CTNS Mutations Causing Autosomal Recessive Cystinosis in a Subset of Iranian Population: Report of Two New Variants.

Background: Nephropathic cystinosis (NC) is an uncommon autosomal recessive disease with abnormality in lysosomal storage that appearances in patients with mutations in the CTNS gene encoding a lysosomal transporter cystinosin. Disrupted function of this transporter is followed by accumulation of cysteine crystals in cells of many various organs. This study aimed to investigate the mutations of the CTNS gene in 20 Iranian patients suffering from NC. Materials and Methods: Twenty Iranian cystinosis patients referring to Imam Hossein Hospital of Isfahan were employed in this case-series study. After extraction of genomic DNA, the promoter and entire coding regions of CTNS were analysed using sanger sequencing in all patients. Gap-Polymerase Chain Reaction was used to detect 57 kb deletion in the CTNS gene. In silico study was performed to analyse variants. Results: The large deletion was not seen in any NC patients. Molecular analysis which conducted to screen the CTNS gene of patients, identified eight different mutations, including two new mutations, c.971_972insC and c.956_956delA, which have not been reported before, and c.681G>A mutation, which was identified as a frequently founded mutation in the Middle East and was observed in 35% of patients. In this study, five other mutations including c.1015G>A, c.922G>A, c.323_323delA, c.433C>T, and c.18_21delGACT were also observed, which have been reported in previous studies. Conclusion: The mutational spectrum in the Iranian patients is the same as previously reported mutations except that two new mutations were found. The present findings will present suggestions for regular molecular diagnosis of cystinosis in Iran.

Time-series bioinformatics analysis of SARS-CoV-infected cells to identify the biological processes associated with severe acute respiratory syndrome.

BACKGROUND: The COVID-19 pandemic, caused by the new virus of the coronavirus family, SARS-CoV-2, could lead to acute respiratory syndrome. The molecular mechanisms related to this disorder are still debatable. METHODS: In this study to understand the pathogenicity mechanism of SARS-CoV-2, using the bioinformatics approaches, we investigated the expression of involved genes, their regulatory, and main signaling pathways during the time on days 1, 2, 3, and 4 of SARS-CoV infected cells. RESULTS: Here, our investigation shows the complex changes in gene expression on days 2 and 3 post-infection. The functional analysis showed that especially related to immune response, response to other organisms, and defense response. IL6-AS1 is the predicted long non-coding RNA and is a key regulator during infection. In this study, for the first time has been reported the role of IL6-AS1. Also, the correlation of differential expression genes with the level of immune infiltration was shown in the relationship of Natural killer cells and T cell CD 4+ with DE genes. CONCLUSION: In the current study, identification of the altered expression pattern of genes in SARS-CoV-infected cells in time course also can help identify and link the molecular mechanisms and explore the holistic view of infection of SARS-CoV-2.

Constructing a novel competing Endogenous RNAs network based on NR3C1 and X-linked inhibitor of apoptosis protein genes reveals potential prognostic biomarkers in colorectal cancer.

Background: Long noncoding RNAs (lncRNAs) have been recognized as the main modulatory molecules in various cancers and perform as competing endogenous RNAs (ceRNAs). The nuclear hormone receptor superfamily of ligand-activated transcription factors (NR3C1) regulates numerous proliferative and metabolic processes such as tumorigenesis and metabolic diseases. Furthermore, X-linked inhibitor of apoptosis protein (XIAP) belongs to a family of the inhibitors of apoptosis proteins, is located downstream of the glucocorticoid receptor (GR or NR3C1) pathway, and cooperates with GR to suppress apoptosis. However, the underlying mechanisms of NR3C1 and XIAP in colorectal cancer (CRC) remain mainly unclear. This research aims to clarify the potential RNA biomarkers and to construct a novel ceRNA network in CRC. Materials and Methods: Multistep bioinformatics methods such as Lnc2cancer and miRDB databases were applied to identify candidate lncRNAs and miRNAs. The interaction energy between lncRNAs, NR3C1, and XIAP genes was analyzed by the LncRRIsearch database. Plus, microRNAs and lncRNA were evaluated via the Diana tools database to select microRNAs with the most binding scores. Quantitative reverse transcription-polymerase chain reaction (QRT-PCR) was applied to verify RNA molecules' expression levels and their association with the clinicopathological factors in 30 CRC tissues compared to 30 adjacent tissues. Results: QRT-PCR showed upregulation of KCNQ1OT1, NR3C1, and XIAP and downregulation of miR-421. The ceRNA network was constructed with 17 lncRNAs, 2 mRNAs, and 42 miRNAs. Thus, we explained the potential interactions between KCNQ1OT1 and miR-421 with NR3C1 and XIAP genes. Conclusion: Our study represents potential prognostic biomarkers and a new ceRNA network for further study in CRC.

miR-574, miR-499, miR-125b, miR-106a, and miR-9 potentially target TGFBR-1 and TGFBR-2 genes involving in inflammatory response pathway: Potential novel biomarkers for chronic lymphocytic leukemia.

MicroARNAs (miRNAs) are linked to a variety of cancers, which resulted in molecular pathway dysregulation in chronic lymphocytic leukemia (CLL). Using five dysregulated miRNAs identified by literature mining and in silico analysis, we were able to demonstrate the critical role that the TGFBR1 and TGFB receptor signaling pathways play in the state of CLL. Assays using real-time PCR were run on 30 patients and 30 healthy controls. This study showed that patient samples have considerably higher levels of miR-574 and miR-499. Notably, the same groups had lower expression levels of miR-125b, miR-106a, and miR-9. Furthermore, we suggested that TGFBR1 and TGFBR2 expression levels were decreased in patients, and we suggested that these genes could be targets for our profile miRNAs. In the current study, we hypothesized that miR-574, miR-499, miR-125b, miR-106a, and miR-9 are likely five new potential biomarkers for early diagnosis. Our research also showed that these profile miRNAs have a role in the formation of CLL, possibly through controlling the TGFBR1 and TGFBR2 pathways. This suggests that these profile miRNAs could serve as biomarkers for the diagnosis and prognosis of CLL.

Study of The Correlation between miR-106a, miR-125b, and miR-330 on Multiple Sclerosis Patients by Targeting TNFSF4 and SP1 in NF-кb/TNF-α Pathway: A Case-Control Study.

OBJECTIVE: Multiple sclerosis (MS) is a complex multifactorial neuro-inflammatory disorder. This complexity arises from the evidence suggesting that MS is developed by interacting with environmental and genetic factors. This study aimed to evaluate the miR-106a, miR-125b, and miR330- expression levels in relapsing-remitting multiple sclerosis (RRMS) patients. The miRNAs' impact on TNFSF4 and Sp1 genes through the NF-кB/TNF-α signaling pathway was analyzed by measuring the expression levels in case and controls. MATERIALS AND METHODS: In this in silico-experimental study, we evaluated the association of miR-106a, miR- 125b, and miR330- with TNFSF4 and SP1 gene expression levels in 60 RRMS patients and 30 healthy controls by real-time polymerase chain reaction (PCR). RESULTS: The expression levels of miR-330, miR-106a, and miR125-b in blood samples of RRMS patients were predominantly reduced. The expression of TNFSF4 in patients demonstrated a significant enhancement, in contrast to the diminishing Sp1 gene expression level in controls. CONCLUSION: Our findings indicated an association between miR-106a and miR-330 and miR125-b expression and RRMS in our study population. Our data suggested that the miR106-a, miR125-b, and mir330- expression are correlated with TNFSF4 and Sp1 gene expression levels.

An experimental in silico study on COVID-19: Response of neutrophil-related genes to antibiotics.

Background and Aims: All components of the immune system are involved in alleviating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Further research is required to provide detailed insights into COVID-19-related immune compartments and pathways. In addition, a significant percentage of hospitalized COVID-19 patients suspect bacterial infections and antimicrobial resistance occurs following antibiotics treatment. The aim of this study was to evaluate the possible effects of antibiotics on the response of neutrophil-related genes in SARS-CoV-2 patients by an experimental in silico study. Methods: The two data sets GSE1739 and GSE21802 including 10 SARS positive patients and 35 influenza A (H1N1) patients were analyzed, respectively. Differentially expressed genes (DEGs) between these two data sets were determined by GEO2R analysis and the Venn diagram online tool. After determining the hub genes involved in immune responses, the expression of these genes in 30 COVID-19 patients and 30 healthy individuals was analyzed by real-time polymerase chain reaction (PCR). All patients received antibiotics, including levofloxacin, colistin, meropenem, and ceftazidime. Results: GEO2R analysis detected 240 and 120 DEGs in GSE21802 and GSE1739, respectively. Twenty DEGs were considered as enriched hub genes involved in immune processes such as neutrophil degranulation, neutrophil activation, and antimicrobial humoral response. The central nodes were attributed to the genes of neutrophil elastase (ELANE), arginase 1 (ARG-1), lipocalin 2 (LCN2), and defensin 4 (DEFA4). Compared to the healthy subjects, the expression of LCN2 and DEFA4 were significantly reduced in COVID-19 patients. However, no significant differences were observed in the ELANE and AGR-1 levels between COVID-19 subjects and the control group. Conclusions: Activation and degranulation of neutrophils were observed mainly in SARS, and H1N1 infection processes and antibiotics administration could affect neutrophil activity during viral infection. It can be suggested that antibiotics can decrease inflammation by restoring the expression of neutrophil-related genes in COVID-19 patients.

Dysregulation of RNA interference components in COVID-19 patients.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the novel coronavirus causing severe respiratory illness (COVID-19). This virus was initially identified in Wuhan city, a populated area of the Hubei province in China, and still remains one of the major global health challenges. RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing that plays a crucial role in innate viral defense mechanisms by inhibiting the virus replication as well as expression of various viral proteins. Dicer, Drosha, Ago2, and DGCR8 are essential components of the RNAi system, which is supposed to be dysregulated in COVID-19 patients. This study aimed to assess the expression level of the mentioned mRNAs in COVID-19patients compared to healthy individuals. RESULTS: Our findings demonstrated that the expression of Dicer, Drosha, and Ago2 was statistically altered in COVID-19 patients compared to healthy subjects. Ultimately, the RNA interference mechanism as a crucial antiviral defense system was suggested to be dysregulated in COVID-19 patients.

A novel carcinogenic PI3Kα mutation suggesting the role of helical domain in transmitting nSH2 regulatory signals to kinase domain.

AIMS: Mutations in PIK3CA, which encodes p110α subunit of PI3K class IA enzymes, are highly frequent in breast cancer. Here, we aimed to probe mutations in exon 9 of PIK3CA and computationally simulate their function. MATERIALS AND METHODS: PCR/HRM and PCR/sequencing were used for mutation detection in 40 breast cancer specimens. The identified mutations were queried via in silico algorithms to check the pathogenicity. The molecular dynamics (MD) simulations were utilized to assess the function of mutant proteins. KEY FINDINGS: Three samples were found to harbor at least one of the E542K, E545K and L551Q mutations of which L551Q has not been reported previously. All mutations were confirmed to be pathogenic and MD simulations revealed their impact on protein function and regulation. The novel L551Q mutant dynamics was similar to that of previously found carcinogenic mutants, E542K and E545K. A functional role for the helical domain was also suggested by which the inhibitory signal of p85α is conducted to kinase domain via helical domain. Helical domain mutations lead to impairment of kinase domain allosteric regulation. Interestingly, our results show that p110α substrate binding pocket of kinase domain in mutants may have differential affinity for enzyme substrates, including anit-p110α drugs. SIGNIFICANCE: The novel p110α L551Q mutation could have carcinogenic feature similar to previously known helical domain mutations.

A Comparison Between Full-COLD PCR/HRM and PCR Sequencing for Detection of Mutations in Exon 9 of PIK3CA in Breast Cancer Patients.

One of the most common somatic mutations in breast cancer is found in PIK3CA with a prevalence rate of 18-45%. Different variants of this gene are considered as resistance markers for treatment with HER2-targeted medicines. Conventional molecular methods such as Sanger sequencing are not able to detect mutations with low abundance in a mixture of wild-type DNA, especially in the early stages of cancer development. In this study, two methods of co-amplification at lower denaturation temperature PCR (COLD-PCR) and high-resolution melting (HRM) were combined for detection of mutations in exon 9 of PIK3CA; DNA, therefore, was extracted from MCF-7 and BT-474 as mutant and wild-type cell lines respectively. Thereafter, serial dilutions of extracted DNA were used to determine sensitivity of full-COLD PCR/HRM in comparison with conventional PCR sequencing as the gold standard method. Cell line experiments resulted in almost 30 fold increase in sensitivity by use of full-COLD PCR/HRM. In addition, 40 patients with primary breast cancer were investigated with the mentioned methods. As a result of this part of study, four mutations were detected by conventional PCR sequencing including E542K and E545K mutations in three and one samples respectively. Whereas, full-COLD PCR/HRM was able to detect one E542K mutation more than gold standard method which caused the percentage of sensitivity to get improved by 2.5% (10 to 12.5%). Our results clearly demonstrated that full-COLD PCR/HRM could detect lower levels of mutations in wild-type background as a sensitive method with simple and cost-effective procedure; therefore, it can prospectively be used in screening of patients with early-stage breast cancers.

Association between (GT)n Repeats in Heme Oxygenase-1 Gene Promoter and 3-Year Survival of Patients with Acute Leukemia: a Controlled, Cross-Sectional Study.

Background: Acute leukemia is a common pediatric cancer. Novel strategies for treatment of acute leukemia have been developed, but treatment resistance is remained as the most problematic issue. It is hypothesized that the HO-1 gene up-regulation is responsible for tumor resistance to chemotherapy or radiotherapy-induced apoptosis. The levels of HO-1 expression are related to (GT)n microsatellite polymorphisms in the location of its promoter. This study designed to compare allelic frequencies of (GT)n microsatellite polymorphisms in HO-1 gene between acute leukemia patients and healthy controls. Indeed, 3-year disease-free survival was also evaluated. Methods: Sixty-three patients with acute leukemia and seventy healthy infants were included in this study. We used the medical records of patients to collect information about survival after chemotherapy. The number of GT repeats in HO-1 promoter was determined by an ABI 3100 sequencer. Results: The HO-1 GT repeats ranged from 14 to 34 with peaks at 27 repeats in both cases and controls. Children with longer alleles ((GT)n ≥ 27) had enhanced 3-year survival rate after treatment with chemotherapy or radiotherapy (P<0.05). Conclusion: Although no significant differences were observed between leukemia patients and controls regarding allelic frequency, we found elevated frequency of "LL" genotype in leukemia patients with good prognosis and 3-year surveillance. Radiotherapy and chemotherapy might elevate the expression levels of HO-1 with subsequent increased resistance of leukemia patients to therapy.