Tagging large CNV blocks in wheat boosts digitalization of germplasm resources by ultra-low-coverage sequencing.

BACKGROUND: The massive structural variations and frequent introgression highly contribute to the genetic diversity of wheat, while the huge and complex genome of polyploid wheat hinders efficient genotyping of abundant varieties towards accurate identification, management, and exploitation of germplasm resources. RESULTS: We develop a novel workflow that identifies 1240 high-quality large copy number variation blocks (CNVb) in wheat at the pan-genome level, demonstrating that CNVb can serve as an ideal DNA fingerprinting marker for discriminating massive varieties, with the accuracy validated by PCR assay. We then construct a digitalized genotyping CNVb map across 1599 global wheat accessions. Key CNVb markers are linked with trait-associated introgressions, such as the 1RS·1BL translocation and 2NvS translocation, and the beneficial alleles, such as the end-use quality allele Glu-D1d (Dx5 + Dy10) and the semi-dwarf r-e-z allele. Furthermore, we demonstrate that these tagged CNVb markers promote a stable and cost-effective strategy for evaluating wheat germplasm resources with ultra-low-coverage sequencing data, competing with SNP array for applications such as evaluating new varieties, efficient management of collections in gene banks, and describing wheat germplasm resources in a digitalized manner. We also develop a user-friendly interactive platform, WheatCNVb ( http://wheat.cau.edu.cn/WheatCNVb/ ), for exploring the CNVb profiles over ever-increasing wheat accessions, and also propose a QR-code-like representation of individual digital CNVb fingerprint. This platform also allows uploading new CNVb profiles for comparison with stored varieties. CONCLUSIONS: The CNVb-based approach provides a low-cost and high-throughput genotyping strategy for enabling digitalized wheat germplasm management and modern breeding with precise and practical decision-making.

VPAC2 Receptor Signaling Promotes Growth and Immunosuppression in Pancreatic Cancer.

Pancreatic cancer (PDAC) harbors a complex tumor microenvironment (TME), and crosstalk between cells in the TME can contribute to drug resistance and relapse. Vasoactive intestinal peptide (VIP) is overexpressed in PDAC, and VIP receptors expressed on T cells are a targetable pathway that sensitizes PDAC to immunotherapy. In this study, we showed that pancreatic cancer cells engage in autocrine VIP signaling through VIP receptor 2 (VPAC2). High co-expression of VIP with VPAC2 correlated with reduced relapse-free survival in PDAC patients. VPAC2 activation in PDAC cells upregulated piwi-like RNA-mediated gene silencing 2 (Piwil2), which stimulated cancer cell clonogenic growth. In addition, VPAC2 signaling increased expression of TGF-ß1 to inhibit T cell function. Loss of VPAC2 on PDAC cells led to reduced tumor growth and increased sensitivity to anti-PD1 immunotherapy in mouse models of PDAC. Overall, these findings expand our understanding of the role of VIP/VPAC2 signaling in PDAC and provide the rationale for developing potent VPAC2-specific antagonists for treating PDAC patients.

Innovative computational tools provide new insights into the polyploid wheat genome.

Bread wheat (Triticum aestivum) is an important crop and serves as a significant source of protein and calories for humans, worldwide. Nevertheless, its large and allopolyploid genome poses constraints on genetic improvement. The complex reticulate evolutionary history and the intricacy of genomic resources make the deciphering of the functional genome considerably more challenging. Recently, we have developed a comprehensive list of versatile computational tools with the integration of statistical models for dissecting the polyploid wheat genome. Here, we summarize the methodological innovations and applications of these tools and databases. A series of step-by-step examples illustrates how these tools can be utilized for dissecting wheat germplasm resources and unveiling functional genes associated with important agronomic traits. Furthermore, we outline future perspectives on new advanced tools and databases, taking into consideration the unique features of bread wheat, to accelerate genomic-assisted wheat breeding.

Prognostic factors in surgically treated tongue squamous cell carcinoma in stage T1-2N0-1M0: A retrospective analysis.

PURPOSE: The study aimed to retrospectively identify the prognostic factors of surgically treated primary tongue squamous cell carcinoma (TSCC) cases and assess the benefits of surgical neck lymph node dissection (LND) in early-stage cancer. METHODS: Patients with primary TSCC with pT1-2N0-1M0 stage without distant metastasis who were treated with surgery during 2014-2016 at Xiangya Hospital, Central South University were included. Univariate and multivariate Cox models were constructed to explore prognostic factors of overall survival (OS), disease-free survival (DFS), and local recurrence-free survival (LRFS). Sub-group analysis was used to assess the effect of adjuvant therapy and the prognostic value of LND for the early-stage patients. RESULTS: In total, 440 patients met the inclusion criteria. During the follow-up period, the 5-year OS, DFS, were 84.4% and 70.0%, respectively. Univariate analysis showed that TNM stage, lymphovascular invasion (LVI), and/or perineural invasion (PNI), pathological differentiation, etc. were significant predictors of OS and DFS. Multivariate analysis showed that TNM stage and the degree of pathological differentiation were independent prognostic factors for all outcomes. Besides, the number of cervical LND could independently predict both DFS and LRFS while LVI/PNI were associated with DFS. And high-quality neck LND (≥30) significantly improved DFS and LRFS for patients of pT1cN0M0 or stage I as compared to those without LND. CONCLUSIONS: TNM stage and pathological differentiation were crucial prognostic factors for postoperative patients with TSCC. Notably, high-quality cervical LND was beneficial for the improvement of DFS and LRFS for patients of pT1cN0M0 or stage I.

Hyperbranched Polymer Dots with Strong Absorption and High Fluorescence Quantum Yield for In Vivo NIR-II Imaging.

In order to improve the fluorescence quantum yield (QY) of NIR-II-emitting nanoparticles, D-A-D fluorophores are typically linked to intramolecular rotatable units to reduce aggregation-induced quenching. However, incorporating such units often leads to a twisted molecular backbone, which affects the coupling within the D-A-D unit and, as a result, lowers the absorption. Here, we overcome this limitation by cross-linking the NIR-II fluorophores to form a 2D polymer network, which simultaneously achieves a high QY by well-controlled fluorophore separation and strong absorption by restricting intramolecular distortion. Using the strategy, we developed polymer dots with the highest NIR-II single-particle brightness among reported D-A-D-based nanoparticles and applied them for imaging of hindlimb vasculatures and tumors as well as fluorescence-guided tumor resection. The high brightness of the polymer dots offered exceptional image quality and excellent surgical results, showing a promising performance for these applications.

The RNA-binding protein LRPPRC promotes resistance to CDK4/6 inhibition in lung cancer.

Kinase inhibitors against Cyclin Dependent Kinase 4 and 6 (CDK4/6i) are promising cancer therapeutic drugs. However, their effects are limited by primary or acquired resistance in virtually all tumor types. Here, we demonstrate that Leucine Rich Pentatricopeptide Repeat Containing (LRPPRC) controls CDK4/6i response in lung cancer by forming a feedback loop with CDK6. LRPPRC binds to CDK6-mRNA, increasing the stability and expression of CDK6. CDK6 and its downstream E2F Transcription Factor 1 (E2F1), bind to the LRPPRC promoter and elevate LRPPRC transcription. The activation of the LRPPRC-CDK6 loop facilitates cell cycle G1/S transition, oxidative phosphorylation, and cancer stem cell generation. Gossypol acetate (GAA), a gynecological medicine that has been repurposed as a degrader of LRPPRC, enhances the CDK4/6i sensitivity in vitro and in vivo. Our study reveals a mechanism responsible for CDK4/6i resistance and provides an enlightening approach to investigating the combinations of CDK4/6 and LRPPRC inhibitors in cancer therapy.

A novel method for the component identification of human blood products: Mass spectrometric analysis of human fibrinogen digested after SDS-PAGE in-gel digestion.

Human fibrinogen, as a blood product of special origin, is relatively simple to prepare and purify. Therefore, completely isolating and removing the relevant impurity proteins is difficult. Further, which impurity protein components are present is not clear. In this study, human fibrinogen products from seven enterprises were collected from the market, and the presence of impurity proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, the major 12 impurity proteins were identified and screened by in-gel enzymolysis mass spectrometry, and 7 major impurity proteins with different peptide coverage were identified by enzyme-linked immunosorbent assay, in agreement with the mass spectrometry results. The seven major impurity proteins included fibronectin, plasminogen, F-XIII, F-VIII, complement factor H, cystatin-A, and α-2-macroglobulin. The final test results were in the range of undetectable to 50.94 µg/mL, with correspondingly low levels of impurity proteins between different companies and a manageable risk. Moreover, we found that these impurity proteins existed in the form of polymers, which might also be an important cause of adverse reactions. This study established a protein identification technique applicable to fibrinogen products, which provided new ideas for studying the protein composition of blood products. In addition, it provided a new means of testing for companies to monitor the flow of proteomic fractions and improve the purification yield and product quality. It laid the foundation for reducing the risk of clinical adverse reactions.

Mitochondrial Protease Targeting Chimeras for Mitochondrial Matrix Protein Degradation.

Targeted protein degradation (TPD) is an emerging technique for protein regulation. Currently, all TPD developed in eukaryotic cells relies on either ubiquitin-proteasome or lysosomal systems, thus are powerless against target proteins in membrane organelles lacking proteasomes and lysosomes, such as mitochondria. Here, we developed a mitochondrial protease targeting chimera (MtPTAC) to address this issue. MtPTAC is a bifunctional small molecule that can bind to mitochondrial caseinolytic protease P (ClpP) at one end and target protein at the other. Mechanistically, MtPTAC activates the hydrolase activity of ClpP while simultaneously bringing target proteins into proximity with ClpP. Taking mitochondrial RNA polymerase (POLRMT) as a model protein, we have demonstrated the powerful proteolytic ability and antitumor application prospects of MtPTAC, both in vivo and in vitro. This is the first modularly designed TPD that can specifically hydrolyze target proteins inside mitochondria.

RuCu Nanosheets with Ultrahigh Nanozyme Activity for Chemodynamic Therapy.

Nanoenzymes have been widely explored for chemodynamic therapy (CDT) in cancer treatment. However, poor catalytic efficiency of nanoenzymes, especially in the tumor microenvironment with insufficient H2 O2 and mild acidity, limits the effect of CDT. Herein, a new ultrathin RuCu nanosheet (NS) based nanoenzyme which has a large specific surface area and abundant channels and defects is developed. The RuCu NSs show superb catalytic efficiency for the oxidation of peroxidase substrate H2 O2 at a wide range of pH and their catalytic efficiency (kcat /Km = 177.2 m-1  s-1 ) is about 14.9 times higher than that of the single-atom catalyst FeN3 P. Besides being an efficient nanozyme as peroxidase, the RuCu NSs possess other two enzyme activities, not only disproportionating superoxide anion to produce H2 O2 but also consuming glutathione to keep a high concentration of H2 O2 in the tumor microenvironment for Fenton reaction. With these advantages, the RuCu NSs exhibit good performance to kill cancer cells and inhibit tumor growth in mice, demonstrating a promising potential as new CDT reagent.

Transcriptomic analysis of the effect of deferoxamine exposure on the growth, photosynthetic activity and iron transfer of Microcystis aeruginosa.

Deferoxamine (DFB) is a trihydroxamic acid siderophore that chelates with iron (Fe) to form iron-siderophore complexes. The existence of siderophores in nature changes the form of iron and affects the absorption and utilization of iron by organisms. However, the relationship between siderophores and the growth of Cyanobacteria is largely unknown. In this study, the cellular and transcriptomic responses to the addition of DFB were investigated. A high concentration of DFB (12 mg/L) significantly inhibited the growth of Cyanobacteria cells, reduced photosynthetic activity, and induced the production of peroxidase, with the highest inhibition rate of algal growth of 74.82%. These indexes were also affected for the low (3 mg/L) and medium concentration (6 mg/L) groups, but this difference is closely related to the growth stage of Cyanobacteria cells. This may be due to competition between the cell-associated iron-binding part/system and the extracellular Fe (Ⅲ)-DFB ligand. Transcriptome results showed that most of the genes involved in iron uptake and transport were down-regulated, and only the fur gene encoding the iron uptake regulator protein was significantly up-regulated. Most genes related to photosynthesis, glycolysis, and fatty acid metabolism were also down-regulated, while the obvious up-regulation of a few genes may be a complex regulation in response to the down-regulation of most genes. These findings will provide important insights into the effects of siderophores on iron bioavailability in algae.

Label-Free SERS Analysis of Serum Using Ag NPs/Cellulose Nanocrystal/Graphene Oxide Nanocomposite Film Substrate in Screening Colon Cancer.

Label-free surface-enhanced Raman scattering (SERS) analysis shows tremendous potential for the early diagnosis and screening of colon cancer, owing to the advantage of being noninvasive and sensitive. As a clinical diagnostic tool, however, the reproducibility of analytical methods is a priority. Herein, we successfully fabricated Ag NPs/cellulose nanocrystals/graphene oxide (Ag NPs/CNC/GO) nanocomposite film as a uniform SERS active substrate for label-free SERS analysis of clinical serum. The Ag NPs/CNC/GO suspensions by self-assembling GO into CNC solution through in-situ reduction method. Furthermore, we spin-coated the prepared suspensions on the bacterial cellulose membrane (BCM) to form Ag NPs/CNC/GO nanocomposite film. The nanofilm showed excellent sensitivity (LOD = 30 nM) and uniformity (RSD = 14.2%) for Nile Blue A detection. With a proof-of-concept demonstration for the label-free analysis of serum, the nanofilm combined with the principal component analysis-linear discriminant analysis (PCA-LDA) model can be effectively employed for colon cancer screening. The results showed that our model had an overall prediction accuracy of 84.1% for colon cancer (n = 28) and the normal (n = 28), and the specificity and sensitivity were 89.3% and 71.4%, respectively. This study indicated that label-free serum SERS analysis based on Ag NPs/CNC/GO nanocomposite film combined with machine learning holds promise for the early diagnosis of colon cancer.

Distribution characteristics of selenium, cadmium and arsenic in rice grains and their genetic dissection by genome-wide association study.

High selenium (Se) and low cadmium (Cd) and arsenic (As) contents in rice grains were good for human health. The genetic basis and relationship of Se, Cd and As concentrations in rice grains are still largely unknown. In the present study, large variations were observed in Se, Cd and As concentrations in brown and milled rice in normal and Se treatment conditions in 307 rice accessions from 3K Rice Genomes Project. Se fertilizer treatment greatly increased Se concentrations but had no obvious changes in concentrations of Cd and As both in brown and milled rice. Total of 237 QTL were identified for Se, Cd and As concentrations in brown and milled rice in normal and Se treatment conditions as well as ratio of concentrations under Se treatment to normal conditions. Only 19 QTL (13.4%) were mapped for concentrations of Se and Cd, Se and As, and Se, Cd and As in the same or adjacent regions, indicating that most Se concentration QTL are independent of Cd and As concentration QTL. Forty-three favorable alleles were identified for 40 candidate genes by gene-based association study and haplotype analysis in 14 important QTL regions. Se-enriched rice variety will be developed by pyramiding favorable alleles at different Se QTL and excluding undesirable alleles at Cd and As QTL, or combining favorable alleles at Se QTL with the alleles at Se-sensitive QTL by marker-assisted selection.

Dispersed emergence and protracted domestication of polyploid wheat uncovered by mosaic ancestral haploblock inference.

Major crops are all survivors of domestication bottlenecks. Studies have focused on the genetic loci related to the domestication syndrome, while the contribution of ancient haplotypes remains largely unknown. Here, an ancestral genomic haploblock dissection method is developed and applied to a resequencing dataset of 386 tetraploid/hexaploid wheat accessions, generating a pan-ancestry haploblock map. Together with cytoplastic evidences, we reveal that domesticated polyploid wheat emerged from the admixture of six founder wild emmer lineages, which contributed the foundation of ancestral mosaics. The key domestication-related loci, originated over a wide geographical range, were gradually pyramided through a protracted process. Diverse stable-inheritance ancestral haplotype groups of the chromosome central zone are identified, revealing the expanding routes of wheat and the trends of modern wheat breeding. Finally, an evolution model of polyploid wheat is proposed, highlighting the key role of wild-to-crop and interploidy introgression, that increased genomic diversity following bottlenecks introduced by domestication and polyploidization.

A butenolide signaling system synergized with biosynthetic gene modules led to effective activation and enhancement of silent oviedomycin production in Streptomyces.

Secondary metabolic gene clusters widely exist in the genomes of Streptomyces but mostly remain silent. To awaken this hidden reservoir of natural products, various strategies concerning secondary metabolic pathways are applied. Here, we describe that butenolide signaling molecule deficiency and glucose addition can interdependently activate the expression of silent oviedomycin biosynthetic gene clusters in Streptomyces ansochromogenes and Streptomyces antibioticus. Since oviedomycin is a promising anti-tumor lead compound, in order to improve its yield, we use the cluster-situated genes (ovmF, ovmG, ovmI and ovmH) encoding the enzymes for acyl carrier protein modification and precursor biosynthesis, and the discrete precursor biosynthetic genes (pyk2, gap1 and accA2) involved in glycolysis to assemble two gene modules (pFGIH and pPGA). Their co-overexpression in ΔsabA (a disruption mutant of sabA encoding SAB synthase) has superimposed effect on the yield of oviedomycin, which can be further increased to 59-fold in the presence of galactose as optimal carbon source. This is the most unambiguous evidence that butenolide signaling system can synergize with the optimization of primary metabolism to regulate the expression of secondary metabolic gene clusters, providing efficient strategies for mining natural products of Streptomyces.

Roles of PTEN inactivation and PD-1/PD-L1 activation in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal cancer in China and developing countries. The purpose of this review is to summarize the roles of inactivation of the tumor suppressor gene, phosphatase and tensin homolog (PTEN), and activation of the programmed cell death protein 1 (PD-1) upon binding to its ligand (PD-L1) in the promotion of ESCC. Studies of ESCC performed in vitro and in vivo indicated that PTEN and PD-L1 function in the regulation of cell proliferation, invasion, and migration; the epithelial-mesenchymal transition; resistance to chemotherapy and radiotherapy; and the PI3K/AKT signaling pathway. Certain genetic variants of PTEN are related to susceptibility to ESCC, and PTEN and PD-L1 also function in ESCC progression and affect the prognosis of patients with ESCC. There is also evidence that the expression of PD-L1 and PTEN are associated with the progression of certain other cancers. Future studies should further examine the relationship of PD-L1 and PTEN and their possible interactions in ESCC.

Artesunate restrains the malignant progression of human cutaneous squamous cell carcinoma cells via the suppression of the PI3K/AKT pathway.

PURPOSE: Artesunate (ART) is recognized for its anticancer activity, but a few studies concentrate on its anti-skin cancer effect. This study emphazied this aspect and preliminarily discussed the impact and mechanism of ART on cutaneous squamous cell carcinoma (CSCC). METHODS: The viability of HaCaT and CSCC cells treated with ART (0, 30, 60, 90, 120, 150, 180, or 210 µmol/L) for 48 h were assessed utilizing cell counting kit-8. Next, the migration, invasion, proliferation, apoptosis, and cell cycle of CSCC cells treated with ART were evaluated by dint of cell function experiments. Then, cell cycle-, apoptosis-, and phosphatidylinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway-related markers were examined via western blot or quantitative real-time PCR. Moreover, the influences of ART and PI3K/AKT agonist IGF-I on CSCC cell biological behaviors were gauged again. RESULTS: The suppressive role of ART (30, 60, 90, 120, 150, 180, or 210 µmol/L) was stronger in viability of CSCC cells than in viability of HaCaT cells. ART evidently attenuated the migration, invasion and proliferation, lessened cell numbers at G2/M phase and triggered apoptosis of CSCC cells. At the molecular level, ART regulated cell cycle-, apoptosis-, and PI3K/AKT pathway-related markers in CSCC cells. Moreover, the suppression of ART on CSCC cell malignant phenotypes was reversed by PI3K/AKT agonist IGF-I. CONCLUSION: ART restrains the malignant progression of CSCC, which may be intensely related to the PI3K/AKT pathway repression.

Self-Assembled Micellar Glutaminase Allosteric Inhibitor for Effective Therapeutic Intervention.

INTRODUCTION: Kidney-type glutaminase (KGA) has been an important anti-tumor drug target, and KGA allosteric inhibitors attracted much interest for their superior enzymatic specificity with good drug safety profiles. For glutaminase allosteric inhibitors such as BPTES, CB-839 and Selen derivatives, the low solubility remains as the main factor that limits in vivo efficacy. The 1,3,4-Selenadiazole compound CPD 23 showed improved in vivo efficacy but worse solubility; however, the graft polymer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol (PVCap-PVA-PEG), Soluplus® (SOL) stood out as an excellent delivery carrier for CPD 23. METHODS: The CPD 23@SOL micelles were prepared, optimized and evaluated through on the basis of solubility improvement and loading capacity. Characterizations of particle size and Zeta potential by dynamic light scattering, morphology by transmission electron microscopy and solid state by X-ray powder diffraction were closely conducted. The biological studies included the tumor cell growth inhibition, blood and liver microsomal stability, in vivo pharmacokinetics and tissue biodistribution. RESULTS: At 1:20 ratio of CPD 23:SOL, CPD 23@SOL micelles were well-dispersed, spherical and stable, with size less than 200 nm with encapsulation efficiency of more than 90%. This SOL micellar system significantly increased the aqueous solubility of CPD 23 by 15,000 folds. Particularly, CPD 23@SOL micelles demonstrated higher stability in blood and liver microsomes, showing approximately 86% remaining at 2 h incubation and about 66% at 4 h, respectively. In addition, with or without micellar formulation, CPD 23 maintained essentially the same inhibitory activity in tumor cells. Interestingly, CPD 23@SOL micelles significantly improved the pharmacokinetic exposure, prolonged the in vivo circulation and dramatically changed tissue biodistributions of CPD 23. CONCLUSION: The current work provided an encouraging and practical delivery system for novel Selenadiazoles and glutaminase allosteric inhibitors whose poor water-soluble characteristic has been a bottleneck for the field.

Extracellular vesicles isolated by size-exclusion chromatography present suitability for RNomics analysis in plasma.

BACKGROUND: Extracellular vesicles (EVs), known as cell-derived membranous structures harboring a variety of biomolecules, have been widely used in liquid biopsy. Due to the complex biological composition of plasma, plasma RNA omics analysis (RNomics) is easily affected, thus it is necessary to select an optimal strategy from exiting methods according to the performance for intended application. METHODS: In this study, four different strategies for EVs isolation were performed and compared (i.e. ultracentrifugation (UC), size exclusion chromatography (SEC), and two most frequently-used commercially available isolation kit (ExoQuick and exoEasy). We compared the yield, purity, PCR quantification of RNAs, miRNA-seq analyses and mRNA-seq analyses of RNAs from EVs isolated using four methods. RESULTS: The results showed that the lowest miRNA binding protein AGO2 (Argonaute-2) and the highest EVs-specific miRNA and lncRNA were observed in EVs obtained through SEC, meanwhile the content of the non-specific miRNA was the lowest. Further RNA-Seq data revealed that RNAs obtained via SEC presented more useful reads for both miRNA and mRNA. Furthermore, the mRNA delivered via SEC tended to have a concentration comparable to the ideal FPKM (Fragments Per Kilobase Million) value. CONCLUSIONS: SEC shall be used as an optimal strategy for the isolation of EVs in plasma RNomics analysis.

Biodegradable self-assembly micelles significantly enhanced the solubility, biological stability and in vivo antitumor efficacy of Hexylselen.

Glutaminolysis inhibitors have shown early promise in cancer therapeutics. Specifically, kidney-type glutaminase (KGA) has been a long-standing anti-tumor drug target; KGA allosteric inhibitors have attracted great attention due to their superior enzyme specificity and good drug safety profiles. However, the main issue with allosteric inhibitors-including BPTES, CB-839, and the recently developed KGA allosteric and glutamate dehydrogenase (GDH) dual inhibitor, Hexylselen (CPD-3B)-is their low solubility; it leads to limited in vivo efficacy. To optimize their formulation, various delivery carriers were screened in the present study. Soluplus® (SOL), an amphiphilic graft polymer, showed an interesting structure-solubility/activity relationship with Selen molecules containing different middle chain sizes. Among these molecules, the long chain molecule CPD-3B showed 3000-fold increased solubility with SOL, forming well-dispersed and stable micelles 60-80 nm in size. Moreover, CPD-3B@SOL micelles exhibited good metabolic stability in both blood and liver microsomes. These advantages significantly enhanced the bioavailability and in vivo antitumor efficacy of CPD-3B@SOL micelles in the H22 hepatocarcinoma xenograft mouse model. Thus, the current study provided a practical delivery system for allosteric inhibitors of glutaminase, which is one of the bottlenecks of targeting tumor glutaminolysis.

Reconstitution of a mini-gene cluster combined with ribosome engineering led to effective enhancement of salinomycin production in Streptomyces albus.

Salinomycin, an FDA-approved polyketide drug, was recently identified as a promising anti-tumour and anti-viral lead compound. It is produced by Streptomyces albus, and the biosynthetic gene cluster (sal) spans over 100 kb. The genetic manipulation of large polyketide gene clusters is challenging, and approaches delivering reliable efficiency and accuracy are desired. Herein, a delicate strategy to enhance salinomycin production was devised and evaluated. We reconstructed a minimized sal gene cluster (mini-cluster) on pSET152 including key genes responsible for tailoring modification, antibiotic resistance, positive regulation and precursor supply. These genes were overexpressed under the control of constitutive promoter PkasO* or Pneo . The pks operon was not included in the mini-cluster, but it was upregulated by SalJ activation. After the plasmid pSET152::mini-cluster was introduced into the wild-type strain and a chassis host strain obtained by ribosome engineering, salinomycin production was increased to 2.3-fold and 5.1-fold compared with that of the wild-type strain respectively. Intriguingly, mini-cluster introduction resulted in much higher production than overexpression of the whole sal gene cluster. The findings demonstrated that reconstitution of sal mini-cluster combined with ribosome engineering is an efficient novel approach and may be extended to other large polyketide biosynthesis.