Recombinant T7 RNA polymerase production using ClearColi BL21(DE3) and animal-free media for in vitro transcription.

In vitro transcription (IVT) using T7 RNA polymerase (RNAP) is integral to RNA research, yet producing this enzyme in E. coli presents challenges regarding endotoxins and animal-sourced toxins. This study demonstrates the viable production and characterization of T7 RNAP using ClearColi BL21(DE3) (an endotoxin-free E. coli strain) and animal-free media. Compared to BL21(DE3) with animal-free medium, soluble T7 RNAP expression is ~50% lower in ClearColi BL21(DE3). Optimal soluble T7 RNAP expression in flask fermentation is achieved through the design of experiments (DoE). Specification and functional testing showed that the endotoxin-free T7 RNAP has comparable activity to conventional T7 RNAP. After Ni-NTA purification, endotoxin levels were approximately 109-fold lower than T7 RNAP from BL21(DE3) with animal-free medium. Furthermore, a full factorial DoE created an optimal IVT system that maximized mRNA yield from the endotoxin-free and animal-free T7 RNAP. This work addresses critical challenges in recombinant T7 RNAP production through innovative host and medium combinations, avoided endotoxin risks and animal-derived toxins. Together with an optimized IVT reaction system, this study represents a significant advance for safe and reliable reagent manufacturing and RNA therapeutics. KEY POINTS: • Optimized IVT system maximizes mRNA yields, enabling the synthesis of long RNAs. • Novel production method yields endotoxin-free and animal-free T7 RNAP. • The T7 RNAP has equivalent specifications and function to conventional T7 RNAP.

Inorganic-Organic Silica/PDMS Nanocomposite Antiadhesive Coating with Ultrahigh Hardness and Thermal Stability.

Antiadhesive surfaces have been gaining continuous attention, because of the scientific and industrial significance. Slippery surfaces and antismudge coatings with antiadhesive behavior have been readily designed and prepared. However, improving robustness of the surfaces, especially the simultaneous demonstration of features of high hardness, excellent adhesion to different substrates, and high thermal stability, is constantly challenging. Herein, we present a silica/polydimethylsiloxane (PDMS) nanocomposite coating (SPNC), wherein silica acts as a consecutive phase and nanophased PDMS is covalently embedded. The nanoconfined PDMS phase exhibits enhanced thermal stability and endows SPNC with slippery behavior; meanwhile, enrichment of PDMS on the surface renders a gradient composition of the coating. Accordingly, the inorganic-organic SPNC simultaneously displays a high nanoindentation hardness of 3.07 GPa and a pencil hardness over 9H, outstanding thermal stability of the slippery performance up to 400 °C, and excellent adhesion strength to different substrates. Additionally, SPNC exhibits high optical transparency, flexibility, resistance to bacterial clone, and chemical corrosion. With the scalable fabrication process, it can be envisioned that the antiadhesive coating with unprecedented comprehensive merits in this work has significant potentials for large-area applications, especially under severe service environments.

Tacrolimus in the treatment of childhood nephrotic syndrome: Machine learning detects novel biomarkers and predicts efficacy.

STUDY OBJECTIVE: The pharmacokinetics and pharmacodynamics of tacrolimus (TAC) vary greatly among individuals, hindering its precise utilization. Moreover, effective models for the early prediction of TAC efficacy in patients with nephrotic syndrome (NS) are lacking. We aimed to identify key factors affecting TAC efficacy and develop efficacy prediction models for childhood NS using machine learning algorithms. DESIGN: This was an observational cohort study of patients with pediatric refractory NS. SETTING: Guangzhou Women and Children's Medical Center between June 2013 and December 2018. PATIENTS: 203 patients with pediatric refractory NS were used for model generation and 35 patients were used for model validation. INTERVENTION: All patients regularly received double immunosuppressive therapy comprising TAC and low-dose prednisone or methylprednisolone. In this observational cohort study of 203 pediatric patients with refractory NS, clinical and genetic variables, including single-nucleotide polymorphism (SNPs), were identified. TAC efficacy was evaluated 3 months after administration according to two different evaluation criteria: response or non-response (Group 1) and complete remission, partial remission, or non-remission (Group 2). MEASUREMENTS: Logistic regression, extremely random trees, gradient boosting decision trees, random forest, and extreme gradient boosting algorithms were used to develop and validate the models. Prediction models were validated among a cohort of 35 patients with NS. MAIN RESULTS: The random forest models performed best in both groups, and the area under the receiver operating characteristics curve of these two models was 80.7% (Group 1) and 80.3% (Group 2). These prediction models included urine erythrocyte count before administration, steroid types, and eight SNPs (ITGB4 rs2290460, TRPC6 rs3824934, CTGF rs9399005, IL13 rs20541, NFKBIA rs8904, NFKBIA rs8016947, MAP3K11 rs7946115, and SMARCAL1 rs11886806). CONCLUSIONS: Two pre-administration models with good predictive performance for TAC response of patients with NS were developed and validated using machine learning algorithms. These accurate models could assist clinicians in predicting TAC efficacy in pediatric patients with NS before utilization to avoid treatment failure or adverse effects.

Novobiocin, a Newly Found TRPV1 Inhibitor, Attenuates the Expression of TRPV1 in Rat Intestine and Intestinal Epithelial Cell Line IEC-6.

Background and Purpose: Novobiocin (NOVO), an ABC transporter inhibitor, decreases intestinal wall permeability of capsaicin (CAP), an ABC transporter substrate. However, the mechanism of this effect is not consistent with the action of NOVO as an ABC transporter inhibitor. We previously found that CAP can also be transported via TRPV1, which was site-specific in the permeability of CAP across the intestine. We explored the regulation by NOVO of TRPV1 in the present study. Methods: Rats and transfected IEC-6 cells were used as the models to assess intestinal permeability and expression of TRPV1. Ussing chamber and intracellular accumulation were used to evaluate the influence of NOVO on the transport of CAP in vitro. The expression of TRPV1 was detected after administration of NOVO by qRT-PCR, western blot and immunofluorescent imaging. In addition, MTT and lactate dehydrogenase (LDH) were used to evaluate the cytotoxicity of NOVO in both rat and cell models. Finally, the effect of NOVO on the absorption of CAP in vivo was studied by LC-MS/MS. Results: In vitro data showed that there existed a dose-dependent relationship in the range of concentration between 5 and 50 µM, and even 5 µM NOVO could decrease intestinal permeability of CAP across the intestine. Meanwhile, cytosolic accumulation of CAP decreased when NOVO was used simultaneously or 24 h in advance. NOVO exhibited an inhibition level similar to that of ruthenium red (RR) or SB-705498, a TRPV1-specific inhibitor. NOVO down-regulated TRPV1 expression in the intestine and in transfected cells in a concentration-dependent fashion, hinting that its inhibition of the permeability of CAP is due to its inhibition of TRPV1 expression. Immunofluorescent imaging data showed that the fluorescence intensity of TRPV1 was reduced after pre-treatment with NOVO and SB-705498. In vivo data further demonstrated that oral co-administration of NOVO decreased Cmax and AUC of CAP in dosage-dependent ways, consistent with its role as a TRPV1 inhibitor. Conclusion: NOVO could be a potential TRPV1 inhibitor by attenuating the expression of TRPV1 and may be used to attenuate permeability of TRPV1 substrates.

Dense, uniform, smooth SiO2/TiO2 hard coatings derived from a single precursor source of tetra-n-butyl titanate modified perhydropolysilazane.

SiO2/TiO2 composite coatings are of great interest due to their optical, photocatalytic, electrical and mechanical properties. There are many methods for preparing SiO2/TiO2 composite coatings. Among these, the sol-gel process is eco-friendly and easily implementable for industrial applications. However, it is still a great challenge to prepare dense, uniform and smooth SiO2/TiO2 composite coatings with a sol-gel process. In this work, we show a modified sol-gel process for achieving dense, uniform and smooth SiO2/TiO2 coatings with tetra-n-butyl titanate modified perhydropolysilazane (PHPS) as a single precursor source, in which PHPS acts as the source for SiO2 instead of a traditional alkoxylsilane compound. The micro-morphology and composition evolution during the preparation process have been investigated; a smooth surface with a roughness average (S a) below 4.5 nm and a uniform distribution of Ti elements over the entire coating are shown. These dense, uniform SiO2/TiO2 coatings exhibit excellent mechanical robustness, with hardness values as high as 9.45 GPa, excellent optical transparency and hydrophilic properties.

The selective effect of glycyrrhizin and glycyrrhetinic acid on topoisomerase IIα and apoptosis in combination with etoposide on triple negative breast cancer MDA-MB-231 cells.

Triple negative breast cancer(TNBC) has generated growing interests due to its aggressive biologic behavior and absence of targeted therapy approach. Glycyrrhizin(GL) from licorice root and its metabolite, glycyrrhetinic acid(GA) have shown extensive bioactivities in clinic. Here, we demonstrate that GL and GA have contrary anti-cancer effect on TNBC MDA-MB-231 cells. Beside its inhibition of cell proliferation, GA at non-cytotoxic concentration showed synergistic effect in combination with anti-cancer drug, etoposide(VP-16). Specifically, GA enhanced cytotoxicity through regulating topoisomerase IIα(TOPO 2A) targeted by etoposide. GA sensitized the cells to etoposide through elevating TOPO 2A with a 2.4 fold rate at 12h. From 12 to 48h, GA halved the expression of TOPO 2A and stimulated apoptosis, which exhibited its antineoplastic effect. Our experiments showed that GSH depletion, modulation of MAPK and AKT pathways accounted for the regulation of topoisomerase IIα and apoptosis. However, GL showed protection and detoxication by decreasing reactive oxygen species generation, maintaining GSH and differentially modulating apoptosis, AKT pathway, ERK and JNK of MAPK pathway. Collectively, our results demonstrate that GA, instead of GL, is a better candidate for TNBC treatment because of its anti-cancer effect and sensitization of topoisomerase IIα inhibitor.

New application of an old drug: Antitumor activity and mechanisms of doxycycline in small cell lung cancer.

Small cell lung cancer (SCLC) remains one of the most aggressive tumors with a poor prognosis. The clinical outcome of SCLC patients has reached its plateau with the existing standard treatment and thus new therapies are urgently required. Accumulating evidences have indicated that doxycycline, a commonly used antibiotic, has antitumor activity against several malignancies. However, whether doxycycline has antitumor activity in SCLC and its underlying mechanisms remain unclear. Our investigation demonstrated that doxycycline could significantly inhibit the proliferation and colony formulation of SCLC cells (p<0.05). Furthermore, both Hoechst 33258 dye staining and TUNEL assays indicated that doxycycline could induce remarkable apoptosis of H446 cells in a concentration-dependent manner. RT-PCR and western blot assays proved that apoptosis induction effect of doxycycline was achieved via inducing the expression of caspase-3 and bax, as well as attenuating the expression of survivin and bcl-2. Moreover, the wound healing assay and Transwell assay indicated that doxycycline could significantly suppress the migration and invasion of H446 cells in a concentration-dependent manner (p<0.05). ELISA assay proved that the inhibitory effect of doxycycline on the migration and invasion of H446 cells was achieved via decreasing the secretion of MMP-2, MMP-9 and VEGF, as well as increasing the secretion of TIMP-2. Taken together, doxycycline dose-dependently suppressed the proliferation, colony formulation, migration and invasion of SCLC cells, as well as induced apoptosis. These findings encourage further investigations on the potential of doxycycline as a candidate drug for the treatment of SCLC.

Formulation and evaluation of novel glycyrrhizic acid micelles for transdermal delivery of podophyllotoxin.

CONTEXT: As the first-line agent for genital warts, podophyllotoxin (POD) could induce extensively skin burning, itching, and erythema. Meanwhile, as a common anti-inflammatory agent, glycyrrhizic acid (GA), also has amphipathic and solubilizing properties, indicating that it might be a promising drug carrier. OBJECTIVE: The objective of this study is to formulate and characterize the POD-loaded GA micelles preparation and to evaluate its drug release characteristics and anti-inflammatory properties. MATERIALS AND METHODS: The novel micelles preparation was prepared by ultrasonic dispersion method and characterized using different scanning calorimetries, dynamic light scattering, and transmission electron microscopies. Subsequently, its encapsulation efficiency (EE), drug-loading content (LC), in vitro skin permeation, in vivo drug retention, and distribution of POD were detected. The anti-inflammatory effect of the preparation was reflected by HE staining and immunohistochemistry in the rat skin. RESULTS: The POD-loaded GA micelles formed spherical shapes (approximately 10 nm) with an EE of 78.53 ± 2.17% and a LC of 7.293 ± 0.42%. Meaningfully, unlike the extensive distribution of the POD tincture throughout the skin tissue, POD released from the POD-loaded GA micelles mainly located in the epidermis and could maintain steady skin retention for 12 h. Moreover, the POD-loaded GA micelles induced less leukocyte infiltration and inflammatory factors (IL-6 and TNF-α) expression when compared with the POD tincture. DISCUSSION AND CONCLUSION: Our results suggested that the POD-loaded GA micelles could achieve a higher POD distribution in the epidermal layer as well as a lighter skin inflammation. This new POD delivery system might be a potential and promising candidate for genital warts and deserved further researches.

Afatinib reverses multidrug resistance in ovarian cancer via dually inhibiting ATP binding cassette subfamily B member 1.

ABCB1-mediated multidrug resistance (MDR) remains a major obstacle to successful chemotherapy in ovarian cancer. Herein, afatinib at nontoxic concentrations significantly reversed ABCB1-mediated MDR in ovarian cancer cells in vitro (p < 0.05). Combining paclitaxel and afatinib caused tumor regressions and tumor necrosis in A2780T xenografts in vivo. More interestingly, unlike reversible TKIs, afatinib had a distinctive dual-mode action. Afatinib not only inhibited the efflux function of ABCB1, but also attenuated its expression transcriptionally via down-regulation of PI3K/AKT and MAPK/p38-dependent activation of NF-κB. Furthermore, apart from a substrate binding domain, afatinib could also bind to an ATP binding domain of ABCB1 through forming hydrogen bonds with Gly533, Gly534, Lys536 and Ala560 sites. Importantly, mutations in these four binding sites of ABCB1 and the tyrosine kinase domain of EGFR were not correlated with the reversal activity of afatinib on MDR. Given that afatinib is a clinically approved drug, our results suggest combining afatinib with chemotherapeutic drugs in ovarian cancer. This study can facilitate the rediscovery of superior MDR reversal agents from molecular targeted drugs to provide a more effective and safer way of resensitizing MDR.

[Effect of capsaicin on intestinal permeation of P-glycoprotein substrate rhodamine 123 and fluorescein sodium in rats].

OBJECTIVE: To investigate the role of capsaicin in regulating permeation of P-gp substrate rhodamine 123 (R123) across the jejunum, ileum and colon membranes of rats. METHODS: The permeability of R123 or fluorescein sodium (CF) across the jejunum, ileum and colon membranes of male SD rats was evaluated using a Ussing chamber. The concentration of R123 or CF in the receptor was determined using fluorospectrophotometry to calculate the apparent permeability coefficient (Papp). RESULTS: Compared with the blank control group, capsaicin increased the permeability of R123 across jejunal membranes in the mucosal-to-serosal (M-S) direction and decreased its permeability in the serosal-to-mucosal (S-M) direction, but produced no obvious effect on R123 transport across the ileum or colon membranes. Capsaicin caused a regional increase in the permeability of CF across the jejunal membranes compared with the control group, but CF transport across the ileum and colon membranes was not affected. CONCLUSION: Capsaicin can affect the transport of R123 and CF across rat jejunal membranes, and this effect is shows an obvious intestine segment-related difference probably because of the different distribution of P-gp or tight junction in the intestines. This finding suggests that capsaicin is a weak P-gp inhibitor and an improver of mucous membrane channels.

Bioavailability enhancement of paclitaxel via a novel oral drug delivery system: paclitaxel-loaded glycyrrhizic acid micelles.

Paclitaxel (PTX, taxol), a classical antitumor drug against a wide range of tumors, shows poor oral bioavailability. In order to improve the oral bioavailability of PTX, glycyrrhizic acid (GA) was used as the carrier in this study. This was the first report on the preparation, characterization and the pharmacokinetic study in rats of PTX-loaded GA micelles The PTX-loaded micelles, prepared with ultrasonic dispersion method, displayed small particle sizes and spherical shapes. Differential scanning calorimeter (DSC) thermograms indicated that PTX was entrapped in the GA micelles and existed as an amorphous state. The encapsulation efficiency was about 90%, and the drug loading rate could reach up to 7.90%. PTX-loaded GA micelles displayed a delayed drug release compared to Taxol in the in vitro release experiment. In pharmacokinetic study via oral administration, the area under the plasma concentration-time curve (AUC0→24 h) of PTX-loaded GA micelles was about six times higher than that of Taxol (p < 0.05). The significant oral absorption enhancement of PTX from PTX-loaded GA micelles could be largely due to the increased absorption in jejunum and colon intestine. All these results suggested that GA would be a promising carrier for the oral delivery of PTX.