Anti-mesothelin CAR-T immunotherapy in patients with ovarian cancer.

Recently, chimeric antigen receptor T cell (CAR-T) therapy has received increasing attention as an adoptive cellular immunotherapy that targets tumors. However, numerous challenges remain for the effective use of CAR-T to treat solid tumors, including ovarian cancer, which is an aggressive and metastatic cancer with a poor therapeutic response. We screened for an effective anti-MSLN single-chain Fv antibody with comparable binding activity and non-off-target properties using human phage display library. A second-generation of anti-MSLN CAR was designed and generated. We demonstrated the efficacy of our anti-MSLN CAR-T cells for ovarian cancer treatment in an in vitro experiment to kill ovarian tumor cell lines. The anti-MSLN CAR-T cells impeded MSLN-positive tumor growth concomitant with a significant increase in cytokine levels compared with the control. Then, we demonstrated the efficacy of anti-MSLN CAR-T cells in an in vivo experiment against ovarian cancer cell-derived xenografts. Furthermore, we herein report three cases with ovarian cancer who were treated with autologous anti-MSLN CAR-T cells and evaluate the safety and effectiveness of adoptive cell therapy. In this investigator-initiated clinical trials, no patients experienced cytokine release syndrome or neurological symptoms over 2 grads. Disease stabilized in two patients, with progression-free survival times of 5.8 and 4.6 months. Transient CAR expression was detected in patient blood after infusion each time. The tumor partially subsided, and the patient's condition was relieved. In conclusion, this work proves the efficacy of the anti-MSLN CAR-T treatment strategy in ovarian cancer and provides preliminary data for the development of further clinical trials.

Antibiotics and antibiotic resistance genes in the water sources of the Wuhan stretch of the Yangtze River: Occurrence, distribution, and ecological risks.

Given the ubiquitous detection of antibiotics and antibiotic resistance genes (ARGs) in waterbodies worldwide and increasing public attention to water resource safety, this study investigated the presence of antibiotics and ARGs in the water sources of the Wuhan stretch of the Yangtze River (YR) as well as potential ecological risks. In this study, 15 antibiotics and 10 ARGs in a source of drinking water were analyzed using solid-phase extraction-ultra performance liquid chromatography-mass spectrometry technology (SPE-UPLC-MS/MS) and real-time fluorescence quantitative polymerase chain reaction (qPCR). Fourteen antibiotics were detected in the samples from 18 water sources, with the highest concentration detected for tetracycline, reaching up to 1708.33 ng/L. The detection rates of norfloxacin, enrofloxacin, ofloxacin, tetracycline, and roxithromycin were 100%. The concentrations of antibiotics were highest in She Shui, followed by the Wuhan stretch of the lower reaches of the YR, whereas the lowest concentrations were found in the Wuhan stretch of the upper reaches of the YR which were approximately equal to those in the Han River (HR). Ofloxacin and roxithromycin presented a substantial threat to aquatic organisms with high sensitivity at the majority of the sampling sites. The overall abundance of ARGs was notably greater in the lower reaches of the YR compared with the upper reaches and the HR. The highest absolute abundance was observed for sulfa ARGs. Integron intl1 strongly correlated with sul1, sul2, ermB, and qnrS, and antibiotics, strongly correlated with multiple ARGs, suggesting that antibiotics and ARGs are present in water sources in Wuhan and may present a plausible hazard to both human and ecological well-being. Hence, regulating the spread and dissemination of antibiotics and ARGs in the environment is imperative. The findings of this research offer significant insights into the stewardship and safeguarding of aquatic reserves in the Wuhan stretch of the YR.

FEN1 inhibitor synergizes with low-dose camptothecin to induce increased cell killing via the mitochondria mediated apoptotic pathway.

Camptothecin has been used in tumor therapy for a long time but its antitumor effect is rather limited due to the side effect and the drug resistance. FEN1, a major component of DNA repair systems, plays important roles in maintaining genomic stability via DNA replication and repair. Here we found that FEN1 inhibitor greatly sensitizes cancer cells to low-dose camptothecin. The combinative treatment of FEN1 inhibitor and 1 nM camptothecin induced a synthetic lethal effect, which synergistically suppressed cancer cell proliferation and significantly mediated apoptosis both in vitro and in vivo. Our study suggested that targeting FEN1 could be a potent strategy for tumor-targeting cancer therapy.

Protective effect of γ-glutamylcysteine against UVB radiation in NIH-3T3 cells.

BACKGROUND: Ultraviolet (UV) radiation-induced oxidative stress is the main cause of photodamage to the skin. Glutathione (GSH) serves important physiological functions, including scavenging oxygen-free radicals and maintaining intracellular redox balance. γ-glutamylcysteine (γ-GC), as an immediate precursor of GSH and harboring antioxidant and anti-inflammatory properties, represents an unexplored option for skin photodamage treatment. PURPOSE: The purpose of this study was to investigate whether γ-GC can reduce UVB-induced NIH-3T3 cell damage. METHODS: The experimental groups were as follows: control, UVB radiation, UVB radiation after pretreatment with γ-GC. Cell counting kit-8 (CCK-8) assays were used to measure cell proliferation, flow cytometry, and immunoblotting to detect the apoptosis rate and apoptosis-associated proteins. The levels of Reactive Oxygen Species (ROS), Superoxide Dismutase (SOD), and GSH/GSSG (oxidized GSH) were measured to assess oxidative stress. Immunoblotting and immunofluorescence were used to detect DNA damage. The members of the MAPK signaling pathways were detected by immunoblotting. RESULTS: UVB irradiation significantly reduced cell viability and destroyed the oxidative defense system. Pretreatment with γ-GC reduced UVB-induced cytotoxicity, restored the oxidation defense system, and inhibited activation of the MAPK pathway. It also reduced the apoptosis rate, downregulated the levels of cleaved caspase 3 and cleaved PARP. Furthermore, pretreatment with γ-GC reduced the accumulation of γH2AX after UVB radiation exposure, indicating that γ-GC could protect cells from DNA damage. CONCLUSION: γ-GC protected NIH-3T3 from damage caused by UVB irradiation. The photoprotective effect of γ-GC is mediated via strengthening the endogenous antioxidant defense system, which prevents DNA damage and inhibits the activation of the MAPK pathway.

32A9, a novel human antibody for designing an immunotoxin and CAR-T cells against glypican-3 in hepatocellular carcinoma.

BACKGROUND: Treatment of hepatocellular carcinoma (HCC) using antibody-based targeted therapies, such as antibody conjugates and chimeric antigen receptor T (CAR-T) cell therapy, shows potent antitumor efficacy. Glypican-3 (GPC3) is an emerging HCC therapeutic target; therefore, antibodies against GPC3 would be useful tools for developing immunotherapies for HCC. METHODS: We isolated a novel human monoclonal antibody, 32A9, by phage display technology. We determined specificity, affinity, epitope and anti-tumor activity of 32A9, and developed 32A9-based immunotherapy technologies for evaluating the potency of HCC treatment in vitro or in vivo. RESULTS: 32A9 recognized human GPC3 with potent affinity and specificity. The epitope of 32A9 was located in the region of the GPC3 protein core close to the modification sites of the HS chain and outside of the Wnt-binding site of GPC3. The 32A9 antibody significantly inhibited HCC xenograft tumor growth in vivo. We then pursued two 32A9-based immunotherapeutic strategies by constructing an immunotoxin and CAR-T cells. The 32A9 immunotoxin exhibited specific cytotoxicity to GPC3-positive cancer cells, while 32A9 CAR-T cells efficiently eliminated GPC3-positive HCC cells in vitro and caused HCC xenograft tumor regressions in vivo. CONCLUSIONS: Our study provides a rationale for 32A9 as a promising GPC3-specific antibody candidate for HCC immunotherapy.

MiR-7, inhibited indirectly by lincRNA HOTAIR, directly inhibits SETDB1 and reverses the EMT of breast cancer stem cells by downregulating the STAT3 pathway.

Epithelial-mesenchymal transition (EMT) contributes to tumor invasion and metastasis in many cancers and correlates highly with the acquisition of cancer stem cell (CSC) characteristics. EMT also correlates with changes in specific microRNAs (miRNAs) that have already been integrated into tumorigenic programs as either oncogenes or tumor suppressor genes. Here, we show that miR-7, which was downregulated in breast CSCs (BCSCs) isolated from the human MCF-7 and MDA-MB-231 cell lines, inhibited cell invasion and metastasis, decreased the BCSC population and partially reversed EMT in MDA-MB-231 cells by directly targeting the oncogene, SETDB1. The conspicuous epigenetic transition induced by miR-7 overexpression was found not only in MDA-MB-231 cells but also in BCSC xenograft tumors. MiR-7 inhibited the metastasis of BCSCs in lungs, kidneys, and adrenal glands of NOD/SCID mice. ChIP-polymerase chain reaction result suggested that the SETDB1 induced STAT3 expression by binding to the promoter of STAT3. MiR-7-mediated downregulation of SETDB1 resulted in the suppression of STAT3, which led to the downregulation of c-myc, twist, and mir-9. In addition, the downregulation of miR-7 in BCSCs may be indirectly attributed to lincRNA HOTAIR by modulating the expression of HoxD10 that promotes the expression of miR-7. These findings demonstrate that miR-7 was a tumor suppressor and that the overexpression of miR-7 might serve as a good strategy for treating highly invasive breast cancer.

Development of active packaging with chitosan, guar gum and watermelon rind extract: Characterization, application and performance improvement mechanism.

The objective of this study was to make a film matrix containing chitosan (CS) and guar gum (GG), and to improve the physicochemical properties of the film using watermelon rind extract (WRE) as a cross-linker and active substance for the preservation of fresh-cut bananas. The results of Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy showed that the WRE and CG matrix formed intermolecular hydrogen bond interactions, which made the structure of the resulting films more compact. With increasing amounts of WRE, the mechanical properties of the films were significantly increased, but the permeability of water vapor and oxygen was significantly decreased (p < 0.05). Notably, when the amount of extract reached 4 wt%, the DPPH radical scavenging activity of the composite film significantly increased to 83.24 %, and the antibacterial activity also reached its highest value. Fresh-cut bananas were stored at room temperature with polyethylene film, CG and CG-WRE. The CG with 4 wt% WRE effectively inhibited the changes in appearance, firmness, weight, color and total soluble solids content of fresh-cut bananas during storage. Therefore, CG-WRE as a novel active food packaging material, has good physicochemical properties and great potential to extend the shelf life of foods.

Intelligent packaging based on chitosan/fucoidan incorporated with coleus grass (Plectranthus scutellarioides) leaves anthocyanins and its application in monitoring the spoilage of salmon (Salmo salar L.).

The innovation of this study was to develop a novel biodegradable intelligent packaging based on chitosan/fucoidan combined with different amounts (1, 3 and 5 wt% on chitosan basis) of coleus grass (Plectranthus scutellarioides) leaves anthocyanins (CGL) to monitor the spoilage of salmon (Salmo salar L.). The addition of fucoidan improved the barrier and mechanical properties of the chitosan films (CS) due to hydrogen bonds and intermolecular electrostatic interactions. Moreover, the addition of CGL not only improved the physical properties but also improved the biological activity of chitosan/fucoidan film (CF). The DPPH and ABTS radical scavenging activity of CF contained 5 wt% CGL was 1.83 and 1.75 times than CF, respectively. The inhibition zone size of CF films containing 5 wt% CGL (CF-5%CGL) was approximately 2.04 (Escherichia coli) and 2.16 (Staphylococcus aureus) times higher than that of CF. Moreover, CF-CGL displayed obvious color changes in different pH environments and is highly sensitive to ammonia gas. The CF-CGL has visible color changes during the monitoring of salmon spoilage and extended the shelf life of salmon. According to our findings, CF-CGL film might be employed as a possible intelligent packaging material for monitoring and preserving salmon in the future.

Investigation of the formation mechanism and ß-carotene encapsulation stability of emulsion gels based on egg yolk granules and sodium alginate.

The formation mechanism of heat-induced egg yolk granules (EYGs)/sodium alginate (SA) emulsion gel was studied under pH 6.2 and 7.5. Particle size, water holding capacity, LF NMR, and protein solubility revealed that pH 7.5 increased the surface charge of EYGs and enhanced non-covalent interaction with SA, and hydrogen bonding dominated of the gel formation process. Microscopy and rheological analysis indicated that samples with 0.75% SA had the smallest particle size and highest G', with chain-like oil droplets. Excess SA (1%) led to depletion flocculation due to SA structural rearrangements around oil droplets caused by the increase in negatively charged, causing uneven network structure. The in vitro release property and storage stability of ß-carotene loaded in the EYGs/SA emulsion gel showed that SA increased storage stability and decreased bioaccessibility of ß-carotene with delayed digestion rate. These results provide a theoretical basis for the nutrient delivery system in gel foods.

Development and characterization of active packaging based on chitosan/chitin nanofibers incorporated with scallion flower extract and its preservation in fresh-cut bananas.

The aim of this study was to develop a novel active packaging using chitosan (CS) and esterified chitin nanofibers (CF) combined with different contents (1, 2 and 4 wt% on CS basis) of scallion flower extract (SFE) to protect banana samples. The addition of CF significantly improved the barrier and mechanical properties of the CS films (p < 0.05) due to hydrogen bonds and electrostatic interactions. Moreover, the addition of SFE not only improved the physical properties of the CS film but also improved the CS film biological activity. The oxygen barrier property and antibacterial ability of CF-4%SFE were approximately 5.3 and 1.9 times higher than those of the CS film, respectively. In addition, CF-4%SFE had strong DPPH radical scavenging activity (74.8 ± 2.3 %) and ABTS radical scavenging activity (84.06 ± 2.08 %). Fresh-cut bananas stored in CF-4%SFE showed less weight loss, starch loss, color and appearance change than those stored in traditional polyethylene film, which indicated that CF-4%SFE was much better at storing fresh-cut bananas than conventional plastic packaging. For these reasons, CF-SFE films have great potential as a candidate to replace traditional plastic packaging and extend the shelf life of packaged foods.

Tuning egg yolk granules/sodium alginate emulsion gel structure to enhance ß-carotene stability and in vitro digestion property.

In this study, emulsion gels were constructed by ionic gelation method using egg yolk granules/sodium alginate bilayers emulsion. In particular, the main driving force of the emulsion gels was controlled by adjusting pH. Compared with pH 7.0, the mechanical properties of EYGs emulsion gel were enhanced at pH 4.0 (G' > G″). The interfacial protein aggregation that occurred at pH 4.0 promoted the compactness of the EYGs emulsion gel structure along with enhanced capillary effect. The emulsion gel structure tended to be complete at 1 % SA of pH 4.0, for the electrostatic interaction required more SA molecules involved in maintaining emulsion gel structural stability. The denser emulsion gel structure of pH 4.0 than pH 7.0 improved storage stability, FFA releasing, and chemical stability of ß-carotenes. Bioaccessibility of ß-carotenes also decreased to achieve sustained release. This study provides a theoretical basis for tuning emulsion gel structure to adjust encapsulation stability and in vitro digestion characteristics of active ingredients.

Understanding the structure, interfacial properties, and digestion fate of high internal phase Pickering emulsions stabilized by food-grade coacervates: Tracing the dynamic transition from coacervates to complexes.

Although protein-polysaccharide complexes have shown tremendous potential in stabilizing high internal phase Pickering emulsions (HIPPEs), it is unclear whether coacervates have the same potential to be used as effective Pickering stabilizers. In this study, HIPPEs were prepared by ovalbumin (OVA)-pectin (PE) coacervates during the transition from coacervates to complexes. The results showed that enhanced OVA-PE interactions significantly affected the wettability and surface-tension reduction ability of the OVA-PE coacervates. At pH 2, the coacervate-stabilized HIPPEs exhibited smaller oil droplet sizes (21.3±2.3 µm), tighter droplet packing, and finer solid-like structures through the bridging of droplets and the generation of stronger gel-like network structures to prevent coalescence and lipid oxidation. The gastrointestinal digestion results proved that the OVA-PE coacervates promoted lipid hydrolysis and improved the bioaccessibility (from 19.7±0.7% to 36.5±2%) of curcumin-loaded HIPPEs. Our work provides new ideas for the development of biopolymer particles as effective Pickering stabilizers in the food industry.

Chitosan/esterified chitin nanofibers nanocomposite films incorporated with rose essential oil: Structure, physicochemical characterization, antioxidant and antibacterial properties.

Active films were developed based on chitosan, esterified chitin nanofibers and rose essential oil (REO). The joint effects of chitin nanofibers and REO on structure and physicochemical properties of chitosan film were investigated. Scanning electron microscopy and Fourier transform infrared spectroscopy showed that the chitin nanofibers and REO had significant effects on the morphology and chemical structure of chitosan composite films. The negatively charged esterified chitin nanofibers formed a compact network structure through intermolecular hydrogen bonding and electrostatic interactions with the positively charged chitosan matrix. Chitin nanofibers and REO synergistically enhanced the water resistance, mechanical properties and UV resistance of chitosan-based films, but the addition of REO increased the oxygen permeability. Furthermore, the addition of REO enhanced the inhibition of ABTS and DPPH free radicals and microorganisms by chitosan-based film. Therefore, chitosan/chitin nanofiber-based active films containing REO as food packaging materials can potentially provide protection to extend food shelf life.

Development and characterization of high internal phase pickering emulsions stabilized by heat-induced electrostatic complexes particles: Growth nucleation mechanism and interface architecture.

In this work, the heat-induced ovalbumin (OVA)-pectin (PE) electrostatic complex particles (HIECP) prepared by different heating sequences (type I particles (I): Heat-treated ovalbumin/pectin complexes at pH 4; type II particles (II): Complexes between pre-heated ovalbumin and pectin at pH 4) and biopolymer ratios were used as stabilizers to form high internal phase Pickering emulsions (HIPPEs). The results showed that I had a more compact structure, higher net surface charge, and smaller particle size than II, due to the different growth nucleation mechanism. II-stabilized HIPPEs exhibited a smaller oil droplet size, stronger gel structure, and better stability than I-stabilized HIPPEs, owing to their suitable wettability, rigid "core-shell" structure, and robust and dense interface architecture. Moreover, the stability and gel-like structure of HIECP-stabilized HIPPEs improved with increasing PE content due to steric barrier and thickening effects. Our findings provide a new perspective for understanding heat-induced biopolymer particles as effective Pickering stabilizers.

Development and characterization of chitosan/guar gum active packaging containing walnut green husk extract and its application on fresh-cut apple preservation.

The aim of this work was to develop active packaging film by using chitosan/guar gum (CG) film matrix and walnut green husk extract (WE), for preservation of fresh-cut apple. WE was used as cross-linking agent to improve physicochemical properties, and as active substances to enhance antioxidant activity of CG films. Fourier transform infrared spectroscopy and scanning electron microscopy results showed WE formed intermolecular hydrogen bond interactions with the film matrix, and microstructures of the film were more compact. With the increase of WE content (0-4 wt%), the mechanical properties of composite films were significantly enhanced, while permeability of water vapor and oxygen was significantly decreased (p < 0.05). When the amount of extract reached 4 wt%, the DPPH radical scavenging activity of composite film was significantly increased to 94.59%. CG-WE and CG films were used as active packaging materials to preserve fresh-cut apple. When stored at 4 °C for 10 days, CG-WE films showed better performance in reducing firmness, weight loss, total soluble solids and inhibiting browning and microbial growth of fresh-cut apples. Therefore, as a new type of active food packaging material, CG-WE films have good physical properties, and great potential in ensuring food quality and extending shelf life.

Fabrication and digestive characteristics of high internal phase Pickering emulsions stabilized by ovalbumin-pectin complexes for improving the stability and bioaccessibility of curcumin.

In this study, ovalbumin (OVA) interacted with pectin (PE) to form soluble electrostatic complexes to improve the functional properties of high internal phase Pickering emulsions (HIPEs) under extreme conditions. The results showed that the stability of the OVA-PE soluble complexes-stabilized HIPEs was significantly better than that of the free OVA-stabilized HIPEs and was modulated by the biopolymer ratio. In particular, the complexes at an OVA:PE ratio of 1:1 (C-1:1) may form particulates with a core-shell structure by a flocculation mechanism. The C-1:1-stabilized HIPEs had the smallest oil droplet size (11.34 ± 1.14 µm) and the best resistance to extreme environmental stresses due to their strong, rigid structure and dense interfacial architecture. The in vitro digestion results showed that the bioaccessibility (from 18.3% ± 0.5% to 38.8% ± 4.8%) of curcumin improved with increasing PE content. Our work is helpful in understanding OVA-PE complexes as stabilizers for HIPEs and their potential applications in food delivery systems.

Encapsulation of catechin into nano-cyclodextrin-metal-organic frameworks: Preparation, characterization, and evaluation of storage stability and bioavailability.

This study, nanoscale α-, ß-, γ-cyclodextrin (CD)-metal-organic frameworks (MOFs) were successfully prepared using solvothermal assisted ultrasound method. CD-MOFs were used as nanocarriers to encapsulate catechin (CA), and their encapsulation capacities were evaluated. Encapsulation capacities of CD-MOFs to incorporate CA followed the order: ß-CD-MOFs > Î³-CD-MOFs > α-CD-MOFs. CA/CD-MOFs were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and differential scanning calorimetry (DSC). DSC and SEM results provided evidence for the formation of CA/CD-MOFs. XRD results indicated the new solid crystalline phases formed in CA/CD-MOFs complex. Results of FT-IR showed that CA was combined with CD-MOFs through hydrogen bonding and van der Waals forces. Current research demonstrated that encapsulation of CA within CD-MOFs provided it against light, oxygen and temperature. Moreover, encapsulation by CD-MOFs improved storage stability and bioavailability of CA. Thus, these CA/CD-MOFs have potential to be used as nutritional supplements and functional foods.

A novel polymerase ß inhibitor from phage displayed peptide library augments the anti-tumour effects of temozolomide on colorectal cancer.

The therapeutic efficacy of TMZ, a common used drug for chemotherapy, is limited by the resistance from colorectal cancer cells. Base excision repair (BER) pathway has been identified as one of the reasons for drug resistance. By blocking Polß-dependent BER (Base Excision Repair) pathway, the efficacy of TMZ treatment can be improved greatly. Several Polß inhibitors that have been identified could not become approved drugs due to lack of potency or specificity. To find therapeutic candidates with exquisite specificity and high affinity to Polß, phage display technology was used in the current research. We screened out a candidate Polß inhibitor, 10 D, that can inhibit the activity of Polßand SP-BER (Short-Patch Base excision Repair) pathway. Co-treatment with 10 D enhanced the sensitivity of colorectal cancer (CRC) cells to TMZ both in vitro and in vivo. Our data suggested that the novel Polß inhibitor we identified can improve TMZ efficacy and optimize CRC chemotherapy.

Human umbilical blood mononuclear cell-derived mesenchymal stem cells serve as interleukin-21 gene delivery vehicles for epithelial ovarian cancer therapy in nude mice.

Ovarian cancer causes more deaths than any other cancer of the female reproductive system, and its overall cure rate remains low. The present study investigated human umbilical blood mononuclear cell (UBMC)-derived mesenchymal stem cells (UBMC-MSCs) as interleukin-21 (IL-21) gene delivery vehicles for ovarian cancer therapy in nude mice. MSCs were isolated from UBMCs and the expanded cells were phenotyped by flow cytometry. Cultured UBMCs were differentiated into osteocytes and adipocytes using appropriate media and then the UBMC-MSCs were transfected with recombinant pIRES2-IL-21-enhancement green fluorescent protein. UBMC-MSCs expressing IL-21 were named as UBMC-MSC-IL-21. Mice with A2780 ovarian cancer were treated with UBMC-MSC-IL-21 intravenously, and the therapeutic efficacy was evaluated by the tumor volume and mouse survival. To address the mechanism of UBMC-MSC-IL-21 against ovarian cancer, the expression of IL-21, natural killer glucoprotein 2 domain and major histocompatibility complex class I chain-related molecules A/B were detected in UBMC-MSC-IL-21 and in the tumor sites. Interferon-γ-secreting splenocyte numbers and natural killer cytotoxicity were significantly increased in the UBMC-MSC-IL-21-treated mice as compared with the UBMC-MSCs or the UBMC-MSC-mock plasmid-treated mice. Most notably, tumor growth was delayed and survival was prolonged in ovarian-cancer-bearing mice treated with UBMC-MSC-IL-21. Our data provide important evidence that UBMC-MSCs can serve as vehicles for IL-21 gene delivery and inhibit the established tumor.