Assessment of databases to determine the validity of ß- and γ-carbonic anhydrase sequences from vertebrates.

BACKGROUND: The inaccuracy of DNA sequence data is becoming a serious problem, as the amount of molecular data is multiplying rapidly and expectations are high for big data to revolutionize life sciences and health care. In this study, we investigated the accuracy of DNA sequence data from commonly used databases using carbonic anhydrase (CA) gene sequences as generic targets. CAs are ancient metalloenzymes that are present in all unicellular and multicellular living organisms. Among the eight distinct families of CAs, including α, ß, γ, δ, ζ, η, θ, and ι, only α-CAs have been reported in vertebrates. RESULTS: By an in silico analysis performed on the NCBI and Ensembl databases, we identified several ß- and γ-CA sequences in vertebrates, including Homo sapiens, Mus musculus, Felis catus, Lipotes vexillifer, Pantholops hodgsonii, Hippocampus comes, Hucho hucho, Oncorhynchus tshawytscha, Xenopus tropicalis, and Rhinolophus sinicus. Polymerase chain reaction (PCR) analysis of genomic DNA persistently failed to amplify positive ß- or γ-CA gene sequences when Mus musculus and Felis catus DNA samples were used as templates. Further BLAST homology searches of the database-derived "vertebrate" ß- and γ-CA sequences revealed that the identified sequences were presumably derived from gut microbiota, environmental microbiomes, or grassland ecosystems. CONCLUSIONS: Our results highlight the need for more accurate and fast curation systems for DNA databases. The mined data must be carefully reconciled with our best knowledge of sequences to improve the accuracy of DNA data for publication.

Combination of Biodata Mining and Computational Modelling in Identification and Characterization of ORF1ab Polyprotein of SARS-CoV-2 Isolated from Oronasopharynx of an Iranian Patient.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an emerging zoonotic viral infection, which was started in Wuhan, China, in December 2019 and transmitted to other countries worldwide as a pandemic outbreak. Iran is one of the top ranked countries in the tables of COVID-19-infected and -mortality cases that make the Iranian patients as the potential targets for diversity of studies including epidemiology, biomedical, biodata, and viral proteins computational modelling studies. RESULTS: In this study, we applied bioinformatic biodata mining methods to detect CDS and protein sequences of ORF1ab polyprotein of SARS-CoV-2 isolated from oronasopharynx of an Iranian patient. Then through the computational modelling and antigenicity prediction approaches, the identified polyprotein sequence was analyzed. The results revealed that the identified ORF1ab polyprotein belongs to a part of nonstructural protein 1 (nsp1) with the high antigenicity residues in a glycine-proline or hydrophobic amino acid rich domain. CONCLUSIONS: The results revealed that nsp1 as a virulence factor and crucial agent in spreading of the COVID-19 among the society can be a potential target for the future epidemiology, drug, and vaccine studies.

Expansion of Single Cell Transcriptomics Data of SARS-CoV Infection in Human Bronchial Epithelial Cells to COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 19 (COVID-19) that was emerged as a new member of coronaviruses since December 2019 in Wuhan, China and then after was spread in all continentals. Since SARS-CoV-2 has shown about 77.5% similarity to SARS-CoV, the transcriptome and immunological regulations of SARS-CoV-2 was expected to have high percentage of overlap with SARS-CoV. RESULTS: In this study, we applied the single cell transcriptomics data of human bronchial epithelial cells (2B4 cell line) infected with SARS-CoV, which was annotated in the Expression Atlas database to expand this data to COVID-19. In addition, we employed system biology methods including gene ontology (GO) and Reactome pathway analyses to define functional genes and pathways in the infected cells with SARS-CoV. The transcriptomics analysis on the Expression Atlas database revealed that most genes from infected 2B4 cell line with SARS-CoV were downregulated leading to immune system hyperactivation, induction of signaling pathways, and consequently a cytokine storm. In addition, GO:0016192 (vesicle-mediated transport), GO:0006886 (intracellular protein transport), and GO:0006888 (ER to Golgi vesicle-mediated transport) were shown as top three GOs in the ontology network of infected cells with SARS-CoV. Meanwhile, R-HAS-6807070 (phosphatase and tensin homolog or PTEN regulation) showed the highest association with other Reactome pathways in the network of infected cells with SARS-CoV. PTEN plays a critical role in the activation of dendritic cells, B- and T-cells, and secretion of proinflammatory cytokines, which cooperates with downregulated genes in the promotion of cytokine storm in the COVID-19 patients. CONCLUSIONS: Based on the high similarity percentage of the transcriptome of SARS-CoV with SARS-CoV-2, the data of immunological regulations, signaling pathways, and proinflammatory cytokines in SARS-CoV infection can be expanded to COVID-19 to have a valid platform for future pharmaceutical and vaccine studies.

Gamma irradiation of ocular melanoma and lymphoma cells in the presence of gold nanoparticles: in vitro study.

The aim of this work was to determine whether conjugation of cultivated choroidal melanoma and Burkitt's lymphoma cells with gold nanoparticles (GNPs) is beneficial for these series of ocular cancer patients. GNPs are radiosensitizers and can sensitize tumors to radiotherapy.This application has been examined in several tumor types, but not in choroidal melanoma. This study shows the results of in vitro study on the choroidal melanoma and also Burkitt's lymphoma cells in the presence of GNPs during continuous gamma irradiation. Cytotoxicity of GNPs were assessed for five different concentrations then cultured melanoma and Burkitt's lymphoma cells were irradiated with a Gamma source in the presence and absence of NPs. Incubation of melanoma cells with GNP concentrations below 100 µg/ml, accompanied by gamma irradiation, increased cell death (P value = 0.016) . In the absence of irradiation, GNPs at these concentrations did not affect cultured melanoma cell metabolism. Reduced cell viability resulted from a significant increase in absorbed energy by the tumor. Moreover, GNP concentrations higher than 200 µg/ml induced cytotoxicity in melanoma cells. Cytotoxicity assay in GNPs-loaded Burkitt's lymphoma cells showed a slight decrease in cell viability at 50 µg/ml and clear cytotoxicity at concentrations higher than 100 µg/ml (P value = 0.035). Concentration and proper injection doses of GNPs in sensitive tissues such as the human eye are important variables yet to be determined.This is the first report of choroidal melanoma dosimetry performed in the presence of GNPs and provides valuable insights into future therapeutic approaches. Further in vitro study with more different sizes and concentrations is needed to determine the optimum size and concentration before any clinical research in this regard.

In Vitro Radiosensitizing Effects of Temozolomide on U87MG Cell Lines of Human Glioblastoma Multiforme.

BACKGROUND: Glioma is the most common primary brain tumor with poor prognosis. Temozolomide (TMZ) has been used with irradiation (IR) to treat gliomas. The aim of the present study was to evaluate the cytotoxic and radiosensitizing effect of TMZ when combined with high-dose and high-dose rate of gamma irradiation in vitro. METHODS: Two 'U87MG' cell lines and skin fibroblast were cultured and assigned to five groups for 24, 48, and 72 hours. The groups were namely, TMZ group (2000 µM/L), IR group (5 Gy), TMZ plus IR group, control group, and control solvent group. MTT assay was applied to evaluate cell viability. Data were analyzed with SPSS 21.0 software using one-way ANOVA and Kruskal-Wallis test. P<0.05 were considered statistically significant. RESULTS: The slope of growth curve U87MG cells in semi-logarithmic scale was equal to 27.36±0.89 hours. The viability of cells was determined for different TMZ and IR treatment groups. In terms of cell viability, there were no significant differences between the control and control solvent groups (P=0.35) and between treated group by IR (5 Gy) alone and TMZ (2000 µM/ml) alone (P=0.15). Data obtained for the cell viability of combined TMZ plus IR in both cell lines compared to TMZ or IR treated group alone showed a significant difference (P=0.002). CONCLUSION: The evaluation of cells viability showed that TMZ in combination with IR on glioma cells led to a significant radiosensitivity compared to IR alone.

Expression of the circulating and the tissue microRNAs after surgery, chemotherapy, and radiotherapy in mice mammary tumor.

The expression of microRNAs (miRNAs), as novel biomarkers, is subject to change in many cancers. Therefore, the overall profile of miRNAs can be used for detection of cancer type, response to therapies, pathological variables, and other factors related to the disease. In this study, to evaluate miRNA expression associated with the tumor progression and response to treatment, 60 BALB/c mice received subcutaneous injections of 4T1 cells. The study includes ten groups: one group as control, six groups were euthanized at different time points to assess the role of miRNA expression in the tumor progression, and three groups received chemotherapy, radiotherapy, and surgery to evaluate miRNA expression in response to treatment. MicroRNAs were extracted from the breast tumor and the plasma samples, and their relative expressions were quantified using qRT-PCR. MiR-155 expression was increased in the plasma in the early weeks after the cell injection but decreased in the plasma after surgery and radiotherapy and also in tumor samples after chemotherapy and radiotherapy. MiR-10b expression was increased in the late weeks both in the plasma and the tumor and was decreased in the plasma after radiotherapy and surgery and in the tumor after radiotherapy. MiR-21 expression was increased in the plasma and the tumor tissue during the disease progression at the third and the fourth weeks following tumor induction but was decreased in the plasma in all the therapy groups. Interestingly, miR-125a showed a significant decrease during the tumor progression, and its expression was increased after the treatment. Our results showed that the candidate miRNAs could be divided into two groups of oncomiRs and tumor suppressor miR based on their deregulation after tumor growth and treatments. It seems that the oncomiRs in the plasma can be an ideal noninvasive candidate biomarker for the early detection of breast cancer and also for following the response of the common therapies.

Identification and characterization of a silent mutation in RNA binding domain of N protein coding gene from SARS-CoV-2.

OBJECTIVE: This study describes the occurrence of a silent mutation in the RNA binding domain of nucleocapsid phosphoprotein (N protein) coding gene from SARS-CoV-2 that may consequence to a missense mutation by onset of another single nucleotide mutation. RESULTS: In the DNA sequence isolated from severe acute respiratory syndrome (SARS-CoV-2) in Iran, a coding sequence for the RNA binding domain of N protein was detected. The comparison of Chinese and Iranian DNA sequences displayed that a thymine (T) was mutated to cytosine (C), so "TTG" from China was changed to "CTG" in Iran. Both DNA sequences from Iran and China have been encoded for leucine. In addition, the second T in "CTG" in the DNA or uracil (U) in "CUG" in the RNA sequences from Iran can be mutated to another C by a missense mutation resulting from thymine DNA glycosylase (TDG) of human and base excision repair mechanism to produce "CCG" encoding for proline, which consequently may increase the affinity of the RNA binding domain of N protein to viral RNA and improve the transcription rate, pathogenicity, evasion from human immunity system, spreading in the human body, and risk of human-to-human transmission rate of SARS-CoV-2.

Potential therapeutic agents to COVID-19: An update review on antiviral therapy, immunotherapy, and cell therapy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in December 2020 and coronavirus disease 19 (COVID-19) was later announced as pandemic by the World Health Organization (WHO). Since then, several studies have been conducted on the prevention and treatment of COVID-19 by potential vaccines and drugs. Although, the governments and global population have been attracted by some vaccine production projects, the presence of SARS-CoV-2-specific antiviral drugs would be an urge necessity in parallel with the efficient preventive vaccines. Various nonspecific drugs produced previously against other bacterial, viral, and parasite infections were recently evaluated for treating patients with COVID-19. In addition to therapeutic properties of these anti-COVID-19 compounds, some adverse effects were observed in different human organs as well. Not only several attentions were paid to antiviral therapy and treatment of COVID-19, but also nanomedicine, immunotherapy, and cell therapy were conducted against this viral infection. In this review study, we planned to introduce the present and potential future treatment strategies against COVID-19 and define the advantages and disadvantages of each treatment strategy.

Gamma-radiated immunosuppressed tumor xenograft mice can be a new ideal model in cancer research.

Tumor xenograft models can create a high capacity to study human tumors and discover efficient therapeutic approaches. Here, we aimed to develop the gamma-radiated immunosuppressed (GIS) mice as a new kind of tumor xenograft model for biomedical studies. First, 144 mice were divided into the control and treated groups exposed by a medical Cobalt-60 apparatus in 3, 4, and 5 Gy based on the system outputs. Then, 144 BALB/c mice were divided into four groups; healthy, xenograft, radiation, and radiation + xenograft groups. The animals in the xenograft and radiation + xenograft groups have subcutaneously received 3 × 106 MCF-7 cells 24 h post-radiation. On 3, 7, 14, and 21 days after cell injection, the animals were sacrificed. Then, the blood samples and the spleen and tumor tissues were removed for the cellular and molecular analyses. The whole-body gamma radiation had a high immunosuppressive effect on the BALB/c mice from 1 to 21 days post-radiation. The macroscopic and histopathological observations have proved that the created clusters' tumor structure resulted in the xenograft breast tumor. There was a significant increase in tumor size after cell injection until the end of the study. Except for Treg, the spleen level of CD4, CD8, CD19, and Ly6G was significantly decreased in Xen + Rad compared to the Xen alone group on 3 and 7 days. Unlike IL-4 and IL-10, the spleen level of TGF-ß, INF-γ, IL-12, and IL-17 was considerably decreased in the Xen + Rad than the Xen alone group on 3 and 7 days. The spleen expressions of the VEGF, Ki67, and Bax/Bcl-2 ratio were dramatically increased in the Xen + Rad group compared to the Xen alone on 3, 7, 14, and 21 days. Our results could confirm a new tumor xenograft model via an efficient immune-suppressive potential of the whole-body gamma radiation in mice.

Identification and characterization of the first fish parvalbumin-like protein data from a pathogenic fungal species, Trichophyton violaceum.

Parvalbumins are the most important fish allergens, which are heat-stable, classified in the family of calcium-binding EF-hand proteins, and contain one magnesium binding site. The functional connection between calcium and parvalbumin gives fish the high-speed swimming ability because of high concentration of Ca2+-binding parvalbumin in fish white muscles. Although parvalbumins are widely studied and conceivably play crucial roles in the physiology and swimming pattern of fishes, still no report is available about their presence in microbes, such as pathogenic fungal species. We detected a DNA sequence in the genome of Trichophyton violaceum and used in silico and polymerase chain reaction (PCR) technique with a designed pair of primers to identify it as parvalbumin-coding gene.

Improving the mechanical behavior of reduced graphene oxide/hydroxyapatite nanocomposites using gas injection into powders synthesis autoclave.

In this study, we show the synthesis of reduced graphene oxide/hydroxyapatite (rGO/HA) composites using a hydrothermal autoclave with argon-15% hydrogen gas injection. This both increases the hydrothermal pressure and uses hydrogen as a reductive agent in the process. The synthesized powders were then consolidated with spark plasma sintering method. The analysis of the consolidated samples included Vickers Indentation technique and cell viability. The results showed that injected gases in the autoclave produced powders with a higher crystallinity compared to synthesis without the gases. Also, hydrogen gas led to increased reduction of GO. The microscopic analysis confirmed existing graphene sheets with folding and wrinkling in the powders and indicated that various preferential directions played a role in the growth of hydroxyapatite crystals. The results showed that in general, graphene sheets increased the mechanical properties of HA. In the samples synthesized with injected gases, this increase was more significant. Interface analysis results indicate that reduced graphene oxide (rGO)/HA interface is likely coherent. These nanocomposites were biocompatible and showed some hydrophobicity compared to pure HA.

Comparison of the effect of argon, hydrogen, and nitrogen gases on the reduced graphene oxide-hydroxyapatite nanocomposites characteristics.

In this study, the effect of the argon, nitrogen, and hydrogen gases on the final properties of the reduced graphene oxide- hydroxyapatite nanocomposites synthesized by gas injected hydrothermal method was investigated. Four samples were synthesized, which in the first sample the pressure was controlled by volume change at a constant concentration. In subsequent samples, the pressure inside the autoclave was adjusted by the injecting gases. The initial pressure of the injected gases was 10 bar and the final pressure considered was 25 bar. The synthesized powders were consolidated at 950 °C and 2 MPa by spark plasma sintering method. The final samples were subjected to Vickers indentation analysis. The findings of this study indicate that the injection of argon, hydrogen, and nitrogen gases improved the mechanical properties of the nanocomposites. Injection of gases increased the crystallinity and particle size of hydroxyapatite, and this increase was greater for nitrogen gas than for others. Injection of these gases increased the rate of graphene oxide reduction and in this case the effect of nitrogen gas was greater than the others.

G2-lymphocyte chromosomal radiosensitivity in patients with LPS responsive beige-like anchor protein (LRBA) deficiency.

Lipopolysaccharide-responsive, beige-like anchor protein (LRBA) deficiency is an autosomal recessive primary immunodeficiency disease characterized by a CVID-like phenotype, particularly severe autoimmunity and inflammatory bowel disease. This study was undertaken to evaluate radiation sensitivity in 11 LRBA-deficient patients. Therefore, stimulated lymphocytes of the studied subjects were exposed to a low dose γ-radiation (100 cGy) in the G2 phase of the cell cycle and chromosomal aberrations were scored. Lymphocytes of age-sex matched healthy individuals used in the same way as controls. Based on the G2-assay, six (54.5%) of the patients had higher radiosensitivity score comparing to the healthy control group, forming the radiosensitive LRBA-deficient patients. This chromosomal radiosensitivity showed that these patients are predisposed to autoimmunity and/or malignancy, and should be protected from unnecessary diagnostic and therapeutic procedures using ionizing radiation and exposure to other DNA damaging agents.

Individual Radiosensitivity Assessment of the Families of Ataxia-Telangiectasia Patients by G2-Checkpoint Abrogation.

OBJECTIVES: Ataxia-telangiectasia (A-T) is an autosomal recessive multisystem disorder characterised by cerebellar degeneration, telangiectasia, radiation sensitivity, immunodeficiency, oxidative stress and cancer susceptibility. Epidemiological research has shown that carriers of the heterozygous ataxia-telangiectasia mutated (ATM) gene mutation are radiosensitive to ionising irradiation and have a higher risk of cancers, type 2 diabetes and atherosclerosis. However, there is currently no fast and reliable laboratory-based method to detect heterozygous ATM carriers for family screening and planning purposes. This study therefore aimed to evaluate the ability of a modified G2-assay to identify heterozygous ATM carriers in the families of A-T patients. METHODS: This study took place at the Tehran University of Medical Sciences, Tehran, Iran, between February and December 2017 and included 16 A-T patients, their parents (obligate heterozygotes) and 30 healthy controls. All of the subjects underwent individual radiosensitivity (IRS) assessment using a modified caffeine-treated G2-assay with G2-checkpoint abrogation. RESULTS: The mean IRS of the obligate ATM heterozygotes was significantly higher than the healthy controls (55.13% ± 5.84% versus 39.03% ± 6.95%; P <0.001), but significantly lower than the A-T patients (55.13% ± 5.84% versus 87.39% ± 8.29%; P = 0.001). A receiver operating characteristic (ROC) curve analysis of the G2-assay values indicated high sensitivity and specificity, with an area under the ROC curve of 0.97 (95% confidence interval: 0.95-1.00). CONCLUSION: The modified G2-assay demonstrated adequate precision and relatively high sensitivity and specificity in detecting heterozygous ATM carriers.

Neuroendocrine carcinoma of the tongue.

Neuroendocrine carcinoma usually originates from lung. Few data exist in the literature regarding neuroendocrine carcinoma of the tongue. Patient data including history, surgical procedure, histology, and radiology investigations were collected and summarized. A 40-year-old woman was referred after partial glossectomy. Squamous mucosa with neoplasm and cells with round nuclei and light cytoplasm was reported in the tongue biopsy. Immunohistochemistry (IHC) staining was positive for cytokeratin, neuron specific enolase, synaptophysin and chromogranin and negative for leukocyte common antigen. This case showed a high proliferative activity (Ki-67 labeling index were 60%). These IHC findings were in favor of poorly differentiated neuroendocrine carcinoma. After surgery, she received chemotherapy and chemoradiation. The diagnosis of neuroendocrine tumors in the present case is based on immunohistochemical markers and cellular shapes. Postoperative chemoradiotherapy is a critical element of therapy for head and neck high-grade neuroendocrine carcinomas, our patient received this treatment after surgery.