISG-15 conjugates (ubiquitin-like molecules) from protein substrates . It is also a type 1 interferon receptor repressor and belongs to a deubiquitinating protease family (DUB). Deficiency of this DUB is known to lead to upregulation of the immune system, and USP18 is degraded by the proteasome when produced in excessive amounts. EIF2AK2 (protein kinase R) is an interferon-induced enzyme which catalyzes autophosphorylation of IκB and can induce cellular apoptosis to prevent viral spread. Collectively, LPS activates gene expression of proteins in UPS mentioned in this paragraph, RES inhibits these inflammatory proteins. Further protein analysis and clinical studies are needed to evaluate the potential therapeutic benefit of RES in reducing inflammation and impacting outcomes in human diseases, such as autoimmune conditions, diabetes, neurological problems, and cardiovascular diseases. Genes for several key enzymes involved in metabolic pathways linked to the proteasomes were also affected as explained above. Importantly, treatment of PBMC with LPS activates gene expression of proteins involved in several pathways, but LPS + RES leads to down-regulation of gene expression of some of the key enzymes involved in metabolism and other functions. These IFN-γ-induced genes include 2′-5′ oligoadenylate synthetase 1  and T cell receptor TCR. There is also a crosstalk between the CD80 (dendritic cells) and CD28 proteins (T cells). In non-diabetic controls IL-12 is released from the macrophages/dendritic cells, resulting in differential induction of cytokines such as IFN-γ, IL-2 (Th1), and IL-4 (Th2), IL-17 A (Th17) and IL-10 (Tregs) as explained above . In contrast, LEC alone upregulated expression of all these genes involved in innate and adaptive immune response. The mechanisms underlying these effects and the signaling pathways need to be further clarified.LPS and LEC activated innate immune response via toll receptors and gene expression of proinflammatory cytokines such as ed above  in respoM1 possess XYZ subunits (in tissue cells or in mice) that are upregulated by LPS and new LMP2, 7 and 10 subunits are biosynthesized via activation of transcription factor, NF-κB by cleavage of IκB by the proteasomes. However, PBMC from controls were more differentiated in that subunit X is almost absent there was induction of LMP7 and LMP2 (but not LMP10), with LPS (active M2). In PBMC there was gene induction of p50/p50 subunits. Then, the gene expression of proteasome subunits is repressed or there is no further change (NC) in proteasome subunits with agonists during tolerance and the cells become refractory to LPS (M3). Proteasome subunits are degraded after ubiquitination, and this may lead to autophagy due to upregulation of Atg7 (an important autophagy gene induced by LPS) and recycling of cellular components [Based on this novel study, we propose a simplified model that describes mechanisms for the beneficial and deleterious effects of dietary components in PBMCs The proteasome’s subunits can be linked to immune responses in the proposed model, LPS and IFN-γ (T cells) activates macrophages/monocytes and T cells by inducing LMP7 and LMP2 subunits of proteasomes and these are put into place within 3–24 h. LPS or LEC activates IL-12 in monocytes/macrophages, and this leads to the activation of Th1 cells via the crosstalk between T cell receptor (TCR) and MHC-II; and between CD28, (CTLA4) to release IFN-γ by Th1 (T) cells . The celmponents . Treatmemponents ,45. p50/p50 homodimers (cause tolerance), and HDAC9 (deacetylase) is expected to cause epigenetic changes that shuts down new gene expression involved during adaptive immunity. This is due to DNA methylation and histone acetylation enzymes that are also regulated by the proteasome. Histone Acetyl Transferases (HATS) acetylate histones and activate the immune cells [HDAC9 gene is the only deacetylase in human PBMCs that is induced by LPS or LEC, and repressed by RES. In addition, histone methylation [LPS also causes tolerance after 8–24 h, and this may also be operative via the dietary compounds that affect gene encoding expression of ne cells ,63,64,65ne cells . Our reshylation ,67,68.PPARγ (for lipids and fatty acid synthesis); LXR/RXR, VDR/RXR, and LXR/PPAR. , and the antioxidant action of vitamin C signaling pathways were downregulated by LPS and LEC (PSMB8 (LMP7) alone. RES also downregulates crucial genes expressed in the LPS-induced Toll receptor and IFN-γ pathways. These results provide support for the hypothesis that dietary nutrients can robustly affect the LPS-induced signaling pathways in ex vivo experiments, however, more research is needed to confirm these important findings and determine which lectins have beneficial effects or adverse effects. We have established here that highly effective proteasome modulators, e.g., RES and LEC, commonly present in foods of human diets ,47,48, c and LEC . RES didE. coli D31m4 was purified as described by Qureshi et al. [Deep rough chemotype LPS (Re-LPS) from i et al. . For tisViability and number of PBMCs were determined by trypan blue dye exclusion test and by using a cell counter. After treatment, cells were washed with PBS and total RNA was extracted by using RNeasy mini kit  as per manufacturer’s instructions. 6/10 mL/plate, duplicates) were treated either with 1. medium only, 2. RES (80 μM), 3. LPS (10 ng/mL), 4. LPS10 ng/mL+ RES 80 μM, 5. LEC10 (10 μg/mL), 6. LPS 10 ng/mL + LEC10 μg, 7. LEC 50 (50 μg/mL) and 8. LPS10 ng/mL + LEC50 μg/mL for 3 h. All samples were adjusted to contain the same final concentration of DMSO (0.2%). At 3 h of treatment, Total RNA was extracted from PBMCs and purified further using an affinity resin column . Viable PBMCs  for RNAseq analyses using the human Illumina system . They assessed the purity of total RNA through the following tests, Nanodrop (OD 260/280), agarose gel electrophoresis for RNA purity, and Agilent 2100 analysis to check RNA integrity again. The following procedures were carried out by Novogene: mRNA enrichment, conversion to double-stranded cDNA, end repair, poly-A adaptor addition, fragment selection and PCR, library quality assessment and Illumina sequencing. An average of 44–61 million raw read counts were obtained. Novogene used the STAR software for alignment for RNA-seq data analysis. Read counts is proportional to gene expression levels, gene length and sequencing depth. The differential gene analysis was carried out on two samples (control vs. treatment group) at a time using the DESeq2 R package. The threshold of differential expression genes is log2 fold change >1, and Gene expression data were first imported in the differentially expressed genes (DEG) program and numbers were corrected for differences in the IIlumina analysis. The numbers presented in PSMB8), IFN-γ. and STAT1. These results are consistent with our previous studies that showed that macrophages and T cells from LMP7/LMP10 double knockouts do not induce IFN-γ, STAT1, or nitric oxide (NO) with LPS [44}. Soybean lectin on the other hand, induces gene expression of mediators that is very similar to that of LPS, suggesting some degree of similarity in the signaling pathways by upregulating PSMB8, STAT1, and IFN-γ with LEC. Low doses of lectins may be beneficial to the immunosuppressed host, due to priming and enhancing IFN-γ and NO for killing bacteria and cancer cells. Lectins can also be used as adjuvants such as monophosphoryl lipid A (MPLA), which is used in vaccines and isolated from bacterial toxins, which was purified and characterized by us previously [Resveratrol may potentially be used for downregulation of chronic inflammation during autoimmune diseases, to inhibit the acquired immunity genes induced by LPS and other plant and viral toxins by downregulating gene expression of LMP7 (eviously ,71. Higheviously ,73. This


ACE2, TMPRSS2, FURIN, and CD147) enhance the likeliness of SARS-CoV-2 infection. The genes for same receptors are overexpressed in the periodontal tissues of periodontitis patients. On the other hand, BMAL1 is recognized to play a crucial role in regulating pulmonary inflammation and enhancing susceptibility to viral infection. Silenced BMAL1 disrupts circadian transcriptional regulations, enhances vulnerability to SARS-CoV-2 infections, and may trigger the further production of TNF-α and other pro-inflammatory cytokines that propagate the cytokine storm and exacerbate periodontal inflammation. Therefore ACE2, TMPRSS2, FURIN, CD147, and BMAL1 are the crossroads between SARS-CoV-2 and Periodontitis genes. The enhanced expression of ACE2, TMPRSS2, FURIN, and CD147 and the diminished expression of BMAL1 may be a strategy to check both ailments simultaneously. In gene manipulation techniques, oligos are introduced, which contain all the necessary information to manipulate gene expression. The data are derived from the studies on genes’ molecular patterns, including nucleotide composition, dinucleotide patterns, relative synonymous codon usage, codon usage bias, codon context, and rare and abundant codons. Such information may be used to manipulate the overexpression and underexpression of the genes at the time of SARS-CoV-2 infection and periodontitis to mitigate both ailments simultaneously; it can be explored to uncover possible future treatments.The overexpression of SARS-CoV-2 primary receptors and co-receptors ( TMPRSS2 [TMPRSS2 is also required for viral spread and pathogenesis [ACE2, TMPRSS2, CD147, and FURIN may be considered receptors for SARS-CoV-2 entry [ACE2, CD147, FURIN, and TMPRSS2 in neurons of SARS-CoV-2-infected cells resulted in the reduction in viral RNA expression to 26.6%, 61.1%, 37.7%, and 38.7% for ACE2, CD147, FURIN, and TMPRSS2 genes, respectively [BMAL1 resulted in increased viral burden and disease severity [COVID-19 was caused by the SARS-CoV-2 virus and had a catastrophic effect on the world’s demographics leading to more than 6 million deaths worldwide up to early 2022 and is emerging as the most critical health crisis. The symptoms of COVID-19 include fever, malaise, dry cough, sore throat, dyspnea, and respiratory complications that can eventually lead to multiple health problems and death . The preTMPRSS2 . TMPRSS2ogenesis . S1 subuogenesis . There a-2 entry ,11. It i-2 entry , who repectively . In variseverity . ACE2, TMPRSS2, FURIN, and CD147, which are SARS-CoV-2 primary receptors and co-receptors, are overexpressed in the periodontal tissues of periodontitis patients, presenting with inflammation, periodontal pathogens, and damage-induced pyroptosis [roptosis , and perBMAL1 gene regulates the innate immune system, and its disruption mediates the release of pro-inflammatory cytokines, which are involved in periodontitis, an inflammatory disorder [The disorder . Diminisdisorder . ACE2, BMAL1, CD147, FURIN, and TMPRSS2, more genes, including CD26(DPP4), IFITM3, HLA, ABO, SLC6A20, GSTT1-M1, and DBP [CD26 has a role in expressing the essential regulatory genes participating in SARS-CoV-2 internalization [CD26 is not significantly altered [IFITM3 gene [IFITMs blocks SARS-CoV-2 infection [Other than  and DBP , have allization ; howeverTM3 gene , while tnfection . Among tnfection . The aggnfection . p < 0.05) between COVID-19 prevalence, mortality rates, and GT genotype among all populations. On the other hand, it showed a significant negative correlation (p < 0.05) between prevalence, mortality rates, and TT genotype at the rs7041 locus among all populations [p < 0.05) of serum DBP were observed in periodontitis patients, with a significant linear relationship (p = 0.005) between the levels of DBP and the severity of periodontitis [GSTM1−/−, GSTT1−/−, and GSTM1−/−/GSTT1−/− is higher in severe COVID-19 patients [GSTM1 and GSTT1 combination [Vitamin D binding protein (DBP) showed a positive correlation (ulations . Signifidontitis . Contrarpatients  and the bination . AADC) gene is delivered that converts L-DOPA to dopamine [In attempts at gene manipulation, gene therapy has been employed. To enhance the gene expression, a codon-optimized copy of the gene may be inserted into the engineered virus as a delivery vector where the engineered virus contains a minimal amount of viral DNA, just sufficient for genetic material packing . Adenovidopamine . To suppdopamine . InformaACE2, BMAL1, CD147, FURIN, and TMPRSS2 genes, respectively  test of independence identifies the preferred and rejected pairs of codons in any transcript [Codon pair context is the possibility of the occurrence of two codons together. Few codon pairs are reported to be greater in number than other codon pairs. The frequency tables were prepared through Anconda2.0 software, which permits the contingency table’s statistical analysis and indicates whether the context is significantly biased. The chi-square  occurrence of CpG, CpA, and TpG dinucleotides has been observed along with a small borderline underrepresentation of TpA and CpG. The composition also influences the odds ratio for a dinucleotide pair, and Megremis and colleagues [p < 0.0001).Odds ratios show the underrepresentation or overrepresentation of certain dinucleotides. For example, in Human Rhinoviruses A, B, and C, significantly different . For four-fold degenerate codons, GTA , GGT (Gly), and CCG (Pro) are rare in four out of five genes .Codon usage bias is ruled by several mechanisms, resulting in various consequences. Optimal and unbiased codons are used in mRNA to be robustly translated . On the There is the presence of a specific bias for a certain combinations of codons . The impACE2, CD147, FURIN, TMPRSS2, and BMAL1 genes, respectively. In CD147, FURIN, and TMPRSS2 genes Val (GTG)-initiated codon pairs were most abundant, while in ACE2 and BMAL1 genes, Gln (CAA)- and Glu (GAA)-initiated codons were most abundant. Among all genes, TMPRSS2 showed preference for Asn -, Phe -, Tyr -, Val -, Gly -, Gly -, Ala -, Asp -, Glu -, Pro -, and Gln -initiated codons. CD147 gene showed 25 codon pairs  to be highly used, followed by FURIN  and BMAL1 .The analysis revealed that CAA-GAA, GTG-CTG, GTG-GCC, GTG-TGC, and GAA-TAT are the most abundantly found codon pair in TMPRSS2 gene, the rejected codon pairs are absent.ACE2, TMPRSS2, CD147, and FURIN act as receptors and coreceptors for SARS-CoV-2 entry. Their overexpression led to the enhanced severity and knocking down the expression of these genes by shRNA strategy, showed a reduction in viral protein expression [BMAL1 shows enhanced viral burden and disease severity [BMAL1 may trigger TNF-α and other pro-inflammatory cytokines production leading to cytokine storm and exacerbating periodontal inflammation [COVID-19, caused by the SARS-CoV-2 virus, emerged as a severe health problem leading to the death of more than 6 million worldwide up to early 2022 [pression . Similarseverity . Diminisammation . ACE2, TMPRSS2, FURIN, CD147, and downregulation of BMAL1 genes predispose towards the ailment. Therefore, if a strategy can be planned to modulate the gene expression of these genes in the future, it might help to prevent and cure the COVID-19 infection and periodontitis. Therefore, based on the above evidence, we studied five genes at the crossroads of periodontitis and SARS-CoV-2. Gene manipulations that alter the gene expression level of these genes might be helpful in simultaneously controlling both the SARS-CoV-2 infection and periodontitis. Here the upregulation of Codon optimization and codon pair optimization are the strategies to increase gene expression . There aCD147, FURIN, and TMPRSS2 genes are found to be GC rich and vice versa for ACE2 and BMAL1 genes. Based on RSCU, it is evident that G/C ending codons preference in CD147, FURIN, and TMPRSS2 gene set, while A/T-ending codons in ACE2 and BMAL1 and our results are in concordance with the results of human albumin superfamily members, which are AT-rich genes and preference for A/T-ending codons [Therefore, we analyzed the different codon patterns in the present study and the knowledge will help modulate the expression of five genes associated with periodontitis and SARS-CoV-2 pathology. Based on the nucleotide composition of the genome, the codon usage of any organism is decided . Organisg codons .E. Coli that not only switches the expression of reporter genes between the On and Off state but also helped in precisely regulating the protein expression at intermediate levels. A proof-of-concept experiment shows that altering the rare codon number might modulate protein expression levels. Similarly, the antibiotic pyoluteorin biosynthetic gene pltL encompasses six AGA rare codons, substituting four with CGC and two with CGG significantly enhanced antibiotic production [Rare codons slow down the translation rate due to the rarity of their cognate tRNAs. A linear relationship has been reported between the gene expression level and the number of rare codons. Wang and colleagues  engineeroduction .E.coli [CD147, FURIN, and TMPRSS2 genes. To attenuate the virus, viral protein production needs to be reduced; therefore, the strategy is to increase the number of rare codons in recoded virus sequences. As a result, particular codons pair with specific codons at a frequency higher than expected, while some codon combinations are avoided. This phenomenon is called codon pair bias. In the present study CGT (Arg), AGA (Arg), CGA (Arg), TCG (Ser), CTA (Leu), GTA , GGT (Gly) and CCG (Pro) codons were rare in at least four out of five genes. AGA and AGG codons are the rarest codons in E.coli , and theACE2, CD147, FURIN, and TMPRSS2 may be deoptimized since, apart from participation in pathogenesis, these genes also contribute to physiological roles, so we cannot wholly knock down the genes. Instead, if using the CRISPR-Cas technique if we can recode the genes, the strategy might be used to ameliorate the ailment. Likewise, we will need to enhance the expression of the BMAL1 gene, which may be carried out using optimized codons in the recoded sequence to be inserted through CRISPR.Various researchers have used codon pair deoptimization to construct the attenuated virus for vaccine candidate development. Examples are poliovirus , avian iCD147, FURIN, and TMPRSS2 genes was observed, while in ACE2 and BMAL1 genes, Gln (CAA)- and Glu (GAA)-initiated codons were most abundant. On the other hand, in the SARS-CoV-2 isolates, a bias towards valine-initiated  and glycine-initiated codon pairs  was observed [UL30 gene, encoding for the catalytic subunit of the viral DNA polymerase, was codon pair deoptimized. First, the gene was divided into three segments of equal length, and then the segments were codon pair deoptimized. A minimum of 224 codon pairs deoptimized in one segment resulted in reduced viral plaque size. In comparison, the introduction of changes in 770 codon pairs appeared highly lethal for the virus and no viable progeny was generated [In the present study, amongst the genes envisaged the highest utilization of Val (GTG)–initiated codon pairs in observed . Consistobserved . The firobserved . They geobserved . While aenerated . In the enerated  in polioContrarily, while deoptimizing HIV, mutant HIV-P minA showed significantly reduced replication only with 15 substitutions . Hence tAPOL-I) and haptoglobin-related protein [SMN2 gene is produced in higher amounts. Risdiplam® is another SMA therapy where the SMN2 gene is modified; however, the drug molecule is orally active. Zolgensma® is a more traditional gene therapy where a single intravenous dose is delivered to correct the missing or non-functional copy of the SMN1 gene [TP53, NOTCH1, CDKN2A, PIK3CA, and FBXW7, are found inactivated in head and neck squamous cell carcinoma (HNSCC). The expression of Tp53 in cancer cells via gene therapy initiates the apoptotic pathways and has antitumor effects.Expression studies have been carried out in mice lungs where IL-1β is transiently expressed using adenoviral gene transfer via intratracheal administration. As a result, the high expression of IL-1β accompanied by a local increase of the pro-inflammatory cytokines IL-6 and TNF-α was observed with evidence of lung injury . The exp protein . The genMN1 gene . The genMN1 gene , harborsACE2, CD147, FURIN, and TMPRSS2, which serve as the receptors and co-receptors for SARS-CoV-2 and are overexpressed during COVID-19 infection and in the periodontal tissues of periodontitis patients too, presenting with inflammation, periodontal pathogens, and damage-induced pyroptosis [BMAL1, an immune system regulator, limits the production of the pro-inflammatory cytokine IL-1β [ACE2, CD147, FURIN, and TMPRSS2 genes and the upregulation of BMAL1 may offer a therapeutic strategy against COVID-19 and periodontitis.In the present case, out of the five genes we envisaged, four genes, roptosis  and periroptosis . BMAL1, ne IL-1β . TherefoIn light of knowledge from gene therapy studies, it is evident now that the desired upregulation or downregulation may be achieved in human tissue using codon usage information. For gene expression reduction, however, many codon changes are required to achieve the desired result, and this is the limitation inherently associated with codon pair deoptimization. The addition of rare codons might help in this context. With that, a uniform strategy cannot be made based on our studies for all the ailments and every disease model; it has to be studied and optimized separately. Synonymous mutation influence phenotypic effects, owing to control over mRNA stability and translational regulation. However, our technique has the limitation that it is challenging to pre-judge the changes in which part of the gene will be most effective . Presently the gene therapy technique is limited to treating genetic disorders and life-threatening diseases, such as cancer; however, in the future, with the advancement of technology, it will be possible to apply this to other conditions as well, such as in the case of COVID-19 and dental infections, and the codon usage and context analysis of genes may help reach this target.ACE2, BMAL1, CD147, FURIN, and TMPRSS2 genes so that the pathological consequences of SARS-CoV-2 infection and periodontitis may be avoided.We believe that our study about codon usage, codon bias, availability of rare codons, and codon context will help modulate the gene expression pattern within the organism and, therefore, simultaneously, the COVID-19 and periodontitis problem may be addressed. However, more study is required to obtain an optimal number of codon pairs or rare codons to be replaced in order to obtain an optimal level of gene expression, which is required to maintain the normal physiological functions of the


Erythromelalgia is a disabling syndrome of paroxysmal vasodilation affecting the feet, hands and face characterised by patient’s cooling behaviour to achieve symptom relief. It can be primary or secondary and although a rare disorder it has been described in children and adolescents. We describe the case of a 14-year-old female diagnosed with primary erythromelalgia successfully treated with aspirin, amitriptyline, and carbamazepine. Figure 1), with erosion of the skin of the palms due to continuous immersion to water . She had no evidence of arthritis, nor any other signs suggestive of a systemic inflammatory disorder. Laboratory testing revealed white blood count 6550 K/μl, haemoglobin 13,4 g/dl, platelets 308 K/μl, erythrocyte sedimentation rate 18 mm/1h, C-reactive protein 0.1 mg/L, AST 11 U/L, ALT 6 U/L, CPK 27 U/L, C3 91 mg/dl and C4 21 mg/dl. Antinuclear antibodies, rheumatoid factor, cyclic citrullinated peptide antibody, antineutrophil cytoplasmic antibodies, lupus anticoagulant, β2 – glycoprotein IgG and IgM, anti-cardiolipin antibodies IgG and IgM were negative. Genetic testing for the SCN9A gene mutation, which encodes the voltage-gated sodium channel Na(v)1.7 was negative. Often, a gain of function mutation of this gene is associated both with familial and sporadic cases of erythromelalgia.1 The diagnosis of primary erythromelalgia was established and managed with behavioural interventions  and per os Aspirin (200 mg/day), Carbamazepine (200 mg/day), Amitriptyline (25 mg/day), and Clobazam (30 mg/day), all initiated simultaneously. Two weeks after starting treatment her symptoms were controlled and her sleep was fully restored. The hand skin healed completely, except for mild dry skin on the dorsal surface .A 14-year-old female was referred due to burning pain of her feet followed 2 weeks later by similar symptoms in her hands, for the last 6 weeks. She was previously treated with non-steroidal anti-inflammatory drugs and oral steroids with no improvement. Her physical examination revealed redness and oedema of the dorsal surface of the fingers, hands and palms, in both hands  and less commonly the hands (25%), legs (23%), face, ear, and nose.4 Symptoms are often symmetrical and during episodes the affected areas become red, hot, and painful, while swelling occasionally occurs.3 A characteristic feature of erythromelalgia is patient’s cooling behaviour to achieve symptom relief. Potential complications of cooling techniques include “windburn” from fans, and signs and symptoms of frostbite secondary to overuse of ice. Chronic immersion of the extremity in water may lead to skin maceration, oedema, and ulcerations as in the case presented.5 The majority of both familial and sporadic cases of primary erythromelalgia are attributed to gain-of-function mutations of SCN9A, which encodes the voltage-gated sodium channel Na(v)1.7.1 Secondary erythromelalgia has been associated with thrombocytosis in children, but not with other myeloproliferative disorders as described in adults, while it can also develop in patients with small fibre neuropathies, autoimmune disorders, malignancies, multiple sclerosis, hypercholesterolemia, hypertension obesity, type 1 diabetes mellitus, and heavy-metal poisoning.7Erythromelalgia has an incidence of less the 2 per 100000 people per year. It is more common in women than in men and exceedingly rare in children.8There is no cure for erythromelalgia and combination of non-pharmacological and pharmacological interventions such as local treatment, aspirin and anti-epileptics are directed towards improving quality of life and reducing symptoms.


Marine microorganisms are an important source of natural polyketides, which have become a significant reservoir of lead structures for drug design due to their diverse biological activities. In this review, we provide a summary of the resources, structures, biological activities, and proposed biosynthetic pathways of the benzyl alcohol/salicylaldehyde-type polyketides. In addition, the total syntheses of these secondary metabolites from their discoveries to the present day are presented. This review could be helpful for researchers in the total synthesis of complex natural products and the use of polyketide bioactive molecules for pharmacological purposes and applications in medicinal chemistry. Polyketides represent a vast group of secondary metabolites that are produced by certain living organisms to provide sustaining advantages for them. In terms of their chemical structures, Baerson and Rimando  describeEmericella, first described by Berkeley . Cl. Cl1, acnd Kumar  (Scheme 1), several other total syntheses were developed. These synthetic approaches usually involve multistep sequences and can be differentiated by the means of the disconnection of individual bonds in the varitriol (1) backbone (A/B) is d-ribose ..56, 57, analysis ,18. The 56) demonstrated the highest antibacterial activity against ESBL-producing E. coli and P. aeruginosa, with MICs of 4 μg/mL for both strains.Wang, Xue, Zhang, and coworkers  performeMagnaphorthe grisea (syn. Pyricularia oryzae) caused massive damage to a rice plant in Japan and other Asian countries. This fungus produces several different phytotoxic compounds 59–66 . Th. ThAscoc121) and agropyrenal (122), while agropyrenone (123) was described as inactive. None of the metabolites showed antibiotic, fungicidal, or zootoxic activity butan-2-one (149) butan-2-one (150) [142) and N (143) and a pair of epimers, heterocornols O (144) and P (145), were isolated from a refermented strain of P. heterocornis in the same culture medium [Genus oworkers  describe A (151) , xylarin1) [147) , agropyr7) [121) , vaccino1) [148) , (R)-3-hne (149) , and (R)ne (150)  , the biosynthesis of which is described ,P. heterocornis were evaluated by an MTT assay for their cytotoxic activities against four human cancer cell lines, including a human gastric carcinoma (BGC-823), a human large-cell lung carcinoma (H460), a human prostate cancer (PC-3), and a human hepatocellular carcinoma (SMMC-7721) [121, 130, 131, 135, 146, and 148 and benzooxepines 132, 136, and 137 showed moderate cytotoxicity, with IC50 values of 15–100 μM with adriamycin assayed as a positive control. In the antimicrobial assay, the MIC values for the same compounds were evaluated between 25 and 100 μg/mL for the pathogens Staphylococcus aureus and Bacillus subtilis. The antifungal properties of the isolated derivatives appeared to be dependent on the presence of a pent-4-ene-2,3-diol block. The derivatives 132, 121, 136, and 148 showed moderate antifungal activity against Candida parapsilosis and Cryptococcus neofromans at 100 μg/mL (Metabolites of MC-7721) . 6-Alkyl00 μg/mL .142–145), together with polyketide derivatives 149, 150, and 152, were evaluated for their cytotoxicity against a carcinoma cell line (Ichikawa), gastric cancer (BGC-823), liver cancer (HepG2), and kidney cancer (7860). The tested metabolites showed activity, with IC50 values of 20.4–94.2 μM. Compounds 143 and 152 showed no cytotoxic activity [Heterocornols M–P  and the subsequent aromatization of the polyketide chain. The resulting salicylaldehyde derivative with an aliphatic polyenyl substituent appears to be key for the biosynthesis of salicylaldehyde- and benzyl alcohol-type metabolites. The reported polyketide derivatives exhibited valuable bioactivities, particularly those of marine origin. The highest cytotoxicity towards the human cell line panel was demonstrated with varitriol (1), while agropyrenol (121) and other polyketides of the salicylaldehyde-type, such as heterocornols 130–132 and 135–137, showed moderate activity. In the antimicrobial test, the MIC values ranged from 25 to 100 μg/mL in the pathogens Staphylococcus aureus and Bacillus subtilis. Interesting results were obtained in tests for lipid-lowering effects against oleic acid in HepG2 liver cells. Varioxiranol A (39), andytriol (6), and preshamixanthone (11) exhibited inhibitory effects at a dose of 10 μM and showed no toxicity up to 100 μM toward the tested cells. Moreover, preliminary tests of agropyrenol (121) and its derivatives 124–129 showed significant zootoxic and slight antimicrobial activities. This article also provides an overview of the laboratory syntheses of polyketides containing benzyl alcohol or salicylaldehyde functionalities (To date, more than 100 polyketides containing either benzyl alcohol or a salicylaldehyde aromatic moiety have been isolated and characterized. These secondary metabolites are mainly produced by the fungus nalities . We beli


Boehmeria nivea L.). In this study, 138 AP2/ERF TFs were identified from the ramie genome and were grouped into five subfamilies, including the AP2 (19), RAV (5), Soloist (1), ERF (77), and DREB (36). Unique motifs were found in the DREB/ERF subfamily members, implying significance to the AP2/ERF TF functions in these evolutionary branches. Segmental duplication events were found to play predominant roles in the BnAP2/ERF TF family expansion. Light-, stress-, and phytohormone-responsive elements were identified in the promoter region of BnAP2/ERF genes, with abscisic acid response elements (ABRE), methyl jasmonate response elements, and the dehydration response element (DRE) being dominant. The integrated transcriptome and quantitative real-time PCR (qPCR) revealed 12 key BnAP2/ERF genes positively responding to waterlogging. Five of the genes are also involved in ramet development, with two (BnERF-30 and BnERF-32) further showing multifunctional roles. The protein interaction prediction analysis further verified their crosstalk mechanism in coordinating waterlogging resistance and ramet development. Our study provides new insights into the presence of AP2/ERF TFs in ramie, and provides candidate AP2/ERF TFs for further studies on breeding varieties with coupling between water stress tolerance and high yield.AP2/ERF transcription factors (TFs) are one of the largest superfamilies in plants, and play vital roles in growth and response to biotic/abiotic stresses. Although the AP2/ERF family has been extensively characterized in many species, very little is known about this family in ramie ( Boehmeria nivea L.) is a multipurpose crop, widely used as fiber, medicinal preparation, forage, phytoremediation, and as biofuel [Ramie  that regulate signaling networks for various plant biological processes . AP2/ERFcis-acting elements and transcription factor binding sites, the conserved motifs, and the phylogenetic relationships of their encoded proteins. In addition, expression patterns of the identified genes in different tissues and in response to water deficit, nitrogen deficit, and waterlogging stresses were analyzed. Key genes are identified and discussed in response to waterlogging stress and ramet development regulation.In this study, we attempted to build an extensive map of the AP2/ERF gene family based on the third-generation sequencing data of ramie. We performed a genome-wide identification of AP2/ERF family genes, gene structure, chromosome distribution, gene duplication, AP2/ERF superfamily TFs were identified from the ramie genome, and their sequence information and physical properties are shown in A total of 138 putative A. thaliana using MEGAX software. The results show that the BnAP2/ERFs were clustered into four major clusters: Soloist, RAV (related to ABI3/VP), AP2 (APETALA2), and ERF (ethylene-responsive factors) subfamilies  had 5’ UTR, 3’UTR, and one intron, with the intron occurring in AP2 conserved domain, which suggests their critical function roles in ramie. Noticeably, all members of the AP2, RAV, and Soloist subfamilies in ramie had introns except BnRAV-04.Gene structure analysis is crucial for understanding the structural diversity of genes, revealing such information as the position of the coding sequence (CDS) and the untranslated regions (UTR) . The numThe putative conserved motifs (CMs), possibly reflecting the functional domains involved in specific protein binding sites, such as nucleases and transcription factors, are shown in BnAP2-07, BnDREB-36, and BnERF-09 genes, which were not assembled  and synonymous substitutions (Ks), which can determine whether the gene encoding the protein is under selection pressure. Ka/Ks >> 1 stands for positive selection, Ka/Ks ≈ 1 represents neutral selection, and Ka/Ks << 1 means purifying selection ,23. Withthan one .Arabidopsis thaliana and Cannabis sativa female) and monocotyledons (Oryza sativa and Zea mays), were also investigated to further understand the evolutionary relationship of AP2/ERF genes , C. sativa (74), O. sativa (33), and Z. mays (38), respectively, among which 20 BnAP2/ERF genes were collinear with all four species . Box 4 (423) was found in all the BnAP2/ERFs promoters, but that of BnDREB-03, BnDREB-30, BnERF-06, BnERF-16, BnERF-73 and BnAP2-01. Among the phytohormone-responsive elements, BnAP2/ERFs contained abundant abscisic acid response elements (ABRE) (405) and methyl jasmonate response elements (CGTCA-motif and TGACG-motif) (398). Moreover, BnAP2/ERFs were found to contain many ARE elements for the stress-responsive elements (315), indicating that most BnAP2/ERF TFs may be induced by low oxygen stress, such as waterlogging. The significant presence of these elements suggests that most BnAP2/ERF TF members are involved in plant development and biotic/abiotic stress responses.elements . Light-rBnAP2/ERF target genes were confirmed. It contained disease-resistance-related PRs genes, transcription factors such as WRKY, NAC, and MYB, as well as F-box proteins, D14L, GLT1, and GRF1/3, which may be related to tiller traits of plants.Based on two approaches, 1700 genes with at least one DRE/CRT in their putative promoters and 1009 genes with at least one GCC-box in their promoters were manually searched from the ramie genome. After removing the redundant genes, 2197 potential The target genes were assigned to 54 functional groups and divided into 3 main ontologies: biological processes, molecular functions, and cellular components A. Some tBnAP2/ERF genes are involved in biotic/abiotic stress responses as well as plant growth and development pathways.The most enriched KEGG pathways included riboflavin metabolism, cellular autophagy, gluconeogenesis, glutathione metabolism, and MAPK signaling pathways B. Many tBnAP2/ERFs varied among tissues, with 70 genes expressed in all tissues (FPKM > 0). Seven genes  had high expression levels in all tissues, and most genes were expressed at higher levels in root and bast fiber than that in the stem and leaf.In this study, based on the expression profiles obtained from RNA-Seq analysis data, we constructed a heat map of the gene expression patterns in different tissues of ramie A. ThirtyBnAP2/ERF genes in response to abiotic stresses, relative expression profiles in ramie leaves under water deficit, nitrogen deficit, and waterlogging stresses were constructed based on transcriptomic data . Seventy-two differentially expressed genes (DEGs) were detected under nitrogen deficiency stress, with 44 being up-regulated and 28 down-regulated. Members of the DREB subgroup showed a stronger response under nitrogen starvation than the other subgroups. Sixty-nine DEGs were detected under waterlogging stress, of which 49 members were up-regulated and 20 members were down-regulated. Some members of the DREB subgroup and most of the ERFs showed significant up-regulation, and most BnAP2/ERF genes showed positive feedback to waterlogging stress. Of particular interest is that the up-regulated genes were mainly from the ERF subfamily, and the down-regulated genes from the DREB subfamily.To determine the involvement of mic data B. Fifty-BnERF-17 and BnERF-18 were up-regulated only under water stress (water deficit and waterlogging), while BnERF-22, BnERF-23, and BnERF-25 in group VII were up-regulated only under waterlogging. BnERF-30 and BnERF-31 of the VIII group were up-regulated under waterlogging, and BnERF-26 and BnERF-32 showed up-regulation under both nitrogen deficit and waterlogging. Some genes up-regulated under waterlogging  showed relative expression patterns in water deficit, further indicating that these genes have essential regulatory roles under water stress. On top of that, BnERF-03, BnERF-07, BnERF-08, BnERF-10, BnERF-11, BnERF-18, BnERF-22, BnRAV-03, BnAP2-03, BnAP2-17, and BnAP2-19 were only expressed under abiotic stress, and no expression was detected under normal development, suggesting that these genes may play roles in response to abiotic stress.Furthermore, AtESR1 (AT1G12980), AtESR2 (AT1G24590), AtPUCHI (AT5G18560), AtERF4 (AT3G15210), and AtERF11 (AT1G28370) from Arabidopsis, have been reported to be involved in axillary meristem and branch development and stem elongation and growth [BnERFs. The results , and some genes showed the exact opposite trend . BnERF-26 showed strongly inhibited expression under stress as well as in the recovery state after stress.Furthermore, qPCR analysis was performed to confirm the expression of the 12 candidate genes. The results  indicateBnERF-21 and BnERF-39), some showed the exact opposite trend under both conditions . The expression of BnERF-26 showed the same trend as that under the light condition. These results imply that BnAP2/ERF genes regulate ramie through a complex network under different conditions.The light-or-dark treatments significantly changed the patterns of gene expression profile under waterlogging. While some genes were not significantly induced by the waterlogging stress under darkness , RAV (related to ABI3/VP), DREB (dehydration-responsive element binding), ERF (ethylene-responsive factors) and Soloist subfamilies, which contain 18, 5, 36, 72, and 1 members, respectively. Our results also support the large difference in the number of DREB and ERF occurrences, while Soloist and RAV are small subfamilies with few members in land plants [BnRAV-04. The insertion of introns may be a special mechanism that developed during the evolution of the RAV subfamily in ramie to better adapt to survival pressures [PRs genes, stress-related genes RAB18, LEA3, TIP2, and POX2, transcription factors such as WRKY, NAC, and MYB, as well as F-box genes, D14L, GLT1, and GRF1/3, which may be related to tiller traits of plants. BnAP2/ERFs may regulate ramie response to environmental pressures and ramet development by binding to the corresponding sites in the promoter regions of these genes.The AP2/ERF superfamily genes are defined by the presence of at least one highly conserved AP2 DNA binding structural domain consisting of three d plants ,30,31,32d plants . Our resd plants ,32,34, hressures . Most meressures . KEGG anArabidopsis had N-terminal structures, and two of them  were anaerobic response proteins. In addition, numerous studies have shown that members in the VII group of the ERF subfamily play a central role in regulating waterlogging tolerance [An exciting feature of the AP2/ERF superfamily is that these members have transcriptional activation or repression activity. Amphipathic repressor (EAR) motifs found at the C-terminus of proteins are responsible for their transcriptional repression activity, which involves biotic and abiotic stress responses, plant internode elongation, and leaf senescence ,39,40,41olerance ,43,44,45Arabidopsis and rice, suggesting that the number of AP2/ERF family members is relatively stable, independent of genome size, and that the difference in number may be due to expansion events during the evolution of different plant species. Nonetheless, gene evolution and duplication induced the differentiation of the numbers of AP2/ERF genes among plants. Thus, we calculated the Ka, Ks, and Ka/Ks ratios of BnAP2/ERF repeating gene pairs, including tandem repeats and fragment repeats, to estimate divergence time and selection pressure. All Ka/Ks values were below 1, suggesting that these genes might have experienced strong purifying selective pressure during evolution [AP2/ERF genes per Mb in ramie, while the values for rice and Arabidopsis were 0.4047 and 1.1760, respectively. These data indicate that the AP2/ERF family genes were retained during the evolution of ramie in the presence of extensive gene loss, which proves the significance of the AP2/ERF family in the growth and development of ramie [In ramie, the number of members is similar to that in volution . In addiof ramie .BnAP2/ERFs were identified. Under different pressures, each subfamily showed different behaviors. For example, under nitrogen deficit, the up-regulated genes were mainly in the DREB subfamily and the down-regulated genes were mainly in the ERF subfamily; this was the opposite for water deficit. However, the response genes under waterlogging were predominantly in the ERF subfamily. Furthermore, we found that BnDREB-25, BnDREB-26, BnDREB-32, BnDREB-33, and BnDREB-34 in the DREB subfamily, BnERF-04, BnERF-05, BnERF-09, BnERF-12, BnERF-13, BnERF-29, BnERF-36, BnERF-37, BnERF-38, BnERF-47, BnERF-53, BnERF-54, BnERF-56, BnERF-57, BnERF-71, and BnERF-72 in the ERF subfamily, BnAP2-06, BnAP2-09, BnAP2-11, BnAP2-14, BnAP2-16, and BnAP2-18 in the AP2 subfamily, and BnRAV-02 were not expressed under either normal developmental or abiotic stress conditions, suggesting that these genes may be pseudogenes. However, the possibility that these genes respond to other stressful environments cannot be excluded.Plants must adapt to a variety of biotic/abiotic pressures because they are immobile throughout their life cycle. AP2/ERF genes play important roles in stress responses, including drought, waterlogging, heat, and salt stress. From the RNA-seq data, the DEGs of AP2/ERF family genes in the waterlogging response has been reported for several crops, with the most attention being given to members of group VII. We found from qPCR results that group VII members such as BnERF-21, BnERF-22, BnERF-24, and BnERF-25 were all up-regulated under waterlogging stress, with the latter three strongly induced even after reoxygenation, indicating their importance in stress tolerance and in growth and development. In addition, plants still have to balance the presence and absence of varying degrees of light under field conditions [cis-element analysis also show that the promoter region of the BnAP2/ERFs was enriched in light-responsive elements, in addition to elements such as ABRE and ARE, in response to biotic/abiotic stresses. This indicates that BnAP2/ERFs also play important roles in regulating plant development in response to photoperiod. Expression pattern analysis showed that some AP2/ERF genes had to be expressed earlier under shade conditions to cope with these stresses to ensure their growth and nutrient accumulation. It has been demonstrated that plants improve their tolerance to stress under different light environments by responding appropriately to abiotic stresses [The involvement of nditions . The resstresses .Plants actively slow down their growth when they are under stress, a complementary strategy they use to cope with adverse conditions . While pBnERF-27, BnERF-38, BnERF-36, BnERF-37, BnERF-32, and BnERF-30 were AtESR1, AtESR2, AtPUCHI, AtERF4, and AtERF11 in Arabidopsis, respectively. However, the analysis of transcriptome data revealed that BnERF-27, BnERF36, BnERF-37, and BnERF38 did not show differential expression in the two ramie varieties with different ramet numbers. Therefore, we predicted that BnERF-30 and BnERF-32 were likely to be involved in the multifunctional role of waterlogging tolerance and ramet development in ramie.Ramie is a vital fiber crop that exhibits defeat, stalk shrinkage, and stunted chloroplast development in response to stress to ensure its growth ,52. MeanProtein interaction analysis is more intuitive and rapid for understanding gene function, and is also important for regulatory network relationships between functional proteins. We used STRING software to map an integrated protein interaction network based on AtERF11 and AtERF4. The results  show thaAlternanthera philoxeroides and Hemarthria altissima with well-developed root systems are more likely to grow new branches after waterlogging [BnAP2/ERF family genes may also act as a more complex crosstalk mechanism in coordinating stress and growth development.Moreover, BnERF-32 (AtERF4) can directly bind to the repressor proteins SPL2 and SPL6 involved in axillary meristematic tissue to regulate plant structure. Studies have shown that the number of branches or tillers of some plants increases or decreases after waterlogging stress, which may be related to the growth regulation of the plants themselves after exposure to adversity. For example, waterlogging-tolerant species rlogging , while wrlogging . Thus, BAP2/ERF gene sequences were identified from the ramie genome derived from the whole genome data sequenced by our laboratory. The hidden Markov model (HMM) file of the AP2 domain (PF00847) was obtained from the Pfam database (http://pfam.xfam.org/ (accessed on 18 April 2022)), and the target genes were searched using HMMER3.0 software with a threshold E-value < 0.05 [https://web.expasy.org/protparam/ (accessed on 28 April 2022)). The sub-cellular location was predicted using the Softberry ProtComp v.9.0 (http://www.softberry.com/ (accessed on 29 April 2022)).The e < 0.05 . To confArabidopsis thaliana AP2/ERF protein sequences (147) were downloaded from TAIR (https://www.arabidopsis.org/ (accessed on 18 April 2022)) and used to establish the phylogeny. Multiple alignments of BnAP2/ERF and AtAP2/ERF genes were performed by MUSCLE with default parameters, and the result was pruned using the Trimmer function in TBtools [https://itol.embl.de/ (accessed on 13 May 2022)) [ TBtools . A phylo TBtools ,67. The y 2022)) .https://meme-suite.org/meme/tools/meme (accessed on 15 May 2022)) was used for predicting the BnDREB/ERF conserved motifs (CMs), with the maximum number of motifs set to 10 and other parameters set to default. The gene structure of BnAP2/ERF and the final overall figure was generated with Gene Structure View in TBtools [The MEME Suite 5.4.1 online tool ) for prediction. The results are summarized in All sequences 2.0 kb upstream of the Target genes with DREB protein binding site elements DRE (TACCGACAT) and CRT (G/ACCGAC) were screened. In addition, genes with AP2/ERF TF binding site elements (GCC-box) with sequence “AGCCGCC” were manually searched and used in the subsequent enrichment analysis. Pathway enrichment analysis for the potential BnAP2/ERF target genes was conducted using the KEGG database . Gene onBnAP2/ERF expression levels per mapped read per million (FPKM) values were calculated from these transcriptome data. Heat maps were generated using TBtools with log2(FPKM + 1) normalization [Transcriptome analysis of tissue-specific expression was conducted using four different tissue samples of “Zhongzhu No.1” sequenced by our laboratory. Transcriptome data of water deficit and nitrogen deficit were obtained from . The addlization .AP2/ERF genes between Arabidopsis and ramie was determined using OrthoVeen2 [Arabidopsis homologs were obtained with high confidence of >0.400 using the STRING v11.5 online program, and used to build a correlation network [The homology of thoVeen2 . The int network ; the netThe ramie seedlings of variety “Zhongzhu No.2”, which has the largest cultivation area in the world, were prepared by hydroponic methods. Seedlings with even sizes were transplanted in earthen pots in the greenhouse under controlled conditions of 60 ± 5% humidity at 30 ± 2 ℃, and cultured for more 3 weeks with only water. Then, the seedlings with similar height, leaf number, and leaf area were selected and divided into four groups with six biological replicates for each treatment. The CK group was grown by applying 100 mL nutrient solution every day and additional water to keep the soil moisture at 80% of soil field capacity for 10 days. The waterlogging group was grown under the same conditions as the CK group, but for 9 days, and the pots were immersed by maintaining 2 cm water level above the underlying soil surface  for 12 h® Green Premix Pro Taq HS qPCR Kit II  Co., Ltd.) on a CFX96 Touch Deep Well Real-Time Quantitative PCR System (Bio-Rad) following standard procedures. Relative transcript levels were calculated using the 2-ΔΔCt formula, and the results were depicted in histograms drawn using GraphPad Prism v8.0 software.Samples from the waterlogging treatment were used for qPCR analysis. Total RNA was extracted using a SteadyPure Plant RNA Extraction Kit  Co., Ltd.). The RNA was reverse transcribed, using an Evo M-MLV One Step RT-PCR Kit  Co., Ltd.), into cDNA, and qPCR analysis was performed using gene-specific primers . The 18sBnAP2/ERF genes were identified using bioinformatics, and further classified into five subfamilies based on conserved sequence characteristics, gene structure, and motif constituent. Synteny analysis revealed that fragment replication events were the key drivers of BnAP2/ERF evolution. Analysis of gene promoter cis-acting elements and target gene predictions indicated that BnAP2/ERF members actively respond to plant growth and stress stimuli such as water stress, mineral substance stress, and temperature stress. Based on the expression patterns analysis of BnAP2/ERFs, we identified some key genes involved in water stress and ramet development. In addition, transcriptome data and results from the protein interaction network analysis suggest that BnERF-30 and BnERF-32 had multifunctional regulatory effects on waterlogging stress and ramet development in ramie. In short, these results guide us a step further towards understanding the basic information of the AP2/ERF genes in ramie, which might serve as a first step toward the comprehensive functional characterization of the AP2/ERF gene family via genetic approaches in the future.A comprehensive analysis of the AP2/ERF family genes in ramie was carried out in this study. A total of 138


Of particular note are two-pore channels (TPCs), a group of recently identified Ca2+-channels, located within the endolysosomal system. TPC2 has recently emerged as an intracellular ion channel of significant pathophysiological relevance, specifically in cancer, and interest in its role as an anti-cancer drug target has begun to be explored. Herein, an overview of the cancer-related functions of TPC2 and a discussion of its potential as a target for therapeutic intervention, including a summary of clinical trials examining the TPC2 inhibitors, naringenin, tetrandrine, and verapamil for the treatment of various cancers is provided.The importance of Ca Dysregulation of Ca2+ signaling is increasingly being demonstrated to contribute to the development of cancer , whereas others are expressed on the membrane of intracellular organelles, such as ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3R) located on the sarcoplasmic and endoplasmic reticulum. Two-pore channels (TPCs) are unique in that they are located on the acidic organelles of the endolysosomal system  and nicotinic acid adenine dinucleotide phosphate (NAADP) [2+ release has been controversial [2+ can inhibit these NAADP-mediated Ca2+ currents, but that when Mg2+ is absent, NAADP can activate TPC2, which may account for the conflicting findings.TPCs comprise a family of voltage- and ligand-gated Na (NAADP) –8. Howevoversial , 10. In oversial  identifiTPC1–3) have been identified, humans and rodents only express two isoforms, TPC1 and TPC2 [2 binds to the first 6 transmembrane domains to activate the channel independently of voltage changes, inducing a structural change at IS6 [While three distantly related genes , which is modulated through the endoplasmic reticulum Ca2+ sensor stromal interaction molecule-1 (STIM1) and plasma membrane Orai1 Ca2+ channels, both of which were found to associate with TPC2 [While NAADP can endogenously regulate Cainterest , and pro release . TPC2 ca release . Althoug release , and the release . These fith TPC2 . A yeastith TPC2 . Hax-1 hith TPC2 , and theith TPC2 , thus po2+ signaling has been shown to play a critical role in a variety of associated pathophysiological processes, including the life cycle of Ebola virus, human immunodeficiency virus (HIV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Parkinson’s disease, Alzheimer’s disease, non-alcoholic fatty liver disease, and cardiac dysfunction [TPC2 plays important roles in various diseases and physiological conditions, including cell proliferation, differentiation, development, autophagy, membrane trafficking, endolysosomal degradation pathway, phagocytosis, angiogenesis, low-density lipoprotein (LDL)-cholesterol trafficking, T cell activation, hormone secretion from the pancreas, and cardiac function –34. Consfunction , 35–42, function , 41, 43,Of particular note, TPC2 is overexpressed or mutated in several cancer types. Additionally, TPC2 has recently been implicated in a variety of cancer-related processes  that underpin the key hallmarks of cancer [TPC2 expression in pulmonary artery smooth muscle cells, MNT-1 human melanoma cells, RIL175 murine hepatocellular carcinoma cells, and 4T1 murine breast cancer cells significantly decreased proliferation in vitro [TPC2 expression in hepatocellular carcinoma cells reduced glycolysis and respiratory activity in vitro [in vivo [TPCN2 gene in murine primary melanoma cells decreased initial proliferation rates by slowing G2 phase progression [TPC2 overexpression in 4T1 breast cancer cells did not influence proliferation [A role for TPC2 in proliferation was first postulated based on the finding that TPC2 function could be regulated by the protein kinases, p38 mitogen activated protein kinase (MAPK), c-jun N-terminal kinase (JNK), and the mTOR complex 1 (mTORC1) . Since tin vitro , 44–46, in vitro , potenti[in vivo . Furthergression . By contferation , suggestTPC2 expression have been observed at various stages in differentiation in multiple cell types, suggesting that TPC2 may control differentiation. For example, TPC2 mRNA expression was highest in the first 3 days of differentiation of C2C12 myoblast cells but was reduced thereafter, and TPC2 knockdown inhibited differentiation of these myoblasts [TPC2 mRNA was increased in osteoclast precursor cells upon receptor activator of nuclear factor kappa B (NFκB) ligand (RANKL)-induced osteoclast differentiation, and downregulation of TPC2 suppressed osteoclastogenesis [TPC2 knockdown accelerated the initial commitment of neural progenitors, but inhibited later neuronal differentiation. Additionally, overexpression of TPC2 induced cell death and prevented embryonic stem cells from differentiating into early neural lineages [TPC2 knockdown had no effect on the differentiation of astrocytes or oligodendrocytes [Changes in yoblasts . Similarogenesis . By contogenesis . As a relineages . Interesdrocytes . Taken tTPC2 expression in T24 bladder cancer and CHL1 and B16-F10 melanoma cells, and pharmacological inhibition in T24, HUH7 liver cancer and 4T1 breast cancer cells, significantly reduced adhesion [TPC2 gene in CHL1 and B16-F10 melanoma cell lines decreased adhesion to collagen type I matrix and was associated with a decrease in α2β1 integrin expression on the plasma membrane [Emerging evidence has also implicated TPC2 in all the major steps of tumor metastasis, namely adhesion, migration, and invasion. Knockdown of adhesion , 51. Mecmembrane . While pmembrane . WhetherTPC2 expression in MNT-2 melanoma cells, T24 bladder cancer cells, RIL175 hepatocellular carcinoma, 4T1 breast cancer, Hela cervical cancer and pulmonary artery smooth muscle cells, and pharmacological inhibition in T24, HUH7 and 4T1 breast cancer cells led to decreased migration in vitro [TPC2 overexpression in 4T1 breast cancer cells had no effect on migration in vitro [in vitro, however, similarly as to what was observed for proliferation, the presence of TPC2, rather than an increased level of expression, may be the only requirement for the regulation of migration.In support of TPC2 having a metastasis promoting role, knockdown of in vitro –46, 51. in vitro . Taken tTPC2 in MNT-1 human melanoma cells significantly reduced invasion in vitro [. By contrast, knockout of the TPC2 gene in CHL1 and B16-F10 primary murine melanoma cells increased invasion, with an associated increase in matrix metalloproteinase 9 (MMP-9) expression [TPC2 has also been implicated in invasion in melanoma cells. Knockout of in vitro , providipression . These cin vitro studies demonstrate the importance of TPC2 in adhesion, migration, and invasion, and suggest that TPC2 may promote metastasis in vivo. Indeed, knockdown of TPC2 expression and pharmacological inhibition in 4T1 orthotopic breast cancer and RIL175 hepatocellular carcinoma mouse xenograft models reduced the formation of lung metastases in vivo [in vivo, particularly for breast and hepatocellular cancer, and suggest that TPC2 may be a potential therapeutic target for preventing cancer metastasis, at least in breast and hepatocellular cancers. Because the expression levels of TPC2 seem to be less important than its presence for most cancer-associated functions, further investigation into TPC2 binding partners in specific cancer cell types will ultimately be required to unravel the pathways responsible for the varying effects observed.Taken together, these  in vivo , 51, pro2+ release [2+ release and angiogenesis in vitro [in vivo [Tpcn2−/− mice or pharmacological inhibition of TPC2 inhibited vascularisation of VEGF-containing matrigel plugs in vivo [Tumor cells are highly dependent on the formation of new blood vessels to sustain their insatiable growth requirements. This pathological process of neoangiogenesis involves stimulation of normally quiescent endothelial cells through their VEGF receptors to induce proliferation, migration, and capillary branch formation. Production of VEGF by tumor cells is the key mediator of this process, but VEGF also affects immune cells in the tumor micro-environment as well as providing an autocrine pro-malignancy signal that promotes epithelial-mesenchymal transition of the cancer cells . VEGF in release  offers ain vitro . Further[in vivo –57. Addi in vivo , 58. TPC in vivo , demonst in vivo . Thus, pAutophagy is a lysosomal dependent mechanism used to naturally rid the cell of dysfunctional or redundant elements and is a critical regulator of cellular homeostasis. In extreme cases of starvation, a cell will use autophagy as a source of energy to maintain survival, and defects in this pathway have been associated with numerous human diseases, including cancer . HoweverTPC2 in astrocytes increased the expression of the autophagic markers LC3-II and beclin-1, as well as acidic vesicular organelle formation [TPC2 construct led to decreased LC3-II levels in these astrocytes, and knockdown of TPC2 expression decreased autophagy in astrocytes in an mTOR-independent manner [TPC2 alone or in combination with TPC1 in nutrient-rich conditions induced autophagy, whereas under starvation, silencing of either TPC1 or TPC2 induced an autophagic block [Several studies have indicated that TPC2 is a positive regulator of autophagic flux. Overexpression of ormation . Converst manner . Furthert manner . Additioic block . This stic block . These Lic block , identifTPC2 knockdown in mouse embryonic stem cells promoted autophagosomal-lysosomal fusion [ATG5) knockdown, but not mTOR activity inhibition, abolished the TPC2-induced accumulation of autophagosomes [Tpcn2−/− mice exhibited enhanced autophagy flux characterized by increased accumulation of autophagosomes upon starvation and treatment with the microtubule inhibitor, colchicine [By contrast, there is also evidence that TPC2 can act as a negative regulator of autophagic flux. Overexpression of TPC2, but not an inactive mutant form of TPC2, in Hela cervical cancer and 4T1 breast cancer cells decreased autophagosomal-lysosomal fusion, resulting in the accumulation of autophagosomes , 65. Conlchicine . Taken tFurther controversy surrounds the mechanisms by which TPC2 may mediate autophagy. While the TPC-mediated effects in astrocytes occurred in an mTOR-independent manner , pharmacTPC2 have been identified as predisposition factors for a range of cancer types [TPC2 coding region appear restricted to rare instances of gene fusions in breast cancer and uterine leiomyomas [TPC2 (11q13.2) is commonly amplified in cancer [TPC2 gene has been identified as the potential driver of this amplification by providing growth advantage in oral squamous cell carcinoma [As TPC2 has been implicated in several cancer related processes, it raises the question as to whether abnormalities in TPC2 gene coding or protein expression may be observed in cancer. Inherited polymorphisms in er types –72, moster types . Howeveriomyomas , 75. Then cancer , and ovearcinoma , 78. Heparcinoma , 79. Addarcinoma .TPC2 expression is also a potential prognostic biomarker for several types of solid tumors. TPC2 was part of a gene signature for biomarkers of lymph node metastasis in oral squamous cell carcinoma, recurrence in prostate cancer, and decreased overall survival in bladder cancer [TPC2 is highly expressed in hepatocellular carcinoma, oral squamous cell carcinoma and blood cancers, however, whether this expression is observed in additional cancer types, remains to be seen. Additionally, increased TPC2 expression may be a biomarker for poor prognosis for several types of cancer, including oral, prostate and bladder cancers. These negative effects on patient prognosis are most likely due to the central role of TPC2 in regulating cancer cell proliferation and metastasis and highlight that inhibiting TPC2 activity is likely to be an anti-cancer therapeutic strategy for a range of cancer types.r cancer , 80, 81,r cancer . Taken tDue to the importance of TPC2 in controlling cancer-related functions , its sui2+ response [Naringenin, a natural flavonoid found in citrus and tomatoes, is one of the main flavonoids present in the human diet . Naringeresponse , and as response , so its in vitro [in vivo [in vitro but combined with 5-fluorouracil synergistically enhanced cell death [in vitro [in vivo [Naringenin exhibits anti-proliferative and pro-apoptotic effects in glioma, melanoma, breast, colorectal, hepatic, prostate, lung, and pancreatic cancer in vitro –114, and[in vivo , 115–121ll death . Additioin vitro , and whe[in vivo . Taken t[in vivo , 125. Itin vitro [in vivo [Further, naringenin inhibits migration of glioma, melanoma, breast, prostate pancreatic, and lung cancer cells in vitro –114, 128[in vivo , 127. AnSeveral studies have investigated the mechanisms underlying naringenin-mediated anti-cancer effects and have identified a variety of mechanisms , includiDalbergia parviflora) block TPC2 activity in melanoma cells and inhibit melanoma cell proliferation, migration, and invasion in vitro [In an attempt to improve the effectiveness of naringenin, derivatives modified at position 7 have been developed. Initial examination has shown that HCT116 colon cancer cells were more sensitive to these derivatives than to naringenin , howeverin vitro . Furtherin vitro, with reported IC50 values ranging from 2.2–178 μmol/L for most cell lines [Despite the abundance of pre-clinical evidence indicating that naringenin is a potent anti-cancer agent with low toxicity , this hall lines . Future Stephania tetrandra and belongs to the class of bisbenzylisoquinoline alkaloids [2+ channel blocker, and recently, tetrandrine was shown to block TPC2 currents elicited by both NAADP and PIP2 [2+-signaling pathways. Tetrandrine can also suppress another Ca2+-mediated process, specifically the nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome activation via Sirt-1 [2+-signaling related processes. Nevertheless, emerging evidence indicates that tetrandrine possesses anti-cancer efficacy against a range of cancer types P2 . AdditioIP2 , a kinasIP2 , 187. Tha Sirt-1 , 189. Taer types .in vitro [in vivo [in vitro, nasopharyngeal carcinoma xenografts were not sensitive to tetrandrine as a monotherapy in vivo, however tetrandrine was able to sensitise these tumors to irradiation [in vitro and in vivo [Tetrandrine displayed anti-proliferative and cytotoxic effects as a monotherapy against AML, glioma, neuroblastoma, osteosarcoma, nasopharyngeal, lung, oral, gastric, prostate, bladder, breast, renal cell, hepatocellular, colorectal, gallbladder, pancreatic, and ovarian cancer cells in vitro –175, an[in vivo , 175 (Tadiation , suggest in vivo –198, inin vitro [in vivo [There is also substantial pre-clinical evidence that tetrandrine may be effective against metastatic disease. Tetrandrine treatment decreases invasion and migration of a range of cancer types in vitro , 172, a[in vivo , 57, 166Several studies have investigated the mechanisms underlying tetrandrine-mediated anti-cancer effects . Althougin vitro and in vivo cancer models (in vitro effects will translate into in vivo or clinical effects, therefore further examination in clinically relevant in vivo models is required before future clinical examination is performed. Additionally, it is likely that tetrandrine will be more beneficial clinically when combined with existing chemotherapeutics.Taken together, these studies highlight that tetrandrine is an interesting drug candidate for the treatment of cancer as a monotherapy and in combination with a variety of chemotherapeutics. While a large amount of pre-clinical evidence demonstrated that tetrandrine is effective in both r models , the majNevertheless, a clinical trial examining the combination of intravenous tetrandrine with daunorubicin, etoposide and cytarabine for the treatment of refractory and relapsed AML showed that this combination was relatively well tolerated. Forty-two percent of patients achieved complete remission or restored chronic phase, 23% achieved a partial response, and 34% failed therapy , this prin vitro [in vivo [Despite these promising results, several animal models have identified that tetrandrine can induce substantial liver and lung damage –203, whiin vitro –206. Tet[in vivo . While tin vitro [Ned-19 is a selective membrane-permeable non-competitive NAADP antagonist , which lin vitro , 177. Thin vitro [in vivo [Further evidence indicates that Ned-19 may be a potential inhibitor of metastasis. Importantly, Ned-19 decreased migration of melanoma, hepatic, bladder, and breast cancer cells in vitro , 177, an[in vivo , 176. Wh2+-channel blocker [2+ release [2+-channel blockers, particularly verapamil, and increased risk for the development of breast cancer [in vitro [ex vivo of tumor cells collected from AML, but not CML patients in blast phase [Verapamil is a Ca blocker , used cl release , and hent cancer . Indeed,in vitro , suggestst phase , 180. Tain vitro evidence spanning several decades indicates that verapamil can sensitise a range of cancer cells to treatment with chemotherapeutics, including doxorubicin, daunorubicin, vincristine, paclitaxel, mitomycin and amlodipine [in vivo models. For example, verapamil + doxorubicin combination treatment did not increase survival or reduce metastatic burden in a 4T1-resistant breast cancer xenograft mouse model in vivo [Despite these conflicting results on cancer cell proliferation, verapamil has been widely established to act as a chemosensitiser, by inhibiting the function of p-glycoprotein –214. Whilodipine –221, thi in vivo . Additio in vivo . Further in vivo . By cont in vivo . Taken t in vivo  was clin in vivo . In addi in vivo . These cin vivo. These agents have all been shown to act as chemosensitizing agents, suggesting that they will be most beneficial clinically when used in combination with existing chemotherapeutics. Indeed, several clinical studies have shown that tetrandrine and verapamil can potentiate the effects of chemotherapeutics in advanced cancer patients. However, both of these drugs can induce toxicity, most likely due to their Ca2+-channel blocking effects, so different routes of administration and analogues with improved safety profiles are being examined. Overall, a large body of evidence indicates that TPC2 is an attractive anti-cancer target that warrants further investigation.TPC2 is an attractive anti-cancer target as it is expressed in a range of cancer types and is vital in the control of cancer cell proliferation, survival, metastasis, and angiogenesis. Several drugs that inhibit TPC2, including the naturally occurring naringenin and tetrandrine, have been identified which demonstrate pre-clinical anti-cancer efficacy, including reducing metastatic burden


Spodoptera frugiperda, is a serious worldwide agricultural pest, threatening food security and crop production. Sex pheromone lures are commonly used in population monitoring and biological control of FAWs. Although the sex pheromone components of the FAW have been successfully identified, there are no reports on the molecular mechanism of FAW sex pheromone biosynthesis. In this study, we identified a total of 99 genes related to the biosynthesis of sex pheromones from the S. frugiperda genome, which belonged to 11 families of genes. Based on gene expression patterns and phylogenetic analysis, several genes had PG-biased expression, indicating that they may play an important role in sex pheromone biosynthesis. These results could lay a solid foundation for understanding the molecular mechanisms of S. frugiperda sex pheromone biosynthesis and provide new targets for developing novel pest control methods based on disrupting sexual communication.The fall armyworm (FAW), Spodoptera frugiperda is an agricultural pest causing substantial damage and losses to commercial crops. Sex pheromones are critical for successful mating in Lepidoptera and have been used for monitoring and control of many pest species. The sex pheromone of S. frugiperda is known, but the genes involved in its biosynthesis have not been identified. We systematically studied 99 candidate sex pheromone genes in the genome of S. frugiperda including 1 acetyl-CoA carboxylase (ACC), 11 fatty acid synthases (FASs), 17 desaturases (DESs), 4 fatty acid transport proteins (FATPs), 29 fatty acyl-CoA reductases (FARs), 17 acetyl-CoA acetyltransferases (ACTs), 5 acyl-CoA dehydrogenase (ACDs), 3 enoyl-CoA hydratases (ECHs), 3 hydroxyacyl-CoA dehydrogenases (HCDs), 6 ethyl-CoA thiolases (KCTs), and 3 acyl-CoA-binding proteins (ACBPs). Based on the comparative transcriptome results, we found 22 candidate sex pheromone biosynthesis genes predominately expressed in pheromone glands (PGs) than abdomens without PGs including SfruFAS4, SfruFATP3, SfruACD5, SfruKCT3, SfruDES2, SfruDES5, SfruDES11, SfruDES13, SfruFAR1, SfruFAR2, SfruFAR3, SfruFAR6, SfruFAR7, SfruFAR8, SfruFAR9, SfruFAR10, SfruFAR11, SfruFAR14, SfruFAR16, SfruFAR29, SfruACT6, and SfruACT10. A combination of phylogenetic and tissue-specific transcriptomic analyses indicated that SfruDES5, SfruDES11, SfruFAR2, SfruFAR3, and SfruFAR9 may be key genes involved in the sex pheromone synthesis of S. frugiperda. Our results could provide a theoretical basis for understanding the molecular mechanisms of sex pheromone biosynthesis in S. frugiperda, and also provide new targets for developing novel pest control methods based on disrupting sexual communication. Female Lepidoptera (moths) release sex pheromones to attract males for mating ,2. Most Spodoptera frugiperda, native to tropical and subtropical America, is an agricultural pest. It has a wide plant host range, strong migratory ability, high reproductive capacity, and invaded sub-Saharan Africa, Asia, and other regions  − 1) × 100 [−ΔΔCT method [Four differentially expressed genes (1) × 100  (Table S1) × 100 . The qRTT method .SfruFATP1, SfruFATP2, and SfruFATP4) and 24 (SfruFATP3) (SfruFAS2 and SfruFAS3), 7 (SfruFAS1), and 17 (SfruFAS4)   . Both ofruFATP3) . Seven FfruFAS4) . Five ACruACD22) . Thirty ruACD22) . DESs anruACD22) .S. frugiperda, S. exigua, S. litura, S. inferens, A. pernyi, O. nubilalis, and H. assulta. The DESs phylogenetic trees showed that all three identified SfruDESs from the S. frugiperda genome were clustered in three different clades of Lepidoptera desaturases: Δ11 desaturase (SfruDES5), Δ9 desaturase (18 C > 16 C) (SfruDES9), and Δ9 desaturase (16 C > 18 C) (SfruDES11)  were clustered within the Lepidoptera pgFAR group, which is a clade with functionally investigated FARs  . In the ted FARs .S. frugiperda, we performed a transcriptome analysis for the genes expressed in female PGs and ABs (SfruFAS4), FATP (SfruFATP3), ACD (SfruACD5), and KCT (SfruKCT3) had significantly higher expression levels in pheromone glands (PGs) than abdomens (ABs) , twelve FARs  and two ACTs (SfruACT6 and SfruACT10) were predominately expressed in the PGs of S. frugiperda  . Five DEugiperda . SfruFARd in PGs I. Among eded 100 . The qRT the PGs .S. frugiperda. Among them, 22 genes were expressed at significantly higher levels in PGs than in the abdomen, and most of them were key genes involved in the sex pheromone biosynthesis pathway of moths such as DESs and FARs. Consistent with this study, DESs and FARs also showed a trend of high PG expression in S. litura and S. exigua [In moths, the PGs are the most important organ for synthesizing and releasing sex pheromones . Thus, t. exigua ,30.S. frugiperda. Among the 11 FAS genes, SfurFAS4 had the highest expression in PG, suggesting its important role in fatty acid synthesis. As an evolutionarily conserved membrane-bound protein, FATPs can bind fatty acids and transport them to PG cells via the hemolymph for pheromone biosynthesis [S. frugiperda, consistent with the number of FATP genes in S. litura and S. exigua [SfurFATP3 had abundant expression levels in the PGs based on the FPKM values.Sex pheromones released by female moths are composed of a mixture of sex pheromone components in specific ratios that show high species specificity ,44. Syntynthesis ,48. Four. exigua , and theS. frugiperda. Except for Z11-16:OAc, containing 16 carbons, Z9-14:OAc, Z7-12:OAc, Z9-12:OAc, and E7-12:OAc are less than 16-carbon chain length unsaturated fatty acid ester derivatives [SfurACD5 and SfurKCT3 had higher expression abundance in PGs than in the abdomen.There are several sex pheromone components of ivatives ,28. TherS. frugiperda have different positions, numbers, and configurations of double bonds, DES is crucial for sex pheromone formation. A total of 17 DESs were identified from the FAW genome. Phylogenetic tree analysis showed that the SfruDES5 was clustered with the DES5 of S. litura and S. exigua, and both were clustered in the ∆11 DES branch. Both transcriptome FPKM values and qRT-PCR showed that SfruDES5 was significantly overexpressed in the PGs of S. frugiperda. SfruDES9 and SfruDES11 are clustered with the corresponding DES9 and DES11 of S. litura and S. exigua and belong to the Δ9 (18 C > 16 C) and Δ9 (16 C > 18 C) desaturase groups, respectively, in which SfruDES11 was specifically expressed in PGs. The Δ9 and Δ11 desaturases are important desaturases in Spodoptera. The Δ9 desaturases and Δ11 desaturases can introduce Δ9-double bonds and Δ11-double bonds in precursors [S. frugiperda sex pheromone, and Δ9 and Δ11 desaturases are key enzymes for introducing the Δ9 and Δ11 double bonds into the pheromone. Therefore, SfurDES5 and SfurDES11 may participate in the desaturation step from saturated to unsaturated acids during sex pheromone synthesis in S. frugiperda.DES is a key enzyme in sex pheromone biosynthesis. It removes hydrogen atoms at specific positions and introduces double bonds to form cis-trans isomers ,49. DES ecursors . The Z9-S. frugiperda, 12 FARs were specifically highly expressed in the PGs. The phylogenetic tree showed that SfruFAR3 was clustered with FAR3 of S. litura and S. exigua, belonging to the PgFAR subfamily of Spodoptera, and had a high expression abundance. This indicated that this gene may play an important role in the synthesis of precursor alcohols. ACT can catalyze the formation of esters from alcohols [S. frugiperda sex pheromone, ACT genes play a key role in sex pheromone biosynthesis. A total of 17 ACT genes were identified in the FAW genome, among which the SfurACT6 and SfurACT10 were highly expressed in the PGs. These two genes may play a role in the process of converting alcohol to esters. Furthermore, two ACTs were unplaced in the chromosome, which might be caused by the genome quality. In Bombyx mori, ACBP functions as acyl-CoA or cell deposition [S. frugiperda genome.The precursor substance forms an intermediate product with a specific length and double bond position after the desaturation reaction and chain shortening reaction. It then needs to be catalyzed by reductases to form alcohols. During this process, FAR is responsible for converting unsaturated fatty acids into the corresponding alcohols ,54. In oalcohols ,56. SincS. frugiperda, we speculated the S. frugiperda sex pheromone biosynthesis pathway. Firstly, ACC catalyzes acetyl-CoA to malonyl-CoA, which is followed by FAS to produce the most saturated palmitic acid (16: CoA). The 16: CoA was desaturated by Δ11 desaturase (Z11/E11) to produce Z11-16: CoA/E11-16: CoA. After the desaturase-induced formation of a double bond, specific β-oxidation enzymes shorten the chains to Z9-14: CoA, Z7-12: CoA, or E7-12: CoA. In addition, the 16: CoA was shortened by β-oxidation enzymes to 12: CoA. Then, 12: CoA generated Z9-12: CoA under Δ9 desaturase (Z9). These acyl-CoA precursors were further reduced by FARs to form corresponding fatty alcohols. Finally, pheromone components were produced after the oxidation by ACTs (According to the sex pheromone components of  by ACTs .S. frugiperda genome. Based on gene expression patterns and phylogenetic analysis, several genes highly expressed in the PGs might play an important role in sex pheromone synthesis. The specific functions of these genes in the process of sex pheromone biosynthesis in S. frugiperda require further study.In conclusion, we identified a total of 99 genes belonging to gene families involved in the biosynthesis of sex pheromones from the