Avicenna Journal of Medical Biotechnology
Volume:12 Issue: 2, Apr-Jun 2020

In recent years, the method of constructing and evaluating the properties of polymer nanocomposite and bioactive ceramics in tissue engineering such as biocompatible scaffolds was studied by some researchers.

In this study, the bio-nanocomposite scaffolds of Chitosan (CS)–Hydroxyapatite (HA)–Wllastonite (WS), incorporated with 0, 10, 20 and 30 wt% of zirconium were produced using a freeze-drying method. Also, the phase structure and morphology of scaffolds were investigated using X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS). By analyzing the SEM images, the porosity of the scaffolds was observed in the normal bone area of the body. In the next step, bioactivity and biodegradability tests of the scaffolds were carried out. Due to the presence of hydrophilic components and the high-water absorption capacity of these materials, the bio-nanocomposite scaffolds were able to absorb water properly. After that, the mechanical properties of the scaffolds were studied.

The mechanical test results showed that the preparation of reinforced bionanocomposites containing 10 wt% of zirconium presented better properties compared to incorporated bio-nanocomposites with different loadings of zirconium.

According to MTT assay results, the prepared scaffolds did not have cytotoxicity at different concentrations of scaffold extracts. Consequently, the investigated scaffold can be beneficial in bone tissue engineering applications because of its similarity to natural bone structure and its proper porosity

Dengue burden is increasing day-by-day globally. A rapid, sensitive, cost-effective early diagnosis kit is the need of the hour. In this study, a label-free electrochemical immunosensor was proposed for dengue virus detection. A modified Polyaniline (PANI) coated Glassy Carbon (GC) electrode, immobilized with DENV NS1 antibody was used to detect the circulating DENV NS1 antigen in both spiked and infected sample.

Cloning, purification and expression of DENV NS1 protein in Escherichia coli (E. coli) was performed and sensor design, PANI modification on GC electrode surface by electrochemical polymerization and immobilization of NS1 antibody on the modified electrode surface was done and finally the analytical performance of the electrochemical immunosensor was done using Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS).

CV and EIS were used to study and quantitate the circulating DENV antigen. The calibration curve showed wide linearity, good sensitivity (Slope=13.8% IpR/ml.ng-1) and distribution of data with a correlation coefficient (R) of 0.997. A lower Limit of Detection (LOD) was found to be 0.33 ng.ml-1 which encourages the applicability of the sensor.

Thus, a PANI based new electrochemical immunosensor has been developed which has the potential to be further modified for the development of cost effective, point of care dengue diagnostic kit.

Emergence and prevalence of multi drug resistance strains such as Methicillin-Resistant Staphylococcus aureus (MRSA) call for new antibacterial option. Endolysins as a new option is suggested. The phage display technique is suggested for production of recombinant endolysins. The recombinant endolysins displayed nano phages specifically lysis bacteria, which penetrate to the depth of tissue and the effective dose is reduced.

CHAPK gene was ligated in T7Select vector arms in T7Select10-3b cloning kit. To produce recombinant nano phages, ligation reaction was added directly to the packaging extract. Recombinant nano phages were amplified by Double Layer Agar assay (DLA). The recombinant nano phages were characterized using TEM. Size of recombinant nano phages was determined using DLS. The spot test was performed to confirm CHAPk -displayed on the surface of nano phages. The turbidimetry was used to investigate lytic activity of recombinant nano phages against MRSA ATCC No. 33591.

The results showed recombinant nano phages belonged to order Caudovirales and family Podoviridae with titer 2×107 PFU/ml. According to the results of DLS, size of recombinant nano phages was 71 nm. Formation inhibition zone confirmed the presence of CHAPk on the surface of nano phage phenotypically. The turbidimetry showed lytic activity recombinant nano phages against MRSA after 5 min.

This study suggests that CHAPk -displayed nano phages can be effective in MRSA infections.

One of the important therapeutic approaches in cancer field is development of compounds which can block the initial tumor growth and the progression of tumor metastasis with no side effects. Thus, the recent study was carried out to design anti-VEGFR2-peptidomimetics as the most significant factor of angiogenesis process- and evaluate their biological activity by in vitro assays.

We designed anti-VEGFR2 peptidomimetics with anti-angiogenic activity, including compound P (lactam derivative) and compound T (indole derivative) by using in silico methods. Then, the inhibitory activity on angiogenesis was evaluated by using angiogenesis specific assays such as Human Umbilical Vein Endothelial Cell (HUVEC) proliferation, tube formation in Matrigel, MTT and Real-Time PCR. IC50 values of the compounds were also determined by cytotoxicity plot in MTT assay.

Compounds P and T inhibited HUVEC cell proliferation and viability in a dose-dependent manner. The IC50 for compound T and compound P in HUVEC cell line were 113 and 115 μg/ml, respectively. Tube formation assay revealed that both compounds can inhibit angiogenesis effectively. The results of Real-Time PCR also showed these compounds are able to inhibit the expression of CD31 gene in HUVEC cell line.

Our study suggested that compounds P and T may act as therapeutic molecules, or lead compounds for development of angiogenesis inhibitors in VEGF-related diseases.

Most of Gastric Cancer (GC) patients are diagnosed at an advanced stage with poor prognosis. Hypermethylations of several tumor suppressor genes in cell-free DNA of GC patients have been previously reported. In this study, an attempt was made to investigate the methylation status of P16, RASSF1A, RPRM, and RUNX3 and their potentials for early diagnosis of GC.

Methylation status of the four tumor suppressor genes in 96 plasma samples from histopathologically confirmed gastric adenocarcinoma patients (Stage I-IV) and 88 healthy controls was determined using methylation-specific PCR method. Receiver operating characteristic curve analysis was performed and Area Under the Curve (AUC) was calculated. Two tailed p<0.05 were considered statistically significant.

Methylated P16, RASSF1A, RPRM, and RUNX3 were significantly higher in the GC patients (41.7, 33.3, 66.7, and 58.3%) compared to the controls (15.9, 0.0, 6.8, and 4.5%), respectively (p<0.001). Stratification of patients showed that RPRM (AUC: 0.70, Sensitivity: 0.47, Specificity: 0.93, and p<0.001) and RUNX3 (AUC: 0.77, Sensitivity: 0.59, Specificity: 0.95, and p<0.001) had the highest performances in detection of early-stage (I+II) GC. The combined methylation of RPRM and RUNX3 in detection of early-stage GC had a higher AUC of 0.88 (SE=0.042; 95% CI:0.793–0.957; p<0.001), higher sensitivity of 0.82 and reduced specificity of 0.89.

Methylation analysis of RPRM and RUNX3 in circulating cell free-DNA of plasma could be suggested as a potential biomarker for detection of GC in early-stages.

Glioblastoma Multiforme (GBM) is the most common and deadly type of primary brain tumor in adults. Magnetic Resonance Spectroscopy (MRS) is a non-invasive imaging technique used to study metabolic changes in the brain tumors. Some metabolites such as Phosphocholine, Creatine, NAA/Cr, and Pcho/Cr have been proven to show a diagnostic role in GBM. The present study was conducted to analyze important metabolites using MRS multivoxel in GBM tumor.

In this study, information was collected from 8 individuals diagnosed with GBM using Siemens multivoxel MRS with a magnetic field strength of 3 T. Data were obtained by Point-Resolved Spectroscopy (PRESS) protocol with TE=135 ms and TR=1570 ms. NAA, Pcho, Cr, Ala, Gln, Gly, Glu, Lac, NAAG, and Tau metabolites were extracted and evaluated statistically.

Given total number of normal voxels and total number of all voxels, levels of Cr, Glu, NAA, NAAG, and Gly/Tau ratio in healthy voxels were significantly higher than tumoral voxels (p=0.005, p=0.03, p<0.001, p<0.001 and p=0.041, respectively). In contrast, levels of Gly, Gln, Tau, Lac/Cr, Pcho/Cr, Pcho/NAA, Lac/NAA, and Gln/Glu ratios in tumoral voxels were significantly more than healthy voxels (p=0.001, p=0.037, p<0.001, p=0.010, p<0.001, p<0.001, and p=0.024, respectively). However, levels of Lac and Pcho had no significant difference in the two types of voxels.

In summary, compared to patients with glioblastoma with 1H-MRS, the Pcho/Cr and Pcho/NAA ratios, and NAAG are the most important parameters to differentiate between tumoral and normal voxels.

Isolation,introduction,producing bioactive compounds from bacteria,especially marine bacteria,is an attractive research area. One of the main challenges of using these metabolites as drug,their industrialization is the optimization of production conditions.

In the present study,the response surface methodology was applied to optimize the production of a cytotoxic extract (C-137-R) by Bacillus velezensis (B. velezensis) strain RP137. Initially,among the three carbon,three nitrogen sources,rice starch,potassium nitrate were selected as the best,with cell toxicity equal to IC50,54.4,45.1 μg,ml in human lung,liver cancer cell lines,respectively (A549,HepG2). In the next step,fractional factorial design was performed to survey effect of seven physical,chemical factors on the amount of production,and the most important factors including carbon,nitrogen sources with the positive effect,the sea salt with negative effect were determined. Finally,using the central composite design with 20 experiments,the best concentrations of rice starch,potassium nitrate (1.5%),sea salt (1%) were obtained.

The average amount of dried extract produced in the optimum conditions was 131.1 mg,L,the best response was 71.45%,which is more than 28-fold better than the pre-optimized conditions.

In general,it can be suggested that the use of modern statistical methods to optimize environmental conditions affecting the growth,metabolism of bacteria can be a highly valuable tool in industrializing the production of bioactive compounds.

Growing antibiotic resistance among urinary opportunistic pathogens such as Klebsiella pneumoniae (K. pneumonia) has created a worrisome condition in the treatment of the Urinary Tract Infections (UTIs) in recent years. Integrons play a significant role in the dissemination of antibiotic resistance genes. The present study was conducted to investigate class 1-3 integrons and the corresponding resistance gene cassettes in urinary K. pneumoniae isolates.

In this study, from December 2015 to September 2016, a total of 196 K. pneumoniae isolates were collected from the patients with UTI referred to medical diagnostic laboratories in Yasouj, Southwestern Iran. Antibiotic susceptibility patterns of isolates were determined using 12 antibiotics by the disc diffusion method. Polymerase Chain Reaction (PCR) was used for detection of integron genes (intI1, intI2, and intI3). The variable regions of integrons were amplified by PCR and sequenced to identify the corresponding gene cassettes.

Thirty-nine different antibiotic resistance profiles were observed among K. pneumoniae isolates. Only 12.2% of K. pneumoniae isolates were found to harbor the intI1 gene. While 17 (60.7%) out of 28 Multidrug Resistance (MDR) K. pneumoniae isolates carried the intI1 gene, only 4.2% of non-MDR isolates harbored intI1 gene. Totally 7 different gene cassette arrays were found in the intI1 gene of K. pneumoniae isolates. The aadA1 was the most prominent gene cassette. Also, high frequency of dfrA containing gene cassettes was observed.

Continuous monitoring and characterization of integrons and their associated gene cassettes could be helpful in controlling the rising rate of antibiotic resistance.

TGF-β1 is known to promote cardiac remodeling and fibrosis during Congestive Heart Failure (CHF). In this study, an attempt was made to investigate expression of Transforming Growth Factor beta1 (TGF-β1) and relative expansion or contraction of regulatory T-cell (Tregs) population in peripheral blood of patients with Chronic Heart Failure (CHF).

Real-time PCR assay was used to investigate expression and post-stimulation levels of TGF-β1 in cell culture supernatant of Peripheral Blood Mononuclear Cells (PBMC) of 42 patients with CHF and 42 controls. Flow cytometry was used to identify relative counts of CD4+CD25+FoxP3+ Tregs.

PBMCs in patients with CHF expressed higher levels of TGF-β1 compared to controls. Post-stimulation levels of TGF-β1 expression were significantly higher in New York Heart Association (NYHA) functional class IV patients compared to stage I patients. Tregs were significantly expanded in PBMC in CHF, while the CD4+ helper T-cells were unchanged. Treg expansion was more significant in NYHA functional class I patients compared to class IV patients.

Expansion of Treg population in CHF provides an extrinsic source for TGF-β1 production to induce reactive fibrosis and cardiac remodeling. Relative decrease in Treg population at advanced stages of CHF is indicative of a loss of regulatory characteristics in these cells and unopposed proinflammatory milieu.

This study aimed to assess construction and expression of CagA recombinant protein of Helicobacter pylori (H. pylori) in Escherichia coli (E. coli) BL21.

Bioinformatics was used in designing the desired gene by Gene Runner. Next, the construct was subcloned to pET21b vector and this process was confirmed by Polymerase Chain Reaction (PCR), enzyme digestion and sequencing techniques. Then, it was cloned in the Escherichia coli BL21 as an expression host. Expression of protein was verified using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting technique. For purification of the protein, the Ni-NTA column was used. Protein concentration was determined by the Bicinchoninic Acid Protein Assay Kit (Parstoos). Finally, Western blotting was performed using CagA antibodies and normal human serum for determining immunogenicity feature with human antiserum.

According to the results of the present study, CagA construct was cloned into the pET21b vector and after confirmation and cloning in host expression, recombinant protein with the size of 38 kDa was successfully expressed and purified. The recombinant CagA protein showed immunogenicity characteristics with human antiserum.

In conclusion, only 5′-end of recombinant protein CagA with high immunogenicity effects was successfully constructed, cloned and expressed. Also, CagA recombinant protein showed good immunogenicity activity with human antiserum.