Biogenic synthesized copper oxide nanoparticles by Bacillus subtilis: Investigating antibacterial activity on the mexAB-oprM efflux pump genes and cytotoxic effect on MCF-7 cells.

One of the main characteristics of Pseudomonas aeruginosa is remarkable intrinsic antibiotic resistance which is associated with production of ß-lactamases and the expression of inducible efflux pumps. Nanoparticles (NPs) are a novel option for coping with this resistant bacteria. Hence, the aim of present study was production of CuO NPs via Bacillus subtilis and applied them to deal with resistant bacteria. For this purpose, first NPs were synthesized and were analyzed with different standard techniques containing scanning electron microscope, Fourier-transform infrared spectroscopy, and X-ray powder diffraction. Microdilution Broth Method and real-time polymerase chain reaction were used to antibacterial properties of the CuO NPs and expression of mexAB-oprM in clinical samples of P. aeruginosa, respectively. The cytotoxic effect of CuO NPs was also evaluated on MCF7 as a breast cancer cell line. Finally, the data were analyzed by one-way analysis of variance and Tukey's tests. The size of CuO NPs was in the range of 17-26 nm and showed antibacterial effect at <1000 µg/mL concentrations. Our evidence noted that the antibacterial effects of the CuO NPs occurred through the downregulation of mexAB-oprM and upregulation of mexR. The interesting point was that CuO NPs had an inhibitory effect on MCF7 cell lines with the optimal inhibition concentration at IC50 = 25.73 µg/mL. Therefore, CuO NPs can be considered as a promising medical candidate in the pharmaceutical industry.

Monophosphoramide derivatives: synthesis and crystal structure, theoretical and experimental studies of their biological effects.

Synthesizing new chemical compounds and studying their biological applications have been important issues in scientific research. In this investigation, we synthesized and characterized ten new N-acetyl phosphoramidate compounds and explored the crystal structure of three others. Furthermore, not only were some kinetic inhibition parameters measured, like IC50, Ki, kp, KD for 7 compounds on human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), but also their hydrophobic parameter was determined by shake-flask technique. All compounds (number 1-10) were investigated for anti-bacterial activity against three Gram-positive and three Gram-negative bacteria, while chloramphenicol was used as a standard antibiotic. In order to find new insecticide, toxicities of 13 acephate (Ace)-derived compounds (number 20-32) were bioassayed on third larval instar of elm leaf beetle and Xanthogaleruca luteola. Additionally, screening in vivo tests revealed that two compounds had had the greatest insecticidal potential in comparison with others. It means these ones inhibited AChE (with mixed mechanisms) and general esterase more than the rest. According to ChE-QSAR models, the inhibitory potency for enzyme and bacteria is directly influenced by the electronic parameters versus structural descriptors. AChE-QSPR model of fluorescence assay indicated that the inhibitory power of AChE is primarily influenced by a set of electronic factors with the priority of: EHB > PL > δ(31P) versus structural descriptor (SA and Mv). Synthesizing new chemical compounds and studying their biological applications have been important issues in scientific research. Toxicities of 13 acephate (Ace)-derived compounds (number 20-32) were bioassayed on third larval instar of elm leaf beetle and Xanthogaleruca luteola. Insect-QSAR equations of these compounds, based on MLR and PCA, showed that non-descriptor net charge nitrogen atom (which was affected by the polarization of N-H group) had the greatest effect on insecticidal potential.

Encapsulation of Vitamin B12 by Complex Coacervation of Whey Protein Concentrate-Pectin; Optimization and Characterization.

Vitamin B12 (VB12) is one of the essential vitamins for the body, which is sensitive to light, heat, oxidizing agents, and acidic and alkaline substances. Therefore, the encapsulation of VB12 can be one of the ways to protect it against processing and environmental conditions in food. In this work, the influence of pectin concentration (0.5−1% w/v), whey protein concentrate (WPC) level (4−8% w/v) and pH (3−9) on some properties of VB12-loaded pectin−WPC complex carriers was investigated by response surface methodology (RSM). The findings showed that under optimum conditions (1:6.47, pectin:WPC and pH = 6.6), the encapsulation efficiency (EE), stability, viscosity, particle size and solubility of complex carriers were 80.71%, 85.38%, 39.58 mPa·s, 7.07 µm and 65.86%, respectively. Additionally, the formation of complex coacervate was confirmed by Fourier-transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM). In addition, it was revealed that the most important factor in VB12 encapsulation was pH; at a pH < isoelectric point of WPC (pH = 3), in comparison with higher pH values (6 and 9), a stronger complex was formed between pectin and WPC, which led to an increase in EE, lightness parameter, particle size and water activity, as well as a decrease in the zeta-potential and porosity of complex carriers.

Synergistic Cytotoxic and Apoptotic Effects of Local Probiotic Lactobacillus Brevis Isolated from Regional Dairy Products in Combination with Tamoxifen.

Tamoxifen (TAM), the most widely used anti-estrogenic drug, inhibits the progression of breast cancer through competing with estrogen for binding to the estrogen receptor (ER). Tamoxifen has been the first-line adjuvant endocrine therapy in pre- and postmenopausal patients with ER + breast cancer for two decades. However, due to its side effects, interest in using anticancer agents derived from natural foods has increased. It has recently been stated that some probiotics can improve the efficacy of anticancer drugs via synergistic effects. Here, Local Probiotic Lactobacillus Brevis were isolated and characterized from dairy products and its supernatant was prepared. Proliferation of MCF-7, a breast cancer cell line, was investigated after treatment with Lactobacillus Brevis supernatant (LBS) solely, and in combination with Tamoxifen. In Vitro trials were performed to assess the LBS potency. Bax and Bcl-2 mRNA expression levels and apoptotic effects after these treatments were examined by qRT-PCR and flow cytometry, respectively. The results indicated that LBS induces apoptosis in MCF-7 at high concentrations. Transcription of Bcl-2 was reduced but Bax mRNA expression was enhanced. Tamoxifen's inhibitory effect on the cell growth was synergistically augmented by LBS. In addition, we found that the Bcl-2 mRNA levels in the cells exposed to TAM/LBS were lower than in those treated with TAM alone. Our findings suggest potential role of LB as an adjuvant therapy in cancer treatment and prevention; along with its underlying mechanism.

Probiotics and Nigella sativa extract supplementation improved behavioral and electrophysiological effects of PTZ-induced chemical kindling in rats.

INTRODUCTION: Epilepsy is a most common neurological disorder that has negative effects on cognition. In the present study, we investigated the protective effect of Nigella sativa (NS) and probiotics on seizure activity, cognitive performance, and synaptic plasticity in pentylenetetrazole (PTZ) kindling model of epilepsy. METHODS: One hundred and forty-four rats were divided into 2 experiments: In experiment 1, animals were grouped and treated as follows: 1) control (PTZ + saline), 2) NS treatment, 3) probiotic treatment, and 4) NS and probiotic treatment. Six weeks after the treatment, PTZ kindling were performed, and 48 h after kindling, spatial learning and memory were measured in Morris water maze (MWM) test. Animals in experiment 2 received the same treatment as experiment 1: in control nonkindled groups, control animals were treated with probiotics, NS, and probiotics + NS. Six weeks after the treatment, PTZ kindling were performed, and 48 h after kindling, field potentials were recorded from the dentate gyrus area of the hippocampus; synaptic transmission and long-term potentiation (LTP) was measured. RESULTS: The results showed that the probiotic and NS supplementation significantly reduces kindling development so that animals in PTZ + NS + probiotic did not show full kindling. In MWM test, the escape latency and traveled path in the kindled group were significantly higher than the control group. In PTZ + NS + probiotics, these parameters were significantly lower than those in the PTZ + saline group. Adding probiotic and NS supplementation significantly reduced population spike (PS)-LTP as compared with the PTZ + saline group. CONCLUSION: Probiotic and NS supplementation have some protection against seizure, seizure-induced cognitive impairment, and hippocampal LTP in kindled rats.

Preparation of Diphtheria and Pseudomonas Exotoxin A Immunotoxins and Evaluation of Their Cytotoxicity Effect on SK-BR-3, BT-474, and MDA-MB-231 Breast Cancer Cell Lines.

Immunotoxin targeted therapy is a promising way of cancer therapy that is made from a toxin attached to an antibody which target a specific protein presented on cancer cells. In this study, we introduce immunotoxins comprising of truncated pseudomonas exotoxin A (PEA) and diphtheria toxin (DT) conjugated to trastuzumab. The effectiveness of 20 and 30 µg/ml immunotoxins and trastuzumab were studied on SK-BR-3 and BT-474 HER2/neu positive breast cancer cell lines by a cell death assay test. The produced immunotoxins have the potential to reduce the therapeutic dose of the trastuzumab and in the same time achieve higher efficiency.

Assessment of biofilm formation among clinical isolates of Acinetobacter baumannii in burn wounds in the west of Iran.

Burn wound infection by A. baumannii is one of the predominant cause of mortality worldwide. The present investigation aimed at determination of antimicrobial resistance profile and expression of the biofilm-related genes in A. baumannii isolated from hospitalized patients with burn wound infection in Kermanshah hospitals. Sixty four isolates of A. baumannii were recovered from burn wound of hospitalized patients at hospitals in Kermanshah. The antimicrobial susceptibility testing (AST) was performed. Biofilm formation was measured and antibiotic resistance was compared between before and after of biofilm formation. The polymerase chain reaction (PCR) and Real-Time PCR were performed to detect of abaI and pgaD genes. The biofilm producer isolates and the most resistant isolates were exposed to ozone gas .More than 70% strains were resistance to Erythromycin, Ofloxacin, Ceftazidime, Ceftriaxone, and Ticarcillin-clavulanic acid and 50% isolates were resistant to Imipenem. Thirty one (48.4%) isolates were biofilm producer. The pgaD and abaI genes were positive in 29 (45.3%) and 9 (14%) isolates, respectively. Real time PCR demonstrated that the copy numbers of the pgaD and abaI genes after biofilm formation were increased. After exposure to ozone, biofilm formation reduced in all very strong biofilm producing isolates. Our results showed that after biofilm formation, an increased resistance was observed in most isolates. Also rising expression of abaI gene was associated with biofilm formation and an increase of antibiotic resistance. In the current study, both biofilm formation and antibiotic resistance were reduced after O3 exposure.

Cytotoxic activity and apoptosis induction by supernatant of Lentilactobacillus buchneri on HT-29 colon cancer cells.

Background and Objectives: Colorectal cancer (CRC) is the fourth most commonly diagnosed cancer and the third most deadly cancer in the world. According to recent experimental reports, probiotics and their derivatives protect CRC patients from treatment-related side effects. Therefore, the present study aimed to investigate the cytotoxic impact of the cell-free supernatant (CFS) of Lentilactobacillus buchneri on the HT-29 cancer cell line. Materials and Methods: In the current study, we used the L. buchneri CFS, which was well isolated and identified in our previous investigation from traditional yogurt in the Arak region of Iran. The apoptosis induction in HT-29 cancer cells was assessed by cell cytotoxicity, flow cytometry, and qRT-PCR. Results: L. buchneri CFS inhibited the proliferation of HT-29 cancer cells in a time- and dose-dependent manner. The apoptotic effect of CFS was further supported by the flow cytometry data, which showed that the maximum incidence of apoptosis was observed in HT-29 cancer cells treated with the IC50 concentration of CFS after 72 hours. CFS of L. buchneri also exerted the up-regulating effect on the expression of pro-apoptotic genes including BAX, CASP9, and CASP3. L. buchneri CFS at an IC50 dose induced cell cycle arrest in the G0/G1 phase in HT-29 cells. Conclusion: This study indicates that L. buchneri CFS can prevent colorectal cancer (CRC) development in patients by inducing cancer cell apoptosis. This finding suggests that the CFS of L. buchneri could be used as a therapeutic agent for the control of CRC.

The inhibition effects of Lentilactobacillus buchneri-derived membrane vesicles on AGS and HT-29 cancer cells by inducing cell apoptosis.

In recent years, probiotics and their derivatives have been recognized as important therapeutic agents in the fight against cancer. Therefore, this study aimed to investigate the anticancer effects of membrane vesicles (MVs) from Lentilactobacillus buchneri strain HBUM07105 probiotic isolated from conventional and unprocessed yogurt in Arak province, Iran, against gastric and colon cancer cell lines. The MVs were prepared from the cell-free supernatant (CFS) of L. buchneri and characterized using field-emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) and SPS-PAGE techniques. The anticancer activity of MVs was evaluated using MTT, flow cytometry, qRT-PCR techniques, and a scratch assay. The study investigated the anti-adenocarcinoma effect of MVs isolated from L. buchneri on a human gastric adenocarcinoma cell line (AGS) and a human colorectal adenocarcinoma cell line (HT-29) at 24, 48, and 72-h time intervals. The results demonstrated that all prepared concentrations (12.5, 25, 50, 100, and 200 µg/mL) of MVs reduced the viability of both types of human adenocarcinoma cells after 24, 48, and 72 h of treatment. The analysis of the apoptosis results revealed that the percentage of AGS and HT-29 cancer cells in the early and late stages of apoptosis was significantly higher after 24, 48, and 72 h of treatment compared to the untreated cancer cells. After treating both AGS and HT-29 cells with the MVs, the cells were arrested in the G0/G1 phase. These microvesicles demonstrate apoptotic activity by increasing the expression of pro-apoptotic genes (BAX, CASP3, and CASP9). According to the scratch test, MVs can significantly decrease the migration of HT-29 and AGS cancer cells after 24, 48, and 72 h of incubation compared to the control groups. The MVs of L. buchneri can also be considered a potential option for inhibiting cancer cell activities.

Bacillus megaterium RTS1 enhances resistance of Lycopersicon esculentum to salinity stress through the improvement of antioxidant defenses.

Background and Objectives: Plant growth-promoting bacteria (PGPB) may reduce the negative effects of salinity stress. The aim of this study was to optimize Bacillus megaterium RTS1 and characterize the effect of the PGPB on the physiological characteristics of tomato (Lycopersicon esculentum). Materials and Methods: The Central composite design (CCD) of response surface methodology (RSM) was used to optimize Bacillus megaterium RTS1 to produce maximum cell biomass and spores. Then the effect of the PGPB on the physiological characteristics of tomato (Lycopersicon esculentum), including membrane stability, leaf relative water content percentage, anthocyanin and carotenoids content, chlorophyll photosynthetic parameters, sugar and starch level, superoxide anion and antioxidant activity under salt stress conditions. The NFB medium was inoculated with 5% bacterial culture and the fermentation was carried out in a 10-lit fermenter. Results: After optimization, the amount of cell biomass by the model was 9.45 log10 CFUs/mL, which showed a 1.2-fold increase compared to the non-optimized medium. Usage of bacteria under the optimal conditions of the culture medium may increase the stability of the membrane and improve the relative water content. Bacteria were able to prevent the excessive increase of anthocyanins. Oxidative stress led to an increase in the content of chlorophyll a, while causing the degradation of chlorophyll b. Bacterial inoculation led to an increase in the level of sugar and starch compared to the control. PGPB showed an increasing effect on the amount of superoxide anion production and caused a significant increase in the antioxidant activity under salinity stress conditions. Conclusion: The PGPB can be a promising way to boost physiological characteristics of tomato plant under salinity stress. Also, sporulation capacity of Bacillus megaterium with high bacterial cell density in fermenter produce a sustainable product for tomato plants.

Adsorption behavior of trace elements of 90Sr on MnO2-ZrO2 loaded with polyacrylonitrile polymer from aqueous solutions.

A MnO2-ZrO2-polyacrylonitrile (MnO2-ZrO2-PAN) composite ion exchanger was produced and its properties were examined by Fourier-transformed infrared spectroscopy, scanning electron microscopy, The BET (Brunauer, Emmett and Teller) surface area, X-Ray diffraction analysis and thermogravimetric analysis. The adsorption of Strontium (Sr) from solutions by MnO2-ZrO2-PAN composite was studied thru batch experiments. The distribution Coefficient of Sr (II) on the composite sorbent was investigated against pH, interaction time, and primary concentration ion. To study the kinetics of adsorption, Pseudo-first-order and Pseudo second-order adsorption kinetics were studied and the results revealed that adsorption kinetics better fit to the pseudo-second-order model. Three iso-temperature models, Langmuir, Freundlich, and Temkin were applied to fit the experimental results. Among those models, Langmuir revealed the most suitable one with minimum deviation. The created composite exhibited strong compatibility to the elimination of Y (III), Ni (II), Pb (II), and Co (II) from radioactive waste streams. On the other, it is evident from the data that the quantifiable extraction of Sr (II) ions from Zr (IV), Mo (VI), and La (III) is feasible. MnO2-ZrO2 Loaded with (PAN) Polymer was figured out to have high ion exchange capacity and thermal stability and selectivity for strontium.

Optimizing of the formation of active BMW-amylase after in vitro refolding.

This study was carried out to determine the optimal folding condition of α-amylase from Bacillus megaterium WHO using response surface methodology (RSM). A first-order model showed that three factors namely glycerol, Ca(2+) and protein concentration had the most significant effect on refolding. Analysis of the results showed that glycerol was better than the other polyols due to its effect on protein stability. Since α-amylases are known to contain calcium ions in their structure, the presence of calcium in the refolding buffer was compulsory. The concentration of protein had the most significant quadratic effect on the response studied. A second-order polynomial model was developed to quantify the relationships between variables. It was shown that the combination of 20%(v/v) glycerol, 25 mM Ca(2+) and 0.3 (mg/ml) protein was the most efficient condition for in vitro refolding of α-amylase. Under the optimal condition the yield of refolding was enhanced up to 7-fold. In order to analysis the size distribution in optimized and basic medium, dynamic light scattering (DLS) was fulfilled. The information gathered in this study showed that the use of solvent engineering and optimization procedure can be a general method for protein refolding.

Possible therapeutic effects of boron citrate and oleoylethanolamide supplementation in patients with COVID-19: A pilot randomized, double-blind, clinical trial.

BACKGROUND: The present study aimed to assess the therapeutic effects of boron citrate and oleoylethanolamide supplementation in patients with COVID-19. METHODS: Forty adult patients with a diagnosis of COVID-19 were recruited in the present study. Patients were randomized in a 1:1:1:1 allocation ratio to 1of 4 treatment groups: (A) 5 mg of boron citrate twice a day, (B) 200 mg of oleoylethanolamide twice a day, (C) both therapies, or (D) routine treatments without any study medications. At pre-and post-intervention phase, some clinical and biochemical parameters were assessed. RESULTS: Supplementation with boron citrate alone or in combination with oleoylethanolamide significantly improved O2 saturation and respiratory rate (p < 0.01). At the end of the study, significant increases in white blood cell and lymphocyte count were observed in the boron citrate and combined groups (p < 0.001). Boron citrate supplementation led to a significant decrease in serum lactate dehydrogenase (p = 0.026) and erythrocyte sedimentation rate (p = 0.014), compared with other groups. Furthermore, boron citrate in combination with oleoylethanolamide resulted in a significant reduction in the high-sensitivity C-reactive protein and interleukin-1ß concentrations (p = 0.031 and p = 0.027, respectively). No significant differences were found among four groups post-intervention, in terms of hemoglobin concentrations, platelet count, and serum interleukin-6 levels. At the end of the study, common symptoms of COVID-19 including cough, fatigue, shortness of breath, and myalgia significantly improved in the supplemented groups, compared to the placebo (p < 0.05). CONCLUSION: Supplementation with boron citrate alone or in combination with oleoylethanolamide could improve some clinical and biochemical parameters in COVID-19 patients.

Selective screening and characterization of plant growth promoting bacteria for growth enhancement of tomato, Lycopersicon esculentum.

BACKGROUND AND OBJECTIVES: Plant Growth-promoting Bacteria (PGPB) can replace the dangerous chemical fertilizers and pesticides. The aim of this study was to isolate the PGPBs for Lycopersicon esculentum plant and to determine the appropriate volume for inoculation. MATERIALS AND METHODS: Plants samples were collected from tomato fields. Nitrogen fixing-PGPBs were isolated from rhizoplane and rhizosphere. Five isolates were screened based on their growth abilities and examined for PGPB traits including phosphate solubilization, and IAA, ammonia and HCN production. After high cell density cultivation, the cells were separated by centrifugation and freeze dried after resuspension in cryoprotectant. The powders were inoculated into sterile soil with a dose of 106, 107 and 108 CFUs/g. Tomato (Lycopersicon esculentum) seeds were sown in soil and after 42 days the shoot length was measured. RESULTS: Most of the potent PGPBs with high growth capacity were isolated from rhizoplane. Maximum phosphate solubilization was 289.7 µg/ml by NFB12 which isolated from rhizoplane. This strain produced the maximum level of IAA. NFB12 produced ammonia without the ability of production of HCN. This strain enhanced shoot length in dosed dependent manner. Surprisingly, inoculation of soil with 108 CFUs/g dramatically decreased the shoot length by 21%. Based on molecular approach NFB12 was identified as Bacillus megaterium. CONCLUSION: Isolation of specific PGPBS is recommended for sustainable plant production. Our results showed that NBF12 improves tomato plant growth and its effect on tomato plant growth is does dependent. Maximum growth rate of tomato was observed with 107 CFUs/g soil inoculation of NFB12 while higher inoculation showed negative effect.

Efficient refolding of recombinant lipase from Escherichia coli inclusion bodies by response surface methodology.

An experimental design was employed to optimize the refolding conditions of a recombinant lipase from Pseudomonas sp. which expressed as inclusion bodies in Escherichia coli. The effects of several variables on the refolding and activation of the enzyme has been studied. Because of the complexity of the reaction with respect to the number of parameters that can affect the refolding efficiency, 2(6-1) half-fractional factorial design (H-FFD) was employed for initial screening of the factors, potentially influencing the response. Experiments were performed in triplicate at two levels. Subsequently, the selected factors were subjected to response surface methodology (RSM) with a four factor-five coded level central composite design (CCD), using Quadratic model for obtaining the optimum values for the factors. The adequacy of the calculated model was confirmed by the coefficient of determination (R(2)) and F value of 0.89 and 9.12, respectively. The optimized condition for the refolding was obtained in the refolding buffer containing unfolded lipase (10 microg/ml) and foldase (3 microg/ml) in combination with glycerol (10%), NaCl (1M) and sucrose (0.5M). Using chemicals in combination with foldase under the optimal condition exhibited a 50% increase in refolding yield over the conventional method.

High-level expression of lipase in Escherichia coli and recovery of active recombinant enzyme through in vitro refolding.

Microbial lipases are widely used for biotechnological applications. In this study we have cloned and sequenced the lipase and lipase specific foldase genes of a Pseudomonas sp., which was isolated from Southern Iran. The lipase was composed of 936 bp which encoded 311 amino acids and the lipase specific foldase gene consisted of 1008 bp which encoded 336 amino acids. The low amount of recombinant lipase was expressed as an active enzyme in Escherichia coli harboring a plasmid with the clustered lipase and lipase specific foldase genes. To increase the enzyme expression level, the lipase and lipase specific foldase genes subcloned into two separate expression vectors. The lipase was expressed as inactive inclusion bodies under the control of the strong T7 promoter. Inclusion bodies were dissolved in 8M urea and 1mM dithiothreitol (DTT) and purified using Ni-nitrilotriacetic acid column. Subsequently, purified lipase diluted in 20mM phosphate buffer (pH 7) containing the lipase specific foldase which was expressed in another clone of E. coli. In the presence of foldase, it was possible to achieve active lipase with a specific activity of up to 240 IU/mg using a simple refolding procedure. Moreover, the effect of different concentrations of various additives was investigated on the refolding of denatured lipase. The best yield of 70 IU/ml with the specific activity of 3000 IU/mg were obtained after incubation of denatured enzyme in a refolding buffer containing lipase specific foldase (0.005 mg/ml), 1M NaCl and 10% glycerol at 4 degrees C.

Assessment of boron-containing compounds and oleoylethanolamide supplementation on the recovery trend in patients with COVID-19: A structured summary of a study protocol for a randomized controlled trial.

OBJECTIVES: In this study, we investigate the effect of boron-containing compounds and oleoylethanolamide supplementation on the recovery trend in patients with COVID-19. TRIAL DESIGN: The current study is a single-center, randomized, double-blind, placebo-controlled clinical trial with parallel groups. PARTICIPANTS: The inclusion criteria include male and female patients≥18 years of age, with a confirmed diagnosis of SARS-CoV-2 infection via polymerase chain reaction (PCR) and/or antibody test and with written informed consent to participate in this trial. The exclusion criteria include regular use of any other supplement, severe and critical COVID-19 pneumonia, pregnancy and breastfeeding. This study is being conducted at Imam Reza Hospital, Tabriz University of Medical Sciences, Tabriz, Iran. INTERVENTION AND COMPARATOR: Patients are randomly assigned to four groups. The first group (A) will take one capsule containing 5 mg of boron compounds twice a day for two weeks. The second group (B) will take one capsule containing 200 mg oleoylethanolamide twice a day for two weeks. The third group (C) will take one capsule containing 5 mg boron compounds with 200 mg oleoylethanolamide twice a day for two weeks, and the fourth group (D) does not receive any additional treatment other than routine treatments. Boron-containing compounds and oleoylethanolamide capsules will be synthesized at Nutrition Research Center of Tabriz University of Medical Sciences. MAIN OUTCOMES: The primary end point of this study is to investigate the recovery rate of clinical symptoms, including fever, dry cough, and fatigue, as well as preclinical features, including complete blood count (CBC), the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) profiles within two weeks of randomization. RANDOMISATION: Patients are randomized into four equal groups in a parallel design (allocation ratio 1:1). A randomized block procedure is used to divide subjects into one of four treatment blocks (A, B, C, and D) by a computer-generated allocation schedule. BLINDING (MASKING): The participants and investigators (enrolling, assessing, and analyzing) are blinded to the intervention assignments until the end of the study and data analysis. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): The calculated total sample size is 40 patients, with 10 patients in each group. TRIAL STATUS: The protocol is Version 1.0, May 17, 2020. Recruitment began May 19, 2020, and is anticipated to be completed by October 19, 2020. TRIAL REGISTRATION: This clinical trial has been registered by the title of "Assessment of boron-containing compounds and oleoylethanolamide supplementation on the recovery trend in Patients with COVID-19: A double-blind randomized placebo-controlled clinical trial" in the Iranian Registry of Clinical Trials (IRCT). The registration number is " IRCT20090609002017N35 ", https://www.irct.ir/trial/48058 . The registration date is 17 May 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Photothermal inactivation of methicillin-resistant Staphylococcus aureus: anti-biofilm mediated by a polypyrrole-carbon nanocomposite.

Widespread resistance to antibiotics amongst pathogens has become a tremendous challenge of high morbidity and mortality rates which increases the needs to exploring novel methods of treatment. An efficient antimicrobial procedure to root out pathogenic bacteria is photothermal therapy. In this study, antimicrobial effects of a polypyrrole-carbon nanocomposite (PPy-C) upon laser irradiation in order to destroy the pathogenic gram-positive bacterium, methicillin-resistant Staphylococcus aureus (MRSA) were assessed. The bacterial cells were incubated with 500, 750 and 1000 µg ml-1 concentrations of PPy-C and irradiated with an 808-nm laser at a power density of 1.0 W cm-2. To indicate the biocompatibility and toxic effect of the nanocomposite without and with laser irradiation, the authors counted the number of CFUs and compared it to an untreated sample. Antibacterial mechanisms of PPy-C were assessed through temperature increment, reactive oxygen species production, and protein and DNA leakages. Photothermal heating assay showed that 26°C temperature increases in the presence of 1000 µg ml-1 PPy-C led to >98% killing of MRSA. Furthermore, 20 min radiation of near-infrared light to PPy-C in different concentrations indicated destruction and reduction in the MRSA biofilm formation. Therefore, PPy-C was introduced as a photothermal absorber with a bactericidal effect in MRSA.

Rapid Identification of Vancomycin Resistant Enterococcus Faecalis Clinical Isolates using a Sugar Fermentation Method.

INTRODUCTION: Vancomycin Resistant Enterococci (VRE) can be found all over the world. Thus, rapid detection of the isolates could be of high importance in the treatment or prevention of the associated disease. AIM: To measure the turanose fermentation in Enterococcus faecalis clinical isolates for rapid differentiation of VRE and Vancomycin-Susceptible E. faecalis (VSE) isolates. MATERIALS AND METHODS: Forty E. faecalis samples were isolated from 200 clinical samples in Tehran Medical Center, Iran, from October 2012 to December 2012. These isolates were detected according to the standard microbial and biochemical tests. Detection of VRE isolates was originally performed by disk diffusion using 1 µg vancomycin disk, followed by Polymerase Chain Reaction (PCR) amplification of the vanA gene. Finally, the turanose consumption in 1%, 0.7% and 0.5% dilutions was detected by a phenotypic method. RESULTS: Among the 40 E. faecalis isolates, 20 vancomycin-susceptible and 20 vancomycin-resistant E. faecalis were isolated according to the disk diffusion and PCR of the vanA gene. There was a considerable difference between VRE and VSE isolates in 0.7% dilution of turanose. However, there was no significant difference between VRE and VSE in 1% and 0.5% dilutions of turanose. CONCLUSION: Since detection of VRE isolates is of high importance, especially in nosocomial infections, phenotypic methods may be highly useful for this purpose. In conclusion, our data indicate that VRE isolated from clinical samples could be distinguished from VSE isolates by turanose fermentation at dilution 0.7%.

Utilizing intein-mediated protein cleaving for purification of uricase, a multimeric enzyme.

Uric acid, a side product of nucleotide metabolism, should be cleared from blood stream since its accumulation can cause cardiovascular diseases and gout. Uricase (urate oxidase) converts uric acid to 5-hydroxyisourate, but it is absent in human and other higher apes. Yet, the recombinant form of uricase, Rasburicase, is now commercially available to cure tumor lysis syndrome by lowering serum uric acid level. Developing new methods to efficiently purify pharmaceutical proteins like uricase has attracted researchers' attention. Self-cleaving intein mediated single column purification is one of these novel approaches. Self-cleaving inteins are modified forms of natural inteins that can excise and join only at one junction site. In this study, the synthetic gene of Aspergillus flavus uricase, a homotetrameric protein, was cloned into pTXB1 vector as a fusion with the N-terminal of Mxe GyrA intein and chitin-binding domain (CBD) for simple purification. Expression was confirmed by western blot analysis. The fusion protein containing uricase-intein-CBD was purified on a chitin column. The cleavage was induced by adding DTT,1 as a reducing agent to release uricase. The purity of uricase and complete excision of the intein and CBD were confirmed by SDS-PAGE2 while its proper folding was proved by circular dichroism and fluorescent emission studies. Isoelectric focusing further confirmed its homogeneity when a single protein band was observed at the predicted pI value. This is the first report of successful purification of a multimeric therapeutic enzyme by intein-mediated protein cleaving using a well-established and facile system.