Amikacin-loaded niosome nanoparticles improve amikacin activity against antibiotic-resistant Klebsiella pneumoniae strains.

Amikacin is an aminoglycoside antibiotic used in drug-resistant bacterial infections. The spread of bacterial infections has become a severe concern for the treatment system because of the simultaneous drug resistance bacteria and SARS-CoV-2 hospitalized patients. One of the most common bacteria in the development of drug resistance is Klebsiella strains, which is a severe threat due to the possibility of biofilm production. In this regard, recent nanotechnology studies have proposed using nanocarriers as a practical proposal to improve the performance of antibiotics and combat drug resistance. Among drug nanocarriers, niosomes are considered for their absorption mechanism, drug coverage, and biocompatibility. In this study, niosomal formulations were synthesized by the thin-layer method. After optimizing the synthesized niosomes, their properties were evaluated in terms of stability and drug release rate. The toxicity of the optimal formulation was then analyzed. The effect of free amikacin and amikacin encapsulated in niosome on biofilm inhibition were compared in multi-drug resistant isolated Klebsiella strains, and the mrkD gene expression was calculated. The MIC and MBC were measured for the free drug and amikacin loaded in the noisome. The particle size of synthesized amikacin-loaded niosomes ranged from 175.2 to 248.3 nm. The results showed that the amount of lipid and the molar ratio of tween 60 to span 60 has a positive effect on particle size, while the molar ratio of surfactant to cholesterol has a negative effect. The highest release rate in amikacin-loaded niosomes is visible in the first 8 h, and then a slower release occurs up to 72 h. The cytotoxicity induced by amikacin-loaded niosome is significantly less than the cytotoxicity of free amikacin in HFF cells (***p < 0.001, **p < 0.01). The mrkD mRNA expression level in the studied strains was significantly reduced after treatment with niosome-containing amikacin compared to free amikacin (***p < 0.001). It was confirmed that in the presence of the niosome, the amikacin antibacterial activity increased while the concentration of the drug used decreased, the formation of biofilm inhibited, and reduced antibiotics resistance in MDR Klebsiella strains.

Exotoxin A-PLGA nanoconjugate vaccine against Pseudomonas aeruginosa infection: protectivity in murine model.

Pseudomonas aeruginosa is the major infectious agent of concern for cystic fibrosis (CF) patients. Therefore, it is necessary to develop appropriate strategies for preventing colonization by this bacterium and/or neutralizing virulence factors. In this study, we formulated the encapsulation of exotoxin A into PLGA nanoparticles. The biological activities of the nanovaccine candidate were also characterized. Based on the results, ETA-PLGA can act as a suitable immunogen to stimulate the humoral and cellular immune response. The antibodies raised against ETA-PLGA significantly decreased bacterial titer in the spleens of the immunized mice after challenge with PAO1 strain, compared to the control groups. The encapsulation of PLGA into ETA led to a significantly higher production of INF-γ, TNF-α, IL-4, and IL-17A cytokine responses compared to the ETA group. ETA-PLGA enhanced IgG responses in immunized mice compared to ETA antigen. We concluded that encapsulation of Pseudomonas aeruginosa ETA to PLGA nanoparticles can increase its functional activity by decreasing the bacterial dissemination.

Replacement of Trypsin by Proteases for Medical Applications.

Background: Cell culture has a crucial role in many applications in biotechnology. The production of vaccines, recombinant proteins, tissue engineering, and stem cell therapy all need cell culture. Most of these activities needed adherent cells to move, which should be trypsinized several times until received on a large scale. Although trypsin is manufactured from the bovine or porcine pancreas, the problem of contamination by unwanted animal proteins, unwanted immune reactions, or contamination to pathogen reagents is the main problem. Objectives: This study investigated microbial proteases as a safe alternative for trypsin replacement in cell culture experiments for the detachment of adherent cells. Methods: The bacteria were isolated from the leather industry effluent based on their protease enzymes. After sequencing their 16S ribosomal deoxyribonucleic acid, their protease enzymes were purified, and their enzyme activities were assayed. The alteration of enzymatic activities using different substrates and the effect of substrate concentrations on enzyme activities were determined. The purified proteases were evaluated for cell detachment in the L929 fibroblast cells compared to trypsin. The separated cells were cultured again, and cell proliferation was determined by the MTT assay. Results: The results showed that the isolated bacteria were Bacillus pumilus, Stenotrophomonas sp., Klebsiella aerogenes, Stenotrophomonas maltophilia, and Bacillus licheniformis. Among the isolated bacteria, the highest and the lowest protease activity belonged to Stenotrophomonas sp. and K. aerogenes, with 60.34 and 11.09 U/mL protease activity, respectively. All the isolated microbial proteases successfully affected L929 fibroblast cells' surface proteins and detached the cells. A significant induction in cell proliferation was observed in the cells treated with K. aerogenes protease and B. pumilus protease, respectively (P < 0.05). Conclusions: The obtained results suggested that microbial proteases can be used as safe and efficient alternatives to trypsin in cell culture in biopharmaceutical applications.

Prediction of Blood miRNA-mRNA Regulatory Network in Gastric Cancer.

BACKGROUND: The aim of the study was to suggest a high specific and sensitive blood biomarker for early GC diagnosis. METHODS: the expression data of miRNAs and mRNAs were collected from the blood samples of the GC patients based on literature mining. Bioinformatics tools and databases (PANTHER, TargetScan, miRTarBase, miRDB, STRING, and Cytoscape) were used to predict the regulatory relationship. Subsequently, expression level of the selected miRNA was evaluated in the blood samples of gastritis patients to recognize the common miRNA between the GC and gastritis patients. RESULTS: Analysis of 40 target genes by MCODE (installed in Cytoscape software) indicated 4 hub genes (WWP1, SKP2, KLHL42, and FBXO11) as a significant cluster in the PPI network related to miR-21, with Node Score Cutoff: 0.2, Degree Cutoff: 2 and K-Core: 2. In addition, the miRNA RT-qPCR results showed that, the expression level of miR-21 was significantly higher in gastritis group compared to the healthy group (p< 0.05). CONCLUSION: the present study clearly demonstrated the increasing level of blood miR-21 among the gastritis patients infected by H. pylori. Therefore, the altered miRNAs, especially overexpression of onco-miRs, may identify a potential link between miRNAs and pathogenesis of the H. pylori-related complications.

High risk genotype distribution of human papillomavirus (HPV) according to age groups in Iranian asymptomatic men.

BACKGROUND: The Human Papillomavirus (HPV) is one of the most common sexually transmitted viruses worldwide. HPV infection in men is a serious clinical issue as they could be considered as a reservoir for inadvertently transmitting infection to women. Moreover, genital HPV infection could be a source for anogenital cancers in men. METHODS: This cross sectional study was conducted from January 2017 to December 2018. Four hundred fifteen asymptomatic men who were visited by specialists, referred to Nilou laboratory in terms of high risk (HR) HPV test testing. HR-HPV genotypes were detected using an approved assay which could discover HPV 16, HPV 18 and a pool of other high risk HPV genotypes as well as 16+ other HR and 18 + other HR (as multiple genotypes). SPSS software was used for statistical analysis. RESULTS: The mean age was 33 ± 8.14 years. Specimens were referred to the laboratory by urologists, (n = 132, 32%, 95%CI: 25.0-39.4), dermatologists, (n = 104, 25, 95% CI: 19.1-30.9), gynecologists, (n = 75, 18, 95%CI: 13.3-29.3) and other specialists (n = 104, 25, 95% CI:19.1-30.9). The overall prevalence of other HR HPV, HPV16, HPV18 and multiple genotypes were 54.2% (45/83), 25.3% (21/83), 3.6% (3/83) and 16.8% (14/83), respectively. The frequency of HR-HPV, HPV16 and HPV18 genotypes was the highest among 30-40 years old. CONCLUSION: The prevalence of HR-HPV infection among Iranian asymptomatic males was relatively high. Investigation on HPV infection in men as reservoir and transmission vehicle of HPV in addition to screening in women will improve the national public health provisions and will contribute to the application of infection control measurements at a national level.

Protective Potential of Conjugated P. aeruginosa LPS -PLGA Nanoparticles in Mice as a Nanovaccine.

BACKGROUND: Pseudomonas aeruginosa has an important role in nosocomial infections. OBJECTIVE: To evaluate biological activity of the detoxified LPS (D-LPS) entrapped into Poly lactic-co-glycolic acid (PLGA) nanoparticles. MATERIALS: LPS was extracted and detoxified from the P. aeruginosa strain PAO1. The D-LPS, conjugated to the PLGA nanoparticles with 1-ethyl-3-dimethyl aminopropyl carbodiimide (EDAC) and N-hydroxy-succinimide (NHS). The connection was evaluated by FTIR (Fourier transform infrared), Zetasizer, and Atomic Force Microscope (AFM). The BALB/c mice injected intramuscularly with the D-LPS-PLGA with two-week intervals and then challenged two weeks after the last immunization. The bioactivity of the induced specific antisera and cytokines responses against D-LPS-PLGA antigen was assessed by ELISA. RESULTS: D-LPS-PLGA conjugation was confirmed by FTIR, Zetasizer, and AFM. The ELISA results showed that D-LPS was successful in the stimulation of the humoral immune response. The immune responses raised against the D-LPS-PLGA, significantly decreased bacterial titer in the spleen of the immunized mice after challenge with PAO1 strain in comparison with the control groups. CONCLUSION: The conjugation of the bacterial LPS to the PLGA nanoparticle increased their functional activity by decrease in bacterial dissemination and increase the killing of opsonized bacteria.

Down-regulatory effects of green coffee extract on las I and las R virulence-associated genes in Pseudomonas aeruginosa.

BACKGROUND: Antibiotic resistant strains of Pseudomonas aeruginosa are the cause of Gram negative nosocomial infections especially among the immunosuppressed patients. The bacteria contains las I and las R genes that play very important roles in the pathogenesis and mechanisms of aggression. These genes can be influenced by the quorum sensing (QS) system and such mechanism is becoming clinically important worldwide. This study aimed to investigate the preventive effects of green coffee extract (GCE) on the expression of pathogenesis-related genes, las I and las R in P. aeruginosa. METHODS: A total of fifty four P. aeruginosa strains were isolated out of 100 clinical samples collected from the infectious wards in different hospitals (Tehran province) using conventional microscopic and biochemical methods. Susceptibility of the isolates to different antibiotics, GCE and chlorogenic acid were elucidated. Multiplex polymerase chain reaction (PCR) and real-time PCR were performed to detect and quantify the expression levels of las I and las R genes. The presence of chlorogenic acid in GCE was confirmed by HPLC. RESULTS: Antibiotic susceptibility tests revealed multidrug resistance among the clinical isolates of those 40 strains were resistant to ciprofloxacin (74.07%), 43 to ceftazidime (79.26%), 29 to amikacin (53.7%), 42 to ampicillin (77.77%), 17 to colistin (31.48%), 40 to gentamicin (74.77%), and 50 to piperacillin (92.59%). PCR outcomes exhibited that the frequency of las I and las R genes were 100% in resistant and sensitive strains isolated from clinical and standard strains of P. aeruginosa (ATCC 15449). Real-time PCR analyses revealed that GCE significantly prevented the expression of las I and las R genes in P. aeruginosa. GCE at concentration level as low as 2.5 mg/mL could prevent the expression of lasI and lasR genes in P. aeruginosa clinical isolates. CONCLUSION: The presence and expression levels of las I and las R genes in P. aeruginosa isolates were investigated when the bacteria was exposed to GCE. Our results tend to suggest that genes involved in pathogenesis of:Pseudomonas aeruginosa are down regulated by quorum sensing effect of chlorogenic acid and therefore GCE could be useful as an adjuvant in combating multidrug resistance strains of Pseudomonas aeruginosa.

Human papilloma virus (HPV) genotypes concordance between Iranian couples referrals.

BACKGROUND: Human Papilloma Virus (HPV) genotypes concordance among sexual couples has been evaluated in many investigations with considerable variations in the concordance. However, no such study has carried out between Iranian couples yet. METHODS: Urogenital specimen from both males and females of couples were taken and transferred to Nilou laboratory for molecular analysis. HPV DNA extraction and typing were carried out using cobas 4800 platform. Demographic and virological data were analyzed afterwards. RESULTS: One hundred fourteen couples were enrolled in the study. The mean age of participants were 36 ± 8 and 32 ± 7 for males and females, respectively. 64 (28%) of specimens were positive for at least one HPV genotype. The positive rates within genders were 30.7 and 25.4% for females and males, respectively with a considerable association (P value 0.021). Within the positive samples, 13(5.7%), 8 (7%) and 31(13.5%) were belonged to 16, 18 and other HR genotypes. 59 (51.8%) couples who were negative for HPV showed negative concordance. Of the total positive HPV patients (55 couples, 48.2%), 9 (16.3%) couples had positive concordance and the rest of 46 (83.7%) couples (either of spouse being negative and the other being positive for HPV) showed neither kinds of concordance. CONCLUSION: Recognition of the dynamics of HPV infection not only in women, but in their sexual partners could impact the implementation of preventive measures like HPV vaccination for cervical cancer and other HPV-related diseases for both sexual partners.

Can phages cause Alzheimer's disease?

UNLABELLED: Alzheimer's disease (AD) is a progressive neurodegenerative disorder with progressive dementia. Multiple processes have been implicated in AD, notably including abnormal beta-amyloid production, tau hyperphosphorylation and neurofibrillary tangles (NFTs), synaptic pathology, oxidative stress, inflammation, protein processing or misfolding, calcium dyshomeostasis, aberrant reentry of neurons into the cell cycle, cholesterol synthesis, and effects of hormones or growth factors. The complexity of the disease, which affects numerous molecules, cells, and systems and impedes attempts to determine which alterations are specifically associated with early pathology. Chlamydia pneumoniae is an obligate intracellular bacterium. Infection with this organism has been suggested to be a risk factor for AD. C. pneumoniae has two phages phiCPAR39 and phage related to phiCPG1. HYPOTHESIS: we propose that these two phages by entering into mitochondria of chlamydia's host cell can work as slow viruses and can initiate AD.

A new Alzheimer's disease cell model using B cells to induce beta amyloid plaque formation and increase TNF alpha expression.

Different cell models have been developed for the study of Alzheimer's disease (AD) pathways. The neuronal dysfunction and cell death mechanisms that are commonly found in this disease are due to the production of high levels of cytokines and the formation of amyloid plaques. In the cell model introduced in the present study, the production of these two important factors is induced by using B cells from an AD patient. The B cells of an Alzheimer's patient and a normal control were immortalized by using EBV (Epstein-Barr virus) to produce a lymphoblastoid cell line (LCL). The amount of TNF alpha cytokine was evaluated at the RNA and protein levels by RT-PCR and ELISA, respectively. The AD LCL was cultured with SKNMC cells with and without treatment of TNF alpha siRNA. Amyloid plaque formation was monitored by Congo-red staining and microscopy. The amount of TNF alpha cytokine was significantly increased in the AD LCL compared to the normal LCL. The AD LCL induced the formation of amyloid plaques in SKNMC cells. The AD LCL treated with TNF alpha siRNA and co-cultured with SKNMC cells decreased the size and number of amyloid plaques in SKNMC cells. This cellular model is an appropriate model for studying Alzheimer's disease and the mechanisms related to it, as well as for research on cytokine inhibitors, especially TNF alpha inhibitors.

The assessment of anti-tumoral activity of polysaccharide extracted from terrestrial filamentous fungus.

Fungal polysaccharides are well-known for the medicinal properties such as antitumor and immunomodulating effects. Hence, this study evaluated antitumor effects of polysaccharide extracted from Fusarium sp. isolated from soil samples of Karaj district, Alborz, Iran along with its taxonomic study. The filamentous fungus strain FK1 was isolated from the soil sample of Karaj, Iran. The strain was identified based on cultural, morphological and 18 S rRNA gene parameters as Fusarium. Further, the strain Fusarium was cultured in fermented broth of modified (PDB) for 10  days at 25 °C. The polysaccharide of strain FK1 was extracted from the mycelium free supernatant by boiling water method and evaluated for antitoxicity effect on two human cancer cell lines: HeLa cell line and Lymphoblastoid cell line (LCL) by MTT method. Findings revealed that water-extracted from mycelia polysaccharide of strain FK1 had the highest cytotoxicity effect against LCL which is the cause of B lymphocyte cancer, at 50  µg/ml concentration dose (114 ± 1.63) followed by 100  µg/ml (105 ± 0.57) and 10  µg/ml (104 ± 0.57), while it did not have a considerable effect on HeLa cell line. Fusarium could be alternative sources as an antitumor component.

Expression and Characterization of Glucose Oxidase from Aspergillus niger in Yarrowia lipolytica.

Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.