Research in Molecular Medicine
Volume:9 Issue: 2, May 2021

Toxoplasmosis is a global public health concern with severe complications, particularly in pregnant women and immunosuppressed patients. Herein, we revealed the first genotypic evidence of Toxoplasma gondii (T. gondii) in diabetic and non-diabetic individuals referred to Imam Sajjad Hospital, Tehran, Iran, using nested-Polymerase Chain Reaction (PCR) and sequencing.

We collected 98, 95, and 94 blood samples from Type-1 Diabetes Mellitus (T1DM), Type-2 Diabetes Mellitus (T2DM), and control subjects from February to August 2018, and their DNA was extracted. After amplification and visualization of dense granule antigen 6 gene (344 bp) using external and internal primer pairs, the positive samples were sent for sequencing.

Among 287 individuals, three T1DM patients (two IgG, one IgM) and one T2DM patient (IgG) were found positive by molecular method, which proved to be type III strain by sequencing.

To the best of our knowledge, this is the first genotyping investigation on T. gondii in diabetic individuals. Further research should be done to better realize the association between Toxoplasma genotypes and the outcome of diabetes mellitus.

The invention of Polymerase Chain Reaction (PCR) marked a breakthrough in biomedical research. Its invention divided the timeline into an era before and after PCR. Because of its multiple applications, it has become a vital tool for clinical researchers and diagnosticians. A effective PCR experiment requires adequate knowledge of each reaction component and step-by-step procedure to attain the optimized results. This research aims to employ optimization strategies that are easy to perform, cost-effective, and do not require PCR kits for the generation of amplicons for TYR, MITF, and SOX10 genes and can be used in sequence analysis.

Whole blood samples were used to extract genomic DNA with an inorganic method. DNA quantification was done by spectrophotometry analysis. Optimization strategies were adopted to generate PCR products of candidate genes and visualized by agarose gel electrophoresis. Sanger sequencing was performed to check the quality and specificity of generated amplicons via optimization strategies.

This study demonstrates a novel approach for troubleshooting failed reactions without the use of PCR kits. The result indicated that use of sterilized material and reagents along with optimum MgCl2 concentration (2.0-3.0 mM), DNA quantity (25 ng/μL), and annealing temperature (54-60°C) are necessary to achieve successful amplification. Sequence analysis revealed no background noise often associated with sequencing results.

Optimizing MgCl2 concentration, DNA quantity, annealing temperature, along with the use of contamination-free material and reagents are essential steps in PCR optimization. Following this guide, anyone lacking proper supervision, and with little or no knowledge of the procedure should attain the desired results.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has currently caused a significant pandemic among worldwide populations. The high transmission and mortality rates of the disease necessitate studies for rapid designing and effective vaccine production. This study aims to predict and design a novel multi-epitope vaccine against the SARS-CoV-2 virus using bioinformatics approaches. 

Coronavirus envelope proteins, Open Reading Frame 7b (ORF7b), Open Reading Frame 8 (ORF8), Open Reading Frame 10 (ORF10), and Nonstructural protein 9 (Nsp9) were selected as targets for epitope mapping using Immune Epitope Data Bank (IEDB) and BepiPred 2.0 servers. Also, molecular docking studies were performed to determine the candidate vaccine’s affinity to Toll-Like Receptor (TLR3, TLR4) and Major Histocompatibility Complex (MHC I and MHC II) molecules. Thirteen epitopes were selected to construct the multi-epitope vaccine. 

We found that the constructed peptide has valuable antigenicity, stability, and appropriate half-life. The Ramachandran and ERRAT plots approved the quality of the predicted model after the refinement process. Molecular docking investigations revealed that antibody-mode in the ClusPro 2.0 server showed the lowest binding energy for MHC I, MHC II, TLR3, and TLR4.

The designed vaccine has a good antigenicity and stability and could be a proper vaccine candidate against the Coronavirus Disease 2019 (COVID-19) infectious disease though, in vitro and in vivo experiments are necessary to complete and confirm our results.

Six pathogen-associated Outer Membrane Iron receptors (OMPs) reside in Uropathogenic strains of E. coli (UPEC): haem-utilization gene (ChuA), Heme acquisition protein (Hma), IrgA homologue adhesin (Iha), Iron-regulated virulence gene (IreA), IroN, and IutA. Cumulative concern over the prevalence of this bacteria in hospital environments, especially in Intensive Care Units (ICUs), highlights the significance of vaccination against this pathogen. In this study, we aimed to develop 3D models of ChuA, Hma, IutA, IreA, Iha, and IroN proteins by invoking various in silico methods and design a chimeric immunogen composed of highly immunogenic regions from these six Escherichia coli antigens as a chimeric vaccine.

In the present study, homology modeling, fold recognition, Ab initio approaches, and their combination were invoked to determine the Three-Dimensional (3D) structures of ChuA, Hma, Iha, IreA, IroN, and IutA. Next, a set of biochemical, immunological, and functional properties were predicted using various bioinformatics tools.

The obtained results indicated that all six modeled proteins fold to a β-barrel structure. The results of biochemical, immunological, and functional analysis determined the regions of each antigen carrying the best immunogenic properties. These regions are employed to construct the final vaccine linked via flexible GGGGS linkers. Intriguingly, re-analyzing the properties of the final vaccine indicated its immunological advantage over individual proteins. 

The strategy of this study to predict the protein 3D structure, followed by epitope prediction, could be adapted to design efficient vaccine candidates. Applying this approach, we designed a vaccine candidate harboring the most promising regions of six OMPs. This approach could lead to better functional, structural, and therapeutic outcomes in the context of vaccine design investigations.

Determining the expression pattern of testis genes in the brain is essential for understanding tissue functions and correlation or inter-correlation between testis and the brain. In this study, we examined spermatogenesis-associated 19 (SPATA19 gene) expression in 10 parts of the brain with bioinformatics analysis.

The public dataset GSE46706, including 1231 samples originated from 134 Caucasian individuals, was downloaded from NCBI Gene Expression Omnibus (GEO). SPATA19 gene expression in the cerebellar cortex, frontal cortex, hippocampus, medulla, occipital cortex, putamen, substantia nigra, temporal cortex, thalamus, and white matter was examined against each other using R software and the t-test. 

Out of 10 brain parts examined, the cerebellar cortex and white matter showed the highest expression, and the temporal cortex showed the lowest expression of the gene. So the cerebellar cortex had a 5.6% and 6.2% increase in gene expression relative to the putamen and temporal cortex with P values of 6.04e-13 and 2.15e-17, respectively. Also, the white matter had a 4% increase in gene expression over the temporal cortex with a P value of 1.89e-13. 

SPATA19 had more expression in the cerebellar cortex and white matter than other brain parts. These two parts make up the cerebellum.

Bone-related osteokines are crucial for bone function and metabolic response to physical activity. The present study aimed to shed light on the effect of different intensities of continuous and interval endurance training on the serum levels of some osteokines that are associated with wingless-related integration site (WNT) signaling pathway and Receptor Activator of Nuclear Factor (NF)-κB Ligand (RANKL) in old male Wistar rats.

A total of 24 old male Wistar rats (mean age: 23 months, mean weight: 437.93 g) were randomly assigned to three groups: interval endurance-training group (n=8), continuous endurance-training group (n=8), and control group (n=8). The continuous and interval training interventions comprised 8 weeks of treadmill exercise, 5 days a week. The continuous endurance-training group started to exercise at 60% of velocity at maximal oxygen uptake (vVO2max) for 16 min during the first week. It continued with 70% of vVO2max for 45 min from the fourth week onward. In contrast, the interval endurance-training group switched on exercise with 40%-80% of vVO2max from the first week and persisted with 30%-110% of vVO2max from the fourth week onward. As the exercise bout was completed, the enzyme-linked immunosorbent assay was applied to measure the study dependent variables. Statistical analysis was further performed using 1-way analysis of variance, considering the significance level of P≤0.05.

The study results demonstrated a significant difference in the levels of Oteoprotegerin (OPG) (P=0.036) and RANKL (P=0.001) in the experimental (namely, interval and continuous training) groups compared with the controls following the exercise bout. However, the level of sclerostin was not significantly changed (P=0.549).

High-intensity endurance training in the RANKL/OPG and WNT pathways decreased sclerostin and RANKL levels, but this decrease was significant at the RANKL level. It was effective with regard to the intensities of different types of endurance exercise.