In-Utero exposure to potential sources of indoor air pollution and umbilical cord blood leukocyte telomere length.

Indoor air pollution (IAP) has been associated with various adverse health effects. However, the evidence regarding such an association with leukocyte telomere length (LTL) in cord blood samples is still scarce. Therefore, the present study aimed to assess the relationship between exposure to indicators of IAP and LTL in umbilical cord blood samples. This cross-sectional study was based on 188 mother-newborn pairs who participated in our study between 2020 and 2022 in Isfahan, Iran. Umbilical LTL was measured by quantitative real-time polymerase chain reaction (qRT-PCR) technique. Linear mixed-effect models were used to assess the relationship between IAP indicators and umbilical LTL, adjusted for relevant covariates. The median (interquartile range (IQR)) of umbilical LTL was 0.92 (0.47). In fully adjusted models, frequency of using degreasing spray during pregnancy (times per month) (ß = -0.047, 95% CI:0.09, -0.05, P-value = 0.02), using air freshener spray during pregnancy (ß = -0.26, 95% CI: -0.5, -0.02, P-value = 0.03) and frequency of using insecticides during pregnancy (times per month) (ß = -0.025, 95% CI: -0.047, -0.003, P-value = 0.02) were significantly associated with shorter umbilical LTL. There was a positive significant relationship between the frequency of using cleaning spray during pregnancy (times per month) with umbilical LTL (ß = 0.019, 95% CI: 0.005, 0.033, P-value = 0.01). Furthermore, the direct connection of the parking with home and the frequency of using barbecue (times per week) were marginally associated with shorter umbilical LTL. For other indicators of IAP, we did not observe any statistically significant associations. Overall, this study suggested a negative association between prenatal exposure to IAP during pregnancy and umbilical LTL.

Unmasking the complex roles of hypocalcemia in cancer, COVID-19, and sepsis: Engineered nanodelivery and diagnosis.

Calcium (Ca2+) homeostasis is essential for maintaining physiological processes in the body. Disruptions in Ca2+ signaling can lead to various pathological conditions including inflammation, fibrosis, impaired immune function, and accelerated senescence. Hypocalcemia, a common symptom in diseases such as acute respiratory distress syndrome (ARDS), cancer, septic shock, and COVID-19, can have both potential protective and detrimental effects. This article explores the multifaceted role of Ca2+ dysregulation in inflammation, fibrosis, impaired immune function, and accelerated senescence, contributing to disease severity. Targeting Ca2+ signaling pathways may provide opportunities to develop novel therapeutics for age-related diseases and combat viral infections. However, the role of Ca2+ in viral infections is complex, and evidence suggests that hypocalcemia may have a protective effect against certain viruses, while changes in Ca2+ homeostasis can influence susceptibility to viral infections. The effectiveness and safety of Ca2+ supplements in COVID-19 patients remain a subject of ongoing research and debate. Further investigations are needed to understand the intricate interplay between Ca2+ signaling and disease pathogenesis.

Unraveling the potential of M13 phages in biomedicine: Advancing drug nanodelivery and gene therapy.

M13 phages possessing filamentous phage genomes offer the benefits of selective display of molecular moieties and delivery of therapeutic agent payloads with a tolerable safety profile. M13 phage-displayed technology for resembling antigen portions led to the discovery of mimetic epitopes that applied to antibody-based therapy and could be useful in the design of anticancer vaccines. To date, the excremental experiences have engaged the M13 phage in the development of innovative biosensors for detecting biospecies, biomolecules, and human cells with an acceptable limit of detection. Addressing the emergence of antibiotic-resistant bacteria, M13 phages are potent for packaging the programmed gene editing tools, such as CRISPR/Cas, to target multiple antimicrobial genes. Moreover, their display potential in combination with nanoparticles inspires new approaches for engineering targeted theragnostic platforms targeting multiple cellular biomarkers in vivo. In this review, we present the available data on optimizing the use of bacteriophages with a focus on the to date experiences with M13 phages, either as monoagent or as part of combination regimens in the practices of biosensors, vaccines, bactericidal, modeling of specific antigen epitopes, and phage-guided nanoparticles for drug delivery systems. Despite increasing research interest, a deep understanding of the underlying biological and genetic behaviors of M13 phages is needed to enable the full potential of these bioagents in biomedicine, as discussed here. We also discuss some of the challenges that have thus far limited the development and practical marketing of M13 phages.

The potential therapeutic impacts of trehalose on cardiovascular diseases as the environmental-influenced disorders: An overview of contemporary findings.

Cardiovascular diseases (CVDs) as environmental-influenced disorders, are a major concern and the leading cause of death worldwide. A range of therapeutic approaches has been proposed, including conventional and novel methods. Natural compounds offer a promising alternative for CVD treatment due to their ability to regulate molecular pathways with minimal adverse effects. Trehalose is natural compound and disaccharide with unique biological functions and cardio-protective properties. The cardio-protective effects of trehalose are generated through its ability to induce autophagy, which is mediated by the transcription factors TFEB and FOXO1. The stimulation of TFEB plays a significant role in regulating autophagy genes and autophagosome formation. Activation of FOXO1 through dephosphorylation of Foxo1 and blocking of p38 mitogen-activated protein kinase (p38 MAPK) also triggers autophagy dramatically. Trehalose has been shown to reduce CVD risk factors, including atherosclerosis, cardiac remodeling after a heart attack, cardiac dysfunction, high blood pressure, and stroke. It also reduces structural abnormalities of mitochondria, cytokine production, vascular inflammation, cardiomyocyte apoptosis, and pyroptosis. This review provides a molecular overview of trehalose's cardioprotective functions, including its mechanisms of autophagy and its potential to improve CVD symptoms based on clinical evidence.

Pathological Mechanisms Induced by TRPM2 Ion Channels Activation in Renal Ischemia-Reperfusion Injury.

Renal ischemia-reperfusion (IR) injury triggers a cascade of signaling reactions involving an increase in Ca2 + charge and reactive oxygen species (ROS) levels resulting in necrosis, inflammation, apoptosis, and subsequently acute kidney injury (AKI).Transient receptor potential (TRP) channels include an essential class of Ca2+ permeable cation channels, which are segregated into six main channels: the canonical channel (TRPC), the vanilloid-related channel (TRPV), the melastatin-related channel (TRPM), the ankyrin-related channel (TRPA), the mucolipin-related channel (TRPML) and polycystin-related channel (TRPP) or polycystic kidney disease protein (PKD2). TRP channels are involved in adjusting vascular tone, vascular permeability, cell volume, proliferation, secretion, angiogenesis and apoptosis.TRPM channels include eight isoforms (TRPM1-TRPM8) and TRPM2 is the second member of this subfamily that has been expressed in various tissues and organs such as the brain, heart, kidney and lung. Renal TRPM2 channels have an important role in renal IR damage. So that TRPM2 deficient mice are resistant to renal IR injury. TRPM2 channels are triggered by several chemicals including hydrogen peroxide, Ca2+, and cyclic adenosine diphosphate (ADP) ribose (cADPR) that are generated during AKI caused by IR injury, as well as being implicated in cell death caused by oxidative stress, inflammation, and apoptosis.

Vitamin D can be effective on the prevention of COVID-19 complications: A narrative review on molecular aspects.

The widespread COVID-19 pandemic has been, currently, converted to a catastrophic human health challenge. Vitamin D (VD) and its metabolites have been used as a palliative treatment for chronic inflammatory and infectious diseases from ancient times. In the current study, some molecular aspects of the potential effects of VD against COVID-19 side-effects have been discussed. An arguable role in autophagy or apoptosis control has been suggested for VD through calcium signaling at the mitochondrial and ER levels. 1,25(OH)2D3 is also an immunomodulator that affects the development of B-cells, T-cells, and NK cells in both innate and acquired immunity. The production of some anti-microbial molecules such as defensins and cathelicidins is also stimulated by VD. The overexpression of glutathione, glutathione peroxidase, and superoxide dismutase, and down-regulation of NADPH oxidase are induced by VD to reduce the oxidative stress. Moreover, the multi-organ failure due to a cytokine storm induced by SARS-CoV2 in COVID-19 may be prevented by the immunomodulatory effects of VD. It can also downregulate the renin-angiotensin system which has a protective role against cardiovascular complications induced by COVID-19. Given the many experimental and molecular evidences due to the potential protective effects of VD on the prevention of the COVID-19-induced morbidities, a VD supplementation is suggested to prevent the lethal side-effects of the infection. It is particularly recommended in VD-deficient patients or those at greater risk of serious or critical effects of COVID-19, including the elderly, and patients with pre-existing chronic diseases, especially those in nursing homes, care facilities, and hospitals.

The impact of neutrophil extracellular trap from patients with systemic lupus erythematosus on the viability, CD11b expression and oxidative burst of healthy neutrophils.

BACKGROUND: NET (neutrophil extracellular trap) has been shown to directly influence inflammation; in SLE (systemic lupus erythematosus), it is reportedly a plausible cause for the broken self-tolerance that contributes to this pathology. Meanwhile, the role of NET is not easily explicable, and there is a serious discrepancy in the role of NET in SLE pathology and generally inflammation; in particular, the interactions of neutrophils with NET have been rarely inspected. This study evaluates the effect of NET on neutrophils in the context of SLE. The neutrophils were incubated by the collected NET (from SLE patients and healthy controls) and their expression of an activation marker, viability and oxidative burst ability were measured. RESULTS: The level of cell mortality, CD11b expression and the oxidative burst capacity were elevated in NET-treated neutrophils. Also, the elevation caused by the SLE NET was higher than that produced by the healthy NET. CONCLUSION: The decreased neutrophil viability was not due to the increase in apoptosis; rather, it was because of the augmentation of other inflammatory cell-death modes. The upregulation of CD11b implies that NET causes neutrophils to more actively contribute to inflammation. The increased oxidative burst capacity of neutrophils can play a double role in inflammation. Overall, the effects induced by NET on neutrophils help prolong inflammation; accordingly, the NET collected from SLE patients is stronger than the NET from healthy individuals.

Generation of HBsAg DNA aptamer using modified cell-based SELEX strategy.

Aptamers as potential alternatives for antibodies could be employed against hepatitis B surface antigen (HBsAg), the great hallmark and first serological marker in HBV, for further theragnostic applications. Therefore, isolation HBsAg specific aptamer was performed in this study with a modified Cell-SELEX method. HEK293T overexpressing HBsAg and HEK293T as target and control cells respectively, were incubated with single-stranded rounds of DNA library during six SELEX and Counter SELEX rounds. Here, we introduced the new modified Cell-SELEX using deoxyribonuclease I digestion to separate single stranded DNA aptamers against the HBsAg. Characterization and evaluation of selected sequences were performed using flow cytometry analysis. The results led to isolation of 15 different ssDNA clones in six rounds of selection which were categorized to four clusters based on common structural motifs. The evaluation of SELEX progress showed growth in aptamer affinity with increasing in the cycle number. Taken together, the application of modified cell-SELEX demonstrated the isolation of HBsAg-specific ssDNA aptamers with proper affinity. Modified cell-SELEX as an efficient method can shorten the selection procedure and increase the success rate while the benefits of cell-based SELEX will be retained. Selected aptamers could be applied in purification columns, diagnostic kits, and drug delivery system against HBV-related liver cancer.

The Molecular Basis of COVID-19 Pathogenesis, Conventional and Nanomedicine Therapy.

In late 2019, a new member of the Coronaviridae family, officially designated as "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), emerged and spread rapidly. The Coronavirus Disease-19 (COVID-19) outbreak was accompanied by a high rate of morbidity and mortality worldwide and was declared a pandemic by the World Health Organization in March 2020. Within the Coronaviridae family, SARS-CoV-2 is considered to be the third most highly pathogenic virus that infects humans, following the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV). Four major mechanisms are thought to be involved in COVID-19 pathogenesis, including the activation of the renin-angiotensin system (RAS) signaling pathway, oxidative stress and cell death, cytokine storm, and endothelial dysfunction. Following virus entry and RAS activation, acute respiratory distress syndrome develops with an oxidative/nitrosative burst. The DNA damage induced by oxidative stress activates poly ADP-ribose polymerase-1 (PARP-1), viral macrodomain of non-structural protein 3, poly (ADP-ribose) glycohydrolase (PARG), and transient receptor potential melastatin type 2 (TRPM2) channel in a sequential manner which results in cell apoptosis or necrosis. In this review, blockers of angiotensin II receptor and/or PARP, PARG, and TRPM2, including vitamin D3, trehalose, tannins, flufenamic and mefenamic acid, and losartan, have been investigated for inhibiting RAS activation and quenching oxidative burst. Moreover, the application of organic and inorganic nanoparticles, including liposomes, dendrimers, quantum dots, and iron oxides, as therapeutic agents for SARS-CoV-2 were fully reviewed. In the present review, the clinical manifestations of COVID-19 are explained by focusing on molecular mechanisms. Potential therapeutic targets, including the RAS signaling pathway, PARP, PARG, and TRPM2, are also discussed in depth.

Tracking antibiotic resistance genes and class 1 integrons in Escherichia coli isolates from wastewater and agricultural fields.

Considering high concentrations of multidrug-resistant bacteria and antibiotic resistance genes (ARGs) in wastewater, agricultural reuse of treated wastewater may be a public health threat due to ARG dissemination in different environmental compartments, including soil and edible parts of crops. We investigated the presence of antibiotic-resistant Escherichia coli as an indicator bacterium from secondary treated wastewater (STWW), water- or wastewater-irrigated soil and crop samples. ARGs including blaCTX-m-32, blaOXA-23, tet-W, sul1, cml-A, erm-B, along with intI1 gene in E. coli isolates were detected via molecular methods. The most prevalent ARGs in 78 E. coli isolates were sul1 (42%), followed by blaCTX-m-32 (19%), and erm-B (17%). IntI1 as a class 1 integrons gene was detected in 46% of the isolates. Cml-A was detected in STWW isolates but no E. coli isolate from wastewater-irrigated soil and crop samples contained this gene. The results also showed no detection of E. coli in water-irrigated soil and crop samples. Statistical analysis showed a correlation between sul1 and cml-A with intI1. The results suggest that agricultural reuse of wastewater may contribute to the transmission of antibiotic-resistant bacteria to soil and crop. Further research is needed to determine the potential risk of ARB associated with the consumption of wastewater-irrigated crops.

Expression and clinicopathological significance of lipin-1 in human breast cancer and its association with p53 tumor suppressor gene.

Breast cancer (BC) is an important cause of female cancer-related death. It has recently been demonstrated that metabolic disorders including lipid metabolism are a hallmark of cancer cells. Lipin-1 is an enzyme that displays phosphatidate phosphatase activity and regulates the rate-limiting step in the pathway of triglycerides and phospholipids synthesis. The objective of this study was to evaluate lipin-1 expression, its prognostic significance, and its correlation with p53 tumor suppressor in patients with BC. In this study, 55 pairs of fresh samples of BC and adjacent noncancerous tissue were used to analyze lipin-1, using quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) staining. The expression of other clinicopathological variables and p53 was also examined using IHC technique. The cell migration was studied in MCF-7 and MDA-MB231 cells following the inhibition of lipin-1 by propranolol. Our results show that the relative expression of lipin-1 messenger RNA was significantly higher in BC tissues compared with the adjacent normal tissue and its inhibition reduced cell migration in cancer cells. This upregulation was negatively correlated with histological grade of tumor and p53 status (p = .001 and p = .034) respectively and positively correlated with the tumor size (p = .006). Our results also seem to indicate that the high lipin-1 expression is related to a good prognosis in patients with BC. The expression of lipin-1 may be considered as a novel independent prognostic factor. The inhibition of lipin-1 may also have therapeutic significance for patients with BC. The correlation between lipin-1 and p53 confirms the role of p53 in the regulation of lipid metabolism in cancer cells.

Bioinformatics prediction and experimental validation of a novel microRNA: hsa-miR-B43 within human CDH4 gene with a potential metastasis-related function in breast cancer.

As a class of short noncoding RNAs, microRNAs (miRNAs) play a key role in the modulation of gene expression. Although, the regulatory roles of currently identified miRNAs in various cancer types including breast cancer have been well documented, there are many as yet undiscovered miRNAs. The aim of the current study was to bioinformatically reanalyze a list of 189 potentially new miRNAs introduced in a previously published paper (PMID: 21346806) and experimentally explore the existence and function of a candidate one: hsa-miR-B43 in breast cancer cells. The sequences of 189 potential miRNAs were re-checked in the miRbase database. Genomic location and conservation of them were assessed with the University of California Santa Cruz (UCSC) genome browser. SSC profiler, RNAfold, miRNAFold, MiPred, and FOMmiR bioinformatics tools were furthermore utilized to explore potential hairpin structures and differentiate real miRNA precursors from pseudo ones. hsa-miR-B43 was finally selected as one of the best candidates for laboratory verification. The expression and function of hsa-miR-B43 were examined by real-time polymerase chain reaction, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and wound-healing assays. DIANA-microT, RNAhybrid and Enrichr tools were used to predict the miRNA target genes and for further enrichment analysis. We could detect the exogenous and endogenous expression of hsa-miR-B43, as a real novel miRNA, in cancer cell lines. Gene Ontology enrichment, pathway analysis and wound-healing assay results furthermore confirmed that a metastasis-related function may be assigned to hsa-miR-B43. Our results introduced hsa-miR-B43, as a novel functional miRNA, which might play a role in the metastatic process. Further studies will be necessary to completely survey the existence and function of hsa-miR-B43 in other cancer types.

The role of Bax in the apoptosis of Leishmania-infected macrophages.

BACKGROUND: Leishmania is a protozoan parasite that nests in macrophages and is responsible for the Leishmaniasis disease. In spite of different defense pathways, last strategy of macrophage for killing parasite is apoptosis process. By permeableizing the mitochondrial outer membrane (MOM). As breaching MOM releases apoptogenic factors like cytochrome-c which activate caspases that result in the destruction of the cell. In this review, we summarized the appropriate manuscripts regarding the bax includes, its different types and the effect of bax on the apoptosis of Leishmania and parasite-infected macrophages. METHODS: Information about the role of BAX in the apoptosis of parasite-infected macrophage of recent articles were surveyed by searching computerized bibliographic database PubMed and Google Scholar entering the keywords BAX and leishmaniasis. RESULTS: The common studies revealed Leishmania use different survival strategies for inhibiting macrophage apoptosis. As Leishmania by preventing homooligomerization or upregulating the anti-apoptotic molecule Bcl-2 can prohibits proteins of host-cell apoptosis such as Bax that is required for mitochondrial permeabilisation during apoptosis. CONCLUSION: With regard to the supportive role of bax in apoptosis and the preventive role of Leishmania in its function, it seems that expression of bax gene in parasite by technologies like transgenic or down regulating of anti-apoptotic molecule Bcl-2 by miRNA could be prompted the apoptosis process of infected-macrophages and inhibited extensive spread of Leishmania and the resulting lesions.

Evaluation of transgenic Leishmania infantum expressing mLLO-BAX-SMAC in the apoptosis of the infected macrophages in vitro and in vivo.

BACKGROUND: Leishmaniasis is an important infectious disease that develops because of escaping parasite from the host immune system or preventing host macrophages apoptosis. Recently, the development of transgenic methods and the manipulation of the parasite genome has provided many advantages. So, in this study, the effect of the transgenic Leishmania infantum expressing mLLO-BAX-SMAC proteins was examined in accelerating host cell apoptosis. METHOD: The entire coding sequence of designed codon-optimized mLLO-Bax-Smac was cloned in the pLexyNeo2 vector and integrated downstream of the 18srRNA locus of L infantum genome by homologous recombination. Next, the expression of mLLO-BAX-SMAC fusion protein was evaluated by the Western blotting technique and the pathogenesis of transgenic parasite was surveyed in vitro and in vivo. RESULTS: The results of PCR and Western blot confirmed proper integration and expression of mLLO-Bax-Smac sequence into the 18srRNA locus of L infantum. Flow cytometry showed accelerating apoptosis of transgenic Leishmania-infected macrophages compared to wild-type parasite. Also, transgenic parasites were less virulent as a fewer parasitic burden was found in the spleen and liver of transgenic-infected mice compared to the control. CONCLUSION: The data suggested that the transgenic L infantum expressing BAX-SMAC can be used as an experimental model for developing vaccination against leishmaniasis.

Single-Strand DNA-Like Oligonucleotide Aptamer Against Proprotein Convertase Subtilisin/Kexin 9 Using CE-SELEX: PCSK9 Targeting Selection.

BACKGROUND: Proprotein convertase subtilisin/kexin 9 (PCSK9) serves a key regulatory function in the metabolism of low-density lipoprotein (LDL)-cholesterol (LDL-C) through interaction with the LDL receptor (LDLR) followed by its destruction that results in the elevation of the plasma levels of LDL-C. The aims of the present study were to separate and select a number of single-stranded DNA (ssDNA) aptamers against PCSK9 from a library pool (n > 1012) followed by their characterization. METHODS: The aptamers obtained from the DNA-PCSK9 complexes which presented the highest affinity against PCSK9 were separated and selected using capillary electrophoresis evolution of ligands by exponential enrichment (CE-SELEX). The selected aptamers were amplified and cloned into a T/A vector. The plasmids from the positive clones were extracted and sequenced. The Mfold web server was used to predict the secondary structure of the aptamers. RESULTS: Following three rounds of CE-SELEX, the identified anti-PCSK9 ssDNA aptamers, namely aptamer 1 (AP-1) and aptamer 2 (AP-2), presented half maximal inhibitory concentrations of 325 and 327 nM, lowest dissociation constants of 294 and 323 nM, and most negative Gibbs free energy values of - 9.17 and - 8.28 kcal/mol, respectively. CONCLUSION: The results indicated that the selected aptamers (AP-1 and AP-2) induced potent inhibitory effects against PCSK9. Further in vivo studies demand to find out AP-1 and AP-2 aptamers as suitable candidates, instead of antibodies, for using in therapeutic purposes in patients with hypercholesterolemia and cardiovascular disease.

Evaluation of Silencing Effect of miR-133a-5p Mimic on TIM-3 Expression in AML (HL-60) Cell Line.

Acute myelogenous leukemia (AML) is a complex blood malignancy leading to immature leukemic stem cells (LSCs) proliferation. T cell immunoglobulin mucin-3 (TIM-3) is known as a biomarker of AML LSCs. Several microRNAs (miRNAs) can affect gene expression in AML. In this study, the silencing effect of miR-133a-5p on TIM-3 expression in AML cell lineage (HL-60) was investigated. It's been hypothesized that miR-133a-5p may suppress the TIM-3 expression in AML cell line. Initially, miRNA-TIM-3 prediction, enrichment, and network analysis were done. Then, miR-133a-5p mimic was transfected into HL-60 cells. The TIM-3 protein and gene expression were measured by flow cytometry analysis and real-time PCR, respectively. MTT assay was also carried out. Based on the Bioinformatics predictions, miR-133a-5p was able to silence TIM-3 expression. Also, significant pathways pertained to miR-133a-5p were obtained using enrichment analysis. According to this, miR-133a-5p was mainly engaged in the MAPK signaling pathway and Nicotine addiction pathway using the KEGG database. The TIM-3 protein expression of the transfected cells was measured as 17.15 ± 8.87% (p = 0.001). A 52.48% significant gene silencing in mRNA level was obtained in comparison to the negative control. Despite of down regulation of TIM-3, HL-60 cell viability has not been significantly changed. It has been finally confirmed that miR-133a-5p could strongly suppress TIM-3 expression in AML cell line. Presumably, down regulation of TIM-3 could affect MAPK and Nicotine addiction signaling pathways.

Development of α4 integrin DNA aptamer as a potential therapeutic tool for multiple sclerosis.

One of the most important molecules for multiple sclerosis pathogenesis is α4 integrin, which is responsible for autoreactive leukocytes migration into the brain. The monoclonal antibody, natalizumab, was introduced to market for blocking the extravasation of autoreactive leukocytes via inhibition of α4 integrin. However, the disadvantages of antibodies provided a suitable background for other agents to be replaced with antibodies. Considering the profound advantages of aptamers over antibodies, aptamer isolation against α4 integrin was intended in the current study. The α4 integrin-specific aptamers were selected using cell-systematic evolution of ligands by exponential enrichment (SELEX) method with human embryonic kidney (HEK)-293T overexpressing α4 integrin and HEK-293T as target and control cells, respectively. Evaluation of selected aptamer was performed through flow cytometric analysis. The selected clones were then sequenced and analyzed for any possible secondary structure and affinity. The results of this study led to isolation of 13 different single-stranded DNA clones in 11 rounds of selection which were categorized to three clusters based on common structural motifs and the equilibrium dissociation constant (K d ) of the most stable structure was calculated. The evaluation of SELEX progress showed growth in aptamer affinity with increasing of the number of cycles. Taken together, the findings of this study demonstrated the isolation of α4-specific single-stranded DNA aptamers with suitable affinity for ligand, which can further be replaced with natalizumab.

Engineered zinc-finger nuclease to generate site-directed modification in the KLF1 gene for fetal hemoglobin induction.

Elevation of hemoglobin F (HbF) ameliorates symptoms of ß-thalassemia, as a common autosomal recessive disorder. In this study, the ability of an engineered zinc-finger nuclease (ZFN) system was assesed to disrupt the KLF1 gene to inhibit the γ to ß hemoglobin switching in K562 cells. This study was performed using a second generation integration-deficient lentiviral vector assigned to transient gene targeting. The sequences coding for zinc finger protein arrays were designed and subcloned in TDH plus as a transfer vector. Transduction of K562 cells was performed with the integrase minus lentivirus containing ZFN. The indel percentage of the transducted cells with lentivirus containing ZFN was about 29%. Differentiation of K562 cell line into erythroid cell lineage was induced with cisplatin concentration of 15 µg/mL. After differentiation, γ-globin and HbF expression were evaluated using real-time reverse-transcription polymerase chain reaction and hemoglobin electrophoresis methods. The levels of γ-globin messenger RNA were nine-fold higher in the ZFN treated cells compared with untreated cells 5 days after differentiation. Hemoglobin electrophoresis method showed the same results for HbF level measurement. Application of the ZFN tool to induce KLF1 gene mutation in adult erythroid progenitors might be a candidate to stimulate HbF expression in ß-thalassemia patients.

Distribution of CYP2D6 polymorphism in the Middle Eastern region.

Cytochrome P450 2D6 (CYP2D6) is an important drug-metabolizing enzyme involved in the pharmacokinetic metabolism of drugs. CYP2D6 gene is highly polymorphic, and the combination of its different alleles yields different phenotypes including extensive metabolizer (EM), intermediate metabolizer (IM), poor metabolizer (PM), and ultrarapid metabolizer (UM). Genotyping of the important alleles for this gene in different ethnicities is of particular importance for assessing the efficacy of various drugs. In this study, we reviewed the CYP2D6 allele and phenotype frequencies predicted from the genotypes of CYP2D6 in the Middle East area. Regardless of different ethnicities, the CYP2D6*41 allele frequency was shown to be higher than that of other reduced functional alleles. In addition, CYP2D6*4 was the most frequent nonfunctional allele in all studied populations in the Middle East. Taken together, our findings illustrated that the frequencies of PM or IM alleles and different genotypes harboring these alleles are relatively high in the Middle Eastern countries. Therefore, the study of CYP2D6 alleles for each patient to detect those that are at risk is of great importance to prevent adverse drug reactions through individualization therapy.

Inducing indel mutation in the SOX6 gene by zinc finger nuclease for gamma reactivation: An approach towards gene therapy of beta thalassemia.

ß-thalassemia is a common autosomal recessive disorder characterized by a deficiency in the synthesis of ß-chains. Evidences show that increased HbF levels improve the symptoms in patients with ß-thalassemia or sickle cell anemia. In this study, ZFN technology was applied to induce a mutation in the binding domain region of SOX6 to reactivate γ-globin expression. The sequences coding for ZFP arrays were designed and sub cloned in TDH plus as a transfer vector. The ZFN expression was confirmed using Western blot analysis. In the next step, using the site-directed mutagenesis strategy through the overlap PCR, a missense mutation (D64V) was induced in the catalytic domain of the integrase gene in the packaging plasmid and verified using DNA sequencing. Then, the integrase minus lentivirus containing ZFN cassette was packaged. Transduction of K562 cells with this virus was performed. Mutation detection assay was performed. The indel percentage of the cells transducted with lenti virus containing ZFN was 31%. After 5 days of erythroid differentiation with 15 µg/mL cisplatin, the levels of γ-globin mRNA were sixfold in the cells treated with ZFN compared to untreated cells. In the meantime, the measurement of HbF expression levels was carried out using hemoglobin electrophoresis and showed the same results. Integrase minus lentivirus can provide a useful tool for efficient transient gene expression and helps avoid disadvantages of gene targeting using the native virus. The ZFN strategy applied here to induce indel on SOX6 gene in adult erythroid progenitors may provide a method to activate fetal hemoglobin expression in individuals with ß-thalassemia.