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Nowadays, mutations in the epidermal growth factor receptor (EGFR) kinase domain are studied in targeted therapy of non-small cell lung cancer (NSCLC) with EGFR tyrosine kinase inhibitors including gefitinib and erlotinib. The present study reports a rare case of a patient harboring three simultaneous EGFR mutations (L718A, Q849H, and L858R). The development of erlotinib resistance was detected in the subsequent treatment. Using a computational approach, the current study investigated the conformational changes of wild-type and mutant EGFR's kinase domains in the interaction with erlotinib. Their binding modes with erlotinib were elucidated during molecular dynamics simulation, where higher fluctuations were detected in the mutated forms of the EGFR tyrosine kinase domain. Prediction of stability and functional effect of mutations revealed that amino acidic substitutions have decreased the protein stability as well as the binding affinity to erlotinib. These results may be useful for a recommendation of EGFR mutational analysis for patients with NSCLC carcinoma.

In the present study, we investigated the synergistic effects of targeted methotrexate-selenium nanostructure containing Myc decoy oligodeoxynucleotides along with X-irradiation exposure as a combination therapy on LNCaP prostate cancer cells. Myc decoy ODNs were designed based on the promoter of Bcl-2 gene and analyzed by molecular docking and molecular dynamics assays. ODNs were loaded on the synthesized Se@BSA@Chi-MTX nanostructure. The physicochemical characteristics of nanostructures were determined by FTIR, DLS, UV-vis, TEM, EDX, in vitro release, and hemolysis tests. Subsequently, the cytotoxicity properties of them with and without X-irradiation were investigated by uptake, MTT, cell cycle, apoptosis, and scratch assays on the LNCaP cell line. The results of DLS and TEM showed negative charge (-9 mV) and nanometer size (40 nm) for Se@BSA@Chi-DEC-MTX NPs, respectively. The results of FTIR, UV-vis, and EDX showed the proper interaction of different parts and the correct synthesis of nanoparticles. The results of hemolysis showed the hemocompatibility of this nanoparticle in concentrations less than 6 mg/mL. The ODNs release from the nanostructures showed a pH-dependent manner, and the release rate was 15% higher in acidic pH. The targeted Se@BSA@Chi-labeled ODN-MTX NPs were efficiently taken up by LNCaP cells by targeting the prostate-specific membrane antigen (PSMA). The significant synergistic effects of nanostructure (containing MTX drug) treatment along with X-irradiation showed cell growth inhibition, apoptosis induction (~57%), cell cycle arrest (G2/M phase), and migration inhibition (up to 90%) compared to the control. The results suggested that the Se@BSA@Chi-DEC-MTX NPs can potentially suppress the cell growth of LNCaP cells. This nanostructure system can be a promising approach for targeted drug delivery and chemoradiotherapy in prostate cancer treatment., Competing Interests: The authors declare that they have no conflicts of interest to report regarding the present study., (© 2024 Ghorbani et al.)

Background: Inborne errors of metabolism are a common cause of neonatal death. This study evaluated the acute early-onset metabolic derangement and death in two unrelated neonates., Methods: Whole-exome sequencing (WES), Sanger sequencing, homology modeling, and in silico bioinformatics analysis were employed to assess the effects of variants on protein structure and function., Results: WES revealed a novel homozygous variant, p.G303Afs*40 and p.R156P, in the pyruvate carboxylase (PC) gene of each neonate, which both were confirmed by Sanger sequencing. Based on the American College of Medical Genetics and Genomics guidelines, the p.G303Afs*40 was likely pathogenic, and the p.R156P was a variant of uncertain significance (VUS). Nevertheless, a known variant at position 156, the p.R156Q, was also a VUS. Protein secondary structure prediction showed changes in p.R156P and p.R156Q variants compared to the wild-type protein. However, p.G303Afs*40 depicted significant changes at C-terminal. Furthermore, comparing the interaction of wild-type and variant proteins with the ATP ligand during simulations, revealed a decreased affinity to the ATP in all the variants. Moreover, analysis of Single nucleotide polymorphism impacts on PC protein using Polyphen-2, SNAP2, FATHMM, and SNPs&GO servers predicted both R156P and R156Q as damaging variants. Likewise, free energy calculations demonstrated the destabilizing effect of both variants on PC., Conclusion: This study confirmed the pathogenicity of both variants and suggested them as a cause of type B Pyruvate carboxylase deficiency. The results of this study would provide the family with prenatal diagnosis and expand the variant spectrum in the PC gene,which is beneficial for geneticists and endocrinologists.

Over-expression of K+ channels has been reported in human cancers and is associated with the poor prognosis of several malignancies. EAG1, a particular potassium ion channel, is widely expressed in the brain but poorly expressed in other normal tissues. Kunitz proteins are dominant in metazoan including the dog tapeworm, Echinococcus granulosus. Using computational analyses on one A-type potassium channel, EAG1, and in vitro cellular methods, including major cancer cell biomarkers expression, immunocytochemistry and whole-cell patch clamp, we demonstrated the anti-tumor activity of three synthetic small peptides derived from E. granulosus Kunitz4 protease inhibitors. Experiments showed induced significant apoptosis and inhibition of proliferation in both cancer cell lines via disruption in cell-cycle transition from the G0/G1 to S phase. Western blotting showed that the levels of cell cycle-related proteins including P27 and P53 were altered upon kunitz4-a and kunitz4-c treatment. Patch clamp analysis demonstrated a significant increase in spontaneous firing frequency in Purkinje neurons, and exposure to kunitz4-c was associated with an increase in the number of rebound action potentials after hyperpolarized current. This noteworthy component in nature could act as an ion channel blocker and is a potential candidate for cancer chemotherapy based on potassium channel blockage., (© 2023. The Author(s).)

Background: Methylmalonic acidemia (MMA) is a rare metabolic disorder resulting from functional defects in methylmalonyl-CoA mutase. Mutations in the MMAB gene are responsible for the cblB type of vitamin B12-responsive MMA., Results: This study used Whole-exome sequencing (WES), Sanger sequencing, linkage analysis, and in-silico evaluation of the variants' effect on protein structure and function to confirm their pathogenicity in a 2-day-old neonate presenting an early-onset metabolic crisis and death. WES revealed a homozygous missense variant on chromosome 12, the NM_052845.4 (MMAB):c.557G > A, p.Arg186Gln, in exon 7, a highly conserved and hot spot region for pathogenic variants. After being confirmed by Sanger sequencing, the wild-type and mutant proteins' structure and function were modeled and examined using in-silico bioinformatics tools and compared to the variant NM_052845.4 (MMAB):c.556C > T, p.Arg186Trp, a known pathogenic variant at the same position. Comprehensive bioinformatics analysis showed a significant reduction in the stability of variants and changes in protein-protein and ligand-protein interactions. Interestingly, the variant c.557G > A, p.Arg186Gln depicted more variations in the secondary structure and less binding to the ATP and B12 ligands compared to the c.556C > T, p.Arg186Trp, the known pathogenic variant., Conclusion: This study succeeded in expanding the variant spectra of the MMAB, forasmuch as the variant c.557G > A, p.Arg186Gln is suggested as a pathogenic variant and the cause of severe MMA and neonatal death. These results benefit the prenatal diagnosis of MMA in the subsequent pregnancies and carrier screening of the family members. Furthermore, as an auxiliary technique, homology modeling and protein structure and function evaluations could provide geneticists with a more accurate interpretation of variants' pathogenicity., (© 2023. The Author(s).)

Keratinocyte growth factor (KGF) is a potential therapeutic factor in wound healing. However, its applications have been restricted due to its low stability, short half-life, and limited target specificity. We aimed to immobilize KGF on collagen-based biomaterials for long-lasting and targeted therapy by designing fusion forms of KGF with collagen-binding domains (CBD) from natural origins. Twelve fusion proteins were designed consisting of KGF and CBDs with different lengths and amino acid compositions. Three-dimensional (3D) structures of the fusions were predicted by homology modeling. Physiochemical properties and secondary structure of the fusions were evaluated by bioinformatics tools. Moreover, the effect of the CBDs on the 3D structure and dynamic behavior of the fusions was investigated by molecular dynamics (MD) simulation. The binding affinity of the fusions to collagen, KGF receptor, and heparin was assessed using docking tools. Our results demonstrated that fusions with small CBDs like CBD of mammalian collagenase and decapeptide CBD of von Willebrand factor (VWF) were more stable and properly folded than those with larger CBDs. On the other hand, the insertion of bulky CBDs, including Fibronectin CBD and CBD of Clostridium histolyticum collagenase, into KGF resulted in stronger binding to collagen. Therefore, very small or large CBDs are inappropriate for constructing KGF fusions because they suffer from low collagen affinity or poor stability. By comparing the results of MD simulation and docking, this study proposed that CBDs belonging to Vibrio mimicus metalloprotease and A3 domain of VWF would be good candidates to produce stable fusions with proper affinities toward collagen and KGF receptors. Moreover, the secondary structure analysis showed that the overall structure of KGF and CBDs was better preserved when CBDs were inserted at the C-terminal of KGF. This computational information about novel KGF fusions may help find the best constructs for experimental studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

One of the most important challenges in the battle against contagious SARS-CoV-2 is subtle identification of the virus pathogenesis. The broad range of COVID-19 clinical manifestations may indicate diversity of virus-host cells. Amongst key manifestations, especially in severe COVID-19 patients, reduction and/or exhaustion of lymphocytes, monocytes, basophils, and dendritic cells are seen.; therefore, it is required to recognize that how the virus infects the cells. Interestingly, angiotensin-converting enzyme 2 (ACE2) as the well-known receptor of SARS-CoV-2 is low or non-expressed in these cells. Using computational approach, several receptor candidates including leukocyte surface molecules and chemokine receptors that expressed in most lineages of immune cells were evaluated as the feasible receptor of spike receptor-binding domain (RBD) of SARS-CoV-2. The results revealed the higher binding affinity of CD26, CD2, CD56, CD7, CCR9, CD150, CD4, CD50, XCR1 and CD106 compared to ACE2. However, the modes of binding and amino acids involved in the interactions with the RBD domain of spike were various. Overall, the affinity of immune receptor candidates in binding to SARS-CoV-2 RBD may offer insight into the recognition of novel therapeutic targets in association with COVID-19., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni-NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.

Hepatitis B virus (HBV) is a global virus responsible for a universal disease burden for millions of people. Various vaccination strategies have been developed using viral vector, nucleic acid, protein, peptide, and virus-like particles (VLPs) to stimulate favorable immune responses against HBV. Given the pivotal role of specific immune responses of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in infection control, we designed a VLP-based vaccine by placing the antibody-binding fragments of HBsAg in the major immunodominant region (MIR) epitope of HBcAg to stimulate multilateral immunity. A computational approach was employed to predict and evaluate the conservation, antigenicity, allergenicity, and immunogenicity of the construct. Modeling and molecular dynamics (MD) demonstrated the folding stability of HBcAg as a carrier in inserting Myrcludex and "a" determinant of HBsAg. Regions 1-50 and 118-150 of HBsAg were considered to have the highest stability to be involved in the designed vaccine. Molecular docking revealed appropriate interactions between the B cell epitope of the designed vaccine and the antibodies. Totally, the final construct was promising for inducing humoral and cellular responses against HBV., (Copyright © 2020 Mobini, Chizari, Mafakher, Rismani and Rismani.)

Background: According to scientific recommendations, paratransgenesis is one of the solutions for improving the effectiveness of the Global Malaria Eradication Programme. In paratransgenesis, symbiont microorganisms are used for distorting or blocking the parasite life-cycle, affecting the fitness and longevity of vectors or reducing the vectorial competence. It has been revealed recently that bacteria could be used as potent tools for double stranded RNA production and delivery to insects. Moreover, findings showed that RNase III mutant bacteria are more competent for this aim. Asaia spp. have been introduced as potent paratransgenesis candidates for combating malaria and, based on their specific features for this goal, could be considered as effective dsRNA production and delivery tools to Anopheles spp. Therefore, we decided to characterize the rnc gene and its related protein to provide the basic required information for creating an RNase III mutant Asaia bacterium., Methods: Asaia bacteria were isolated from field-collected Anopheles stephensi mosquitoes. The rnc gene and its surrounding sequences were characterized by rapid amplification of genomic ends. RNase III recombinant protein was expressed in E. coli BL21 and biological activity of the purified recombinant protein was assayed. Furthermore, Asaia RNaseIII amino acid sequence was analyzed by in silico approaches such as homology modeling and docking to determine its structural properties., Results: In this study, the structure of rnc gene and its related operon from Asaia sp. was determined. In addition, by performing superimposition and docking with specific substrate, the structural features of Asaia RNaseIII protein such as critical residues which are involved and essential for proper folding of active site, binding of magnesium ions and double stranded RNA molecule to protein and cleaving of dsRNA molecules, were determined., Conclusions: In this study, the basic and essential data for creating an RNase III mutant Asaia sp. strain, which is the first step of developing an efficient RNAi-based paratransgenesis tool, were acquired. Asaia sp. have been found in different medically-important vectors and these data are potentially very helpful for researchers studying paratransgenesis and vector-borne diseases and are interested in applying the RNAi technology in the field.

Reteplase is a segment of tissue plasminogen activator used for the removal of thrombi in blood vessels. In the present study the cloned reteplase gene was used for its expression in competent E. coli. The recombinant plasmid, pET15b/reteplase (rpET-BL21), was transformed into competent E. coli strain BL21 (DE3) cells. Overnight culture of the transformed bacteria was induced by the addition of isopropylthio-ß-Dgalactoside (IPTG) to the final concentrations of 0.25, 0.5, 1 and 1.5 mM. Also, the effects of different temperatures(25, 30, 37 and 39°C), shaking speeds (100, 170 and 190 rpm), and various glucose concentrations (0.25, 0.5, 0.75 and 1 mM) on the expression of reteplase were examined. Samples were analyzed by SDS-PAGE. Maximum amount of protein production was obtained by the addition of 1 mM IPTG at 37°C, 100 rpm of shaking speed in the absence of glucose.