NAIP Gene Deletion and SMN2 Copy Number as Molecular Tools in Predicting the Severity of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is one of the most prevalent autosomal recessive illnesses with type I being the most severe type. Genomic alterations including survival motor neuron (SMN) copy number as well as deletions in SMN and Neuronal Apoptosis Inhibitory Protein (NAIP) are greatly implicated in the emergence of SMA. However, the association of such alterations with the severity of the disease is yet to be investigated. This study was directed to elucidate the molecular assessment of NAIP and SMN genomic alterations as a useful tool in predicting the severity of SMA among patients. This study included 65 SMA pediatric patients (30 type I and 35 type II) and 65 healthy controls. RFLP-PCR was employed to determine the genetic polymorphisms of the SMN1, SMN2, and NAIP genes. In addition, qRT-PCR was used to identify the expression of the SMN1 and SMN2 genes, and serum levels of creatine kinase were measured using a colorimetric method. DNA sequencing was performed on some samples to detect any single nucleotide polymorphisms in SMN1, SMN2, and NAIP genes. All SMA patients had a homozygous deficiency of SMN1 exon 7. The homozygous deficiency of SMN1 exons 7 and 8, with the deletion of NAIP exon 5 was found among the majority of Type I patients. In contrast, patients with the less severe condition (type II) had SMN1 exons 7 and 8 deleted but did not have any deletions in NAIP, additionally; 65.7% of patients had multiple copies of SMN2. Analysis of NAIP deletion alongside assessing SMN2 copy number might enhance the effectiveness of the diagnosis that can predict severity among Spinal Muscular Atrophy patients.

Renal histological alterations induced by acute exposure of titanium dioxide nanoparticles / Alteraciones histológicas renales inducidas por la exposición aguda de nanopartículas de dióxido de titanio

SUMMARY: Titanium dioxide nanoparticles (TiO2 NPs) are widely used in many commercial products, nanomedicine, agriculture, personal care products, different industries and pharmaceutical preparations with potential risk in human health and the environment. The current work was conducted to investigate the renal damage that might be induced by the acute toxicity TiO2 NPs. A total of 40 healthy male adult Wistar albino rats (Rattus norvegicus) were exposed to TiO2 NPs (126, 252, 378 mg/kg bw) for 24 and 48 h. Fresh portions of the kidneys from each rat were processed for histological and histochemical alterations. In comparison with respective control rats, exposure to TiO2 NPs has marked the following glomerular, tubular and interstitial alterations including the followings: glomerular congestion, Bowman's capsule swelling and dilatation, inflamed glomeruli, renal tubules cloudy swelling, karyorrhexis, karyolysis, infiltration of inflammatory cells, congestion, necrosis, hydropic degeneration, dilatation and congestion of blood vessels, hyaline droplets and hyaline casts precipitation, interstitial edema and fibrosis. From the findings of the current work one may conclude that TiO2 NPs are capable of inducing kidney damage with more insulation in the cortex and the proximal convoluted tubules than the medulla and the distal ones respectively. In addition, it might be concluded that renal damage induced by these nanomaterials is dose and duration of exposure dependent. Further hematological, biochemical, immunohistochemical, and ultra-structural studies are recommended.

RESUMEN: Las nanopartículas de dióxido de titanio (TiO2 NP) se usan ampliamente en muchos productos comerciales, nanomedicina, agricultura, productos para el cuidado personal, diferentes industrias y preparaciones farmacéuticas con riesgo potencial para la salud humana y el medio ambiente. El trabajo actual se realizó para investigar el daño renal que podría ser inducido por la toxicidad aguda NP de TiO2. Un total de 40 ratas Wistar albinas adultas sanas (Rattus norvegicus) fueron expuestas a TiO2 NP (126, 252, 378 mg / kg de peso corporal) durante 24 y 48 h. Las muestras de los riñones de las ratas se procesaron para estudios histológicos e histoquímicos. En comparación con las ratas control, la exposición de las ratas a TiO2 NP presentaron las siguientes alteraciones glomerulares, tubulares e intersticiales: congestión glomerular, dilatación de la cápsula de Bowman, inflamación glomerular, túbulos renales aumentados, cariorrexis, cariólisis, infiltración de células inflamatorias, congestión, necrosis, degeneración hidrópica, dilatación y congestión de vasos sanguíneos, gotas y precipitaciones hialina, edema intersticial y fibrosis. A partir de los hallazgos del trabajo actual, se puede concluir que las NP de TiO 2 son capaces de inducir daño renal con más aislamiento en la corteza y en los túbulos contorneados proximales que en la médula y los túbulos contorneados distales, respectivamente. Además, se podría concluir que el daño renal inducido por estos nanomateriales depende de la dosis y la duración de la exposición. Se recomiendan estudios adicionales hematológicos, bioquímicos, inmunohistoquímicos y ultraestructurales.

Multiplex bioimaging of piRNA molecular pathway-regulated theragnostic effects in a single breast cancer cell using a piRNA molecular beacon.

Recently, PIWI-interacting small non-coding RNAs (piRNAs) have emerged as novel cancer biomarkers candidate because of their high expression level in various cancer types and role in the control of tumor suppressor genes. In this study, a novel breast cancer theragnostics probe based on a single system targeting the piRNA-36026 (piR-36026) molecular pathway was developed using a piR-36026 molecular beacon (MB). The piR-36026 MB successfully visualized endogenous piR-36026 biogenesis, which is highly expressed in MCF7 cells (a human breast cancer cell line), and simultaneously inhibited piR-36026-mediated cancer progression in vitro and in vivo. We discovered two tumor suppressor proteins, SERPINA1 and LRAT, that were directly regulated as endogenous piR-36026 target genes in MCF7 cells. Furthermore, multiplex bioimaging of a single MCF7 cell following treatment with piR-36026 MB clearly visualized the direct molecular interaction of piRNA-36026 with SERPINA1 or LRAT and subsequent molecular therapeutic responses including caspase-3 and PI in the nucleus.

Distribution of Glutathione S-Transferase Omega Gene Polymorphism with Different Stages of HBV Infection Including Hepatocellular Carcinoma in the Egyptian Population.

BACKGROUND: Infection with hepatitis B virus (HBV) is a major global public health problem, with a wide spectrum of clinical manifestations. Human cytosolic glutathione-S-transferases (GSTs) include several classes such as alpha (A), mu (M), pi (P), sigma (S), zeta (Z), omega (O) and theta (T). The present study aimed to investigate the role of GST omega genes (GSTO1 and GSTO2) in different groups of patients infected with HBV. MATERIALS AND METHODS: HBV groups were classified according to clinical history, serological tests and histological analysis into normal carriers (N), acute (A), chronic (CH), cirrhosis (CI) and hepatocellular carcinoma (HCC) cases. The study focused on determination of the genotypes of GST omega genes (GSTO1 and GSTO2) and GST activity and liver function tests. RESULTS: The results showed that GSTO1 (A/A) was decreased in N, A, CH, CI and HCC groups compared to the C-group, while, GSTO1 (C/A) and GSTO1(C/C) genotypes were increased significantly in N, A, CH, CI and HCC groups. GSTO2 (A/A) was decreased in all studied groups as compared to the C-group but GSTO2(A/G) and GSTO2(G/G) genotypes were increased significantly. In addition, GST activities, albumin and TP levels were decreased in all studied groups compared to the C-group, while the activities of transaminases were increased to differing degrees. CONCLUSIONS: The results indicate that GSTO genetic polymorphisms may be considered as biomarkers for determining and predicting the progression of HBV infection.

Isolation and evaluation of biological efficacy of quercetol in human hepatic carcinoma cells.

Quercetol is a polyphenolic molecule present in vegetables and fruits, and is beneficial to human and animal health. The current work aimed to test cytotoxic and apoptotic effects of quercetol on HepG2 cells. Quercetol was isolated from Ocimum sanctum and characterized by gas chromatography-tandom mass spectrometry (GC-MS/MS), nuclear magnetic resonance spectroscopy, and Fourier transform infrared spectroscopy. Quercetol (50-600 µg/mL) was examined for cytotoxic activity by tetrazolium salt and neutral red uptake tests and comet assay for genotoxicity, using HepG2 cells, over 24 hours. Data from 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and neutral red uptake tests demonstrated quercetol-induced cytotoxicity in HepG2 cells in a concentration-dependent manner. With 4',6-diamidino-2-phenylindole staining, a significant induction of chromosomal condensation was observed at 300 µg/mL of quercetol. DNA fragmentation analysis showed that quercetol produced cell death in HepG2 cells in a concentration-dependent manner. Thus, our study suggests that an environmentally relevant concentration of quercetol, which was a chemically standardized extract from O. sanctum, induced cell death and DNA damage in HepG2 cells.

Multimodal imaging probe for targeting cancer cells using uMUC-1 aptamer.

For adequate cancer therapy, newer imaging modalities with more specific ligands for unique targets are crucial. Underglycosylated mucin-1 (uMUC-1) antigen is an early marker of tumor development and is widely overexpressed on most tumors. A combination of nanotechnology with optical, radionuclide, and magnetic resonance (MR) imaging has great potential to improve cancer diagnosis and therapy. In this study, a multimodal nanoparticle imaging system was developed that can be used for optical, MR and positron emission tomography (PET) imaging. Cobalt ferrite magnetic nanoparticles surrounded by fluorescent rhodamine (designated MF) within a silica shell matrix were conjugated with an aptamer targeting uMUC-1 (designated MF-uMUC-1) and further labeled by (68)Ga (designated MFR-uMUC-1) with the help of a p-SCN-bn-NOTA chelating agent, resulting in single multimodal nanoparticles. The resultant nanoparticles are spherical and monodispersed, as revealed by transmission electron microscopy. The MFR-uMUC-1 nanoparticle showed specific and dose-dependent fluorescent, radioisotope and MR signals targeting BT-20 cells expressing uMUC-1. In vivo targeting and multimodal imaging in tumor-bearing nude mice also showed great specificity for targeting cancers with MFR-uMUC-1. The MFR-uMUC-1 probe could be used as a single multimodal probe to visualize cancer cells by means of optical, radionuclide and MR imaging.

Bioimaging of microRNA124a-independent neuronal differentiation of human G2 neural stem cells.

Evaluation of the function of microRNAs (miRNAs or miRs) through miRNA expression profiles during neuronal differentiation plays a critical role not only in identifying unique miRNAs relevant to cellular development but also in understanding regulatory functions of the cell-specific miRNAs in living organisms. Here, we examined the microarray-based miRNA expression profiles of G2 cells (recently developed human neural stem cells) and monitored the expression pattern of known neuron-specific miR-9 and miR-124a during neuronal differentiation of G2 cells in vitro and in vivo. Of 500 miRNAs analyzed by microarray of G2 cells, the expression of 90 miRNAs was significantly increased during doxycycline-dependent neuronal differentiation of G2 cells and about 60 miRNAs showed a gradual enhancement of gene expression as neuronal differentiation progressed. Real-time PCR showed that expression of endogenous mature miR-9 was continuously and gradually increased in a pattern dependent on the period of neuronal differentiation of G2 cells while the increased expression of neuron-specific mature miR-124a was barely observed during neurogenesis. Our recently developed miRNA reporter imaging vectors (CMV/Gluc/3×PT_miR-9 and CMV/Gluc/3×PT_miR-124a) containing Gaussia luciferase, CMV promoter and three copies of complementary nucleotides of each corresponding miRNA showed that luciferase activity from CMV/Gluc/3×PT_miR-9 was gradually decreased both in vitro and in vivo in G2 cells induced to differentiate into neurons. However, in vitro and in vivo bioluminescence signals for CMV/Gluc/3×PT_miR-124a were not significantly different between undifferentiated and differentiated G2 cells. Our results demonstrate that biogenesis of neuron-specific miR-124a is not necessary for doxycycline-dependent neurogenesis of G2 cells.

Bioimaging of transcriptional activity of microRNA124a during neurogenesis.

OBJECTIVES: A special vector system was developed to monitor the in vitro and in vivo endogenous level of a primary transcript of miR124a during neuronal differentiation RESULTS: The upstream regions of miR124a were fused with luciferase (Gluc) and their activity was measured. During neurogenesis of P19 cells, the primary transcript level of miR124a was increased 1.5-times compared to the undifferentiated P19 cells. P19 cells grafted to nude mice exhibited the same pattern of luciferase activity in vivo as they did in vitro. CONCLUSION: The expression of primary miR124a during neurogenesis was successfully imaged by in vitro and in vivo luciferase reporter gene-based method.

Sperm DNA-mediated reduction of nonspecific fluorescence during cellular imaging with quantum dots.

Salmon sperm DNA was used as a blocking agent to reduce background fluorescence signals from gelatin-coated cell culture dishes.

Quantum dot-based molecular beacon to monitor intracellular microRNAs.

Fluorescence monitoring of endogenous microRNA (miRNA or miR) activity related to neuronal development using nano-sized materials provides crucial information on miRNA expression patterns in a noninvasive manner. In this study, we report a new method to monitor intracellular miRNA124a using quantum dot-based molecular beacon (R9-QD-miR124a beacon). The R9-QD-miR124a beacon was constructed using QDs and two probes, miR124a-targeting oligomer and arginine rich cell-penetrating peptide (R9 peptide). The miR124a-targeting oligomer contains a miR124a binging sequence and a black hole quencher 1 (BHQ1). In the absence of target miR124a, the R9-QD-miR124a beacon forms a partial duplex beacon and remained in quenched state because the BHQ1 quenches the fluorescence signal of the R9-QD-miR124a beacon. The binding of miR124a to the miR124a binding sequence of the miR124a-targeting oligomer triggered the separation of the BHQ1 quencher and subsequent signal-on of a red fluorescence signal. Moreover, enhanced cellular uptake was achieved by conjugation with the R9 peptide, which resulted in increased fluorescent signal of the R9-QD-miR124a beacons in P19 cells during neurogenesis due to the endogenous expression of miR124a.

Simultaneous imaging of two different cancer biomarkers using aptamer-conjugated quantum dots.

Studying gene expression profile in a single cancer cell is important because multiple genes are associated with cancer development. Quantum dots (QDs) have been utilized as biological probes for imaging and detection. QDs display specific optical and electrical properties that depend on their size that can be applied for imaging and sensing applications. In this study, simultaneous imaging of the cancer biomarkers, tenascin-C and nucleolin, was performed using two types of aptamer-conjugated QDs. The simultaneous imaging of these two different cancer markers in three cancer cell lines was reliable and cell line-specific. Current requirements for cancer imaging technologies include the need for simple preparation methods and the ability to detect multiple cancer biomarkers and evaluate their intracellular localizations. The method employed in this study is a feasible solution to these requirements.

Bioimaging of the microRNA-294 expression-dependent color change in cells by a dual fluorophore-based molecular beacon.

A dual fluorophore-based color-tunable molecular beacon visualized the microRNA-294 expression-dependent color change in cells.

Histologic and apoptotic changes induced by titanium dioxide nanoparticles in the livers of rats.

Titanium dioxide (TiO2) nanoparticles are among the top five nanoparticles used in consumer products, paints, and pharmaceutical preparations. Given that exposure to such nanoparticles is mainly via the skin and inhalation, the present study was conducted in male Wistar albino rats (Rattus norvegicus). Our aim was to investigate the effect of TiO2 nanoparticles on hepatic tissue in an attempt to understand their toxicity and the potential effect of their therapeutic and diagnostic use. To investigate the effects of TiO2 nanoparticles on liver tissue, 30 healthy male Wistar albino rats were exposed to TiO2 nanoparticles at doses of 63 mg, 126 mg, and 252 mg per animal for 24 and 48 hours. Serum glutamate oxaloacetate transaminase and alkaline phosphatase activity was altered. Changes in hepatocytes can be summarized as hydropic degeneration, cloudy swelling, fatty degeneration, portal and lobular infiltration by chronic inflammatory cells, and congested dilated central veins. The histologic alterations observed might be an indication of hepatocyte injury due to the toxicity of TiO2 nanoparticles, resulting in an inability to deal with accumulated residues from the metabolic and structural disturbances caused by these nanoparticles. The appearance of cytoplasmic degeneration and destruction of nuclei in hepatocytes suggests that TiO2 nanoparticles interact with proteins and enzymes in hepatic tissue, interfering with antioxidant defense mechanisms and leading to generation of reactive oxygen species which, in turn, may induce stress in hepatocytes, promoting atrophy, apoptosis, and necrosis. More immunohistochemical and ultrastructural investigations are needed in relation to TiO2 nanoparticles and their potential effects when used as therapeutic and diagnostic tools.

Cytotoxicity and genotoxicity of copper oxide nanoparticles in human skin keratinocytes cells.

The wide scale use of copper oxide nanoparticles (CuONPs) due to their unique properties and important applications in magnetic, thermal, electrical, sensor devices, and cosmetics makes human beings more prone to the exposure of CuONPs and its potential adverse effects. Exposure to such nanoparticles is mainly through skin and inhalation. Therefore, the aim of the present study was to assess the apoptotic and genotoxic potential of CuONPs (50 nm) in the human skin epidermal (HaCaT) cells and its underlying mechanism of cellular toxicity. Significant decreases in cell viability were observed with CuONPs exposure in a dose- and time-dependent manner and also induced significant reduction in glutathione and induction in lipid peroxidation, catalase, and superoxide dismutase in HaCaT cells. A significant increase in caspase-3 activity was observed with CuONPs exposure in HaCaT cells indicating apoptosis. Apoptosis or necrosis was confirmed with fluorescent staining (acridine orange and propidium iodide). The CuONPs also induced DNA damage that was mediated by oxidative stress. This study investigating the effects of CuONPs in human skin cells has provided valuable insights into the mechanism of potential toxicity induced by CuONPs.

Arsenic trioxide-mediated oxidative stress and genotoxicity in human hepatocellular carcinoma cells.

BACKGROUND: Arsenic is a ubiquitous environmental toxicant, and abnormalities of the skin, lung, kidney, and liver are the most common outcomes of long-term arsenic exposure. This study was designed to investigate the possible mechanisms of genotoxicity induced by arsenic trioxide in human hepatocellular carcinoma cells. METHODS AND RESULTS: A mild cytotoxic response of arsenic trioxide was observed in human hepatocellular carcinoma cells, as evident by (3-(4,5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) and lactate dehydrogenase assays after 24 and 48 hours of exposure. Arsenic trioxide elicited a significant (P < 0.01) reduction in glutathione (15.67% and 26.52%), with a concomitant increase in malondialdehyde level (67.80% and 72.25%; P < 0.01), superoxide dismutase (76.42% and 81.09%; P < 0.01), catalase (73.33% and 76.47%; P < 0.01), and reactive oxygen species generation (44.04% and 56.14%; P < 0.01) after 24 and 48 hours of exposure, respectively. Statistically significant (P < 0.01) induction of DNA damage was observed by the comet assay in cells exposed to arsenic trioxide. It was also observed that apoptosis occurred through activation of caspase-3 and phosphatidylserine externalization in human hepatocellular carcinoma cells exposed to arsenic trioxide. CONCLUSION: The results demonstrate that arsenic trioxide induces apoptosis and genotoxicity in human hepatocellular carcinoma cells through reactive oxygen species and oxidative stress.

Dual optical biosensors for imaging microRNA-1 during myogenesis.

Dual optical microRNA (miRNA) imaging systems, bioluminescent reporter gene (a signal-off mechanism)- or fluorescent molecular beacon (MB) (a signal-on mechanism)-based miRNA imaging system, have individually allowed us to sense miRNA biogenesis in a noninvasive and iterative manner. Both of these imaging systems have shortcomings with respect to image quality. Therefore, we designed a dual optical imaging systems by simultaneous imaging of a miRNA-1 reporter gene (CMV/Gluc/3xPT_miR-1) and miRNA-1 MB in a single cell to overcome these limitations and used it to visualize miRNA-1, a highly expressed miRNA in cardiac and skeletal muscle. During myogenic differentiation of C2C12 cells, the bioluminescence intensity from CMV/Gluc/3xPT_miR-1 revealed a miRNA-1-dependent gradual decrease and the fluorescence intensity from miR-1 MB demonstrated a miRNA-1-dependent gradual increase both in vitro and in vivo. The dual miRNA-1 imaging systems, which provides the complementary imaging information about miRNA biogenesis, could be useful to sense miRNA expression during various biologic processes.

A reverse complementary multimodal imaging system to visualize microRNA9-involved neurogenesis using peptide targeting transferrin receptor-conjugated magnetic fluorescence nanoparticles.

Multimodal imaging systems may eliminate the disadvantages of individual imaging modality by providing complementary information about cellular and molecular activites. In this sutdy, we developed a reverse complementary multimodal imaging system to image microRNAs (miRNA, miR) during neurognesis using transferrin receptor (TfR) and a magnetic fluorescence (MF) nanoparticle-conjugated peptide targeting TfR (MF targeting TfR). Both in vitro and in vivo imaging demonstrated that, in the absence of miR9 during pre-differentiation of P19 cells, the MF targeting TfR nanoparticles greatly targeted TfR and were successfully internalized into P19 cells, resulting in high fluorescence and low MR signals. When the miR9 was highly expressed during neurogenesis of P19 cells, the MF targeting TfR nanoparticles were hardly targeted due to the miR9 function, which represses the expression and functional activity of TfR from the miRNA TfR reproter gene, resulting in low fluorescence and high MR signals. The reverse complementary multimodal miRNA imaging system may serve as a new imaging probe to montior miRNA-involved cellular developments and diseases.

Revealing the history of sheep domestication using retrovirus integrations.

The domestication of livestock represented a crucial step in human history. By using endogenous retroviruses as genetic markers, we found that sheep differentiated on the basis of their "retrotype" and morphological traits dispersed across Eurasia and Africa via separate migratory episodes. Relicts of the first migrations include the Mouflon, as well as breeds previously recognized as "primitive" on the basis of their morphology, such as the Orkney, Soay, and the Nordic short-tailed sheep now confined to the periphery of northwest Europe. A later migratory episode, involving sheep with improved production traits, shaped the great majority of present-day breeds. The ability to differentiate genetically primitive sheep from more modern breeds provides valuable insights into the history of sheep domestication.