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Hypomyelinating leukodystrophy-17 is a neurodevelopmental disorder caused by autosomal recessive mutations in the AIMP2 gene, resulting in a lack of myelin deposition during brain development, leading to variable neurological symptoms. Research on brain function in these disorders is challenging due to the lack of access to brain tissue. To overcome this problem, researchers have utilized different cell and animal models. The CRISPR-Cas9 system is considered the most optimal and effective method for genetic modification and developing cell models. We studied the efficacy of the CRISPR-Cas9 technology in inducing mutations in the AIMP2 gene in HEK293 cell lines. The study involved transfecting HEK293 cells with recombinant PX458 plasmids targeting spCas-9 and AIMP2 sgRNA. The cells were evaluated using fluorescent microscopy and enriched using serial dilution. The CRISPR/Cas9 plasmids were validated through PCR and Sanger sequencing. After serial dilution, AS-PCR, Sanger sequencing, and TIDE program analysis showed the construct successfully induces an indel mutation in HEK cells. Our findings demonstrated the great efficacy of the CRISPR system and produced a construct for inducing mutations in the AIMP2 gene, which can be utilized to edit the AIMP2 gene in nerve cells and create a cellular model of the HLD17 disease., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Generating animal models can explore the role of new candidate genes in causing diseases and the pathogenicity of a specific mutation in the underlying genes. These animals can be used to identify new pharmaceutical or genetic therapeutic methods. In the present experiment, we developed a rpe65a knock out (KO) zebrafish as a retinitis pigmentosa (RP) disease model. Using the CRISPR/Cas9 system, the rpe65a gene was KO in zebrafish. Two specific single-guide RNAs (sgRNAs) were designed for the zebrafish rpe65a gene. SgRNAs were cloned into the DR274 plasmid and synthesized using in vitro transcription method. The efficiency of Ribonucleoprotein (synthesized sgRNA and recombinant Cas9) was evaluated by in vitro digestion experiment. Ribonucleoprotein complexes were microinjected into one to four-celled eggs of the TU zebrafish strain. The effectiveness of sgRNAs in KO the target gene was determined using the Heteroduplex mobility assay (HMA) and Sanger sequencing. Online software was used to determine the percent of mosaicism in the sequenced samples. By examining the sequences of the larvae that showed a mobility shift in the HMA method, the presence of indels in the binding region of sgRNAs was confirmed, so the zebrafish model for RP disease established. Zebrafish is an ideal animal model for the functional study of various diseases involving different genes and mutations and used for evaluating different therapeutic approaches in human diseases. This study presents the production of rpe65a gene KO zebrafish models using CRISPR/Cas9 technology. This model can be used in RP pathophysiology studies and preclinical gene therapy experiments., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Glioblastoma (GBM) stands out as the most prevalent and aggressive intracranial tumor, notorious for its poor prognosis. The current standard-of-care for GBM patients involves surgical resection followed by radiotherapy, combined with concurrent and adjuvant chemotherapy using Temozolomide (TMZ). The effectiveness of TMZ primarily relies on the activity of O 6 -methylguanine DNA methyltransferase (MGMT), which removes alkyl adducts from the O 6 position of guanine at the DNA level, thereby counteracting the toxic effects of TMZ., Method: In this study, we employed fusions of catalytically-inactive Cas9 (dCas9) to DNA methyltransferases (dCas9-DNMT3A) to selectively downregulation MGMT transcription by inducing methylation at MGMT promoter and K-M enhancer., Result: Our findings demonstrate a significant reduction in MGMT expression, leading to intensified TMZ sensitivity in the HEK293T cell line., Conclusion: This study serves as a proof of concept for the utilization of CRISPR-based gene suppression to overcome TMZ resistance and enhance the lethal effect of TMZ in glioblastoma tumor cells., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Duchenne Muscular Dystrophy (DMD) is a progressive muscle wasting disorder caused by loss-of-function mutations in the dystrophin gene. Although the search for a definitive cure has failed to date, extensive efforts have been made to introduce effective therapeutic strategies. Gene editing technology is a great revolution in biology, having an immediate application in the generation of research models. DMD muscle cell lines are reliable sources to evaluate and optimize therapeutic strategies, in-depth study of DMD pathology, and screening the effective drugs. However, only a few immortalized muscle cell lines with DMD mutations are available. In addition, obtaining muscle cells from patients also requires an invasive muscle biopsy. Mostly DMD variants are rare, making it challenging to identify a patient with a particular mutation for a muscle biopsy. To overcome these challenges and generate myoblast cultures, we optimized a CRISPR/Cas9 gene editing approach to model the most common DMD mutations that include approximately 28.2% of patients. GAP-PCR and sequencing results show the ability of the CRISPR-Cas9 system to efficient deletion of mentioned exons. We showed producing truncated transcript due to the targeted deletion by RT-PCR and sequencing. Finally, mutation-induced disruption of dystrophin protein expression was confirmed by western blotting. All together, we successfully created four immortalized DMD muscle cell lines and showed the efficacy of the CRISPR-Cas9 system for the generation of immortalized DMD cell models with the targeted deletions., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Major histocompatibility complex class II (MHC-II) deficiency is a rare inborn error of immunity (IEI). Impaired antigen presentation to CD4 + T cells results in combined immunodeficiency (CID). Patients typically present with severe respiratory and gastrointestinal tract infections at early ages. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy., Methods: We describe the clinical, immunologic, and genetic features of eighteen unrelated Iranian patients with MHC-II deficiency., Results: Consanguinity was present in all affected families. The median age at the initial presentation was 5.5 months (range 7 days to 18 years). The main symptoms included failure to thrive, persistent diarrhea, and pneumonia. Autoimmune and neurologic features were also documented in about one-third of the patients, respectively. Thirteen patients carried RFXANK gene mutations, two carried RFX5 gene mutations, and three carried a RFXAP gene mutation. Six patients shared the same RFXANK founder mutation (c.162delG); limited to the Iranian population and dated to approximately 1296 years ago. Four of the patients underwent HSCT; three of them are alive. On the other hand, nine of the fourteen patients who did not undergo HSCT had a poor prognosis and died., Conclusion: MHC-II deficiency is not rare in Iran, with a high rate of consanguinity. It should be considered in the differential diagnosis of CID at any age. With the limited access to HSCT and its variable results in MHC-II deficiency, implementing genetic counseling and family planning for the affected families are mandatory. We are better determined to study the c.162delG RFXANK heterozygous mutation frequency in the Iranian population., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Acetyl-L-carnitine has been shown to exert neuroprotection against neurodegenerative diseases. The present study was performed to evaluate neuroprotection effects of acetyl-L-carnitine against lipopolysaccharide (LPS) -induced neuroinflammation and clarify possible mechanisms. A single dose (500 µg/kg) of LPS was intraperitoneally injected to rats to induce model. The animals were intraperitoneally treated with different doses of acetyl-L-carnitine (30, 60, and 100) for 6 days. Y-maze task, single-trial passive avoidance and novel object recognition tests were used to evaluate memory impairments. ELISA assay was used to evaluate the expression of TLR4/NFκB, autophagic and oxidative stress markers. Our result showed that intraperitoneal injection of LPS resulted in initiation of neuroinflammation by activation of TLR4/NFκB, suppression of autophagic markers such as LC3 II/ LC3 I ratio and becline-1, and excessive production of ROS and MDA. Intraperitoneal administration of acetyl-L-carnitine contributed to neuroprotection against LPS -induced neuroinflammation by suppression of TLR4/NFκB pathway, restoring activity of autophagy and inhibition of oxidative stress. Collectively, our findings show that acetyl-L-carnitine attenuated LPS-induced neuroinflammation by targeting TLR4/NFκB pathway, autophagy and oxidative stress., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Insulin-like growth factor-binding protein acid-labile subunit (IGFALS) encodes a protein which binds to IGF1 and IGFBP-3 to regulate the growth, differentiation, and other physiological processes. The aim of this study was the identification of allelic polymorphisms of the IGFALS gene using the PCR-RFLP technique and evaluation of their association with growth traits. For this end, 120 blood samples were randomly collected from each breed. Following amplification of an 1113-bp fragment of exon 1 and a part of intron 1 of the IGFLAS gene, genotyping was conducted by three restriction enzymes including HinfI, MscI, and PvuII. The results showed that only one allele was observed in IGFALS-PvuII site, while in IGFLAS-MscI site, three AA, AB, and BB genotypes with the frequencies of 17.5%, 32%, and 50.5% and 11%, 37.5%, and 51.5% were observed in Makouei and Ghezel sheep breeds, respectively. Additionally, in the IGFLAS-HinfI site, two AB and BB genotypes with the frequency of 34.2% and 65.8% were observed in Makouei sheep and AA, AB, and BB genotypes with the frequency of 9%, 21%, and 70% were observed in Ghezel sheep. So that, Makouei sheep with AB genotype had more chest girth (CG) compared with other genotypes. Furthermore, a significant association was observed between the genotypes of IGFLAS-HinfI with birth weight (BW) in Ghezel and BW, weaning weight (BW3), and CG in Makouei sheep. Haplotype analysis revealed an association between paternal haplotypes and BW in both Ghezel and Makouei breeds. So that, AAB and ABB haplotypes showed more BW than others in Makouei and Ghezel sheep, respectively.

Objectives: In clinical practice, internal urethrotomy is an easy procedure and is offered as a first modality for treatment of short urethral strictures. Internal urethrotomy refers to any procedure that opens the stricture by incising or ablating it transurethrally. The most common complication of internal urethrotomy is stricture recurrence. The curative success rate of internal urethrotomy is approximately 20%. Triamcinolone has antifibroblast and anticollagen properties. This study evaluated the efficacy of triamcinolone in the prevention of anterior urethral stricture recurrence after internal urethrotomy., Methods: Fifty male patients with anterior urethral stricture were randomized to undergo internal urethrotomy with or without urethral submucosal injection of triamcinolone. Using general anesthesia urethrotomy was performed. Triamcinolone (40 mg) was injected submucosally at the urethrotomy site in 25 patients. The patients were followed for at least 12 months and the stricture recurrence rate was compared between the two groups., Results: 23 patients in the triamcinolone group and 22 in the control group completed the study. There were no significant differences in the baseline characteristics of the patients or the etiology of the stricture between the two groups. Mean follow-up time was 13.7 ± 5.5 months (range: 1-25 months). Urethral stricture recurred in five patients (21.7%) in the triamcinolone group and in 11 patients (50%) in the control group (P = 0.04)., Conclusions: Injection of triamcinolone significantly reduced stricture recurrence after internal urethrotomy. Further investigations are warranted to confirm its efficacy and safety.