A novel 355-357delGAG mutation and frequency of connexin-26 (GJB2) mutations in Iranian patients

The common form of autosomal recessive non-syndromic deafness is caused by the mutation in gap junction beta 2 (GJB2) gene (GenBank M86849, OMIM# 121011) which is located at the DFNB1 locus at 13q11. GJB2 is a small gene about 5500-bp length with two exons, of which only one contains the coding region (Kelley et al. 2000). The sequence of the coding region consists of 681 bp, encoding a gap-junction protein with 226 amino acids (Schrijver 2004). The genetics of hearing loss is highly heterogeneous and more than 100 mutations in connexin 26 (GJB2) genes are reported to be responsible for 30%–40% of hereditary hearing loss in deaf subjects (Ballana et al. 2001; Schrijver 2004). The most frequent mutation 35delG has been detected in different populations; especially in European countries where it is established to be due to founder effect (Van Laer et al. 2001; Rothrock et al. 2003). In this study, we performed mutation screening in 33 families who met clinical criteria of non-syndromic hereditary hearing loss (NSHHL) to evaluate the type and frequency of GJB2 mutations in Iranian population

The Influence of Exposure to Stress of Pregnant Rats on the Adrenal Gland Structure of their Offspring. An Unbiased Stereological Study

Many factors may interact with normal differentiation and growth of tissues and cells. Stress might be  experienced during pregnancy and it has been shown that stress may cause low birth weight. The effect of  prenatal manipulations on the HPA axis has been focused on physiological and biochemical alteration of  the adrenal gland. A stereological examination of the influences of prenatal stress on the structure of the  developing adrenal gland of one day and 21 day-old rats has now been performed. In this study experiments were conducted to test the hypothesis that exposure to restraint stress during pregnancy  in rat results in structural changes in the developing adrenal gland of their pups. Female rats were exposed to restraint stress from the first day of pregnancy throughout gestation. Male offspring  of stressed rats (PS= experimental) and of unstressed mothers (C= control) who were one day and 21  days of age were selected. Their body weight (BW), crown-rump length (CRL), biparietal diameter (BPD),  volume of the gland and the cortical layers and medulla were estimated using stereological methods. The results showed that the prenatal stress led to a decrease in BW, but CRL and BPD remained unchanged.  Also, a significant increase in volume of the adrenal gland and cortical layers in one day and 21 day-old  offspring were observed. The volume of the medulla of the adrenal gland of neonate rats remained  unchanged but the volume of the medulla in 21 day-old rats was decreased. Therefore, it can be concluded that prenatal stress alters the structure of the developing adrenal gland.

Promoter Hypermethylation of Estrogen Receptor Alpha Gene Is Correlated to Estrogen Receptor Negativity in Iranian Patients with Sporadic Breast Cancer

Objective: Breast Cancer is the most common cancer in Iranian women. Breast tumors are classified based on the estrogen receptor alpha (ERα) expression status into ER negative and ER positive tumors. ER negative tumors tend to have worse prognosis and less likely to respond to endocrine therapy. Aberrant methylation of gene promoter is one of the mechanisms for gene silencing in breast tumors. Because of its reversible nature, promoter methylation is a good target for new therapeutic strategies. We aimed to evaluate the frequency of this epigenetic event in ERα gene and its association to clinicopathological features in Iranian breast cancer patients.Materials and Methods: In this case control study the patient series consisted of 100 sporadic primary breast cancer cases (51 ER negative and 49 ER positive tumors). None of the participants had chemo or radiotherapy before surgery. In breast tumors ERα promoter methylation were assessed with methylation specific polymerase chain reaction (MSP). Data was collected on clinicopathological features of the patients. Correlation between ERα methylation and clinicopathological characteristics of the patients was investigated by Pearson Chi-Square and Fisher’s exact test.Results: ERα methylation was detected in 98% of ER negative and 65% of ER positive breast tumors. A strong correlation was found between ERα methylation and ER negativity in tumors (p<0.0001). Also, ERα methylation has associated to progesterone receptor negativity (p<0.008) and double receptor negative status (p<0.0001) in breast tumors.Conclusion: ERα methylation occurs with high frequency in the breast tumors of Iranian breast cancer patients and may play a considerable role in pathogenesis of ERα negative tumors as a poor prognosis and more aggressive category. The reversible nature of DNA methylation may provide new therapeutic possibilities in ER negative breast tumors

Molecular Analysis of HS-111 and 3`HS1 Variations in β-Thalassemia Intermedia Patients with High Levels of HbF

Objective: To study the possible association between high levels of fetal haemoglobin(HbF) in β-thalassemia intermedia patients and HS-111 and 3`HS1 sequence variations.Materials and Methods: In this study, the 3' HS-1 and HS-111 regions of 30 ß-thalassaemiaintermedia patients (ß°/ß°) with high levels of HbF, 21 ß-thalassemia major patientsand 40 normal Iranian individuals were analyzed by single-strand conformation polymorphism(SSCP) and polymerase chain reaction (PCR) sequencing.Results: Two nucleotide variations in 3' HS111 (-21A>G) and 3`HS1 (179C>T) wereidentified. The most frequent sequence variation was 3' HS111 (-21A) in the intermediapatients and 3`HS111 (-21G) in the major thalassemia patients. In contrast to the 3`HS1marker, both 3'HS111 A and G variants showed a correlation with each studied group.Conclusion: The HS111 marker in conjunction with other parameters could be used asappropriate genetic markers to discriminate β-thalassemia intermedia patients (β°/β°) withhigh levels of HbF from β-thalassemia major patients

Expression analysis data of BCL11A and γ-globin genes in KU812 and KG-1 cell lines after CRISPR/Cas9-mediated BCL11A enhancer deletion

The data presented in this article are related to the research article entitled as "Targeted deletion of the BCL11A gene by CRISPR-Cas9 system for fetal hemoglobin reactivation: A promising approach for gene therapy of beta-thalassemia disease " [1]. BCL11A is a master regulator of γ-globin gene silencing, and suppresses fetal hemoglobin expression by association with other γ-globin suppressors, and also interacts with human beta-globin locus control region as well as intergenic region between the Aγ and δ-globin genes to reconfigure beta-globin cluster. Thus, HbF reactivation has been proposed to be an approach for the treatment of β-thalassemia through knockout of BCL11A. Accordingly, an erythroid enhancer sequence was identified that, when inactivated, led to repression of BCL11A and induction of γ-globin in the erythroid lineage [2-7]. This article describes data that obtained from BCL11A gene enhancer modification in KU812 and KG-1 cell lines using the CRISPR-Cas9 genome editing system in order to reactivate γ-globin gene expression

The Efficacy of a Protocolized Nursing Care on Nasal Skin Breakdown in Preterm Neonates Receiving Nasal Continuous Positive Airway Pressure

Background: Nasal continuous positive airway pressure (NCPAP) is an effective method of ventilation in newborns with respiratory distress syndrome (RDS). Using an appropriate nasal skin care protocol is identical to prevention or reduction of nasal skin breakdown in those who receive NCPAP. This study aimed to investigate the effectiveness of an evidence-based clinical care protocol on nasal skin integrity in preterm newborns who receive NCPAP.Materials and Methods: A Randomized Controlled Trial was used to conduct the study. A cohort of 110 preterm newborns with a gestational age (GA) of 25 to 36 weeks, receiving nasal continuous positive airway pressure (NCPAP) for RDS in the neonatal intensive care unit of a university teaching hospital were selected to perform the study. They were randomly assigned to a protocolized nasal skin care (group A) or to a group receiving the routine care (group B). Nasal skin integrity of the preterm neonates, were measured on a daily basis for 10 days using the Neonatal Skin Condition Scale (NSCS) 24 hours after placement of NCPAPs in both groups.Results: Each intervention and control group included 55 neonates. 65.50% of neonates in the control group and 47.30% of neonates in the intervention group were male. Repeated measures analysis showed thatNSCS scores were significantly lower in intervention group receiving nasal skin care in accordance with the protocol than the control group receiving the routine nasal skin care (P=0.000). Conclusion: In this study, the protocolized care reduced nasal skin breakdown in the preterm newborns receiving NCPAP. Therefore, it can be used as an effective method in nasal skin care in neonates who are treated by NCPAP

Structural Insight into Anaphase Promoting Complex 3 Structure and Docking with a Natural Inhibitory Compound

Background: Anaphase promoting complex (APC) is the biggest Cullin-RING E3 ligase and is very important in cell cycle control; many anti-cancer agents target this. APC controls the onset of chromosome separation and mitotic exit through securin and cyclin B degradation, respectively. Its APC3 subunit identifies the APC activators-Cdh1 and Cdc20. Materials and Methods: The structural model of the APC3 subunit of APC was developed by means of computational techniques; the binding of a natural inhibitory compound to APC3 was also investigated. Results: It was found that APC3 structure consists of numerous helices organized in anti-parallel and the overall model is superhelical of tetratrico-peptide repeat (TPR) domains. Furthermore, binding pocket of the natural inhibitory compound as APC3 inhibitor was shown. Conclusion: The findings are beneficial to understand the mechanism of the APC activation and design inhibitory compounds

Improvement of K562 Cell Line Transduction by FBS Mediated Attachment to the Cell Culture Plate

Lentiviral vectors have been used for gene therapy in the clinical phase in recent years. These vectors provide a tool for gene insertion, deletion, or modification in organisms. The K562 human cell line has been used extensively in hematopoietic research. Despite its broad application, it is hard-to-transfection and transduction. So, this study presents a simple method to increase the transduction efficiency of K562 cells with a low multiplicity of infection (MOI) of the virus particle. For this purpose, 24-well plate was coated by 300 μl fetal bovine serum (FBS) before seeding. Then 2×104 K562 cells were seeded in each FBS coated plate. After 24h, K562 cells were attached and doubled. Different amount of lentivirus-based GFP vector according to MOI (5, 10, 15, and 20) along with 8 μg polybrene was added to the attached K562 cells and after 6h cells and viral particle complex were spinfected. Then cells were returned to the plate and incubated in 37°C overnight. After 48h transduction efficiency was established by measuring the GFP-expressing cells by flow cytometry. Flow cytometry analysis showed that, after plate treatment by FBS, 64.5% transduction rate in K562 cells was achieved at MOI=20. Therefore, this method can be an effective and simple way to increase the lentiviral transduction rate for suspended cells such as K562

Rational design of DKK3 structure-based small peptides as antagonists of Wnt signaling pathway and in silico evaluation of their efficiency.

Dysregulated Wnt signaling pathway is highly associated with the pathogenesis of several human cancers. Dickkopf proteins (DKKs) are thought to inhibit Wnt signaling pathway through binding to lipoprotein receptor-related protein (LRP) 5/6. In this study, based on the 3-dimensional (3D) structure of DKK3 Cys-rich domain 2 (CRD2), we have designed and developed several peptide inhibitors of Wnt signaling pathway. Modeller 9.15 package was used to predict 3D structure of CRD2 based on the Homology modeling (HM) protocol. After refinement and minimization with GalaxyRefine and NOMAD-REF servers, the quality of selected models was evaluated utilizing VADAR, SAVES and ProSA servers. Molecular docking studies as well as literature-based information revealed two distinct boxes located at CRD2 which are actively involved in the DKK3-LRP5/6 interaction. A peptide library was constructed conducting the backrub sequence tolerance scanning protocol in Rosetta3.5 according to the DKK3-LRP5/6 binding sites. Seven tolerated peptides were chosen and their binding affinity and stability were improved by some logical amino acid substitutions. Molecular dynamics (MD) simulations of peptide-LRP5/6 complexes were carried out using GROMACS package. After evaluation of binding free energies, stability, electrostatic potential and some physicochemical properties utilizing computational approaches, three peptides (PEP-I1, PEP-I3 and PEP-II2) demonstrated desirable features. However, all seven improved peptides could sufficiently block the Wnt-binding site of LRP6 in silico. In conclusion, we have designed and improved several small peptides based on the LRP6-binding site of CRD2 of DKK3. These peptides are highly capable of binding to LRP6 in silico, and may prevent the formation of active Wnt-LRP6-Fz complex