Lactobacillus facilitated the metabolism of heterocyclic flavor chemicals such as 2-acetylpyrrole, 2,3,5-trimethylpyrazine, and 2-acetylfuran. Moreover, we found that the intraspecific genomic diversity and microbial structure were two biotic factors that contributed to dynamic microbiome assembly. Based on the assembly pattern, adjusting the composition and distribution of initial species was one option to regulate the formation of diverse flavor characteristics. Our study provided a rationale for developing a microbiome design to achieve a defined flavor goal.Fermented foods often have attractive flavor characteristics to meet various human demands. An ever-challenging target is the production of fermented foods with equal flavor profiles outside the product’s origin. However, the formation of geography-dependent flavor in high-complexity fermentations remains poorly understood. Here, taking Chinese liquor (baijiu) fermentation as an example, we collected 403 samples from 9 different locations in China across a latitude range of 27°N to 37°N. We revealed and validated the geography-dependent flavor formation patterns by using culture-independent  and culture-dependent tools. We found that the baijiu microbiomes along with their metabolites were flavor related and geography dependent. The geographical characteristics were determined mainly by 20 to 40 differentiated chemical markers in metabolites and the latitude-dependent fungal structure of the microbiome. About 48 to 156 core microbiota members out of 735 bacterial genera and 290 fungal genera contributed to the chemical markers. The contributions of both fungi and bacteria were greater than those from either bacteria or fungi alone. Representatively, we revealed that dynamic interdependent interactions between yeasts and IMPORTANCE People consume many spontaneously fermented foods and beverages with different flavors on a daily basis. One crucial and hotly discussed question is how to reproduce fermented food flavor without geographical limitations to meet diverse human demands. The constantly enriched knowledge of the microbial contribution to fermented flavor offers valuable insights into flavor biotechnological development. However, we still have a poor understanding of what factors limit the reproduction of fermented flavor outside the product’s origin in high-complexity spontaneous fermentations. Here, taking baijiu fermentation as an example, we revealed that geography-dependent flavor was contributed mainly by fungus-bacterium cooperative metabolism. The distinct initial microbial composition, distribution, and intraspecific genomic diversity limited reproducible microbial interactions and metabolism in different geographical areas. The abundant microbial resources and predicted fungus-bacterium interactions found in baijiu fermentation enable us to design a synthetic microbial community to reproduce desired flavor profiles in the future. Fermented foods accompany human civilization and development not only because of the primary purpose for food preservation but also for the many attractive and distinguishing flavors , 2. Ferm7–So far, researchers have clarified the key causes of geography-dependent flavor formation in less complex food fermentations . Geographical microbial variations, phage infection, facility-specific parameters, and local environments  are essential for the generation of flavor-related chemicals , 12–14. 12–Among high-complexity food fermentations, Chinese liquor fermentation is a representative complex process with saccharification and spontaneous fermentation simultaneously , and mor24–To further understand the mechanism of geography-dependent flavor formation, we explored Chinese liquor fermentation to assess (i) the geographical characteristics of the chemical and microbial compositions, (ii) why microbial geographical diversity can contribute to distinct flavor formation; and (iii) how to regulate the microbial generation of flavor-related chemicals. Here, we collected 403 samples of baijiu fermentations from 9 different locations in China across a latitude range of 27°N to 37°N, belonging to 3 aroma types of Chinese liquor . We used gas chromatography-mass spectrometry (GC-MS) to measure the volatile metabolites of fermented samples. In addition, marker metabolites of flavor types at geographical scales were identified by partial least-squares regression discriminant analysis. We used high-throughput amplicon sequencing to profile the bacterial and fungal geographical diversity of fermented samples. Microbial geographical diversity was assessed at the latitude and longitude levels. We then revealed the distinct associations of microbial genera with marker metabolites by molecular ecological networks in three groups. Furthermore, we clarified the molecular mechanisms of partial associations in the JX group. Time series metatranscriptome sequencing was used to illustrate pathways and genes that contribute to the generation of marker metabolites during the fermentation process. What is more, we identified the trigger factors underlying the distinct microbiome assemblies by comparing the initial microbial structure and the initial genomic diversity. Metagenome sequencing and metagenome-assembled genomes (MAGs) were used to assess microbial genomic diversity. Based on trigger factors, we then validated two optional approaches for regulating flavor formation. Our findings provide novel ecological and molecular insights into the formation of geography-dependent flavor in high-complexity spontaneous food fermentations and advance our ability to develop a microbiome design to achieve a defined flavor goal.R = 0.714; P < 0.001).In total, GC-MS detected 471 volatile compounds (not including ethanol) from 403 baijiu fermented samples that may contribute to baijiu flavor. Among 471 volatiles, 56 metabolites were shared by all three groups  in the samples. We found 69 unique volatile chemicals in QX, 206 in NX, and 62 in JX . The main chemicals included esters, alcohols, acids, aldehydes, and multiple aromatic compounds (Data Set S1). Based on the relative concentrations of metabolites at the end of the fermentation, esters were the same most abundant volatile metabolites in QX (89%), NX (92%), and JX (73%). Besides, NX and JX contained more acids than QX, while QX and JX contained more alcohols than NX . Based on the relative concentration of metabolites during the whole fermentation process, a principal-component analysis plot shows clear clusters of samples . We founP < 0.0001]). We found a significant linear correlation between metabolic profiles and geographical distance . Table S2 shows the contribution and confidence of each chemical variable from the established partial least-squares model. We found that the different geographical characteristics of chemical compositions were contributed mainly by 81 flavor-related metabolites . The 81 flavor-related metabolites contained 37 esters, 11 alcohols, 9 acids, 7 aldehydes, and 17 other chemicals. For these 81 chemicals, QX samples presented 54 flavor-related metabolites, including several long-carbon-chain alcohols, such as 3-methyl-1-butanol, 3-methylthiopropanol, 1-nonanol, and 3-octanol. JX samples presented 53 flavor-related metabolites, mainly short-carbon-chain acids, such as propanoic acid, butanoic acid, and 3-methylbutanoic acid. NX samples exhibited high relative concentrations of 62 flavor-related metabolites, primarily esters, such as ethyl caproate, ethyl butyrate, ethyl caprylate, ethyl valerate, ethyl 3-phenylpropionate, propyl caproate, and ethyl heptanoate . We confirmed the significant geographical difference in volatile chemical compositions among the three groups by partial least-squares regression discriminant analysis  in QX, 1,851 bacterial ASVs in NX, and 3,032 bacterial ASVs in JX. The numbers of fungal ASVs were 648, 832, and 1,108 in QX, NX, and JX, respectively. In total, we annotated 735 bacteria and 290 fungi to the genus taxonomic level within the baijiu fermentation ecosystem (Table S3).Lactobacillus, Weissella, Pseudomonas, Bacillus, and Pediococcus were the most abundant bacteria, whereas Saccharomycopsis, Saccharomyces, Candida, Pichia, and Aspergillus were the most abundant fungi. For NX samples, Lactobacillus, Pseudomonas, and Bacillus were the most abundant bacteria, whereas Pichia, Kazachstania, and Aspergillus were the most abundant fungi. For JX samples, Lactobacillus, Kroppenstedtia, Bacillus, and Lentibacillus were the most abundant bacteria, whereas Issatchenkia, Pichia, Byssochlamys, Thermomyces, and Saccharomyces were the most abundant fungi. Interestingly, the dominant genera were similar among the three groups but with different relative abundances, such as Lactobacillus, Saccharomyces, and some non-Saccharomyces yeasts (Data Set S2). Rare (relative abundance of <0.1%) microbial genera comprised over 90% of the unique bacterial or fungal genera. JX included the largest number of unique bacterial and fungal genera in fermented grains, followed by NX and QX . Statistically, the three groups represented significantly different (P < 0.001) microbial structures at the genus taxonomic level (We found that the most abundant (relative abundance of >0.1%) microbial genera were shared among geography-dependent aroma groups, comprising over 90% of the total bacterial or fungal relative abundance . For QX ic level . For funR2 = 0.494; P < 0.001) than with longitude , indicating that fermentation fungal constituents were latitude dependent  correlation with longitude. Besides, the correlation between the bacterial structure and longitude was insignificant, indicating that fermentation bacterial genera were not geographically dependent.Both fungal and bacterial structures were significantly related to latitude rather than longitude . The funP < 0.01 by a Mantel test). We modeled the associations between them individually in the three groups through two-way orthogonal partial least-squares (O2-PLS) modeling. The Q2 values of the models were 0.375 for the QX group, 0.219 for the NX group, and 0.262 for the JX group. In total, we identified 212 bacterial genera and 106 fungal genera as volatile chemical-associated microbial genera in QX, 79 bacterial genera and 69 fungal genera in NX, and 97 bacterial genera and 39 fungal genera in JX (Data Set S3). We then characterized the core microbial genera that contribute to geographically flavor-related chemical markers. After filtering microbial genera based on coverage in the samples (>20%), it was determined that 46 chemical markers were significantly associated with 69 core bacteria and 87 core fungi in QX, 44 chemical markers were significantly associated with 39 core bacteria and 34 core fungi in NX, and 35 chemical markers were significantly associated with 35 core bacteria and 13 core fungi in JX (Data Set S3).We found that both bacteria and fungi contributed to chemical markers. Table S4 shows that bacteria and fungi were significantly correlated with volatile chemicals . Generally, the QX and NX groups showed more redundant associations between aldehydes and core microbial genera than did the JX group. The NX and JX groups showed more redundant associations between acids and core microbial genera than did the QX group .Figure S5 shows detailed significant associations between chemical markers and the core microbial community at the phylum and genus taxonomic levels. The Ascomycota, Basidiomycota, Zygomycota, and Mucoromycota were the fungal phyla that contributed to chemical markers. The Hyphopichia, Pichia, and Lactobacillus were related mainly to esters such as ethyl 3-phenylpropionate, ethyl palmitate, and ethyl octadecenoate. In the NX group, the interactions among Pichia, Zygosaccharomyces, Candida, and Lactobacillus were related primarily to esters and acids such as hexanoic acid, butyl hexanoate, and ethyl caproate. In the JX group, the interactions among Pichia, Zygosaccharomyces, and Lactobacillus were significantly related to diverse chemicals such as 2-acetylpyrrole, tetramethylpyrazine, β-ethylphenethyl alcohol, and benzoic acid . Metabolism-related genes were involved mainly in functional categories of carbon, nitrogen, and sulfur metabolism during the fermentation process. During the early phase of fermentation, fungi like non-Saccharomyces yeasts were active in sulfur metabolism, terpenoid and polyketide metabolism, glyoxylate and dicarboxylate metabolism, lipid metabolism, and the tricarboxylic acid (TCA) cycle. During the medium and later phases, bacteria such as Lactobacillus were active in carbon metabolism, butanoate metabolism, propanoate metabolism, methane metabolism, galactose metabolism, nitrogen metabolism, and amino acid metabolism. We found that 7 core species were involved in the metabolism of 15 chemical markers or precursors of chemical markers in JX fermentation. Specifically, Zygosaccharomyces bailii contributed to the metabolism of 1,2-propanediol, benzoic acid, ethyl lactate, 2,3-butanediol, β-ethylphenethyl alcohol, and precursors of 2-acetylpyrrole, ethyl phenylacetate, ethyl nonanoate, and 2,3,5-trimethylpyrazine. Schizosaccharomyces pombe generated the precursors of 2,3-butanediol, 1-(furan-2-yl)ethenone, 1,2-propanediol, 2,3,5-trimethylpyrazine, ethyl lactate, ethyl phenylacetate, and ethyl nonanoate. Pichia kudriavzevii, Torulaspora delbrueckii, and Saccharomyces cerevisiae generated several organic acids and the precursors of 2,3-butanediol, 1-(furan-2-yl)ethenone, 1,2-propanediol, and β-ethylphenethyl alcohol. Limosilactobacillus panis and Limosilactobacillus reuteri generated the precursors of 1,2-propanediol, benzoic acid, ethyl lactate, 2,3-butanediol, and 2-acetylpyrrole. Based on the correlations between species and the corresponding chemical markers, we found abundant cometabolism interactions between fungi and bacteria across fermentation phases . Representatively, both Zygosaccharomyces bailii and Limosilactobacillus panis contributed to the metabolism of 1,2-propanediol, benzoic acid, ethyl lactate, 2,3-butanediol, and 2-acetylpyrrole. Both Pichia kudriavzevii and Limosilactobacillus reuteri contributed to the metabolism of 2,3,5-trimethylpyrazine, ethyl lactate, and precursors of the Maillard reaction. Some cometabolism patterns, such as the interdependent interactions between Zygosaccharomyces and Lactobacillus, facilitated the synthesis of 1-(furan-2-yl)ethenone, 2-acetylpyrrole, 2,3-butanediol, 2,3,5-trimethylpyrazine, and tetramethylpyrazine  values of metabolism-related genes (Pearson’s correlation Lactobacillus) participated in the metabolism of chemical markers. Although yeasts and Lactobacillus coexisted during the entire fermentation process, the cometabolism process happened mainly in the early and medium phases. Specifically, during the first 10 days of fermentation, Zygosaccharomyces, Torulaspora delbrueckii, and other yeasts converted raw materials into glucose, acetoin, l-2-amino-acetoacetate, glutamate-5-semialdehyde, and other flavor precursors. Yeasts exhibited high FPKM values for SGA1 (glucoamylase), INV (beta-fructofuranosidase), BDH (butanediol dehydrogenase), dfG (3-hydroxy acid dehydrogenase), and OAT (ornithine-oxoacid transaminase). Next, Lactobacillus, along with abiotic reactions, converted these flavor precursors into chemical markers. We found the Lactobacillus exhibited high FPKM values for INV, butA (butanediol dehydrogenase), pepN (aminopeptidase N), and LAP3 (cytosol aminopeptidase) at 10 days of fermentation. After 25 days of fermentation, bacterial expression of INV, butA, pepN, and LAP3 gradually increased, while the metabolic functions of yeasts gradually diminished  differences in the initial fungal and bacterial structures among the three groups . The high initial abundance of Hyphopichia enhanced its possibility of encountering other species in the QX group. As a result, Hyphopichia showed more interactions with bacteria in the QX group than in the NX and JX groups   to show the effect of biotic factors on the assembly patterns . We founSaccharomyces cerevisiae, respectively. Combinations 10 and 11 generated the highest concentrations of acids, which were 5.371 and 4.385 times higher than those with Saccharomyces cerevisiae, respectively. Combinations 6 and 8 generated the highest concentrations of aromatic chemicals, which were 5.415 and 4.488 times higher than those with Saccharomyces cerevisiae, respectively. We found that combination 11 (PK (Pichia kudriavzevii)/SC (Saccharomyces cerevisiae)/SP (Schizosaccharomyces pombe)/ZB (Zygosaccharomyces bailii)/TD (Torulaspora delbrueckii) ratio of 3:3:1:2:4) showed the highest accumulated concentrations of flavor-related chemicals and was regarded as the most efficient yeast combination of the simulated fermentation. Second, Saccharomyces cerevisiae and Fructilactobacillus fructivorans . The concentrations of nonanal, ethyl 2-hydroxypropionate, 1-pentanol, hexanoic acid, isobutyl phthalate, and ethyl butyrate all decreased with increasing strain distance.We created and validated two optional approaches for regulating microbial flavor metabolism based on the characteristics of the geographical baijiu microbiome assembly. First, p-cresol . In JX fermentation, the baijiu microbiome was active in the expression of over 32,373 genes (Data Set S6). Compared with less complex fermentations .For most spontaneous fermentations, fermentation microbiomes are usually artificially isolated or enriched from the local environmental microbial pools , 37–39 t37–The geographical characteristics of the baijiu microbiome could be ascribed to the enrichment of species from various microbial pools . At a laThe changeable strength of microbial interactions could affect the efficiency of chemical marker metabolism . Our resPichia and Monascus is driven by the biosynthesis of 2-phenylethanol. Volatile flavor chemical markers such as 2-phenylethanol will inhibit conidium germination and mycelial growth by filamentous fungi . The mechanisms of many microbial interactions through volatile chemicals in the network  that are related to the initial microbial inoculum for fermentation is the key step in the regulation of fermentation flavor . Here, wChinese liquor has three typical aroma types, named Qingxiang (QX) (light-aroma type), Nongxiang (NX) (strong-aroma type), and Jiangxiang (JX) (sauce aroma type). The spontaneous solid-state fermentation of Chinese liquor is done in sealed pits (a sort of fermentation chamber) and lasts approximately 30 days for QX and JX and 60 days for NX , 18, 36.To assess the geographical characteristics of the chemical and microbial compositions, we collected 403 fermented samples, including 101 samples of QX, 98 samples of NX, and 204 samples of JX, from 9 representative distilleries in China  in 50-mL centrifuge tubes to collect the supernatants after centrifugation at 5,000 × g for 10 min. The supernatant (8 mL) was added to headspace bottles and mixed with 3 g of NaCl and an internal standard (10 μL menthol).Volatile chemicals of samples were identified by headspace solid-phase microextraction–gas chromatography–mass spectrometry (HS-SPME-GC-MS) as described previously . All samg for 5 min. Next, the supernatants were centrifuged at 11,000 × g for 5 min to obtain sediments.To reveal why microbial geographical diversity can contribute to the formation of distinct flavors, we extracted the total DNA and RNA from samples for further sequencing and bioinformatic analysis. The samples were pretreated with sterile phosphate-buffered saline (PBS)  and centrifuged at 300 × For DNA extraction, the sediments were cooled, milled in liquid nitrogen, and extracted using sodium laurate buffer  with phenol-chloroform-isoamyl alcohol (25:24:1) to obtain total DNA. The quality of the total DNA was assessed by 1% agarose gel electrophoresis with a NanoDrop 8000 spectrophotometer  (260-nm/280-nm ratio). All genomic DNA of the samples was stored at −80°C for further procedures.For RNA extraction, the sediments were milled with liquid nitrogen, and total RNA was extracted with sodium laurate buffer  containing TRIzol . A Ribo-Zero rRNA removal kit (bacteria) and a Ribo-Zero magnetic gold kit (yeast)  were used to remove rRNA from the total RNA. The RNA of the samples was then stored at −80°C for further procedures.For DNA sequencing of marker genes, the V3-V4 hypervariable region of the 16S rRNA gene and the internal transcribed spacer 1 (ITS1)/ITS2 region were PCR amplified as previously described , 54. TheFor DNA sequencing of the metagenome, the genomic DNA was randomly broken into fragments with a length of about 350 bp by a sonicator. Next, the whole library was prepared by steps of terminal repair, A-tail addition, ligation of the adaptors to the fragments, purification, and PCR amplification. Illumina  HiSeq sequencing was performed after qualified library pooling.For RNA sequencing, metatranscriptomic libraries were constructed according to the instructions of the NEBNext Ultra RNA library prep kit (Illumina)  and sequenced on the Illumina  HiSeq 2500 platform, which was conducted by the Allwegene Technology Company .Q20 quality standard  for taxonomic identification.For raw DNA sequencing reads, we trimmed low-quality sequences according to the average standard . Next, ostandard . The bacQ20 value. Clean reads were assembled using the MEGAHIT (v1.0.6)   assembly(v1.0.6)  and filt(v1.0.6)  (with se(v1.0.6)  and normP value threshold in multiple tests  of ≤0.05 and a |log2 fold change| of >1 were used as the thresholds for identifying significant differences in gene expression in this study.For RNA sequences, we then performed species classification analysis, species complexity analysis, as well as gene expression abundance analysis. We compared the high-quality reads to the reads in the nonredundant (Nr) protein, metabolic pathway (KEGG), gene ontology (GO), protein family (Pfam), homologous gene cluster (eggNOG), and carbohydrate-active enzyme (CAZy) databases to obtain functional annotation information. The sequencing reads for each sample were remapped to the reference sequences using RSEM software . Gene exle tests . An FDR-To assess major microbial contributors of flavor-related metabolites, molecular ecological network analysis was applied with online tools using default parameters . All intThe beta nearest-taxon index (βNTI) was calculated according to a protocol described previously . We exclFructilactobacillus fructivorans were isolated from pooled fermented samples of JX. To isolate microbial strains, 50 g of fermented samples was homogenized in 250 mL of sterile PBS in a shaking incubator at 200 rpm for 30 min at 30°C. Next, 1.0 mL of the homogenate was diluted 10-fold in a sterile saline solution (0.9% [wt/vol] NaCl). Using methods similar to the ones described in our previous study  in liquid medium containing sorghum extract. Each simulated community had three replications of fermentation. The volatile compounds of fermented samples were analyzed by GC-MS using the same process as the one described above. Next, we screened out the most efficient yeast combinations according to the total concentration of flavor-related chemicals.We created and validated two optional approaches for the regulation of the microbial generation of flavor-related chemicals based on the geographical characteristics of baijiu fermentation. The first simulated fermentation aimed to validate that different initial microbial structures can generate different flavor-related chemical profiles. If we have different demands for flavor-related chemicals, the best microbial combination design could be different even with the same microbial members. We established 21 synthetic microbial communities of one yeast, two yeasts, or five yeasts (Table S5) to compare the metabolic profiles after 3 days of fermentation. The simulated still fermentations were conducted at 30°C with the same initial biomass (2 × 10Saccharomyces cerevisiae and Fructilactobacillus fructivorans were isolated from JX fermentation samples as flavor producers. We inoculated the two strains into a solid fermentation matrix (sorghum extract medium with agar added) with the same biomass (2 × 106 CFU) at the start of fermentation . After 10 days of simulated fermentations, we harvested and smashed the fermented material to conduct further volatile compound analysis.We assumed that different abundances of strains  in solid fermentation material would provide different possible opportunities to interact with other strains. The speed and possibility of fungus-bacterium interactions may impact microbial flavor metabolism. Thus, the second simulated fermentation aimed to validate that different speeds and possibilities of fungus-bacterium interactions could affect fungus-bacterium cometabolism and, thus, flavor formation in solid-state fermentations. We designed a fungus-bacterium fermentation system under four cell spatial distances  to create different microbial interaction possibilities. http://vegan.r-forge.r-project.org/). P values were adjusted for nonparametric analysis using the Statistical Package for the Social Sciences (SPSS) (version 22).The dynamics of physical and chemical factors were fitted with OriginPro2019. Principal-component analysis, two-way orthogonal partial least-squares modeling, and variable importance in projection analysis were done via SIMCA-P (version 13.0). Mantel tests, ANOVA, ANOSIM, microbial correlation analysis, canonical correspondence analysis, and virtual population analysis were carried out using the vegan package in R .PRJNA377357, PRJNA396629, and PRJNA837950. The reads of metatranscription sequencing can be accessed in the PRJNA837634. The reads of metagenome sequencing can be accessed in the PRJNA837610.The reads of marker genes sequencing can be accessed in the


Cancer remains a serious health problem owing to its high morbidity and mortality. Immunotherapy, represented by anti-programmed death-1D1/anti-PD-Ligand 1 treatments, has become one of the most important methods for cancer treatment. However, there are still some challenges in this method such as low response rate, limited therapeutic targets, and unclear underlying molecular mechanisms of immune checkpoints. Thus, in this review, we aim to focus on the proliferative role of fifteen immune checkpoints that occur in various tumors. This has provided more clarity on the functions and mechanisms of immune checkpoints in tumors, especially on their proliferation role. This may provide better insights in developing more therapeutic targets and strategies in tumor immunotherapy. Cancer remains a serious social health problem, and immunotherapy has become the major treatments in tumor treatment. Additionally, improving the efficiency and safety of treatment is necessary. Further, more therapy targets are warranted for future tumor treatments. In this review, in addition to examining the currently recognized role of immune regulation, we focus on the proliferative role of 15 immune checkpoints in various tumors, including PD1, PD-L1, FGL1, CD155, CD47, SIRPα, CD276, IDO1, SIGLEC-15, TIM3, Galectin-9, CD70, CD27, 4-1BBL, and HVEM. We managed to conclude that various immune checkpoints such as PD1/PD-L1, FGL1, CD155, CD47/SIRPα, CD276, and SIGLEC-15 all regulate the cell cycle, and specifically through Cyclin D1 regulation. Furthermore, a variety of signal pathways engage in proliferation regulation, such as P13K, AKT, mTOR, and NK-κB, which are also the most common pathways involved in the regulation of immune checkpoint proliferation. Currently, only PD1/PD-L1, CD47/SIRPα, TIM3/Galectin-9, and CD70/CD27 checkpoints have been shown to interact with each other to regulate tumor proliferation in pairs. However, for other immune checkpoints, the role of their receptors or ligands in tumor proliferation regulation is still unknown, and we consider the enormous potential in this area. An increasing number of studies have validated the various role of immune checkpoints in tumors, and based on this literature review, we found that most of the immune checkpoints play a dual regulatory role in immunity and proliferation. Therefore, the related pathways in proliferation regulation can served the role of therapy targets in tumor therapy. Further, great potential is displayed by IDO1, SIGLEC-15, 4-1BBL, and HVEM in tumor proliferation regulation, which may become novel therapy targets in tumor treatment. According to the report of 2022 cancer statistics , cancer + T cells [PD1 is a type I transmembrane protein expressed on the surface of various immune cells such as B cells, T cells, NK cells, NKT cells, and macrophages . PD-L1 i T cells . Further T cells . The act T cells . Moreove T cells . Certain T cells .+ and CD8+ T cells through the Wnt/β-catenin signaling pathway (related with the proliferation and EMT of cancer stem cells) [+PD-L1+ T lymphoma cells promote tumor growth by activating TCR signaling [Both PD1 and PD-L1 are involved in the proliferative regulation of tumors . In headm cells) , by inhim cells) . In uveam cells) . In thyrm cells) . In NSCLm cells) . Conversm cells) . Thus, Pignaling .A. formosanus extracts  can significantly inhibit the accumulation of PD-L1 and various proliferative genes such as MMP-2, c-Myc, and Cyclin D1. This further proves that the proliferation inhibition of AFE is related to the downregulation of PD-L1 [Many studies have shown that various agents that block PD-1/PD-L1 are associated with proliferation inhibition. A single block of PD1 and PD-L1 can effectively inhibit proliferation and induce apoptosis of esophageal adenocarcinoma cells. This may result from a decreased expression of DNA repair genes . In oralof PD-L1 .PD1 and PD-L1 are ubiquitous intrinsically within various tumors by immunohistochemical staining ,16. Addi+ T cells, CD8+ T cells, and Treg cells [LAG3 and FGL1 form a pair of immune checkpoints, with LAG3 being the most promising immune checkpoint besides PD1/PD-L1 and CTLA-4. LAG3 is a type I transmembrane protein with an extracellular region consisting of four domains—D1, D2, D3, and D4—among which D1 and D2 are responsible for binding the FD domain of FGL1, exerting an inhibitory immune regulation. LAG3 is mainly expressed on the surface of lymphocytes and stored in lysosomes, the surface of NK cells, NKT cells, CD4eg cells . The proeg cells . Furthereg cells ,25,26,27eg cells ,29. The eg cells . Both LAeg cells ,31.There are few reports about the proliferative role of LAG3 in tumors, but many studies have reported that FGL1 regulates the proliferation of various tumors either positively or negatively. FGL1 may be positively associated with the growth of gastrointestinal tumors. In MC38 colon cancer mouse models, the inhibition of FGL1 by anti-FGL1 mAb and the knockout of FGL1 in cancer cells result in a potent antitumor effect, and the proliferation of cancer cells is significantly inhibited . SimilarCD155 is a member of the nectin-like family with a similar structure to the immunoglobulin superfamily , which iCD155 is associated with tumor growth, mainly by regulating the cell cycle and the activation of signaling pathways . In meduCD47 is a glycoprotein consisting of three domains: a short cytoplasmic region, a five-time transmembrane spanning domain, and a single extracellular V-set immunoglobulin . SIRP-α Both CD47 and SIRP-α regulate apoptosis and proliferation in various tumors. In prostate cancer, the overexpression of SIRP-α induces the apoptosis of prostate cancer cells, and its silencing exerts the opposite effect. However, SIRP-α inhibits the activation of the NF-κB pathway and p38 mitogen-activated protein kinase, and further suppresses the expression of cyclooxygenase-2 (COX-2) . In nasoCD276 is a type I transmembrane protein with two extracellular IgC and variable (IgV) domains , mainly CD276 mainly regulates the proliferation of oral cancer, hepatocellular carcinoma, colorectal cancer, hematologic tumor, gynecological oncology, and lung adenocarcinoma. In oral squamous cell carcinoma (OSCC), CD276 is involved in tumor proliferation regulation, mainly via glycometabolism. The knockdown of CD276 inhibits tumor growth, as it was also found that CD276 in Ca9-22 oral cancer cells can better interact with DC-SIGN with terminal α-galactose and higher fucosylation. The structure differences in CD276 in OSCC may induce the proliferation regulation in OSCC ; in Cal2Inherently, IDO1 is a type of rate-limiting enzyme, and works in converting tryptophan to kynurenines. IDO1 is always expressed on the surface of antigen-presenting cells, myeloid-derived suppressor cells (MDSCs), and various tumor cells, including sarcoma, breast cancer, and chronic lymphocytic leukemia . The actIDO1 promotes the progression of various tumors via diverse signaling pathways. In CRC, the IDO1–kynurenine pathway (KP) is directly associated with the proliferation of tumors, as the metabolites from the pathway induce the activation of the PI3K-Akt signal to enhance the nuclear translocation of β-catenin, which is correlated with CRC proliferation and apoptosis resistance . In HCC,SIGLEC-15 is a member of the Siglec gene family. SIGLEC-15 binds to Sialyl-Tn antigen with the help of the sialic acid-binding immunoglobulin-type lectin structure . SIGLEC-SIGLEC-15 may be involved in tumor proliferation regulation, but more evidence is warranted for further validation. In MNNG/HOS and 143B osteosarcoma cells, the overexpression of SIGLEC-15 promotes the proliferation, migration, and invasion of tumor cells. The silencing of SIGLEC-15 exerts the opposite effect, as the expression of dual-specificity phosphatase 1 (DUSP1) is downregulated conformably, and the JNK/MAPK and p38/MAPK signals are both activated simultaneously . SimilarTIM3 is a member of the TIM family and is characterized by five tyrosine residues in the cytoplasmic domain . GalectiBoth TIM3 and Galectin-9 are potent proliferative regulatory factors in various tumors, simultaneously affecting the function of migration and invasion while regulating the content of matrix metalloproteinase (MMP), N-cadherin, and E-cadeherin. In esophageal squamous cell carcinoma (ESCC), it was found that the downregulation of TIM3 inhibits the invasion, migration, and proliferation of tumors. Diverse mechanisms are involved in this regulation, as the expression of TIM3 decreases and the content of MMP-9 and E-cadherin increases, while that of TIMP-1, vimentin, and N-cadherin decreases with the inactivation of p-Akt, p-GSK-3β, and SNAIL pathways . SimilarNF-κB signaling pathway is a critical regulator in TIM3 proliferation regulation. The overexpression of TIM3 in breast cancer induces the upregulation of various factors, including VEGF, TWIST, MMP1, c-Myc, and Cyclin D1, which promote tumor proliferation, tubal formation, invasion, migration, and tight junction deterioration. Further, the NF-κB/STAT3 signaling pathway is activated and E-cadeherin is downregulated during the regulation . In osteGalectin-9 exerts a proliferation regulation role in various gastrointestinal tumors. Galectin-9 plays an important role in the regulation of cell apoptosis. In esophageal adenocarcinoma (EAC), Galectin-9 regulates the proliferation of SK-GT4, OE33, OE19, and OACM5.1c cell lines via inducing cell apoptosis and autophagy, and by affecting the cell cycle. The administration of Galectin-9 elevates the level of cleaved PARP and cleaved caspase-3/9, consequently inducing the apoptosis of EAC cells. Furthermore, the autophagy markers SQSTM1/p62 and LC3-II are both upregulated, and cell-cycle-related molecules such as CDK4, Cyclin D1, and Cyclin E are all decreased during treatment with Galectin-9 . SimilarThe upregulation of expression of caspase-cleaved keratin 18  is one of the characteristics of Galectin-9 in regulating the proliferation of various gastrointestinal tumors. In cholangiocarcinoma, Galectin-9 induces cell apoptosis and inhibits the proliferation of TFK-1 and HuH-28 cholangiocarcinoma cell lines, with an increase in CCK-18 and cytochrome c . In coloCD70/CD27 are mainly involved in lymphoma/leukemia proliferation regulation. Particularly, blocking CD70 promotes the proliferation of leukemic cells during the remission phase . In acutImmune checkpoints have become the major targets in tumor immunotherapy. PD1 and PD-L1 are mature treatment targets, but the response rate is just about 30% in lung cancer immunotherapy . FurtherImmune checkpoints may not only serve as an immune regulators but as proliferation regulators, especially for intrinsic tumor genes ,59, whicFurthermore, PD1/PD-L1 serve as a pair of mature immune checkpoints in tumor immunotherapy. In fact, their proliferative role has been proven to be present in various tumors, Similarly, FGL1 and CD155 can regulate the cell cycle and exert their function by activating the AKT, mTOR, and MYC signal pathways. CD47/SIRPα may affect tumor growth intrinsically. Anti-CD47 therapy was shown to promote the effect of chemotherapy by inhibiting tumor proliferation. The number of studies on CD276 is the largest among the 15 immune checkpoints. On the other hand, the number of studies regarding IDO1 and SIGLEC-15 is the lowest, indicating that the research potential of these two immune checkpoints is significant. NK-κB is the critical signal pathway in TIM3 regulation. Galectin-9 mainly plays a role in growth regulation in gastrointestinal tumors, characteristically by upregulating CCK-18. CD70/CD27 has been found to be involved in hematologic tumors, 4-1BBL in colon cancers, and HVEM in ovarian cancers. However, whether these genes play a role in other tumors is still unknown, warranting further exploration.Interestingly, only PD1/PD-L1, CD47/SIRPα, TIM3/Galectin-9, and CD70/CD27 checkpoint have proved to work in pairs. All other checkpoints work individually. Whether other pairs of checkpoints work in tumor proliferation is unknown and warrants further exploration.Our review elucidates the proliferative role of immune checkpoint genes and their immune regulation, elaborating the diverse mechanisms of immune checkpoints on carcinogenesis in various tumors, providing novel research insight and possible theoretical basis for the occurrence and development of tumors. Most importantly, PD1/PD-L1 serves as a milestone in tumor immunotherapy, and also regulates proliferation in tumors, if there are immune checkpoints that exert a similar function of regulation of tumor proliferation, this particular function may act as a link that provides a strong theoretical basis to develop clinical combination therapy in the future. In this post-PD1 era, more therapy targets may be discovered in tumor therapy with the proliferative regulation by immune checkpoints opening a whole novel epoch for tumor combination therapy.Various immune checkpoints are emerging in this post-PD1 era, with a huge potential to be immunotherapy targets. However, the specific mechanisms of these immune checkpoints are not fully elucidated. Therefore, we have reviewed the literature on the mechanism of seventeen pairs of immune checkpoints and found that fifteen immune checkpoints  exert proliferative and immunologic regulatory effects in tumors. This review mainly focuses on the regulation of proliferation that immune checkpoints mediate, and it is found that immune checkpoints mostly affect cell cycle and apoptosis by affecting some classical signaling pathways, and finally regulate cell proliferation. The most significant finding of this review is that on the one hand, the dual regulation function of the immune checkpoints is able to provide a new direction and insight for research on immune checkpoints, and on the other hand, owing to the role of PD1/PD-L1 that also functions to regulate tumor proliferation, it can provide the theoretical basis for developing clinical combination therapy in the future.


Allergen-specific immunotherapy (AIT) is applied as treatment to rise tolerance in patients with food allergies. Although AIT is thoroughly used, the underlying epigenetic events related to tolerant induction are still unknown. Thus, we aim to investigate epigenetic changes that could be related to tolerance in dendritic cells (DCs) from anaphylactic mice to lipid transfer proteins, Pru p 3, in the context of a sublingual immunotherapy (SLIT) with a glycodendropeptide (D1ManPrup3) that has demonstrated tolerant or desensitization responses depending on the treatment dose.Changes in DNA methylation in CpG context were determined comparing Sensitized (Antigen-only) animals and two groups receiving SLIT with the D1ManPrup3 nanostructure (D1ManPrup3-SLIT): Tolerant (2nM D1ManPrup3) and Desensitized (5nM D1ManPrup3), against anaphylactic animals. DNA from lymph nodes-DCs were isolated and then, Whole Genome Bisulphite Sequencing was performed to analyze methylation.Most differentially methylated regions were found on the area of influence of gene promoters (DMPRs). Compared to the Anaphylactic group, the highest value was found in Desensitized mice , followed by Tolerant  and Sensitized  mice. Moreover, many of these epigenetic changes were found in genes involved in immune and tolerance responses  as shown by functional enrichment .In conclusion, different doses of D1ManPrup3-SLIT induce different DNA methylation changes, which are reflected in the induction of distinct responses, tolerance, or desensitization. Over recent years, food allergy (FA) has experienced a relevant increase worldwide and constitutes a health and socio-economic burden . In most4 and IL10+ Treg cells, while decreasing allergen-specific IgE (sIgE) and Th2/Th9 effector cells (FA to non-specific lipid transfer protein (nsLTP), especially to Pru p 3, the major peach allergen, can lead to severe reactions and cross-reactivity with other nsLTPs from numerous foods and pollens, producing a complex clinical pattern: LTP syndrome . Food avor cells , 9. Furtor cells . Additioor cells , 12.Cd14), Th2 responses, mast cell activation, as well as diverse cytokines and cell recruitment  that could discriminate desensitized animals from long-term tolerant animals . An initmals DCs . Such spEpigenetic regulation, to which DNA methylation belongs, has been linked to FA pathogenesis as another component . LikewisThe novelty and strengths of our work lie in the use of Whole Genome Bisulphite sequencing (WGBS-seq) to study DNA methylation changes that could either differentiate desensitized and tolerant animals or explain the divergence of the underlying mechanisms of a dose-dependent AIT immunomodulatory treatment, aiming to obtain steady prognostic biomarkers to detect long-term tolerance induction after D1ManPrup3-SLIT.BALB/c female mice of four to five week of age  were used. Experimental animal procedures were performed following the international standards of animal welfare. All protocols were approved by the Animal Experimentation Ethics Committee of Andalusian Centre for Nanomedicine and Biotechnology .Animals were divided into four groups: 1) Sensitized, non-anaphylactic (Antigen-only) (n = 7); 2) Anaphylactic, non-treated (Anaphylactic) (n = 7); 3) Anaphylactic, treated with 2 nM D1ManPrup3 (Tolerant) (n = 7); 4) Anaphylactic, treated with 5 nM D1ManPrup3 (Desensitized) (n = 7); as previously described  and accoPreparation of synthetic glycodendropeptide D1ManPrup3 was performed as described previously . After tOnce the sensitization was completed, at 6-week, D1ManPrup3-SLIT was started following the procedure described , 21 and One week after completing SLIT, mice were challenged with one intraperitoneal dose of 100 μg of natural Pru p 3  in a final volume of 100 µl. Anaphylaxis or tolerance were evaluated within 30-40 min after the challenge measuring changes in body temperature with a rectal probe, and assessing physical and behavioral symptoms according to a scoring system . Additioin vivo evaluation, mice were anaesthetized intraperitoneally and bled from the retro-orbital plexus; sera were stored at -20°C for humoral studies. Immediately after bleeding, and while anesthetized, they were sacrificed by cervical dislocation by applying pressure to the neck and dislocating the spinal column from the skull, being performed by trained and authorized personnel. Then, the spleen and lymph nodes  were removed aseptically and homogeneously through a cell strainer 40 μm  to obtain a single-cell suspension.After the challenge and + cell fraction was maintained in RLT Buffer at -80°C until use.DCs from axillary, maxillary, and mesenteric lymph nodes were purified by positive selection using CD11c MicroBeads Ultrapure Mouse kit , following manufacturer’s recommendations. The CD11cDNA was extracted from isolated DCs with DNeasy Blood & Tissue Kit  according to manufacturer’s guidelines. DNA quality control, bisulphite conversion, library construction, and sequencing were performed by Novogene  .Sequenced libraries were processed to obtain the methylation ratio of each cytosine in CpG context. First, in the quality control step, low-quality or adapter containing reads were trimmed with Trimmomatic . Next, WFollowing all these processing steps, differential methylation analysis was conducted with MethylKit R package , using aLater, with those annotated genes, a functional enrichment in gene ontology terms and KEGG pathways was conducted aided by R package clusterProfiler . Additiopost hoc test were used to compare multiples groups. Statistical analyses were performed using GraphPad Prism 8 .Data is expressed as mean ± standard deviation (SD) for parametric data, or median and interquartile range (Q1-Q3) for nonparametric data. Normality was analyzed using the Shapiro-Wilk test. Mann-Whitney test was used to compare paired groups, and Kruskal-Wallis test with Dunn’s p < 0.001)  and Desensitized/5 nM  mice in both parameters evaluated. Moreover, it was observed that Pru p 3-sIgE serum levels in Antigen-only group were similar to Anaphylactic group , in line with our previous results  suffered a significant drop in the body temperature , but not in Group 4 treated with 5 nM D1ManPrup3 (data not shown). These results are in line with our previous findings .All the 26 mice’s DCs DNA samples (5 for Anaphylactic group and 7 per each other group) that passed the quality control criteria, with bisulphite conversion of > 99%, were sequenced to obtain ~ 300,000,000 reads/sample, of which on average more than 95% aligned to the genome, and more than 80% were retained after deduplication. This resulted in a mean coverage of 20 for each chromosome  with an 80% of mean methylation on the CpG context (data not shown).The principal purpose was to examine the epigenomic changes arising from anaphylactic mice after receiving SLIT (Groups 3 and 4). Methylation differences were obtained comparing Antigen-only, Tolerant, and Desensitized mice groups against Anaphylactic group; upmethylated/downmethylated regions exhibit a higher/lower percentage of methylation than the Anaphylactic group, respectively. Besides, the analysis was focused on DMPRs, as they are potentially more prone to influence gene expression.First, the greatest number of DMPRs was found in Desensitized group , followed by Tolerant  and Antigen-only  groups Table 1.Analyzing in detail the affected genes and pathways, via MHC class II  and proinflammatory transcription factors . In However, we have to take into account that at the time-point when the samples were collected, D1ManPrup3-SLIT mice, independently of the dose, tolerated Pru p 3 although this response diverge over the time towards tolerance or desensitization 5 weeks later. This indicates the possibility to find shared mechanisms of immune tolerance as well as some other different that will lead to either tolerance or desensitization at this early time point. To elucidate this phenomenon, a functional enrichment of the genes affected by the DMPRs present in both comparisons was performed. Il12b and Il18r1 are noteworthy because they are related to Th1 response as seen in the GO terms enrichment  linked to histone deacetylation were found. On the other hand, 39 DEGs with at least one DMPR in Desensitized group were observed  and the unique functional enriched terms found in each SLIT comparison. However, focusing on D1ManPrup3-SLIT comparisons, 1,666 common DMPRs were found and could be related to common triggered mechanisms providing tolerance near the end of treatment. The common core of DMPRs found in our study affects genes involved in the regulation of Th1 skewed immune response such as d Ifngr2 –34. More and Tnf –42, MHC-nd H2-Q5 , antigen  in an RNA-seq study by our group . Specifieactions . In thiseactions ; Il12b desponses  and geneesponses . As a reTlr4, Tlr3, Tlr8, Tlr5), and other genes linked to its function including Cd180 and Tirap  and the proinflammatory transcription factor (Cebpb)  . The fir (Cebpb) , 65. FinIl13  which mediates high affinity binding of Il18 to its receptor , which is involved in oral tolerance  with a tolerant response, could be used as biomarkers to predict the future response to treatment, long-lasting tolerance, or desensitization. Therefore, these particular methylation marks affecting DC effector genes could be used as potential biomarkers to distinguish a successful AIT from a temporary desensitization.In conclusion, we found that different D1ManPrup3-SLIT dosage induces diverse methylation changes that could be linked to long-term phenotype differences. Some of these dose-exclusive differentially methylated promoter regions found in the long-term protected mice against a Pru p 3 are related to tolerant Th1 response such as https://www.ncbi.nlm.nih.gov/bioproject/PRJNA905809).The data presented in the study are deposited in the Bioproject repository, accession number PRJNA905809 .Conceptualization: MT, JC and CM. Methodology: RN, MR, CL-M, MM-A, FP, JR-S, JR and JC. Formal analysis: RN, MR and JC. Data curation: RN, MR, FP, JC and CM. Original draft preparation: RN, CL-M, MM-A, JC and CM. Review and editing: MT, JC and CM. Supervision: JR, MT, JC and CM. Funding acquisition: MT, JR and CM. All authors have read and agreed to the published version of the manuscript. All authors contributed to the article and approved the submitted version.


R/qtl software by combining phenotypic data, and the genotyping by sequencing (GBS) method to produce a high-density genetic linkage map. Twenty QTLs were detected during QTL mapping, of which seven QTLs simultaneously detected a consistent 197.10–202.30 Mb segment on chromosome 1. The primary segment was named cQTL1-2, with a phenotypic variation of 5.18–25.96% and a physical distance of 5.2 Mb. This combines the phenotype and genotype with the identification of seven chromosome segment substitution lines (CSSLs), which were derived from Ye478*Qi319 and related to cQTL1-2. The physical distance of cQTL1-2 was reduced to approximately 1.9 Mb. The consistent meta-QTL mQTL1 was located at 619.06 cM on chromosome 1, had a genetic distance of 7.27 cM, and overlapped with cQTL1-2. This was identified by combining the results of previous QTL studies assessing maize tolerance to low temperatures at the germination stage. An assessment of the results of the RIL population, CSSLs, and mQTL1 found the consistent QTL to be LtQTL1-1. It was identified in bin1.06-1.07 at a confidence interval of between 200,400,148 and 201,775,619 bp. In this interval, qRT-PCR found that relative expression of the candidate genes GRMZM2G082630 and GRMZM2G115730 were both up-regulated in low-temperature tolerant lines and down-regulated in sensitive lines (P < 0.01).Low temperatures in the spring often lead to a decline in the emergence rate and uniformity of maize, which can affect yield in northern regions. This study used 365 recombinant inbred lines (RILs), which arose from crossing Qi319 and Ye478, to identify low-temperature resistance during the germination stage by measuring eight low-temperature-related traits. The quantitative trait locis (QTLs) were mapped using  Zea mays L.) is an important crop, representing 40% of global cereal production1  and CRF3, serve vital functions and regulate the growth of the lateral roots of A. thaliana under low-temperature conditions  inbred line with a cold-tolerant line (EP42), both of which were related to tolerance to low temperatures at the germination and seedling stages in small populations  were detected in previous studies, such as a primary QTL on chromosome 6 that is related to cold tolerance. This QTL explains 37.4% of the differences in phenotypes during photo inhibition at low temperatures, and is related to the expression of six other traits . Five meulations . PreviouArabidopsis thaliana, and other plants, including CBFs, MYBs, and MPKs  analyze QTLs related to low-temperature tolerance using R/qtl software and identify the primary QTL linked by multiple traits, (2) verify the consistent primary QTL linked by multiple traits using the contig substitution mapping method combining genotype and phenotype data of chromosome segment substitution lines (CSSLs), (3) analyze the consistent meta-mQTL data from previous studies, and (4) predict and verify candidate genes in the primary QTL confidence interval.This study used the genotypic and phenotypic data from 365 maize RILs, which were F11. The two parent lines had significant differences in eight traits related to cold tolerance, including relative root volume (RRV), relative total length (RTL), relative shoot length (RSL), relative germination rate (RGR), relative root average diameter (RRAD), relative root length (RRL), relative root superficial area (RRSA), and relative simple vigor index (RSVI). Ye478, a dent maize, had an average RGR of 0.845 and an average RSVI of 0.715. In contrast, Qi319, a flint maize, was sensitive to cold, with averages of 0.449 and 0.257 for RGR and RSVI, respectively. Seven CSSLs were selected from the CSSL with donor parent Qi319 and recipient parent Ye478 and were used to verify the QTLs. The details of these seven CSSLs were displayed in Total of 365 lines were obtained from a hybrid of two well-known inbred maize strains, the cold-tolerant line Ye478 and the sensitive line Qi319, via single-seed origin of Fn = 3) taken under low-temperature treatment conditions and normal temperature conditions (The seeds from both lines were sterilized with 1% sodium hypochlorite (NaClO) for 5 min and washed with distilled water. They were then soaked in tap water for 6 h and grown in paper rolls at 10°C chambers (treatment) and 25°C chambers (control) for 30 and 6 days in a dark environment, respectively. The chamber was ARC-36L2-E from PERCIVAL. A completely randomized design with three replicates was used for the germination experiment, with each replicate containing 50 seeds. Eight cold-related traits were measured after the seeds were placed in the chambers. The germination rate (GR) was expressed as the percentage of germinating plants out of the total number of seeds used. The root scanner (Epson Perfection V800 scanner) and analysis software (Regent WinRHIZO from Canada) were used to measure the following seven cold-related traits in germinated seedlings: shoot length (SL), root length (RL), root volume (RV), root superficial area (RSA), root average diameter (RAD), simple vigor index (SVI), and total length (TL). The mean of 10 seedlings were used to measure all these seven traits. For evaluation of seed germination ability at low temperatures, the ratio  of eight traits  were used as indicators for low-temperature tolerance in order to eliminate the differences in genetic background of the different materials. The ratios (relative performance) were calculated as the ratios of the mean values of measurements  for each characteristic. Pearson correlation coefficients (r) between different traits were determined by linear regressions at the significance level p = 0.05, and calculated using SPSS20.0 . The following equation was used to calculate the coefficients of variation : CV = s/x. In this equation, “x” equals each trait’s mean in a population and “s” is equals the standard deviation.The analysis of variance (ANOVA), as well as QTL mapping, was performed using the mean of all replicates. A combined ANOVA spanning several environments with the Mixed Linear Model procedure (PROC MLM) and Statistical Analysis System (SAS) software version 9.2  were performed, which allowed us to approximate the variance. Linear regressions with significance levels of 2 under the accession PRJNA627044), were used with an Illumina 2500 platform and methods previously described to characterize the RIL population (R/qtl package. The threshold of the logarithm of the odds (LOD) scores were determined using 1,000 permutations and a significance level of p = 0.05. These were used to evaluate the effects of the QTL. The QTLs with LOD figures higher than the threshold, which was 2.5, warranted additional study. The ftqtl function from the R/qtl package was used to assess the phenotypic variation of the identified QTLs. The consistent QTLs influencing multiple traits were named with the initial “c,” which represents consistent, and the numbers in the name indicate chromosome and number.The GBS technology . The original QTL maps to the reference map IBM2 2008 Neighbors were compared, which shares enough markers with other maps to make an accurate projection. As such, the IBM2 2008 Neighbors integrated QTLs from other populations. A homothetic function were used to project the QTLs to the reference map by estimating the most likely position and CI. The projected QTLs related to cold tolerance were used to construct a consensus map of cold-related traits with the BioMercator ver.2.1 software . A meta-3, NCBI database (see Text Footnote 2), and UniPort4, the gene annotation function of B73 (B73 RefGen_v3) was searched for this major QTL segment. Well-annotated genes related to low-temperature tolerance and other abiotic stresses were obtained from A. thaliana, Sorghum bicolor, and Oryza sativa. Two genes were selected from our confident QTL interval in order to validate them with quantitative PCR (qPCR). A total of six maize inbred lines of Ye478 (tolerant), Qi319 (sensitive), ZYQ219 (tolerant), ZYQ011 (sensitive), CL082 (tolerant), and CL018 (sensitive) were used as test materials. Their relative germination rate phenotypes were listed in TransZol™ Up Plus RNA Kit [TransGen Biotech ]. They were then subjected to reverse transcription reaction of cDNA via RT MasterMix [TransGen Biotech ], while qPCR analysis was performed on a TransStart® Tip Green qPCR SuperMix kit [TransGen Biotech ]. Ct–ΔΔ analytical method. And the gene expression was translated to log2(fold change).Based on the comprehensive analysis results of RIL population, CSSLs and Meta-QTL analysis, combined with MaizeGDBP < 0.01) in seven traits , except RRAD that showed a significant difference (P < 0.05). All traits were normally and continuously distributed in all 365 RILs, which also displayed quantitative inheritance. For example, RGR ranged from 0.088 to 0.993 and had a mean of 0.680 in the RIL population, and RSVI ranged from 0.036 to 0.765 and had a mean of 0.343. RSVI had the highest CV (0.365) in the RIL population, while RRAD (0.035) had the lowest CV. Within the RIL population, the broad-sense heritability (H2) related to eight characteristics related to the germination stages spanned from 0.824 for RSVI to 0.907 for RRL , RSL , RTL , and RRSA . RRSA displayed significant positive correlations with RSL , RRL , and RTL . Three traits  displayed significant positive correlations with each other (P < 0.01)  .Total of 19 QTLs were associated with eight traits in the control group, while 2, 4, 4, 2, 1, 2, 2, and 2 QTLs were associated with normal germination rate (NGR), normal shoot length (NSL), normal root length (NRL), normal total length (NTL), normal root superficial area (NRSA), normal root average diameter (NRAD), normal root volume (NRV), and normal simple vigor index (NSVI), respectively. These QTLs were detected on chromosomes 1, 2, 3, 4, 5, 7, 8, and 10, with LOD values ranging from 2.65 to 6.34, and the physical lengths from 0.25 to 2.80 Mb .qRRL3-1) to 25.96%  for eight traits  possessed seven QTLs related to germination, with consistent QTLs of RGR, RSL, RRL, RTL, RRSA, RRV, and RSVI. These explained phenotypic variances from 5.18 to 25.96%, suggesting a close genetic relationship between the roots of the germinates and the indicators, possibly due to pleiotropy. cQTL1-1 was found on chromosome 1 at position 63.85 to 68.45 Mb. It accounted for 7.80 and 13.52% of the respective total phenotypic variance for RSV and RGR (cQTL3-1 with qRRL3-1 and qRTL3-1 were detected on chromosome 3 (cQRL7-1) on chromosome 7, from 1.45 to 8.75, included three QTLs of qRRL7-1, qRTL7-1, and qRSVI7-1. These phenotypic variances ranged from 3.59 to 7.41% (Four stable or consistent QTLs were detected for at least two traits. Two consistent QTLs influencing multiple traits were found on chromosome 1. The  and RGR . The cQTmosome 3 . One conto 7.41% .cQTL1-2 (chr1: 197.10–202.30 Mb) of a CSSLs population with 180 families which was constructed with Ye478 as the female parent and Qi319 as the male parent. In these CSSLs, the genotypes of CL174, CL017, CL006, and CL018 introduced Qi319 fragment from markers umc1254 to umc2237. However, the genotypes of CL014, CL009, and CL173 were still from the recurrent parent Ye478 fragment, and the imported Qi319 fragment was located near cQTL1-2 . cQTL1-2, which controlled ability to germinate at low temperatures, was between the umc1254 and umc2237 markers . Combined with cQTL1-2, the major QTL was from markers umc1254 and umc2505 , with a confidence interval of 1,899,852 bp  was detected on chromosome 1, with 11 QTLs. These QTLs were co-located and distributed in clusters. The mQTL1 was located on chromosome 1, from 199,674,463 to 201,775,619 bp in bin 1.06–1.07 with molecular markers of ereb172 and tena2. Combined with the results of cQTL1-2 and mQTL1, the consistent major low-temperature tolerance QTL (LtQTL1-1), which controls ability to germinate in maize, was on bin 1.06–1.07 on chromosome 1 at a range of 200,400,148–201,775,619 bp  within LtQTL1-1 were similar to the genes relating to low-temperature adaptation published by BLAST analysis  (Referring to B73 in MaizeGDB (see Text Footnote 3) RefGen_v3 genome annotation information, there were 66 genes in the analysis . qRT-PCR < 0.01) .Recent studies on plant stress resistance suggested that when plants are subjected to abiotic stress, their root structure changes to improve stress tolerance . Therefo2 offspring were obtained by crossing the varieties SG-7 and SG-5 and genotyping them via GBS   . HoweverqRSL3-2, qRRAD8-1, qRGR9-1, and qRGR10-1. The remaining 16 QTLs overlapped with QTLs known to be related to low-temperature tolerance, and this study narrowed the confidence interval of their positioning. This study assessed the QTLs of RGR, RSL, RRL, RTL, RRSA, RRV, and RSVI: qRGR1-2, qRSL1-1, qRRL1-1, qRTL1-1, qRRSA1-1, qRRV1-1, and qRSVI1-2. The mapped cQTL1-2 segment of chromosome 1 (197.10–202.30 Mb) was consistent with QTLs (58.66 Mb) for shoot length, root length, and total length . LtQTL1-1 is the major QTL linked to multiple low-temperature tolerance traits and had a phenotypic contribution rate from 5.18 to 25.96%. Additionally, the SNP related to the chlorophyll content at low temperatures (PZE-101159230)   was alsoour QTLs . The SNP(qRRL7-1 .GRMZM2G082630 was the protein that codes for superfamily of basic Helix Loop Helix (bHLH) domain. The bHLH proteins were transcriptional regulators, and members of this superfamily with two functionally distinct regions, which were highly conserved: a basic DNA binding region and a helix-loop-helix (HLH) region. The characteristics of superfamily of bHLHs in A. thaliana were play important function of stress responses, light signal transduction, plant growth and development  domain. The domain was a portion of structurally related ENTH, ANTH, or VHS domain in the N-terminal region and a variable C-terminal region, with the functions of transport vesicle  from that of materials with different low-temperature tolerances. Both genes were up-regulated in low-temperature-tolerant varieties and down-regulated in low-temperature-sensitive varieties.This study performed QTL mapping of the 365 RILs which obtained from crossing Qi319 and Ye478. Major QTL was verified by seven CSSLs derived from Ye478*Qi319. And a meta-QTL analysis were performed of the ability of maize to tolerate low temperatures at the germination stage. The QTL https://www.ncbi.nlm.nih.gov/, PRJNA627044.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: YZ, QL, and DW performed the experiments and wrote the manuscript. JM, XL, HD, LZ, LD, XJL, XZ, and ZZ took participate in the experiments. ZW, JW, and YZ designed the experiments and revised the manuscript. All authors read and approved the manuscript.


Bee venom (BV), a type of defensive venom, has been confirmed to have favorable activities, such as anti-tumor, neuroprotective, anti-inflammatory, analgesic, anti-infectivity effects, etc. This study reviewed the recent progress on the pharmacological effects and mechanisms of BV and its main components against cancer, neurological disorders, inflammatory diseases, pain, microbial diseases, liver, kidney, lung and muscle injury, and other diseases in literature during the years 2018–2021. The related target proteins of BV and its main components against the diseases include Akt, mTOR, JNK, Wnt-5α, HIF-1α, NF-κB, JAK2, Nrf2, BDNF, Smad2/3, AMPK, and so on, which are referring to PI3K/Akt/mTOR, MAPK, Wnt/β-catenin, HIF-1α, NF-κB, JAK/STAT, Nrf2/HO-1, TrkB/CREB/BDNF, TGF-β/Smad2/3, and AMPK signaling pathways, etc. Further, with the reported targets, the potential effects and mechanisms on diseases were bioinformatically predicted via Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, disease ontology semantic and enrichment (DOSE) and protein-protein interaction (PPI) analyses. This review provides new insights into the therapeutic effects and mechanisms of BV and its main components on diseases. Apis mellifera) (2 (PLA2) and other components referring to amines, sugars and minerals  . BV is allifera) . BV contminerals .Based on these active components, BV has multiple diverse pharmacological effects. Some reviews have retrieved the pharmacological progress on one or a few aspects of BV, mainly referring to anti-tumor , neuropr1, Re and Tanshinone IIA have been analyzed systematically and deeply through mining their reported targets and pathways from literature, which indeed provide a lot of inspiration and clues for the future study of these ingredients  pathway, disease ontology semantic and enrichment (DOSE) and protein-protein interaction (PPI) analyses, etc., have been widely used in the investigation fields of genomics and proteomics, due to that they can comprehensively discover the biological mysteries of large and complex biological data accounting for physiological and pathological alternations of organism, or changes of organism in response to external stimuli . For theredients . Reasona2 and apamin. The reported action targets and pathways of them against cancer, neurological disorders, inflammatory diseases, pain, microbial diseases, liver, kidney, lung and muscle injury, and other diseases were summarized. Further, the possible anti-ill mechanisms of BV and its main components were comprehensively and systematically studied through DOSE, KEGG pathway, and PPI analyses according to the reported targets. The present study has deeply understood the pharmacological effects and mechanisms of BV and its main components against ills, which will help to promote the development and clinical application for BV.Hence, in this paper, articles published from 2018 to 2021 and archived in Web of Science and PubMed databases were searched mainly using the keywords “bee venom and pharmacology,” supplemented with the keywords “bee venom and cancer” and “melittin and cancer,” and the duplicate articles were excluded. Based on these articles, we reviewed the current progress mainly from year 2018–2021 on the investigation of pharmacological effects and mechanisms of BV and its main components, mainly MEL, bvPLA2 and apamin are three main components in BV, which are the important material basis for BV to exert its pharmacological effects, and their structures are shown in MEL, bvPLAMEL is the dominant component, which is consisting of 26-residue peptide and representing about 40%–60% BV’s dry weight . The car2 is a 128 amino acids single polypeptide chain containing four disulfide bridges. The bvPLA2 pertains to the group III secretory PLA2 (sPLA2) enzymes, accounting for 12%–15% of BV’s dry weight  were suppressed; after vaccinating A549/DDP into Balb/c athymic nude mice and treating them via intraperitoneal (i.p.) injection of MEL, their tumor and cell sensitivity to cisplatin was enhanced and tumor size and mass were controlled  systems have considerably enhanced the security of MEL mination . Such drmination  and lipimination  increaseBreast cancer, as the most commonplace tumor in female population, has a very strong time variant tumor metastasis and spatial heterogeneity associated with genotype and phenotypic differences, leading to a continuous change in the evaluation and treatment process of breast cancer .BV and MEL induced strongly selective cell death in triple-negative breast carcinoma and human epidermal growth factor receptor 2 (HER2) enriched breast cancer with little effect in routine cells, through interfering with growth factor-dependent receptor tyrosine kinase interactions critical for receptor phosphorylation and activation of phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signaling. Besides, in an allograft model, the effect of docetaxel in suppressing breast tumor growth was potentiated by the administration of MEL, and the programmed death ligand-1 (PD-L1) protein expression, phosphorylated HER2 and epidermal growth factor receptor (p-EGFR), were significantly reduced . Researc2O2 production, blocked G1 cycle and inhibited breast cancer proliferation in MCF-7 cells  signaling pathway was inhibited by MEL and the expression of NF-κB, HIF-1α, vascular endothelial growth factor A (VEGFA) and lactate dehydrogenase A (LDHA) decreased, while the expression of Bax and tumor necrosis factor-α (TNF-α) was reversed, ultimately disrupting the tumor microenvironment (TME) of cancer cells and activating the phenomenon of apoptosis (BV reduced the expression of nuclear translocation of nuclear factor-κB (NF-κB) and Cyclin D1, enhanced H-7 cells . MEL red-7 cells . MEL als-7 cells . After 5poptosis .In vitro, BV inhibited the growth of MCF7 and T47D cells, while blocking the cell cycle in the G2/M phase. BV alone or in combination with tamoxifen, hesperidin, and piperine resulted in reduced expression of Bcl-2, EGFR, and estrogen receptors α (ERα) receptors, and elevated expression of Bax, which eventually led to apoptosis. The anticancer and anti-drug resistance effects of tamoxifen were enhanced by the synergistic effect of BV , NF-κB, TNF-α, malondialdehyde (MDA), elevated Caspase-3, superoxide dismutase (SOD), catalase (CAT), and ovarian histopathological changes due to mammary carcinoma were improved in mothers and offspring rats. Additionally, calreticulin and p53 protein response in the ovarian stroma switched from positive to negative  -polyelectrolyte nanocarriers (PENs) , nanograCA-MNPs) , and actCA-MNPs) . BesidesCA-MNPs) .Cervical cancer is one of the most common cancers in the global female population . BV demo3O4)/MEL complexes caused time-dependent toxic effects on HeLa cells with deformation lysis, membrane breakage and other abnormal cellular states. The experimental results demonstrated that this material achieved the long-lasting release and effect enhancement of MEL, while preventing the degradation or denaturation of MEL, ensuring the anti-cervical cancer effect of MEL , overexpression of NONHSAT105177 in long non-coding RNAs is associated with activities such as cell proliferation and migration . This RNMEL exhibited inhibitory effects on human gastric cancer AGS cell viability, adhesion, colony-forming ability, EMT, and a limiting effect on MMP-2, MMP-9 and MMP-13 proteins related to cell migration and invasion ability. In addition, MEL tended to act more in a variety of signaling pathways, containing bone morphogenetic protein (BMP)/Smad, Wnt/β-catenin and PI3K/Akt pathways .2 inhibited HCT116 cell proliferation in a synergistic manner, demonstrating synergistic utility: MEL promoted the effect of bvPLA2 on cell membranes, and pretreatment of cells with bvPLA2 enhanced the inhibitory effect of MEL on cells  increased when BV treated HCT-116 cells and decreased when BV treated SW-480 cells. After MEL treatment, the expression of Bax decreased and MRP-2 increased in HCT-116 cells, while the expression of Bax and MRP-2 decreased in SW-480 cells . Besideson cells . Besideson cells .Meanwhile, the derivation of the side chain of oligopeptide-alginate NPs provided the basis for the specific binding of MEL, ultimately achieving potent killing ability on human cloned colon adenocarcinoma Caco-2 cells .2) with EMT can also be inhibited by applying MEL. The hypoxia model caused upregulation of the expression of HIF-1α, VEGF, MMP-2 and MMP-9 in SMMC-7721, Huh7, and HepG2 cells, and MEL reversed this trend. In addition, in the presence of MEL, it decreased SMMC-7721 cell viability, inhibited EMT induced by CoCl2, upregulated E-cadherin, and downregulated p-Akt, vimentin and N-cadherin expression. An in vivo tumor treatment model of MEL was established by s.c. injecting SMMC-7721 cells into male BALB/c nude mice, which showed the significantly inhibited HIF-1α expression and tumor growth , Caspase-3, and Caspase-9 expression, predicting that MEL may rely on the mitochondrial apoptotic pathway to induce tumor injury, and the ratio of apoptosis to necrosis in cancer cells was positively relative to the MEL concentration. On the other hand, MEL achieved its autophagy-inhibiting effect on HepG2 cells by downregulating p62 and upregulating Beclin 1 and LC3 expression. The anti-tumor effect of MEL was enhanced when the autophagy inhibitor chloroquine was applied; the enhanced autophagic effect of MEL on hepatocellular carcinoma cells was diminished after the application of the autophagy activator rapamycin . The shar growth .Sorafenib had unsatisfactory effects in the treatment of advanced hepatocellular carcinoma, while BV and MEL had certain efficacy in inhibiting hepatocellular carcinoma. Therefore, BV and MEL alone or in combination with sorafenib, respectively, showed synergistic effects in adjuvant inhibition of HepG2 cell proliferation. The expression of p53, Bax, Caspase-3, Caspase-7 and PTEN was elevated, meanwhile the expression of Bcl-2, Cyclin D1, HIF-1α, VEGF, Ras-related C3 botulinum toxin substrate 1 (Rac1), MMP-9 and NF-κB decreased in HepG2 cells. The promotion or suppression effects on the above genes were strengthened under the crosstalk conditions .In an investigation of the Gene Expression Omnibus database of bladder cancer, MEL regulated and inhibited the expression of key module genes in the PI3K-Akt and TNF signaling pathways, referring to LPAR1, COL5A1, COL6A2, CXCL1, CXCL2 and CXCL3 in human bladder cancer cell lines T24 and 5637, and suppressed cell proliferation and migration activities, revealing the potential role of these genes as targets of MEL in bladder cancer . SimilarProstate cancer is divided into metastatic/non metastatic prostate cancer. As one of the familiar type diseases in masculinity, it faces several problems with drug resistance of cancer cells and inability to control the progress and spread of the disease. BV and MEL have certain effects on a variety of prostate cancer and xenotransplantation . For exaApis florea (MEL-AF) similarly showed a proliferation inhibitory effect on A375 cells, where MEL-AF, upon binding to the cell membrane, caused an elevation of intracellular F-actin with a decrease in EGFR, ultimately resulting in apoptosis through the induced expression of Cyt C, Caspase-3 and Caspase-9 in the mitochondrial apoptotic pathway  level. In addition, when MEL was combined with temozolomide, the growth and invasion inhibition of A375SM and SK-MEL-28 cells elevated . MEL fro pathway .MEL alone or in combination with 5-fluorouracil was able to damage A431 cells of skin squamous cell carcinomas, causing morphological alternations, e.g., cell shedding, shrinkage, and plasma membrane damage. Besides, the combination of MEL and 5-fluorouracil caused a more significant decrease in terms of cell number and cell cycle arrest in both phases S and G2/M. More importantly, the drug combination re-sensitized A431 cells to 5-fluorouracil .Head and neck squamous cell carcinoma (HNSCC) is also a type of skin cancer. Four types of HNSCC cells viability such as UMSCC12, UMSCC29, UMSCC38 and UMSCC47 were inhibited by BV alone or combined with cisplatin. Besides, mitosis was blocked in G2/M phase, during which the Bcl-2 and EGFR expression was significantly reduced, while the expression of Bax was significantly elevated. It is worth noting that different drugs and ratios of treatment were shown to significantly reduce the number of the S-phase cells .Melecta albifrons, by virtue of its α-helical structure, inhibited cell proliferation by interfering with the cell membrane of leukemic cells K562, decreasing the viability of various cells such as K562, K562/ADM and HL-60 while enhancing LDH output  were used as an anti-degradation delivery system for MEL on U87 glioma cells cultured in vitro contributing to the growth inhibitory effect. Besides, inoculation of U87 cells in female BALB/c nude mice and intravenous (i.v.) administration of 9G-A7R-Disk/PTX/MEL co-loaded liposomes resulted in increased apoptosis, tissue damage, and reduced angiogenesis at the glioma, demonstrating their targeted anti-tumor effects  and Alzheimer’s disease (AD), belonging to neurodegenerative diseases, are caused by nervous system abnormalities, involving neurotransmitter abnormalities, the accumulation of false proteins, etc. . BV and 2 against PD have also been studied. Purified bvPLA2 showed dose-dependent neuroprotective effects on PD in mice, relating to the induction of CD4+CD25+Foxp3+ regulatory T cells (Tregs), which to some extent suppressed the polarization of T helper 1 (Th1) and Th17, and the microglia activation  expression, and increased dopamine (DA) content and butyrylcholinesterase (BuChE) activity in a rotenone-induced PD mice model . Besidestivation . Fraterntivation .Besides, current evidence was summarized and supported the therapeutic effects of acupuncture in treating PD patients and animal models of PD . Therefo2 also exerted neuroprotective effects against AD. It alleviated memory impairments, reduced Aβ burdens, showed high cerebral glucose uptake, and eliminate central nervous system inflammation through reducing TNF-α and interferon-γ (IFN-γ) level and elevating IL-10 level in a 3xTg-AD mouse model , ionised calcium binding adaptor molecule 1 (IBA-1), inducible nitric oxide synthase (iNOS), COX-2, p-IκB-α, p50 and p65, and modulated Tregs infiltration through the upregulation of Foxp3 in a lipopolysaccharide (LPS)-induced AD mouse model brain. Besides, it reduced amyloidogenesis and neuroinflammation by reducing the level of amyloid precursor protein (APP), β-amyloid precursor protein-cleaving enzyme-1 (BACE1), iNOS, COX-2, IBA-1, p-IκB-α, p50, p65, TNF-α, IL-6, IL-1β, and the activity of β-secretase in LPS-treated microglial BV-2 cells. The in vivo and in vitro results indicated that BV sPLA2 inhibited inflammatory responses and amyloidogenesis via blockage of NF-κB signaling  activity  accumulation in U87MG AD mimic cells, as well as suppressed inflammatory reaction through inhibiting the mRNA expression of IL-1, TNF-α and cyclooxygenase-2 (COX-2), and prevented apoptosis by reducing the expression level of Caspase-3, indicating that BV could be a potential AD therapeutic drug . Besidesse model . BV sPLAignaling . In addiactivity .BV and its main components, such as MEL and apamin, also had neuroprotective effects against other neurological disorders, including epilepticus, blood brain barrier damage and neurobehavioral changes, memory-deficit, laceration injury in cortical neurons, neuroinflammation and multiple sclerosis.BV rebalanced neurotransmitters and blood electrolytes, ameliorated alterations of voltage-gated channels expression, and regulated pro- and anti-inflammatory cytokines levels in a pilocarpine-induced epilepticus rat model, which demonstrated that BV could slow down the development of epilepticus as a combined treatment with other antiepileptic drugs . Besides25–35 through activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme oxygenase-1 (HO-1), and tropomyosin-related kinase receptor B (TrkB)/cAMP response element-binding (CREB)/BDNF signaling pathways. Additionally, MEL restored exhausted learning and memory abilities in an Aβ25–35-induced cognitive deficits mouse model. The above results showed that MEL could be a candidate agent for neurodegenerative disorders  treatment; furthermore, BV and MEL inactivated the complement system, especially via the CD55 induction, which downregulated the membrane attack complex (MAC) and C3 convertase (C3C) levels  levels .2 treatment suppressed atopic dermatitis-related skin swelling, improved ear thickness, reduced IgE and the expression of Th1 and Th2 cytokines , induced Treg, and decreased epidermal and dermal thickness and macrophage and mast cell infiltration in Dermatophagoides farinae extract/DNCB-treated mice. However, the effects were eliminated in CD206−/− mice after D. farinae extract/DNCB treatment. The above data suggested that bvPLA2 could alleviate atopic skin inflammation through interaction with CD206  concentration, its anti-apoptotic property by downregulating Bax and Caspase-3 levels, as well as protection of gastric tissue by upregulating Hsp70 . Besidesin vitro and in vivo. Molecular docking analysis confirmed that MEL targeted PKM2, and knockout of PKM2 produced anti-inflammatory effects similar to MEL in vitro. The above findings highlighted a new strategy of MEL in treating acute liver failure  mediated NF-κB and p38 MAPK signaling . Besides failure .2 and apamin, also exerted anti-inflammatory effects on other diseases, such as allergic chronic rhinosinusitis  . In rece1-and α2-adrenergic receptors , caused by SARS-CoV-2, has infected people in 210 countries and was declared a pandemic by WHO on 12 March 2020 . BV has activity .Human immunodeficiency virus (HIV) has been a major social and health problem for about 30 years. BV was safe and maybe therapeutic in a specified dose, owing to MEL uptake by HIV infected cells which resulted in reduced HIV gene expression and replication . A recenHepatitis C virus (HCV) infection is a worldwide health concern, which can lead to serious liver diseases. BV inhibited the entry stages of HCV infection cycle by interacting with virus particles, through a non-common peptide(s), which demonstrated that the BV could be a potential candidate for characterizing and developing new natural anti-HCV agents .Besides, chitosan/alginate nanoparticle encapsulated BV (CH/AL-BV) was developed, and nasal administration of CH/AL-BV induced non-specific immune stimulating effects, and protected against porcine reproductive and respiratory syndrome virus (PRRSV) infection, suggesting that nasal-delivered CH/AL-BV could produce effective protective immunity against PPRSV infection .In vitro, BV showed strong anti-infectivity effects against two MRSA strains, ATCC 33591 and USA300, and decreased the hla and agrA genes expression and the α-hemolysin production in ATCC 33591. BV alleviated the secretion of TNF-α and IL-6 in MRSA-stimulated 264.7 RAW macrophage. Besides, BV protected A549 cells from MRSA. In vivo, BV increased mouse survival rates, decreased the bacteria number in the lungs and relived symptoms of pneumonia induced by MRSA in mice. The above results indicated that BV could be a candidate for treating pneumonia caused by MRSA infection  ranged from 256 to 1024 μg/ml. In addition, BV inhibited biofilm formation in 14 strains. The above findings indicated that BV was a potential anti-infectivity agent against food-borne pathogens  Acinetobacter spp., caused fluorescence release from XDR isolates which severed as a marker of membrane damage, and had membranolytic effect. In vivo, MEL eradicated the colonized XDR bacteria on infected mouse model with third-degree burn, and no toxicity at the therapeutic dose was observed  P. aeruginosa , glutathione peroxidase (GPx), Nrf2 and GSH in rats induced by high-fat diet (HFD) was reversed by BV, and the levels of adiponectin and TNF-α were restored. The above results suggested that BV is a promising alternative treatment option for non-alcoholic fatty liver disease . BV and ollidine .BV and its main components, such as MEL and apamin, showed protect effects on acute kidney injury and renal fibrosis, as listed in via the suppression of oxidative stress, inflammation and cell death in mice  and myosin heavy chain (MHC), was elevated in mice. The results suggested that MEL could be a promising candidate for the muscle injury treatment  Lee J. .Bee wax coated water-soluble fraction of BV (BWCBVA) and MEL-loaded NPs showed anti-diabetic efficacy in diabetic animal models, as shown in 2 could be a potential agent for atherosclerosis through inducing Treg cells , triglycerides (TG), LDL-C, VLDL-C, creatine kinase (CK)-MB, LDH, troponin I, and heart MDA, NF-κB levels, and heart vascular cell adhesion molecule-1 (VCAM-1) and galectin-3 expression, as well as increased bodyweight, plasma insulin, serum HDL-C and heart total antioxidant capacity (TAC) levels in diabetic hyperlipidemic rats, which indicated that BV could reduce oxidative stress and downregulate NF-κB pathway to improve cardiac dysfunction . Besideseg cells . The aboIn vitro, BV suppressed lipid accumulation, and inhibited adipogenesis by reducing the expression of CCAAT/enhancer-binding proteins (C/EBPs), peroxisome proliferator-activated receptor γ (PPARγ), and p-p38, as well as by upregulating the expression of p-ERK, p-JNK, p-adenosine monophosphate–activated protein kinase (AMPK), and p-acetyl-coenzyme A carboxylase (ACC) during differentiation of 3T3-L1 preadipocytes. In vivo, BV inhibited fat and lipid accumulation and reduced body weight, through downregulating the expression of adipogenic markers, PPARγ and C/EBPα, and upregulating the expression of p-AMPK and p-ACC in HFD-induced obese mice, suggesting BV an effective preventive and therapeutic candidate for obesity , upregulated the expression of p-ERK, p-JNK, p-IKKαβ and p-IκB-α. It enhanced the intracellular poly (I:C) uptake, and the enzymatic activity was essential. These findings indicated that bvPLA2 could ameliorate wound healing via the enhancement of TLR3 responses (Diabetic wound formation is one of the serious complications of diabetes. BV treatment improved wound closure in streptozotocin-induced mice diabetic model through enhancing β-defensin-2 (BD-2) and collagen expression and restoring angiopoietin-1 (Ang-1) and Nrf2 levels, and thereby increasing the Tie-2 downstream signaling, including the phosphorylation of endothelial nitric oxide synthase (eNOS), Akt and ERK. Furthermore, BV restored wounded tissue antioxidant enzymes GSH Px, mitochondrial SOD and CAT activities, and the chemokines CCL2, CCL3 and CXCL2 levels, and then rescued wound macrophages from apoptosis induced by mitochondrial membrane potential. The above results provided a new example for BV treatment to stimulate angiogenesis and improve the wound healing process of diabetes . Besidesesponses . The aboBV could improve reproductive performance and growth performance, as shown in 2 had therapeutic and ameliorative effects on asthma, radial bone defect, dry eye disease and abortion, as well as eliminated teratogenic human induced pluripotent stem cells (iPSCs), as shown in Moreover, BV and bvPLA2+ inward flow and accumulation to the cytoplasm, elevated calpain activity and reactive oxygen species (ROS) generation, selective induction and reduction of viability and adhesion capacity of undifferentiated stem cell iPSCs, and increased cell death. In addition, BV treatment before in ovo transplantation suppressed the formation of iPSC-derived teratoma. Moreover, BV was neither cytotoxic nor genotoxic to iPSC-derived differentiated cells. The above findings indicated that BV possessed anti-teratoma effect by getting rid of residual iPSCs, and could be a safe and effective cell preparation agent for stem cell therapy (BV prevented forkhead box A2 (FOXA2)-regulating mucin 5AC (MUC5AC) expression in IL-13-treated A549 cells not only through inhibiting Akt activation but also by suppressing enhanced SAM-pointed domain containing Ets-like factor (SPDEF), which demonstrated that BV could help prevent mucus metaplasia in asthma . Althoug therapy .BV alongside chitosan scaffold (CS) displayed high radiographic outcomes compared with the untreated and CS groups at day 56 postoperatively, and showed high osseous tissue density and high counts of osteocytes and osteoblasts compared with the CS and autograft groups, which highlighted the capability of CS-BV as a potential alternative and enhancer of bone graft substitute material . The nat2 prevented fetal loss with growth restriction in the remaining live fetuses in abortion mouse model induced by LPS. It increased Treg cells, and decreased TNF-α and IFN-γ expression. The prophylactic effect of bvPLA2 was not found in abortion prone mice lacking Treg. The above results indicated that bvPLA2 might have effects on LPS-induced abortion mice through regulating Treg populations (bvPLAulations .An important concern with BV has been its safety. BV can cause anaphylaxis . It is rVenom immunotherapy (VIT) could prevent severe allergic reactions in 80–100% subjects allergic to Hymenoptera venom. An immunological study on the early phase of VIT performed on forty individuals indicated that cytokines (CCL5/RANTES and IL-17E/IL-25) and eosinophils were conducive to the immune response .2 has attenuated several immune system-related diseases. In a recent study, bvPLA2 decreased apoptotic Treg cells, reduced early apoptosis in splenocytes and CD4+CD25+ Treg cells, and raised the expression of CTLA-4 and PD-1 on CD4+CD25+ Treg cells, which showed that bvPLA2 could induce Treg expansion by changing apoptotic signal (MEL could be used in adjuvant immunotherapy. MEL combined with LPS produced an increase of IL-1β and IL-6 levels and a decrease of IL-10 level in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells. Besides, the response to MEL and LPS was characterized by metabolic profiling, and the clearest effects were on glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and purine, pyrimidine, and fatty acid metabolism . Similarc signal .As experts have commented, if the venom is selected correctly, and adequate venom preparation and maintenance dose are used, VIT may be a treatment method of significant value in preventing the systemic reaction of Hymenoptera stings. Besides, the duration of VIT is usually 5 years, which can provide long-term protection from stinging after treatment interruption, but it recommends indefinite (possibly life-long) treatment for patients with mastocytosis or other high-risk patients .in vitro, resulting in cellular instability , with an IC50 value of 2.35 μM at 48 h , HDFa , and MCF-12A  cells was more delayed and attenuated, with an IC at 24 h .50 values were 95.73 μg/ml in the BV group and 78.38 μg/ml in the MEL group, both of which were significantly higher than the IC50 values of treated HepG2 , which showed negligible effects of BV and MEL on hepatocytes  for 24 h, the ICatocytes .50 values reaching 6.72 and 6.38 μg/ml for the two time periods, respectively; the IC50 values of MEL-treated HaCaT were greater than 10 μg/ml exceeding the maximum MEL concentration used in the experiment  with 0.1–10 μg/ml of BV and MEL for 24 and 72 h, the toxic effects possessed by BV were more pronounced, with ICperiment .BV and Wasp venom were non-toxic to BV-2 murine microglial cells at concentrations of ≤12 μg/ml and ≤160 μg/ml for 24 h, respectively . And 1 μ50 of mice with i.p. injection of MEL was 7.4 mg/kg  (BV at 0.1 and 1 mg/kg was an effective concentration and had no side effects on rats . Similar6129627) .In order to decrease the toxic and side effects such as hemolysis of BV and MEL, and improve the characteristics of drug targets and release, besides of NPs mentioned above , other nBV and its main constituents have multiple biological activities and applied to treat several diseases, such as cancer, neurological disorders, inflammatory diseases, pain, microbial diseases, liver, kidney, lung and muscle injury, etc. In this paper, we reviewed the recently published reports (2018–2021) on BV and its main constituents. These articles have indicated that BV and its main constituents exerted the above protections and were contained in a variety of signaling tranduction pathways –4.in vitro and in vivo. The mechanisms of action of BV were: upregulation of Caspase-3, Caspase-7, Caspase-9, Bax, p21, p27, p53, Rb, PTEN, and 15-lipoxygenase-1 expression, downregulation of MITF, PARP, Bcl-2, Bcl-xL, ERα, EGFR, VEGF, MMP-1, MMP-2, MMP-9, TNF-α, NF-κB, HIF-1α, Cyclin D1, Cyclin A, Cyclin B, and Rac1, etc. BV inhibited the mTOR pathway, PI3K/Akt and MAPK signaling pathway, and mitotic signaling pathway by reducing the expression of p-mTOR, Akt, p-Akt, p38, p-p38, JNK, p-JNK, ERK, p-ERK, p-PI3K, p-EGFR, and p-HER2, which in turn impaired cancer cell viability, reduced migration and invasion activities, and promoted cell death. BV activated the mitochondrial apoptotic pathway in cancer cells by increasing the expression of apoptosis signaling molecules such as Fas and Caspase-9. On the other hand, it inhibited EMT in cancer cells by decreasing the expression of vimentin, ZEB2, and Slug and elevating the expression of E-cadherin  in treating neurological disorders. They inhibited hepatic and renal fibrosis via suppressing TGF-β/Smad2/3 signaling pathway. They also downregulated the expression of C/EBPs and PPARγ, and activated AMPK signaling pathway in the treatment of obesity.Except cancer, BV and its main components can treat a variety of other diseases, including neurological disorders, inflammatory diseases, liver, kidney, lung and muscle injury, etc., mainly via inhibiting the release of inflammatory cytokines and chemokines, regulating the redox balance in the body, modulating the expression of apoptosis related factors, and so on. The most common points were: first, BV and its main components reduced pro-inflammatory cytokines  and raiseed anti-inflammatory cytokines (IL-10 and IL-4); second, they improved antioxidant capacity by upregulating the levels of GSH, GPx, SOD, CAT, glutamate-cysteine ligase (GCL), GST, etc., and downregulating the levels of lipid peroxidation (LPO), MPO, MDA, protein carbonyl (PCO), ROS, etc.; third, they downregulated pro-apoptotic factors , Calpain, Cyt C, etc.) and upregulated anti-apoptotic factors (Bcl-2 and Survivin); fourth, they reduced chemokines, such as CCL17, CCL22, thymic stromal lymphopoietin (TSLP), macrophage inflammatory protein-1α (MIP-1α), MIP-1β, MCP-1, growth related oncogene-α (GRO-α), and so on; finally, they often downregulated MAPK, TLR4/NF-κB, Akt/mTOR and JAK/STAT signaling pathways, and upregulated Nrf2/HO-1 signaling pathway thereby producing corresponding pharmacological effects on different pathophysiological environments. Besides, BV and its main components upregulated TrkB/CREB/BDNF signaling pathway and the expression of growth-associated factors  and growth-associated protein 43 (GAP43)), as well as downregulated the expression of amyloidogenic proteins . As shown in The potential effects of BV and its main components on ills and their potential mechanisms were predicted through bioinformatic analysis, referring to DOSE analysis and KEGG pathway enrichment of the targets reported using bioinformatics tools in the light of our previous methods . As showanalysis  shows th(1) First, although all the reported targets were proved to be correlated with the interfere effects of BV and its main components mainly on cancers, the DOSE analysis strongly suggest that BV and its main components could have an effect on lung disease and obstructive pulmonary disease, which could be worth exploring in the current COVID-19 epidemic. Therefore, next, it is suggested to further validate the effect of BV and its main components on lung disease and obstructive pulmonary disease for seeking hope for the treatment of lung diseases, especially COVID-19.in vitro and in vivo disease models to confirm the unreported pathways in the future study.(2) Second, the present KEGG enrichment analysis suggests that lipid and atherosclerosis, Human cytomegalovirus infection and Human papillomavirus infection pathways could be involved in the disease intervention, but these pathways are not mentioned in the previous reports for BV and its main components. For integratively understanding the intervention and mechanisms of BV and its main components on diseases, proteomics or genomics investigations should be performed using (3) Last, TRAF6 is predicted as one core target and XIAP, TNF, PIK3R1, TP53, SQSTM1, TLR4 and TGFBR1 are predicted as subcentral proteins for the therapeutic effects of BV and its main components. Accordingly, it will be very meaningful to validate the target roles of these proteins for the further drug development of BV and its main components by gene silencing, knockout experiments, etc.Ultimately, based on the previous results and the present bioinformatics and PPI analysis, it gives the following perspectives.In conclusion, these ideas provide valuable clues or perspectives to further study on the pharmacological effects and mechanisms of BV and its main components, so as to the promoting the drug development and clinical applications for BV.