Genomic characterization and related functional genes of γ- poly glutamic acid producing Bacillus subtilis.

γ- poly glutamic acid (γ-PGA), a high molecular weight polymer, is synthesized by microorganisms and secreted into the extracellular space. Due to its excellent performance, γ-PGA has been widely used in various fields, including food, biomedical and environmental fields. In this study, we screened natto samples for two strains of Bacillus subtilis N3378-2at and N3378-3At that produce γ-PGA. We then identified the γ-PGA synthetase gene cluster (PgsB, PgsC, PgsA, YwtC and PgdS), glutamate racemase RacE, phage-derived γ-PGA hydrolase (PghB and PghC) and exo-γ-glutamyl peptidase (GGT) from the genome of these strains. Based on these γ-PGA-related protein sequences from isolated Bacillus subtilis and 181 B. subtilis obtained from GenBank, we carried out genotyping analysis and classified them into types 1-5. Since we found B. amyloliquefaciens LL3 can produce γ-PGA, we obtained the B. velezensis and B. amyloliquefaciens strains from GenBank and classified them into types 6 and 7 based on LL3. Finally, we constructed evolutionary trees for these protein sequences. This study analyzed the distribution of γ-PGA-related protein sequences in the genomes of B. subtilis, B. velezensis and B. amyloliquefaciens strains, then the evolutionary diversity of these protein sequences was analyzed, which provided novel information for the development and utilization of γ-PGA-producing strains.

Preparation of lignin-based full-color carbon quantum dots and their multifunctionalization with waterborne polyurethanes.

Lignin is a popular material for energy transition and high-value utilization due to its low cost, non-toxicity, renewability, and widespread availability. However, its complex structure has hindered its application. Waterborne polyurethane (WPU) uses water as a dispersion medium, which is safer for humans and the environment but also leads to disadvantages such as poor mechanical properties and water resistance. In this study, we prepared multicolor photoluminescent carbon quantum dots (CQDs) in a wide range of wavelengths from lignin. We successfully prepared panchromatic CQDs by additive mixing. The redshift of the emission wavelength is attributed to the synergistic effect of the sp2 conjugated structure and the surface functional groups. The full-color solid-state luminescence of the CQDs was successfully achieved, and most importantly, the application of full-color CQDs in light-emitting diodes was realized. Moreover, the embedding of the multicolor CQDs in WPU not only makes WPU luminescent but also improves the water resistance and mechanical properties of WPUs. The hydrogen-bonding interactions between the functional groups on the surface of the CQDs and the urethane were responsible for the high performance of the composite. We investigated the UV and strong blue light shielding abilities of WPU/yellow CQDs films, which resulted from the unique absorption peaks of yellow CQDs in the UV region and the strong blue light region. This work provides an efficient method for the high-value utilization of biomass materials and paves the way for the multifunctional application of WPU.

The assessment of circulating tumor DNA associated with Wnt/ß-catenin signaling pathway as a diagnostic tool for liver cancer: a systematic review and meta-analysis.

BACKGROUND: Circulating tumor DNA (ctDNA) in peripheral blood has become a promising noninvasive biomarker. However, the diagnostic potential of Wnt/ß-catenin signaling pathway-related ctDNA for liver cancer is controversial. Here, we aimed to access the diagnostic potential and clinicopathological features of Wnt/ß-catenin signaling pathway-related ctDNA in liver cancer and provide data support for its clinical diagnosis and treatment. METHODS: A comprehensive literature search was conducted to identify the relevant studies. The methodological quality of the included studies was evaluated using the QUADAS-2 tool. The bivariate linear mixed models were used. RESULTS: The AUC (area under the curve), pooled sensitivity and specificity were 0.77, 0.42 and 0.98, respectively. The findings suggested that control type, sample source, research methods and thresholds were the potential sources of heterogeneity (p < 0.05). Additionally, this study also found that there were significant correlations between the hypermethylation of Wnt/ß-catenin signaling pathway-related ctDNA and tumor size, TNM stage, distant metastasis, and HBV infection(p < 0.05). CONCLUSION: This study confirmed that Wnt/ß-catenin signaling pathway-related ctDNA had the better diagnostic potential for liver cancer and might be an effective complementary tool for serum AFP assays in the early diagnosis of liver cancer. PROSPERO: (No. CRD42023404984).[Figure: see text].

Split-Cas9-based targeted gene editing and nanobody-mediated proteolysis-targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells.

BACKGROUND: Precise regulation of partial critical proteins in cancer cells, such as anti-apoptotic proteins, is one of the crucial strategies for treating cancer and discovering related molecular mechanisms. Still, it is also challenging in actual research and practice. The widely used CRISPR/Cas9-based gene editing technology and proteolysis-targeting chimeras (PROTACs) have played an essential role in regulating gene expression and protein function in cells. However, the accuracy and controllability of their targeting remain necessary. METHODS: Construction of UMUC-3-EGFP stable transgenic cell lines using the Sleeping Beauty system, Flow cytometry, quantitative real-time PCR, western blot, fluorescence microplate reader and fluorescence inverted microscope analysis of EGFP intensity. Characterization of Survivin inhibition was done by using Annexin V-FITC/PI apoptosis, calcein/PI/DAPI cell viability/cytotoxicity assay, cloning formation assay and scratch assay. The cell-derived xenograft (CDX) model was constructed to assess the in vivo effects of reducing Survivin expression. RESULTS: Herein, we established a synergistic control platform that coordinated photoactivatable split-Cas9 targeted gene editing and light-induced protein degradation, on which the Survivin gene in the nucleus was controllably edited by blue light irradiation (named paCas9-Survivin) and simultaneously the Survivin protein in the cytoplasm was degraded precisely by a nanobody-mediated target (named paProtacL-Survivin). Meanwhile, in vitro experiments demonstrated that reducing Survivin expression could effectively promote apoptosis and decrease the proliferation and migration of bladder cancerous cells. Furthermore, the CDX model was constructed using UMUC-3 cell lines, results from animal studies indicated that both the paCas9-Survivin system and paProtacL-Survivin significantly inhibited tumour growth, with higher inhibition rates when combined. CONCLUSIONS: In short, the coordinated regulatory strategies and combinable technology platforms offer clear advantages in controllability and targeting, as well as an excellent reference value and universal applicability in controlling the fate of cancer cells through multi-level regulation of key intracellular factors.

Construction and application of a multifunctional CHO cell platform utilizing Cre/lox and Dre/rox site-specific recombination systems.

During the development of traditional Chinese hamster ovary (CHO) cell lines, target genes randomly integrate into the genome upon entering the nucleus, resulting in unpredictable productivity of cell clones. The characterization and screening of high-yielding cell lines is a time-consuming and expensive process. Site-specific integration is recognized as an effective approach for overcoming random integration and improving production stability. We have designed a multifunctional expression cassette, called CDbox, which can be manipulated by the site-specific recombination systems Cre/lox and Dre/rox. The CDbox expression cassette was inserted at the Hipp11(H11) locus hotspot in the CHO-K1 genome using CRISPR/Cas9 technology, and a compliant CHO-CDbox cell platform was screened and obtained. The CHO-CDbox cell platform was transformed into a pool of EGFP-expressing cells using Cre/lox recombinase-mediated cassette exchange (RMCE) in only 2 weeks, and this expression remained stable for at least 75 generations without the need for drug stress. Subsequently, we used the Dre/rox system to directly eliminate the EGFP gene. In addition, two practical applications of the CHO-CDbox cell platform were presented. The first was the quick construction of the Pembrolizumab antibody stable expression strain, while the second was a protocol for the integration of surface-displayed and secreted antibodies on CHO cells. The previous research on site-specific integration of CHO cells has always focused on the single functionality of insertion of target genes. This newly developed CHO cell platform is expected to offer expanded applicability for protein production and gene function studies.

Antiproliferative and apoptotic activity of gemcitabine-lauric acid conjugate on human bladder cancer cells.

Objectives: Gemcitabine is a first-line drug for the treatment of bladder cancer. One of the most important mechanisms of gemcitabine resistance is the low expression of cellular membrane transporter hENT1. Various derivatives containing fatty acid side chains have been developed in order to facilitate gemcitabine uptake and prolong its retention in cells, such as CP-4126. In this study, the anti-tumor effect and mechanism of a new derivative of gemcitabine named SZY-200 on bladder cancer cells were investigated. SZY-200 was assembled from the gemcitabine-lauric acid conjugate. Materials and Methods: Antiproliferative activities of SZY-200 and lauric acid were evaluated using CCK-8 assay and clonogenic survival assay. The hENT1 inhibitor NBMPR was employed to determine the role of hENT1 in the apoptotic activity of GEM, CP-4126, and SZY-200. RT-qPCR, flow cytometry, fluorescence microscope, western blotting, and wound healing assay were used to study the mechanisms of SZY-200. The target genes were predicted using the BATMAN-TCM database. Results: Our data showed that SZY-200 could inhibit the proliferation of bladder cancer cells by inducing cell cycle arrest and apoptosis. The inhibitory effects were comparable to gemcitabine and CP-4126. SZY-200 does not rely on hENT1 to help it enter bladder cancer cells. Also, we found that lauric acid could inhibit the proliferation of bladder cancer cells. SZY-200 could down-regulate the expressions of PPARG and PTGS2 which were related to the occurrence and development of bladder cancer. Conclusion: SZY-200 has the same or more advantages as CP-4126 and could be an ideal candidate drug for further in vivo investigation.

Multistage targeting and dual inhibiting strategies based on bioengineered tumor matrix microenvironment-mediated protein nanocages for enhancing cancer biotherapy.

Regulation of the apoptotic pathway plays a critical role in inducing tumor cell death and circumventing drug resistance. Survivin protein is the strongest inhibitor of apoptosis found so far. It is highly expressed in several cancers and is a promising target for cancer therapy. However, clinical applications are limited by incomplete inhibition of survivin expression. Here, we present a novel strategy that extended the release of YM155 (an effective survivin inhibitor that works by inhibiting the activity of survivin promoter) and TATm-survivin (T34A) (TmSm) protein (survivin protein mutant with penetrating peptide, a potential anticancer protein therapeutic) via tumor matrix microenvironment-mediated ferritin heavy chain nanocages (FTH1 NCs), enabling significant inhibition of survivin activity at both transcript and protein levels. FTS (FTH1-matrix metalloproteinase-2-TmSm)/YM155 NC synthesis was easily scaled up, and these NCs could sequentially release TmSm protein through matrix metalloproteinase-2 and promote YM155 to enter the nucleus via transferrin receptor 1 (TfR1) binding, which increased the cytotoxicity and apoptosis of Capan-2 and A549 cells compared to that with individual drugs. Moreover, FTS/YM155 NCs enhanced drug accumulation at tumor sites and had a higher tumor inhibition rate (88.86%) than the compounds alone in A549 tumor-bearing mice. In addition, FTS/YM155 NCs exerted significant survivin downregulation (4.43-fold) and caspase-3 upregulation (4.31-fold) and showed better therapeutic outcomes without inducing organ injury, which highlights their promising future clinical application in precision therapy. This tumor microenvironment-responsive platform could be harnessed to develop an effective therapy via multilevel inhibition of cancer targets.

A nanobody-based molecular toolkit for ubiquitin-proteasome system explores the main role of survivin subcellular localization.

Targeted protein degradation is a powerful tool for determining the function of specific proteins nowadays. Survivin is the smallest member of the inhibitor of the apoptosis protein (IAP) family. It exists in the cytoplasm and nucleus of cells, but the exact function of survivin in different subcellular locations retained unclear updates due to the lack of effective and simple technical means. In this study, we created a novel nanoantibody-based molecular toolkit, namely, the ubiquitin-proteasome system (Nb4A-Fc-T2A-TRIM21), that can target to degrade survivin localized in cytoplasmic and cell nuclear by ubiquitinating, and by which to verify the potential roles of survivin subcellular localization. Also, the results showed that the cytoplasmic survivin mainly plays an anti-apoptotic function by directly or indirectly inhibiting the caspase pathway, and the nuclear survivin mainly promotes cell proliferation and participates in the regulation of the cell cycle. In addition, the Nb4A-Fc-T2A-TRIM21 system can degrade the endogenous survivin protein in a large amount by the ubiquitin-proteasome pathway, and the system can provide theoretical support for ubiquitination degradation targeting other endogenous proteins.

Cannabidiol Effectively Promoted Cell Death in Bladder Cancer and the Improved Intravesical Adhesion Drugs Delivery Strategy Could Be Better Used for Treatment.

Cannabidiol (CBD), a primary bioactive phytocannabinoid extracted from hemp, is reported to possess potent anti-tumorigenic activity in multiple cancers. However, the effects of CBD on bladder cancer (BC) and the underlying molecular mechanisms are rarely reported. Here, several experiments proved that CBD promoted BC cells (T24, 5637, and UM-UC-3) death. For example, T24 cells were treated with 12 µM CBD for 48 h, flow cytometry analysis demonstrated that early and late apoptotic cells were accounted for by 49.91%, indicating CBD enhanced cell apoptosis ability. To deeper explore molecular mechanisms, the CBD-treated T24 cell transcriptome libraries were established. KEGG analysis implied that the significantly changed genes were enriched in the PI3K/Akt pathway. qRT-PCR and Western blot assays verified that CBD regulated BC cells growth and migration and induced apoptosis by inactivating the PI3K/Akt pathway. Meanwhile, the developed chitosan to wrap CBD-loaded PLGA nanoparticles can significantly enhance the adhesion of the material to the mouse bladder wall, and the binding efficiency of mucin to chitosan-PLGA nanoparticles reached 97.04% ± 1.90%. In summary, this work demonstrates that CBD may become a novel reliable anticancer drug and the developed intravesical adhesion system is expected to turn into a potential means of BC chemotherapy drug delivery.

Discovering and Characterizing of Survivin Dominant Negative Mutants With Stronger Pro-apoptotic Activity on Cancer Cells and CSCs.

Survivin as a member of the inhibitor of apoptosis proteins (IAPs) family is undetectable in normal cells, but highly expressed in cancer cells and cancer stem cells (CSCs) which makes it an attractive target in cancer therapy. Survivin dominant negative mutants have been reported as competitive inhibitors of endogenous survivin protein in cancer cells. However, there is a lack of systematic comparative studies on which mutants have stronger effect on promoting apoptosis in cancer cells, which will hinder the development of novel anti-cancer drugs. Here, based on the previous study of survivin and its analysis of the relationship between structure and function, we designed and constructed a series of different amino acid mutants from survivin (TmSm34, TmSm48, TmSm84, TmSm34/48, TmSm34/84, and TmSm34/48/84) fused cell-permeable peptide TATm at the N-terminus, and a dominant negative mutant TmSm34/84 with stronger pro-apoptotic activity was selected and evaluated systematically in vitro. The double-site mutant of survivin (TmSm34/84) showed more robust pro-apoptotic activity against A549 cells than others, and could reverse the resistance of A549 CSCs to adriamycin (ADM) (reversal index up to 7.01) by decreasing the expression levels of survivin, P-gp, and Bcl-2 while increasing cleaved caspase-3 in CSCs. This study indicated the selected survivin dominant negative mutant TmSm34/84 is promising to be an excellent candidate for recombinant anti-cancer protein by promoting apoptosis of cancer cells and their stem cells and sensitizing chemotherapeutic drugs.

NET1 promotes HCC growth and metastasis in vitro and in vivo via activating the Akt signaling pathway.

Neuroepithelial cell transforming gene 1 (NET1), a member of the guanine nucleotide exchange factor family, is involved in various cancers, including gastric cancer, breast cancer and glioma. However, the role of NET1 in hepatocellular carcinoma (HCC) remains largely uncovered. In this study, we found that NET1 expression was upregulated in HCC, and that upregulated NET1 expression was closely associated with poor prognosis and some clinical characteristics in HCC patients. Whilst forced expression of NET1 in HCC cells was observed to significantly promote cell growth and metastasis in vitro and in vivo; downregulation of NET1 was shown to exhibit an opposite inhibitory effect. RNA-seq analysis and gene set enrichment analysis demonstrated that knockdown of NET1 significantly suppressed the level of Akt phosphorylation level and the expression of Akt downstream genes in HCC cells. Moreover, MK2206, a potent Akt inhibitor was shown to block the NET1-induced effects in HCC. Taken together, this study demonstrated that, through the Akt signaling pathway, NET1 plays an oncogenic role in HCC progression and metastasis. Hence, NET1 may potentially be used as a potential therapeutic target and prognostic marker of HCC.

Designing and constructing a phage display synthesized single domain antibodies library based on camel VHHs frame for screening and identifying humanized TNF-α-specific nanobody.

Tumor necrosis factor (TNF-α) is an important clinically tested cytokine that could induce autoimmune diseases and inflammation. Therefore, the anti-TNF-α therapy strategy was developed and used therapeutically in various diseases, especially in the cytokine storm associated chimeric antigen receptor (CAR) T-cell therapy and antiviral therapy. Compare with other anti-TNF-α inhibitors, anti-TNF-α Nb (nanobody) has many unique advantages. Herein, we reported a novel humanized scaffold for library construction, which could be soluble and expressed in Escherichia coli (E.coli), and the efficiency capacity could reach as high as 2.01 × 109. Meanwhile, an anti-TNF-α Nb was selected for further study after 4 rounds of screening, NT-3, as the optimal Nb could effectively inhibit TNF-mediated cytotoxicity. The IC50 of NT-3 was determined as 0.804 µM, and its apoptosis inhibition rate was 62.47 % in L929 cells. Furthermore, the molecular docking results showed that complementarity-determining regions (CDRs) of NT-3 could connect to TNF for blocking function through strong hydrogen bonds and salt bridges. In general, our study not only provided a good Nb screening platform in vitro without animal immunization, but also generated a series of novel humanized anti-TNF-α Nb candidates with potential applications.

The Establishment of Quantitatively Regulating Expression Cassette with sgRNA Targeting BIRC5 to Elucidate the Synergistic Pathway of Survivin with P-Glycoprotein in Cancer Multi-Drug Resistance.

Quantitative analysis and regulating gene expression in cancer cells is an innovative method to study key genes in tumors, which conduces to analyze the biological function of the specific gene. In this study, we found the expression levels of Survivin protein (BIRC5) and P-glycoprotein (MDR1) in MCF-7/doxorubicin (DOX) cells (drug-resistant cells) were significantly higher than MCF-7 cells (wild-type cells). In order to explore the specific functions of BIRC5 gene in multi-drug resistance (MDR), a CRISPR/Cas9-mediated knocking-in tetracycline (Tet)-off regulatory system cell line was established, which enabled us to regulate the expression levels of Survivin quantitatively (clone 8 named MCF-7/Survivin was selected for further studies). Subsequently, the determination results of doxycycline-induced DOX efflux in MCF-7/Survivin cells implied that Survivin expression level was opposite to DOX accumulation in the cells. For example, when Survivin expression was down-regulated, DOX accumulation inside the MCF-7/Survivin cells was up-regulated, inducing strong apoptosis of cells (reversal index 118.07) by weakening the release of intracellular drug from MCF-7/Survivin cells. Also, down-regulation of Survivin resulted in reduced phosphorylation of PI3K, Akt, and mTOR in MCF-7/Survivin cells and significantly decreased P-gp expression. Previous studies had shown that PI3K/Akt/mTOR could regulate P-gp expression. Therefore, we speculated that Survivin might affect the expression of P-gp through PI3K/Akt/mTOR pathway. In summary, this quantitative method is not only valuable for studying the gene itself, but also can better analyze the biological phenomena related to it.

PEGylated-PLGA Nanoparticles Coated with pH-Responsive Tannic Acid-Fe(III) Complexes for Reduced Premature Doxorubicin Release and Enhanced Targeting in Breast Cancer.

Biodegradable poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) have been widely used as delivery vehicles for chemotherapy drugs. However, premature drug release in PLGA NPs can damage healthy tissue and cause serious adverse effects during systemic administration. Here, we report a tannic acid-Fe(III) (FeIII-TA) complex-modified PLGA nanoparticle platform (DOX-TPLGA NPs) for the tumor-targeted delivery of doxorubicin (DOX). A PEGylated-PLGA inner core and FeIII-TA complex outer shell were simultaneously introduced to reduce premature drug release in blood circulation and increase pH-triggered drug release in tumor tissue. Compared to the unmodified NPs, the initial burst rate of DOX-TPLGA NPs was significantly reduced by nearly 2-fold at pH 7.4. Moreover, the cumulative drug release rate at pH 5.0 was 40% greater than that at pH 7.4 due to the pH-response of the FeIII-TA complex. Cellular studies revealed that the TPLGA NPs had enhanced drug uptake and superior cytotoxicity of breast cancer cells in comparison to free DOX. Additionally, the DOX-TPLGA NPs efficiently accumulated in the tumor site of 4T1-bearing nude mice due to the enhanced permeability and retention (EPR) effect and reached a tumor inhibition rate of 85.53 ± 8.77% (1.31-fold versus DOX-PLGA NPs and 3.12-fold versus free DOX). Consequently, the novel TPLGA NPs represent a promising delivery platform to enhance the safety and efficacy of chemotherapy drugs.

A novel liposome-polymer hybrid nanoparticles delivering a multi-epitope self-replication DNA vaccine and its preliminary immune evaluation in experimental animals.

DNA vaccine is an attractive immune platform for the prevention and treatment of infectious diseases, but existing disadvantages limit its use in preclinical and clinical assays, such as weak immunogenicity and short half-life. Here, we reported a novel liposome-polymer hybrid nanoparticles (pSFV-MEG/LNPs) consisting of a biodegradable core (mPEG-PLGA) and a hydrophilic shell (lecithin/PEG-DSPE-Mal 2000) for delivering a multi-epitope self-replication DNA vaccine (pSFV-MEG). The pSFV-MEG/LNPs with optimal particle size (161.61 ±â€¯15.63 nm) and high encapsulation efficiency (87.60 ±â€¯8.73%) induced a strong humoral (3.22-fold) and cellular immune responses (1.60-fold) compared to PBS. Besides, the humoral and cellular immune responses of pSFV-MEG/LNPs were 1.58- and 1.05-fold than that of pSFV-MEG. All results confirmed that LNPs was a very promising tool to enhance the humoral and cellular immune responses of pSFV-MEG. In addition, the rational design and delivery platform can be used for the development of DNA vaccines for other infectious diseases.

Anti-Inflammatory Activity and Mechanism of Hydrostatin-SN1 From Hydrophis cyanocinctus in Interleukin-10 Knockout Mice.

Biopeptides derived from marine species have garnered significant research interest owing to their anti-inflammatory, antibacterial, and anticancer activities. In our previous study, Hydrostatin-SN1, a bioactive peptide extracted from the Hydrophis cyanocinctus venom gland T7 phage display library, demonstrated anti-inflammatory activity in a dextran sulfate sodium-induced murine colitis model. In this study, we investigated the anti-inflammatory activity and the underlying mechanism of Hydrostatin-SN1 in lipopolysaccharide (LPS)-induced bone marrow-derived macrophage (BMDM) cells and interleukin (IL)-10 knockout mice. The results showed that Hydrostatin-SN1 inhibited phosphorylation of JNK, ERK1/2, and p38 and decreased the mRNA expression of tumor necrosis factor-α (TNF-α), IL-6, and IL-1ß in LPS-stimulated BMDM cells in a dose-dependent manner. In LPS-induced acute shock model, a significant higher survival rate of Hydrostatin-SN1-treated mice was observed. Furthermore, Hydrostatin-SN1 reduced body weight loss, decreased disease activity index, reduced spleen index, prevented histological injury, and inhibited the expression of IL-ß and phosphorylation of JNK, ERK1/2, and p38 in the colon tissue of IL-10 knockout mice. Additionally, the positive expression rate of TNF-α in mice colon was decreased. Overall, our results suggest that Hydrostatin-SN1 has significant anti-inflammatory effects, both in vitro and in vivo.

ABA-responsive transcription factor bZIP1 is involved in modulating biosynthesis of phenolic acids and tanshinones in Salvia miltiorrhiza.

Phenolic acids and tanshinones are major bioactive ingredients in Salvia miltiorrhiza, which possess pharmacological activities with great market demand. However, transcriptional regulation of phenolic acid and tanshinone biosynthesis remains poorly understood. Here, a basic leucine zipper transcription factor (TF) named SmbZIP1 was screened from the abscisic acid (ABA)-induced transcriptome library. Overexpression of SmbZIP1 positively promoted phenolic acid biosynthesis by enhancing expression of biosynthetic genes such as cinnamate-4-hydroxylase (C4H1). Furthermore, biochemical experiments revealed that SmbZIP1 bound the G-Box-like1 element in the promoter of the C4H1 gene. Meanwhile, SmbZIP1 inhibited accumulation of tanshinones mainly by suppressing the expression of biosynthetic genes including geranylgeranyl diphosphate synthase (GGPPS) which was confirmed as a target gene by in vitro and in vivo experiments. In contrast, the phenolic acid content was reduced and tanshinone was enhanced in CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9]-mediated knockout lines. In addition, the previously reported positive regulator of tanshinone biosynthesis, SmERF1L1, was found to be inhibited in SmbZIP1 overexpression lines indicated by RNA sequencing, and was proven to be the target of SmbZIP1. In summary, this work uncovers a novel regulator and deepens our understanding of the transcriptional and regulatory mechanisms of phenolic acid and tanshinone biosynthesis, and also sheds new light on metabolic engineering in S. miltiorrhiza.

Multiple targeting strategies achieve novel protein drug delivery into proapoptosis lung cancer cells by precisely inhibiting survivin.

Therapeutic recombinant proteins have numerous advantages and benefits over chemical drugs, particularly high specificity and good biocompatibility. However, the therapeutic potential and clinical application of current anticancer protein drugs are limited as most biomarkers are located within cells, and multiple physiological barriers exist between the point of administration and the intracellular biomarker. Herein, we report a novel strategy to accurately deliver a cell-permeable dominant-negative TATm-Survivin (TmSm) protein (T34A) to intracellular survivin in cancer cells by overcoming multiple barriers in vivo. A poly(d,l-lactide-co-glycolide) (PLGA) inner core, a polyethylene glycol (PEG) modification, and a TATm peptide were simultaneously introduced to mediate tumor tissue targeting and response to pH-triggered TmSm release. Compared to free TmSm, the PEGylated-PLGA nanoparticle platform achieved a significantly higher cellular uptake efficiency (1.79-fold for A549 and 1.77-fold for Capan-2), effectively decreased IC50 (1.22-fold for A549 and 1.17-fold for Capan-2), and largely elevated apoptosis in different cancer cells (1.17-fold for A549 and 1.15-fold for Capan-2). Besides, this newly developed nanoplatform showed increased protein drug accumulation in the tumor site in A549-bearing nude mice and reached a tumor inhibition rate of 55.81% (1.35-fold versus free TmSm) by reducing the expression of intracellular survivin. All these results confirmed that our newly developed delivery strategy is a very promising tool, which helps protein drugs to cross multiple barriers in vivo and achieves precise targeting to intracellular biomarkers. This strategy could also be applied to other types of protein drugs to further improve their clinical anticancer therapeutic efficacy.

SmMYB2 promotes salvianolic acid biosynthesis in the medicinal herb Salvia miltiorrhiza.

MYB transcription factors play vital roles in plant growth and metabolism. The phytohormone methyl jasmonate (MeJA) promotes phenolic acid accumulation in the medicinal herb Salvia miltiorrhiza, but the regulatory mechanism is poorly understood. Here, we identified the MeJA-responsive R2R3-MYB transcription factor gene SmMYB2 from a transcriptome library produced from MeJA-treated S. miltiorrhiza hairy roots. SmMYB2 expression was tightly correlated with the expression of key salvianolic acid biosynthetic genes including CYP98A14. SmMYB2 was highly expressed in the periderm of S. miltiorrhiza and SmMYB2 localized to the nucleus. Overexpressing SmMYB2 in S. miltiorrhiza hairy roots significantly increased the levels of salvianolic acids (including rosmarinic acid and salvianolic acid B) by upregulating salvianolic acid biosynthetic genes such as CYP98A14. SmMYB2 binds to the MYB-binding motifs in the promoter of CYP98A14, as confirmed by a dual-luciferase assay and electrophoretic mobility shift assays. Anthocyanin contents were significantly higher in SmMYB2-overexpressing hairy root lines than the control, primarily due to the increased expression of CHI, DFR, and ANS. These findings reveal the novel regulatory role of SmMYB2 in MeJA-mediated phenolic acid biosynthesis, providing a useful target gene for metabolic engineering and shedding light on the salvianolic acid regulatory network.

Transforming growth factor-ß signaling, a potential mechanism associated with diabetes mellitus and pancreatic cancer?

Pancreatic cancer is a common malignant digestive disease. Epidemiological and clinical studies have demonstrated that pancreatic cancer is closely related to diabetes mellitus. Diabetic patients are more likely to develop pancreatic cancer, which is linked with poor outcomes. Pancreatic cancer is complicated with abnormal blood sugar and insulin resistance and promotes the development of diabetes mellitus. Understanding the molecular mechanisms linking diabetes mellitus and pancreatic cancer is essential for the treatment of diabetes cancer patients. The transforming growth factor-ß (TGF-ß) signaling pathway is deregulated in cancer and has a dual role in different stages of cancer as a suppressor or a promoter. More important, The TGF-ß signaling pathway is also another important reason for diabetic complications. This review summarizes the relationship between diabetes and pancreatic cancer, in particular, focusing on the role of the TGF-ß signaling pathway. It is possible to find drugs like metformin that can prevent and treat pancreatic cancer by targeting the TGF-ß signaling pathway.