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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.

Carboxypeptidase B (CPB) in Anopheles spp. breaks down blood and releases free amino acids, which promote Plasmodium sexual development in the mosquito midgut. Our goal was to computationally assess the inhibitory effectiveness of carboxypeptidase inhibitors obtained from tomato, potato (CPiSt), and leech against the Anopheles stephensi CPBAs1 and CPBAs2 enzymes. The tertiary structures of CPB inhibitors were predicted and their interaction mode with CPBAs1 and CPBAs2 were examined using molecular docking. Next, this data was compared with four licensed medications that are known to reduce the Anopheles' CPB activity. Molecular dynamics simulations were used to evaluate the stability of complexes containing CPiSt and its mutant form. Both CPiSt and its mutant form showed promise as possible candidates for further evaluations in the paratransgenesis technique for malaria control, based on the similar bindings of CPiSt and CPiSt-Mut to the active sites of CPBAs1 and CPBAs2, as well as their binding affinity in comparison to the drugs., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: Cancer testis antigens (CTAs) are a family of proteins typically expressed in male testicles but overexpressed in various cancer cell types. Transmembrane Phosphatase with Tensin homology (TPTE) is expressed only in the testis of healthy individuals and is a member of the family of CTAs. The current study, for the first time, examined the significance of TPTE expression in prostate cancer (PCa) tissues by generating a novel antibody marker targeting TPTE protein., Methods: Polyclonal antibodies were prepared for TPTE-p1 and TPTE-p2 peptides, which are derived from the extracellular domains of TPTE. Anti-TPTE-p2 antibody was then used to study the extent and pattern of TPTE expression in 102 PCa and 48 benign prostatic hyperplasia (BPH) tissue samples by immunohistochemistry. The viability of cancer cell lines (PC-3 and MCF-7 cells) was also evaluated in the presence of anti-TPTE-p2 antibody using the MTT test., Results: The immunohistochemical analysis demonstrated a significant increase in cytoplasmic and membrane TPTE expression in the PCa samples compared to the BPH group (both P < 0.0001). Cytoplasmic TPTE expression was positively correlated with Gleason score and PSA levels (P = 0.03 and P = 0.001, respectively). Significant correlations were identified between the levels of PSA and perineural invasion and the membrane expression (P = 0.01, P = 0.04, respectively). Moreover, anti-TPTE-p2 antibody inhibited PC-3 and MCF-7 cells proliferation compared to the control group for 24 h (P < 0.001 and P = 0.001, respectively) as well as for 48 h (P = 0.001 and P = 0.001, respectively)., Conclusion: Our findings indicate that increased TPTE expression is associated with progression of disease. The ability of anti-TPTE-p2 antibody to recognize and target the TPTE protein makes it a potential biomarker to assess and/or target the PCa., (© 2024. The Author(s).)

The lack of effective medicines or vaccines, combined with climate change and other environmental factors, annually subjects a significant proportion of the world's inhabitants to the risk of dengue virus (DENV) infection. These conditions increase the likelihood of exposure to mosquito-borne diseases such as dengue fever. Hence, many research approaches tend to develop efficient vaccine candidates against the dengue virus. Therefore, we used immunoinformatics and bioinformatics to design a construction for developing a candidate vaccine against dengue virus serotypes. In this study, the in silico structure, containing the non-structural protein 1 region (NS1) (consensus and epitope), the envelope domain III protein (EDIII) as the structural part of the virus construction, and the bc-loop of envelope domain II (EDII) as the neutralizing and protected epitope, were employed. We utilized in silico tools to enhance the immunogenicity and effectiveness of dengue virus vaccine candidates. Evaluations included refining and validating physicochemical characteristics, B and T-cell epitopes, homology modeling, and the three-dimensional structure to assess the designed vaccine's quality. In silico results for tertiary structure prediction and validation revealed high-quality modeling for all vaccine constructs. Additionally, the instructed model demonstrated stability throughout molecular dynamics simulation. The results of the immune simulation suggested that the titers of IgG and IgM could be raised to desirable values following injection into in vivo models. It can be concluded that the designed construct effectively induce humoral and cellular immunity and can be proposed as effective vaccine candidate against four dengue serotypes.Communicated by Ramaswamy H. Sarma.

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).)

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, with some known classical factors. Cicer arietinum (Leguminosae) is a source of protein for humans and contains albumin, globulin, glutelin, and prolamin. The protein content of two cultivars of C. arietinum, Hashem and Mansour, was isolated to evaluate their inhibition activity against acetylcholinesterase (AChE), butyrylcholine esterase (BChE), and β-amyloid peptide (βA) aggregation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and molecular docking were also applied to evaluate the content and determine the potential of each chickpea protein to interact with AChE, respectively. Obtained data showed that proteins from both cultivars could inhibit AChE with IC50 of 17.73 (0.03) and 22.20 (0.06) μg/mL, respectively, with no activity on BChE. The 50 μg/mL protein concentration of each cultivar suppressed βA accumulation (Mansour: 25.66% and Hashem: 21.69%) and showed biometal chelating activity. SDS-PAGE analysis revealed relatively different protein patterns, though the Mansour cultivar contained some protein bands with molecular weights of 18, 24, and 70 kDa were estimated to belong to vicilin and legumin, which were absent in the Hashem protein mass. Molecular docking showed that legumin and especially vicilin have good potential to interact with AChE. The chickpea proteins showed inhibitory activity against AChE, which might be due to the vicilin and legumin fractions. The characterization of the inhibitory effect of each protein band could be promising in finding new therapeutic peptide candidates to treat Alzheimer's in the future, although more experimental work is needed in this issue., (© 2023 Wiley Periodicals LLC.)

Whole-exome sequencing (WES) is an excellent method for the diagnosis of diseases of uncertain or heterogeneous genetic origin. However, it has limitations for detecting structural variations such as InDels, which the bioinformatics analyzers must be aware of. This study aimed at using WES to evaluate the genetic cause of the metabolic crisis in a 3-day-old neonate admitted to the neonatal intensive care unit (NICU) and deceased after a few days. Tandem mass spectrometry (MS/MS) showed a significant increase in propionyl carnitine (C3), proposing methylmalonic acidemia (MMA) or propionic acidemia (PA). WES demonstrated a homozygous missense variant in exon 4 of the BTD gene (NM&#95;000060.4(BTD):c.1330G > C), responsible for partial biotinidase deficiency. Segregation analysis of the BTD variant revealed the homozygous status of the asymptomatic mother. Furthermore, observation of the bam file, around genes responsible for PA or MMA, by Integrative Genomics Viewer (IGV) software displayed a homozygous large deletion in the PCCA gene. Comprehensive confirmatory studies identified and segregated a novel outframe deletion of 217,877 bp length, "NG&#95;008768.1:g.185211&#95;403087delinsTA", extended from intron 11 to 21 of the PCCA, inducing a premature termination codon and activation of nonsense-mediated mRNA decay (NMD). Homology modeling of the mutant PCCA demonstrated eliminating the protein's active site and critical functional domains. Thereupon, this novel variant is suggested as the largest deletion in the PCCA gene, causing an acute early-onset PA. These results could expand the PCCA variants spectrum, and improve the existing knowledge on the molecular basis of PA, as well as provide new evidence of pathogenicity of the variant (NM&#95;000060.4(BTD):c.1330G > C., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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&#95;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&#95;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).)

Since the onset of the global epidemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), whole genome sequencing of virus in all countries has been considered to track and predict virus transmission and variation patterns. In the current study we reported a novel complete genome sequence of SARS-CoV-2 isolated from Iran. Genomics variations and protein sequences were evaluated for the isolated sequence and seven Iranian complete genome sequences of SARS-CoV-2 from NCBI using the reference genome of the SARS-CoV-2 Wuhan-Hu-1. The results showed six nucleotide substitutions. The multiple sequence alignment of the spike protein of the Wuhan-Hu-1 strain and the emerging variants indicated similar its residue pattern in the current sequence to the Wuhan-Hu-1 strain. There were relatively similar binding affinity and residues involved in the interactions of the spike receptor-binding domain (RBD) of the Wuhan-Hu-1 strain, the variants and Hormozgan With angiotensin-converting enzyme 2 (ACE2). Tracing the phylogeny of virus indicated distinct clustering of Iranian variants in branches close to the Asian countries. The mutation effect study on the function of proteins predicted neutral impact of all six nucleotide substitutions. However, the free energy calculations indicated a decreasing the protein stability related to the mutations. This data, consistent with similar studies, showed that despite the high similarity in the nucleotide sequence of the SARS-CoV-2, the mutation pattern varies from country to country. Therefore, any country can benefit from these studies to track and find appropriate strategies for treating and controlling the epidemic.Communicated by Ramaswamy H. Sarma.

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.)

Aberrant activation of Wnt/β-catenin signaling pathway, due to the genetic or epigenetic changes, is responsible for tumorigenesis in epithelial cells of different types of cancer such as colorectal cancer. Secreted Frizzled-Related Protein-1 (SFRP1), as one of the antagonist proteins of this pathway, is hyper-methylated in colorectal cancer leading to the formation of Wnt-Fz-LRP and activation of Wnt/β-catenin signaling pathway. We aimed to design antagonist peptides based on SFRP1 structure against wingless-type 2 (Wnt2), a highly expressed ligand in different cancers like colorectal cancer, to inhibit the formation of the initial triple complex of Wnt-Fz-LRP. After homology modeling of SFRP1, molecular docking showed that Wnt2 and SFRP1 interact in the same mode of xWnt8-mFz8 and hWnt3-mFz8 through the thumb and finger binding sites. These binding sites were selected for designing peptides using either substitution or deep learning-based approaches. The efficiency of each designed peptide in interacting with Wnt2 was evaluated by molecular docking. Stability assessment of Wnt2-peptide complexes via molecular dynamic (MD) revealed that the designed peptides could effectively interact with Wnt2 binding sites during the simulation. However, the designed peptides against the thumb site had higher binding affinity and hydrogen bonds compared to the initial sequence. The secondary structure of the designed peptides indicated an alpha-helix structure which is a favorable structure for peptide drugs. Computing the physicochemical properties of peptides predicted a fairly acceptable structure which made them promising candidates in the treatment of cancers like CRC.

The development of bacterial resistance toward antibiotics has been led to pay attention to the antimicrobial peptides (AMPs). The common mechanism of AMPs is disrupting the integrity of the bacterial membrane. One of the most accessible targets for α-defensins human neutrophil peptide-1 (HNP-1) is lipid II. In the present study, we performed homology modeling and geometrical validation of human neutrophil defensin 1. Then, the conformational and physicochemical properties of HNP-1 derived peptides 2Abz 14 S 29 , 2Abz 23 S 29 , and HNP1ΔC18A, as well as their interaction with lipid II were studied computationally. The overall quality of the predicted model of full protein was -5.14, where over 90% of residues were in the most favored and allowed regions in the Ramachandran plot. Although HNP-1 and HNP1ΔC18A were classified as unstable peptides, 2Abz 14 S 29 and 2Abz 23 S 29 were stable, based on the instability index values. Molecular docking showed similar interaction pattern of peptides and HNP-1 to lipid II. Molecular dynamic simulations revealed the overall stability of conformations, though the fluctuations of amino acids in the modified peptides were relatively higher than HNP-1. Further, the binding affinity constant (Kd) of HNP-1 and 2Abz 23 S 29 in complex with lipid II was 10 times stronger than 2Abz 14 S 29 and HNP1ΔC18A. Overall, computational studies of conformational and interaction patterns have signified how derived peptides could have displayed relatively similar antimicrobial results compared to HNP-1 in the reported experimental studies. Chemical modifications not only have improved the physicochemical properties of derived peptides compared to HNP-1, but also they have retained the similar pattern and binding affinity of peptides. Communicated by Ramaswamy H. Sarma.

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.)

Pertussis, caused by Bordetella pertussis is still one of the controversial diseases worldwide due to its high prevalence in both the developed and the developing countries, especially among young children. As currently approved vaccines are not protective enough and provide Th2-type immune responses, there is an urgent need to develop new vaccines. In the current study, we applied the C-terminal fragment of Clostridium perferingens enterotoxin (C-CPE) as a delivery system and F1S1 fragment (Filamentous hemagglutinin (F1) and subunit 1 of pertussis toxin (S1) of B. pertussis to design a novel chimeric protein in silico , to target Claudin-4 receptors in mice lung cells. To achieve this goal, the primary, secondary and tertiary structures of the fusion protein were evaluated and the interaction of this protein with Claudin-4 receptors was studied. Molecular dynamic (MD) simulation analysis was performed to investigate the physical movement of atoms in a fixed period. According to the results; the full-length fusion protein has consisted of 807 amino acid residues which could be classified as a stable protein. There was a convenient consistency between the 3D predicted structure and the secondary structure prediction. An acceptable percentage of the residues were also detected in the most favored and allowed regions for the model. Based on HADDOCK results, there were no considerable differences between the interactions and MD simulation analysis, indicating that the predicted structures were stable during the simulation. Altogether, the data reported in this study represents the first step toward developing a nasal vaccine candidate against B. pertussis infection. Communicated by Ramaswamy H. Sarma.

Signal transducer and activator of transcription 3 (STAT3) is a critical regulator for angiogenesis, cell cycle progression, apoptosis, and drug resistance. Resistance toward EGF receptor (EGFR) inhibitors is a significant clinical concern for metastatic colon cancer patients. The present study aimed to evaluate the blocking influences of STAT3 decoy oligodeoxynucleotides (ODNs) on the STAT3 survival signaling pathway in nonresistant and erlotinib-resistant SW480 colon cancer cells. First, STAT3 decoy and scramble ODNs were designed according to STAT3 elements in the promoter region of MYCT1 gene and tested for the interaction of STAT3 protein with designed ODNs via in silico molecular docking study. Then, the efficiency of transfection and subcellular localization of ODNs were assessed using flow cytometry and fluorescence microscopy, respectively. Cell viability, cell cycle, and apoptosis tests, scratch and colony formation assays, and real-time PCR were also used to study the cancerous properties of cells. A considerable decrease in proliferation of colon cancer cells was observed with blockade of STAT3 signaling due to cell cycle arrest and induced apoptosis via downregulation of cyclin D1 and Bcl-XL, respectively. Furthermore, upon transfecting STAT3 decoy ODNs, colony formation potential and migration activity in both SW480 colon cancer cell lines were decreased compared to the control groups. From this study, it could be concluded that STAT3 is critical for cell growth inhibition and metastatic properties reduction of resistant SW480 colon cancer cells; therefore, STAT3 decoy ODNs could be considered as potential therapeutics along with current remedies for treating drug-resistant colon cancer., (© 2020 International Federation for Cell Biology.)

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.

Recently, advances in the synthesis and development of multifunctional nanoparticle platforms have opened up great opportunities and advantages for specifically targeted delivery of genes of interest. BSA-coated niosome structures (NISM@B) can potentially improve the efficiency in vitro delivery of nucleic acid molecules and the transfection of genes. Few studies have reported the combined use of niosomes with nucleic acid as therapeutic agents or decoy oligodeoxynucleotides (ODNs). Herein, we synthesized NISM@B to encapsulate NANOG decoy ODN (NISM@B-DEC), after which the physicochemical characteristics and in vitro and in vivo properties of NISM@B-DEC were investigated. Our results regarding physicochemical characteristics revealed that the stable niosome nanocarrier system was successfully synthesized with a regular spherical shape and narrow size distribution with proper zeta-potential values and had an appropriate biocompatibility. The ODN release from the niosome nanocarrier system exhibited controlled and pH-dependent behavior as the best models to explain the ODN release profile. NISM@B-DEC was efficiently taken up by human glioblastoma cells (U87) and significantly inhibited cell growth. Finally, blockage of the NANOG pathway by NISM@B-DEC resulted in G1 cell cycle arrest, apoptosis, and cell death. In addition, NISM@B-DEC caused a significant decrease in tumor formation and improved wound-healing efficiency of the U87 cells. These findings confirm that NISM@B-DEC could potentially suppress the metastatic ability of these cells. It can be concluded that the presented nanocarrier system can be a promising approach for targeted gene delivery in cancer therapy.

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.)

Due to the presence of cancer stem cells (CSCs), breast cancer often relapsed after conventional therapies. Strategies that induce differentiation of CSCs will be helpful in eradication of tumor cells, so we designed an oligodeoxynucleotide (ODNs) for targeting of signal transducer and activator of transcription 3 (STAT3) transcription factor which is involved in stemness, and constitutively activated in triple-negative breast cancer. Molecular docking and electrophoretic mobility shift assay analysis showed that decoy ODN bound specifically to the DNA binding site of STAT3 protein. The prevalent uptake of Cy3-labeled ODNs is in the cytoplasm and the nucleus of MDA-MB-231 treated cells. STAT3 decoy ODNs treatment showed cell growth inhibition by decreasing cell viability (17%), increasing the percentage of arrested cells in G0/G1 phases (18%), and triggering apoptosis (29%). Migration and invasion potential decreased from 10.77 to 6.76 µm/hr, by wound closure rate, and migrated/invaded percentage by 26.4% and 15.4% in the transwell assays, respectively. CD44 protein expression level on the cell surface also decreased, while CD24 increased. Mammosphere formation efficiency reduced in terms of tumorsphere size by 47%, while the required time increased. Cells morphology was changed, and lipid droplets were accumulated in the cytoplasm compared to the control and scrambled groups, in all assays (repeated triplicate). Furthermore, the gene expression of all downstream targets significantly decreased owing to suppressing the STAT3 transcription factor. Overall, the results confirmed the antitumor effects of STAT3 decoy in MDA-MB-231 cells. Thus, it seems that STAT3 decoy ODNs might be considered as an auxiliary tool for breast cancer eradicating by the differentiation therapy approach., (© 2020 Wiley Periodicals, Inc.)

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.

Expression of master transcriptional regulators of stem cells (Oct4 and Sox2) is associated with mediating tumor proliferation and tumor differentiation. The main goal of this study is the investigation of specific binding of designed Oct4-Sox2 transcription factors decoy oligodeoxynucleotides (ODNs) sequence to their nucleus-extracted proteins in HT29-ShE cells containing enriched cancer stem-like cells (SCLCs). First, gene expression of Oct4, Sox2, and E-cadherin revealed the overexpression of Oct4 and Sox2 and downregulation of E-cadherin in HT29-ShE cells compared with HT29 wild-type and HT29-ShC cells. Next, Oct4-Sox2 complex decoy ODNs were designed according to their elements in the promoter region of Sox2 gene. Then, the interactions of Oct4 and Sox2 proteins to designed ODNs were evaluated in silico. Finally, DNA-protein interactions of decoy ODNs and their corresponding proteins were examined by electrophoretic mobility shift assay (EMSA). Analysis of gel shift retardation assay admitted the specific binding of designed ODNs sequence to the nuclear extracted Oct4 and Sox2 proteins. The results will be a promising approach to target cancer stem cells for potential use in differentiation therapy before chemotherapy and radiotherapy of cancers., (© 2019 Wiley Periodicals, Inc.)

The transcription factor T-cell factor 3 (TCF3), one component of the Wnt pathway, is known as a cell-intrinsic inhibitor of many pluripotency genes in embryonic stem cells (ESCs) that influences the balance between pluripotency and differentiation. In this study, the effects of inhibition of TCF3 transcription factor on the stemness of mouse ESCs (mESCs) were investigated using the decoy oligodeoxynucleotides (ODNs) strategy. The TCF3 decoy and its scramble ODNs were designed and synthesized. The interaction specificity of the TCF3 decoy with the TCF3 transcription factor was evaluated by the electrophoretic mobility shift assay. Subcellular localization was carried out using fluorescence and confocal microscopy. Self-renewal and pluripotency of mESCs were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), cell cycle and apoptosis, alkaline phosphatase (ALP), embryoid body (EB) formation, and real-time assays. All experiments were performed in triplicate. The results showed that knockdown of TCF3 by decoy ODNs transfection in mESCs led to an increase in the cell proliferation, ALP enzyme activity, and master regulatory stemness genes and a decrease in the number and diameter of EBs. These results supported TCF3 as a potential target to maintain the pluripotency and self-renewal capacity of mESCs. Knockdown of the TCF3 transcription factor using decoy ODNs can be a promising method to maintain the stemness of stem cells in regenerative medicine and cell therapy researches., (© 2019 International Federation for Cell Biology.)

Indirubin, an ingredient in traditional Chinese medicine, is considered as an anti-cancer agent. However, due to its hydrophobic nature, clinical efficiency has been limited. Drug delivery via nanotechnology techniques open new windows toward treatment of cancerous patients. Glioblastoma multiforme (GBM) is the most severe and common type of brain primary tumors. Of common problems in targeting therapies of glioblastoma is the availability of drug in tumoric tissues. In this study, Indirubin loaded solid lipid nanoparticles were prepared and their therapeutic potentials and antitumoric effects were assessed on GBM cell line (U87MG). The SLNs were prepared with Cetyl palmitate and Polysorbat 80 via high-pressure homogenization (HPH) methods in hot mode. Then, properties of SLNs including size, zeta potential, drug encapsulation efficacy (EE %) and drug loading were characterized. SLNs morphology and size were observed using SEM and TEM. The crystalinity of formulation was determined by different scattering calorimetry (DSC). The amount of drug release and antitumor efficiency were evaluated at both normal brain pH of 7.2 and tumoric pH of 6.8. The prapared SLNs had mean size of 130 nm, zeta potential of -16 mV and EE of 99.73%. The results of DSC showed proper encapsulation of drug into SLNs. Drug release assessment in both pH displayed sustain release property. The result of MTT test exhibited a remarkable increment in antitumor activity of Indirubin loaded SLN in comparison with free form of drug and blank SLN on multiform GB. This study indicated that Indirubin loaded SLNs could act as a useful anticancer drugs., (© 2018 International Federation for Cell Biology.)

Background: Colorectal cancer (CRC) is known as one of the most leading causes of death from cancer in the world. Wnt signaling pathway is the most important signaling pathway activated in this type of cancer. The low-density lipoprotein (LDL) receptor-related protein-6 (LRP6) functions as a co-receptor in order to trigger this signaling pathway., Objective: The expression of LRP6 was studied in cell lines, tumoral and normal adjacent tissues from patients affected with colorectal cancer., Materials and Methods: The expression of LRP6 was assayed by quantitative Real time PCR., Results: LRP6 is overexpressed at the transcript and protein level in HCT116 and SW480 in comparison to HUVEC as the non-cancerous cell. Furthermore, LRP6 was significantly up-regulated in 61% of human malignant tissues (p-value = 0.0008). In inherited type of disease, this upregulation was lower than other types of colorectal cancer, while in rectal cancer the overexpression was significantly higher (p-value = 0.039). Gene expression was not correlated with the age, gender and the stage of disease., Conclusion: Due to the profound overexpression of LRP6 in sporadic and rectal types of cancer compared to normal colonic ones, antagonist related approaches can be promising for targeted therapies of cancer.

An ultrathin bilayer overcoat of silicon nitride and carbon (SiNx/C) providing low friction, high wear resistance, and high corrosion resistance is proposed for future generation hard disk media. The 16 Å thick SiNx/C overcoat consists of an atomically thin SiNx underlayer (4 Å) and a carbon layer (12 Å), fabricated by reactive magnetron sputtering and filtered cathodic vacuum arc (FCVA), respectively. When compared with monolithic overcoats of FCVA-deposited carbon (16 Å) and sputtered SiNx (16 Å), the SiNx/C bilayer overcoat demonstrated the best tribological performance with a coefficient of friction < 0.2. Despite showing marginally less electrochemical corrosion protection than monolithic SiNx, its ability to protect the magnetic media from corrosion/oxidation was better than that of an ∼27 Å thick commercial hard disk overcoat and 16 Å thick monolithic FCVA-deposited carbon. From X-ray photoelectron spectroscopy and Raman spectroscopy analyses, it was found that the introduction of the 4 Å SiNx underlayer facilitated higher sp(3) hybridization within the carbon layer by acting as a barrier and promoted the formation of strong bonds at the SiNx/C and the SiNx/media interfaces by acting as an adhesion layer. The higher sp(3) carbon content is expected to improve the thermal stability of the overcoat, which is extremely important for future hard disk drives employing heat assisted magnetic recording (HAMR).

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.