Gene Expression Patterns of Colorectal Cancer Stem Cells Following Ibuprofen and Hyperthermia Treatment.

Background: Cancer stem cells (CSCs) substantially influence the development of colorectal cancer (CRC), metastasis, relapse, and resistance to therapy. Ibuprofen and hyperthermia can be effective in the treatment of cancer. Herein, we evaluated the effects of hyperthermia and ibuprofen on the isolated-CSCs of CRC. Methods: This experimental study was conducted between Sep 2020 and Jan 2022 at the Department of Pathology, School of Medicine, Shiraz University of Medical Sciences, Iran. A non-adhesive culture system was used to isolate CSCs from HT-29 cells. To confirm the stemness nature of isolated-CSCs, the expression of stemness genes and protein markers was evaluated by quantitative Real-time PCR (qRT-PCR) and flow cytometry assay. The isolated-CSCs were treated with hyperthermia and ibuprofen. The cell viability was determined by MTT assay and trypan blue staining. The expression of stemness, proliferation, Wnt signaling pathway and apoptosis genes was assessed by qRT-PCR. Results: CSCs were isolated within 14 days. The expression of CD-133 marker and OCT3/4, C-MYC, KLF4, and NANOG genes in isolated-CSCs was higher than HT-29 cells (P<0.05). Cell viability of treated-CSCs were considerably reduced (P<0.05). Hperthermia reduced the expression of OCT3/4, NANOG, PCNA, WNT1 and CTNNB1 genes and increased the expression of P53, BAX, and KLF4 genes (P<0.05). Ibuprofen decreased the expression of OCT3/4, BCL2, NANOG, PCNA, WNT1, and CTNNB1 genes and increased the expression of P53, BAX, and KLF4 genes in treated-CSCs (P<0.05). Conclusion: Hyperthermia and ibuprofen treatment demonstrate an inhibitory effect on colorectal CSCs. However, using combination therapy is remaining to be tested.

A novel missense variant in CDK5RAP2 associated with non-obstructive azoospermia.

OBJECTIVE: The most severe type of male infertility is non-obstructive azoospermia (NOA), where there is no sperm in the ejaculate due to failure of spermatogenesis, affecting 10%-20% of infertile men with azoospermia. Genetic studies have identified dozens of NOA genes. The main aim of the present study is to identify a novel monogenic mutation that may cause NOA. MATERIALS AND METHODS: We studied the pedigree of a consanguineous family with three NOA and one fertile brother by a family-based exome-sequencing, segregation analysis, insilico protein modeling and single-cell RNA sequencing data analysis. RESULTS: Bioinformatics analysis followed by sanger sequencing revealed that three NOA brothers were homozygous for a rare missense variant in Cyclin Dependent Kinase Regulatory Subunit Associated Protein 2 (Centrosomin) CDK5RAP2 (NM_018249:exon26:c.A4003T:p.R1335W, rs761196443). Protein modeling demonstrated that CDK5RAP2, Arg1335Trp resided nearby the Microtubule Associated Protein RP/EB Family Member 1 (EB1/MAPRE1) interaction site. As a consequence of the R1335W mutation, the positively charged Arginine was replaced by to the hydrophobic tryptophan residue, possibly leading to local instability in the structure and perturbation in the CDK5RAP2-MAPRE1 interaction. CONCLUSION: Our study reports a novel missense variant of CDK5RAP2 that segregates in homozygosity with male infertility and NOA in a consanguineous family. In silico structural predictions and gene expression data indicate a potential role of the CDK5RAP2 variant in causing defective centrosomic maturation during spermatogenesis.

ADCY10 frameshift variant leading to severe recessive asthenozoospermia and segregating with absorptive hypercalciuria.

STUDY QUESTION: Can whole exome sequencing (WES) reveal a novel pathogenic variant in asthenozoospermia in a multiplex family including multiple patients? SUMMARY ANSWER: Patients were discovered to be homozygous for a rare 2-bp deletion in the ADCY10 coding region (c.1205_1206del, rs779944215). WHAT IS KNOWN ALREADY: ADCY10 encodes for soluble adenylyl cyclase (sAC), which is the predominant adenylate cyclase in sperm. It is already established that proper sAC activity and a constant supply of cAMP are crucial to sperm motility regulation, and knockout mouse models have been reported as severely asthenozoospermic. ADCY10 is a susceptibility gene for dominant absorptive hypercalciuria (OMIM#143870); however, no ADCY10 variations have been confirmed to cause human asthenozoospermia to date. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of a highly consanguineous pedigree of asthenozoospermia. The subject family was recruited in Iran in 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: The two patients were diagnosed as asthenozoospermic through careful clinical investigations. Both patients, respective parents, and an unaffected brother were subjected to WES. The discovered variant was validated by Sanger sequencing and segregated with the phenotype. To confirm the pathogenicity of the variant, sperm samples from both patients, 10 normozoospermic men and 10 asthenozoospermic patients not representing the variation, were treated with a cAMP analogue dissolved in human tubal fluid medium, followed by computer-assisted sperm analysis and statistical analyses. MAIN RESULTS AND THE ROLE OF CHANCE: The discovered homozygous variant occurs at 10 amino acids upstream of the ADCY10 nucleotide binding site leading to a premature termination (p.His402Argfs*41). Treatment of the patients' sperm samples with a cell-permeable cAMP analogue resulted in a significant increase in sperm motility, indicating the pathogenic role of the variant. Moreover, absorptive hypercalciuria, segregating within the family, was also associated with the same variant following a dominant inheritance. LIMITATIONS, REASONS FOR CAUTION: Though nonsense-mediated decay is highly likely to occur in the mutated transcripts, we were not able to confirm this due to low RNA levels in mature sperm. WIDER IMPLICATIONS OF THE FINDINGS: Our finding enlarges the phenotypic spectrum associated with the ADCY10 gene, previously described as a susceptibility gene for dominant absorptive hypercalciuria. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Royan Institute, Tehran, Iran, and San Raffaele Hospital, Milan, Italy. The authors have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Cloning and expression of nlpA gene as DNA vaccine candidate against Acinetobacter baumannii.

Acinetobacter baumannii is one of the highly antibiotic-resistant bacteria that cause infections with high rate of death. This bacterium is one the common causes of infection worldwide leading to endemic and epidemic nosocomial infections. Despite many efforts, there is no effective vaccine against A. baumannii. As NlpA is one of the important antigenic factors in biogenesis of outer membrane vesicles, and OMV-based reported vaccines in A. baumannii stimulated the immune responses, this study was aimed to clone and express nlpA gene in eukaryotic HDF cells and evaluate the induced immunization following the administration of resulting construct as DNA vaccine in BALB/c mice. The nlpA gene of A. baumannii was amplified using PCR. The PCR product was then cloned and subcloned into the pTZ57R/T and pEGFP-C2 vectors respectively. The cloning was confirmed by PCR, restriction enzyme digestion and DNA sequencing. The pEGFP-C2-nlpA recombinant plasmid was transferred into the HDF cells using electroporation and the expression of target gene was validated by RT-PCR. The recombinant construct was injected to BALB/c mice through three IM injections and the levels of IgG, IgM, INF-γ, IL-2, IL-4, and IL-12 were determined using ELISA assay. The A. baumannii nlpA gene was amplified during PCR as 867 bp band which was successfully cloned in pEGFP-C2-nlpA vector. Obtained data from RT-PCR and presence of the 867 bp fragment in transformed HDF cells confirmed the nlpA gene expression. Following the injection of pEGFP-C2-nlpA showed the increased level of IgG, IgM, INF-γ, IL-2, IL-4, and IL-12 in serum of immunized mice. Overall, through this study recombinant pEGFP-C2-nlpA was generated and successfully expressed the A. baumannii nlpA gene in eukaryotic cells. Additionally, our in vivo study confirmed that the recombinant construct capable to induce the immune response in immunized mice. These findings suggest the pEGFP-C2-nlpA may be considered as DNA vaccine candidate against A. baumannii.

Cloning of ompA gene from Acinetobacter baumannii into the eukaryotic expression vector pBudCE4.1 as DNA vaccine.

Antibiotic resistant features of Acinetobacter baumannii is partly due to the decreased outer membrane proteins (OMPs) permeability. The OmpA is one of the most conserved proteins among A. baumannii with a considerable antigenic potential to stimulate the multidimensional immune system responses. The present study was aimed to clone the ompA gene into the eukaryotic expression vector with potential as DNA vaccine. The ompA gene of A. baumannii was amplified using polymerase chain reaction (PCR). The target DNA was cloned and sub-cloned into the pTZ57R/T and pBudCE4.1 vectors, respectively. The recombinant vectors containing ompA were then validated using colony PCR, vector sequencing and double-digestion strategies. The pBudCE4.1-ompA recombinant plasmid was transfected into the human dermal fibroblast cells (HDF) and presence of ompA transcript and protein was evaluated using reverse transcribed-PCR (RT-PCR) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Our finding from colony PCR, sequencing and enzyme double digestion result confirmed that target gene has been successfully inserted into the pTZ57RT and pBudCE4.1. The presence of an expected band (1112 bp) in RT-PCR as wells as a ~ 38 kDa band during SDS-PAGE showed that the recombinant pBudCE4.1-ompA construct was efficiently transfected into the HDF cells and expressed. Altogether, our observation demonstrated that the recombinant pBudCE4.1-ompA construct was successfully produced although further experiments are needed.

Silencing mutations in JAG1 gene may play crucial roles in the pathogenesis of Tetralogy of Fallot.

JAG1 gene through Notch signaling is implicated in cell fate decisions in early cardiac development, and mutations in several proteins in the pathway have been involved in various disorders. Tetralogy of Fallot (TOF) is the most frequent form of complicated congenital heart disease. The abnormality of TOF begins through the first eight weeks of fetal growth and is confused with ventricular septal defects, obstruction to right ventricular outflow tract, aortic dextroposition, and right ventricular hypertrophy. Hence the existence of mutations in JAG1 gene in Iranian patients with TOF is evaluated. The clinical data and peripheral blood samples were collected from 44 sporadic nonsyndromic patients with TOF and compared to 44 healthy individuals. DNA was extracted, and the exon 6 of the JAG1 gene was amplified by PCR then the PCR products were purified and sequenced. The age range in patients and the control group was 2-36 years, and the mean and standard deviation (SD) of the age in patients was (11.69 ± 7.85 years) and in control group (11.63 ± 7.99 years).  Finally, the samples were successfully sequenced, then analyzed and one synonymous variant (c.765C>T; p.Y255Y) was observed in 38 patients with frequency (86.4%) and three controls with frequency (6.8%). The c.765C>T variant is significantly associated with the pathogenesis of TOF in Iranian population.

A Novel Loss-of-Function Mutation in HOXB1 Associated with Autosomal Recessive Hereditary Congenital Facial Palsy in a Large Iranian Family.

Hereditary congenital facial palsy (HCFP) is a rare congenital cranial dysinnervation disorder, recognisable by non-progressive isolated facial nerve palsy (cranial nerve VII). It is caused by developmental abnormalities of the facial nerve nucleus and its nerve. So far, 4 homozygous mutations have been identified in 5 unrelated families (12 patients) with HCFP worldwide. In this study, a large Iranian consanguineous kindred with 5 members affected by HCFP underwent thorough clinical and genetic evaluation. The candidate gene HOXB1 was screened and analysed by Sanger sequencing. As in previous cases, the most remarkable findings in the affected members of the family were mask-like faces, bilateral facial palsy with variable sensorineural hearing loss, and some dysmorphic features. Direct sequencing of the candidate gene HOXB1 identified a novel homozygous frameshift mutation (c.296_302del; p.Y99Wfs*20) which co-segregated with the disease phenotype within the extended family. Our findings expand the mutational spectrum of HOXB1 involved in HCFP and consolidate the role of the gene in the development of autosomal recessive HCFP. Moreover, the truncating mutation identified in this family leads to a broadly similar presentation and severity observed in previous patients with nonsense and missense mutations. This study characterises and defines the phenotypic features of this rare syndrome in a larger family than has previously been reported.