Unveiling the hidden world: How arbuscular mycorrhizal fungi and its regulated core fungi modify the composition and metabolism of soybean rhizosphere microbiome.

BACKGROUND: The symbiosis between arbuscular mycorrhizal fungi (AMF) and plants often stimulates plant growth, increases agricultural yield, reduces costs, thereby providing significant economic benefits. AMF can also benefit plants through affecting the rhizosphere microbial community, but the underlying mechanisms remain unclear. Using Rhizophagus intraradices as a model AMF species, we assessed how AMF influences the bacterial composition and functional diversity through 16 S rRNA gene sequencing and non-targeted metabolomics analysis in the rhizosphere of aluminum-sensitive soybean that were inoculated with pathogenic fungus Nigrospora oryzae and phosphorus-solubilizing fungus Talaromyces verruculosus in an acidic soil. RESULTS: The inoculation of R. intraradices, N. oryzae and T. verruculosus didn't have a significant influence on the levels of soil C, N, and P, or various plant characteristics such as seed weight, crude fat and protein content. However, their inoculation affected the structure, function and nutrient dynamics of the resident bacterial community. The co-inoculation of T. verruculosus and R. intraradices increased the relative abundance of Pseudomonas psychrotolerans, which was capable of N-fixing and was related to cry-for-help theory (plants signal for beneficial microbes when under stress), within the rhizosphere. R. intraradices increased the expression of metabolic pathways associated with the synthesis of unsaturated fatty acids, which was known to enhance plant resistance under adverse environmental conditions. The inoculation of N. oryzae stimulated the stress response inside the soil environment by enriching the polyene macrolide antifungal antibiotic-producing bacterial genus Streptomyces in the root endosphere and upregulating two antibacterial activity metabolic pathways associated with steroid biosynthesis pathways in the rhizosphere. Although inoculation of pathogenic fungus N. oryzae enriched Bradyrhizobium and increased soil urease activity, it had no significant effects on biomass and N content of soybean. Lastly, the host niches exhibited differences in the composition of the bacterial community, with most N-fixing bacteria accumulating in the endosphere and Rhizobium vallis only detected in the endosphere. CONCLUSIONS: Our findings demonstrate that intricate interactions between AMF, associated core fungi, and the soybean root-associated ecological niches co-mediate the regulation of soybean growth, the dynamics of rhizosphere soil nutrients, and the composition, function, and metabolisms of the root-associated microbiome in an acidic soil.

Impact of dual Bt-transgenic maize (2A7) on soil microbial communities and enzyme activities: A comparative study with control variety Z58.

The impacts of transgenic crops on soil microbiology and fertility are critical in determining their biosafety. While transgenic crops can alter soil microbes, their effects are often context-dependent; therefore, the ecological importance of these changes remains a topic of ongoing research. Using high-throughput sequencing, we investigated the effects of Bacillus thuringiensis (Bt) maize expressing the mcry1Ab and mcry2Ab genes (2A7) on soil nutrient dynamics, as well as the diversity and function of soil microbial communities, including bacteria and fungi, within different soil compartments. Our findings revealed a plant-shaped rhizosphere (RS) microbial community as a result of the selective recruitment of microorganisms from the surrounding environment. The transgene insertion had a significant impact on the RS niche, and several species eventually became associated with Z58 and 2A7 plants. For example, Neocosmospora rubicola fungal and Pantoea dispersa bacterial microorganisms were significantly decreased in the dual Bt-transgenic 2A7 rhizosphere but enriched in the Z58 rhizospheres. The activity of soil enzymes such as urease, invertase, and alkaline phosphatase was boosted by Bt-transgenic 2A7. LefSe analysis identified significant bacterial and fungal biomarker species that were responsible for the differential effects of Bt-transgenic 2A7 and control Z58 within rhizosphere soils. Mantel analysis further demonstrated that the root exudates of 2A7 altered nutrient-acquisition enzymes by influencing biomarker taxa. PICRUSt2 functional characterization revealed a significantly higher abundance of the phosphate-starvation-inducible protein in control Z58 than in Bt-transgenic 2A7. Furthermore, taxonomy, alpha (Shannon diversity), and beta diversity analyses all revealed niche-driven microbial profile differentiation. Niche partitioning also had a significant impact on N- and P-related COGs as well. Our findings suggests that Bt-transgenic 2A7 modulates rhizosphere microbial communities by affecting biomarker taxa and soil enzyme activity. These findings will promote sustainable agriculture practices by advancing our knowledge of the ecological effects of Bt crops on soil microbial communities.

In vivo transgenic studies confirm the critical acylation function of LeBAHD56 for shikonin in Lithospermum erythrorhizon.

KEY MESSAGE: LeBAHD56 is preferentially expressed in tissues where shikonin and its derivatives are biosynthesized, and it confers shikonin acylation in vivo. Two WRKY transcriptional factors might regulate LeBAHD56's expression. Shikonin and its derivatives, found in the roots of Lithospermum erythrorhizon, have extensive application in the field of medicine, cosmetics, and other industries. Prior research has demonstrated that LeBAHD1(LeSAT1) is responsible for the biochemical process of shikonin acylation both in vitro and in vivo. However, with the exception of its documented in vitro biochemical function, there is no in vivo genetic evidence supporting the acylation function of the highly homologous gene of LeSAT1, LeBAHD56(LeSAT2), apart from its reported role. Here, we validated the critical acylation function of LeBAHD56 for shikonin using overexpression (OE) and CRISPR/Cas9-based knockout (KO) strategies. The results showed that the OE lines had a significantly higher ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than the control. In contrast, the KO lines had a significantly lower ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than controls. As for its detailed expression patterns, we found that LeBAHD56 is preferentially expressed in roots and callus cells, which are the biosynthesis sites for shikonin and its derivatives. In addition, we anticipated that a wide range of putative transcription factors might control its transcription and verified the direct binding of two crucial WRKY members to the LeBAHD56 promoter's W-box. Our results not only confirmed the in vivo function of LeBAHD56 in shikonin acylation, but also shed light on its transcriptional regulation.

Harnessing the power of microbes: Enhancing soybean growth in an acidic soil through AMF inoculation rather than P-fertilization.

The low phosphorus (P) availability of acidic soils severely limits leguminous plant growth and productivity. Improving the soil P nutritional status can be achieved by increasing the P-content through P-fertilization or stimulating the mineralization of organic P via arbuscular mycorrhizal fungi (AMF) application; however, their corresponding impacts on plant and soil microbiome still remain to be explored. Here, we examined the effects of AMF-inoculation and P-fertilization on the growth of soybean with different P-efficiencies, as well as the composition of rhizo-microbiome in an acidic soil. The growth of recipient soybean NY-1001, which has a lower P-efficiency, was not significantly enhanced by AMF-inoculation or P-fertilization. However, the plant biomass of higher P-efficiency transgenic soybean PT6 was significantly increased by 46.74%-65.22% through AMF-inoculation. Although there was no discernible difference in plant biomass between PT6 and NY-1001 in the absence of AMF-inoculation and P-fertilization, PT6 had approximately 1.9-2.5 times the plant biomass of NY-1001 after AMF-inoculation. Therefore, the growth advantage of higher P-efficiency soybean was achieved through the assistance of AMF rather than P-fertilization in available P-deficient acidic soil. Most nitrogen (N)-fixing bacteria and some functional genes related to N-fixation were abundant in endospheric layer, as were the P-solubilizing Pseudomonas plecoglossicida, and annotated P-metabolism genes. These N-fixing and P-solubilizing bacteria were positive correlated with each other. Lastly, the two most abundant phytopathogenic fungi species accumulated in endospheric layer, they exhibited positive correlations with N-fixing bacteria, but displayed negative interactions with the majority of the other dominant non-pathogenic genera with potential antagonistic activity.

A modified natural small molecule inhibits triple-negative breast cancer growth by interacting with Tubb3.

BACKGROUND: Triple-negative breast cancer (TNBC) is a malignant tumor without specific therapeutic targets and a poor prognosis. Chemotherapy is currently the first-line therapeutic option for TNBC. However, due to the heterogeneity of TNBC, not all of TNBC patients are responsive to chemotherapeutic agents. Therefore, the demand for new targeted agents is critical. ß-tubulin isotype III (Tubb3) is a prognostic factor associated with cancer progression, including breast cancer, and targeting Tubb3 may lead to improve TNBC disease control. Shikonin, the active compound in the roots of Lithospermun erythrorhizon suppresses the growth of various types of tumors, and its efficacy can be improved by altering its chemical structure. PURPOSE: In this work, the anti-TNBC effect of a shikonin derivative (PMMB276) was investigated, and its mechanism was also investigated. STUDY DESIGN/METHODS: This study combines flow cytometry, immunofluorescence staining, immunoblotting, immunoprecipitation, siRNA silencing, and the iTRAQ proteomics assay to analyze the inhibition potential of PMMB276 on TNBC. In vivo study was performed, Balb/c female murine models with or without the small molecule treatments. RESULTS: Herein, we screened 300 in-house synthesized analogs of shikonin against TNBC and identified a novel small molecule, PMMB276; it suppressed cell proliferation, induced apoptosis, and arrested the cell cycle at the G2/M phase, suggesting that it could have a tumor suppressive role in TNBC. Tubb3 was identified as the target of PMMB276 using proteomic and biological activity analyses. Meanwhile, PMMB276 regulated microtubule dynamics in vitro by inducing microtubule depolymerization and it could act as a tubulin stabilizer by a different process than that of paclitaxel. Moreover, suppressing or inhibiting Tubb3 with PMMB276 reduced the growth of breast cancer in an experimental mouse model, indicating that Tubb3 plays a significant role in TNBC progression. CONCLUSION: The findings support the therapeutic potential of PMMB276, a Tubb3 inhibitor, as a treatment for TNBC. Our findings might serve as a foundation for the utilization of shikonin and its derivatives in the development of anti-TNBC.

Genome-Wide Comparison and Functional Characterization of HMGR Gene Family Associated with Shikonin Biosynthesis in Lithospermum erythrorhizon.

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), as the rate-limiting enzyme in the mevalonate pathway, is essential for the biosynthesis of shikonin in Lithospermum erythrorhizon. However, in the absence of sufficient data, the principles of a genome-wide in-depth evolutionary exploration of HMGR family members in plants, as well as key members related to shikonin biosynthesis, remain unidentified. In this study, 124 HMGRs were identified and characterized from 36 representative plants, including L. erythrorhizon. Vascular plants were found to have more HMGR family genes than nonvascular plants. The phylogenetic tree revealed that during lineage and species diversification, the HMGRs evolved independently and intronless LerHMGRs emerged from multi-intron HMGR in land plants. Among them, Pinus tabuliformis and L. erythrorhizon had the most HMGR gene duplications, with 11 LerHMGRs most likely expanded through WGD/segmental and tandem duplications. In seedling roots and M9 cultured cells/hairy roots, where shikonin biosynthesis occurs, LerHMGR1 and LerHMGR2 were expressed significantly more than other genes. The enzymatic activities of LerHMGR1 and LerHMGR2 further supported their roles in catalyzing the conversion of HMG-CoA to mevalonate. Our findings provide insight into the molecular evolutionary properties and function of the HMGR family in plants and a basis for the genetic improvement of efficiently produced secondary metabolites in L. erythrorhizon.

Triple-transgenic soybean in conjunction with glyphosate drive patterns in the rhizosphere microbial community assembly.

Plant roots continuously influence the rhizosphere, which also serves as a recruitment site for microorganisms with desirable functions. The development of genetically engineered (GE) crop varieties has offered unparalleled yield advantages. However, in-depth research on the effects of GE crops on the rhizosphere microbiome is currently insufficient. We used a triple-transgenic soybean cultivar (JD606) that is resistant to insects, glyphosate, and drought, along with its control, ZP661, and JD606 treated with glyphosate (JD606G). Using 16S and ITS rDNA sequencing, their effects on the taxonomy and function of the bacterial and fungal communities in the rhizosphere, surrounding, and bulk soil compartment niches were determined. Alpha diversity demonstrated a strong influence of JD606 and JD606G on bacterial Shannon diversity. Both treatments significantly altered the soil's pH and nitrogen content. Beta diversity identified the soil compartment niche as a key factor with a significant probability of influencing the bacterial and fungal communities associated with soybeans. Further analysis showed that the rhizosphere effect had a considerable impact on bacterial communities in JD606 and JD606G soils but not on fungal communities. Microbacterium, Bradyrhizobium, and Chryseobacterium were found as key rhizobacterial nodes. In addition, the LEfSe analysis identified biomarker taxa with plant-beneficial attributes, demonstrating rhizosphere-driven microbial recruitment. FUNGuild, Bugbase, and FAPROTAX functional predictions showed that ZP661 soils had more plant pathogen-associated microbes, while JD606 and JD606G soils had more stress-tolerance, nitrogen, and carbon cycle-related microbes. Bacterial rhizosphere networks had more intricate topologies than fungal networks. Furthermore, correlation analysis revealed that the bacteria and fungi with higher abundances exhibited varying degrees of positive and negative correlations. Our findings shed new light on the niche partitioning of bacterial and fungal communities in soil. It also indicates that following triple-transgenic soybean cultivation and glyphosate application, plant roots recruit microbes with beneficial taxonomic and functional traits in the rhizosphere.

Design, synthesis and biological evaluation of novel modified dual-target shikonin derivatives for colorectal cancer treatment.

Warburg effect provides energy and material essential for tumor proliferation, the reverse of Warburg effect provides insights into the development of a novel anti-cancer strategy. Pyruvate kinase 2 (PKM2) and pyruvate dehydrogenase kinase 1 (PDK1) are two key enzymes in tumor glucose metabolism pathway that not only contribute to the Warburg effect through accelerating aerobic glycolysis, but also serve as druggable target for colorectal cancer (CRC). Considering that targeting PKM2 or PDK1 alone does not seem to be sufficient to remodel abnormal glucose metabolism and achieve significant antitumor activity, a series of novel benzenesulfonyl shikonin derivatives were designed to regulate PKM2 and PDK1 simultaneously. By means of molecular docking and antiproliferative screen, we found that compound Z10 could act as the combination of PKM2 activator and PDK1 inhibitor, thereby significantly inhibited glycolysis that reshaping tumor metabolism. Moreover, Z10 could inhibit proliferation, migration and induce apoptosis in CRC cell HCT-8. Finally, the in vivo anti-tumor activity of Z10 was evaluated in a colorectal cancer cell xenograft model in nude mice and the results demonstrated that Z10 induced tumor cell apoptosis and inhibited tumor cell proliferation with lower toxicity than shikonin. Our findings indicated that it is feasible to alter tumor energy metabolism through multi-target synergies, and the dual-target benzenesulfonyl shikonin derivative Z10 could be a potential anti-CRC agent.

Construction and Identification of an NLR-Associated Prognostic Signature Revealing the Heterogeneous Immune Response in Skin Cutaneous Melanoma.

Background: Skin cutaneous melanoma (SKCM) is the deadliest dermatology tumor. Ongoing researches have confirmed that the NOD-like receptors (NLRs) family are crucial in driving carcinogenesis. However, the function of NLRs signaling pathway-related genes in SKCM remains unclear. Objective: To establish and identify an NLRs-related prognostic signature and to explore its predictive power for heterogeneous immune response in SKCM patients. Methods: Establishment of the predictive signature using the NLRs-related genes by least absolute shrinkage and selection operator-Cox regression analysis (LASSO-COX algorithm). Through univariate and multivariate COX analyses, NLRs signature's independent predictive effectiveness was proven. CIBERSORT examined the comparative infiltration ratios of 22 distinct types of immune cells. RT-qPCR and immunohistochemistry implemented expression validation for critical NLRs-related prognostic genes in clinical samples. Results: The prognostic signature, including 7 genes, was obtained by the LASSO-Cox algorithm. In TCGA and validation cohorts, SKCM patients with higher risk scores had remarkably poorer overall survival. The independent predictive role of this signature was confirmed by multivariate Cox analysis. Additionally, a graphic nomogram demonstrated that the risk score of the NLRs signature has high predictive accuracy. SKCM patients in the low-risk group revealed a distinct immune microenvironment characterized by the significantly activated inflammatory response, interferon-α/γ response, and complement pathways. Indeed, several anti-tumor immune cell types were significantly accumulated in the low-risk group, including M1 macrophage, CD8 T cell, and activated NK cell. It is worth noting that our NLRs prognostic signature could serve as one of the promising biomarkers for predicting response rates to immune checkpoint blockade (ICB) therapy. Furthermore, the results of expression validation (RT-qPCR and IHC) were consistent with the previous analysis. Conclusion: A promising NLRs signature with excellent predictive efficacy for SKCM was developed.

Mycorrhizae Enhance Soybean Plant Growth and Aluminum Stress Tolerance by Shaping the Microbiome Assembly in an Acidic Soil.

Strongly acidic soils are characterized by high aluminum (Al) toxicity and low phosphorus (P) availability, which suppress legume plant growth and nodule development. Arbuscular mycorrhizal fungi (AMF) stimulate rhizobia and enhance plant P uptake. However, it is unclear how this symbiotic soybean-AMF-rhizobial trio promotes soybean growth in acidic soils. We examined the effects of AMF and rhizobium addition on the growth of two soybean genotypes, namely, Al-tolerant and Al-sensitive soybeans as well as their associated bacterial and fungal communities in an acidic soil. With and without rhizobial addition, AMF significantly increased the fresh shoot and root biomass of Al-tolerant soybean by 47%/87% and 37%/24%, respectively. This increase in plant biomass corresponded to the enrichment of four plant growth-promoting rhizobacteria (PGPR) in the rhizospheric soil, namely, Chitinophagaceae bacterium 4GSH07, Paraburkholderia soli, Sinomonas atrocyanea, and Aquincola tertiaricarbonis. For Al-sensitive soybean, AMF addition increased the fresh shoot and root biomass by 112%/64% and 30%/217%, respectively, with/without rhizobial addition. Interestingly, this significant increase coincided with a decrease in the pathogenic fungus Nigrospora oryzae as well as an increase in S. atrocyanea, A. tertiaricarbonis, and Talaromyces verruculosus (a P-solubilizing fungus) in the rhizospheric soil. Lastly, the compartment niche along the soil-plant continuum shaped microbiome assembly, with pathogenic/saprotrophic microbes accumulating in the rhizospheric soil and PGPR related to nitrogen fixation or stress resistance (e.g., Rhizobium leguminosarum and Sphingomonas azotifigens) accumulating in the endospheric layer. IMPORTANCE Taken together, this study examined the effects of arbuscular mycorrhizal fungi (AMF) and rhizobial combinations on the growth of Al-tolerant and Al-sensitive soybeans as well as their associated microbial communities in acidic soils and concluded that AMF enhances soybean growth and Al stress tolerance by recruiting PGPR and altering the root-associated microbiome assembly in a host-dependent manner. In the future, these findings will help us better understand the impacts of AMF on rhizosphere microbiome assembly and will contribute to the development of soybean breeding techniques for the comprehensive use of PGPR in sustainable agriculture.

PKM2/PDK1 dual-targeted shikonin derivatives restore the sensitivity of EGFR-mutated NSCLC cells to gefitinib by remodeling glucose metabolism.

Pyruvate kinase 2 (PKM2) and pyruvate dehydrogenase kinase 1 (PDK1) are two key enzymes in tumor glucose metabolism pathway that not only promote tumor growth and proliferation through accelerating aerobic glycolysis, but also contribute to drug resistance of non-small cell lung cancer (NSCLC). Considering that targeting PKM2 or PDK1 alone seems insufficient to remodel abnormal glucose metabolism to achieve significant antitumor activity, we proposed a "two-step approach" that regulates PKM2 and PDK1 synchronously. Firstly, we found that the combination of ML265 (PKM2 activator) and AZD7545 (PDK1 inhibitor) could synergistically inhibit proliferation and induce apoptosis in H1299 cells. Base on this, we designed a series of novel shikonin (SK) thioether derivatives as PKM2/PDK1 dual-target agents, among which the most potent compound E5 featuring a 2-methyl substitution on the benzene ring exerted significantly increased inhibitory activity toward EGFR mutant NSCLC cell H1975 (IC50 = 1.51 µmol/L), which was 3 and 17-fold more active than the lead compound SK (IC50 = 4.56 µmol/L) and the positive control gefitinib (IC50 = 25.56 µmol/L), respectively. Additionally, E5 also showed good anti-tumor activity in xenografted mouse models, with significantly lower toxicity side effects than SK. Moreover, E5 also inhibited the entry of PKM2 into nucleus to regulate the transcriptional activation of oncogenes, thus restoring the sensitivity of H1975 cell to gefitinib. Collectively, these data demonstrate that E5, a dual inhibitor of PKM2/PDK1, may be a promising adjunct to gefitinib in the treatment of EGFR-TKIs resistant NSCLC, deserving further investigation.

Discrepancies in rhizobacterial assembly caused by glyphosate application and herbicide-tolerant soybean Co-expressing GAT and EPSPS.

There are concerns that the innovation of genetically modified herbicide-tolerant (GMHT) plants, as well as the application of herbicide to such GMHT plants, could have an impact on ecological interactions and unintentionally harm non-targeted organisms. Consequently, we intend to use full-length 16 S rDNA amplicon sequencing to examine changes in the bacterial community in the rhizosphere of GMHT soybean (Z106) harboring 5-enolpyruvylshikimate-3-phosphate synthase and Glyphosate N-acetyltransferase genes and GMHT soybean treated with glyphosate (Z106G). Glyphosate application significantly impacted bacterial alpha diversity (species richness, and Shannon diversity). Permutational multivariate analysis of variance of beta diversity demonstrated that soil compartments and growth stages had a substantial impact on soybean rhizobacterial communities (soil compartments, growth stages, P = 0.001). Community composition revealed that Z106G soils were abundant in Taibaiella and Arthrobacter pascens at maturity, while Chryseobacterium joostei and Stenotrophomonas maltophilia predominated in Z106 soils during flowering. Nitrogen-fixing and phosphate-solubilizing microbes were found in higher proportions in the rhizosphere than in bulk soil, with Sinorhizobium being more abundant in Z106 and Bacillus and Stenotrophomonas being more prevalent in Z106G rhizosphere soils. Collectively, our findings suggest glyphosate application and glyphosate-tolerant soybean as potential regulators of soybean rhizobacterial composition.

The host niches of soybean rather than genetic modification or glyphosate application drive the assembly of root-associated microbial communities.

Plant roots significantly influence soil microbial diversity, and soil microorganisms play significant roles in both natural and agricultural ecosystems. Although the genetically modified (GM) crops with enhanced insect and herbicide resistance are thought to have unmatched yield and stress resistance advantages, thorough and in-depth case studies still need to be carried out in a real-world setting due to the potential effects of GM plants on soil microbial communities. In this study, three treatments were used: a recipient soybean variety Jack, a triple transgenic soybean line JD321, and the glyphosate-treated JD321 (JD321G). Three sampling stages (flowering, seed filling and maturing), as well as three host niches of soybean rhizosphere [intact roots (RT), rhizospheric soil (RS) and surrounding soil (SS)] were established. In comparison to Jack, the rhizospheric soil of JD321G had higher urease activity and lower nitrite reductase at the flowering stage. Different treatments and different sampling stages existed no significant effects on the compositions of microbial communities at different taxonomic levels. However, at the genus level, the relative abundance of three plant growth-promoting fungal genera (i.e. Mortierella, Chaetomium and Pseudombrophila) increased while endophytic bacteria Chryseobacterium and pathogenic bacteria Streptomyces decreased from the inside to the outside of the roots (i.e. RT → RS → SS). Moreover, two bacterial genera, Bradyrhizobium and Ensifer were more abundant in RT than in RS and SS, as well as three species, Agrobacterium radiobacter, Ensifer fredii and Ensifer meliloti, which are closely related to nitrogen-fixation. Furthermore, five clusters of orthologous groups (COGs) associated to nitrogen-fixation genes were higher in RT than in RS, whereas only one COG annotated as dinitrogenase iron-molybdenum cofactor biosynthesis protein was lower. Overall, the results imply that the rhizosphere host niches throughout the soil-plant continuum largely control the composition and function of the root-associated microbiome of triple transgenic soybean.

Genome-Wide Identification of LeBAHDs in Lithospermum erythrorhizon and In Vivo Transgenic Studies Confirm the Critical Roles of LeBAHD1/LeSAT1 in the Conversion of Shikonin to Acetylshikonin.

The BAHD acyltransferase family is a unique class of plant proteins that acylates plant metabolites and participates in plant secondary metabolic processes. However, the BAHD members in Lithospermum erythrorhizon remain unknown and uncharacterized. Although the heterologously expressed L. erythrorhizon BAHD family member LeSAT1 in Escherichia coli has been shown to catalyze the conversion of shikonin to acetylshikonin in vitro, its in vivo role remains unknown. In this study, the characterization, evolution, expression patterns, and gene function of LeBAHDs in L. erythrorhizon were explored by bioinformatics and transgenic analysis. We totally identified 73 LeBAHDs in the reference genome of L. erythrorhizon. All LeBAHDs were phylogenetically classified into five clades likely to perform different functions, and were mainly expanded by dispersed and WGD/segmental duplication. The in vivo functional investigation of the key member LeBAHD1/LeSAT1 revealed that overexpression of LeBAHD1 in hairy roots significantly increased the content of acetylshikonin as well as the conversion rate of shikonin to acetylshikonin, whereas the CRISPR/Cas9-based knockout of LeBAHD1 in hairy roots displayed the opposite trend. Our results not only confirm the in vivo function of LeBAHD1/LeSAT1 in the biosynthesis of acetylshikonin, but also provide new insights for the biosynthetic pathway of shikonin and its derivatives.

Novel shikonin derivatives suppress cell proliferation, migration and induce apoptosis in human triple-negative breast cancer cells via regulating PDK1/PDHC axis.

AIMS: PDK1 is one of the key enzymes in the glucose metabolism pathway, which is abnormally high expressed in breast cancer tissues and can promote tumor proliferation and metastasis. PDK1 and the PDHC/PDK axis are important targets for regulating glucose metabolism and anti-tumor activity. In this study, we evaluated the anti-tumor activities of a series of semi-synthesized shikonin (SK) derivatives against human breast cancer cells. MAIN METHODS: The anti-proliferation activity of SK derivatives against human breast cancer cell lines was tested by CCK-8 and EdU assay. Flow cytometry was utilized to evaluate cell apoptosis, reactive oxygen species and cell cycle distribution. Cell migration ability was determined by wound healing and trans-well assay. PDK1 targeting effect was confirmed by western bolting, molecular docking, bio-layer interferometry and PDK1 enzyme activity assay. Nude-mouse transplanted tumor model was used to evaluate their anti-tumor effect in vivo. KEY FINDINGS: Findings revealed that SK derivatives had good anti-proliferation ability against MDA-MB-231 cell. They induced cell apoptosis by regulating the mitochondrial apoptosis and death receptor pathway. They also inhibited cell migration by suppressing EMT progression. Molecular docking, PDK1 affinity and enzyme activity demonstrated their PDK1 targeting. In vivo antitumor experiment showed that E2 could significantly inhibit tumor growth with lower side-effect on mice than SK. SIGNIFICANCE: In conclusion, the novel SK derivatives E2 and E5 inhibited tumor glycolysis by targeting PDK1 and ultimately induced apoptosis. Our data demonstrated that E2 would be a good lead compound for the treatment of human TNBC as a novel PDK1 inhibitor.

Differential assembly of root-associated bacterial and fungal communities of a dual transgenic insect-resistant maize line at different host niches and different growth stages.

Transgenic technology has been widely applied to crop development, with genetically modified (GM) maize being the world's second-largest GM crop. Despite the fact that rhizosphere bacterial and fungal populations are critical regulators of plant performance, few studies have evaluated the influence of GM maize on these communities. Plant materials used in this study included the control maize line B73 and the mcry1Ab and mcry2Ab dual transgenic insect-resistant maize line 2A-7. The plants and soils samples were sampled at three growth stages (jointing, flowering, and maturing stages), and the sampling compartments from the outside to the inside of the root are surrounding soil (SS), rhizospheric soil (RS), and intact root (RT), respectively. In this study, the results of alpha diversity revealed that from the outside to the inside of the root, the community richness and diversity declined while community coverage increased. Morever, the different host niches of maize rhizosphere and maize development stages influenced beta diversity according to statistical analysis. The GM maize line 2A-7 had no significant influence on the composition of microbial communities when compared to B73. Compared to RS and SS, the host niche RT tended to deplete Chloroflexi, Gemmatimonadetes and Mortierellomycota at phylum level. Nitrogen-fixation bacteria Pseudomonas, Herbaspirillum huttiense, Rhizobium leguminosarum, and Sphingomonas azotifigens were found to be enriched in the niche RT in comparison to RS and SS, whilst Bacillus was found to be increased and Stenotrophomonas was found to be decreased at the maturing stage as compared to jointing and flowering stages. The nitrogen fixation protein FixH (clusters of orthologous groups, COG5456), was found to be abundant in RT. Furthermore, the pathogen fungus that causes maize stalk rot, Gaeumannomyces radicicola, was found to be abundant in RT, while the beneficial fungus Mortierella hyalina was found to be depleted in RT. Lastly, the abundance of G. radicicola gradually increased during the development of maize. In conclusion, the host niches throughout the soil-plant continuum rather than the Bt insect-resistant gene or Bt protein secretion were primarily responsible for the differential assembly of root-associated microbial communities in GM maize, which provides the theoretical basis for ecological agriculture.

Natural shikonin and acetyl-shikonin improve intestinal microbial and protein composition to alleviate colitis-associated colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) and inflammatory bowel disease (IBD) are the most common diseases of human digestive system. Nowadays, the influence of the inflammatory microenvironment on tumorigenesis has become a new direction, and the exploration of relative molecular mechanism will facilitate the discovery and identification of novel potential anti-cancer molecules. METHODS: Natural shikonin (SK) and acetyl-shikonin (acetyl-SK) was administered to azoxymethane (AOM)/dextran sodium sulphate (DSS)-induced colitis-associated colorectal cancer (CAC) mice model by gavage to investigate their therapeutic effects. Moreover, fresh feces and colon tissues were collected for determining the function of SK and acetyl-SK on the gut microbes and protein expression, respectively. RESULTS: Both SK and acetyl-SK decreased AOM/DSS-induced CAC, and regulated the intestinal flora structure in CAC mouse model. They, especially SK, improved species richness, evenness and diversity of intestinal flora, recovered the upregulated ratio of Firmicutes to Bacteroidota (F/B ratio) which symbolizes gut microbiota dysbiosis. SK and its derivative increased the beneficial bacteria g__norank_f__Muribaculaceae, Lactobacillus, Lachnospiraceae_NK4A136_Group, and reduced those harmful ones including Ileibacterium and Coriobacteriaceae UCG-002. Notably, AOM/DSS caused significant increase in the abundance of Ileibaterium valens and g__norank_f__norank_o__Clostridia_UCG-014, which were not previously reported in studies of colonic inflammation or cancer, and the disorder was reversed by 20 mg/kg of SK. In our current study, the action of SK and acetyl-SK is dose-dependent, and 20 mg/kg SK exhibited the most effective functions, even better than the positive drug mesalazine. Moreover, differential proteomics and ELISA results showed that SK could recover the increase of pro-inflammatory cytokines (including IL-1ß, IL-6 and TNF-α), the upregulation of pyruvate kinase isozyme type M2 (PKM2) and some other proteins (mainly concentrated in transcriptional mis-regulation in cancer and IL-17 signaling pathways), and the downregulation of Aldh1b1-Acc3-Maoa and Μgt2b34-Aldh1a1-Aldh1a7 involved in Wnt/ß-catenin signaling pathway. CONCLUSION: Our study identified SK and acetyl-SK, especially SK, as potential preventive agents for CAC through regulating both gut microbes and pathways involved in inflammation and cancer such as Wnt/ß-catenin signaling pathway.

Anti-microbial efficacy, mechanisms and druggability evaluation of the natural flavonoids.

AIMS: This study was conducted to evaluate 35 natural flavonoids for their in vitro susceptibility against E. coli (ATCC 25922), Ps. aeruginosa (ATCC 27853), B. subtilis (ATCC 530) and Staph. aureus (ATCC 6538) in search of a potential broad-spectrum antibiotic. METHODS AND RESULTS: Glabridin, a natural isoflavonoid isolated from Glycyrrhiza glabra L., was identified to be highly active with a MIC of 8-16 µg ml-1 against Staph. aureus, B. subtilis and E. coli. By the results of the docking simulation, we located the potential targets of glabridin as DNA gyrase and dihydrofolate reductase (DHFR). The subsequent DNA gyrase inhibition assays (glabridin: IC50  = 0.8516 µmol L-1 , ciprofloxacin: IC50  = 0.04697 µmol L-1 ), DHFR inhibition assays (glabridin: inhibition ratio = 29%, methotrexate: inhibition ratio = 45% under 100 µmol L-1 treatment) and TUNEL confirmed that glabridin acted as DNA gyrase inhibitor and DHFR mild inhibitor, exerting bactericidal activity by blocking bacterial nucleic acid synthesis. CCK-8 and in silico calculations were also conducted to verify the low cytotoxicity and acceptable druggability of glabridin. CONCLUSION: These findings suggest that glabridin represents the prototypical member of an exciting structural class of natural antimicrobial agents. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a novel mechanism of bactericidal activity of glabridin against Staph. aureus.

PLA2G6 Silencing Suppresses Melanoma Progression and Affects Ferroptosis Revealed by Quantitative Proteomics.

Although phospholipase A2 group VI (PLA2G6) is involved in oncogenesis in several human tumors, its expression and role in cutaneous malignant melanoma (CMM) pathogenesis remains unclear. Here, by using the Oncomine and CCLE online database, immunohistochemistry, RT-qPCR, and western blotting analysis, we revealed that PLA2G6 was markedly up-regulated in CMM tissues compared to nevus tissues, as well as remarkably increased in vitro in SK-MEL-28 and M14 melanoma cell lines compared to human melanocytes. In vivo, PLA2G6 was also elevated in nine melanoma tissues compared to adjacent tissues. To investigate the malignant behaviors of PLA2G6 in CMM, SK-MEL-28 and M14 cell lines with PLA2G6 stable knockdown by RNAi strategy were constructed. Through CCK8 and colony formation assays in vitro and xenograft tumor experiment in vivo, we found that knockdown of PLA2G6 dramatically inhibited cell proliferation. The results of scratch-wound and transwell assays suggested that the migration and invasion of melanoma cells were prominently suppressed after silencing PLA2G6. In addition, flow cytometry showed that the knockdown of PLA2G6 promoted the apoptosis rate of melanoma cells. To further explore the potential molecular mechanism, we used liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) proteomic and bioinformatics analysis. The GO and KEGG analysis suggested that the underlying mechanism of PLA2G6 in CMM might be associated with the ferroptosis pathway, and ferroptosis-related proteins were validated to be differentially expressed in PLA2G6 knockdown SK-MEL-28 and M14 cells. Together, these results suggested that PLA2G6 knockdown significantly inhibited cell proliferation, metastasis, and promoted apoptosis in melanoma. Our findings on the biological function of PLA2G6 and the underlying association between PLA2G6 and ferroptosis in melanoma may contribute to developing a potential therapeutic strategy for melanoma.

Shikonin N-benzyl matrinic acid ester derivatives as novel telomerase inhibitors with potent activity against lung cancer cell lines.

In this study, a series of novel shikonin N-benzyl matrinic acid ester derivatives (PMMB-299-PMMB-310) were synthesized and tested for their ability to inhibit the proliferation of cancer cells. Compared with shikonin and matrine, some of the ester derivatives were found to exhibit better anti-proliferative activity against seven different cancer cell lines, with less cytotoxicity toward non-cancerous cells. The strongest anti-proliferative activity was exhibited by PMMB-302, which had an IC50 value of 2.71 µM against A549 cells. The compound caused cell cycle arrest in the G2/M phase and induced apoptosis. Effects on the expression of apoptosis-related molecules such as Bcl2, Bcl-XL, caspase-3, caspase-9 and FADD suggested that PMMB-302 has tumor suppressive roles in lung cancer cells. In addition, PMMB-302 inhibited expression of telomerase core proteins, dyskerin and NHP2, and telomerase reverse transcriptase RNA. Moreover, molecular docking of PMMB-302 was subsequently conducted to determine the probable binding mode with telomerase. Taken together, the results indicate that PMMB-302 acts as a tumor suppressor in lung cancer cells by negatively regulating telomerase expression.