Generation of Murine Chimeric Antigen Receptor-Modified T Cells for In Vivo Studies in Syngeneic Tumor Models.

CAR-T cell therapy has emerged as a potent and effective tool in the immunotherapy of refractory cancers. However, challenges exist in their clinical application, necessitating extensive preclinical research to optimize their function. Various preclinical in vitro and in vivo models have been proposed for such purpose; among which immunocompetent mouse models serve as an invaluable tool in studying host immune interactions within a more realistic simulation of the tumor milieu. We hereby describe a standardized protocol for the generation of high-titer γ-retroviral vectors through transfection of the HEK293T packaging cell line. The virus-containing supernatant is further concentrated using an inhouse concentrator solution, titrated, and applied to mouse T cells purified via a convenient and rapid method by nylon-wool columns. Using the method presented here, we were able to achieve high titer γ-retrovirus and highly pure mouse T cells with desirable CAR transduction efficiency. The mouse CAR T cells produced through this protocol demonstrate favorable CAR expression and viability, thus making them suitable for further in vitro/in vivo assays. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Production of γ-retroviral vectors from retrovirus-backbone plasmids Basic Protocol 2: Concentration of γ-retrovirus-containing supernatants Basic Protocol 3: Titration of concentrated γ-retrovirus Basic Protocol 4: Isolation and activation of mouse T cells Basic Protocol 5: Transduction of activated mouse T cells, assessment of CAR expression, and expansion of CAR T cells for further in vitro/in vivo studies Support Protocol: Surface staining of cells for flow cytometric assessment of CAR expression.

Ascorbic Acid and α-Tocopherol in the Inactivated SARS-CoV-2 Vaccine Formulation: Induction of the Th1 Pattern in Aged Mice.

Aging is physiologically associated with a decline in the function of the immune system and subsequent susceptibility to infections. Interferon-gamma (IFN-γ), a key element in the activation of cellular immunity, plays an important role in defense against virus infections. Decreased levels of IFN-γ in the elderly may explain their increased risk for viral infectious diseases such as COVID-19. There is accumulating evidence that ascorbic acid (vitamin C [VitC]) and α-tocopherol together help improve the function of the immune system in the elderly, control infections, and decrease the treatment duration. A SARS-CoV-2 strain was isolated from a patient and then cultured in the Vero cell line. The isolated and propagated virus was then inactivated using formalin and purified by the column chromatography. The inactivated SARS-CoV-2 was formulated in the Alum adjuvant combined with VitC or α-tocopherol and/or both of them. The vaccines were injected twice to young and aged C57BL/6 mice. Two weeks later, IFN-γ, IL-4, and IL-2 cytokines were assessed using ELISA Kits. Specific IgG and IgG1/IgG2a were assessed by an in-house ELISA. In addition, the expression of PD1 and TERT genes in the spleen tissue of the mice was measured using real-time PCR. IL-4 and IFN-γ cytokines showed a significant increase in both aged and young mice compared with the Alum-based vaccine. In addition, our results exhibited a significant decrease and increase in specific total IgG and the IgG2a/IgG1 ratio, respectively. Furthermore, the vaccine formulated in α-tocopherol + VitC led to decreased PD1 and increased TERT gene expression levels. In conclusion, our results demonstrated that α-tocopherol + VitC formulated in the inactivated SARS-CoV-2 vaccine led to a shift toward Th1, which may be due to their effect on the physiology of cells, especially aged ones and changing their phenotype toward young cells.

Characterization and genome analysis of a broad host range lytic phage vB_SenS_TUMS_E19 against Salmonella enterica and its efficiency evaluation in the liquid egg.

Salmonella enterica serovars are zoonotic bacterial that cause foodborne enteritis. Due to bacteria's antibiotic resistance, using bacteriophages for biocontrol and treatment is a new therapeutic approach. In this study, we isolated, characterized, and analyzed the genome of vB_SenS_TUMS_E19 (E19), a broad host range Salmonella bacteriophage, and evaluated the influence of E19 on liquid eggs infected with Salmonella enterica serovar Enteritidis. Transmission electron microscopy showed that the isolated bacteriophage had a siphovirus morphotype. E19 showed rapid adsorption (92% in 5 min), a short latent period (18 min), a large burst size (156 PFU per cell), and a broad host range against different Salmonella enterica serovars. Whole-genome sequencing analysis indicated that the isolated phage had a 42 813 bp long genome with 49.8% G + C content. Neither tRNA genes nor those associated with antibiotic resistance, virulence factors, or lysogenic formation were detected in the genome. The efficacy of E19 was evaluated in liquid eggs inoculated with S. Enteritidis at 4 and 25 °C, and results showed that it could effectively eradicate S. Enteritidis in just 30 min and prevented its growth up to 72 h. Our findings indicate that E19 can be an alternative to a preservative to control Salmonella in food samples and help prevent and treat salmonellosis.

Decoding the Structure-Function Relationship of the Muramidase Domain in E. coli O157.H7 Bacteriophage Endolysin: A Potential Building Block for Chimeric Enzybiotics.

Bacteriophage endolysins are potential alternatives to conventional antibiotics for treating multidrug-resistant gram-negative bacterial infections. However, their structure-function relationships are poorly understood, hindering their optimization and application. In this study, we focused on the individual functionality of the C-terminal muramidase domain of Gp127, a modular endolysin from E. coli O157:H7 bacteriophage PhaxI. This domain is responsible for the enzymatic activity, whereas the N-terminal domain binds to the bacterial cell wall. Through protein modeling, docking experiments, and molecular dynamics simulations, we investigated the activity, stability, and interactions of the isolated C-terminal domain with its ligand. We also assessed its expression, solubility, toxicity, and lytic activity using the experimental data. Our results revealed that the C-terminal domain exhibits high activity and toxicity when tested individually, and its expression is regulated in different hosts to prevent self-destruction. Furthermore, we validated the muralytic activity of the purified refolded protein by zymography and standardized assays. These findings challenge the need for the N-terminal binding domain to arrange the active site and adjust the gap between crucial residues for peptidoglycan cleavage. Our study shed light on the three-dimensional structure and functionality of muramidase endolysins, thereby enriching the existing knowledge pool and laying a foundation for accurate in silico modeling and the informed design of next-generation enzybiotic treatments.

Enzymatic dual-faced Janus structures based on the hierarchical organic-inorganic hybrid matrix for an effective bioremoval and detoxification of reactive blue-19.

A novel, dual-faced, and hierarchical type of Janus hybrid structures (JHSs) was assembled through an in situ growing of lipase@cobalt phosphate sheets on the laccase@copper phosphate sponge-like structures. The chemical and structural information of prepared JHSs was investigated by Scanning electron microscopy-energy dispersive X-ray analysis (SEM-EDX), Fourier Transform Infrared Spectroscopy (FTIR), and X-ray diffraction analysis (XRD). The catalytic activity, storage stability, and reusability of JHSs were then investigated. The SEM-EDX analysis clearly confirmed the asymmetric morphology of the fabricated JHSs with two distinct metal distributions. Under optimized synthesis conditions, the prepared JHSs showed 97.8 % and 100 % of laccase and lipase activity, respectively. Compared to the free biocatalysts, the immobilization resulted in ~ a 2-fold increase in laccase and lipase stability at temperatures of >40 °C. The fabricated JHSs maintained 61 % and 90 % of their original laccase and lipase activity upon 12 successive repetition cycles. Up to 80 % of Reactive Blue-19 (RB-19), an anthraquinone-based vinyl sulphone dye, was removed after 5 h treatment with the prepared JHSs (50 % higher than the free forms of laccase and lipase). The dye removal data fitted very well on the pseudo-second-order kinetic model with a rate constant of 0.8 g mg-1 h-1. Following the bioremoval process, bacterial toxicity also decreased by about 70 %. Therefore, the prepared JHSs provide a facile and sustainable approach for the decolorization, biotransformation, and detoxification of RB-19 by integrating enzymatic oxidation and hydrolysis.

Isolation, characterization, and genome analysis of a broad host range Salmonella phage vB_SenS_TUMS_E4: a candidate bacteriophage for biocontrol.

Salmonella enteritidis is one of the most important foodborne pathogens that cause numerous outbreaks worldwide. Some strains of Salmonella have become progressively resistant to antibiotics, so they could represent a critical threat to public health and have led to the use of alternative therapeutic approaches like phage therapy. In this study, a lytic phage, vB_SenS_TUMS_E4 (E4), was isolated from poultry effluent and characterized to evaluate its potential and efficacy for bio-controlling S. enteritidis in foods. Transmission electron microscopy revealed that E4 has a siphovirus morphotype, with an isometric head and non-contractile tail. Determining the host range showed that this phage can effectively infect different motile as well as non-motile Salmonella enterica serovars. The biological characteristics of E4 showed that it has a short latent period of about 15 min and a large burst size of 287 PFU/cell, and is also significantly stable in a broad range of pHs and temperatures. The E4 whole genome contains 43,018 bp and encodes 60 coding sequences (CDSs) but no tRNA genes. Bioinformatics analysis revealed that the genome of E4 lacks any genes related to lysogeny behavior, antibiotic resistance, toxins, or virulence factors. The efficacy of phage E4 as a bio-control agent was assessed in various foodstuffs inoculated with S. enteritidis at 4°C and 25°C, and the resulting data indicated that it could eradicate S. enteritidis after a very short time of 15 min. The findings of the present study showed that E4 is a hopeful candidate as a bio-control agent against S. enteritidis and has the potential to be used in various foodstuffs.

Isolation, characterization, and genomic analysis of vB_PaeS_TUMS_P81, a lytic bacteriophage against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that can lead to nosocomial infections which are in turn life threatening. The increase in antibiotic resistance, at an alarming rate, has resulted in a pressing need for alternative therapeutic approaches such as phage therapy, which hold promise according to several studies. This study featured the isolation and characterization of vB_PaeS_TUMS_P81, a new lytic Pseudomonas phage. The whole-genome sequencing indicated that it has a genome of 73,167 bp containing 93 predicted coding sequences. Genes involved in virulence or lysogeny pathway were nowhere to be found in the genome, so it is potentially safe when it comes to therapeutic applications. Genomic and phylogenetic analysis indicated that vB_PaeS_TUMS_P81 is a member of the genus Litunavirus, belonging to Schitoviridae family. The present study lays the groundwork for further research on treatment of P. aeruginosa infections.

Isolation, characterization, and genomic analysis of vB_PaeP_TUMS_P121, a new lytic bacteriophage infecting Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause life-threatening nosocomial infections. The alarming increase in antibiotic resistance has led to an urgent need for alternative therapeutic approaches, such as phage therapy, which has shown promising results in many studies. In this study, P121, a new lytic Pseudomonas phage, was isolated and characterized. Whole-genome sequencing showed that it has a genome of 73,001 bp that contains 91 predicted coding sequences. No genes involved in virulence or lysogeny were found in the genome, thus making it potentially safe for therapeutic applications. Genomic and phylogenetic analysis indicated that P121 is a member of the genus Litunavirus, family Schitoviridae. The present study provides some basic information for further research on treatment of P. aeruginosa infections.

Wound healing and anti-inflammatory effects of bacterial cellulose coated with Pistacia atlantica fruit oil.

BACKGROUND: Biological activities of Pistacia atlantica have been investigated for few decades. The fruit oil of the plant has been used for treatment of wounds, inflammation, and other ailments in Traditional Persian Medicine (TPM). OBJECTIVES: The main objectives of this study were to analyze the chemical composition of Pistacia atlantica fruit oil and to study wound healing and anti-inflammatory effects of oil-absorbed bacterial cellulose in an in vivo burn wound model. METHOD: Bacterial cellulose membrane was prepared from Kombucha culture and Fourier-transform infrared was used to characterize the bacterial cellulose. Cold press technique was used to obtain Pistacia atlantica fruit oil and the chemical composition was analyzed by gas chromatography. Bacterial cellulose membrane was impregnated with the Pistacia atlantica fruit oil. Pistacia atlantica hydrogel was prepared using specific Carbopol. Burn wound model was used to evaluate in vivo wound healing and anti-inflammatory effects of the wound dressings containing either silver sulfadiazine as positive control, Pistacia atlantica hydrogel or bacterial cellulose membrane coated with the Pistacia atlantica fruit oil. Blank dressing was used as negative control. RESULTS: FT-IR analysis showed that the structure of the bacterial cellulose corresponded with the standard FT-IR spectrum. The major components of Pistacia atlantica fruit oil constituted linoleic acid (38.1%), oleic acid (36.9%) and stearic acid (3.8%). Histological analysis showed that bacterial cellulose coated with fruit oil significantly decreased the number of neutrophils as a measure of inflammation compared to either negative control or positive control (p < 0.05). Wound closure occurred faster in the treated group with fruit oil-coated bacterial cellulose compared to the other treatments (p < 0.05). CONCLUSION: The results showed that bacterial cellulose coated with Pistacia atlantica fruit oil can be a potential bio-safe dressing for wound management.

Fabrication of Calcium Sulfate Coated Selenium Nanoparticles and Corresponding In-Vitro Cytotoxicity Effects Against 4T1 Breast Cancer Cell Line.

BACKGROUND: The inhibitory effect of selenium nanoparticles (SeNPs) on cancer cells has been reported in many studies. In this study, the purpose was to compare the in vitro effects of SeNPs and calcium sulfate coated selenium nanoparticles (CaSO4@SeNPs) on breast cancer cells. METHODS: CaSO4@SeNPs and SeNPs were chemically synthesized and characterized with Field Emission Scanning Electron Microscope (FESEM) and energy-dispersive X-ray spectroscopy (EDX). By applying MTT assay, the cytotoxicity effect of both nanomaterials on the 4T1 cancer cells was investigated. RESULTS: While LD50 of SeNPs on 4T1 cancer cells was 80 µg, the LD50 of CaSO4@SeNPs was reported to be only 15 µg. The difference between the inhibition rates obtained for SeNPs and CaSO4@SeNPs was statistically significant (p=0.05). In addition, at higher concentrations (50 µg) of CaSO4@SeNPs, the cytotoxicity was 100% more than SeNPs alone. CONCLUSION: According to the result of the present work, it can be concluded that decoration of SeNPs with calcium sulfate leads to an increase in potency by decreasing the effective dose. This effect can be attributed to activation of intrinsic apoptosis signaling and/or pH regulatory properties of CaSO4@SeNPs. However, further studies are still needed to determine the exact corresponding mechanisms of this synergistic effect.

Comparison of Cytokine Expression in Human PBMCs Stimulated with Normal and Heat-Shocked Lactobacillus plantarum Cell Lysate.

Regulation of immune responses is among the beneficial effects of probiotic bacteria on human health. In this study, we aim to investigate the effect of normal and heat-shocked Lactobacillus plantarum PTCC 1058 cell lysate on cytokine expression by human PBMCs. The mid-exponential phase L. plantarum (108 CFU/mL) were used to prepare cell lysate. Isolated PBMCs were stimulated with 100 µg/mL of each normal and heat-shocked L. plantarum cell lysate for 72 h. Non-stimulated PBMCs were also evaluated as negative control. The mRNA expression of IL-6, IL-10, IFN-É£, TNF-α, and TGF-ß genes was determined by quantitative RT-PCR amplification of total RNA extracted from PBMCs. Both types of cell lysate were able to increase pro-inflammatory cytokines and decrease anti-inflammatory cytokines. However, this effect was significantly stronger in heat-shocked cell lysate-treated PBMCs. Moreover, comparison of IFN-É£/IL-10, IFN-É£/TGF-ß, IL-6/IL-10, IL-6/TGF-ß, and TNF-α/IL-10 ratios in both conditions demonstrated that in the heat-shocked group, all of the above ratios were significantly higher than normal lysate treatment (p˂0.001), suggesting that heat-shocked probiotics are a potent inducer of the immune system in comparison to intact probiotics. Regarding these results, it may be possible to develop a new postbiotic product for the stimulation of immune responses of cancer patients or individuals who suffer from an immune defect.

PerioVax3, a key antigenic determinant with immunoprotective potential against periodontal pathogen.

Treponema (T.) denticola is one of the key etiological agents in the development of periodontitis. The major outer sheath protein (Msp) of T. denticola has been shown to mediate pathogenesis and to facilitate adhesion of T. denticola to mucosal surfaces. This study aimed to find short polypeptides in the amino acid sequence of Msp which may be immunogenic and might elicit protective antisera against T. denticola. The complete msp sequence was divided into six fragments and the corresponding genes were cloned and expressed. Antisera against the polypeptides were raised in rabbits and fragment 3 (F3), hereinafter called PerioVax3 was the most potent fragment of the Msp in terms of yielding high titer antiserum. An adhesion assay was done to examine the inhibitory effects of antisera on the attachment of T. denticola to human gingival fibroblasts (HGFs) and human fibronectin. Antiserum against PerioVax3 significantly inhibited attachment of T. denticola to the substratum. Also, antiserum against PerioVax3 inhibited detachment of HGFs upon T. denticola exposure. To begin examining the clinical relevance of this work, blood samples from 12 sever periodontitis patients were collected and the sera were used in western blotting against the recombinant polypeptides. Periodontitis patient antisera exclusively detected PerioVax3 in western blotting. The data suggest that PerioVax3 carries epitopes that may trigger humoral immunity against T. denticola, which may protect against its adhesion functions. The complexity of periodontitis suggests that PerioVax3 may be considered for testing as a component of an experimental multivalent periodontal vaccine in further preclinical and clinical studies.

Characterization of Folic Acid Surface-Coated Selenium Nanoparticles and Corresponding In Vitro and In Vivo Effects Against Breast Cancer.

BACKGROUNDS AND AIMS: Selenium nanoparticles (SeNPs) have been reported to exhibit an inhibitory effect on cancer cells. In the present study, we aimed to compare the in vitro and in vivo effects of SeNPs and folic acid surface-coated selenium nanoparticles (FA@SeNPs) on breast cancer. METHODS: FA@SeNPs and SeNPs were chemically synthesized and characterized with different instrumental techniques. The cytotoxicity of both nanomaterials was evaluated against 4T1 cells. In addition, the intravenous administration effect of these nanomaterials (300 µg/week) on the lifespan and tumor size of cancer-bearing mice was investigated. RESULTS: Although the SeNPs showed an antiproliferative effect against the cell line, the cytotoxicity of the FA@SeNPs was higher than that of the SeNPs. A low concentration of FA@SeNPs (25 µg/mL corresponding to 8.75 µg/mL of elemental SeNPs) caused approximately 68% cell mortality. In the in vivo study, the nanomaterials decreased the tumor growth rate in cancerous mice in relation to the control group. FA@SeNPs were more effective than SeNPs. CONCLUSIONS: The combination of SeNPs and FA has a potent antiproliferative effect against 4T1 cells, significantly increases the lifespan, and prevents tumor growth.

Targeted Intracellular Heat Transfer in Cancer Therapy: Assessment of Asparagine-laminated Gold Nanoparticles in Cell Model of T cell Leukemia.

BACKGROUND: High temperatures have destructive effects on cancer cells by damaging proteins and structures within cells. Gold nanoparticles (AuNPs) can act as drug delivery vehicles, especially for cancer therapy. Due to the selective intake of asparagine molecules into malignant cells, AuNPs were coated with asparagine; and CCRF-CEM human T-cell leukemia cells were treated with the new combination, Asn-AuNPs, at 39 °C. METHODS: The co-authors from a number of collaborative labs located at Tehran University of Medical Sciences, Tehran, Iran, have initiated the idea and preliminary design of this study in 2011. Hydroxyl surfaced AuNPs were preliminary prepared by tannin free ethanol extract of black tea leaves. These biogenic AuNPs were further capped with asparagines to form asparagine-gold nanoparticle conjugates (Asn-AuNP conjugates). Then CCRF-CEM human T-cell leukemia cells were separately treated with different concentrations of AuNPs and Asn-AuNP conjugates (3, 30, 300 µg/mL). MTT assay and zymography analysis were carried out, and the apoptotic and necrotic effects of Asn-AuNPs were determined in comparison with AuNPs, using flow cytometry assay. RESULTS: Asn-AuNP conjugates at 300 µg/mL significantly inhibited MMPs at 39 °C, compared to AuNPs. In terms of cytotoxicity, a remarkable decrease was observed in the percentage of viable cells treated with Asn-AuNP conjugates, rather than AuNPs. Moreover, the AuNPs and Asn-AuNP conjugates enhanced the level of apoptosis at almost similar rates. CONCLUSION: AuNPs are coated with asparagine molecules and the temperature is slightly increased by 2 °C, the apoptosis is not only enhanced among cells but also shifts to necrosis in higher concentrations of Asn-AuNP conjugates. More investigations should be carried out to explain the exact mechanism underlying the necrotic effects of Asn-AuNPs.

Tracing Tellurium and Its Nanostructures in Biology.

Tellurium (Te) is a semimetal rare element in nature. Together with oxygen, sulfur (S), and selenium (Se), Te is considered a member of chalcogen group. Over recent decades, Te applications continued to emerge in different fields including metallurgy, glass industry, electronics, and applied chemical industries. Along these lines, Te has recently attracted research attention in various fields. Though Te exists in biologic organisms such as microbes, yeast, and human body, its importance and role and some of its potential implications have long been ignored. Some promising applications of Te using its inorganic and organic derivatives including novel Te nanostructures are being introduced. Before discovery and straightforward availability of antibiotics, Te had considered and had been used as an antibacterial element. Antilishmaniasis, antiinflammatory, antiatherosclerotic, and immuno-modulating properties of Te have been described for many years, while the innovative applications of Te have started to emerge along with nanotechnological advances over the recent years. Te quantum dots (QDs) and related nanostructures have proposed novel applications in the biological detection systems such as biosensors. In addition, Te nanostructures are used in labeling, imaging, and targeted drug delivery systems and are tested for antibacterial or antifungal properties. In addition, Te nanoparticles show novel lipid-lowering, antioxidant, and free radical scavenging properties. This review presents an overview on the novel forms of Te, their potential applications, as well as related toxicity profiles.

Isolation, one-step affinity purification, and characterization of a polyextremotolerant laccase from the halophilic bacterium Aquisalibacillus elongatus and its application in the delignification of sugar beet pulp.

The aim of the present work was to study the ability of a halophilic bacterial laccase to efficient delignification in extreme conditions. Here, a highly stable extracellular laccase showing ligninolytic activity from halophilic Aquisalibacillus elongatus is described. The laccase production was strongly influenced by NaCl and CuSO4 and under optimal conditions reached 4.8UmL-1. The monomeric enzyme of 75kDa was purified by a synthetic affinity column with 68.2% yield and 99.8-fold purification. The enzyme showed some valuable features viz. stability against a wide range of organic solvents, salts, metals, inhibitors, and surfactants and specificity to a wide spectrum of substrates diverse in structure and redox potential. It retained more than 50% of the original activity at 25-75°C and pH 5.0-10.0. Furthermore, the enzyme was found to be effective in the delignification of sugar beet pulp in an ionic liquid that makes it useful for industrial applications.

Blockage of both the extrinsic and intrinsic pathways of diazinon-induced apoptosis in PaTu cells by magnesium oxide and selenium nanoparticles.

Diazinon (DZ) is an organophosphorus insecticide that acts as an acetylcholinesterase inhibitor. It is important to note that it can induce oxidative stress, lipid peroxidation, diabetic disorders, and cytotoxicity. Magnesium oxide (MgO) and selenium nanoparticles (Se NPs) showed promising protection against oxidative stress, lipid peroxidation, cytotoxicity, and diabetic disorders. Therefore, this study was conducted to explore the possible protective mechanisms of MgO and Se NPs against DZ-induced cytotoxicity in PaTu cell line. Cytotoxicity of DZ, in the presence or absence of effective doses of MgO and Se NPs, was determined in human pancreatic cancer cell line (PaTu cells) after 24 hours of exposure by using mitochondrial activity and mitochondrial membrane potential assays. Then, the insulin, proinsulin, and C-peptide release; caspase-3 and -9 activities; and total thiol molecule levels were assessed. Determination of cell viability, including apoptotic and necrotic cells, was assessed via acridine orange/ethidium bromide double staining. Furthermore, expression of 15 genes associated with cell death/apoptosis in various phenomena was examined after 24 hours of contact with DZ and NPs by using real-time polymerase chain reaction. Compared to the individual cases, the group receiving the combination of MgO and Se NPs showed more beneficial effects in reducing the toxicity of DZ. Cotreatment of PaTu cell lines with MgO and Se NPs counteracts the toxicity of DZ on insulin-producing cells.

Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis.

Lipases as significant biocatalysts had been widely employed to catalyze various chemical reactions such as ester hydrolysis, ester synthesis, and transesterification. Improving the activity and thermostability of enzymes is desirable for industrial applications. The lipase of Serratia marcescens belonging to family I.3 lipase has a very important pharmaceutical application in production of chiral precursors. In the present study, to achieve improved lipase activity and thermostability, using computational predictions of protein, four mutant lipases of SML (MutG2P, MutG59P, Mut H279K and MutL613WA614P) were constructed by site-directed mutagenesis. The recombinant mutant proteins were over-expressed in E. coli and purified by affinity chromatography on the Ni-NTA system. Circular dichroism spectroscopy, differential scanning calorimetry and kinetic parameters (Km and kcat) were determined. Our results have shown that the secondary structure of all lipases was approximately similar to one another. The MutG2P and MutG59P were more stable than wild type by approximately 2.3 and 2.9 in T1/2, respectively. The catalytic efficiency (kcat/Km) of MutH279K was enhanced by 2-fold as compared with the wild type (p<0.05). These results indicate that using protein modeling program and creating mutation, can enhance lipase activity and/or thermostability of SML and it also could be used for improving other properties of enzyme to the desired requirements as well as further mutations.

Adjuvant Effect of Biogenic Selenium Nanoparticles Improves the Immune Responses and Survival of Mice Receiving 4T1 Cell Antigens as Vaccine in Breast Cancer Murine Model.

The modification of tumor-associated antigen-based vaccine to elicit a more robust immune response has been addressed in several ways. In the present work, we aimed to investigate the immunomodulatory effect of selenium nanoparticles as an immunoadjuvant in formulation of a tumor-associated antigen-based vaccine in a preventive form. Fortyfive female inbred BALB/c mice five-to-seven weeks old were used and divided into three groups of test and control, each containing fifteen mice. Group one injected by PBS and used as a control. Group two injected by breast tumor cell lysate alone as vaccine. Group three injected by SeNPs with tumor cell lysate as vaccine. All injections were carried out on day fourteen, twentyone and twentyeight of the study. Tumor induction was done at day thirty. Twenty days after tumor induction serum samples were gathered to measure the cytokine assay. Tumor growth and weight of mice as well as delayed type hyper sensitivity (DTH) response were monitored during the study. Results of the present work showed a significant increase in the level of serum IFN-γ, IL-2, IL-12 and decreased TGF-ß in SeNPs/vaccine injected mice as well as lower tumor volume, more potent DTH responses and longer survival rate in comparison to control and tumor lysate vaccine. Taken together, it can be deduced from this work that SeNPs can be considered as an adjuvant in vaccine in triggering robust immune response against breast cancer. But further evaluations are still needed to find the best formula for this agent in antitumor vaccines.

Biosynthesis of tellurium nanoparticles by Lactobacillus plantarum and the effect of nanoparticle-enriched probiotics on the lipid profiles of mice.

Hypercholesterolemia is an important risk factor contributing to atherosclerosis and coronary heart disease. Lactic acid bacteria have attracted much attention regarding their promising effect on serum cholesterol levels. Tellurium (Te) is a rare element that has also gained considerable interest for its biological effects. There have been some recent in vivo reports on the reduction effect of Te on cholesterol content. In this study, Lactobacillus plantarum PTCC 1058 was employed for the intracellular biosynthesis of Te NPs. The UV-visible spectrum of purified NPs showed a peak at 214 nm related to the surface plasmon resonance of the Te NPs. Transmission electron microscopy showed that spherical nanoparticles without aggregation had the average size of 45.7 nm as determined by the laser scattering method. The energy dispersive X-ray pattern confirmed the presence of Te atoms without any impurities. A significant reduction was observed in group which received L. plantarum with or without Te NPs during propylthiouracil and cholesterol diet in compare with the control group which received just propylthiouracil and cholesterol. The levels of triglycerides also remarkably decrease (p<0.05) in mice given L. plantarum with intracellular Te NPs.