Glioma is the common, most aggressive and poorest prognostic tumor type in the brain. More and more biomarkers associated with glioma treatment, prognosis, and immunity are being discovered. Here, we aimed to explore the underlying biological functions and prognostic predictive value of Apolipoprotein L4 (APOL4) in glioma. We downloaded the expression data of APOL4 and clinical information from several databases and used R software for preprocessing. The clinical significance of APOL4 in a glioma outcome was explored by the Cox regression analysis and Kaplan–Meier survival analysis. In addition, immune infiltrates and microenvironmental indicators were assessed by CIBERSORT and TIMER. GO and KEGG analyses were used to analyze the potential functions of APOL4 in gliomas. APOL4 expression was increased in glioma specimens compared to normal tissues and correlated dramatically with the WHO grade. A survival analysis showed a shorter overall survival (OS) in glioma patients with APOL4 overexpression, and a Cox regression analysis showed that APOL4 was an independent prognostic factor for the OS of glioma patients. GSEA, GO, and KEGG enrichment analyses showed remarkable enrichment in immune-related pathways. APOL4 expression was positively correlated with immune infiltration  and microenvironmental parameters  in gliomas. Glioma patients with a higher expression of APOL4 may be more sensitive to immune checkpoint inhibitors (ICI). In conclusion, these findings suggest that APOL4 is associated with the tumor grade and immune infiltrates; APOL4 may be a new and potential biomarker for therapeutic and prognostic evaluations that may further suggest the therapeutic efficacy of immunotherapy. A glioma is the most common primary tumor in the brain, accounting for over 70% of malignant tumors of the central nervous system (CNS) . Gliomashttps://www.ncbi.nlm.nih.gov/gene/) (accessed on 11 January 2022). The APOL gene family is thought to be innate immune genes that have evolved rapidly in the primate lineage, and their expression is upregulated by pro-inflammatory signals such as interferon, tumor necrosis factor alpha and viral mimics [Apolipoprotein L4 (APOL4), a member of the lipoprotein L family, encodes a protein that may exert an action in reverse cholesterol transport from peripheral cells to the liver, as well as in lipid transport and exchange throughout the body  and cells is crucial for normal tissue homeostasis and tumor growth, and tumor-infiltrating immune cells (TIICs) are essential components of TME . Cancer In this study, we downloaded APOL4-related genetic and clinical information from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Chinese Glioma Genome Atlas (CGGA). After data preprocessing, we performed correlation, survival, and Cox regression analyses to explore the potential functions of APOL4. Our goal is to verify a new diagnostic biomarker for gliomas that will assist in disease stratification and precision treatment.http://www.cgga.org.cn/) (accessed on 21 February 2022) and TCGA (https://cancergenome.nih.gov/) (accessed on 25 February 2022). The TCGA database divides gliomas into LGG (low grade glioma) and GBM. In TCGA, the three cohorts  were named TCGA_ LGG_GBM, TCGA_LGG, and TCGA_GBM, respectively. We applied R software to preprocess the gene expression profiles, including background correction, normalization, and log2 transformation. To evaluate the expression of APOL4 in the tissues of gliomas and other tumors, we employed the TIMRE database (https://cistrome.shinyapps.io/timer/) (accessed on 13 May 2022) to compare the expression of APOL4 in tumor and corresponding normal tissues. Additionally, the GEPIA (http://gepia.cancer-pku.cn/index.html) (accessed on 21 June 2022) was also used to explore the APOL4 expression in LGG, GBM, and normal tissues. The expression of APOL4 in different World Health Organization (WHO) grades (WHO 2021) was also examined, the same as we did [https://www.proteinatlas.org) (accessed on 28 June 2022). Moreover, the location of the APOL4 protein was also explored by the HPA.We obtained the mRNA expression profile datasets containing the clinical information from the public databases CGGA  groups, as we previously studied . The corhttps://cn.string-db.org/) (accessed on 1 July 2022) and GeneMANIA (https://cn.string-db.org/) (accessed on 18 June 2022). GSEA, GO, and KEGG analyses were used to explore the potential function of APOL4 in gliomas.The PPI network of APOL4 was conducted by STRING (p < 0.0001) (To explore the APOL4 expression in 33 human cancers (The full names and abbreviations are in  0.0001) C. Additi 0.0001) E. These p < 0.0001, p = 2.9 × 10−7, p = 1 × 10−15, p = 6.3 × 10−6, To explore whether the overexpression of APOL4 can predict the prognosis of LGG and GBM. Univariate and multivariate Cox regression analyses were carried out, and the results indicated that APOL4 can be served as an independent prognostic factor A,B. The p = 1.73 × 10−2) (p = 4.23 × 10−64), neutrophils , macrophages , CD4+ T cells , CD8+ T cells , and B cells  (p = 2.4 × 10−63) (p = 1.5 × 10−58) (p = 2.8 × 10−47) (p = 1.2 × 10−5) (p = 1.3 × 10−6) (p = 7.9 × 10−11) (p = 6.6 × 10−15) (p = 4.3 × 10−15) (p = 2.6 × 10−4)  A. In LGG× 10−34) A. The AP× 10−63) B, stroma× 10−58) C, and ES× 10−47) D. Anothe × 10−5) E, M2 (Co × 10−6) F, and re× 10−11) I but neg× 10−15) G and mem× 10−15) J in LGG. × 10−4) K. These p = 1.96 × 10−41), CTLA4 (p = 6.51 × 10−20), HAVCR2 (p = 1.23 × 10−58), LAG3 (p = 6.66 × 10−15), PDCD1 (p = 2.06 × 10−42), PDCD1LG2 (p = 2.86 × 10−62), and SIGLEC15 (p = 1.68 × 10−12), were highly expressed in an APOL4-high group of gliomas (p < 0.05)  D.p < 0.05)  . These fA glioma is a common primary malignant brain tumor in adults, and GBM is the most aggressive and incurable type, with a high recurrence rate and a high mortality rate . The advAPOL4 is a member of the lipoprotein L family, which encodes a protein that may play a role in the reverse transport of cholesterol from peripheral cells to the liver and in the transport and exchange of lipids throughout the body. The APOL gene family is thought to be innate immune genes that evolved rapidly in the primate lineage, and their expression is upregulated by proinflammatory signals ,6. It haIn the current study, we found that APOL4 mRNA expression was elevated in gliomas and closely correlated with the tumor grade, with the highest expression in GBM and higher WHO grade 3 than WHO grade 2. APLO4 is highly expressed in GBM and may serve as a predictor. This suggests that APOL4 is a marker of poor prognostic outcomes. The analysis of the clinical survival data also confirmed that patients with gliomas with high APOL4 expression had significantly shorter survival times than those with low expression. Moreover, the Cox regression analysis confirmed that APOL4 was an independent prognostic biomarker for gliomas. These findings indicated that APOL4 may be engaged in the malignant biological process of gliomas, and its expression may be severed to predict the outcome of gliomas. However, it is not clear exactly how APOL4 mediates the development of gliomas.By analyzing the association of APOL4 with TIICs in the tissues of LGG and GBM, we found that APOL4 was positively related to the immune, stromal, and ESTIMATE scores. In GBM, APOL4 was positively associated with the macrophages and B-cell infiltration. In LGG, APOL4 was positively correlated with several immune cells, especially DCs, neutrophils, macrophages, CD4+ T cells, and B cells. These findings illustrate that APOL4 may modulate the immune microenvironment of gliomas. However, the exact mechanism has not been clarified. Additionally, we found that the expression of several immune checkpoints was higher in the APOL4-high group than that in the APOL4-low group of gliomas. Moreover, we revealed a positive connection between APOL4 and CTLA4, PDCD1, PDCCD1LG2, HAVCR2, and CD274. The expression of these immune checkpoints indicated the poor prognosis of glioma patients. Tumor cells tend to evade cytotoxic T-lymphocyte destruction by upregulating immune checkpoints ligands, such as PD-L1, which can lead to the suppression of lymphocyte activation . APOL4 mThe biological process GO analysis demonstrated that APOL4 was involved in numerous immune-related processes, including IFN-γ production, response to type I interferon, adaptive immune response, T-cell activation, neutrophil mediated immunity, granulocyte activation, humoral immune response, leukocyte proliferation, response to virus, lymphocyte mediated immunity, cytokine metabolic process, type I interferon production, leukocyte cell–cell adhesion, cellular defense response, positive regulation of immune effector process, regulation of innate immune response, leukocyte migration, positive regulation of cytokine production, and the regulation of leukocyte activation. The KEGG pathway analysis illustrated that APOL4 was associated with antigen processing and presentation, DNA replication, cytokine–cytokine receptor interaction in glioma, revealing the potential functions of APOL4 in gliomas. The GSEA analysis indicated that several immune-related pathways were positively correlated to a high APOL4 expression, including apoptosis, IL2-STAT5 signaling, IL6-JAK-STAT3 signaling, allograft rejection and complement, epithelial–mesenchymal transition, interferon gamma response, TNFα signaling via NF-κB, hypoxia, inflammatory response, angiogenesis, and glycolysis. As previously reported, the IL6/JAK/STAT3 pathway was involved in the B7-H4(B7x)-mediated cross-talk between glioma-initiating cells and macrophages . AdditioIn conclusion, we found that APOL4 is aberrantly expressed in LGG and GBM than normal tissues, and high APOL4 expression predicts a poor outcome of glioma patients. This work also revealed that APOL4 may be a novel biomarker for the prognosis and ICI treatment of gliomas.


Triticum aestivum L.) is a staple upland crop species that is highly sensitive to low Fe stresses. Although some studies have been previously conducted on the responses of wheat plants to Fe deficiency, the key mechanisms underlying adaptive responses are still unclear in wheat due to its large and complex genome.Plants worldwide are often stressed by low Fe availability around the world, especially in aerobic soils. Therefore, the plant growth, seed yield, and quality of crop species are severely inhibited under Fe deficiency. Fe metabolism in plants is controlled by a series of complex transport, storage, and regulatory mechanisms in cells. Allohexaploid wheat  were identified in the shoots and roots between high-Fe and low-Fe conditions, respectively. These DEGs were mainly involved in the Fe chelator biosynthesis, ion transport, photosynthesis, amino acid metabolism, and protein synthesis. Gene coexpression network diagrams indicated that These results might help us fully understand the morpho-physiological and molecular responses of wheat plants to low-Fe stress, and provide elite genetic resources for the genetic modification of efficient Fe use.The online version contains supplementary material available at 10.1186/s12870-022-03627-4. Althougpressure , 2. Therpressure . Therefo+-ATPase) is expressed in large quantities, pumping a large number of protons (H+) to acidify the rhizosphere environment, thereby improving the rhizosphere Fe availability. Second, the expression of Fe reductase (FRO) genes is upregulated, and then the Fe3+ adsorbed on the root epidermis is reduced to Fe2+. The expression of iron-regulated transporter (IRT) genes involved in the transport of Fe2+ into root cells is significantly induced by low Fe stress [3+ with high affinity, then it is secreted into the rhizosphere and combines with Fe3+ to form a Fe3+-PS complex. Under the action of the YSL (YS1-like protein) family transporters, the Fe3+-PS complex near the root epidermal cells is absorbed into monocotyledonous grasses [Faced with different Fe nutritional statuses in the environment, higher plants have evolved two different strategies to adapt themselves to external Fe changes: a reduction strategy (Strategy I) and a chelation strategy (Strategy II) . Monocote stress . First,  grasses . The eluTriticum aestivum L.) is one of the most important upland crop species in the world [Wheat  concentrations were significantly increased in both shoots and roots. Fig. A, D. MDA2O2 in boththe shoots and roots were significantly higher under low Fe than under the control condition  in wheat plants, we performed DAB and NBT staining on the shoots and roots of wheat plants after different Fe treatments , cell component (CC), and molecular function (MF). GO entries with a ess Fig. E, while ess Fig. E; in theess Fig. E. KEGG aess Fig. F.p value < 0.001 were considered to be significantly enriched. We found that most DEGs were related to plant metabolism and biosynthesis processes, such as the tricarboxylic acid biosynthetic process, nicotianamine synthase activity, the nicotianamine metabolic process, the nicotianamine biosynthetic process, nicotianamine aminotransferase activity, and the L − methionine biosynthetic process. These results indicated that Fe chelator biosynthesis and Fe transport were implicated in maintaining Fe homeostasis. Significantly enriched GO terms related to ion transport and ion homeostasis, which included Fe transport and intracellular Fe sequestration In addition, the GO terms related to photosynthesis, such as photosynthesis and light harvesting in photosystem I, were also highly accumulated. These results emphasized the importance of membrane and/or membrane-localized metal ion transporters and regulatory and metabolic proteins under Fe deficiency.To further deepen our understanding of the functionality of these DEGs, we conducted a more detailed GO enrichment analysis Fig. G. GO terThe KEGG database was used to further determine the pathways involved in the responses of wheat to low-Fe stress Fig. H. Previo3+ chelate reductase (FRO), iron-regulated transporter (IRT), natural resistance-associated macrophage protein (NRAMP), and yellow stripe-like (YSL), involved in efficient Fe uptake belonging toStrategy I, were significantly upregulated under Fe deficiency. Among them, TaFRO2-2A , and TaDMAS1-4A   is a polypeptide transporter located on the plasma membrane of plant epidermal cells. Transcriptomic analysis showed that the TaOPT homologues were significantly induced by low Fe, and the expression of TaOPT3-5B increased by 44 fold under low Fe , nicotinamide synthase (NAS), nicotinamide aminotransferase (NAAT), and deoxygenate synthase (DMAS). Differential gene expression showed that these four key enzyme genes were significantly induced under low Fe stress. Among them,  Figure SD, TaNAS1 Figure SI, TaNAAT Figure SG, and Ta Figure SG, and Ta Figure SG, and Ta Figure SG, and TaVIT)-mediated vacuolar loading of Fe into seed embryos not only promotes seed development but is also crucial for seed germination. NRAMP, located on the vacuole membrane, mediates the transport of Fe from the vacuole of the seed embryo to the cytoplasm and participates in the supply of Fe during seed germination. The expression levels of most VIT genes were significantly downregulated under low Fe stress, particularly TaVIT2-5B, which was downregulated threefold  is mainly involved in the process of exporting Fe-S to mitochondria. TaATM-5A had the highest expression abundance and might play an important role in maintaining mitochondrial Fe balance  was the first protein identified to participate in chloroplast Fe transport, and its expression was not found to be regulated by Fe in other crop species. PIC was significantly induced by low Fe in the shoots of wheat plants, and TaPIC-1A had the highest expression abundance , located in the chloroplast, can regulate intracellular Fe content by participating in the transport of NA or the Fe-NA complex into the chloroplast. FPN was significantly induced by low Fe in the roots, and TaFPN1-7A was significantly upregulated by 60-fold  located on the inner chloroplast membrane encodes a non-plasma membrane-localized nucleotide-binding domain subunit of the ABC transporter. NAP was significantly induced by low Fe in the shoots, and TaNAP-3A was significantly upregulated , chlorophyll b reductase (CBR), chlorophyll synthase (CS), and chlorophyllase play an important role in the conversion between chlorophyll a and chlorophyll b. Among the DEGs involved in photosynthesis, we found that the genes encoding these enzymes showed an increased expression pattern under low Fe stress , β-carotene hydroxylase (β-OHase), zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), LUT5, and violaxanthin de-epoxidase (VDE), was significantly reduced under low Fe stress , particularly NHX2, which is involved in the vacuolar Na+ compartment, and SOS1/NHX7, which regulates cellular Na+ exclusion, were significantly upregulated  regulates Na+ influx, and its expression level was significantly inhibited by low Fe stress, particularly TaBASS1D-1D , specifically expressed in adventitious roots and lateral root primordia, is a transcription factor that regulates root elongation [To study whether the inhibitory effect of Fe deficiency on root growth was related to the degree of cell division and differentiation, we utilized transcriptome data to analyse the expression of cell cycle-related genes Figure SA. Cyclinongation . Under lRBOH) encoding NADPH oxidase play key roles in the production of ROS [SOD and CAT, as well as genes in the shoots and roots, was increased under low Fe stress, which might be necessary for ROS scavenging under low Fe stress  protein is a vacuolar Fe transporter that transports Fe to the vacuole under conditions of excess Fe [AtVTL1 gene was positively correlated with the increase in Fe concentrations in storage roots. This result shows that the VIT gene is involved in mediating vacuolar Fe retention [Fe homeostasis in plants is jointly modulated by a variety of Fe ligands, transporters, and regulatory factors. Usually, Fe is combined with ligands for transportation or storage to reduce the risk of toxic ROS produced by free Fe . NA and  Figure S, Fig. S4r vit1-1 , AtVTL1,xcess Fe . The pasetention . The aboOsVIT2, OsFER2, OsFER2 and other genes in rice were significantly upregulated under low Fe stress, thereby allowing the plants to respond to low Fe stress, which is consistent with the results of this study. Fe deficiency can significantly inhibit maize growth, resulting in decreased chlorophyll and active Fe content [Abiotic stress can negatively affect plant growth and development, resulting in yield loss. It is estimated that more than 90% of the world's agricultural land is affected by abiotic stressors. However, different crops have different responses to low Fe stress, such as Arabidopsis , wheat , 50 and  content , 51. In Fe nutrients play important roles not only in plant physiological functions but also in maintaining various cell processes. In the past few decades, increasing progress has been made in understanding how plants adapt to low Fe availability in soils. However, observing the dynamics of Fe nutrient status in plants is still a significant challenge. The genes responsible for Fe absorption, translocation from roots to shoots, storage in cells and even their regulation at the transcriptional and posttranscriptional levels are being characterized in crop species. However, further studies are still needed to reveal the further connection and integration between Fe-deficient signalling pathways and developmental and physiological networks. Our study analysed the morphological and physiological changes in the shoots and roots under low Fe stress and utilized the transcriptome to analyze the expression profiles of genes related to Fe uptake and transport, photosynthesis, and other ion transporter-related genes. Finally, key genes affecting the plant responses to low Fe stress were identified. In summary, these results might provide excellent genetic resources for the regulatory mechanisms underlying adaptability to low Fe stress and high Fe use efficiency in wheat.Triticum aestivum L.) cultivar, Zhengmai 1860 that is bred by Henan Academy of Agricultural Sciences , was used in this study. The seeds of Zhengmai 1860 on wet gauze were germinated for 7 days, and then the seedlings with uniform sizes were selected for hydroponic culture for 10 days in black plastic boxes containing 10 L of Hoagland nutrient solution (pH 5.8). Half of the seedlings were grown in a nutrient solution containing sufficient Fe, and half were grown in a nutrient solution containing low Fe stress. The regimes of hydroponic solutions were as follows: 1.0 mM KH2PO4, 5.0 mM KNO3, 5.0 mM Ca(NO3)2·4H2O, 2.0 mM MgSO4·7H2O, 9.0 μM MnCl2·4H2O, 0.80 μM ZnSO4·7H2O, 0.30 μM CuSO4·5H2O, 0.10 μM Na2MoO4·2H2O, and 46 μM H3BO3 with different Fe(III)-EDTA concentrations . The growth condition was as follows: light intensity of 200 μmol m−2 s−1, room temperature of 25 °C daytime/22 °C night, light period of 16 h photoperiod/8 h dark, and relative humidity of 70%. The nutrient solution was refreshed every 5 days, and its pH was constantly adjusted to 5.8 with NaOH.The bread wheat  in 5 mL 0.4 M mannitol at 25 °C for 4 h, and then we used the conductivity meter  to measure the solution conductivity as the initial leakage rate. Then, the samples in a water bath were put at 85 °C for 20 min, the solution leakage rate was measured and recorded as the total leakage rate. The ion leakage rate was calculated as the ratio of the initial leakage rate to the total leakage rate .http://www.cominbio.com) was used to determine the content of proline, superoxide anion (OFR), hydrogen peroxide (H2O2), and malondialdehyde (MDA) in the shoots and roots of wheat plants under different Fe treatments. Five biological replicates were prepared for each treatment.A root scanner  was used to measure total root length, maximal root length, root surface area, root volume, root tip number, lateral root length, and average root diameter of wheat plants. Root activity of wheat plants under different treatments was determined by the tetrazolium (TTC) reduction method. The Comin biological kit (2O. The morphology of root hairs was observed with a stereoscopic microscope (Leica M125C).After exposed to 10-d low Fe stress, the roots of wheat plants were taken and placed in a glass container filled with ddHParaffin sections were used to investigate cell sizes in wheat root tips. First, fresh wheat root tips were added into FAA fixative . It was then embedded after dehydration, transparency, and wax dipping. The slice thickness was approximately 8–10 μm, and a typical section was selected for microscopy (Leica M125C) .The shoots of wheat plants under different treatments were fixed in fresh 4% glutaraldehyde fixative for 12 h. Then, the samples were rinsed with 0.1 mol/L phosphate-buffered solution (PBS) of pH 6.8, and then the rinsed samples were put into 1% osmium acid solution for fixation at 4℃ for 1 h, and then were dehydrated with 100% ethanol. The resultant samples were subjected to infiltration, resin embedding and polymerization, and be stored in a desiccator. The samples were cut into ultrathin sections of 50–70 nm, double-stained with uranyl acetate-lead citrate, observed, and photographed by a transmission electron microscope  , 56.2O2 and O2¯ [2O2 in the presence of peroxidase to form a dark brown polymer product, thereby detecting the H2O2 content in plant tissues [2¯ to produce dark blue insoluble formazan, which can be used to detect the O2¯ content in plant tissues [A common consequence of abiotic stress in plants is ROS production, and the accumulation of ROS can be estimated from the histochemical staining intensity of total H and O2¯ . 3′3’-Di tissues . Nitro b tissues .3/HClO4 mixture  at 200℃ until completed digestion. The diluted supernatant was determined to quantify the concentrations of mineral elements using an inductively coupled plasma mass spectrometry . Each sample contained five independent biological replicates for the ionomic analyses under low Fe stress.Uniform wheat seedlings after 7-d seed germination were hydroponically grown under sufficient Fe and low Fe conditions for 10 days until sampling. Over-dried shoot and root tissues were subsequently transferred to an HNOP values < 0.05 were used to identify the DEGs [http://www.pantherdb.org/data/) [http://www.kegg.jp/) [http://www.tm4.org/mev.html) [To understand the molecular basis of Fe tolerance in wheat seedlings under low Fe stress, we constructed RNA-seq libraries of the wheat shoots and roots , 61. Undthe DEGs . PANTHERg/data/)  and KEGGegg.jp/)  were useev.html) .Reverse transcription quantitative PCR (RT-qPCR) assays were performed to validate the accuracy of transcriptome sequencing data. After removing genomic DNA from RNA samples with RNase-free DNase I, total RNA was used as RT templates for cDNA synthesis using the PrimeScript™ RT Reagent Kit Eraser . The RT-qPCR assays were performed to detect relative gene expression using SYBR®Premix Ex Taq™ II (TliRNaseH Plus)  using a Bio-Rad C1000 Touch Thermal Cycler of CFX96™ Real-time PCR detection system.−ΔΔCT method [The RT-qPCR program was as follows: 95ºC for 3 min, 40 cycles of 95ºC for 10 s, and 60ºC for 30 s. The melting curve was plotted as follows to analyse the primer gene-specificity: 95ºC for 15 s, 60ºC for 1 min, and 60-95ºC for 15 s (+ 0.3ºC/cycle). The expression data of target genes were normalized, and the relative expression levels were calculated according to the 2T method . Each saPearson correlation value was set according to the default settings , and then gene coexpression networks were constructed by CYTOSCAPE 3.2.1  (http://www.cytoscape.org/) [Gene coexpression networks were constructed to identify gene interactions and locate core genes that connect most neighboring genes involved in the responses of wheat plants to low Fe stress. For each pair of genes, the threshold of the pe.org/) .P < 0.05; **, P < 0.01; ***, P < 0.001) were determined by unpaired two-tailed Student’s t-test using Statistical Productions and Service Solutions 17.0 .Significant differences . Genomic distribution of DEGs in the shoots (A)and roots (B). Figure S3. Molecularmodel of Fe absorption and transport related genes. A molecular model of genesinvolved in Fe absorption and transport in plant roots (A), chloroplasts,mitochondria, and vacuoles (B). FigureS4. Differential expression profiles of genes related to Fe absorption andtransport in wheat plants under low Fe stress. (A) Fe3+ chelatereductase (FRO), (B) naturalresistance-associated macrophage protein (NRAPM),(C) Yellow stripe-like (YSL), (D) methionine synthetase (SAM), (E) iron-regulated transporter (IRT), (F) Deoxyergate synthase (DMAS), (G) Nicotinamide aminotransferase(NAAT), (H) Mitochondrial m-typethioredoxin in chloroplast (ATM), (I)Nicotinamide synthase (NAS), (J)transporter of mugnetic acid (TOM),(K) multidrug and toxin efflux family (MATE),(L) Permease in chioroplasts (PIC),(M) Oligopeptide transporter (OPT),(N) mitoferrin, (O) mitoferrin-like, (P) Iron efflux transporter ferroportin (FPN), (Q) Nonintrinsic ABC protein (NAP), (R) vacuolar iron transporterginseng (VIT). For transcriptomesequencing, selected uniform wheat plants after germination, half of which weretransplanted into a nutrient solution with normal Fe concentration forcultivation, and half were transplanted into a nutrient solution with low Fefor cultivation, and samples were taken 10 days later. The heat map shows thegene expression level indicated by the TPM value. Differentially expressedgenes that show higher expression levels under the control and treatment (low Fe: 2 μM) are indicated by asterisks. Figure S5. Differential expression profile ofphotosynthesis-related genes in wheat plants under low Fe stress. Differentialexpression profile of genes related to photosynthesis-antenna proteins (A) andcarotenoid biosynthesis (B). The expression pattern of genes involved in thetransformation of chlorophyll a and chlorophyll b (C) and the synthesis ofcarotenoids under low Fe stress (D). The expression pattern of chlorophyll a/bbinding proteins (E), Photosynthesis II reaction proteins (F), RuBisCo subunitbinding proteins (G), Mg2+ chelatase (H), Ribulose bisphosphatecarboxylase/oxygenase activase (I). For transcriptome sequencing, selecteduniform wheat plants after germination, half of which were transplanted into anutrient solution with normal Fe concentration for cultivation, and half weretransplanted into a nutrient solution with low Fe for cultivation, and sampleswere taken 10 days later. The heat map shows the gene expression levelindicated by the TPM value. Differentially expressed genes that show higherexpression levels under he control  and treatment (low Fe: 2μM) are indicated by asterisks. FigureS6. Heat map of expression of genes involved incell cycle and ROS metabolism. (A) Graph of expression abundance of cellcycle-related genes. Differential expression profile of ROS synthesis (B) andclearance related genes (C). For transcriptome sequencing, selected uniformwheat plants after germination, half of which were transplanted into a nutrientsolution with normal Fe concentration for cultivation, and half weretransplanted into a nutrient solution with low Fe for cultivation, and sampleswere taken 10 days later. The heat map shows the gene expression levelindicated by the TPM value. Differentially expressed genes that show higher expressionlevels under he control  and treatment (low Fe: 2 μM) areindicated by asterisks. Figure S7. Co-expressionnetwork analysis of Fe absorption and transport related genes under low Festress conditions. Cycle nodes represent genes, and the size of the nodesrepresents the power of the interrelation among the nodes by degree value.Edges between two nodes represent interactions between genes. Figure S8. Natural variation in wheatresistance to low Fe stress and morphological identification of resistant andsensitive genotypes. (A) Variation in low Fe stress in a panel comprising 386wheat accessions. The root length and chlorophyll value of wheat were used forpopulation screening. After germination, uniform seedlings were selected andgrown for 10 d in Hoagland nutrient solution. Then Half of the seedlings werecultivated in a nutrient solution containing sufficient Fe, and half werecultivated in a nutrient solution containing low Fe stress. Min, minimum; max,maximum; CV, coefficient of variation. (B) Growth performance of ‘zhengmai1860’and ‘zhoumai32’ under low Fe stress. For the low Fe treatment, the wheat plantswere grown hydroponically under high Fe (50 μM) and low Fe (2 μM) conditionsfor 10 d, respectively. Bar: 5cm. (C) Relative expression of TaIRT1b-4A,TaNAS2-6D, TaNAS1a-6A, TaNAS1a-6B, and TaNAAT1b-1D in the shoots and roots ofthe two genotypes grown under normal Fe and low Fe conditions. For the low Fetreatment, the wheat plants were grown hydroponically under high Fe (50 μM) andlow Fe (2 μM) conditions for 10 d, respectively. Data are means (±SD), n=3. Thesignificant difference was determined using Student’s t-test: **P<0.01. Table S2. RT-qPCR primersequences.


Recombinant adeno-associated viruses (rAAV) are commonly used vectors for gene delivery in both basic neuroscience and clinical applications due to their nonpathogenic, minimally immunogenic, and sustained expression properties. However, several challenges remain for the wide-scale rAAV applications, including poor infection of many clinically important cell lines, insufficient expression at low titers, and diffusive transduction in vivo.In this work, PEG, which is a safe and non-toxic polymer of ethylene oxide monomer, was applied as an auxiliary transduction agent to improve the expression of rAAV. In detail, a small dose of PEG was added into the rAAV solution for the transgene expression in cell lines in vitro, and in the central nervous system (CNS) in vivo. The biocompatibility of PEG enhancer was assessed by characterizing the immune responses, cell morphology, cell tropism of rAAV, neuronal apoptosis, as well as motor function of animals.The results show that small dose of PEG additive can effectively improve the gene expression characteristics of rAAV both in vitro and in vivo. Specifically, the PEG additive allows efficient transgene expression in cell lines that are difficult to be transfected with rAAV alone. In vivo studies show that the PEG additive can promote a spatially confined and efficient transgene expression of low-titer rAAV in the brain over long terms. In addition, no obvious side effects of PEG were observed on CNS in the biocompatibility studies.This spatially confined and efficient transduction method can facilitate the applications of rAAV in fundamental research, especially in the precise dissection of neural circuits, and also improve the capabilities of rAAV in the treatment of neurological diseases which originate from the disorders of small nuclei in the brain.The online version contains supplementary material available at 10.1186/s40824-022-00322-1. Adeno-associated virus (AAV) is a nonpathogenic and replication-deficient parvovirus with a ca. 4.7-kilobase single-stranded DNA genome , 2. CombPrevious efforts to improve the transduction efficacy of rAAV vectors have focused primarily on refining the vector system through capsid screening and genome modification , 24, whiPolyethylene glycol (PEG) is a safe and non-toxic polymer of ethylene oxide monomers and has been approved for human use by U.S. food and drug administration (FDA) –30. PEG HeLa, HEK293T, K562, PC12, and C8-D1A cells were purchased from the Cell Resource Center of Chinese Academy of Medical Sciences. Male C57BL/6 mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. The mice were individually housed in a temperature and humidity controlled environment under a 12 h:12 h light:dark cycle, with ad libitum access to food and water. All animal procedures were approved by the Animal Care and Use Committee of the National Center for Nanoscience and Technology, China. The mice were 7–8 weeks old and weighed 20–30 g at the time of viral injection. The rAAV vectors, including rAAV9-CMV::GFP, rAAV9-hSyn::EGFP, and rAAV9-GFAP::EGFP were purchased from Hanbio Co., Ltd.The phosphate-buffered saline was purchased from HyClone Laboratories (AE29246295) and the polyethylene glycol with molecular weight of 4000 was purchased from Sigma-Aldrich Co., LLC. (95904-250G-F). Dulbecco’s modified eagle medium, fetal bovine serum, penicillin, streptomycin and heat-inactivated horse serum were all purchased from Gibco Company. 4% paraformaldehyde was purchased from Biosharp Company (BL539A) and fluoromount G was purchased from Southern Biotechnology Associates, Inc. (A3420-MM70). Mouse primary antibody for staining NeuN and rabbit primary antibody for staining GFAP were purchased from Sigma-Aldrich Co., LLC. . The goat anti-mouse IgG (H + L) secondary antibody 633 and the goat anti-rabbit IgG (H + L) secondary antibody 594 were purchased from ThermoFisher (A-21050 and A-11012). All reagents were of analytical grade and used without further purification. Cell Counting Kit-8  was purchased from Dojindo, Japan.Stereotaxic apparatus frame was purchased from RWD Life Science Co. Ltd. The rAAV solutions were injected through a 10-μL microinjector  assembled with a G34 syringe needle (Hamilton) under the control of a PUMP 11 ELITE Nanomite (Harvard apparatus). Brian slices were sectioned from a freezing microtome (Leica Biosystem). The fluorescence images were acquired from a confocal microscope . The movements of mice were recorded and analyzed with SuperMaze video tracking software purchased from Shanghai XinRun Information Technology Co., Ltd.2. In our study, although different growth conditions were used for different cell types, the serotype and MOI of rAAV for each cell type were used under the same conditions, thus providing an internal control for comparative analysis.HEK293T, HeLa, and C8-D1A cell lines were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin. K562 cell was cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin. PC12 was cultured in RPMI-1640 supplemented with 5% fetal bovine serum, 10% heat-inactivated horse serum and 1% Penicillin/Streptomycin. The cell cultures were grown in a humidified incubator at 37 °C with 5% CO5 v·g/cell, and with PEG concentration of 3.6% (wt/vol). In the meanwhile, the cells of the control group were infected with rAAV9-CMV::GFP solutions alone at the MOI of 5 × 105 v·g/cell. After 3-day infection, GFP expression was analyzed with confocal microscope using a 488-nm laser as the excitation source. To estimate the percentages of the infected cells, 20× magnification images were taken across multiple regions for HEK293T, HeLa, K562, and PC12 cells in random. The number of GFP-positive cells was counted with ImageJ software and three parallel tests were taken for each concentration of PEG. In each test, three random regions with over 500 cells for PC12, and over 1000 cells for HEK293T, HeLa, and K562 were counted for each well.In cell transduction experiments, rAAV9-CMV::GFP vectors were used for HEK293T, HeLa, K562, and PC12 cells, and rAAV9-GFAP::EGFP was used for C8-D1A cell. About 10,000 cells per well were plated in 1 mL of media in a 24-well plate . Immediately after plating, the cells were infected with rAAV9-CMV::GFP/PEG solutions at the MOI of 5 × 105 v·g/cell, while the concentrations of the PEG were 0.2, 0.8, 1.6, and 3.6% (wt/vol), respectively. GFP expression was analyzed after 3-day infection as described above. For C8-D1A cell, the MOI of rAAV9-GFAP::EGFP was kept at 2 × 106 v·g/cell, and the concentration of PEG additives were 1.6, 3.6, 5.4, and 7.2% (wt/vol), respectively.To investigate transduction efficiency of rAAV9-CMV::GFP on the dependence of PEG additive concentration, we infected HeLa, HEK293T, K562, and PC12 cells by rAAV9-CMV::GFP solutions accompanied with different amounts of PEG. The MOIs of the rAAV9-CMV::GFP in all culture wells were kept at 5 × 10. 1 mm2 were drilled into the skull above the left and right M2 cortexes  and the dura was removed carefully. rAAV and rAAV/PEG solutions were injected into the targeted brain region with an injection rate of 0.1 μL·min− 1, respectively. After injection, the syringe was left in place for 10 min before retracting the needle to avoid solution backflow. Then the holes on the skull were filled with silicon elastomer Kwik-Sil before sewing up the scalp. After surgery, the mice were allowed to recover on a 37 °C heating pad for 1 hour before returning to their home cages and an intraperitoneal injection of antibiotic drug was given to the mice for 3 days after the surgery.Mice were anesthetized with intraperitoneally administered pentobarbital sodium and fixed in a standard rodent stereotaxic apparatus frame with two ear bars. Chlortetracycline hydrochloride eye ointment was applied to both eyes to prevent eye drying. A heating pad was placed under the mouse to maintain its body temperature at 36–37 °C, and the anesthesia depth was monitored by testing the paw pinch reflex before the start of surgery. Two square holes of caAt 3 or 8 weeks after rAAV injection and gene expression, the mice were anesthetized with an intraperitoneal injection of pentobarbital sodium, and transcardially perfused with 1× PBS and 4% paraformaldehyde (wt/vol), successively. After that, the mice were decapitated, and the brains were carefully removed. The brains were incubated in 4% paraformaldehyde overnight for post fixation and sectioned into 30-μm-thick slices using a freezing microtome. After that, the slices were mounted on glass slides, and fluoromount G was used to protect the fluorescent proteins from quenching. Last, confocal microscope images were acquired using a 488-nm laser as the excitation source.For NeuN and GFAP staining, the brain slices were prepared following the same procedures described above. Then the slices were incubated in 0.3% Triton X-100 (wt/vol) at 25 °C for 15 min to increase the permeability of the membrane, and blocked with 3% bull serum albumin (wt/vol) for 1 h at room temperature to occupy the nonspecific binding sites. After that, the slices were transferred into primary antibodies (1:200 dilution for NeuN and 1:1000 dilution for GFAP) overnight at 4 °C. After being rinsed three times with 1× PBS, the slices were incubated in fluorophore-conjugated antibodies (1:1000 dilution) for 2 hours at room temperature. Then the slices were rinsed 3 times with 1× PBS and mounted on glass slides. As described above, fluoromount G was used to protect the fluorophores from quenching. Finally, the fluorescence images were acquired on a confocal microscope, and analyzed by ZEN and ImageJ software.2) and allowed to move freely for 15 minutes to adapt the experiment environment. After the acclimation, the movements of mice were recorded for 15 minutes at day and night individually with SuperMaze video tracking software. When replacing a mouse, spraying 75% alcohol to remove the smell inside the behavioral chamber was necessary. Finally, the average locomotion speeds and resting time were analyzed with SuperMaze video tracking software and used to assess the motor function of the mice.Eight wild-type mice were randomly divided into two groups. The control group of mice received an injection of untreated rAAV9-hSyn::EGFP solution, but the other group received an injection of 40% PEG-treated rAAV9-hSyn::EGFP solution in the M2 cortex . At 3 weeks after injection and gene expression, the mice were placed in a standard open field behavioral chamber (50 × 50 cmWe chose rAAV serotype 9 (rAAV9) to evaluate the effects of PEG additive on transgene expression because it has been widely used for gene delivery in the mammalian brain , especia5 v·g /cell. Meanwhile, untreated rAAV9-CMV::GFP solutions were diluted to the same titer and added into cell cultures as a control. After a 3-day infection, the transduction efficiency was analyzed by measuring GFP fluorescence. As shown in Fig. 5 v·g/cell, the percentages of GFP-positive cells (defined as transduction efficiency) in HeLa and HEK293T cultures were 20 and 55%, respectively, similar to previous reports [To evaluate the effects of PEG additive, rAAV9-CMV::GFP/PEG transduction solutions were added into cell cultures at an multiplicity of infection (MOI) of 5 × 10 reports . On the 5 v·g/cell with various amounts of PEG additive. As shown in Fig. We then examined the PEG concentration dependence of the transduction efficiency of rAAV virus. Cells were cultured with rAAV9-CMV::GFP solutions at an MOI of 5 × 10Finally, we investigated the mechanism of PEG-enhanced transduction efficiency of rAAV vectors. We performed transmission electron microscope (TEM) to determine whether PEG and rAAV formed complexes in mixed transduction solutions. As shown in Supplementary Fig. 11 v·g/mL rAAV9 solutions with 40% (wt/vol) PEG were injected to the right hemispheres of the mouse brains, whereas the control rAAV9 solutions without PEG were injected into the contralateral hemispheres   under the control of neuron-specific human synapsin-1 promoter (rAAV9-hSyn::EGFP), were injected into the secondary motor cortex M2) of mice. 300 nL of 1.4 × 10 of mice. μL Fig. –7, EGFP 11 v·g/mL, the transduction areas are 0.61 ± 0.20 mm2 and 1.18 ± 0.31 mm2 for 300-nL and 1000-nL injection, respectively, in the absence of PEG. With 40% PEG additive, the transduction areas are reduced to 0.17 ± 0.09 mm2 and 0.27 ± 0.01 mm2 for 300-nL and 1000-nL injection, respectively. To determine the transduction efficiency, we measured the ratio of EGFP+NeuN+ cells to NeuN+ cells within the expression areas of EGFP  % in the absence of PEG, compared to (86.6 ± 3.8) % in the presence of 40% PEG. With 1000-nL intraparenchymal administration, the transduction efficiency of rAAV was (15.2 ± 2.1) % in the absence of PEG and (93.9 ± 2.3) % in the presence of 40% PEG. These results confirm that the PEG additive can result in highly efficient and localized transduction of rAAV vectors in the brain.We labeled neuronal cells in the brain with the neuron-specific marker NeuN to quantitatively evaluate the effects of PEG on rAAV transduction Fig. a-b. It w11 v·g/mL, respectively. As shown in Fig. Next, we examined time dependent characteristics of PEG-assisted rAAV transduction in the brain. EGFP fluorescence was characterized after 3- or 8-week expressions of untreated and 40% PEG-treated rAAV9-hSyn::EGFP at a titer of 4.2 × 1011 v·g/mL were injected into the M2 of mice with or without 40% PEG. After 8 weeks of expression, the brains were sectioned, and astrocytes were labeled by staining glial fibrillary acid protein (GFAP) to analyze the immune responses. As shown in Fig. + cells and astrocytes, as indicated by the white arrows. These results show that no EGFP expression was leaked into astrocytes under the hSyn promoter control, confirming the good tropism of rAAV on cell types.In previous studies, a series of compounds have been used as rAAV transduction enhancer, including topoisomerase and proteasome inhibitors, epipodophyllotoxins, inducers of DNA damage, effectors of epigenetic modification, and anthracyclines , 40, 41.11 v·g/mL. In the experiment group, 300-nL rAAV9-hSyn::EGFP treated with 40% PEG was injected at the same titer. After a 3-week expression, the motor functions of the two groups of mice were assessed by performing 15-minute open field tests. In order to eliminate the impact of rhythm, the movements of mice were recorded by automatic video-tracking software during the day and night, respectively. As shown in Fig. P values of the average locomotion speeds between the two groups were 0.635 and 0.685 at day and night, respectively. The P values of resting time between the two groups were 0.750 and 0.519 at day and night, respectively. These results demonstrated that PEG did not result in alterations of motor function in mice. In conclusion, the PEG additive at a dose that can significantly improve rAAV transduction shows good biocompatibility in the CNS.To examine the activities of PEG on neuronal apoptosis, the nuclei of all cells, apoptotic neurons, and mature neurons were labeled by staining DAPI, TUNEL and NeuN, respectively Fig. a. The imThe transduction efficiency of rAAV is largely depended on the cellular internalization and the following downstream events after the internalization of rAAV , 43. Spe+ or Mg2+ salts [After internalization, two main steps still need to go through before the transgene expression occurs in the host cells, including the release of virion core into the cytoplasm, and the transcription of the viral single-stranded RNA to double-stranded DNA. It was reported that the PEG can induce the condensation and packaging of DNA in the presence of Na2+ salts , 46, and2+ salts . Taken tFinally, the enhanced transduction efficiency of rAAV could benefit partially from physical processes. For the in vivo transduction, the addictive of PEG could increase the viscosity of rAAV solutions, thus decreasing the diffusion behavior of rAAV in tissue.In this work, we developed an easy and effective method by using PEG as auxiliary agent to improve the rAAV transduction characteristics both in vitro and in vivo. Our results showed that the in vitro expression of rAAV was significantly enhanced by adding a small dose of PEG. Especially, an efficient transgene expression of rAAV in was achieved in the presence of PEG for cell lines that are difficult to be transfected with rAAV alone. For in vivo transduction experiments, rAAV9-hSyn::EGFP solutions with or without PEG were injected into mouse brains at low titers. After both 3 and 8 weeks of expression, the PEG additive promoted a spatially confined and efficient transduction in the mouse brains. In addition, the PEG additive did not cause any detectable side effects in the brain, including aggravated immune responses, abnormal cell morphology, alteration of rAAV tropism, cytotoxicity, and animal behavior alteration. To summary, we demonstrated a simple strategy of using PEG as transduction auxiliary to improve the expression of rAAV in vitro and in vivo, especially in the CNS. We also demonstrated that the improvement of rAAV transduction in vivo could persist for at least 2 months without measurable side effects. Our strategy broadens the applications of rAAV in both scientific research and clinical therapy, especially when it is necessary to transfer genes to small nuclei of brain with high precision and efficiency.Additional file 1: Supplementary Fig. 1. PEG-enhanced infection of rAAV-CMV::GFP vectors in HEK293T, K562 and PC12 cells. (a) Transgene expression of rAAV9-CMV::GFP in HEK293T (left), K562 (middle) and PC12 (right) cells with different concentrations of PEG excipient. The MOI was kept at 5 × 105 v·g/cell, and the concentrations of PEG excipient were 0.2, 0.8, 1.6, and 3.6% (wt/vol) from top to bottom, respectively. Columns from left to right: green fluorescence, bright field, and overlay images. Scale bars, 100 μm. (b) Dependence of the transduction efficiency of rAAV9-CMV::GFP in HEK293T (left), K562 (middle) and PC12 (right) cells on the concentration of PEG excipient. The MOI was kept at 5 × 105 v·g/cell. n = 3 wells in each group. Data are represented as mean ± SD. Supplementary Fig. 2. Molecular weight dependence of infection ability of PEG/rAAV vectors in vitro. (a) GFP expression in HeLa cell line transduced with untreated rAAV9-CMV::GFP (top) and rAAV9-CMV::GFP with PEG2000, PEG4000, and PEG10000 (bottom), respectively, at the MOI of 5 × 105 v·g/cell. Scale bars, 200 μm. (b) Dependence of the transduction efficiency of rAAV9-CMV::GFP in HeLa cells on the concentration of different PEG additives. n = 3 wells in each group. Data are represented as mean ± SD. (c) CCK-8 assay of HeLa cell line that incubated with culture medium with different concentration of PEG2000, PEG4000, and PEG10000 additives, respectively. Supplementary Fig. 3. PEG-enhanced infection of rAAV-GFAP::EGFP vectors in C8-D1A cell line in vitro. (a) EGFP expression in C8-D1A cell line transduced with untreated rAAV9-GFAP::EGFP and rAAV9-GFAP::EGFP treated with 1.6, 3.6 and 5.4% (wt/vol) PEG additive, respectively, at the MOI of 2 × 106 v·g/cell. Scale bar, 200 μm. (b) Abnormal cell morphology occurred in C8-D1A when the concentration of PEG ratio was 7.2%. Scale bar, 200 μm. (c) Dependence of the transduction efficiency of rAAV9-GFAP::EGFP in C8-D1A cell on the concentration of PEG additive. The MOI was kept at 2 × 106 v·g/cell. n = 3 wells in each group. Data are represented as mean ± SD. Supplementary Fig. 4. TEM characterizations of rAAV9-CMV::GFP virus. Negative stain of rAAV9-CMV::GFP virus from rAAV9-CMV::GFP (left) and rAAV9-CMV::GFP/PEG solutions (right), respectively. TEM samples of rAAV vectors were prepared by loading the copper grids  with 10 μL pure rAAV solution or rAAV/PEG solution at a titer of 1011 v·g/mL, and then stained with 5% uranyl acetate. The concentrations of PEG4000 in the rAAV/PEG solution were 20%. Scale bar, 200 nm. Supplementary Fig. 5. 3-week expression of 300-nL rAAV9-hSyn::EGFP in mice at a titer of 1.4 × 1011 v·g/mL. The concentrations of PEG from left to right are 0, 10, 20, 30, and 40%, respectively. Scale bar, 100 μm. Supplementary Fig. 6. 3-week expression of 300-nL rAAV9-hSyn::EGFP in mice at a titer of 4.2 × 1011 v·g/mL. The concentrations of PEG from left to right are 0, 20, and 40%, respectively. Scale bar, 200 μm. Supplementary Fig. 7. 3-week expression of 1000-nL rAAV9-hSyn::EGFP in mice at a titer of 4.2 × 1011 v·g/mL. The concentrations of PEG in the left and right are 0 and 40%, respectively. Scale bar, 200 μm. Supplementary Fig. 8. Transduction comparison of rAAV9-hSyn::EGFP at 3 weeks (a) and 8 weeks (b) after intraparenchymal injection. For precise comparison, the same tube of rAAV or rAAV/PEG solutions were injected to all the mouse in 3-week group and 8-week group on the same day. The injection volume is 300 nL and the titer of rAAV vectors is 4.2 × 1011 v·g/mL. The concentrations of PEG in the left and right are 0 and 40%, respectively. Scale bars, 200 μm.


Lactococcus lactis strain AH1, isolated from viili, a Finnish dairy product. This strain is known for the extreme viscosity it imparts to fermented milk due to its production of exopolysaccharides. The complete sequence was obtained by combining Illumina and Nanopore data, revealing a chromosome with a length of 2,421,519 bp and eight plasmids ranging from 5,773 to 55,958 bp.Here, we report the complete genome sequence of  Lactococci  supplemented with 1% lactose. After 24 h at 30°C, single colonies were purified, and one was stocked as L. lactis AH1. For extraction of genomic DNA from L. lactis AH1 using the Easy-DNA kit , a single colony was inoculated in M17 broth with 1% glucose and grown overnight at 30°C. Genomic libraries were prepared using the Nextera XT DNA sample preparation kit  and then sequenced using v3, 2 × 300-bp chemistry on the Illumina MiSeq platform. For Nanopore sequencing, the genomic DNA was purified using the PureLink genomic DNA kit (Thermo Fisher Scientific); DNA libraries were prepared using the rapid sequencing kit  and sequenced on a MinION instrument using a FLO-MIN106 flow cell. In total, 2,999,192 paired-end Illumina reads were generated and subsequently trimmed based on quality (Q20) using Trimmomatic v0.38. MinKNOW v19.12.2 (Oxford Nanopore) was used to base call 199,948 Nanopore reads with a value greater than Q10 . Read quality checks were performed using FastQC v0.11.7 and NanoStat v1.1.2. Unicycler v0.4.6 was used to carry out a hybrid genome assembly from both the Illumina and Nanopore reads. A complete circular chromosome  was assembled and rotated using Unicycler, with the starting gene being dnaA. Moreover, eight complete circular plasmids ranging from 5,773 to 55,958 bp were found (To isolate an exopolysaccharide-producing re found .Lactococcus lactis strain, as 100% identity to several Lactococcus lactis strains was found. Moreover, the annotation suggested that the genes relevant for exopolysaccharide biosynthesis were plasmid-encoded on pAH1-3 and associated with several insertion sequences. Default software parameters were used throughout unless otherwise stated.The chromosome and plasmid sequences were submitted to NCBI and subjected to the associated annotation pipeline, PGAP. A BLASTN search, using the 16S rRNA sequence as the query, confirmed that the strain indeed was a Lactococcus lactis strain AH1 has been deposited at GenBank under the accession numbers posted in PRJNA814528, whereas the raw sequence data are available under the SRA accession number SRR18888551.The genome sequence of


Ipomoea pes-caprae. In this research, 12 NAC TFs, named IpNAC1–IpNAC12, were selected from transcriptome data. The homologous evolution tree divided IpNACs into four major categories, and six IpNACs were linearly associated with Arabidopsis ANAC genes. From the gene structures, protein domains, and promoter upstream regulatory elements, IpNACs were shown to contain complete NAC-specific subdomains (A–E) and cis-acting elements corresponding to different stress stimuli. We measured the expression levels of the 12 IpNACs under abiotic stress  and hormone treatment , and their transcription levels differed. IpNAC5/8/10/12 were located in the nucleus through subcellular localization, and the overexpressing transgenic Arabidopsis plants showed high tolerance to salt stress. The cellular Na+ homeostasis content in the mature and elongation zones of the four IpNAC transgenic sweetpotato roots showed an obvious efflux phenomenon. These conclusions demonstrate that IpNAC5/8/10/12 actively respond to abiotic stress, have significant roles in improving plant salt tolerance, and are important salt tolerance candidate genes in I. pes-caprae and sweetpotato. This study laid the foundation for further studies on the function of IpNACs in response to abiotic stress. It provides options for improving the stress resistance of sweetpotato using gene introgression from I. pes-caprae.Adverse environmental stress is a major environmental factor threatening food security, which is why improving plant stress resistance is essential for agricultural productivity and environmental sustainability. The NAC  transcription factors (TFs) play a dominant role in plant responses to abiotic and biotic stresses, but they have been poorly studied in  Ipomoea pes-caprae (Linn.) R. Br. (Convolvulaceae 2n = 2x = 30) mainly grows on tropical and subtropical beaches , and it has anti-inflammatory , and maintain the homeostasis of the Na+ ion. Therefore, GmNAC06 overexpression in hairy roots led to significant salt tolerance in the whole composite plant. MfNACs combine with the MtGlyl promoter to improve plant drought tolerance by regulating glutathione back to the reduction state.NAC  proteins comprise a unique TF family in plants . It has thaliana , 151 in a sativa , and 152 tabacum . As one  tabacum , plant d tabacum , and hor tabacum . In resp tabacum . Ju et aI. pes-caprae and its related species. Many genes in wild relatives provide genetic resources for germplasm improvement . Then, after 4–5 weeks, we selected plants with similar growth characteristics for experiments.The experimental materials—I. pes-caprae NAC differentially expressed genes under salt stress, 12 TFs were preliminarily screened as research targets and named IpNAC1–12. The IpNAC nucleotide sequence was converted into an amino acid sequence by NCBIThe amount of expressed genes was obtained from transcriptome data . CombineIpNACs were compared with the full-length sequences by SnapGene 4.36 to determine the exon–intron structure and were visualized using TBtools software. Collinearity between I. pes-caprae (IpNACs) and A. thaliana (TAIR)ANACs) was determined using MCScanX . Then, the PlantCAREMost gene regulatory regions are located 2 kb upstream of the translation start site. The I. pes-caprae to a 45 °C climate box, and the roots were submerged in 20% PEG 6000 for 48 h for dehydration treatment. The leaves and adventitious roots were harvested from the plants. The roots of I. pes-caprae were submerged in a salt solution at three concentrations of 300, 600, and 900 mM NaCl, and adventitious roots were harvested. For hormone treatment, adventitious roots were submerged in 0.1 mM abscisic acid (ABA), 2 mM methyI jasmonate (MeJA), or 2 mM salicylic acid (SA) for 0, 3, 6, 12, 24, or 48 h, and the adventitious roots were collected. The roots were collected from three separate plants at each time point of each treatment and combined to form one sample. All experiments were performed independently and in triplicate. The collected samples were immediately frozen in liquid nitrogen and stored at −80 °C.We selected plants with similar growth rates for plant hormone and abiotic stress treatments. Untreated plants were used as controls. The leaves or adventitious roots were collected after 0, 3, 6, 12, 24, or 48 h of treatment. Heat treatment was performed by transferring 2O, and 1 µL cDNA template (diluted at 1:10). Specific primers for each gene are listed in Actin gene of I. pes-caprae was used as an internal control. Each measurement was performed with three biological replicates. Data were analyzed using the 2-ΔΔCt method.Following the manufacturer’s instructions, the FastPure Plant Total RNA Isolation Kit  was used to isolate the total RNA in the leaves and roots. The quality of the RNA was determined using a NanoDrop2000c spectrophotometer , and total RNA integrity was identified by electrophoresis on 1.0% agarose gel. The HiScript II 1st Strand cDNA Synthesis kit  was used to obtain cDNA from total RNA. Real-time quantitative PCR was carried out using SYBR green  on an ABI QuantStudio 6 Flex  with a final volume of 10 µL per reaction. Each reaction mixture contained 5 µL SYBR Green Realtime PCR Master Mix , 0.4 µL forward primer (10 μM), 0.4 µL reverse primer (10 μM), 3.2 µL ddHIpNAC1–12 were obtained from the transcriptome datasets of I. pes-caprae. We designed the specific primers ; forward primer (10 μM); reverse primer (10 μM); 1 μL Phanta Max Super-Fidelity DNA Polymerase; 1 μL cDNA) were used to amplify the IpNAC1–12 genes from the cDNA of I. pes-caprae. The PCR parameters were set as follows: initial denaturation at 95 °C for 3 min, 30 cycles of denaturation at 95 °C for 15 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min 30 s, and final extension at 72 °C for 10 min. The PCR products were subcloned into the pHB-GFP vector with a Hind III restriction site to form pHB-IpNAC1-12-GFP. pHB-IpNAC1-12-GFP and pHB (set as a control) were transformed into Arabidopsis. In addition, using pCAMBIA0390-DsRed as the backbone vector, the coding regions of IpNAC1–12 were inserted into the pCAMBIA0390-DsRed expression vector constructing pUBI.U4:: IpNAC1-12-CaMV35S::DsRed for Agrobacterium rhizogenes-mediated transformation. pCAMBIA0390-DsRed was used as a control. Each experiment was conducted using more than three biological replicates.The coding DNA sequences of Agrobacterium tumefaciens strain GV3101 for plant transformation. Transient expression in tobacco leaves was determined according to a published method  seeds were germinated on 1/2 MS medium with 50 mg L−1 hygromycin for screening. Then, the WT and overexpressing transgenic seeds were cultivated on 1/2 MS medium with 180 mM NaCl at 22 °C under 16 h of daylight. After 10 d, the primary root length was measured and the growth state was observed.The recombinant vectors pHB-IpNAC5/IpNAC8/IpNAC10/IpNAC12-GFP were transferred into A. rhizogenes strain K599. ZiShu 8 with the same growth state was selected for Agrobacterium infection according to the published method , and statistically significant differences were assessed using Dunnett’s test at I. pes-caprae, named IpNAC1–12. We found that IpNAC1–12 had high homology with Arabidopsis, sweetpotato, and their wild species. IpNAC2 is one of the reported genes, and NAC1 was screened from the cDNA library of I. pes-caprae, but its function was not clear. We verified that IpNACs contained a complete ORF. Analysis of the physicochemical properties revealed amino acid lengths of IpNACs ranging from 242 to 344. The molecular weight varied from 27.58 to 39.21 KDa, and the pI ranged from 5.24 to 8.52. Except for IpNAC2, IpNAC3, and IpNAC6, IpNAC proteins were weakly acidic and were predicted to be located in the nucleus  to establish an unrooted phylogenetic tree  and one ANAC gene (ANAC072) were associated with three or two syntenic blocks, respectively. At the same time, other IpNAC genes have a close relationship with ANAC genes, indicating that their functions may be similar.To investigate the taxonomic and phylogenetic relationships of IpNACs, we used the model plant IpNACs had three exons, separated by two introns  was detected in four IpNAC genes, and a defense and stress response cis-acting element (TC-rich) was detected in five IpNAC genes. Anaerobic induction cis-acting regulatory elements (AREs) were identified in 11 IpNAC genes. Interestingly, there were five IpNAC genes with low-temperature responsive cis-acting elements (LTRs). Many hormone-responsive elements were found in the IpNAC promoter sequence. GA responsive elements were found in three IpNAC genes. SA responsive elements were found in four IpNAC genes. IAA responsive elements were found in 11 IpNAC genes, and ABA responsive elements were found in 12 IpNAC genes. This indicates that plant hormones play a central role in the regulation of the salt stress response.Motifs are key functional units. The IpNAC motif components were analyzed using MEME online software. All IpNAC proteins contained the NAC family characteristic subdomains (A–E), named motif1/2/3/4/5, located at positions 0–210. Except for IpNAC1/4/7/9, other protein sequences had more than five motifs, with a maximum of eight. The 12  introns Figure 1Ipomoea pes-caprae is a highly salt-tolerant crop, and the expression pattern of IpNACs was determined after treatment with different concentrations of NaCl solution. Among the 12 genes, there were significant differences  at 6 and 24 h treatment periods. IpNAC11 was upregulated significantly, reaching the maximum change (47-fold) at 3 h, and returned to baseline over time. In contrast, IpNAC2 reached maximum expression at 48 h (6-fold), and the trend was downregulated and only slightly upregulated throughout the treatment period. IpNAC5, IpNAC8, and IpNAC12 expression were induced 19–300-fold at 6 h. The expressions of IpNAC3, IpNAC6, and IpNAC9 were increased 15–300-fold at 12 h, and those of IpNAC4 and IpNAC7 expression reached 25–130-fold at 24 h. Under 600 mM NaCl treatment, the 12 genes were not strongly induced at 3 and 6 h. IpNAC1 and IpNAC2 were upregulated after 12 h (16–19-fold) and remained stable or were downregulated in the early stage. The induction level of IpNAC5, IpNAC8, IpNAC11, and IpNAC12 increased 10- to 60-fold (24 h) and were upregulated steadily throughout the period. The expressions of IpNAC3, IpNAC4, IpNAC6, IpNAC7, and IpNAC9 reached 15- to 280-fold (48 h). Interestingly, IpNAC6, IpNAC7, and IpNAC9 were upregulated, but the trend was high–low–high. The IpNAC genes were generally downregulated after 900 mM NaCl solution treatment, and IpNAC2, IpNAC3, IpNAC4, IpNAC6, IpNAC7, IpNAC8, IpNAC9, IpNAC11, and IpNAC12 reached the maximum significance level at 48 h, among which IpNAC2, IpNAC3, and IpNAC4 were downregulated (3–24 h), and IpNAC5 and IpNAC10 maintained a high induction at 6 h. Interestingly, IpNAC1 did not respond to 900 mM NaCl solution treatment.Ipomoea pes-caprae is a high temperature- and drought-tolerant crop. The expression level of most IpNAC genes was increased under heat stress (45 °C) and drought stress (20% PEG 6000), but the change in the expression level under drought stress was more obvious than that under high temperature stress (IpNAC5 induction level was the highest (451-fold), followed by that of IpNAC1, IpNAC2, IpNAC3, and IpNAC9, which reached the highest significant expression (10–90-fold) at 24 h. However, the maximum induction of IpNAC4, IpNAC6, IpNAC7, IpNAC8, IpNAC9, IpNAC10, IpNAC11, and IpNAC12 was less than 10-fold. Interestingly, all IpNAC genes were strongly induced at 24 h under heat stress. Most IpNAC genes were near or below the baseline at 3–24 h and near the baseline at 48 h. Under drought stress, IpNAC12 reached a maximum induction at 48 h (442-fold), followed by IpNAC1, IpNAC2, IpNAC3, IpNAC5, IpNAC8, IpNAC9, and IpNAC11, reaching a maximum at 12 h (1–80-fold). IpNAC4, IpNAC6, and IpNAC10 increased 10- to 300-fold at 3 and 48 h. However, the expression trends of IpNAC2 and IpNAC3 after high temperature and drought stress were consistent  at 12–24 h, and the early response was weak or downregulated. Under SA stress, IpNAC1, IpNAC8, and IpNAC12 were significantly upregulated throughout the treatment period. The induced expression of IpNAC12 reached 2064-fold at 48 h. IpNAC2, IpNAC3, IpNAC4, IpNAC5, IpNAC6, IpNAC7, IpNAC9, IpNAC10, and IpNAC11 were mainly upregulated (2–500-fold) at 6–24 h, and except for the highest induction time, the induction times were close to baseline or downregulated. Based on the results of the MeJA treatment, the transcript level of IpNAC9 reached a peak of 241-fold at 48 h. IpNAC4, IpNAC6, IpNAC7, IpNAC8, IpNAC11, and IpNAC12 also maintained a high level (5–150-fold) throughout the period. Most trends continuously increased from 3 to 24 h and steadily decreased from 24 to 48 h. In contrast, IpNAC1, IpNAC2, IpNAC3, IpNAC5, and IpNAC10 were downregulated (2–60-fold) in different periods (3–12 h). At the same time, IpNAC5 did not respond to MeJA induction and was not sensitive throughout the period  play an important role in the stress signal network. We studied the relationship between the transcriptional levels of the 12 hormones Figure 6in vivo  after 24 h of NaCl stress (150 mM). Na+ efflux in the elongation and mature regions of AR and TR differed  and had many important regulatory functions. In addition, AT4G27410 had a collinear relationship with IpNAC6 and IpNAC10. At4G27410 encodes a dehydration-induced NAC transcription factor and acts as a transcriptional activator in the ABA-mediated dehydration response , indicating that IpNACs are highly conserved  among IpNACs  (IpNAC12 was the most strongly induced after drought stress (~500-fold) and SA stress (~2000-fold), but the expression level under high temperature treatment showed low sensitivity (IpNAC8 significantly responded to ABA (~200-fold) and MeJA stress (~150-fold). We speculate that IpNAC10 and IpNAC8 accomplish stress signal transduction not only through the ABA pathway but also through the SA and MeJA stress mechanisms. As a subtropical coastal plant, a high temperature is an essential element for I. pes-caprae. Under induction at 45 °C, IpNAC5 had the highest expression (~500-fold). IpNAC5 shared a relatively high homology with ANAC036 (ANAC036 is induced by osmotic stress and salt stress. In terms of natural factors, salt and high temperature are inseparable. As a result, we speculated that high temperatures accelerated the evapotranspiration rate and increased the loss of water, leading the IpNAC5 gene to be highly expressed in response to stress. Therefore, based on this analysis, we speculate that IpNAC5/8/10/12 are important candidate genes for stress response. Further biological proof is necessary. Hence, we performed subcellular localization of IpNAC5, IpNAC8, IpNAC10 and IpNAC12 and found that they were located in the nucleus (Arabidopsis verified the gene function. We obtained transgenic seeds by overexpressing IpNAC5/8/10/12 in Arabidopsis. T3 homozygous seeds were selected for the salt tolerance test. There was no significant difference between transgenic plants and WT plants in the no-salt medium (IpNAC5/8/10/12 genes, IpNAC5 and IpNAC10 had the strongest salt tolerance in the post-germination stages of Arabidopsis. Cellular Na+ homeostasis and metabolic processes significantly affect plant salt tolerance (+ homeostasis in the transgenic sweetpotato roots, we found that the TR of IpNAC10 had obvious Na+ efflux in the mature and elongation zones compared with AR (IpNAC5 and IpNAC12 showed 3.3- and 2.2-fold more Na+ efflux in the elongation zone than AR, while there was no significant difference in the maturation zone (IpNAC8 showed significant Na+ efflux in the mature region compared to AR (IpNAC5/8/10/12 are highly responsive and protective in sweetpotato, resisting salt stress through Na+ ion channels, and were activated, leading to Na+ efflux in different regions of the sweetpotato roots.The influence of extreme weather is aggravating. Enhancing crop tolerance to respond to various abiotic stressors is essential for improving agricultural production efficiency. olerance  and resiolerance , and otholerance . Comparetressors . HoweverAC genes Table 1.is clear . Based ois clear  and leafis clear . IpNAC2,is clear , indicatis clear . The OsNis clear . The ONAis clear . In addiis clear . As showresponse . IpNAC6  be slow  (Figure be slow . The IpNg IpNACs . Plant rg IpNACs . We specolerance . By measI. pes-caprae as a basis, we utilized biotechnology to explore salt tolerance genes to provide relevant genetic resources and strategies for the genetic improvement of sweetpotato. At the same time, it is important to analyze the molecular regulatory mechanism of salt tolerance in I. pes-caprae to further plant adaptation to extreme climates.Using IpNAC genes in I. pes-caprae. Evolutionary collinearity, gene structure, and promoter cis-acting elements of IpNACs were analyzed. Different responses under a variety of abiotic and hormone stressors indicate that IpNAC genes are controlled by a variety of regulatory mechanisms. IpNAC5/8/10/12 were screened as candidate genes. IpNAC5, IpNAC8, IpNAC10 and IpNAC12 localized in the nucleus. In addition, the root length and growth status of transgenic Arabidopsis under salt stress and the Na+ ion content in sweetpotato roots showed that IpNAC5, IpNAC8, IpNAC10 and IpNAC12 significantly enhanced plant salt tolerance. This study lays the foundation for further studies on the function of IpNACs under stress in I. pes-caprae. The candidate genes may contribute to the functional characterization of salt tolerance genes for breeding abiotic stress-resistant varieties of sweetpotato or other species. Taken together, these results provide new insights into the molecular response of I. pes-caprae and sweetpotato to salt stress.In this study, by combining transcriptome data with bioinformatics analysis, we identified 12 The original contributions presented in the study are included in the article/QC planned and designed the research. YS, YL, and SX wrote the manuscript. YS, YuW, YD, LZ, YaW, DZ, XD, and ZZ conducted the research and analyzed the data. QC supervised the research and the manuscript. All authors read and approved the final manuscript. All authors contributed to the article and approved the submitted version.


Eruca sativa) loaded in the frankincense oil-based nanoemulsion formulation. The choice of frankincense oil for the current study was based on reported research investigations stating its magnificient therapeutic potential against breast cancer. Optimized nanoemulsion of paclitaxel (PTX) and erucin (ER) combination (EPNE) provided sustained release and exhibited enhanced cytotoxicity towards human epithelial breast cancer cells (T-47D) as compared to individual ER and PTX. EPNE was further assessed for its antitumor activity in the 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer mice model. EPNE significantly decreased the levels of hepatic and renal parameters along with oxidative stress in breast cancer mice. Furthermore, EPNE also showed decreased levels of inflammatory cytokines TNF-α, IL-6. Histopathological examinations revealed restoration of the tumorous breast to normal tissues in EPNE-treated breast cancer mice. Therefore, EPNE can act as a viable lead and therapeutic option for drug-resistant breast cancer.Nanoformulation-based combinational drug delivery systems are well known to overcome drug resistance in cancer management. Among them, nanoemulsions are well-known and thermodynamically stable drug delivery systems suitable for carrying hydrophobic drugs and phytoconstituents to tackle drug-resistant cancers. In the present study, we have investigated the effect of paclitaxel in combination with erucin (natural isothiocyanate isolated from the seeds of  Breast cancer is a heterogeneous type of illness that results in the body’s aberrant cells multiplying and dividing out of control, spreading to other body tissues . The numEruca sativa according to our previously described method ], Tween 80 was obtained from Merck, India. Transcutol P was acquired from Sigma Aldrich. Frankincense oil was purchased from Devinez (India). All other chemicals and reagents used were of analytical grade. Erucin, used in this study was isolated from the seeds of d method .v/v) of Frankincense oil, 36% (v/v) of pre-warmed Smix i.e. Tween 20: Transcutol P (3 : 1), and 60% double distilled water. The ratio between oils to surfactants (O/S) was maintained at 1: 9, as this formulation exhibited optimal stability. The resulting mixture was then vortexed for approximately 5 min at room temperature followed by heating and sonication until a clear and transparent solution is obtained (Sonics VCX500 (U.S.A)). The sonication was carried out for approximately 20 min until a clear and transparent phase formed indicating the production of blank nanoemulsion (B-NE). Each experiment was performed in triplicate (n = 3). Furthermore, B-NE was incorporated with a combination of active components (ER + PTX) in 1:1 to produce EPNE.Phase diagrams were constructed by using the water titration method to recognize the type of structure for emulsification and to typify the behavior of the mixtures with dilution paths. The blank oil-in-water (O/W) NE was prepared under optimal conditions using the water titration method followed by probe sonication. NE was prepared by adding 4% (The average droplet sizes (z-average diameter), polydispersity index (PDI), and surface charge  values of B-NE and EPNE samples were identified by dynamic light scattering (DLS) technique through Malvern Particle and Zeta Sizer Analyzer . The experiment was performed with a light scattering angle of 173 and at 25°C. All samples were measured in triplicates.−1 with a 4 cm−1 spectral resolution. The data acquisition was performed in Assistat 7.7en.Fourier Transform Infrared Spectroscopic (FTIR) study was done to locate any integral chemical interactions in the test samples ER, PTX, B-NE, and EPNE. The spectra were recorded in Alpha FTIR spectrophotometer  equipped with a deuterated triglycine sulfate detector and a single reflection diamond ATR sampling module. The spectrographs were scanned at the fingerprint region of 400–4,000 cmThe reconstituted optimized nanoemulsion (EPNE) was subjected to globule morphology using TEM . The optimized nanoemulsion (EPNE) (0.1 ml) was reconstituted 100-fold with deionized water. The prepared sample was placed on a carbon-coated grid in a Petri dish and allowed to air dry for about 1 h. The grids were placed in the specimen cabinet and the beam of electrons focused by an electron gun interacted with a small aperture condensed lens. The transmitted samples were focused on charge-coupled device (CCD) camera and images were generated at a magnification of ×15,000 with a fixed X-ray source of 100 cm.0) was first measured by using UHPLC  at 244 nm and 230 nm for ER, PTX respectively. Then, the centrifugation of 1.0 ml for each sample was performed at 3,000 rpm for 10 min, followed by removing the supernatants. To obtain the sample absorbance (A), the bottom sample was then measured. The Ks value was then calculated as the following equation: Ks = (A0–A)/A0. It should be noted that a smaller Ks value shows less precipitation of developed nanoemulsion which demonstrates good stability.The stability constant (KS) was used to assess the stability of optimized nanoemulsion (EPNE). It was determined by the centrifugation-spectrophotometric method as previously described with modifications. The original absorbance of B-NE samples  and erucin (1.25 mg/mL) were loaded into dialysis bags and placed in 50 mL beaker containing the release medium . Tween 80 was added to maintain sink conditions. The beakers were then placed for 24 h at 37°C while rotating at 100 rpm using temperature controlled magnetic stirrer. At predetermined time points, 1 mL of each sample was withdrawn and replaced with an equal volume of releasing buffer. The samples were analyzed for paclitaxel and erucin content by HPLC analysis  and normal mouse fibroblast (L929) cells were purchased from the National Centre for Cell Science (NCCS), Pune, India. These cells were cultured in RPMI-1640 and Dulbecco’s Modified Eagle Media (DMEM) respectively with 10% fetal bovine serum (FBS) and maintained at a CO2 incubator (5%) at 37°C. Media was exchanged with fresh media at regular intervals. The cytotoxicity of ER, PTX, ER + PTX, B-NE, EPNE was measured against the proliferation of paclitaxel-resistant breast cancer cells (T-47D) and normal mouse fibroblast cells (L929) (4 cells/well) and treated with different concentrations of ER, PTX, ER + PTX, B-NE, EPNE for 24 h. Following treatment, MTT (5 mg/mL) was added to each well and incubated for 4 h in a CO2 (5%) incubator. After incubation, each well-received DMSO (100 μL) solubilized formazan crystals that had settled to the bottom. Absorbance was recorded at 570 nm using a multi-well plate reader . The percentage of growth inhibition was calculated as follows:Human epithelial breast cancer cell line (s (L929) . In a 9650 of combined drugs ER and PTX were taken in 1:1 to calculate the combination index (CI). The CI values for each dose and the corresponding effect level were calculated formula given below:To calculate synergism between erucin with paclitaxel, a combination index was calculated according to The resulting CI values indicated a quantitative definition for an additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) in drug combinations . The Fa–Female Balb/c mice were procured from Guru Nanak Dev University, Amritsar, Punjab, India, 143005. The mice were housed in the Central Animal Facility under standard animal husbandry conditions. The experimental protocol was approved by the Animal Ethical Committee of Guru Nanak Dev University (GNDU), Amritsar. The rules and regulations of control and supervision of experimental animals (CPCSEA) were followed according to the Ministry of Environment and Forests (File No. 226/CPCSEA/2022/09), Government of India. Pharmacokinetic studies were carried out using balb/c mice. Animals weighing 25 ± 2 g were fasted for 12 ± 0.5 h with free access to water before initiating the experiment. A dose of 10 mg/kg erucin (dissolved in sunflower oil) as a carrier system, 10 mg/kg of paclitaxel, and developed nanoemulsion were administered by oral gavage. Blood was collected by a retro-orbital route at different time intervals of 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h. To obtain plasma blood was centrifuged at 15,000 rpm for 10 min. Deproteinization was done by adding acetonitrile to plasma. The supernatant was collected in fresh vials. Erucin and paclitaxel concentrations were analyzed using UHPLC-PDA . In UHPLn = 42) weighing 24.5 ± 0.5 g were procured from Guru Nanak Dev University, Amritsar, Punjab, India, 143,005. The mice were housed in the Central Animal Facility under standard animal husbandry conditions. The experimental protocol was approved by the Animal Ethical Committee of Guru Nanak Dev University (GNDU), Amritsar. The rules and regulations of control and supervision of experimental animals (CPCSEA) were followed according to the Ministry of Environment and Forests (File No. 226/CPCSEA/2022/09), Government of India. All experiments were performed following ethical standards. After 2 weeks of acclimatization, mice were divided into Groups Ⅰ to VII (n = 6). Group I was normal control (does not receive any treatment), Group II was negative control (received DMBA only), Group III was DMBA + placebo (received blank formulation), Group IV received DMBA + erucin , Group V received DMBA + paclitaxel , Group VI received DMBA + erucin: paclitaxel , Group VII received DMBA + nanoemulsion formulation . DMBA  was administered weekly for 4–week. After 90 days of DMBA administration (r3 (where r is the mean radius of tumor in mm). At the end of the experiment, the blood sample was withdrawn through retro-orbital plexus puncture and collected for the assessment of biochemical parameters, followed by the sacrifice of all animals through cervical dislocation. The liver and breast tissue were excised from all experimental animals and stored at −20°C for various biochemical and histological analyses , serum glutamic pyruvic transaminase (SGPT), and kidney parameters  and lipid profile  by using readymade analytical kits (ERBA) .Homogenate (10% w/v) of liver tissue was prepared in chilled Tris-HCl buffer (pH 8.0) for 5 min, centrifuged at 8,000 rpm for 15 min at 4°C. The supernatant was stored at -80°C for further experimentation. Estimation of oxidative stress parameters in hepatic tissues was done by measuring antioxidant enzymes such as superoxide dismutase (SOD) by following the method of IL-6 and TNF-α were estimated in fresh plasma using respective ELISA kits  according to the manufacturer’s instructions.Breast tissues were excised from all experimental animals and preserved in 10% formalin solution for various histological analyses. All collected organs were examined for histopathological changes with hematoxylin and eosin (H & E) staining after full fixation in 10% formalin. The tissues were trimmed, embedded in paraffin, sectioned, mounted on microscope slides, and stained with hematoxylin and eosin. Formalin-fixed organs were processed for histopathological examinations .Assistat 7.7en). The probability p ≤ 0.001, 0.01, and 0.05 was carried out to check the statistical significance of all the values at a level of 0.1%, 1%, and 5%.The mean standard error is used to express all of the results (SE). The analysis of variances and interactions, with the help of Tukey’s test, was done using a one-way analysis of variance (ANOVA)  in ratios of 1:1, 2:1, 3:1, 4:1, 5:1, 1:2, and 1:3 and the obtained combination was further used for characterizing various parameters . The zeteraction . Furthereraction . The obt50 values of 0.31 mg/ml against T-47D  as shown in 50 of ER to ½ times decreases the IC50 of synthetic chemotherapy drug paclitaxel (PTX) to ten times (IC50 = 0.005 mg/ml). Hence this analysis showed a strong synergistic effect for the combination of ER and PTX , and PTX (2.29 folds) while Tmax was enhanced by four folds of ER and two folds of PTX. thalf of both erucin (1.95 folds) and paclitaxel (2.75 folds) was also observed elevated  as compared to the free mixture of ER + PTX (49.4 mm3), ER (58.9 mm3), PTX (67.1 mm3) while DMBA treated group showed a tumor of (145.1 mm3) as shown in viz. SGOT (81.88%), SGPT (85.69%), total bilirubin (169.83%), renal parameters such as urea (116.14%), creatinine (135.74%), and other parameters such as triglyceride (94.15%), cholesterol (103.55%) were significantly enhanced in mice treated with DMBA (group Ⅱ) compared to other groups of ER, PTX, a mixture of ER + PTX and B-NE  while EPNE significantly inhibited lipid peroxidation resulting in the decrease in the level of malondialdehyde (2.52 nmole) as compared to the DMBA groups treated with the combination of ER + PTX (2.99 nmole), ER (3.62 nmole), PTX (5.51 nmole), B-NE (7.55 nmole). On the other hand, DMBA generated oxidative stress in the hepatic tissues which in turn results in the decrease of antioxidant enzymes such as SOD (8.44 U/mg of protein), CAT (4.19 U/mg of protein). However, in group Ⅶ, EPNE significantly enhanced the level of antioxidant enzymes by SOD (13.74 U/mg protein), CAT (10.19 U/mg protein) as shown in Inflammatory cytokines were also elevated by exposure to DMBA. IL-6, TNF- α are inflammatory markers and their elevated level promotes cancer progression. In our study, DMBA enhanced the level of IL-6 (111.8%), TNF- α (164.76%) in group Ⅱ as compared to the control group . EPNE siIn histopathological analysis, mammary tissue of the control group showed normal acini and ductules. However DMBA administered group showed excessive proliferation of epithelial lining of acini and ductules with hyperchromatic nuclei (black arrow); mammary tissue of DMBA + Placebo nanoemulsion administered group showed slight improvement while mammary tissue of DMBA + erucin administered group, mammary tissue of DMBA + paclitaxel administered group and DMBA + (ER + PTX) mixture administered group showed little more improvement in the proliferation of epithelial lining of acini and ductules with less hyperchromatic nuclei as compare to only DMBA treated group. Furthermore, DMBA + nanoemulsion administered group showed restoration to normal tissue with normal acini (black arrow) .In vitro release profile further demonstrated a sustained release pattern of paclitaxel and erucin from nanoemulsion formulation when compared with erucin and paclitaxel suspension alone. EPNE showed excellent growth inhibition (97.44 ± 0.52%) of paclitaxel-resistant estrogen-positive human breast cancer cells T-47D and showed minimum cytotoxicity in normal cell line L929 as compared to T-47D cells.In the present study paclitaxel (PTX), erucin (ER), and frankincense oil were the therapeutically efficient components decorating together the optimized nanoemulsion. The developed nanoemulsion (EPNE) was evaluated for its antitumor efficacy. The prepared nanoemulsion showed zeta size of B-NE was 29 ± 0.71 nm, EPNE was 18.1 ± 0.5 nm and zeta potential of B-NE was −0.107 mV, EPNE was −17.3 mV. TEM analysis of EPNE revealed the spherical and nanosize emulsion globules efficiently encapsulating paclitaxel and erucin within a layer of frankincense oil further surrounded by the continuous aqueous phase. In vivo, pharmacokinetic studies showed enhanced plasma concentrations of both erucin as well as paclitaxel. Potent results of in vitro cytotoxicity studies and in vivo pharmacokinetic outcomes established the solid ground for further assessment of EPNE for its in vivo antitumor potential against breast cancer. DMBA anthracene) induced breast cancer model is an important preclinical animal model as it mimics morphological, histopathological, and biochemical features of human breast cancer (3) as compared to free PTX (67.1 mm3), ER (58.9 mm3), and the mixture of ER + PTX (49.4 mm3) while the DMBA treatment group showed tumor volume of 145.1 mm3. During treatment, significant improvements in body weight were observed in the EPNE-treated groups as compared to groups of DMBA, ER, and PTX solution. Furthermore, histopathological analysis confirmed the restoration of breast tumor in the EPNE-treated group to normal tissues at the end of the experiment. The findings of the current study also found that DMBA elevated the levels of liver serum transaminases i.e., SGOT, and SGPT in the mice serum, resulting in hepatic injury. From the previous study, it was found that DMBA-induced liver damage triggers the release of SGOT, SGPT, in the bloodstream which indicated hepatocellular damage. Similarly in the present study, the level of SGOT, and SGPT were significantly increased in group Ⅱ by 81.88%, 85.69% resp. However, EPNE treatment altered this effect by decreasing the level of SGOT, and SGPT by 41.2%, and 41.36% respectively in the serum. DMBA toxicity induces damage in the kidney tissues resulting in elevated urea, creatinine, total bilirubin, cholesterol, and triglycerides level in the bloodstream, but EPNE treatment significantly decreases the level of kidney parameters by 51.06%, 49.06%, 61.77%, 49.12%, and 48.31% respectively in the serum which in turn showed the renal protective effect. However, the promising renal-protective effect is related to the efficacy of EPNE. The process of DMBA carcinogenesis comprises alteration in the tissue redox balance, resulting in oxidative stress which is responsible for biochemical and pathophysiological disturbances in rats  exhibited an improved pharmacokinetic profile resulting in enhanced bioavailability of both paclitaxel and erucin. Enhanced C