Recombinant Production of TP4-LYC1, A New Chimeric Peptide with Targeted Cytotoxicity to HeLa Cells.

Background: Tilapia Piscidin 4 (TP4) showed potential anti-tumor effects against various cancer cells. Lycosine-1 (LYC1), is another Antimicrobial Peptides (AMP) from spider venom with targeted penetration to cancer cells without any adverse effects on normal cells. The aim of this study was to produce a soluble recombinant fusion peptide in order to diminish the cytotoxicity of TP4 against normal cells. Methods: In order to express of TP4-LYC-1, TP4, and LYC1 in fusion to the inteins1/2 of pTWIN-1 vector, induction condition was optimized to earn soluble peptides. Auto-cleavage induction of inteins1/2 was performed based on IMPACT® manual and their effect on cell viability of HeLa and HUVEC cells was surveyed by MTT assay. Results: The best condition for accessing the most soluble peptide in fusion to the inteins was approximately similar for all three peptides (0.1 mM of IPTG, at 22°C). After the induction of self-cleavage of inteins, a band in 3, 3, and 6 kDa was observed on tricine-SDS-PAGE. The IC50 values of TP4-LYC1 and TP4 against HeLa cells were calculated as 0.83, and 2.75 µM, respectively. Conclusion: In the present study, a novel chimeric peptide, TP4-LYC1, was successfully produced. This fusion protein can act as a safe bio-molecule with potent cytotoxic effects against cancer cells, but the penetration ability and determination of cell death mechanism must be performed in order to have more precise view on the apoptosis induction of this recombinant peptide.

Providing a framework for evaluation disease registry and health outcomes Software: Updating the CIPROS checklist.

BACKGROUND AND AIMS: Properly designed and implemented registry systems play an important role in improving health outcomes and reducing care costs, and can provide a true representation of clinical practice, disease outcomes, safety, and efficacy. Therefore, the aim of this study was to redesign and develop a checklist with items for a patient registry software system (CIPROS) Checklist. METHOD: The study is descriptive-cross-sectional. The extraction of the data elements of the checklist was first done through a comprehensive review of the texts in PubMed, Science Direct and Scopus databases and receiving articles related to the evaluation of registry systems. Based on the extracted data, a five-point Likert scale questionnaire was created and 30 experts in this field were asked for their opinions using the two-step Delphi method. RESULTS: A total of 100 information items were determined as a registry software evaluation checklist. This checklist included 12 groups of software architecture factors, development, interfaces and interactivity, semantics and standardization, internationality, data management, data quality and usability, data analysis, security, privacy, organizational, education and public factors. CONCLUSION: By using the results of this research, it is possible to identify the defects and possible strengths of the registry software and put it at the disposal of the relevant officials to make a decision in this field. In this way, among the designers and developers of these softwares, the best and most appropriate ones are selected with the needs of the registry programs.

Invasive fungal infections in hematologic malignancies: Incidence, management, and antifungal therapy.

The incidence of invasive fungal infections (IFIs) has increased in recent years as a result of increasing the incidence of hematologic malignancies (HMs). IFIs, as the opportunistic diseases, are the most important concern in these patients with a high mortality rate. These infections are one of the leading causes of morbidity and mortality in HM patients and an important factor in increasing the costs of patients' management because of the prolonged hospitalization and the inevitable need to use antifungal agents. Due to the changes in the pattern of organisms causing IFIs, unavailability of effective and safe antifungal drugs, and high rate of drug resistance as well as lack of fast and accurate diagnostic methods, these infections have become a serious and life-threatening problem necessitating effective prevention and treatment strategies using suitable antifungal agents, especially in high-risk patients. The aim of the present study was to review the pathogens causing various types of IFIs, diagnostic methods, and novel prophylactic and therapeutic antifungal regimens in HM patients according to the new published studies and clinical trials.

Protective effects of protocatechuic acid against doxorubicin- and arsenic trioxide-induced toxicity in cardiomyocytes.

Background and purpose: Some chemotherapeutic drugs are associated with an increased risk of cardiotoxicity in patients. Protocatechuic acid (PCA) is a phenolic acid with valuable cardiovascular, chemo-preventive, and anticancer activities. Recent studies have shown the cardioprotective effects of PCA in several pathological conditions. This investigation aimed to assess the possible protective effects of PCA on cardiomyocytes against toxicities caused by anti-neoplastic agents, doxorubicin (DOX), and arsenic trioxide (ATO). Experimental approach: H9C2 cells were exposed to DOX (1 µM) or ATO (35 µM) after 24 h pretreatment with PCA (1-100 µM). MTT and lactate dehydrogenase (LDH) tests were used to define cell viability or cytotoxicity. Total oxidant and antioxidant capacities were evaluated by measuring hydroperoxides and ferric-reducing antioxidant power (FRAP) levels. Expression of the TLR4 gene was also quantitatively estimated by real-time polymerase chain reaction. Findings/Results: PCA showed a proliferative effect on cardiomyocytes and significantly enhanced cell viability and reduced cytotoxicity of DOX and ATO during MTT and LDH assays. Pretreatment of cardiomyocytes with PCA significantly decreased hydroperoxide levels and elevated FRAP value. Moreover, PCA meaningfully decreased TLR4 expression in DOX-and ATO-treated cardiomyocytes. Conclusions and implications: In conclusion, antioxidant and cytoprotective activities were found for PCA versus toxicities caused by DOX and ATO in cardiomyocytes. However, further in vivo investigations are recommended to assess its clinical value for the prevention and treatment of cardiotoxicity induced by chemotherapeutic agents.

The effectiveness of colistin/levofloxacin compared to colistin/meropenem in the treatment of ventilator-associated pneumonia (VAP) caused by carbapenem-resistant Acinetobacter baumannii: a randomized controlled clinical trial.

Background and purpose: The treatment of ventilator-associated pneumonia (VAP) caused by carbapenem-resistant Acinetobacter baumannii (CRAB) is still a great challenge. This study evaluated the effectiveness of the colistin/levofloxacin regimen compared to the usual colistin/meropenem regimen in the treatment of patients with VAP caused by CRAB. Experimental approach: The patients with VAP were randomly assigned to experimental (n = 26) and control (n = 29) groups. The first group received IV colistin 4.5 MIU every 12 h + levofloxacin 750 mg IV daily, and the second group received IV colistin with the same dose + meropenem 1 g IV every 8 h for 10 days. The clinical (complete response, partial response, or treatment failure) and microbiological responses at the end of the intervention were recorded and compared between the two groups. Findings/Results: The complete response rate was higher (n = 7; 35%) and the failure rate was lower (n = 4; 20%) in the experimental group than in the control group (n = 2; 8%, and n = 11; 44%, respectively), but the differences were not statistically significant. Even though the microbiological response rate was higher in the experimental group (n = 14; 70%) than in the control group (n = 12; 48%), the difference was not statistically significant. The mortality rate was 6 (23.10%) and 4 patients (13.8%) in the experimental and control groups, respectively (P = 0.490). Conclusion and implication: The levofloxacin/colistin combination can be considered an alternative regimen to meropenem/colistin in the treatment of VAP caused by CRAB.

Design and construction of scFv-PE35KDEL as a novel immunotoxin against human epidermal growth factor receptor 2 for cancer therapy.

PROPOSE: Human epidermal growth factor receptor 2 (HER2) is overexpressed on the surface of some kinds of cancer cells including breast cancer. In this study, we designed and produced a novel immunotoxin consisting anti-HER2 single-chain Fv (scFv) from pertuzumab and a modified form of Pseudomonas exotoxin (PE35KDEL). METHODS: The three-dimensional (3D) structure of the fusion protein (anti-HER IT) was predicted by MODELLER 9.23 and its interaction with HER2 receptor was assessed using HADDOCK web server. Anti-HER2 IT, anti-HER2 scFv, and PE35KDEL proteins were expressed by Escherichia coli BL21 (DE3). After purification of the proteins using Ni2+ affinity chromatography and refolding through dialysis, the cytotoxicity of proteins against breast cancer cell lines was examined by MTT assay. RESULTS: In-silico studies showed that (EAAAK)2 linker can efficiently prevent the formation of salt bridges between two functional domains and the constructed fusion protein has a high affinity to HER2 receptor. The optimum condition of anti-HER2 IT expression was 25 °C and 1 mM IPTG. The protein was successfully purified and refolded by dialysis with a final yield of 45.7 mg per 1 L of bacterial culture. The cytotoxicity results showed that anti-HER2 IT was much more toxic on HER2-overexpressing cells, BT-474 (IC50 ~ 95 nM) compared with HER2-negative cells, MDA-MB-23 (IC50 ˃ 200 nM). CONCLUSION: This novel immunotoxin has the potential to be applied as a therapeutic candidate for HER2-targeted cancer therapy. However further in vitro and in vivo evaluations are still required to confirm the efficacy and safety of this protein.

Andrographolide protects against doxorubicin-and arsenic trioxide-induced toxicity in cardiomyocytes.

BACKGROUND: Andrographolide (AG) is a lactone diterpene with valuable biological activities. This in vitro study evaluated whether AG can protect cardiomyocytes under toxicities triggered with anti-cancer chemotherapeutic agents, doxorubicin (DOX) and arsenic trioxide (ATO). METHODS AND RESULTS: H9C2 cells were pretreated with AG (0.5-10 µM) for 24 h and then exposed to DOX (1 µM) or ATO (35 µM) for another 24 h period. For determination of cell viability or cytotoxicity, MTT and lactate dehydrogenase (LDH) assay were used. Total oxidant and antioxidant capacities were estimated by determining hydroperoxides and ferric reducing antioxidant power (FRAP) levels. Real time-polymerase chain reaction was also used for quantitative evaluation of TLR4 gene expression. AG inhibited cardiomyocytes proliferation at the concentrations of more than 20 µM. However, it considerably enhanced cell viability and decreased cytotoxicity of DOX and ATO at the concentration range of 2.5-10 µM in MTT and LDH assays. AG significantly declined hydroperoxides concentration in ATO-treated cardiomyocytes and raised FRAP value in DOX- and ATO-treated cells. Furthermore, AG notably lessened TLR4 expression in H9C2 cells after exposure to DOX- and ATO. CONCLUSION: In conclusion, these data presented that AG was able to reverse DOX- and ATO-induced cardiotoxicity in vitro. The cardiomyocyte protective activities of AG may be due to the decrease in TLR4 expression and total oxidant capacity and increase in total antioxidant capacity.

The association of dietary antioxidant index (DAI) with breast cancer among Iranian women.

Recent studies have reported that dietary antioxidants can influence the risk of breast cancer (BC). Therefore, this study aimed to investigate the association of dietary antioxidant index (DAI) with BC among Iranian women. This case-control study was conducted on 180 women with breast cancer and 360 healthy women who were referred to the cancer clinic of Shohadaye Tajrish Hospital in Tehran, Iran. A 168-item validated food frequency questionnaire (FFQ) was used to assess dietary intake. The DAI score was calculated based on the intake of antioxidant vitamins and minerals derived from the FFQ. The control group had a significantly higher intake of vitamin D (1.79±1.56 vs. 1.05±0.84 µg/d; P=0.01) and lower intake of calorie (2315±1066 vs. 2737±925 kcal/d; P=0.01), carbohydrate (311±170 vs. 402±124 g/d; P=0.01), iron (15.4±12.1 vs. 19.7±6.4 mg/d; P=0.01), thiamine (1.5±0.7 vs. 2.3±0.9 mg/d; P=0.01), niacin (18.2±9.2 vs. 24.3±7.9 mg/d; P=0.01), folic acid (465±308.7 vs. 673±205.2 µg/d; P=0.01), and selenium (82.6±41.7 vs. 98.7±40.8 µg/d; P=0.01) compared to the case group. No significant association was found between DAI with breast cancer after adjustments for age. DAI had a negative association with breast cancer after additional adjustments for BMI, the number of pregnancies, duration of breastfeeding, menopause age, and total energy intake (OR: 0.91, 95% CI: 0.90-.93, and all P<0.001). The present study identified a negative association between DAI and the risk of BC, indicating the importance of antioxidants in preventing BC. Longitudinal studies should be conducted to confirm this association.

Development and evaluation of wafer loaded with sertaconazole solid dispersion for the treatment of oral candidiasis

Abstract Candidiasis is one of the most common fungal infections of oral cavity in humans, causing great oral discomfort, pain and aversion to food. To develop more effective antifungal systems for the treatment of oral candidiasis, an oral mucoadhesive wafer containing sertaconazole solid dispersion (STZ-SD) was developed in this study. Dispersion of STZ in Soluplus® as a solubility enhancement excipient was done by melting, solvent evaporation and freeze drying method at various STZ to Soluplus® ratios. The optimized STZ-SD was then incorporated in the sodium carboxymethyl cellulose (SCMC) gel, xanthan gum gel, or their combination to prepare the lyophilized wafers. The swelling capacity, porosity, and mechanical, release and mucoadhesive properties of the wafers, together with their antifungal activity, were then evaluated. The melting method sample with the ratio of 8:1 showed the best results in terms of saturation solubility and dissolution rate. The STZ-SD-composite wafer exhibited higher hardness and mucoadhesion, as compared to those made of the SCMC polymer. The STZ-SD-wafer also exhibited a greater antifungal effect when compared to the STZ-wafer. The present study, thus, suggested that the STZ-SD-wafer could serve as a novel effective delivery system for oral candidiasis treatment.

Antibiotic resistance pattern of Helicobacter pylori strains isolated from patients in Isfahan, Iran.

Background: The objective of this study was to evaluate the antibiotic resistance pattern of Helicobacter pylori strains isolated from patients in Isfahan province. Materials and Methods: Gastric antrum biopsy specimens of patients undergoing endoscopy were cultured. The samples with the growth of H. pylori underwent antibiotic susceptibility test by disk diffusion method. Reaults: Of 96 samples, 50 samples (53%) were positive for H. pylori. The rates of antibiotic resistance were as follows: amoxicillin, 6%; azithromycin, 20%; furazolidone, 22%; levofloxacin, 16%; metronidazole, 20%; rifampin, 12%; and tetracycline, 22%. Conclusion: H. pylori strains in our area have high rates of resistance to azithromycin, levofloxacin, metronidazole, tetracycline, and furazolidone.

Intake of food groups and cervical cancer in women at risk for cervical cancer: A nested case-control study.

Background: The risk of cervical cancer was reported to be influenced by dietary components. This study aimed to illustrate the association between cervical cancer with the intake of food groups in women with a history of cervical neoplasia. Methods: This nested case-control study was conducted in 558 people with a history of cervical intraepithelial neoplasia (CIN), including 279 women with cervical cancers and 279 controls with low-grade squamous intraepithelial lesions (LSIL). A validated food frequency questionnaire (FFQ) was used to assess the intake of food groups. Results: The intake of fruits and vegetables in the case group was significantly lower than the control group (P=0.001). Low intake of dairy products, vegetables, and fruits was associated with cervical cancer risk (OR=4.67; 95% CI 1.2-9.49, P=0.001; OR=9.75, 95% CI 1.36-19. 51, P=0.001; and OR=4.82, 95% CI 1.09-7.25, P=0.001, respectively). After adjusting for age, family history, age at first menstruation, number of children, history of vaginal infection, and age at first sexual intercourse, the results were still significant. Additional adjustments to BMI did not change the results. Conclusion: The results indicate that the risk of cervical cancer can be affected by the intake of certain food groups. Further longitudinal studies are needed to confirm these findings and determine the underlying mechanism of the influence of dietary components on cervical cancer risk.

Recombinant Production of a Mutant Form of Soluble IL-6 Receptor with Inhibitory Effects against Interleukin-6.

Background: Interleukin-6 (IL-6) has undeniable roles in inflammatory processes due to autoimmune diseases. In this regard, soluble receptors are considered a potential approach to mitigate its inflammatory effects and modulate its physiological effects by reducing the IL-6 binding to cell surface-specific receptors. Objective: This study aimed to produce IL-6 receptor (IL-6R) in soluble form with enhanced affinity to IL-6 without signal transduction ability. Materials and Methods: The 3D structure of IL-6R with the selective mutations for enhancing the IL-6 binding, with minimum ability to signal transduction (mIL-6R), was predicted using Modeller 9.19. This mutated form was docked to IL-6 and gp130 (a part of the native IL-6 receptor involved in signal transduction) by the HADDOCK2.2 web server. The expression of mIL-6R was performed in E. coli BL21 (DE3), using pTWIN-1 plasmid as its linkage to the Ssp Intein. IMPACT system manual was used to purify the protein at 25 °C overnight. Next, ELISA was performed to compare the affinity of mutated and native IL-6R to IL-6. Finally, A549 cells were used to compare the inhibition of cytotoxic effects of native and mutated IL-6R. Results: In the silico section, results established the stability of mutant's structure with more and less affinity to IL-6 and gp130, respectively. The expression and purification results showed bands of about 50 and 23 kDa, representing the correct size of the Intein1-mIL-6R fusion protein and cleavaged mIL-6R in SDS-PAGE, respectively. Furthermore, a significant enhancement in the affinity of mutated IL-6R to IL-6 was observed compared to the native receptor. Finally, A549 cells showed more cytotoxic effects followed by treating with mutated IL-6R in comparison to cells treated with native soluble IL-6R. Conclusion: The recombinant production of a mutated form of IL-6R with the potential ability to antagonize the IL-6 inflammatory effects confirmed with in silico studies was successfully performed for the first time to create a new drug candidate for suppressing the inflammatory effects of IL-6.

Resveratrol supplementation efficiently improves endothelial health: A systematic review and meta-analysis of randomized controlled trials.

We perform a systematic review and meta-analysis of randomized controlled trials (RCTs) to quantify the effect of resveratrol supplementation on endothelial function. A comprehensive search was performed in electronic databases including PubMed, Scopus, Web of Science, and Cochrane Library up to February 2021 with no limitation in time and language. A meta-analysis of eligible studies was performed using a random-effects model to estimate the pooled effect size of flow-mediated dilation (FMD), intracellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), fibrinogen, and plasminogen activator inhibitor-1 (PAI-1). In total, 21 arms from 17 studies were included. The meta-analysis results showed that resveratrol significantly change the concentrations of FMD (WMD: 1.43%; 95% CI: 0.98 to 1.88, p < .001) and ICAM-1 (WMD: -7.09 ng/ml, 95% CI: -7.45 to -6.73, p < .001). However, VCAM-1, fibrinogen, and PAI-1 did not change significantly after resveratrol supplementation. In conclusion, the results of this study suggest that resveratrol supplementation can improve endothelial function which could be important, especially in patients with cardiovascular diseases.

FTO genotype was associated with breast cancer in HER2 negative patients.

BACKGROUND: The fat mass and obesity-associated (FTO) gene may influence the risk of breast cancer (BC). The single nucleotide polymorphisms (SNPs) of FTO gene may exert different impacts on different types of BC. In this study, we investigated the association between FTO SNP rs9939609 and the status of estrogen receptor (ER), progesterone receptor (PR), P53, and human epidermal growth factor receptor-2 (HER-2) in BC patients. METHODS: Our case-control study was included 540 Iranian participants aged 35 to 70 (180 women with BC as the case group and 360 healthy controls). After genotyping for risk allele rs9939609 of the FTO gene, a logistic regression was applied to elucidate the association between FTO SNP rs9939609 and BC risk based on the receptor status. RESULTS: The number of HER-2 negative patients was significantly higher in FTO rs9939609 risk allele carrier group (61.5% vs. 41.4%, P < 0.05). A significant association was found between BC and rs9939609 FTO gene polymorphism only in HER2 negative BC patients (OR = 1.79, CI95%: 1.2-3.56, P = 0.03). No association was identified between FTO rs9939609 polymorphism and the status of ER, PR, and P53. CONCLUSION: We indicated that FTO SNP rs9939609 can be a potential therapeutic target particularly in HER-2 negative BC cases. The importance of this risk allele in BC pathogenesis needs to be further highlighted.

Pramlintide: An Amylin Analogue Protects Endothelial Cells against Oxidative Stress through Regulating Oxidative Markers and NF-κb Expression.

Background: Oxidative stress has a prominent role in the pathogenesis of diabetes complications. Pramlintide is an injectional amylin analogue used for the treatment of type 1 and type 2 diabetic patients. The present investigation evaluated the effect of pramlintide against oxidative damage induced by hydrogen peroxide (H2O2) in human umbilical vein endothelial cells (HUVECs). Methods: Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Hydroperoxides level, ferric reducing antioxidant power (FRAP), and expression of transcription factor NF-κB were measured in HUVECs that pretreated with pramlintide and, then exposed to H2O2. Results: Pramlintide significantly decreased the cytotoxicity caused by H2O2 at the concentrations of 5 and 10 µg/mL. Pretreatment of HUVECs with pramlintide reduced hydroperoxides and increased FRAP value in intra- and extra-cellular mediums at different concentration ranges compared with H2O2 stimulated cells. Pramlintide (10 µg/mL) remarkably ameliorated the expression of NF-κB gene after 1, 3 and 24 h exposure to H2O2. Conclusions: Findings of the current investigation displayed that pramlintide may act as a protective against oxidative conditions in endothelial cells through modulation of oxidative markers and transcription factor NF-κB.

Internalizing RGD, a great motif for targeted peptide and protein delivery: a review article.

Understanding that cancer is one of the most important health problems, especially in advanced societies, is not difficult. The term of targeted cancer therapy has also been well known as an ideal treatment strategy in the recent years. Peptides with ability to specifically recognize the cancer cells with suitable penetration properties have been used as the targeting motif in this regard. In the present review article, we focus on an individual RGD-derived peptide with ability to recognize the integrin receptor on the cancer cell surface like its ancestor with an additional outstanding feature to penetrate to extravascular space of tumor and ability to penetrate to cancer cells unlike the original peptide. This peptide which has been named "internalizing RGD" or "iRGD" has been the focus of researches as a new targeting motif since it was discovered. To date, many types of molecules have been associated with this peptide for their targeted delivery to cancer cells. In this review article, we have discussed a summary of penetration mechanisms of iRGD and all introduced peptides and proteins attached to this attractive cell-penetrating peptide and have expressed the results of the studies.

Rational design of a new variant of Reteplase with optimized physicochemical profile and large-scale production in Escherichia coli.

Structural engineering of the recombinant thrombolytic drug, Reteplase, and its cost-effective production are important goals in the pharmaceutical industry. In this study, a single-point mutant of the protein was rationally designed and evaluated in terms of physicochemical characteristics, enzymatic activity, as well as large-scale production settings. An accurate homology model of Reteplase was used as the input to appropriate tools to identify the aggregation-prone sites, while considering the structural stability. Selected variants underwent extensive molecular dynamic simulations (total 540 ns) to assess their solvation profile and their thermal stability. The Reteplase-fibrin interaction was investigated by docking. The best variant was expressed in E. coli, and Box-Behnken design was used through response surface methodology to optimize its expression conditions. M72R mutant demonstrated appropriate stability, enhanced enzymatic activity (p < 0.05), and strengthened binding to fibrin, compared to the wild type. The optimal conditions for the variant's production in a bioreactor was shown to be 37 ºC, induction with 0.5 mM IPTG, for 2 h of incubation. Under these conditions, the final amount of the produced enzyme was increased by about 23 mg/L compared to the wild type, with an increase in the enzymatic activity by about 2 IU/mL. This study thus offered a new Reteplase variant with nearly all favorable properties, except solubility. The impact of temperature and incubation time on its large-scale production were underlined as well.

L-carnitine supplementation ameliorates insulin resistance in critically ill acute stroke patients: a randomized, double-blinded, placebo-controlled clinical trial.

BACKGROUND AND PURPOSE: Insulin resistance (IR) can negatively affect clinical outcomes in acute ischemic stroke (IS) patients. Safe and cost-saving interventions are still needed to improve glycemic indices in this population. The primary objective was to evaluate L-carnitine (LC) effects in acute IS patients' homeostatic model assessment of IR (HOMA-IR). EXPERIMENTAL APPROACH: In this randomized, double-blind placebo-controlled clinical trial, critically ill IS patients were allocated to receive daily oral L-carnitine (1.5 g) or a placebo for six days. Fasting serum levels of glucose, insulin, C-reactive protein, LC, and HOMA-IR were measured on days 1 and 7. Mechanical ventilation duration, ICU/hospital duration, illness severity score, sepsis, and death events were assessed. FINDINGS/RESULTS: Forty-eight patients were allocated to the research groups, 24 patients in each group, and all were included in the final analysis. LC administration showed a decrease in mean difference of HOMA-IR and insulin levels at day 7 compared to placebo, -0.94 ± 1.92 vs 0.87 ± 2.24 (P = 0.01) and -2.26 ± 6.81 vs 0.88 ± 4.95 (P = 0.03), respectively. However, LC administration did not result in significant improvement in clinical outcomes compared to placebo. The short duration of intervention and low sample size limited our results. CONCLUSION AND IMPLICATION: Supplementation of L-carnitine improved HOMA-IR index in acute IS patients admitted to the critical care unit. Supplementation of LC would be a potential option to help to control IR in critically ill acute IS patients.

DFF40-iRGD, a novel chimeric protein with efficient cytotoxic and apoptotic effects against triple-negative breast cancer cells.

PURPOSE: DNA fragmenting factor (DFF40), an endonuclease inducing irreversible apoptosis protein, is down-regulated in many types of tumor cells. iRGD is a tumor-penetrating peptide with high affinity to cancer cells overexpressing αVß3 receptor. The aim of this study was to produce the recombinant DFF40-iRGD protein as a new molecule to selectively induce cytotoxicity in cancer cells and evaluate its biological effects. METHODS: The three-dimensional structure of DFF40-iRGD was predicted using Modeller software and its interaction with αVß3 receptor was evaluated by HADDOCK web-server. Recombinant DFF40 and DFF40-iRGD proteins were produced using intein fusion system in Escherichia coli BL21 (DE3). To improve the soluble expression, the inducer concentration, temperature and incubation time were optimized. After purification of DFF40 and DFF40-iRGD using chitin column, the cytotoxic and apoptotic effects of the proteins against MDA-MB-231 (αVß3 positive) and MCF-7 (αVß3 negative) cell lines were evaluated using cell viability assay and flow cytometric analysis. RESULTS: The results of molecular docking indicated the proper interaction of DFF40-iRGD with the integrin receptor comparable to iRGD. The optimum conditions of soluble expression of proteins were the induction by 0.5 mM and 0.1 mM of IPTG for DFF40 and DFF40-iRGD, respectively, at 7 °C for 24 h. After 48 h of incubation, DFF40-iRGD exhibited significantly higher cytotoxic effect against MDA-MB-231 cells than MCF-7 cells as IC50 values of 19.25 and 41 nM were found for MDA-MB-231 and MCF-7 cells, respectively. However, DFF40 cytotoxicity was not significantly different in two cell lines. Furthermore, Flow cytometry results showed that the fusion protein can induce remarkably apoptotic cell death in cancer cells. CONCLUSION: In this study, DFF40-iRGD protein was produced in soluble form and its inhibitory effects on cancer cell survival and induction of apoptosis were established; therefore, it has the potential to be used as a drug candidate for targeted treatment of breast cancer, especially Triple Negative Breast Cancer Cells.

Recombinant Production and One-Step Purification of IL-1Ra in Escherichia coli and Evaluation of its IL-1 Antagonizing Efficacy.

BACKGROUND: Anakinra (Kineret®), an IL-1 receptor antagonist, is the first FDA-approved biologic drug for antagonizing IL-1 in patients with Rheumatoid arthritis. The less expensive production of this drug might help reduce the final therapeutic costs. OBJECTIVES: To evaluate the possibility of producing biologically active recombinant IL-1Ra by a single-step purification procedure mediated by a self-cleavable intein. METHODS: Soluble expression of the rIL-1Ra was performed in E. coli BL21 (DE3) infusion to intein1 of pTWIN-1 vector and its cleavage induction using an elution buffer (pH 6.8) at room temperature. Evaluation of the antagonizing efficacy of this protein in various concentrations was performed on A375 and HEK293 cells treated by a constant concentration of IL-1ß (2 ng/mL). RESULTS: IPTG induction of E. coli BL21 (DE3) transformed with the recombinant pTWIN-1, revealed a band approximately in 45 kDa, which is related to the intein1-rIL-1Ra fusion protein in the SDS-PAGE. Moreover, protein purification was confirmed by observing a band in 18 kDa. Finally, the percentage of inhibition effects of rIL-1Ra and Kineret® against IL-1ß was not statistically significant in IL-1-responsive A375 cells. The inhibition percentage was calculated as 86% in cells treated with 15µg/mL of rIL-1Ra, which was 96% for the inhibitory effects of the standard drug. CONCLUSION: In this study, biologically active soluble rIL1-Ra was successfully produced with high purity through a one-step procedure. This method can reduce the cost and time of production for this protein and might be applicable other biological products.