Cellular Toxic Effects Research Articles

Novel pH-Responsive Structural Rearrangement of Myristic Acid-Conjugated Quetiapine Nanosuspension for Enhanced Long-Acting Delivery Performance.

Quetiapine myristate (QM), an ester-bonded lipophilic prodrug of quetiapine (QTP), is synthesized and converted into an amphiphilic structure in acidic pH to trigger a novel self-assembled QM nanosuspension (QMN). Following injection, this QMN rearranges within physiological pH to form nanoaggregates in structure, resulting in enhanced physicochemical properties and in vivo therapeutic performance without an initial burst release. The 200-nm-sized QMN exhibits less invasive injection, higher drug content, and better storage stability profile than conventional poly(lactide-co-glycolide) (PLGA) nanosuspensions containing QTP or QM. Following a single intramuscular injection to beagle dogs (35mg kg-1 QTP), QMN undergoes pH-responsive nanoaggregation to form the lipophilic prodrug, providing esterase-oriented sustained release for five weeks compared with the two-week period of PLGA nanosuspensions. Notably, QMN exhibits improved in vivo pharmacokinetic performance with long-acting delivery while minimizing issues associated with polymeric PLGA formulations, including the initial massive burst release, cellular toxicity, and adverse side effects. These results support the further development of QMN as a novel long-acting injectable to improve patient compliance and dosing frequency.

A Life Saving Exchange: A Case of Severe Direct Hyperbilirubinemia And Liver Failure in Leptospirosis Successfully Treated with Plasma Exchange

Leptospirosis is an infection with multiorgan involvement and a high mortality rate. Extreme hyperbilirubinemia in leptospirosis has been reported to exert multiple cellular toxic effects on kidney and liver cells. We describe a young man with leptospirosis who had extreme hyperbilirubinemia, acute liver injury, coagulopathy, hepatic encephalopathy, and pulmonary haemorrhages. Early initiation of therapeutic plasma exchange (PEX) dramatically improved patient’s clinical and biochemical parameters. We conclude that, in addition to the mortality benefit provided by PEX in severe pulmonary hemorrhage syndrome in leptospirosis, PEX is also beneficial in reducing systemic toxic insults of hyperbilirubinemia.

Atrazine: cytotoxicity, oxidative stress, apoptosis, testicular effects and chemopreventive Interventions.

Atrazine (ATZ) is an environmental pollutant that interferes with several aspects of mammalian cellular processes including germ cell development, immunological, reproductive and neurological functions. At the level of human exposure, ATZ reduces sperm count and contribute to infertility in men. ATZ also induces morphological changes similar to apoptosis and initiates mitochondria-dependent cell death in several experimental models. When in vitro experimental models are exposed to ATZ, they are faced with increased levels of reactive oxygen species (ROS), cytotoxicity and decreased growth rate at dosages that may vary with cell types. This results in differing cytotoxic responses that are influenced by the nature of target cells, assay types and concentrations of ATZ. However, oxidative stress could play salient role in the observed cellular and genetic toxicity and apoptosis-like effects which could be abrogated by antioxidant vitamins and flavonoids, including vitamin E, quercetin, kolaviron, myricetin and bioactive extractives with antioxidant effects. This review focuses on the differential responses of cell types to ATZ toxicity, testicular effects of ATZ in both in vitro and in vivo models and chemopreventive strategies, so as to highlight the current state of the art on the toxicological outcomes of ATZ exposure in several experimental model systems.

Identification of Novel Peptides as Potential Modulators of Aβ42 Amyloidogenesis: An in silico Approach.

Alzheimer's disease is a neurodegenerative disease for which no cure is available. The presence of amyloid plaques in the extracellular space of neural cells is the key feature of this fatal disease. The proteolysis of Amyloid Precursor Protein by presenilin leads to the formation of Amyloid-beta peptides (Aβ 42/40). Deposition of 42 residual Aβ peptides forms fibril's structure, disrupting neuron synaptic transmission, inducing neural cell toxicity, and ultimately leading to neuron death. Various novel peptides have been investigated via molecular docking and molecular dynamic simulation studies to investigate their effects on Aβ amyloidogenesis. The sequence-based peptides were rationally designed and investigated for their interaction with Aβ42 monomer and fibril, and their influence on the structural stability of target proteins was studied. Analyzed docking results suggest that the peptide YRIGY (P6) has the highest binding affinity with Aβ42 fibril amongst all the synthetic peptides, and the peptide DKAPFF (P12) similarly shows a better binding with the Aβ42 monomer. Moreover, simulation results also suggest that the higher the binding affinity, the better the inhibitory action. These findings indicate that both the rationally designed peptides can modulate amyloidogenesis, but peptide (P6) has better potential for the disaggregation of the fibrils. In contrast, peptide P12 stabilizes the native structure of the Aβ42 monomer more effectively and hence can serve as a potential amyloid inhibitor. Thus, these peptides can be explored as therapeutic agents against Alzheimer's disease. Experimental testing of these peptides for immunogenicity, stability in cellular conditions, toxic effects and membrane permeability can be the future research scope of this study.

Investigation of Cellular Response to the HSP90 Inhibition in Human Cells Through Thermal Proteome Profiling

Heat shock proteins are chaperones, and they are responsible for protein folding in cells. Heat shock protein 90 (HSP90) is one of the most important chaperones in human cells, and its inhibition is promising for cancer therapy. However, despite the development of multiple HSP90 inhibitors, none of them has been approved for disease treatment due to unexpected cellular toxicity and side effects. Hence, a more comprehensive investigation of cellular response to HSP90 inhibitors can aid in a better understanding of the molecular mechanisms of the cytotoxicity and side effects of these inhibitors. The thermal stability shifts of proteins, which represent protein structure and interaction alterations, can provide valuable information complementary to the results obtained from commonly used abundance-based proteomics analysis. Here, we systematically investigated cell response to different HSP90 inhibitors through global quantification of protein thermal stability changes using thermal proteome profiling, together with the measurement of protein abundance changes. Besides the targets and potential off-targets of the drugs, proteins with significant thermal stability changes under the HSP90 inhibition are found to be involved in cell stress responses and the translation process. Moreover, proteins with thermal stability shifts under the inhibition are upstream of those with altered expression. These findings indicate that the HSP90 inhibition perturbs cell transcription and translation processes. The current study provides a different perspective for achieving a better understanding of cellular response to chaperone inhibition.

Identification and characterization of a heme exporter from the MRP family in Drosophila melanogaster

BackgroundThe heme group constitutes a major functional form of iron, which plays vital roles in various biological processes including oxygen transport and mitochondrial respiration. Heme is an essential nutrient, but its pro-oxidant nature may have toxic cellular effects if present at high levels, and its synthesis is therefore tightly regulated. Deficiency and excess of heme both lead to pathological processes; however, our current understanding of metazoan heme transport is largely limited to work in mammals and the worm Caenorhabditis elegans, while functional analyses of heme transport in the genetically amenable Drosophila melanogaster and other arthropods have not been explored.ResultsWe implemented a functional screening in Schneider 2 (S2) cells to identify putative heme transporters of D. melanogaster. A few multidrug resistance-associated protein (MRP) members were found to be induced by hemin and/or involved in heme export. Between the two plasma membrane-resident heme exporters CG4562 and CG7627, the former is responsible for heme transit across the intestinal epithelium. CG4562 knockdown resulted in heme accumulation in the intestine and lethality that could be alleviated by heme synthesis inhibition, human MRP5 (hMRP5) expression, heme oxygenase (HO) expression, or zinc supplement. CG4562 is mainly expressed in the gastric caeca and the anterior part of the midgut, suggesting this is the major site of heme absorption. It thus appears that CG4562 is the functional counterpart of mammalian MRP5. Mutation analyses in the transmembrane and nucleotide binding domains of CG4562 characterized some potential binding sites and conservative ATP binding pockets for the heme transport process. Furthermore, some homologs in Aedes aegypti, including that of CG4562, have also been characterized as heme exporters.ConclusionsTogether, our findings suggest a conserved heme homeostasis mechanism within insects, and between insects and mammals. We propose the fly model may be a good complement to the existing platforms of heme studies.

Chromatographic Determination of the Mycotoxin Patulin in 219 Chinese Tea Samples and Implications for Human Health.

Patulin (PAT) is a mycotoxin, with several acute, chronic, and cellular level toxic effects, produced by various fungi. A limit for PAT in food of has been set by authorities to guarantee food safety. Research on PAT in tea has been very limited although tea is the second largest beverage in the world. In this paper, HPLC−DAD and GC−MS methods for analysis of PAT in different tea products, such as non-fermented (green tea), partially fermented (oolong tea, white tea, yellow tea), completely fermented (black tea), and post-fermented (dark tea and Pu-erh tea) teas were developed. The methods showed good selectivity with regard to tea pigments and 5-hydroxymethylfurfural (5-HMF) and a recovery of 90–102% for PAT at a 10–100 ppb spiking level. Limit of detection (LOD) and limit of quantification (LOQ) in tea were 1.5 ng/g and 5.0 ng/g for HPLC−UV, and 0.25 ng/g and 0.83 ng/g for GC−MS. HPLC was simpler and more robust, while GC−MS showed higher sensitivity and selectivity. GC−MS was used to validate the HPLC−UV method and prove its accuracy. The PAT content of 219 Chinese tea samples was investigated. Most tea samples contained less than 10 ng/g, ten more than 10 ng/g and two more than 50 ng/g. The results imply that tea products in China are safe with regard to their PAT content. Even an extreme daily consumption of 25 g of the tea with the highest PAT content (124 ng/g), translates to an intake of only 3 μg/person/day, which is still an order of magnitude below the maximum allowed daily intake of 30 µg for an adult.

Novel Lentiviral Vectors for Gene Therapy of Sickle Cell Disease Combining Gene Addition and Gene Silencing Strategies

Sickle cell disease (SCD) is due to a mutation in the β-globin (HBB) gene causing the production of the sickle β S-globin chain. The sickle Hb (HbS, a 2β S2) polymerizes, leading to the formation of sickle-shaped red blood cells that cause vaso-occlusions and organ damage. Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LV) expressing an anti-sickling β-globin transgene (βAS LV) is a promising curative treatment; however, it is partially effective in SCD patients, who still present elevated HbS levels.Here, we aim to improve LVs to boost therapeutic β-like globin levels without increasing the mutagenic vector load in HSPCs. We developed 2 novel LVs expressing βAS together with an artificial microRNA (amiR) targeting either the fetal Hb (HbF) repressor BCL11A (βAS/amiRBCL11A) or the β S-globin (βAS/amiRHBB). By downregulating BCL11A, amiRBCL11A re-activates the expression of the endogenous anti-sickling fetal γ-globin, which, together with βAS, should improve the clinical course of SCD; β S-globin downregulation should favor βAS incorporation in Hb tetramers, increase therapeutic Hb levels and ameliorate the SCD phenotype.First, we developed βAS/amiRBCL11A LV by inserting the amiR in multiple position of the βAS intron 2 under the control of HBB promoter/enhancers to limit BCL11A downregulation to the erythroid lineage and reduce potential amiR-related cellular toxicity and off-target effects. We showed that amiR insertion site did not affect LV titer nor βAS expression in a human erythroid cell line (HUDEP2). BCL11A downregulation in HUDEP2 led to γ-globin gene de-repression and a high proportion of HbF + cells (RTqPCR, HPLC, flow cytometry). Importantly, the total amount of therapeutic β-like globins was substantially higher in βAS/amiRBCL11A LV- than in βAS LV-transduced cells, with no impairment in cell viability or erythroid differentiation.In parallel, we designed 17 amiRs targeting HBB and generated the corresponding βAS/amiRHBB LVs. We tested these LVs in HUDEP2 and selected 2 amiRs efficiently downregulating β-globin at mRNA and protein levels (RT-qPCR and Western Blot). Of note, we modified the βAS transgene by inserting silent mutations that prevent its recognition by the amiR (βASm).Finally, we tested βAS/amiRBCL11A and βAS/amiRHBB LVs in HSPCs from SCD patients. HSPC-derived erythroid cells transduced with βAS/amiRBCL11A LV showed increased HbF levels, although HbS levels remained high. To further reduce β S-globin levels, we targeted the β S-globin mRNA using the βAS/amiRHBB LV. Efficient HSPC transduction by βASm/amiRHBB LV led to a substantial decrease of β S-globin transcripts in HSPC-derived erythroid cells compared to the βAS LV-transduced cells (RTqPCR) at a VCN/cell of 2. Notably, the amiR specifically down-regulated β S-globin, without affecting βAS expression. In βASm/amiRHBB- vs βAS LV-transduced cells, HPLC analysis showed that β S-globin downregulation led to a significant decrease of HbS, which represented 58% and 71% of the total Hb, respectively). This was associated with a significant increase of the therapeutic Hb in βASm/amiRHBB LV- vs βAS LV-transduced erythroid cells (38% and 27% of the total Hb, respectively). Importantly, we observed a substantial reduction of the proportion of HbS-positive cells in βASm/amiRHBB- vs βAS LV-transduced samples (from 96% to 70%; Figure 1A). The increased incorporation of βAS in Hb tetramers and the decrease in β S-globin led to a better correction of the sickling phenotype in mature RBCs derived from HSPCs transduced with βASm/amiRHBB LV- compared to βAS LV (55% and 84% of sickling cells, respectively; Figure 1B). A clonal assay of hematopoietic progenitors showed no impairment in HSPC viability and differentiation towards the erythroid and myeloid lineages upon transduction with bifunctional LVs. βASm/amiRHBB LV showed a standard lentiviral integration profile. Finally, we performed RNAseq to further evaluate the safety of our therapeutic strategy.In conclusion, we created a LV able to concomitantly silence the β S-globin and express βAS, achieving clinically relevant levels of therapeutic Hb and efficient correction of the sickling phenotype. Therefore, the combination of gene addition and gene silencing strategies can improve the efficacy of current therapeutic approaches, representing a novel treatment for SCD. [Display omitted] DisclosuresCavazzana: Smart Immune: Other: co-founder.

Cocaine-induced pulmonary complications

Pulmonary complications of cocaine among users are common. Manifestations include lung congestion, intra-alveolar edema, and diffuse alveolar hemorrhage (DAH). Direct cellular toxicity, eosinophilia, barotrauma, and vasoactive effects of cocaine are believed to induce DAH. We present a rare case of cocaine-associated focal alveolar hemorrhage mimicking malignancy on imaging. Initially contemplated biopsy was avoided based on rapid growth of concerning lung lesion, with subsequent near resolution on follow-up. This case illustrates the importance of epidemiologic and temporal multimodality correlation when evaluating indeterminate lung lesions.

Biological toxicity assessment of carbamate pesticides using bacterial and plant bioassays: An in-vitro approach

In recent times, agricultural practices mainly rely on agrochemicals and pesticides to safe-guard edible crops against various pests and to ensure high yields. However, their indiscriminate use may cause severe environmental hazards that directly and negatively affect soil microorganisms and crop productivity. Considering these, present study was aimed to assess the toxicity of carbamate pesticides namely carbamoyl (CBL), methomyl (MML) and carbofuran (CBN) using bacterial and plant (Vigna mungo L.) bioassays. All pesticide doses (25–100 μg mL−1) showed negative effect on bacteria as well as plant. Growth, morphology, survival, cellular respiration and inner membrane permeability of Sinorhizobiumsaheli was hampered when exposed to pesticides. Pesticide induced morphological changes viz. aberrant margins; cellular cracking and distortion/damage in S. saheli were obvious under scanning electron microscope (SEM). The 100 μgCBNmL−1 had maximum inhibitory effect and it reduced survivability of S. saheli by 75%. In addition, biofilm formation ability of S. saheli was inhibited in a pesticides-dose dependent manner and it was statistically (p ≤ 0.05) significant. Pesticides indorsed significant changes in biomarker enzymatic assays and oxidative stress parameters towards S. saheli. Furthermore, at 100 μgCBNmL−1, germination efficiency, root, shoot length, plant survival and tolerance index of V. mungo were decrease by 50, 75, 65, 70 and 66%, respectively over control. Staining of pesticide treated roots with fluorescently labeled dyes propidium iodide (PI) and acridine orange (AO) showed increased oxidative stress, ROS generation and membrane permeability as revealed under confocal laser scanning microscope (CLSM). Furthermore, stressor metabolites and antioxidant enzymes in plant seedlings were progressively enhanced with increasing concentration of pesticides. Conclusively, present finding bestow an insights into a mechanistic approach of carbamate pesticide induced phyto, morpho and cellular toxic effects towards soil bacterium as well as plant with forthcoming implications for designing the pesticides to reduce their toxic/harmful effects.

In vitro cytotoxicity of different dental resin-cements on human cell lines

Adhesive resin-cements are increasingly used in modern dentistry. Nevertheless, released substances from resin materials have been shown to cause cellular toxic effects. Disc-shaped specimens from 12 different resin cements and one conventional zinc phosphate cement were prepared and used for direct stimulation of five different human cell lines via transwell cell culture system or in an indirect way using conditioned cell culture media. Cytotoxicity was determined using LDH and BCA assays. All tested cements led to a decrease of cell viability but to a distinct extent depending on cell type, luting material, and cytotoxicity assay. In general, cements exhibited a more pronounced cytotoxicity in direct stimulation experiments compared to stimulations using conditioned media. Interestingly, the conventional zinc phosphate cement showed the lowest impact on cell viability. On cellular level, highest cytotoxic effects were detected in osteoblastic cell lines. All resin cements reduced cell viability of human cells with significant differences depending on cell type and cement material. Especially, osteoblastic cells demonstrated a tremendous increase of cytotoxicity after cement exposure. Although the results of this in vitro study cannot be transferred directly to a clinical setting, it shows that eluted substances from resin cements may disturb osteoblastic homeostasis that in turn could lead to conditions favoring peri-implant bone destruction. Thus, the wide use of resin cements in every clinical situation should be scrutinized. A correct use with complete removal of all cement residues and a sufficient polymerization should be given the utmost attention in clinical usage.

Wheat phytase can alleviate the cellular toxic and inflammatory effects of lipopolysaccharide.

The objective of this study was to characterize the enzymatic hydrolysis of lipopolysaccharide (LPS) by wheat phytase and to investigate the effects of wheat phytase-treated LPS on in vitro toxicity, cell viability and release of a pro-inflammatory cytokine, interleukin (IL)-8 by target cells compared with the intact LPS. The phosphatase activity of wheat phytase towards LPS was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine. In vitro toxicity of LPS hydrolyzed with wheat phytase in comparison to intact LPS was assessed. Cell viability in human aortic endothelial (HAE) cells exposed to LPS treated with wheat phytase in comparison to intact LPS was measured. The release of IL-8 in human intestinal epithelial cell line, HT-29 cells applied to LPS treated with wheat phytase in comparison to intact LPS was assayed. Wheat phytase hydrolyzed LPS, resulting in a significant release of inorganic phosphate for 1 h (p < 0.05). Furthermore, the degradation of LPS by wheat phytase was nearly unaffected by the addition of L-phenylalanine, the inhibitor of tissue-specific alkaline phosphatase or L-homoarginine, the inhibitor of tissue-non-specific alkaline phosphatase. Wheat phytase effectively reduced the in vitro toxicity of LPS, resulting in a retention of 63% and 54% of its initial toxicity after 1–3 h of the enzyme reaction, respectively (p < 0.05). Intact LPS decreased the cell viability of HAE cells. However, LPS dephosphorylated by wheat phytase counteracted the inhibitory effect on cell viability. LPS treated with wheat phytase decreased IL-8 secretion from intestinal epithelial cell line, HT-29 cell to 14% (p < 0.05) when compared with intact LPS. In conclusion, wheat phytase is a potential therapeutic candidate and prophylactic agent for control of infections induced by pathogenic Gram-negative bacteria and associated LPS-mediated inflammatory diseases in animal husbandry.

Stabilization of Mitochondrial Function by Ellagic Acid Prevents Celecoxib-induced Toxicity in Rat Cardiomyocytes and Isolated Mitochondria.

The possible action of polyphenolic compounds in the reduction of reactive oxygen species (ROS) and mitochondrial toxicity may suggest them as putative agents for the treatment of drug-induced mitochondrial dysfunction and cardiotoxicity. This study was designed to explore protective effect of ellagic acid (EA) against celecoxib-induced cellular and mitochondrial toxicity in cardiomyocytes and their isolated mitochondria. In order to do this, isolated cardiomyocytes and mitochondria were pretreated with 3 different concentrations of EA (10, 50 and 100 µM), after which celecoxib (16 µg/ml) was added to promote deleterious effects on cells and mitochondria. Using flow cytometry and biochemical methods, the parameters of cellular and mitochondrial toxicity were investigated. Our results showed that celecoxib (16 µg/ml) caused a significant decrease in cell viability, mitochondrial membrane potential (MMP), glutathione (GSH) in intact cardiomyocytes and succinate dehydrogenase (SDH) activity, MMP collapse, and mitochondrial swelling, and a significant increase in reactive oxygen species (ROS) formation, lipid peroxidation (LP) and oxidative stress in isolated mitochondria. Also, our results revealed that co-administration of EA (50 and 100 µM) with celecoxib significantly attenuated the cellular and mitochondrial toxicity effects. In this study, we showed that simultaneous treatment with of EA ameliorated the cellular and mitochondrial toxicity induced by celecoxib, with cardiomyocytes presenting normal activity compared to the control group, and mitochondria retaining their normal activity.

Recognizing Radiation-induced Changes in the Central Nervous System: Where to Look and What to Look For.

Radiation therapy (RT) continues to play a central role as an effective therapeutic modality for a variety of tumors and vascular malformations in the central nervous system. Although the planning and delivery techniques of RT have evolved substantially during the past few decades, the structures surrounding the target lesion are inevitably exposed to radiation. A wide variety of radiation-induced changes may be observed at posttreatment imaging, which may be confusing when interpreting images. Histopathologically, radiation can have deleterious effects on the vascular endothelial cells as well as on neuroglial cells and their precursors. In addition, radiation induces oxidative stress and inflammation, leading to a cycle of further cellular toxic effects and tissue damage. On the basis of the time of expression, radiation-induced injury can be divided into three phases: acute, early delayed, and late delayed. Acute and early delayed injuries are usually transient and reversible, whereas late delayed injuries are generally irreversible. The authors provide a comprehensive review of the timeline and expected imaging appearances after RT, including the characteristic imaging features after RT with concomitant chemotherapy. Specific topics discussed are imaging features that help distinguish expected posttreatment changes from recurrent disease, followed by a discussion on the role of advanced imaging techniques. Knowledge of the RT plan, the amount of normal structures included, the location of the target lesion, and the amount of time elapsed since RT is highly important at follow-up imaging, and the reporting radiologist should be able to recognize the characteristic imaging features after RT and differentiate these findings from tumor recurrence. ©RSNA, 2020.

Silver Sulfadiazine's Effect on Keratin-19 Expression as Stem Cell Marker in Burn Wound Healing.

BackgroundBurn wounds are one of the causes of cutaneous injury that involve both epidermal and dermal layers of skin. Silver sulfadiazine (SSD) has been widely used to treat burn wounds, however recent studies have found the treatment to have some drawbacks, such as cellular toxicity effects. Cutaneous wound regeneration is known to start from the basal layer of the epidermal epithelial cells, which are enriched with highly proliferative cells. Keratin-19 (K19) is one of the epidermal stem cell biomarkers found in the skin. This study aims to explore the expression of K19 in burn wound tissue and to investigate the effect of SSD on its expression.MethodsWe created a burn wound model in Sprague Dawley rats and randomly divided them into control and SSD groups. Wound closure was evaluated (visitrak) overtime series followed by histological evaluation of K19 expression in the wound tissue (immunohistochemistry staining).ResultsOur model successfully represents full-thickness damage caused by a burn wound. The SSD group showed a faster reduction of wound surface area (wound closure) compared to the control group with the peak at day 18 post wounding (p < 0.05). K19 expression was found in both groups and was distributed on epidermal layers, hair follicles and dermis of granulation tissue showing similar patterns.ConclusionTopical application of SSD on burn wounds showed superiority in wound closure and is likely to have no harmful effect on epidermal stem cells. However, further study is required to investigate the effect of silver species on cell viability and toxicity effects during long term treatment.

CRISPR-Cas9 Gene Editing of Hematopoietic Stem Cells from Patients with Friedreich’s Ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by expansion of GAA repeats in intron 1 of the frataxin (FXN) gene, leading to significant decreased expression of frataxin, a mitochondrial iron-binding protein. We previously reported that syngeneic hematopoietic stem and progenitor cell (HSPC) transplantation prevented neurodegeneration in the FRDA mouse model YG8R. We showed that the mechanism of rescue was mediated by the transfer of the functional frataxin from HSPC-derived microglia/macrophage cells to neurons/myocytes. In this study, we report the first step toward an autologous HSPC transplantation using the CRISPR-Cas9 system for FRDA. We first identified a pair of CRISPR RNAs (crRNAs) that efficiently removes the GAA expansions in human FRDA lymphoblasts, restoring the non-pathologic level of frataxin expression and normalizing mitochondrial activity. We also optimized the gene-editing approach in HSPCs isolated from healthy and FRDA patients’ peripheral blood and demonstrated normal hematopoiesis of gene-edited cells in vitro and in vivo. The procedure did not induce cellular toxic effect or major off-target events, but a p53-mediated cell proliferation delay was observed in the gene-edited cells. This study provides the foundation for the clinical translation of autologous transplantation of gene-corrected HSPCs for FRDA.

Synergistic cellular toxicity and uptake effects of iodixanol conjugated to anionic linear globular dendrimer G2

Objective(s): Early diagnosis of cancer using noninvasive imaging techniques has been discussed in several recent studies. The present study aimed to assess the synergistic effects of iodixanol-conjugated polyethylene glycol (PEG)-citrate (anionic linear globular) dendrimer G2 on MCF-7 breast cancer cells and human embryonic kidney 293 (HEK293) cells. Materials and Methods: PEG-citrate dendrimer G2 was synthesized and purified. The product was characterized using atomic force microscopy (AFM), electron energy loss spectroscopy (EELS), dynamic light scattering (DLS). At the next stage, the product was conjugated to iodixanol, purified and lyophilized. The cytotoxic effects of the iodixanol, plain PEG-citrate dendrimer G2, and iodixanol-PEG-citrate dendrimer G2 complex were evaluated using methylthiazole-tetrazolium (MTT) assay on the MCF-7 and HEK293 cells. Inductively coupled plasma mass spectrometry (ICP MS) is a mass spectrometry technique, which applies inductively coupled plasma to ionize samples.Results: According to the obtained results, the uptake of PEG-citrate dendrimer G2 iodixanol increased significantly compared to iodixanol alone (P<0.05), indicating the importance of lack of significant in-vitro toxicity. Moreover, in the particle size and higher negative zeta potential confirmed the loading of iodixanol in dendrimer G2. Increase, the loading of iodixanol in dendrimer was confirmed by the chemical shifts in HNMR. Conclusion: Therefore, it was concluded that the addition of anionic linear globular dendrimer G2 to iodixanol affected the cellular uptake of the drug with no significant toxicity. Recent findings also confirmed that this novel complex could be applied as an effective cancer imaging agent for molecular biology and molecular imaging applications.

Prediction of paclitaxel pharmacokinetic based on in vitro studies: Interaction with membrane models and human serum albumin.

Interactions of paclitaxel (PTX) with models mimicking biological interfaces (lipid membranes and serum albumin, HSA) were investigated to test the hypothesis that the set of in vitro assays proposed can be used to predict some aspects of drug pharmacokinetics (PK). PTX membrane partitioning was studied by derivative spectrophotometry; PTX effect on membrane biophysics was evaluated by dynamic light scattering, fluorescence anisotropy, atomic force microscopy and synchrotron small/wide-angle X-ray scattering; PTX distribution/molecular orientation in membranes was assessed by steady-state/time-resolved fluorescence and computer simulations. PTX binding to HSA was studied by fluorescence quenching, derivative spectrophotometry and dynamic/electrophoretic light scattering. PTX high membrane partitioning is consistent with its efficacy crossing cellular membranes and its off-target distribution. PTX is closely located in the membrane phospholipids headgroups, also interacting with the hydrophobic chains, and causes a major distortion of the alignment of the membrane phospholipids, which, together with its fluidizing effect, justifies some of its cellular toxic effects. PTX binds strongly to HSA, which is consistent with its reduced distribution in target tissues and toxicity by bioaccumulation. In conclusion, the described set of biomimetic models and techniques has the potential for early prediction of PK issues, alerting for the required drug optimizations, potentially minimizing the number of animal tests used in the drug development process.

In Vitro Labeling Mesenchymal Stem Cells with Superparamagnetic Iron Oxide Nanoparticles: Efficacy and Cytotoxicity.

Mesenchymal stem cell (MSC) therapy has emerged as a potential therapeutic option for several diseases due to their unique properties of releasing important bioactive factors. Despite the advances in stem cell therapy, it is still difficult to accurately determine the mechanisms of cell activities after in vivo transplantation. The application of noninvasive cell tracking approaches is important to determine tissue distribution and the lifetime of stem cells following their injection, which consequently provides knowledge about the mechanisms of stem cell tissue repair. Superparamagnetic iron oxide nanoparticles (SPION)can provide a very useful tool for labeling and tracking stem cells by magnetic resonance imaging without causing toxic cellular effects and do not elicit any other side effects. Here we describe how to use SPIONs to label mesenchymal stem cells and evaluate efficacy and potential cytotoxicity in vitro.

Quantitative assessment of cellular uptake and differential toxic effects of HgSe nanoparticles in human cells

A comparative study of the cytotoxicity of HgSe nanoparticles and that induced by inorganic mercury and methylmercury in two cell lines.