Paynantheine more effectively reverses multidrug resistance in malignant EPG85.257RDB and MCF7MX cells than morphine and speciociliatine.

The possible interactions of morphine, paynantheine and speciociliatine alkaloids with ATP-binding cassette (ABC) transporters was investigated. The compounds were docked against ABCG2 and ABCB1 to predict the binding mode of alkaloids in active binding sites. The cytotoxicity of morphine, paynantheine and speciociliatine for EPG85.257RDB and MCF7MX cells was determined and ABCB1 and ABCG2 gene and protein expression were determined. The binding score of paynantheine to ABCB1 was higher in the docking studies. Paynantheine and speciociliatine had similar binding scores to ABCB1, but higher binding scores to ABCG2 than did morphine. Paynantheine and speciociliatine were more effective against MCF7MX and EPG85.257RDB cells and showed greater cyctotoxicity in the MTT assay. The effect of morphine and paynantheine on the ABCB1 gene and protein expression suggests these compounds can reduce resistance in cancer patients, but that speciociliatine may not be a suitable candidate because of its increased ABCB1 expression while speciociliatine decreased the expression of ABCG2 in MCF7MX cells. This indicates that speciociliatine is a better candidate for reducing drug resistance in this cell line. Structural modification, drug-metabolizing enzymes and differences in the binding sites could cause functional differences between these compounds.

Valproate modulates the activity of multidrug resistance efflux pumps, as a chemoresistance factor in gastric cancer cells.

BACKGROUND: Drug resistance is one of the most critical problems in gastric cancer therapy. This study was performed to investigate the valproic acid effects on the proliferation of sensitive and resistant cell lines of human gastric cancer, and to explore the mechanism of the agent on multi drug resistance and apoptosis genes. METHODS: The cytotoxicity effect of valproic acid on the EPG85.257 and EPG85.257RDB cells was assessed by the MTT assay, and the IC50 concentration was evaluated. Apoptosis, genotoxicity, and drug resistance pump activity were evaluated using comet assay, Real-time PCR, and flow cytometry, respectively. Cell proliferation was assayed using a scratch test. RESULTS: Dose-dependent toxicity was recorded after treatment of cells with valproic acid. Valproic acid represented a significant growth inhibition on EPG85.257 cells with IC50 values of 5.84 µM and 4.78 µM after 48 h and 72 h treatment, respectively. In contrast, the drug-resistant counterpart represented 8.7 µM and 7.02 µM IC50 values after the same treatment time. Valproic acid induced PTEN, Bcl2, P53, Bax, P21, and caspase3 expression in EPG85.257 cells, whereas p21, p53, PTEN, and ABCB1 were overexpressed in EPG5.257RDB. Valproic acid hindered cell migration in both cell lines (P < 0.01). Valproate genotoxicity was significantly higher in the parent cells than in their resistant EPG85.257RDB counterparts. Valproate led to a 62% reduction in the daunorubicin efflux of the MDR1 pump activity. CONCLUSIONS: Valproate can affect drug resistance in gastric cancer via a unique mechanism independent of MDR1 expression.

Enzyme-sensitive nanoparticles, smart TAT and cetuximab conjugated immunoliposomes to overcome multidrug resistance in breast cancer cells.

Due to recent advances in the field of small molecule-based drugs, designing an efficient siRNA delivery system seems essential. Here, modified sets of lipids conjugated with cell-penetrating TAT peptide, MMP2 enzyme-sensitive moiety, and cetuximab antibodies against the EGF receptor were synthesized, purified and verified on HPLC, TLC, SEM, and DLS analyses. Different cellular and molecular experiments were designed to evaluate the transfection efficiency, targeting properties, and functions, including cytotoxicity assay, resensitization assessments, flow cytometry-based uptake assay, BCRP silencing efficiency, real-time PCR, and western blotting. The final targeted liposomes represented an average diameter of 160 nm; zeta-potential and siRNA encapsulation rates were respectively around -28.9 ± 3.16 mV and 88.3 ± 0.9 w/w. The siBCRP carried by the TAT+Cetuximab+ liposome led to an increase in the tumoricidal effect of mitoxantrone by a reduction in IC50 value by 4-fold (*** P < 0.001). Flow cytometry results showed that the cellular uptake rate of final immunoliposomes was significantly higher than the naked liposomes (*** P < 0.001). The Targeted siRNA encapsulating liposomes decreased BCRP transcript and protein levels in MCF7-MX cells by 0.24 and 0.2-fold after 48 h, respectively. Due to the silencing results of the BCRP by the encapsulated siRNA and the inhibitory effects of cetuximab on the EGFR, this formulation could widely be utilized as a carrier for tumor-directed gene delivery.

Silencing of α-N-acetylgalactosaminidase in the gastric cancer cells amplified cell death and attenuated migration, while the multidrug resistance remained unchanged.

Although the elevated level of the α-N-acetylgalactosaminidase enzyme (encoded by the NAGA gene) is a well-recognized feature of cancer cells; little research works have been undertaken on the cancer malignancy mechanisms. The effects of NAGA gene downregulation on cancer cells' features such as drug resistance, impaired programmed cell death, and migration were analyzed in this study. The cells grew exponentially with a doubling time of 30 h in an optimal condition. Toxicity of daunorubicin chemotherapy drug on NAGA-transfected EPG85.257RDB cells was evaluated in comparison to control cells and no significant change was recorded. Quantitative transcript analyses and protein levels revealed that the MDR1 pump almost remained unchanged during the study. Moreover, the NAGA gene downregulation enhanced the late apoptosis rate in EPG85.257RDB cells at 24 h posttransfection. The investigated expression level of genes and proteins involved in the TNFR2 signaling pathway, related to cancer cell apoptosis, showed considerable alterations after NAGA silencing as well. MAP3K14 and CASP3 genes were downregulated while IL6, RELA, and TRAF2 experienced an upregulation. Also, NAGA silencing generally diminished the migration ability of EPG85.257RDB cells and the MMP1 gene (as a critical gene in metastasis) expression decreased significantly. The expression of the p-FAK protein, which is located in the downstream of the α2 ß1 integrin signaling pathway, was reduced likewise. It could be concluded that despite drug resistance, NAGA silencing resulted in augmentative and regressive effects on cell death and migration.

Butylcycloheptylprodigiosin and undecylprodigiosin are potential photosensitizer candidates for photodynamic cancer therapy.

BACKGROUND: Prodiginines are bacterial red polypyrrole pigments and multifaceted secondary metabolites. These agents have anti-proliferative, immunosuppressive, antimicrobial, and anticancer effects. Recent analysis revealed that prodigiosin hypersensitizes Serratia marcescens to gamma radiation. In the present study, we report the cytotoxicity and genotoxicity properties of undecylprodigiosin and butylcycloheptylprodigiosin in the presence and absence of radiation through the MTT and alkaline comet experiments. METHODS AND RESULTS: Findings demonstrated that undecylprodigiosin was at least a fivefold more cytotoxic at low radiation doses (1 and 3 Gy) on both MCF7 and HDF lines rather than in the absence or high radiation doses (5 Gy) (P value < 0.05). Although butylcycloheptylprodigiosin toxicity on MCF7 and HDF was dose-dependent, it was not influenced by any radiation doses (P value > 0.05). Comet findings confirmed that these compounds' genotoxicity is only dose-dependent. Radiation had no significant effects on DNA damage on any of the cells (P value > 0.05). CONCLUSIONS: In general, it can be concluded that the prodiginines are cytotoxic agents that act as a double-edged sword, radiosensitizers and radio-protective, respectively at low and high radiation doses in cancer treatment process. As the results they could be used in antitumor therapies very soon.

Improvement of cytotoxicity of mitoxantrone and daunorubicin by candidone, tephrosin, and bavachinin.

BACKGROUND: Flavonoids have been demonstrated to have the ability of sensitizing cancer cells to chemotherapy and inverse multidrug resistance via various mechanisms, such as modulating of pumps. The therapeutic effect of candidone, tephrosin, and bavachinin in treatment of cancer, particularly to overcome multidrug resistance (MDR) is largely unknown. The capacity of these agents in sensitization of MDR cells is investigated in the current work. METHODS AND RESULTS: We analyzed the impact of candidone, tephrosin, and bavachinin, as chemosensitizer on cell cytotoxicity, P-gp and ABCG2 mRNA expression level on two multidrug resistant cells, ABCG2 overexpressing human epithelial breast cancer cell line (MCF7/MX), and P-gp overexpressing human gastric adenocarcinoma cell line (EPG85.257RDB). The inhibitory concentration of 50% (IC50) of daunorubicin in EPG85.257RDB cells in combination with IC10 of Bavachinin, Tephrosin, and Candidone were 6159 ± 948, 4186 ± 665, 730 ± 258 nM, and this data in MCF7/MX cell were 1773 ± 534, 7160 ± 405 and 3340 ± 622 nM respectively. These three flavonoids dose-dependently decreased the viability of MCF7/MX and EPG85.257RDB and significantly (p < 0.05) decreased IC50 of daunorubicin and mitoxantrone except Tephrosin in MCF7/MX cells. Candidone and Bavachinin were the most potent chemosensitizer in EPG85.257RDB and MCF7/MX cells respectively. Flavonoids did not reduce mRNA expression of P-gp and ABCG2 after 72 h treatment, except Candidone in EPG85.257RDB and Bavachinin in MCF7/MX cells. CONCLUSIONS: This effect is not time-dependent, and flavonoids have their own patterns that are cell-dependent. In general, tephrosin, candidone, and bavachinin had the potential of sensitizing MDR cells to mitoxantrone and daunorubicin.

Pharmacodynamic mechanisms of anti-inflammatory drugs on the chemosensitization of multidrug-resistant cancers and the pharmacogenetics effectiveness.

Drug resistance as a remarkable issue in cancer treatment is associated with inflammation which occurs through complex chemical reactions in the tumor microenvironment. Recent studies have implicated that glucocorticoids and NSAIDs are mainly useful combinations for inflammatory response modulation in chemotherapeutic protocols for cancer treatment. Immunosuppressive actions of glucocorticoids and NSAIDs are mainly mediated by the transrepression or activation regulation of inflammatory genes with different DNA-bound transcription factors including AP-1, NFAT, NF-κB, STAT and also, varying functions of COX enzymes in cancer cells. Interestingly, many investigations have proved the benefits of these anti-inflammatory agents in the quenching of multidrug resistance pathways. Numerous analyses on the ABC transporter promoters showed conserved nucleotide sequences with several DNA response elements that participate in transcriptional regulation. Furthermore, genetic variations in nucleotide sequences of membrane transporters were strongly associated with changes in these transporters' expression or function and a substantial impact on systemic drug exposure and toxicity. It appeared that several polymorphisms in MDR transporter genes especially MDR1 have influenced the regulatory mechanisms and explained differences in glucocorticoid responses.

Does survivin overexpression enhance the efficiency of fibroblast cell-based wound therapy?

Cell-based wound therapy is faced with some limiting factors that decrease the therapeutic efficacy of transplanted cells. In this study, we aimed to genetically modify fibroblast cells with anti-apoptotic Survivin gene (Birc5) before cell transplantation. In vitro, pIRES2-eGFP-Survivin plasmid was transfected into the fibroblast cells and the growth curve was evaluated for transfected and normal cells performing MTT assay. In vivo, two 6-diameter cutaneous wounds were created at mice dorsal skin. Fibrin clot was used as a delivery vehicle to transfer cells into the wound bed. The effects of four treatment groups including (a) Cell-SVV-Clot (b) Cell-GFP-Clot, (c) Normal cell-Clot and, (d) Clot alone were evaluated. After 1,2,3,7 and 14 days post-transplantation, the wounds were photographed for evaluating the wound closure rate and wound samples were obtained. Angiogenesis and formation of granulated tissue were assessed via H&E staining for wound samples. The expression levels of Survivin, VEGF, and bFGF genes were also determined using qRT-PCR. The MTT assay showed similar proliferation potential of transfected cells with normal cells verifying that Survivin had no detrimental effect. Compared to the Normal cell-Clot group, the Survivin overexpression was seen for 3 days in the Cell-SVV-Clot group verifying the cell survival during the early stage of wound healing. The Survivin further upregulated VEGF and bFGF expressions resulting in more angiogenesis and formation of granulated tissue by day 3 and 14. The treated wounds with Cell-SVV-Clot were regenerated with a higher wound closure rate by day 7 compared to Normal cell-Clot and Clot groups. Survivin enhanced wound healing through induction of VEGF and bFGF at particular times post-wounding that led to a more structured-epidermis with higher angiogenesis and granulation tissue formation rate.

Design and evaluation of biological gate circuits and their therapeutic applications in a model of multidrug resistant cancers.

OBJECTIVES: Synthetic biology is primarily an emerging research field that consists of designing new synthetic gene circuits dedicated to targeted functions and therapies such as cancer therapy. In this study, a genetic logic NOT-IF gate is used to reduce the multidrug resistance and facilitate the malignant cancer therapy. MCF7 cancer cells were cultured in RPMI-1640 medium and transfected with lentiviral vectors including MDR1 gene and the corresponding shRNA against MDR1 with controllable promoters. Transcript levels and protein levels of MDR1 gene were quantified. RESULTS: Our results showed that when doxycycline (DOX) and sodium butyrate were present and IPTG was absent, these led to a 74,354-fold increase in MDR1 gene expression. Upon IPTG treatment, the MDR1 gene expression was not detected due to the lack of the inducer. In addition, following IPTG induction in the presence of DOX and sodium butyrate and expressing shRNA, there was a 75% reduction in MDR1 gene expression compared to those cells treated only with sodium butyrate and DOX. CONCLUSIONS: We successfully designed and implemented the genetic logic NOT-IF gate at the transcriptional level using the inducible expression of both MDR1 drug resistance pump and its specific shRNA in MCF7 cancer cells, using the third generation lentiviral vectors.

Intelligent TAT-coupled anti-HER2 immunoliposomes knock downed MDR1 to produce chemosensitize phenotype of multidrug resistant carcinoma.

Gene therapy using biocompatible cationic liposomes is amongst promising approaches that decreases death from cancers. Here an invasive multidrug resistant cell model has been developed by lentiviral transfection. In parallel phospholipids have been covalently conjugated to TAT, MMP2, and Herceptin. The functional lipids have been mixed to generate intelligent liposome harboring small interfering RNA (siRNA) with high efficiency. The final liposomal complex was uniformly monodisperse and particle dimension and zeta-potential were respectively around 200 nm and -42.21 mV. Minimal cytotoxic effects have been reported for nanocarriers due to good biocompatibility of the selected phospholipids. Flourescence-activated cell sorter (FACS) analyses have been represented that surface trastuzumab and TAT specifically promote cellular uptake of liposomes in the malignant tumor cells. Assessment of MDR1 transcript and protein expression has been exhibited maximum significant downregulation around of 128-fold and 50-fold, respectively after 48 hr of liposome exposure. As it has been concluded, targeted liposomes may become a potential tool in gene delivery for improving chemotherapeutic efficiency in cancer treatment.

ABC-transporter blockage mediated by xanthotoxin and bergapten is the major pathway for chemosensitization of multidrug-resistant cancer cells.

Furanocoumarins derived from herbal and citrus extracts can act as antibacterial, antioxidant, immunomodulator, apoptotic, and selective anticancer agents, prompting a biological investigation to determine and predict their clinical therapeutic significance. Here, the cell cytotoxic effects of bergapten and xanthotoxin were analyzed alone and in combination with standard chemotherapeutics on three multidrug resistant cells and their nonresistant parental counterparts. The furanocoumarins modulatory effects on MDR1, BCRP, and MRP pump expression and function were investigated. Although quantitative real time PCR demonstrated that the MDR transcript level changes in a time dependent manner, flow cytometric analyses using fluorescent-labeled antibodies have indicated that bergapten and xanthotoxin had no significant effect on the protein levels. FACS analyses indicated that these prominent anticancer agents significantly blocked MDR1, BCRP, and MRP transporter function. Maximum furanocoumarin-mediated pump activity blockage in the MDR-resistant cells was quantified as 87% of normal and consequently, chemotherapeutic accumulation increased up to 2.7-fold and cytotoxicity tension increased 104-fold. MDR1 efflux kinetics also revealed that the maximum velocity and the pump affinity to daunorubicin were uncompetitively decreased. We conclude that bergapten and xanthotoxin are cytotoxic agents capable of preventing daunorubicin, mitoxantrone, and cisplatin binding to ABC-transporters and subsequently inhibiting their efflux out of cells and they may be a potential combination therapy for malignant cancers.

High-throughput bioaccumulation, biotransformation, and production of silver and selenium nanoparticles using genetically engineered Pichia pastoris.

A genetically modified Pichia pastoris strain overexpressing a metal-resistant variant of cytochrome b5 reductase enzyme was developed for silver and selenium biosorption and for nanoparticle production. The maximum recombinant enzyme expression level was approximately 31 IU/ml in the intercellular fluid after 24 h of incubation, and the capacity of the recombinant biomass for the biosorption of silver and selenium in aqueous batch models were measured as 163.90 and 63.71 mg/g, respectively. The ions were reduced in the presence of enzyme, leading to the formation of stable 70-180 nm metal nanoparticles. Various instrumental analyses confirmed the well-dispersed and crystalline nature of the spherical nanometals. The purified silver and selenium nanoparticles exhibited at least 10-fold less cytotoxicity toward HDF, EPG85-257, and T47D cells than silver nitrate and selenium dioxide. These results revealed that the engineered Pichia strain is an eco-friendly, rapid, high-throughput, and versatile reduction system for nanometal production.

Human cytochrome b5 reductase: structure, function, and potential applications.

Cytochrome b5 reductase is a flavoprotein that is produced as two different isoforms that have different localizations. The amphipathic microsomal isoform, found in all cell types with the exception of erythrocytes, consists of one hydrophobic membrane-anchoring domain and a larger hydrophilic flavin catalytic domain. The soluble cytochrome b5 reductase isoform, found in human erythrocytes, is a truncated protein that is encoded by an alternative transcript and consists of the larger domain only. Cytochrome b5 reductase is involved in the transfer of reducing equivalents from the physiological electron donor, NADH, via an FAD domain to the small molecules of cytochrome b5. This protein has received much attention from researchers due to its involvement in many oxidation and reduction reactions, such as the reduction of methemoglobin to hemoglobin. Autosomal cytochrome b5 reductase gene deficiency manifests with the accumulation of oxidized Fe+3 and recessive congenital methemoglobinemia in humans. In this article, we provide a comprehensive overview of the structure and function of cytochrome b5 reductase from different eukaryotic sources and its potential use in the food industry, biosensor, and diagnostic areas.

Innovative genetic scissor strategies and their applications in cancer treatment and prevention: CRISPR modules and challenges.

There are lots of gene editing tools for targeting genome sequences. Some are almost known, and most are a complete mystery and undiscovered. CRISPR/Cas editing tools have brought about a major revolution in medicine. Researchers have shown that CRISPR can modify DNA much more accurately, economically and easily than previous methods. CRISPR has proven itself effective for the deletion, replacement and insertion of DNA fragments into cell types, tissues and organisms. Recently, combining CRISPR/Cas with factors (transcription factors/repressors, exonucleases, endonucleases, transposons, caspase, fluorescent proteins, oxidoreductive enzymes, DNA/RNA polymerases), and elements (aptamers, barcodes, fluorescent probes, Trigger) have provided genome, transcriptome, proteome and epigenome modification. These modules are being investigated for cancer prevention and therapy and this review focuses on such innovative combinations that hopefully will become a clinical reality in the near future.

Epigenetic disruption of histone deacetylase-2 accelerated apoptotic signaling and retarded malignancy in gastric cells.

Background: The objective of this research was to determine whether HDAC2 function is associated with gastric cancer progression. Methods: HDAC2 was knocked out in EPG85.257 cells using CRISPR/Cas9 and tumorigenesis pathways were evaluated. Results: Cell proliferation, colony formation, wound healing and transwell invasion were inhibited in ΔHDAC2:EPG85.257 cells. Quantitative analyses revealed a significant downregulation of MMP1, p53, Bax, MAPK1, MAPK3, pro-Caspase3, ERK1/2, p-ERK1/2, AKT1/2/3, p-AKT1/2/3, p-NF-κB (p65), Twist, Snail and p-FAK transcripts/proteins, while SIRT1, PTEN, p21 and Caspase3 were upregulated in ΔHDAC2:EPG85.257 cells. Conclusion: These results indicated that HDAC2 enhanced migration, colony formation and transmigration ability. HDAC2 inhibition may improve gastric cancer chemotherapy pathways.

DNA changes are the main causes of cancer. Therefore, finding easy ways to manipulate and correct DNA changes has been the biggest medical concern in cancer treatment. Researchers have introduced CRISPR/Cas9 as the newest technology for gene editing that precisely and easily changes the genome of any cell. In our study, histone deacetylase-2 was disrupted in gastric cancer cells using CRISPR technology. This modification reduced growth kinetics and invasion of cancer cells. On the other hand, cell death (also called apoptosis) was induced. Sensitization of the cancer cells to chemotherapeutic agents is noticeable in this research. This study needs to uncover more signaling pathways in vitro and in vivo.

Advances in skin gene therapy: utilizing innovative dressing scaffolds for wound healing, a comprehensive review.

The skin, serving as the body's outermost layer, boasts a vast area and intricate structure, functioning as the primary barrier against external threats. Disruptions in the composition and functionality of the skin can lead to a diverse array of skin conditions, such as wounds, burns, and diabetic ulcers, along with inflammatory disorders, infections, and various types of skin cancer. These disorders not only exacerbate concerns regarding skin health and beauty but also have a significant impact on mental well-being. Due to the complexity of these disorders, conventional treatments often prove insufficient, necessitating the exploration of new therapeutic approaches. Researchers develop new therapies by deciphering these intricacies and gaining a thorough understanding of the protein networks and molecular processes in skin. A new window of opportunity has opened up for improving wound healing processes because of recent advancements in skin gene therapy. To enhance skin regeneration and healing, this extensive review investigates the use of novel dressing scaffolds in conjunction with gene therapy approaches. Scaffolds that do double duty as wound protectors and vectors for therapeutic gene delivery are being developed using innovative biomaterials. To improve cellular responses and speed healing, these state-of-the-art scaffolds allow for the targeted delivery and sustained release of genetic material. The most recent developments in gene therapy techniques include RNA interference, CRISPR-based gene editing, and the utilization of viral and non-viral vectors in conjunction with scaffolds, which were reviewed here to overcome skin disorders and wound complications. In the future, there will be rare chances to develop custom methods for skin health care thanks to the combination of modern technology and collaboration among disciplines.

Recent advances in mesoporous silica nanoparticles formulations and drug delivery for wound healing.

Wound healing is a natural process that can be disrupted by disease. Nanotechnology is a promising platform for the development of new therapeutic agents to accelerate acute and chronic wound healing. Drug delivery by means of nanoparticles as well as wound dressings have emerged as suitable options to improving the healing process. The characteristics of mesoporous silica nanoparticles (MSNs) make them efficient carriers of pharmaceutical agents alone or in combination with dressings. In order to maximize the effect of a drug and minimize its adverse consequences, it may be possible to include targeted and intelligent release of the drug into the design of MSNs. Its use to facilitate closure of adjacent sides of a cut as a tissue adhesive, local wound healing, controlled drug release and induction of blood coagulation are possible applications of MSNs. This review summarizes research on MSN applications for wound healing. It includes a general overview, wound healing phases, MSN formulation, therapeutic possibilities of MSNs and MSN-based drug delivery systems for wound healing.

Intelligent berberine-loaded erythrocytes attenuated inflammatory cytokine productions in macrophages.

Erythrocytes are impressive tools for drug delivery, especially to macrophages. Therefore, berberine was loaded into erythrocytes using both hypotonic pre-swelling and endocytosis methods to target macrophages. Physicochemical and kinetic parameters of the resulting carrier cells, such as drug loading/release kinetics, osmotic fragility, and hematological indices, were determined. Drug loading was optimized for the study using Taguchi experimental design and lab experiments. Loaded erythrocytes were targeted to macrophages using ZnCl2 and bis-sulfosuccinimidyl-suberate, and targeting was evaluated using flow cytometry and Wright-Giemsa staining. Differentiated macrophages were stimulated with lipopolysaccharide, and the inflammatory profiles of macrophages were evaluated using ELISA, western blotting, and real-time PCR. Findings indicated that the endocytosis method is preferred due to its low impact on the erythrocyte's structural integrity. Maximum loading achieved (1386.68 ± 22.43 µg/ml) at 1500 µg/ml berberine treatment at 37 °C for 2 h. Berberine successfully inhibited NF-κB translation in macrophages, and inflammatory response markers such as IL-1ß, IL-8, IL-23, and TNF-α were decreased by approximately ninefold, sixfold, twofold, eightfold, and twofold, respectively, compared to the LPS-treated macrophages. It was concluded that berberine-loaded erythrocytes can effectively target macrophages and modulate the inflammatory response.

Human health risk assessment of toxic heavy metals in summer crops and vegetables: a study in Ilam Province, Iran.

Purpose: The presence of toxic heavy metals (HMs) in agricultural crops can be considered as a noteworthy threat for consumers. The aim of this study was to assess the content of HMs (Pb, As, Cr, Cd, Co, Hg, and Ag) and their potential health risk in summer crops and vegetables (watermelon, cantaloupe, cucumber, melon, tomato, onion, potato, raw and stewed vegetables) in Ilam province, Iran. Methods: Totally, 31 crop samples were collected from local farms during the 2019 harvest season and the elements content were evaluated using inductively coupled plasma-mass spectrometry (ICP-MS). The non-carcinogenic health risk of HMs to the adults and children was estimated by Monte Carlo simulation method and target hazard quotients (THQs). Results: In general, the results showed that the concentration of Cr in the studied agricultural crops was higher than other HMs. As well as, the carcinogenic risk (CR) obtained for adults and children were more than the acceptable range for As. Also, CR for As in raw vegetable was the most ( 8.19E-1) and violated the threshold risk limit. The total carcinogenic risk of HMs in children was higher than that in adults. Conclusion: These results suggest that the agricultural crops were not safe for human consumption with potential risks associated. Due to the possible health effects of such products consumption, proper action should be taken to avoid chronic exposure, prevention of further pollution and consequent adverse health implications.

Novel adjuvant nano-vaccine induced immune response against Acinetobacter baumannii.

Developing adjuvant vaccines to combat rising multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) infections is a promising and cost-effective approach. The aim of this analysis was to construct a pDNA-CPG C274-adjuvant nano-vaccine and investigate its immunogenicity and protection in BALB/c mice. The CPG ODN C274 adjuvant was chemically synthesized and cloned into pcDNA3.1( +), and the cloning was verified using PCR and BamHI/EcoRV restriction enzyme digestion. Then, utilizing a complex coacervation approach, pDNA-CPG C274 was encapsulated by chitosan (CS) nanoparticles (NPs). TEM and DLS are used to explore the properties of the pDNA/CSNP complex. TLR-9 pathway activation was investigated in human HEK-293 and RAW 264.7 mouse cells. The vaccine's immunogenicity and immune-protective effectiveness were investigated in BALB/c mice. The pDNA-CPG C274/CSNPs were small (mean size 79.21 ± 0.23 nm), positively charged (+ 38.87 mV), and appeared to be spherical. A continuous slow release pattern was achieved. TLR-9 activation was greatest in the mouse model with CpG ODN (C274) at concentrations of 5 and 10 µg/ml with 56% and 55%, respectively (**P < 0.01). However, in HEK-293 human cells, by increasing the concentration of CpG ODN (C274) from 1 to 50 µg/ml, the activation rate of TLR-9 also increased, so that the highest activation rate (81%) was obtained at the concentration of 50 µg/ml (***P < 0.001). pDNA-CPG C274/CSNPs immunized BALB/c mice produced increased amounts of total-IgG, as well as IFN-γ and IL-1B in serum samples, compared to non-encapsulated pDNA-CPG C274. Furthermore, liver and lung injuries, as well as bacterial loads in the liver, lung, and blood, were reduced, and BALB/c mice immunized with pDNA-CPG C274/CSNPs showed potent protection (50-75%) against acute fatal Intraperitoneal A. baumannii challenge. pDNA-CPG C274/CSNPs evoked total-IgG antibodies, Th1 cellular immunity, and the TLR-9 pathway, as well as protection against an acute fatal A. baumannii challenge. Our findings suggest that this nano-vaccine is a promising approach for avoiding A. baumannii infection when used as a powerful adjuvant.