Cell Electrophoresis Research Articles

Intelligent sensing technology inspired by cell electrophoresis: Dielectric polarization sensor for monitoring the electrical equipment status of IoT nodes

The rapid development of the Internet of Things (IoT) era has led to the emergence of various types of information sensing devices. The operation and maintenance of IoT devices require a large-scale use of sensors at the expense of high energy consumption. In this work, we develop a self-powered voltage monitoring sensor (VSM-S) based on the principle of dielectric polarization, which is inspired by the cell electrophoresis phenomenon. This sensor imitates the structure of biological cells and takes advantage of the easier polarization characteristics of liquid medium with high dielectric constant in an external electric field. It collects electric field energy to achieve self-powering function and can monitor the position and status of the electric field source in real-time through output signals. Experiments show that the designed VSM-S has the characteristics of fast response (10−9-10−15 s), high sensitivity (0.3 nA/V), and wide application range. Furthermore, its integration with monitoring systems enables applications in smart industries, smart homes, and smart grids. This work has considerable development prospects in the fields of wearable sensors and the IoTs.

Two palladium (II) complexes derived from halogen-substituted Schiff bases and 2-picolylamine induce parthanatos-type cell death in sensitive and multi-drug resistant CCRF-CEM leukemia cells

The use of cisplatin and its derivatives in cancer treatment triggered the interest in metal-containing complexes as potential novel anticancer agents. Palladium (II)-based complexes have been synthesized in recent years with promising antitumor activity. Previously, we described the synthesis and cytotoxicity of palladium (II) complexes containing halogen-substituted Schiff bases and 2-picolylamine. Here, we selected two palladium (II) complexes with double chlorine-substitution or double iodine-substitution that displayed the best cytotoxicity in drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells for further biological investigation. Surprisingly, these compounds did not significantly induce apoptotic cell death. This study aims to reveal the major mode of cell death of these two palladium (II) complexes. We performed annexin V-FITC/PI staining and flow cytometric mitochondrial membrane potential measurement followed by western blotting, immunofluorescence microscopy, and alkaline single cell electrophoresis (comet assay). J4 and J6 still induced neither apoptosis nor necrosis in both leukemia cell lines. They also insufficiently induced autophagy as evidenced by Beclin and p62 detection in western blotting. Interestingly, J4 and J6 induced a novel mode of cell death (parthanatos) as mainly demonstrated in CCRF-CEM cells by hyper-activation of poly(ADP-ribose) polymerase 1 (PARP) and poly(ADP-ribose) (PAR) using western blotting, flow cytometric measurement of mitochondrial membrane potential collapse, nuclear translocation of apoptosis-inducing factor (AIF) by immunofluorescence microscopy, and DNA damage by alkaline single cell electrophoresis (comet assay). AIF translocation was also observed in CEM/ADR5000 cells. Thus, parthanatos was the predominant mode of cell death induced by J4 and J6, which explains the high cytotoxicity in CCRF-CEM and CEM/ADR5000 cells. J4 and J6 may be interesting drug candidates and deserve further investigations to overcome resistance of tumors against apoptosis. This study will promote the design of further novel palladium (II)-based complexes as chemotherapeutic agents.

Diversity in mitotic DNA repair efficiencies between commercial inbred maize lines and native Central American purple landraces

BackgroundHomologous recombination allows plants to repair double strand breaks in DNA, which if unrepaired may lead to cell death. In this project, we determined the relative DNA repair efficiency of the US inbred lines B73 and Mo17 and Central American purple landraces from Guatemala and Costa Rica with the purpose to uncover genetic differences that may allow for the breeding of new lines better adapted to tolerate DNA damage caused by environmental factors.MethodsSingle cell electrophoresis was used to analyze the relative DNA repair ability of several lines from the US and Central America exposed to radiomimetic agent Zeocin, and these results were in turn compared with High Resolution Melting analyses of key genes for homologous DNA recombination. The significance of differences between treatments was evaluated with the Di Rienzo, Guzmán and Casanoves (DGC) test, while High Resolution Melting and difference curves were generated with the R package “HRM.curve”. Curves were created as a negative first derivative (−d(RFU)/d(T)) using normalized relative fluorescence values (RFUs) after background removal. The kinase gene ZeaATM1 was amplified and sequenced in B73, Mo17, P1 and P2. Multiple sequence alignment of DNA and aminoacid sequences was performed using ClustalW. Protein sequence analysis was done in UniProt to compare the resulting aminoacid sequences from maize to the available sequences from Arabidopsis thaliana ecotype Col-0 (ATM protein code: Q9M3G7).ResultsSingle-cell electrophoresis results of statistical significance suggested that the landrace P1-Pujagua Santa Cruz is resistant to damage caused by the radiomimetic agent Zeocin, and landrace P2-Pujagua La Cruz was able to repair all DNA damage after 24 h of treatment and 1 h of recovery time. In contrast, line Mo17 was unable to repair the damage, but B73 and the landraces Jocopilas (Guatemalan), Orotina Congo, and Talamanca were partially able to repair the DNA damage. High resolution melting analysis of the putative homologous DNA repair gene ZeaATM1 revealed that landraces P1 and P2 may harbor polymorphisms for this gene, and P1 may harbor other polymorphisms for the transcription factor ZeaSOG1 as well as the tumor suppressor ZeaRAD51 and recombinase ZeaBRCA1. The kinase gene ZeaATM1 was sequenced, and results indicate that in lines P1 and P2 there are polymorphisms near and within the FATC domain, a domain required for the activation of ATM1-mediated repair of DNA damage.ConclusionsSignificant differences in DNA repair efficiency exist between inbred lines and landraces of maize and may be linked to allelic diversity in key genes for homologous recombination. Thus, Central American landraces could be used for breeding tolerance to genotoxic stress.

Evaluation of the effect of the neurotoxin MPTP and the antiparkinsonian drug hemantane on the DNA integrity in the brain structures of C57BL/6 mice

Hemantane (N-adamant-2-yl-hexamethylenimine hydrochloride) is an antiparkinsonian drug with a multicomponent mechanism of action, including a modulating effect on the activity of dopamine and serotonergic mediator systems, a selective inhibitory effect on MAO-B, properties of a low-affinity non-competitive channel blocker of glutamate NMDA receptors, has moderate antiradical and anti-inflammatory activity. The aim of this study was to assess the effect of the neurotoxin MPTP, which is used for modeling of parkinsonian syndrome, and the hemantane on the DNA integrity in the striatum and frontal cortex of the brain of C57BL/6 mice by the DNA comet assay – gel electrophoresis of DNA single cells. Results. In the first experiment, hemantane was administered once a day for 5 days before the MPTP (20 mg/kg, i.p.), then together with MPTP once a day for 5 days. In the second experiment hemantane 10 mg/kg was injected preliminarily for 4 days and 40 minutes before MPTP 30 mg/kg. The obtained results confirm the absence of an effect on DNA of hemantane at a therapeutic dose of 10 mg / kg. In the experimental schemes used, we did not reveal the expected increase in the level of DNA damage under the influence of MPTP, and, accordingly, we were not able to assess the protective effect of hemantane.

Short- and long-term reproducibility of the COMET assay for measuring DNA damage biomarkers in frozen blood samples of the EPIC-Heidelberg cohort

The comet assay is widely used for quantification of genomic damage in humans. Peripheral blood derived mononuclear cells (PBMCs) are the most often used cell type for this purpose. Since the comet assay can be performed in an enhanced throughput format, it can be applied to large sample collections such as biobanks. The European Prospective Investigation into Cancer and Nutrition (EPIC) study is one of the largest existing prospective cohort studies, and the German Cancer Research Institute (DKFZ) in Heidelberg is a participating center with 25.000 frozen blood samples stored from around 25 years ago, enabling retrospective assessment of disease risk factors. However, experience with decades long frozen samples in the comet assay is so far missing. In Heidelberg, 800 study participants were re-invited twice between 2010 and 2012 to donate further blood samples. Here, we analyzed 299 Heidelberg-EPIC samples, compiled from frozen PBMC and buffy coat preparations selected from the different sampling time points. In addition, 47 frozen PBMC samples from morbidly obese individuals were included. For buffy coat samples, we observed a poor correlation between DNA damage in the same donors assessed at two sampling time points. Additionally, no correlation between DNA damage in buffy coat samples and PBMCs was found. For PBMCs, a good correlation was observed between samples of the same donors at the two time points. DNA damage was not affected by age and smoking status, but high BMI (>30; obesity) was associated with increased DNA damage in PBMCs. There was no indication for a threshold of a certain BMI for increased DNA damage. In conclusion, while 25 year-long stored buffy coat preparations may require adaptation of certain experimental parameters such as cell density and electrophoresis conditions, frozen PBMC biobank samples can be analyzed in the comet assay even after a decade of storage.

Comet Assay for the Detection of Single and Double-Strand DNA Breaks.

DNA damage is a common event that occurs during the life span of the majority of cells, as a result of aberrant energy metabolism, exposure to radiation, DNA damaging chemicals, and viral infections. Rapid, sensitive, and economical methods for the detection of DNA damage offer valuable insights into DNA repair, drug genotoxicity, and biomonitoring. The comet assay, or single cell electrophoresis, has emerged as a popular method for detecting single- and double-stranded DNA breaks in single cells. This experimental technique is based on the fact that, when exposed to an electric current, damaged DNA fragments in a gel, migrate farther from the nucleus toward the anode resulting in the shape of a comet. This assay is versatile, quantitative, simple to perform and exhibits high sensitivity; however, consistent experimental conditions must be maintained to ensure assay reproducibility. Electrophoresis can be performed under neutral conditions to detect solely double stranded breaks, or under alkaline conditions to detect both single- and double-stranded breaks. This chapter describes the protocol for both alkaline and neutral comet assays.

The role of the RNA binding protein HnRNP K in the pathogenesis of frontotemporal lobar degeneration.

HnRNP K is an RNA-binding protein belonging to the family of heterogeneous nuclear ribonucleoproteins (hnRNPs). Like other hnRNPs such as the better known TDP-43, hnRNP K is predominantly nuclear where it plays an important role in pre-mRNA splicing. HnRNPs can also shuttle to the cytoplasm and we find that hnRNP K is predominantly cytoplasmic in pyramidal neurons in Frontotemporal Lobar Degeneration (FTLD). The loss of nuclear hnRNPs in disease states is directly linked to splicing misregulation and aberrant RNA processing. Indeed, loss of nuclear TDP-43 leads to cryptic exon inclusion in gene targets and the generation of truncated proteins. We aimed to investigate changes in RNA processing and presence of cryptic events upon hnRNP K mislocalisation in FTLD. Bioinformatic analysis of a neuronal cell line with hnRNP K knockdown identified a set of cryptic events. We have validated these events using PCR amplification and gel electrophoresis in both SH-SY5Y cells with hnRNP K knockdown and in post-mortem human brain tissue. The loss of nuclear hnRNP K results in the inclusion of cryptic exons in multiple gene targets in both SH-SHY5Y cells with hnRNP K knockdown and FTLD brain regions exhibiting hnRNP K nuclear loss. The mislocalisation of hnRNP K from the nucleus to the cytoplasm in FTLD is associated with the inclusion of cryptic exons in multiple gene targets. This suggests that similarly to TDP-43, hnRNP K plays a crucial role in RNA processing and splicing regulation which when impaired could lead to transcriptional dysregulation and disease pathogenesis.

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Tollens Reaction‐Based Integration of Thin Film Wall Electrodes into Microfluidic PDMS Devices

AbstractThe integration of electrodes into microfluidic devices is a prerequisite for several key technologies such as electrophoresis, dielectrophoresis, electrowetting, or analysis and manipulation of biological cells in organ‐on‐a‐chip applications. However, conventional sputtering or metal deposition methods result in electrodes on the top or the bottom of the microfluidic device leading to either an inhomogeneous electrical field or considerable restrictions regarding optical analysis methods. Here, a novel method for integrating electrodes into microfluidic devices based on the deposition of silver films on the sidewalls of microfluidic channels by Tollens reaction is presented. Aldehydes diffuse readily through polydimethylsiloxane walls and reduce noble metal ions such as silver, which in turn precipitate and deposit on the phase border as homogeneous thin layers. The process results in sidewall electrodes that adopt the channel's geometry and offer a highly homogeneous electrical field in the case of a rectangular channel. The electrodes were analyzed by dissipative X‐ray (EDX) and electrical impedance spectroscopy, while the electrical field was visualized by particle image velocimetry. The method can fabricate electrodes in different shapes rendering the process highly promising for a wide range of different electrochemical applications in the field of microfluidics, while simultaneously enable optical analysis methods.

Comparative Toxicity of Fly Ash: An In Vitro Study.

Fly ash produced during coal combustion is one of the major sources of air and water pollution, but the data on the impact of micrometer-size fly ash particles on human cells is still incomplete. Fly ash samples were collected from several electric power stations in the United States (Rockdale, TX; Dolet Hill, Mansfield, LA; Rockport, IN; Muskogee, OK) and from a metallurgic plant located in the Russian Federation (Chelyabinsk Electro-Metallurgical Works OJSC). The particles were characterized using dynamic light scattering, atomic force, and hyperspectral microscopy. According to chemical composition, the fly ash studied was ferro-alumino-silicate mineral containing substantial quantities of Ca, Mg, and a negligible concentration of K, Na, Mn, and Sr. The toxicity of the fly ash microparticles was assessed in vitro using HeLa cells (human cervical cancer cells) and Jurkat cells (immortalized human T lymphocytes). Incubation of cells with different concentrations of fly ash resulted in a dose-dependent decrease in cell viability for all fly ash variants. The most prominent cytotoxic effect in HeLa cells was produced by the ash particles from Rockdale, while the least was produced by the fly ash from Chelyabinsk. In Jurkat cells, the lowest toxicity was observed for fly ash collected from Rockport, Dolet Hill and Muscogee plants. The fly ash from Rockdale and Chelyabinsk induced DNA damage in HeLa cells, as revealed by the single cell electrophoresis, and disrupted the normal nuclear morphology. The interaction of fly ash microparticles of different origins with cells was visualized using dark-field microscopy and hyperspectral imaging. The size of ash particles appeared to be an important determinant of their toxicity, and the smallest fly ash particles from Chelyabinsk turned out to be the most cytotoxic to Jukart cells and the most genotoxic to HeLa cells.

Molecular and Hematological Analysis of Alpha- and Beta-Thalassemia in a Cohort of Mexican Patients.

Introduction: Alpha- and beta-thalassemia are caused by reduced or absent synthesis of hemoglobin (Hb) subunits α and/or β. HBA2, HBA1, and HBB mutations are the main cause of thalassemias. The aim of this article is to analyze molecular and hematological features of α- and β-thal in a cohort of Mexican patients. Methods: One hundred forty-one thalassemia patients were studied. Peripheral blood was collected for blood cell count, electrophoresis, Hb quantification, and molecular testing. Molecular screening was performed by Gap-PCR, ARMS-PCR, Sanger sequencing, and MLPA. Results: Fifty-four patients had α-thal, 75 β-thal, and 12 patients were complex cases, we observed 13 α- and 18 β-thal alleles in 43 genotypes, -α3.7/αα and βCd39C>T/β were the most frequent. Four α-thal deletions (-Mex4 included HBA2 and HBA1, whereas (αα)Mex5, Mex6 and Mex7 involved MCS-R), a hereditary persistence of fetal hemoglobin-2 like (HPFH-2 like) deletion and six alleles not previously reported in Mexicans (α-59C>Tα, -α4.2, αPlasenciaα, β-32C>T, βInitCdA>C and βFSCd71/72+A) were identified. Conclusion: The observed alleles denote the high heterogeneity and multiple origin admixture of Mexican population. Hematological data are consistent with genotypes, variability in simple carriers, from asymptomatic forms to mild or moderate anemia, was ascertained. We emphasize the importance to consider hematological parameters to establish adequate molecular screening strategies.

Design of a novel electrospun PVA platform for gene therapy applications using the CHAT peptide

The electrospinning of polymers has previously shown excellent potential for localised gene therapy. Thus, it was proposed that for the first time, the cell-penetrating CHAT peptide could be utilised to deliver DNA via electrospun nanofibres for localised gene therapy treatment. CHAT is an effective delivery system that encapsulates pDNA to form nanoparticles with the physicochemical characteristics for cellular uptake and protein generation. In this study, the production of smooth, bead-free PVA nanofibres by electrospinning was optimised through a Design of Experiments approach. Bead-free PVA nanofibres were consistently produced using the optimised parameters as follows: applied voltage (8 kV); collector-emitter distance (8 cm); polymer flow rate (4 µL/min); polymer concentration (9 wt% polymer); PVA MW (146–180 kDa). PVA nanofibres were subsequently crosslinked in 1 vol% glutaraldehyde in methanol to confer stability under aqueous conditions with minimal change to morphology, and no compromise to biocompatibility. Nanoparticles of CHAT/pDNA were synthesised and incorporated into the crosslinked nanofibres via soak-loading. Evaluation studies indicated that 100% of the loaded cargo was released within 48 h from the nanofibres. Furthermore, the released pDNA retained structural integrity and functionality as confirmed by gel electrophoresis and transfection studies in NCTC-929 fibroblast cells. Taken together, this data demonstrates that delivery of CHAT/pDNA nanoparticles from electrospun PVA nanofibres represents a solution for localised gene therapy.

Neutral Comet Assay to Detect and Quantitate DNA Double-Strand Breaksin Hematopoietic Stem Cells.

In vertebrates, hematopoietic stem cells (HSCs) regulate the supply of blood cells throughout the lifetime and help to maintain homeostasis. Due to their long lifespan, genetic integrity is paramount for these cells, and accordingly, a number of stem cell-specific mechanisms are employed. However, HSCs tend to show more DNA damage with increasing age due to an imbalance between proliferation rates and DNA damage responses. The comet assay is the most common and reliable method to study DNA strand breaks at the single-cell level. This procedure is based on the electrophoresis of agarose-embedded lysed cells. Following the electrophoretic mobilization of DNA, it is stained with fluorescent DNA-binding dye. Broken DNA strands migrate based on fragment size and form a tail-like structure called "the comet," whereas intact nuclear DNA remains a part of the head of the comet. Since the alkaline comet assay fails to differentiate between single and double-strand breaks (DSBs), we used a neutral comet assay to quantitate the DSBs in HSCs upon aging and other physiological stresses. The protocol presented here provides procedural details on this highly sensitive, rapid, and cost-effective assay, which can be used for rare populations of cells such as HSCs. Graphical abstract: The neutral comet assay is an extremely useful tool that allows the detection and quantitation of double-strand DNA breaks at the single-cell level. The graphical abstract represents a flowchart for the neutral comet assay procedure.

Dual-modal fullerenol probe containing glypican-3 monoclonal antibody for electron paramagnetic resonance/fluorescence imaging

The multi-modal imaging contrast agent has been attracted an increased interest to the tumor detecting and diagnosis in these recent years since the multi-modal imaging technology combines the advantages of several medical imaging modalities. A water-soluble fullerenol probe containing glypican-3 monoclonal antibody (GPC3-mAb) group mAb-GPC3-C60(OH)10-(TMIO)3-(NI)3 was synthesized by the incorporation of 5-formyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (FTMIO), naphthalimide (NI) and GPC3-mAb as a tumor-targeting group into fullerenol C60(OH)11. This nanomaterial was further characterized by FTIR, UV, MS, fluorescence, etc., and then its properties including electron paramagnetic resonance (EPR), particle size, electrophoresis, electrochemical assay, cell cytotoxicity, cell uptake and fluorescent imaging of HeLa tumor cells in vitro were also evaluated. Experiment results indicated that mAb-GPC3-C60(OH)10-(TMIO)3-(NI)3 had similar fluorescent properties as NI and similar electrochemical and redox properties as 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO). Moreover, mAb-GPC3-C60(OH)10-(TMIO)3-(NI)3 possessed low cell cytotoxicity, good fluorescence and good tumor-targeting property. Meanwhile, mAb-GPC3-C60(OH)10-(TMIO)3-(NI)3 can be taken up highly by HeLa tumor cells, achieve good green fluorescent imaging in tumor cells and highly enhance the contrast of fluorescent images. Therefore, mAb-GPC3-C60(OH)10-(TMIO)3-(NI)3 can disperse homogeneously in aqueous solution and be used as a potential tumor-targeting dual-modal contrast agent for EPR imaging/fluorescence imaging (EPRI/FI).

APLICACIÓN DE LA CALIDAD POR DISEÑO (QBD) EN EL DESARROLLO DE UN MÉTODO DE ANALISIS DE ERITROCITOS HUMANOS POR CITOFEROMETRÍA: COMPARACIÓN CON LA METODOLOGÍA DE DESARROLLO TRADICIONAL

Quality by Design (QbD) is defined as a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management, according to ICH Q section guidelines definition. Although this definition does not specifically mention analytical methods, since 2007 there have been authors who have defended the possibility of applying the principles of QbD to the development and validation of analytical methods in order to ensure their ruggedness and robustness, especially in the field of physical and physicochemical methods related to the quantitative drug analysis. However, in our experience, the real scope of application of QbD for analytical methods can include not only the quantitative analysis of drugs, but also other analytical methods in which other properties are determined, such as cytoferometry, a specific type of cell and particle suspension electrophoresis, with which it is possible to determine the electrophoretic mobility of normal and pathological erythrocytes. Data of the original development of a cytopherometric method carried out in 1988 have been reprocessed according to current QbD principles, and the results obtained with the traditional methodology and with the QbD methodology have been compared. This comparison demonstrates that the results processed following QbD show an enhancement in the knowledge and control of the cytopherometric method, giving more flexibility on physical and physico-chemical parameters management and appropriate tools to control the principal sources of analytical variability.

Усиленная радиосенсибилизация опухолевых клеток с помощью сочетанного воздействия гипертермии и ингибиторов протеасом

Hyperthermia is used in combination with radiation therapy to enhance the radiation response of the target tumor. However, many tumors are thermoresistant, which makes the thermo-radiosensitization ineffective. In this work, we studied a possibility of enhancing the radiosensitiz-ing effect of hyperthermia on cancer cells in the case of combining heating and treatments with inhibitors of proteasomal protein degradation. The objects of the study were HeLa, MCF-7, and PC-3 cell lines derived from human carcinomas of various localization. Before irradiation (2-6 Gy), cell cultures were subjected to heat stress (42-44 °C for 20-60 min) without or in the pres-ence of proteasome inhibitors (MG132, bortezomib). The death and survival of the treated cells was evaluated by the level of apoptosis/necrosis, by clonogenicity, and in the MTT test. The pro-teotoxicity of the exposures and the subcellular redistribution of heat shock proteins were ana-lyzed using cell fractionation, electrophoresis, and immunoblotting. The results show that, com-pared with hyperthermia, the combined effect (hyperthermia + proteasome inhibitor) significantly increased the radiosensitization of cancer cells, which was manifested in the intensification of their postradiation death and significant suppression of clonogenicity. Thus, the combination of hyperthermia with proteasome inhibitors (for example, with bortezomib, which is clinically ap-proved) can help sensitize radioresistant tumors to radiation therapy.

Estimation of amyloid aggregate sizes with semi-denaturing detergent agarose gel electrophoresis and its limitations

ABSTRACT Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was proposed by Vitaly V. Kushnirov in the Michael D. Ter-Avanesyan’s laboratory as a method to compare sizes of amyloid aggregates. Currently, this method is widely used for amyloid investigation, but mostly as a qualitative approach. In this work, we assessed the possibilities and limitations of the quantitative analysis of amyloid aggregate size distribution using SDD-AGE results. For this purpose, we used aggregates of two well-characterized yeast amyloid-forming proteins, Sup35 and Rnq1, and developed a protocol to standardize image analysis and process the result. A detailed investigation of factors that may affect the results of SDD-AGE revealed that both the cell lysis method and electrophoresis conditions can substantially affect the estimation of aggregate size. Despite this, quantitative analysis of SDD-AGE results is possible when one needs to estimate and compare the size of aggregates on the same gel, or even in different experiments, if the experimental conditions are tightly controlled and additional standards are used.

In vivo Characterization of the Radiosensitizing Effect of a Very Low Dose of BrdU in Murine Cells Exposed to Low-Dose Radiation.

The aim of the present study was to characterize the in vivo radiosensitizing effect of a very low dose of bromodeoxyuridine (BrdU) in mice exposed to low-dose radiation by establishing the following: (1) the radiosensitizing effect during DNA synthesis using single-cell gel electrophoresis (SCGE) in murine bone marrow cells, and (2) the number and timing of the mechanisms of genotoxicity and cytotoxicity, as well as the correlation of both end points, using flow cytometry analysis of the kinetics of micronucleus induction in reticulocytes. Groups of mice received intraperitoneal injections of 0.125 mg/g of BrdU 24 h prior to irradiation with 0.5 Gy of 60 Co gamma rays. DNA breaks measured using SCGE were determined at 30 min after exposure to radiation. The kinetics of micronucleated reticulocyte (MN-RET) induction was determined every 8 h after irradiation up to 72 h. The results from both experimental models indicated that low-level BrdU incorporation into DNA increased the sensitivity to 0.5 Gy of radiation, particularly in the S phase. The formation of micronuclei by gamma rays was produced at three different times using two main mechanisms. In the BrdU-substituted cells, the second mechanism was associated with a high cytotoxic effect that was absent in the irradiated BrdU-unsubstituted cells. The third mechanism, in which micronucleus formation was increased in irradiated substituted cells compared with the irradiated nonsubstituted control cells, was also related to an increase in cytotoxicity. Environ. Mol. Mutagen. 60:534-545, 2019. © 2019 Wiley Periodicals, Inc.

Evaluation of Serum Free Light Chain in Diagnosis and Monitoring of Plasma Cell Disorders

To determine diagnostic accuracy of serum free light chain assay compared to serum and urine protein electrophoresis in plasma cell disorders. Descriptive cross-sectional study. This study was conducted in the Immunology Department, Armed Forces institute of Pathology (AFIP), Rawalpindi, from May 2017 to May 2018. Patients referred to AFIP for diagnosis of plasma cell disorders or for monitoring while receiving treatment were included in study. They were tested for serum protein electrophoresis (SPE), urine protein electrophoresis (UPE), immunofixation (IF), and serum free light chain assay (sFLC). IF was used as the reference standard. Test results were compared in terms of sensitivity, specificity, positive or negative predictive value, and accuracy index. During the study period 220 patients were tested for plasma cell disorders. One hundred and sixty-seven patients tested positive. One hundred twenty-nine patients had multiple myeloma, 13 plasmacytoma, 11 monoclonal gammopathy of undetermined significance, 6 amyloidosis, 6 POEMS, and 2 Waldenstrom macroglobulinemia. SPE had a sensitivity of 70.5%, specificity of 100%; sFLC had a sensitivity of 87%, specificity of 81%; and UPE had a sensitivity of 23.5%, specificity of 97%. Accuracy index was 80.5% for SPE, 85% for sFLC, and 54% for UPE. When taken together, SPE and UPE had a combined sensitivity of 72%, specificity 97%, and accuracy index 80.5%. SPE and sFLC had combined sensitivity of 98.6%, specificity 84.3%, and accuracy index 94%. Combination of SPE and sFLC had the highest sensitivity and accuracy index for diagnosis and monitoring of plasma cell disorders compared with conventional tests.

Development of the 1,2,4-triazole-based anticonvulsant drug candidates acting on the voltage-gated sodium channels. Insights from in-vivo, in-vitro, and in-silico studies

The treatment of epilepsy remains difficult mostly since almost 30% of patients suffer from pharmacoresistant forms of the disease. Therefore, there is an urgent need to search for new antiepileptic drug candidates. Previously, it has been shown that 4-alkyl-5-substituted-1,2,4-triazole-3-thione derivativatives possessed strong anticonvulsant activity in a maximal electroshock-induced seizure model of epilepsy. In this work, we examined the effect of the chemical structure of the 1,2,4-triazole-3-thione-based molecules on the anticonvulsant activity and the binding to voltage-gated sodium channels (VGSCs) and GABAA receptors. Docking simulations allowed us to determine the mode of interactions between the investigated compounds and binding cavity of the human VGSC. Selected compounds were also investigated in a panel of ADME-Tox assays, including parallel artificial membrane permeability assay (PAMPA), single cell gel electrophoresis (SCGE) and cytotoxicity evaluation in HepG2 cells. The obtained results indicated that unbranched alkyl chains, from butyl to hexyl, attached to 1,2,4-triazole core are essential both for good anticonvulsant activity and strong interactions with VGSCs. The combined in-vivo, in-vitro and in-silico studies emphasize 4-alkyl-5-substituted-1,2,4-triazole-3-thiones as promising agents in the development of new anticonvulsants.