Avicenna Journal of Medical Biotechnology
Volume:15 Issue: 2, Apr-Jun 2023

Vimentin is a prominent Intermediate Filaments (IFs) protein expressed in different mesenchymal origin cell types. Besides a wide range of cellular function roles associated with vimentin expression, its dysregulation and cell surface expression in the induction of malignancy properties have been reported extensively, making it a promising cancer-specific target. Therefore, this study aimed to generate and characterize anti-vimentin monoclonal antibodies.

A 14-mer synthetic peptide from vimentin was conjugated to Keyhole Limpet Hemocyanin (KLH) and used for immunization of Blab/C mice and monoclonal production by conventional hybridoma technology. The monoclonal antibody was purified using affinity chromatography of supernatants from the selected hybridoma cells. ELISA, Immunoprecipitation-Western blotting (IP-WB), Immunocytochemistry (ICC), and flow cytometry were employed to characterize the produced monoclonal antibody in terms of interaction with vimentin immunizing peptide as well as vimentin protein.

Amid the several obtained producing anti-vimentin antibody hybridomas, the 7C11-D9 clone (IgG1 isotype with kappa light chain) showed higher reactivity with the immunizing peptide, and led to its selection for purification and characterization. The purified antibody could detect vimentin protein in IP-WB, ICC and flow cytometry of the normal and cancerous cells with different origin. No vimentin expression was found in normal healthy Peripheral Blood Mononuclear Cell (PBMC).

Taken together, 7C11-D9 anti-vimentin monoclonal antibody might be used as immune diagnostic or immune therapeutic tool where detection or targeting of vimentin in a wide range of organisms is required.

Stem cells are used to treat numerous diseases; however, their lifespan is rather short. Factors such as probiotics affect and improve various cell lineage efficacies. The aim of this study was to investigate the effects of probiotics-conditioned media on dental pulp stem cell potentials in osteogenesis.

The experiment was initiated by culturing Lactobacillus casei and Lactobacillus acidophilus probiotics as well as DPS-7 cells. Bacterial supernatants were separated and concentrated as the conditioned media. The DPS-7 cells were treated with various concentrations of the conditioned media. Furthermore, MTT assay and alkaline phosphatase activity were used. The mRNA expression of three genes (bFGF, EGF-β and BMP-2) involved in osteogenesis was analyzed using a real-time polymerase chain reaction.

The response of dental pulp stem cells to probiotics preconditioning promoted cell proliferation, increased alkaline phosphatase activity and upregulated bFGF and BMP-2 gene expression. Increased expression was significant for BMP-2 and moderate for bFGF; however, it was non-significant for EGF-β. The use of the two probiotics was the most effective.

In general, synergism of the combined probiotics preconditioning induces differentiation of DPS-7 cells into osteoblasts most effectively.

Anti-tumor effects of N-myc Downstream Regulated Gene2 (NDRG2) have been demonstrated in many tumors. In the present study, NDRG2 was specifically overexpressed in lung cancer cell line using Survivin Promoter (Sur-P). Then, the effects of NDRG2 overexpression on viability, apoptosis, migration, and invasion of A549 cells were evaluated.

Recombinant pAdenoVator-Sur-P-NDRG2-IRES-GFP plasmid harboring NDRG2 gene under transcriptional control of Sur-P and mock plasmid were constructed. A549 lung tumor cells and LX-2 cells (non-tumor cell line) were transfected with pAdenoVator-Sur-P-NDRG2-IRES-GFP, pAdenoVator-CMV-NDRG2-IRES-GFP, or mock plasmids. Tumor specificity of Sur-P was evaluated using fluorescent microscopy for GFP expression. The effects of NDRG2 overexpression on cell viability, apoptosis, and migration of A549 cells were measured using MTT, annexinV/7-AAD flow cytometry, and transwell migration assay, respectively. NDRG2 and matrix metalloproteinase- 2 (MMP-2) expression were measured using real time- PCR.

pAdenoVator-Sur-P-NDRG2-IRES-GFP transfection resulted in a huge GFP expression in A549 cells, but not in LX-2 cells. The results of real time-PCR analysis also showed that pAdenoVator-Sur-P-NDRG2-IRES-GFP transfection led to an abundant NDRG2 expression in A549 cells. NDRG2 overexpression decreased A549 cell viability through increasing cell apoptosis. In addition, migration, invasion, and MMP-2 expression decreased following NDRG2 overexpression in A549 cells.

The findings indicate that the targeted overexpression of NDRG2 using Sur-P can reduce the viability and invasiveness of A549 cells, suggesting possible benefits of this approach in lung cancer therapy.

External factors have the potential to act as immunostimulants in order to influence the body's protection from many foreign antigens. We intended to investigate the ethanol extract Formulary of F-MaCg effect as an immunostimulant.

A purely experimental with a completely randomized design was used on twenty-four white male rats. They were divided into four groups:1) G0 [given aquades (5 ml)]; 2) G1 [given F-MaCg-75 mg/gr BW (Body Weight)]; 3) G2 (F-MaCg -150 mg/gr plus Hepatitis B vaccine at the beginning and the end of treatment); and 4) G3 (FMaCg -300 mg/gr BW plus hepatitis B vaccine at the end of treatment). The rat's spleen lymphocyte blast transformation was evaluated on the 15th and 37th days. Lymphocytes were examined using microtetrazolium assays. Optical Density (OD) was measured using an ELISA reader [493 nμ (nanomicro)]. Observation of lymphocyte viability by a counting chamber using a light microscope and trypan blue 1% before being cultured with Phytohaemoaglutinin.

Lymphocyte cell viability in the hepatitis B vaccine-induced group on the 15th day showed the highest average value in the G2 (1,484/mcl of blood); on the 37th day, it was in G3 (1,578/mcl of blood). The proliferative activity of spleen lymphocytes indicated by the difference in the OD values of the four treatment groups was 0.467, 0.913, 1.619, and 1.473 nμ, respectively. Histological observations of the spleen showed differences at all given formulary dose concentrations.

F-MaCg could be an immunostimulant because of its ability to trigger a cellular immune response.

Methicillin-resistant Staphylococcus aureus (MRSA) has become a worldwide concern as an epidemic bacterium and a cause of nosocomial and community- acquired infections. One of the major problems in the prevention and treatment of infections caused by MRSA strains is their multi-drug resistant trait, which causes the spread of infections and increases the mortality rate. Therefore, a rapid and accurate method is needed to identify MRSA strains, initiate appropriate antibiotic therapy, and control its infection. The aim of this study was to develop a twin lateral flow immunoassay system to detect methicillin-resistant Staphylococcus aureus (MRSA).

First, BSA blocked AuNPs-anti-peptidoglycan antibody and AuNPs-anti- BSA antibody were used to detect Staphylococcus aureus (S. aureus). Then, AuNPsanti- PBP2a antibody was used to specifically detect MRSA. Sensitivity, specificity and limit of detection of this twin immunoassay system were assessed using MRSA, methicillin susceptible S. aureus and clinical samples. Results were compared to those of cefoxitin disc diffusion (FOX30) and Polymerase Chain Reaction (PCR) as gold standards.

The Limit of Detection (LOD) of this twin system were 103 and 104 CFU/ml for the first and second strips, respectively. Sensitivity and specificity of this innovative assay in detecting MRSA were 92.30 and 97.36%, compared to FOX30 and PCR, respectively.

High rates of sensitivity and specificity of this initiative system show its high potentials for rapid and accurate detection of MRSA.

Breast carcinogenesis involves both genetic and epigenetic changes. DNA methylation, as well as micro-RNA regulations, are the significant epigenetic phenomena dysregulated in breast cancer. Herein, the expression of DACH1 as a tumor suppressor gene and its promoter methylation status was analyzed in breast cancer tumors. Also, the expression of three micro RNAs (miR-217, miR-6807-3p, and miR- 552), which had been previously reported to target DACH1, was assessed.

The SYBR green-based Real-Time reverse transcription-PCR was used to determine DACH1 and micro-RNAs (miR-217, miR-6807-3p, and miR-552) expression in 120 ductal breast cancer tumors compared with standard control. Also, the promoter methylation pattern of DACH1 was investigated using the Methylation-specific PCR technique.

DACH1 expression was significantly down-regulated in breast tumors (p< 0.05). About 33.5% of tumors showed DACH1 promoter hyper-methylation. The studied micro-RNAs, expression was negatively correlated with DACH1 expression. The highest expressions of miRNAs and higher DACH1 promoter methylation were observed in advanced cancer situations. The Kaplan-Meier survival curves indicated that the overall survival was significantly poor in higher miRNAs and lower DACH1 expression in breast cancer patients (p<0.002).

DACH1 down-regulation may be associated with a poor breast cancer prognosis. The DACH1 down-regulation may be due to epigenetic regulations such as promoter methylation, especially in triple-negative cases. Other factors, such as micro- RNAs (miR-217, miR-6807-3p, and miR-552), may also have an impact. The elevated expression of miR-217, miR-6807-3p, and miR-552, maybe candidates as possible poor prognostic biomarkers in breast cancer management for further consideration.

Bitter taste-sensing type 2 receptor (T2Rs or TAS2Rs) found on ciliated epithelial cells and solitary chemosensory cells have a role in respiratory tract immunity. T2Rs have shown protection against SARS-CoV-2 by enhancing the innate immune response. The purpose of this review is to outline the current sphere of knowledge regarding this association.

A narrative review of the literature was done by searching (T2R38 OR bitter taste receptor) AND (COVID-19 OR SARS-CoV-2) keywords in PubMed and google scholar.

T2R38, an isoform of T2Rs encoded by the TAS2R38 gene, may have a potential association between phenotypic expression of T2R38 and prognosis of COVID-19. Current studies suggest that due to different genotypes and widespread distributions of T2Rs within the respiratory tract and their role in innate immunity, treatment protocols for COVID-19 and other respiratory diseases may change accordingly. Based on the phenotypic expression of T2R38, it varies in innate immunity and host response to respiratory infection, systemic symptoms and hospitalization.

This review reveals that patients’ innate immune response to SARS-COV- 2 could be influenced by T2R38 receptor allelic variations.

Hearing Loss (HL) is the most common sensory disorder. HL commonly ranges from mild to severe. Persons with HL face difficulty in hearing conversations or sounds through one ear or both ears, which impacts one’s ability to interact with others. Hence it is a communicable disorder that makes people socially isolated, lonely, and frustrated. HL in children severely affects language development. The people who are referred to as 'Deaf' with very little or no hearing capabilities, are considered as having profound hearing loss. More than 124 genes are causative for Non-Syndromic HL (NSHL) with varying inheritance, among which the SLC26A4 mutations are the second commonest cause of hereditary HL across the globe.

Samples from 70 NSHL patients were analyzed through Next-Generation Sequencing (NGS) and generated five pathogenic variants [N246fs (rs918684449), K564fs (rs746427774), F122fs, V239D (rs111033256), T721M (rs121908363)] each with frequency of 1.42%. Three missense variants [S399P (rs747431002), L597S (rs55638457), and G6V (rs111033423)] were reported under the "uncertain" category. All the collected samples were further genotyped to look for the possibility of having GJB2 and HLassociated mutations.

Out of five SLC26A4 pathogenic mutations N246fs (rs918684449) and K564fs (rs746427774) were observed in samples which were positive for GJB2-HL associated candidate mutations [W24X (rs104894396), Q124X (rs397516874) and W77X (rs80338944)]. Similarly, pathogenic variants F122fs, V239D (rs111033256) and T721M (rs121908363) were observed in patient samples which were negative for GJB2-HL associated mutations.

Our data will expand the list of variants underlying NSHL and encourage further genotype SLC26A4 gene concerning the south Indian population with a large sample size.