Using a translanguaging framework to examine language production in a trilingual person with aphasia.

When language abilities in aphasia are assessed in clinical and research settings, the standard practice is to examine each language of a multilingual person separately. But many multilingual individuals, with and without aphasia, mix their languages regularly when they communicate with other speakers who share their languages. We applied a novel approach to scoring language production of a multilingual person with aphasia. Our aim was to discover whether the assessment outcome would differ meaningfully when we count accurate responses in only the target language of the assessment session versus when we apply a translanguaging framework, that is, count all accurate responses, regardless of the language in which they were produced. The participant is a Farsi-German-English speaking woman with chronic moderate aphasia. We examined the participant's performance on two picture-naming tasks, an answering wh-question task, and an elicited narrative task. The results demonstrated that scores in English, the participant's third-learned and least-impaired language did not differ between the two scoring methods. Performance in German, the participant's moderately impaired second language benefited from translanguaging-based scoring across the board. In Farsi, her weakest language post-CVA, the participant's scores were higher under the translanguaging-based scoring approach in some but not all of the tasks. Our findings suggest that whether a translanguaging-based scoring makes a difference in the results obtained depends on relative language abilities and on pragmatic constraints, with additional influence of the linguistic distances between the languages in question.

First detection and characterisation of sub-genotype XIII.2.1 Newcastle disease virus isolated from backyard chickens in Iran.

BACKGROUND: Newcastle disease (ND) is an economically significant poultry disease worldwide. During field surveillance for ND in 2010 in Iran, a backyard chicken flock showed clinical signs of ND with 100% mortality. OBJECTIVES: We aimed to characterise genetically, biologically and epidemiologically an exotic virulent ND virus (NDV) detected in Iran. METHODS: After observing high mortality, dead birds were sampled and then disposed of by burial, and the chicken house was disinfected. Tissue samples were molecularly tested for NDV. The genetic homogeneity of the isolate RT30/2010 was tested by plaque assay, and then a large virus plaque was used for the second step of plaque purification. Fusion and matrix complete genes were sequenced and used for genotyping and epidemiological tracing. We tested biological pathotypes using mean death time (MDT) and intracerebral pathogenicity index (ICPI) assays. RESULTS: The isolate formed heterogeneous plaques in chicken embryo fibroblast cells. The second step of plaque purification produced homogeneous and large plaques. Phylogenetic analysis using both genes classified the virus into sub-genotype XIII.2.1. Nucleic acid and amino acid identities of RT30/2010 fusion gene with the closest available isolate SPVC/Karachi/NDV/43 are 97.95% and 98.73%, respectively. Isolate has 112 RRRKRF117 motif at the fusion cleavage site, and pathogenicity tests showed MDT of 56.4 h and ICPI of 1.85. CONCLUSIONS: This study presents the first detection and characterisation of a velogenic NDV of sub-genotype XIII.2.1 from Iran. Our follow-up surveillance for ND shows that timely virus detection and carcass management have led to the cessation of virus transmission in Iran.

Aphasia in Multilingual Patients.

PURPOSE OF REVIEW: We summarize recent published work concerning assessment and treatment of aphasia in bilingual and multilingual people and review current related models of treatment outcomes. As well, we discuss studies that address the recently debated topic of cognitive processes in bilingual individuals with aphasia, with a focus on the effects of bilingualism on aphasia recovery and its potential protective effects. RECENT FINDINGS: Providing assessment and treatment tools that best serve multilingual individuals with aphasia and unpacking the variables and mechanisms that underlie response to treatment have emerged as goals of several recent studies. Additionally, while findings are still contradictory, some empirical studies reported that aphasia may manifest less severely in multilingual individuals and may improve faster compared to in monolingual counterparts. The findings of recent studies with the focus of aphasia in multilingual individuals are crucial to understanding theoretical and clinical aspects of brain-related language impairment in multilingual people and to the study of language representation and processing in the brain.

TRIM21 Is Targeted for Chaperone-Mediated Autophagy during Salmonella Typhimurium Infection.

Salmonella enterica serovar Typhimurium (S Typhimurium) is a Gram-negative bacterium that induces cell death of macrophages as a key virulence strategy. We have previously demonstrated that the induction of macrophage death is dependent on the host's type I IFN (IFN-I) response. IFN-I signaling has been shown to induce tripartite motif (TRIM) 21, an E3 ubiquitin ligase with critical functions in autoimmune disease and antiviral immunity. However, the importance and regulation of TRIM21 during bacterial infection remains poorly understood. In this study, we investigated the role of TRIM21 upon S Typhimurium infection of murine bone marrow-derived macrophages. Although Trim21 expression was induced in an IFN-I-dependent manner, we found that TRIM21 levels were mainly regulated posttranscriptionally. Following TLR4 activation, TRIM21 was transiently degraded via the lysosomal pathway by chaperone-mediated autophagy (CMA). However, S Typhimurium-induced mTORC2 signaling led to phosphorylation of Akt at S473, which subsequently impaired TRIM21 degradation by attenuating CMA. Elevated TRIM21 levels promoted macrophage death associated with reduced transcription of NF erythroid 2-related factor 2 (NRF2)-dependent antioxidative genes. Collectively, our results identify IFN-I-inducible TRIM21 as a negative regulator of innate immune responses to S Typhimurium and a previously unrecognized substrate of CMA. To our knowledge, this is the first study reporting that a member of the TRIM family is degraded by the lysosomal pathway.

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.

Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a protein that is secreted immediately upon endothelial injury, and thereby it plays a key role in inflammation via recruitment of leucocytes to the site of inflammation at the beginning and throughout the inflammatory processes. Aim of this study was to develop two separate cell lines displaying either human MCP-1 (HMCP-1) or rabbit MCP-1 (RMCP-1) on their surface. A DNA fragment containing HMCP-1- or RMCP-1-encoding sequence was inserted into a pcDNA plasmid. Escherichia coli cells strain TOP 10F' was separately transformed with the pcDNA/RMCP-1 or /HMCP-1 ligation mixture. Following the cloning and construct verification, human embryonic kidney cell line (HEK 293T) was transfected with either of the linearized plasmids. Plasmid integration into the genomic DNA of HEK 293T cells was verified by polymerase chain reaction (PCR). HMCP-1 and RMCP-1 expression was evaluated at RNA and protein levels by real-time PCR and flow cytometry, respectively. PCR products of the expected sizes were amplified from the chromosomal DNA of transfected HEK 293T cells, i.e. 644 bp for H-MCP1 and 737 bp for RMCP-1 constructs. Real-time PCR revealed that the copy numbers of RMCP1 and HMCP1 mRNA per cell were 294 and 500, respectively. Flow cytometry analysis indicated 85% for RMCP-1 and 87% for HMCP-1 expression levels on the surface of transfected cells, when compared with an isotype control. The experiments thus confirmed that the MCP-1 genes were integrated into the HEK 293T genomic DNA and the encoded proteins were stably expressed on the cell surface.

Disruption of SOX6 gene using CRISPR/Cas9 technology for gamma-globin reactivation: An approach towards gene therapy of ß-thalassemia.

Elevation of Hemoglobin F ameliorates symptoms of ß-thalassemia, a common autosomal recessive disorder. The transcription factor SOX6 plays a key role in the γ to ß-globin gene switching. In the current investigation, a mutation was induced using the CRISPR/Cas9 technology in the binding domain region of SOX6 to reactivate γ-globin expression. Three CRISPR/Cas9 cassettes were provided, whose single-guide RNAs targeted different regions in the SOX6 gene-binding domain. After transfection of K562 cells with CRISPR a, b and c, and subsequent erythroid differentiation, the indel percentage of the cells was about 30%, 25%, and 24%, respectively. Relative quantification showed that the γ-globin mRNA level increased to 1.3-, 2.1-, and 1.1-fold in the cells treated with CRISPR/Cas9 a, b, and c, respectively, compared with untreated cells. Our results show that mutation induction in the binding site of the SOX6 gene leads to γ-globin reactivation. These findings support the idea that CRISPR interrupts the SOX6 binding site, and, as a result, SOX6 is incapable of binding the γ-globin promoter. In conclusion, SOX6 disruption could be considered as a therapeutic approach for ß-thalassemia treatment. CRISPR/Cas9 was selected for this purpose as it is the most rapidly evolving technology.

Ectopic Expression of Human DPPA2 Gene in ESCC Cell Line Using Retroviral System.

BACKGROUND: Cancer/Testis Antigens (CTAs) are a sub-group of tumor-associated antigens which are expressed normally in germ line cells and trophoblast, and aberrantly in a variety of malignancies. One of the most important CTAs is Developmental Pluripotency Associated-2(DPPA2) with unknown biological function. Considering the importance of DPPA2 in developmental events and cancer, preparing a suitable platform to analyze DPPA2 roles in the cells seems to be necessary. METHODS: In this study, the coding sequence of DPPA2 gene was amplified and cloned into the retroviral expression vector to produce recombinant retrovirus. The viral particles were transducted to Esophageal Squamous Cell Carcinoma (ESCC) cell line (KYSE-30 cells) and the stable transducted cells were confirmed for ectopic expression of DPPA2 gene by real-time PCR. RESULTS: According to the critical characteristics of retroviral expression system such as stable and long time expression of interested gene and also being safe due to deletion of retroviral pathogenic genes, this system was used to induce expression of DPPA2 gene and a valuable platform to analyze its biological function was prepared. Transduction results clearly showed efficient overexpression of the gene in target cells in protein level due to high level of GFP expression. CONCLUSION: Such strategies can be used to produce high levels of desired protein in target cells as a therapeutic target. The produced recombinant cells may present a valuable platform to analyze the effect of DPPA2 ectopic expression in target cells. Moreover, the introduction of its potential capacity into the mouse model to evaluate the tumorigenesis of these cancer cells in vivo leads to an understanding of the biological importance of DPPA2 in tumorigenesis. In addition, our purified protein can be used in a mouse model to produce specific antibody developing a reliable detection of DPPA2 existence in any biological fluid through ELISA system.

The effects of maternal diabetes and insulin treatment on neurogenesis in the developing hippocampus of male rats.

Diabetes in pregnancy is associated with an increasing risk of congenital malformations and central nervous system disorders (CNS) especially hippocampal neuronal circuitry disruption as a discreet region involved in neurogenesis phenomenon. This study aimed to investigate the effect of maternal diabetes and insulin treatment on the expression and distribution pattern of NeuN and DCX as two important markers of neurogenesis paradigm in developing rat hippocampus. All animals were randomly divided into three groups as follows: Control group, Diabetic (STZ-D), Diabetic treated with insulin (STZ-INS). Diabetes was induced in Wistar female rats by Sterptozotocin intraperitoneal injection (single does). Following confirmation of diabetes, animals were mated with non-diabetic males. Four to six units of protamine-Zinc insulin were delivered subcutaneously (SC) in insulin treated group. At the post-natal day 14 (P14), the brain of male offspring's were removed for further study. In fact Immunofluorescence staining and Real time - PCR assays are used for evaluation of neurogenesis phenomenon. Our results showed a significant higher level of hippocampal DCX expression and an increase in the mean number of DCX positive cells in the DG of diabetic group male offspring (P < 0.05). We also found an insignificant up-regulation in the expression of DCX and the mean number of positive cells in the insulin-treated diabetic group neonates as compared to control group (P > 0.05). Nevertheless the results of immunofluorescence staining for NeuN also indicated that the mean number of NeuN+ cells was significantly lower in dentate gyrus of diabetic group male offspring (P < 0.05). Besides, there were significant down- regulation in the hippocampal mRNA expression of NeuN in diabetic pups compare to control (P < 0.05 each). Our results revealed that diabetes during pregnancy has an adverse effect on the hippocampal neurogenesis in rat neonates. Furthermore, the control of glycemia by insulin is sufficient to prevent the alterations in expression of neurogenesis markers.

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.

A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease.

BACKGROUND: Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods. MATERIALS AND METHODS: At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F' that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry. RESULTS: The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) (P < 0.0001). CONCLUSION: According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.

Interleukin-33 plasma levels in patients with relapsing-remitting multiple sclerosis.

Cytokines are implicated in the immunopathogenesis of multiple sclerosis (MS). Interleukin (IL)-33, one of the recently discovered members of the IL-1 superfamily, is a dual functional cytokine involved in various autoimmune disorders. In a case-control study, venous blood was collected from healthy subjects categorized as control group (n=44) and MS patients (n=44). All recruited patients were clinically diagnosed with relapsing-remitting MS (RRMS), including patients without treatment (new identified cases, n=16) and those treated with interferon beta (IFN-ß) (n=28). The plasma levels of IL-33 in subjects were measured with ELISA. Significantly elevated IL-33 plasma levels were observed in RRMS patients (p=0.005). Furthermore, IFN-ß-treated MS patients had lower levels of IL-33 compared to the untreated patients (p<0.001). Increased IL-33 plasma levels in the patient group might be associated with development of MS. These results could contribute to our better understanding about the role of IL-33 in the immunopathogenesis of MS.

The Increase in Protein and Plasmid Yields of E. coli with Optimized Concentration of Ampicillin as Selection Marker.

Background:Escherichia coli is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression in E. coli, but, it seems that external factors such as selection marker concentration can drastically affect the yield of protein and plasmid. Objectives: Alterations of protein expression and plasmid yields of E. coli in different concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized. Materials and Methods: The expression cassette of codon optimized EGFP for E. coli was synthesized in pUC57. The pUC57-GFP was transformed into E. coli Top10F'. The expression of GFP was verified by SDS-PAGE and flow cytometry after induction by IPTG (0.5 mM) and incubation with 0, 100, 200 and 300 µg.mL-1 ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve. Results: GFP expressing clones formed fair green colonies on LB agar supplemented with 0.5 mM IPTG and showed fluorescence in FL1 filter of flow cytometry and an extra protein band on SDS-PAGE gel. The fluorescent intensity of GFP in 0, 100, 200 and 300 µg.mL-1 ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07×109 , 3.21×109 , 2.32×1010 , 8.11×108 , respectively. The plasmid yields were 55 ng.µL-1, 69 ng.µL-1, 164 ng.µL-1 and 41 ng.µL-1, respectively. Conclusion: Protein and plasmid yields of E. coli are variable in different concentrations of ampicillin and need to be optimized in newly designed expression systems. Protein and plasmid yield in the optimized concentration (200 µg.mL-1) was significantly (p < 0.01) higher than other doses.

Real-Time PCR: an Appropriate Approach to Confirm ssDNA Generation from PCR Product in SELEX Process.

Background: Aptamers are single stranded DNA (ssDNA) or RNA molecules. The potential of aptamers for binding to the different targets has made them be widely used as the preferred diagnostic and therapeutic tools. DNA aptamers present several advantages over the RNA oligonucleotides due to their higher stability, easier selection, and production. Selection of DNA aptamers which is facilitated through a systematic evolution of ligand by exponential enrichment (SELEX) method is much dependent on the successful conversion of double stranded DNA (dsDNA) to ssDNA. Objective: There are different methods available for ssDNA generation. While visualization of ssDNA is limited to the gelbased method, the method is not applicable in the initial rounds of SELEX due to more than 1015 different sequences. This study was designed to evaluate the effi ciency of another technique for confi rming the ssDNA generation in comparison to the polyacrylamide electrophoresis (PAGE) analysis. Materials and Methods: Real-time PCR was employed in the present study for PCR amplifi cation of the initial library that was followed by enzymatic digestion of the dsDNA. Subsequently melting curve analysis was carried out to evaluate ssDNA generation from dsDNA. Moreover, PAGE analysis was performed and the results were compared with the melt curve analysis. Results: The melt curves, revealed dsDNA conversion to the ssDNA based on a significant reduction of Tm from 73.8 to 41.5 °C. Applying PAGE analysis, it was not effectively feasible to show ssDNA generation from the corresponding initial dsDNA library, while, it was effi cient enough to confirm ssDNA generation in accordance with the increasing the number of SELEX rounds. Conclusion: The present study has proven the applicability of the real-time PCR as a suitable confirmatory technique for validating ssDNA generation in the DNA aptamer selection process for the initial library preparation.

Complementary and Alternative Medicine for Osteoporosis.

BACKGROUND: A systemic skeletal disease is characterized by low bone mass and micro-architectural deterioration with a consequent increase in bone fragility and susceptibility to fracture. Asia has the highest increment in the elderly population; therefore, osteoporotic fracture should be a noticeable health issue. The incidence rate of hip fractures in Asia could rise to 45% by the year 2050. Complementary and alternative medicine (CAM) is a group of various medical and health care systems, practices, and products that are not presently considered as part of formal medicine. CAMs have been described as "diagnosis, treatment, and/or prevention which complements mainstream medicine as a holistic, subjective and various natural approaches to medical problems by contributing to a common whole, satisfying claims not met by orthodoxy, or diversifying the conceptual frameworks of medicine". METHODS: Peer-reviewed publications were identified through a search in Scopus, Science Direct, Cochrane, PubMed, and Google scholar using keywords "osteopenia", "osteoporosis", "menopause", "CAM", "phytoestrogens", "phytotherapy" and "herbal medicine". The search was completed in July 2015 and was limited to articles published in English. Relevant articles were identified based on the expertise and clinical experience of the authors. RESULTS: We categorized our results in different classifications including: lifestyle modifications (cigarette, alcohol, exercise and food regimen), supportive cares (intake supplements including vitamin D, C and K), treatments synthetic (routine and newer options for hormone replacement and none hormonal therapies) and natural options (different types of CAM including herbal medicines, yoga and chiropractic). CONCLUSION: Established osteoporosis is difficult to treat because bone density has fallen below the fracture threshold and trabecular elements may have been lost. Antiresorptive agents can be used to prevent further bone loss and stimulation of new bone formation by the use of anabolic steroids or fluoride may increase the overall amount of bone.

Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemia.

OBJECTIVES: ß-thalassemia is one of the most common genetic disorders in the world. As one of the promising treatment strategies, fetal hemoglobin (Hb F) can be induced. The present study was an attempt to reactivate the γ-globin gene by introducing a gene construct containing KLF1 binding sites to the K562 cell line. MATERIALS AND METHODS: A plasmid containing a 192 bp sequence with two repeats of KLF1 binding sites on ß-globin and BCL11A promoters was constructed and used to transfect the K562 cell line. Positive selection was performed under treatment with 150 µg/ml hygromycin B. The remaining cells were expanded and harvested on day 28, and genomic DNA was extracted. The PCR was carried out to verify insertion of DNA fragment to the genome of K562 cells. The cells were differentiated with 15 µg/ml cisplatin. Flowcytometry was performed to identify erythroid differentiation by detection of CD235a+ cells. Real-time RT-PCR was performed to evaluate γ-globin expression in the transfected cells. RESULTS: A 1700 bp fragment was observed on agarose gel as expected and insertion of DNA fragment to the genome of K562 cells was verified. Totally, 84% of cells were differentiated. The transfected cells significantly increased γ-globin expression after differentiation compared to untransfected ones. CONCLUSION: The findings demonstrate that the spongy effect of KLF1-binding site on BCL11A and ß-globin promoters can induce γ-globin expression in K562 cells. This novel strategy can be promising for the treatment of ß-thalassemia and sickle cell disease.

Effect of maternal diabetes on gliogensis in neonatal rat hippocampus.

BACKGROUND: Diabetes in pregnancy is a common metabolic disorder associated with various adverse outcomes in the offspring including impairments in attention and memory and alterations in social behavior. Glial cells are proven to have a critical role in normal function of neurons, and alteration in their activity could contribute to disturbance in the brain function. The aim of this study was to investigate the effect of maternal diabetes on hippocampal mRNA expression and distribution pattern of glial fibrillary acidic protein (GFAP) immunoreactive glial cells in the dentate gyrus (DG) of rat neonate at postnatal day 14 (P14). MATERIALS AND METHODS: Wistar female rats were randomly allocated in control, diabetic, and insulin-treated diabetic groups. Diabetes was induced by injection of streptozotocin from 4 weeks before gestation until parturition. After delivery, the male offspring was euthanized at P14. RESULTS: Our results showed a significant higher level of hippocampal GFAP expression and an increase in the mean number of GFAP positive cells in the DG of diabetic group offspring (P < 0.05). We also found an insignificant up-regulation in the expression of GFAP and the mean number of positive cells in the insulin-treated diabetic group neonates as compared to control group (P > 0.05). CONCLUSION: The present study revealed that diabetes during pregnancy strongly increased the glial cells production in the developing rat hippocampus.

Genetic disruption of the KLF1 gene to overexpress the γ-globin gene using the CRISPR/Cas9 system.

BACKGROUND: ß-thalassemia comprises a major group of human genetic disorders involving a decrease in or an end to the normal synthesis of the ß-globin chains of hemoglobin. KLF1 is a key regulatory molecule involved in the γ- to ß-globin gene switching process directly inducing the expression of the ß-globin gene and indirectly repressing γ-globin. The present study aimed to investigate the ability of an engineered CRISPR/Cas9 system with respect to disrupting the KLF1 gene to inhibit the γ- to ß-hemoglobin switching process in K562 cells. METHODS: We targeted three sites on the KLF1 gene, two of which are upstream of codon 288 in exon 2 and the other site being in exon 3. RESULTS: The average indel percentage in the cells transfected with CRISPR a, b and c was approximately 24%. Relative quantification was performed for the assessment of γ-globin expression. The levels of γ-globin mRNA on day 5 of differentiation were 8.1-, 7.7- and 1.8-fold in the cells treated with CRISPR/Cas9 a, b and c, respectively,compared to untreated cells. The measurement of HbF expression levels confirmed the same results. CONCLUSIONS: The findings obtained in the present study support the induction of an indel mutation in the KLF1 gene leading to a null allele. As a result, the effect of KLF1 on the expression of BCL11A is decreased and its inhibitory effect on γ-globin gene expression is removed. Application of CRISPR technology to induce an indel in the KLF1 gene in adult erythroid progenitors may provide a method for activating fetal hemoglobin expression in individuals with ß-thalassemia or sickle cell disease.