Cloning and expression of recombinant arazyme with anti-inflammatory and anti-breast cancer potential.

Arazyme is an extracellular metalloprotease which is secreted by a Gram-negative symbiotic bacterium called Serratia proteomaculans. There are limited studies on various biological activities of arazyme. This preliminary study was designed to investigate the anti-cancer and anti-inflammatory capacities of recombinant arazyme (rAra) in vitro and in vivo. Arazyme gene, araA was cloned and expressed in E. coli BL21 (DE3) using pET-28a as a vector. Nickel column purification was used to obtain pure rAra. SDS-PAGE and protein assay were used to identify the product and to measure protein content, respectively. Skimmed milk test and casein assay were carried out to assess protease activity. MCF7 cells as a breast cancer cell model were exposed to different concentrations of rAra to study anti-breast cancer potentials using MTT assay. The anti-inflammatory property of rAra was investigated using a murine air-pouch model. PCR and SDS-PAGE data showed that cloning and expression of rAra was successful and the enzyme of interest was observed at 52 KDa. Protein assay indicated that 1 mg/ml of rAra was obtained through purification. A clear zone around the enzyme on skimmed milk agar confirmed the proteolytic activity of rAra and the enzymatic activity was 320 U/mg protein in the casein assay. Cytotoxic effects of rAra reported as IC50 were 16.2 µg/ml and 13.2 mg/ml after 24 h and 48 h, respectively. In the air-pouch model, both the neutrophil count and myeloperoxidase activity, which are measures of inflammation, were significantly reduced. The results showed that rAra can be used in future mechanistic studies and R&D activities in the pharmaceutical industry to investigate the safety and efficacy of the recombinant arazyme.

Effect of goji berry incorporation on the texture, physicochemical, and sensory properties of wheat bread.

The regular intake of Lycium barbarum (goji berry) is supposed to play an important role in the promotion of human health. Regarding, its incorporation into staple foods, including bread, seems to be effective. However, it requires the evaluation of dough behavior and final product quality. This study investigated the effect of goji berry incorporation at levels of 10, 15, 20, 25, and 30% ww-1 on the textural, physicochemical, and sensory properties of wheat bread. Results indicated a significant enhancement of water absorption and gelatinization temperature in composite flour via the inclusion of goji berry powder (p < 0.05). Using goji berry powder up to 20% ww-1 has shown to obtain the structure able to restore gases through the baking process and provide enhancement in a specific volume at about 10%. Alongside, the hardness of composite bread decreased, and the optimal hardness was observed at formulations containing 20% w/w goji berry powder with a value equal to 1199.95 ± 0.05 g, which is supposed to be induced by the higher specific volume and lower moisture content of bread samples. Moreover, color and sensory perception have been found to be significantly changed by goji berry substitution. Goji berry substitution up to 20% ww-1 is found to be preferred by the consumer, and a drop in overall acceptability was observed at its higher inclusion. The technological characteristic changes induced by goji berry incorporation are induced by its gluten dilution impact. However, the gel-like structure formed by the high fiber content of goji berries compensates for this adverse impact up to 20% w/w substitution level.

Hydro- and aerogels from quince seed gum and gelatin solutions.

The composite hydro/aerogels were designed using gelatin and quince seed gum (QSG) at total polymer concentration (TPC) of 1, 1.5 and 2% and gelatin/QSG ratio of 1:0, 1:0.5 and 1:1. The gel syneresis decreased significantly with increase in TPC and QSG. Although, hydrogels with 2% TPC had remarkably higher gel strength and elasticity than 1% TPC ones, the addition of high levels of QSG to the gelatin (i.e., gelatin/QSG 1:1) led to a decrease in its gel strength (∼0.97-fold) and elasticity (∼3,463-fold). The temperature-sweep test showed higher melting points in gelatin/QSG hydrogels (>60 °C) compared to the gelatin ones (∼58 °C). Additionally, QSG addition to the gelatin led to more porous networks with higher gel strength, thermal stability, and crystallinity, as observed by scanning electron microscopy, differential scanning calorimetry, and X-ray diffractometer. Therefore, QSG could be used as a natural hydrocolloid to modify gelatin functionality.

Changes in fat uptake, color, texture, and sensory properties of Aloe vera gel-coated eggplant rings during deep-fat frying process.

There is a widespread use of deep-fat frying in both domestic and industrial sections, and deep-fat fried foods are extremely popular due to their taste, color, and crispy texture. Human health can be, however, seriously compromised by the excessive consumption of oil, especially saturated fats and trans fatty acids. The use of hydrocolloids in inhibiting oil absorption has garnered considerable attention. This study was therefore aimed to lower the oil absorption in eggplant rings during the deep-fat frying process with the aid of Aloe vera gel coating. The effects of gel concentration (0%, 50%, and 100%), frying time (2, 5, and 8 min), and frying temperature (160°C and 180°C) on the oil uptake, moisture content, texture, color, and sensory properties of the eggplant rings were evaluated. The gel coating led to a decrease in oil uptake (up to 50%), hardness (up to 0.98-fold), ΔE (up to 0.89-fold), and overall acceptance (up to 0.85-fold), and an increase in moisture content (up to 1.47-fold) and lightness (up to 1.14-fold) of the samples. The frying time and temperature also influenced the physiochemical and sensory properties of the eggplant rings. The sample coated with 50% gel and fried at 180°C for 8 min had lower oil content and water loss with the highest acceptance rate in terms of taste, color, odor, texture, and appearance. The Aloe vera gel could be, therefore, a good candidate with high nutritional and economic value to reduce oil uptake in fried food products.

The role of miRNA-377 as a tumor suppressor in lung cancer by negative regulation of genes belonging to ErbB signaling pathway.

BACKGROUND: The ErbB signaling pathway plays important role in the pathogenesis of lung cancer. We explored the role of miRNA-377 as a tumor suppressor in NSCLC through silencing of some genes in the ErbB pathway. METHODS AND RESULTS: The targeting effect of miRNA-377 on EGFR, MAPK1, ABL2, and PAK2 was evaluated. The expression levels of these genes and miRNA-377 were surveyed in NSCLC and normal human tissues, Calu-6, and A549 cells. Real-time PCR was used to figure out whether miRNA-377 could decrease the target genes mRNAs in transfected lung cancer cell lines. The effects of miRNA-377 on apoptosis cell and proliferation were analyzed. We showed that miRNA-377 targets EGFR, MAPK1, and PAK2 mRNAs in in-silico and luciferase reporter assay. The expression of miRNA-377 was significantly downregulated in human NSCLC tissues, Calu-6 and A549 cells compared to their controls. We observed a negative correlation between EGFR, MAPK1, PAK2, and miRNA-377 expression in human NSCLC tissues. A significant reduction in EGFR, MAPK1, and PAK2 mRNA levels was detected, following miRNA-377 transfection in Calu-6 and A549 cells. The higher levels of miRNA-377 in Calu-6, and A549 cells induced apoptosis and reduced proliferation, significantly. CONCLUSIONS: All these data reveal that miRNA-377 functions as a tumor suppressor in NSCLC and may serve as a potential therapeutic target for the treatment of NSCLC.

Phytocatalytic and cytotoxic activity of the purified laccase from bled resin of Pistacia atlantica Desf.

This study reports an efficient and fast procedure for the purification of laccase (PaL) obtained from the resin of Pistacia atlantica Desf. It was purified by one-step affinity chromatography and showed the specific activity of 393 U/mg with 81.9-fold purification. The molecular weight of PaL was estimated to be approximately 60 kDa using gel electrophoresis SDS-PAGE. Moreover, it depicted diphenolase activity and high affinity towards 2,6-dimethoxy phenol (Km = 10.01 ± 0.5 mM) and syringaldazine (Km = 6.57 ± 0.2 mM) comparing with plant-origin polyphenol oxidases reported in the literature. It should be noted that PaL possessed optimal activity at pH 7.5 and 45 °C. It also remained stable under different conditions of pH (6.5-8.0), temperature (25-45 °C), and when it was exposed to several metal ions. The MTT and flow cytometry assays demonstrated that the enzyme treatment significantly affected growth of HeLa, HepG2, and MDA-MB-231 cells with LC50 values of 4.83 ± 0.02, 61 ± 0.31, and 26.83 ± 0.11 µM after 72 h, respectively. NOVELTY STATEMENT: This is the first attempt to isolate and characterize a new oxidoreductase from the resin of Pistacia atlantica Desf., native species of Iran, to recruit it in cytotoxicity researches. In the purification process by an efficient affinity column (SBA-NH2-GA), the enzyme was eluted promptly with a satisfied yield. The purified laccase exerted higher affinity to diphenolic compounds and pH-thermal stability compared to other plant-derived polyphenol oxidases. The purified enzyme was found to show anti-oxidant capacity and significantly inhibited the growth of cancerous cells in vitro. PaL showed more cytotoxic activity towards HeLa and MDA-MB-231 cells by induction of apoptosis. The cytotoxic activity of the laccase was measured by flow cytometry.

Purification and study of anti-cancer effects of Serratia marcescens serralysin.

BACKGROUND AND OBJECTIVES: Serralysin is an extracellular metalloprotease from Serratia marcescens which has been the subject of extensive biological investigations. The goal of this study was to extract and purify serralysin from S. marcescens and to investigate its cytotoxic activity on the colorectal cancer cell line. MATERIALS AND METHODS: The presence of the serralysin gene was confirmed using PCR. The supernatant of bacterial culture was collected and precipitated using ammonium sulfate. The precipitated protein was dialyzed and subjected to ion exchange chromatography for further purification. Casein assay and skim milk assay was used to confirm the enzymatic activity. SDS-PAGE was used to visualize the presence of serralysin. Metalloprotease inhibition activity was performed using 50 mM EDTA. Cytotoxic activity of serralysin was assessed on MTT assay. RESULTS: The PCR product corresponding to serralysin was estimated to be approximately 1500 bp. A transparent zone around the bacterial colonies on skim milk agar and casein digestion confirmed the proteolytic activity of serralysin. A 52 kDa band in SDS-PAGE corresponding to serralysin was observed before and after purification processes. MTT assay showed IC50 values 24.78 µg/ml and 19.16 µg/ml after 24 h and 48 h exposure of Caco-2 cells to serralysin, respectively. CONCLUSION: Our results showed that native serralysin has anticancer potential and may be a candidate for further pharmaceutical research and development. Further in vivo and in vitro mechanistic studies are suggested to confirm the biological activities.

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.

Silencing the wild-type and mutant K-ras increases the resistance to 5-flurouracil in HCT-116 as a colorectal cancer cell line.

Colon cancer is the second to third common cancer worldwide. Several efforts have been made to reveal the pathways responsible for drug resistance in this type of cancer. We aimed to investigate the effect of silencing both mutant and wild-type Kristen Rous sarcoma (k-ras) on the response of human colorectal tumor 116 (HCT-116) as a colon cancer cell line to the cytotoxic effect of 5-flurouracil (5-FU). One oligonucelotide against mutant k-ras (12th codon, namely 207) and two against wild-type k-ras (namely 535 and 689) were cloned into pSilencer neo2.1. The linearized vectors besides the negative control plasmid were stably transfected into HCT-116. The proliferation rates of these cells in different concentrations of 5-FU and the apoptosis rates of the cells after treatment with lethal doses of 5-FU were studied. Moreover, the cell cycle in these cells was also analyzed by staining the cells with propidium iodide. Stably transfected cells were named HCT207ks, HCT535ks, HCT689ks, and HCT-Sc (transfected with the negative control plasmid). Decreased expression of k-ras in HCT207ks, HCT535ks, and to a lesser extent in HCT689ks was proved by quantitative real-time PCR. Although in HCT207ks the cells were mostly in G0/G1 and G2/M phases, in HCT535ks and HCT689ks, the cells in the S phase were higher in comparison with nontransfected HCT-116. Lethal doses of 5-FU in HCT-116 and HCT-Sc were 2.5-3 and 3-3.5 µmol/l, whereas in HCT207ks, HCT535ks, and HCT689ks, they were 35-40, 37.5-40, and 22.5-25 µmol/l. In conclusion, silencing mutant and wild-type k-ras would increase the resistance of HCT-116 cell line as a model of colorectal cancer to 5-FU. The degree of resistance was related directly to the k-ras mRNA level. Therefore, both mutant and wild-type k-ras may play a role in sensitizing colorectal cancer cells to 5-FU as a common chemotherapeutic drug.

Bevacizumab for choroidal neovascularization secondary to age-related macular degeneration and pathological myopia.

INTRODUCTION: Many retinal specialists have utilized intravitreal bevacizumab as an anti-VEGF to treat choroidal neovascularization (CNV), secondary to age-related macular degeneration (AMD) and pathological myopia, with favorable results. Bevacizumab is currently approved only for the systemic treatment of colon carcinoma, whereas it is widely used off-label for treating ocular neovascular diseases. AREAS COVERED: In this review, after thorough search, 33 relevant studies conducted in the last 4 years were found. These articles comprised 14 studies about use of bevacizumab alone or in combination with other therapeutic agents to treat exudative AMD, and 19 studies on the use of myopic CNV. EXPERT OPINION: Although bevacizumab is widely used as an anti-VEGF agent for the treatment of exudative AMD, data on its systemic side effects are limited because of studies' short follow-up periods, absence of appropriate controls, limitation in reporting outcomes, and lack of controlled clinical trials in Phase III. Some safety studies demonstrated no difference between bevacizumab and ranibizumab in occurrence of heart attacks or stroke. Conducting proper randomized clinical trials with long-term follow-up is crucial to make sure about efficacy and safety of bevacizumab.