Lysis Solution Research Articles

Formation and Properties of Persister Cells of Staphylococcus capitis and Staphylococcus epidermidis, Bacteria Inhabiting Human Skin

Persisters (P) are the cells surviving, but not reproducing in the presence of lethal doses of antibiotics (AB). These are the cells of a scarce (0.001–1.0%) subpopulation, emerging due to a phenotypic transition, which may be stochastic or induced by stress impacts. Growing in fresh medium, P reproduce the structure of the parent population, which is considered the cause of: (a) recurrent infections; (b) inefficiency of antibiotic treatment; and (c) development of antibiotic resistance, as was discussed in numerous articles and reviews. The goal of the present work was to investigate P formation by bacteria contaminating human skin and their properties, which have not been studied previously. Two approaches were used to measure the frequency of occurrence was determined for P in the populations of stationary-phase cultures of Staphylococcus capitis Р205-1 (UNIQEM, Russia), which is sensitive to polymyxin, Na sulfacyl, and co-trimoxazole, and Staphylococcus epidermidis 14990 АТСС, which is resistant to these antibiotics: (1) traditional, by application of lethal AB doses and (2) by using a lysis solution (LS), which killed the ordinary vegetative cells and selected the persister cells resistant to AB and LS (PAB and PLS, respectively). This is the first report on applicability of the method involving the lysis solution for determination of P abundance in strains with multidrug AB resistance (for the S. epidermidis strain). Unlike most bacterial strains retrieved from collections , the share of P in stationary-phase cultures of staphylococci inhabiting human skin was quite high: ~10% for both S. epidermidis PLS and S. capitis. Heterogeneity in resistance to the lysis solution was revealed in the PAB obtained by the standard procedure: PLS share in S. capitis was ~1% of PAB abundance. These PLS were resistant to AB cross-action. The differences in PLS abundance in the studied strains (10% for S. epidermidis and below 1.0% for S. capitis) correlated with their AB resistance. Long-term incubation (4 months) under standard conditions resulted in the cycle of staphylococci development ending by emergence of dormant forms (DF); their share for both strains was ~0.1% of the number of viable cells in the stationary-phase cultures. The DF of staphylococci had all characteristics of dormant cells: emerged in the cycle of culture development; preserved viability under nongrowth conditions; had no experimentally detectable metabolism (absence of respiration); had specific ultrastructural organization; and were heat-resistant (200‒600 times higher than the vegetative cells at 90°C for 15 min). The correlation between PLS abundance in the cultures of staphylococci and the share of heat-resistant DF confirms our earlier suggestion on persisters as DF percursors.

Comet assay on one- and two-cell mouse embryos

DNA damage quantified as the comet tail length was assessed using in vitro and in vivo comet assay on one- and two-cell mouse embryos obtained by natural mating. The use of a protocol with three layers of agarose reduces the embryo loss and makes it possible to study a small number of embryos. A significantly lower level of basal, but not induced DNA damage was found in embryos with cleaved zona pellucida compared to embryos with intact zona pellucida. There were no significant differences in the length of the comet's tail between embryos lysed in different lysis solutions, both in cases of basal and induced DNA damage. A significant increase in the comet tail length was detected in one-cell embryos of mice treated with methyl methanesulfonate and etoposide compared to the control. The data show that DNA damage induced in maternal germ cells persists, which can be detected in embryos using the comet assay.

Relationship between Higher Atherogenic Index of Plasma and Oxidative Stress of a Group of Patients Living with Sickle Cell Anemia in Cameroon.

Dyslipidemia is highly prevalent in sickle cell anemia (SCA) patients and is one of the major risk factors for cardiovascular diseases induced by oxidative stress in Africa. The aim of this research was to investigate the correlation between higher atherogenic index of plasma (API) and oxidative stress in a group of patients living with SCA in Cameroon. Methods. A group of 85 homozygote SS patients (male and female) were enrolled at the Central hospital of Yaounde in Cameroon between May and October 2017. After informed consent through the signature of a consent form was obtained, the plasma was collected to determine the lipid profile while the lysate solution of RBC was used to explore some markers of oxidative stress using spectrophotometric methods. Results. Among the 85 patients included in our study, the mean age was 30 ± 5 years and the female to male ratio was 0.97. The majority of the patients (52–81%) had dyslipidaemia, and 22.4% of the patients demonstrated a higher level of atherogenic index of plasma. The patients with a higher level of total cholesterol (TC) (>240 mg/dl) and low-density lipoprotein (LDL-C) (>159 mg/dl) had at least 1,334 fold of malondialdeheyde (MDA) concentration than those with normal level. Also in the same patients, the higher atherogenic plasmatic index (API) significantly (p < 0.05) increased with the concentration of MDA. Except HDL-C, the other parameters of lipid profile had significant (p < 0.05) correlation with reduced glutathione (GsH) and total antioxidant capacity (TAC). The significant (p < 0.05) and linear regression was found between the increased MDA and higher API. Conclusion. Dyslipidemia increases oxidative stress and higher API which leads to coronary vascular disease in patients with SCA.

Exosomal Sonic Hedgehog derived from cancer-associated fibroblasts promotes proliferation and migration of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common and aggressive malignancies in China. Cancer‐associated fibroblasts (CAFs) can actively communicate with and stimulate tumor cells, thereby contributing to the development and progression of tumors. Yet, whether CAFs‐derived exosomes have a role in the progression of ESCC is largely unknown. Here, we find that Sonic Hedgehog (SHH) is highly expressed in CAFs lysis solution, conditioned medium of cultured CAFs (CAF‐CM) and CAFs‐derived exosomes, and esophageal cancer cell lines educated by CAF‐CM and CAFs‐derived exosomes can improve their growth and migration abilities in vitro and in vivo. Besides, those effects can be partly neutralized by cyclopamine, inhibitor of the Hedgehog signaling pathway. Thus, our research elucidates the crucial role of CAFs‐derived exosomes in the growth and progression of ESCC, and may open up new avenues in the treatment of ESCC.

Evaluation and Optimization of Genomic DNA Extraction from Food Sample for Microfluidic Purpose

Contamination of various food samples became one of the critical issues in food pathogen infection. Food pathogen can be detected by using digital polymerase chain reaction (PCR) and sequencing. These methods were reliable but consuming and take a longer time for detection. The present work describes the innovation to develop a technology to extract double-stranded deoxyribonucleic acid (dsDNA) from food samples and then denatured dsDNA into and single-strand DNA (ssDNA) for further use on the chip using microfluidic device. Microfluidic device is a lab-on-chip device that consist of microfluidic channels that provide paths for biomolecules to flow to individual point of care. DNA extraction is the process by which DNA is separated from proteins, membranes, and other cellular material contained in the cell from which it is recovered. Lysis solution is used in the process of extraction the DNA to break up the cells containing DNA from protein and other cellular materials. This extraction firstly be done in the most labour-intensive in obtaining the DNA biomolecules. Extraction methods may require an overnight incubation, may be a protocol that can be completed in minutes or a couple of hours by using a commercial kit. The disadvantages of the laboratory and commercial kit is due to time-consuming, poor cost-effectiveness, the need to use big laboratory and a complicated process which need an expertise to conduct the experiment and interpret the data. This research is proposed to design and fabricate a microfluidic device that has DNA extraction capabilities. In this research DNA extraction using a commercial kit will be used as a comparison for the quality of the result. The microfluidic device can be used in health care delivery system and will help the doctors in diagnostic process to identify disease of a patient rapidly. Other than that, the output extracted from microfluidic device will be used for DNA probe target interaction for diagnostic kit. The major advantage of microfluidic device is that it consumes less time compared to the conventional chemical methods.

Flash-comet assay

In the present paper, we present a substantially revised protocol of the widely used SCGE assay performed under alkaline conditions. In our updated version of the comet assay, which we call the Flash-comet, LiOH is used instead of NaOH during the unwinding and electrophoresis. This allows a higher voltage during the electrophoresis (5 V/cm instead of 0.7 V/cm), making it possible to reduce the unwinding time from 20 to 40 to 2.5 min, and the electrophoresis time from 10 to 20 to 1 min. Still, the Flash-comet was found to detect DNA strand breaks and alkali-labile sites with a higher degree of sensitivity than the conventional protocol in cells that had been exposed to H2O2 or ionizing radiation. In order to prevent alkaline hydrolysis of DNA, the wash and lysis solutions have been modified in the Flash-comet protocol.•By using an alkaline LiOH-based medium, the Flash-comet allows for much shorter times for both unwinding and electrophoresis than the conventional comet assay without compromising the sensitivity.•The reduced run-times of the unwinding and electrophoresis steps in the Flash-comet should also reduce the risk of laboratory-induced alkaline hydrolysis of DNA when evaluating the potential DNA-damaging effects of different types of xenobiotics.

Corrigendum to: Standardisation of the in vitro comet assay: influence of lysis time and lysis solution composition on the detection of DNA damage induced by X-rays

The alkaline comet assay, in vivo and in vitro, is currently used in several areas of research and in regulatory genotoxicity testing. Several efforts have been made in order to decrease the inter-experimental and inter-laboratory variability and increase the reliability of the assay. In this regard, lysis conditions are considered as one of the critical variables and need to be further studied. Here, we tested different times of lysis (from no lysis to 1 week) and two different lysis solutions in human lymphoblast (TK6) cells unexposed or exposed to X-rays. Similar % tail DNA values were obtained independently of the time of lysis employed for every X-ray dose tested and both lysis solutions. These results, taken together with our previous ones with methyl methanesulfonate and H2O2, which showed clear lysis-time dependence, support that the influence of the lysis time in the comet assay results depends on the type of lesion being detected; some DNA lesions may spontaneously give rise to apurinic or apyrimidinic (AP) sites during the lysis period, which can be converted into strand breaks detectable with the comet assay. Testing different times of lysis would be useful to increase the sensitivity of the comet assay and to ensure the detection of DNA lesions of an unknown compound, thereby providing some insight into the chemical nature of the lesions induced. However, the same lysis conditions (i.e. lysis time and lysis solution) should be used when comparing results between different experiments or laboratories.

Validation of an extraction method for microplastics from human materials.

Since the beginning of industrial production in 1950, plastic production has continued to grow strongly worldwide and is now at 322 million tonnes in the year 2015. From these very high production volumes ever larger quantities are found in the environment. There the plastics degradate to microplasticity and spread ubiquitously in the world. The present work deals with the possible uptake of microplastic particles in human organisms. For the detection of these plastic particles, an extraction method was developed and validated. Biological materials consist of human blood (healthy volunteers, n = 4) and different tissues of pigs and cattles. Various lysis solutions were tested for degradation efficiency of biological material and for effects on the plastics. The mass loss, surfaces and structure variations as well as the physicochemical spectrum of the material were observed after treatment by atomic force (AFM) and electron microscopy (EM) and Fourier transform infrared spectrometry (FTIR). The different plastic types as polyamide (PA), polycarbonate (PC), polyethylene (PE), polypropylene (PP), polystyrene (PS) and polyvinyl chloride (PVC) could be clearly differentiated and identified by FTIR. Regarding the surface control, especially PVC showed detectable alterations: After extraction an irregular surface structure caused by protuberances or bubbles could be observed. However, instead of these alterations an equivalent count of plastic particles was found in correlation to the applied plastic amount (recovery rate overall was 99,12±0,67%). The applied method can be used for plastic extractions from human or animal tissues without remarkable effects on the plastics.

Evaluation of five preservation methods for recovery of DNA from bone

The effectiveness of five methods for the preservation of bone were assessed: cell lysis solution (with 1% sodium azide), dehydration/freeze-drying, ethanol (96%), freezing, and room temperature storage. Preserved bone samples were extracted using five optimised extraction methods. These preservation methods were tested for their efficiency for storage of fresh and degraded bone samples for 6 weeks, 6 months and 1 year. Freezing was found to be the best preservation method for longer-term storage of bone samples; this was followed by ethanol (96%), dehydration/freeze-drying, and room temperature storage. Full profiles were obtained from bone samples using all these preservation methods.

Microfluidic platform for rapid screening of bacterial cell lysis

Over the past decade significant progress has been found in the upstream production processes, shifting the main bottlenecks in current manufacturing platforms for biopharmaceuticals towards the downstream processing. Challenges in the purification process include reducing the production costs, developing robust and efficient purification processes as well as integrating both upstream and downstream processes. Microfluidic technologies have recently emerged as effective tools for expediting bioprocess design in a cost-effective manner, since a large number of variables can be evaluated in a small time frame, using reduced volumes and manpower. Their modularity also allows to integrate different unit operations into a single chip, and consequently to evaluate the effect of each stage on the overall process efficiency.This paper describes the development of a diffusion-based microfluidic device for the rapid screening of continuous chemical lysis conditions. The release of a recombinant green fluorescent protein (GFP) expressed in Escherichia coli (E. coli) was used as model system due to the simple evaluation of cell growth and product concentration by fluorescence. The concept can be further applied to any biopharmaceutical production platform. The microfluidic device was successfully used to test the lytic effect of both enzymatic and chemical lysis solutions, with lysis efficiency of about 60% and close to 100%, respectively, achieved. The microfluidic technology also demonstrated the ability to detect potential process issues, such as the increased viscosity related with the rapid release of genomic material, that can arise for specific lysis conditions and hinder the performance of a bioprocess. Finally, given the continuous operation of the lysis chip, the microfluidic technology has the potential to be integrated with other microfluidic modules in order to model a fully continuous biomanufacturing process on a chip.

Aberrant Function of the C-Terminal Tail of HIST1H1E Accelerates Cellular Senescence and Causes Premature Aging

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

Redesigning the T-probe for mass spectrometry analysis of online lysis of non-adherent single cells

Single cell mass spectrometry (SCMS) allows for molecular analysis of individual cells while avoiding the inevitable drawbacks of using cell lysate prepared from populations of cells. Based on our previous design of the T-probe, a microscale sampling and ionization device for SCMS analysis, we further developed the device to perform online, and real time lysis of non-adherent live single cells for mass spectrometry (MS) analysis at ambient conditions. This redesigned T-probe includes three parts: a sampling probe with a small tip to withdraw a whole cell, a solvent-providing capillary to deliver lysis solution (i.e., acetonitrile), and a nano-ESI emitter in which rapid cell lysis and ionization occur followed by MS analysis. These three components are embedded between two polycarbonate slides and are jointed through a T-junction to form an integrated device. Colon cancer cells (HCT-116) under control and treatment (using anticancer drug irinotecan) conditions were analyzed. We detected a variety of intracellular species, and structural identification of selected ions was conducted using tandem MS (MS2). We further conducted statistical analysis (e.g., PLS-DA and t-test) to gain biological insights of cellular metabolism. Our results indicate that the influence of anticancer drugs on cellular metabolism of live non-adherent cells can be obtained using the SCMS experiments combined with statistical data analysis.

MASP-1 of the complement system alters fibrinolytic behaviour of blood clots

BackgroundThe lectin pathway serine protease mannan-binding lectin-associated serine protease 1 (MASP-1) has been demonstrated to be a major link between complement and coagulation, yet little is known about its interactions with the fibrinolytic system. The aim of this work was to assess the effects of MASP-1 on fibrin clot lysis in different experimental settings. MethodsRotational thrombelastometry was used to evaluate the effect of MASP-1 on the lysis of clots formed in whole blood under static conditions. Whole blood clots were also formed in the presence and absence of MASP-1 under flow conditions in the Chandler loop and their lysis was analysed separately by fluorescence release of incorporated labelled fibrin. Real-time observation by laser scanning confocal microscopy was used to investigate the lysis of plasma clots where MASP-1 was present either during clot formation or lysis. Cleavage of tPA or plasminogen by MASP-1 was analysed by gel electrophoresis. We performed a turbidimetric clot lysis assay in the presence and absence of the MASP-1 inhibitor SGMI-1 (Schistocerca gregaria protease inhibitor (SGPI)-based MASP inhibitor-1) to evaluate the effect of endogenous MASP-1 in normal plasma and plasma samples from sepsis patients. ResultsIn the thrombelastometric experiments, where MASP-1 was present during the entire clotting and lysis process, MASP-1 had a significant profibrinolytic effect and accelerated clot lysis. When clots were formed in the presence of MASP-1 under flow in the Chandler loop, the effects on fibrinolysis were heterogenous with impaired fibrinolysis in some individuals (n = 5) and no (n = 3) or even the opposite effect (n = 2) in others. In plasma clot lysis observed by confocal microscopy, lysis was prolonged when MASP-1 was added to the lysis solution, yet there was no difference in lysis time when MASP-1 was present during clot formation. When MASP-1 was incubated with tPA or plasminogen, respectively, cleavage of single-chain tPA into two-chain tPA and a slight reduction of plasminogen were observed. SGMI-1 significantly prolonged clot lysis in the turbidimetric clot lysis assay suggesting that MASP-1 accelerated lysis in plasma samples. ConclusionMASP-1 is able to alter the susceptibility of blood clots to the fibrinolytic system. MASP-1 has complex, mostly promoting effects on fibrinolysis with high inter-individual variation. Interactions of MASP-1 with the fibrinolytic system may be relevant in the development and therapy of cardiovascular and thrombotic diseases.

Peptidomic and Bioinformatic Analysis to Evaluate the Application of Peptides from Latex of Hevea brasiliensis Clone BPM24

Peptidomic analysis coupled with mass spectrometry identification was employed to investigate the constitutively expressed peptides in latex serum Phytophthora-resistant rubber tree (Hevea brasiliensis), BPM24. The utilization of proteins and peptides as a strategy to study new methods can lead to a therapeutic application. However, a high-efficiency method to extract peptides from latex serum is not well established. After optimization of latex serum isolation from natural rubber latex, using of lysis solution for peptide extraction exhibited superior serum peptide recovery than using other chemical reagents. Ultrafiltration followed by solid-phase extraction was selected to achieve high extraction yield of low molecular weight proteins and peptides. Mass spectrometric and bioinformatics analysis characterized and predicted the identified protein and peptide contents. All latex serum peptide fractions from C18 reversed-phase chromatography possessed effectiveness of antifungal activity against three tested plant fungal including A. brassicicola, C. lunata and M. grisea with different MIC in the range 250-2,000 μg/ml from different peptide fraction. Moreover, Etotal (E1-E5 pooled together) from lysis solution extraction was found to able to inhibit three tested plant fungal at MIC 500 μg/ml. Moreover, B. cereus and E. faecium were the sensitive gram-positive bacteria with MIC in the range 250-4,000 μg/ml. However, all gram-negative bacteria were not affected by 4,000 μg/ml. Among these tested pathogens, plant fungal was quite sensitive to our peptide product. Etotal from lysis solution extraction will be the best candidate for using further in antifungal testing of other plant fungal both in vitro and in vivo. The further investigation and application will lead to possible use of latex serum as a great biotechnological resource to develop peptide –based product for the treatment of plant infection.

Current approaches for RNA-labelling to identify RNA-binding proteins.

RNA is involved in all domains of life, playing critical roles in a host of gene expression processes, host-defense mechanisms, cell proliferation, and diseases. A critical component in many of these events is the ability for RNA to interact with proteins. Over the past few decades, our understanding of such RNA-protein interactions and their importance has driven the search and development of new techniques for the identification of RNA-binding proteins. In determining which proteins bind to the RNA of interest, it is often useful to use the approach where the RNA molecule is the "bait" and allow it to capture proteins from a lysate or other relevant solution. Here, we review a collection of methods for modifying RNA to capture RNA-binding proteins. These include small-molecule modification, the addition of aptamers, DNA-anchoring, and nucleotide substitution. With each, we provide examples of their application, as well as highlight their advantages and potential challenges.

64 Culture of bovine oocytes and embryos with metabolic hormones concentrations associated with equine metabolic syndrome

Maternal metabolic status could affect fertility and early embryo development due to altered concentrations of metabolic hormones. Equine metabolic syndrome (EMS) is a condition in horses associated with obesity and insulin resistance. Equine metabolic syndrome is accompanied by increased concentrations of insulin and leptin and decreased concentrations of adiponectin, in ovarian follicular fluid (FF) and in systemic circulation (SYST). We sought to determine how altered concentrations of insulin, leptin, and adiponectin (ILA), consistent with those in mares with EMS (EMS) or normal mares (normal), would affect blastocyst formation rates, blastocyst gene expression for metabolism and inner cell mass formation (OCT4, SOX2, COX2, DNMT3a1, HK2, LDH, PDH, and GLUT1), and metabolite uptake from culture media. Because equine oocytes are not available for large-scale study, a bovine model was used in this preliminary study to determine the impact of altered ILA on oocytes and embryos. Bovine ovaries were obtained from an abattoir and embryos produced as previously described using chemically defined media (CDM; Barcelo-Fimbres and Seidel 2007Mol. Reprod. Dev. 74, 1406-1418). Briefly, oocytes were cultured in in vitro maturation medium (IVM), fertilized in FCDM, presumptive zygotes were placed into CDM-1 for ~56h. Cleavage rates were assessed, and embryos were moved to CDM-2 for ~122 additional hours. Treatments consisted of 5 groups: (1) standard oocyte IVM, FCDM and embryo production (EP) system (control), (2) IVM with normal FF ILA and control FCDM and EP, (3) IVM with normal FF ILA and FCDM and EP with normal SYST ILA, (4) IVM with EMS FF ILA and control FCDM and EP, and (5) IVM with EMS FF ILA and FCDM and EP with EMS SYST ILA. Seven days after fertilization, blastocysts were pooled in groups of 5 and placed into 50mL of CDM-2 for 24h. Embryos were removed, and medium was frozen and stored at −80°C to determine metabolite usage via gas chromatography mass spectroscopy. Pooled embryos were washed and placed into RNA lysis solution for relative quantitative PCR. Statistical comparisons were performed using ANOVA with a post-hoc Tukey test. Blastocyst formation rates and gene expression of viability markers were not significantly different among groups. However, aspartate was lower (P=0.02) in spent media from Group 3 (normal FF and SYST ILA) and tended (P=0.09) to be lower in media from Group 5 (EMS FF and SYST ILA) when compared with controls (Group 1). The ILA during early embryo development but not oocyte maturation appeared to be associated with increased uptake of aspartate, a nonessential amino acid, thought to be involved in osmoregulation, cellular signalling, and in mouse embryos, facilitate the metabolism of lactate. In conclusion, the addition of ILA in concentrations observed in normal horses and EMS horses did not affect blastocyst formation rates or markers of embryo viability, although embryo metabolism could have been altered.

A novel chemical lysis method for maximum release of DNA from difficult-to-lyse bacteria

Molecular detection of microorganisms requires releasing DNA from cells. However, since certain microbial organisms are refractory to lysis by chemical or enzymatic methods, mechanical lysis by bead-beating is typically employed to disrupt difficult-to-lyse microbes. A newly developed chemical lysis method called sporeLYSE enables release of DNA from difficult-to-lyse microbes without bead-beating. The sporeLYSE method was compared to bead-beating and an alkaline/detergent lysis solution for releasing DNA from microbes grown in vitro, including surrogates of Category A bioterrorism agents. sporeLYSE released 83% to 100% of DNA from Mycobacterium smegmatis, Francisella philomiragia, Yersinia enterocolitica, Bacillus thuringiensis, Pseudomonas aeruginosa, Moraxella catarrhalis and Klebsiella pneumoniae. qPCR results indicated that sporeLYSE extracted an equal or greater amount of DNA than either bead-beating or alkaline/detergent lysis from Gram-positive and Gram-negative bacteria. When sporeLYSE was used to extract DNA from saliva and sputum spiked with M. smegmatis and M. tuberculosis, respectively, the qPCR Ct values were 4–8 cycles lower than those for extractions via alkaline/detergent lysis and heat. Mean Ct values for sporesLYSE extractions from spores of Clostridium difficile and C. botulinum were approximately two cycles lower than those of MagNA Pure DNA extractions. Our results suggest that sporeLYSE is an easy-to-use liquid reagent that can efficiently release large amounts of DNA from a variety of bacteria, including spores.

A Sensitive Blood Test for Rapid Estimation of Absorbed Radiation Dose

Discovery of a panel of circulating miRNA biomarkers of which changes are dependent on radiation doses in rodent and non-human primate models as well as their validation in human AML/ALL patients receiving total body irradiation (TBI) allowed us to develop a novel radiation biodosimetry test. The available biodosimetry methods such as complete blood count (CBC) assessing lymphocyte depletion and dicentric chromosome assay (DCA) have limitations with regard to its feasibility, robustness and throughput. This novel assay allows rapid estimation of dose of exposure so that victims of mass casualty accidents could be triaged quickly. Male and female mice from different genetic background and age groups were subjected to TBI (0.5 to 10 Gy gamma rays, 137Cs source, 0.94 Gy/min). A single drop of blood was collected into a lysis solution; RNA was isolated and changes in miR150-5p and miR23a-3p were compared by qPCR using ΔΔCT method. The decrease in miR150-5p (blood cell origin) with miR23a-3p (not expressed at significant level in lymphocytes, but abundant in plasma) as internal reference were extrapolated to develop algorithms for dose estimation. Blinded studies and mathematical modelling were used for dose calibration and to evaluate the accuracy and the influence of variables such as age, gender and other potential confounders. Dose-dependent decrease in miR150-5p with miR23a-3p as internal reference was extrapolated that allowed dose estimation with ±0.5 Gy precision at 1 to 3 Gy and with ± 1 Gy precision up to 6 Gy at 24-96 hours, the dose range and time points relevant for triage (two-way ANOVA, p<0.05). Significant correlation was noted between miR150-5p/miR23a-3p assay and lymphocyte depletion kinetics (Pearson correlation coefficient r2 0.83; p<0.0001), confirming the reliability of new molecular biodosimetry assay. Algorithms were developed for dose reconstruction and the blinded studies showed precision in dose estimation (1% uncertainty). Linear regression model was developed for each time point indicated a good agreement between actual and calculated doses (r2>0.9, slope>=0.9, lack of fit test p>0.05). Robustness and sensitivity of the assay has been validated by comparing the values in human healthy volunteers and in leukemia patients prior, during and after radiation treatment and on marrow reconstitution after hematopoietic stem cell transplantation (HSCT). We have developed an internally controlled and simple two miRNA biomarker blood test for radiation biodosimetry with broad translational utility. The assay demonstrates superior capability in dose estimation and distinguishing the unexposed from 2 Gy exposed individuals (triage criteria) using a drop of blood that can be collected by finger-stick. Furthermore, our data demonstrate the utility of the assay in clinical radiation oncology, especially for evaluation of the marrow ablation following radiation and its reconstitution after bone marrow transplantation.

A combined treatment with erythrocyte lysis solution and Sudan Black B reduces tissue autofluorescence in double-labeling immunofluorescence.

The autofluorescence of animal tissues complicates the results obtained using fluorescence microscopy. Many techniques have been used to reduce autofluorescence; however, all these techniques have the disadvantage of reducing the intensity of immunofluorescence staining. We observed the features of autofluorescence in formalin-fixed paraffin-embedded (FFPE) vascularized liver and kidney sections and assessed the effects of an intravascular treatment with erythrocyte lysis solution (ELS) before a routine perfusion with normal saline (NS) and Sudan Black B (SBB) treatment after antigen retrieval on reducing autofluorescence reduction and the visualization of antigens to establish an optimal method for reducing autofluorescence. Erythrocytes exhibited bright autofluorescence in FFPE liver and kidney sections, which altered the results of actin and destrin immunofluorescence staining. The SBB treatment significantly reduced background autofluorescence and exerted a moderate effect on reducing the autofluorescence of erythrocytes, and the intravascular ELS treatment eliminated erythrocyte autofluorescence in FFPE liver and kidney sections. A combined treatment with ELS and SBB further reduced autofluorescence but did not decrease actin and destrin immunofluorescence staining in double-labeled FFPE liver and kidney sections. In conclusion, the application of an intravascular ELS treatment before the NS perfusion combined with an SBB treatment after antigen retrieval is a simple and efficient strategy for reducing autofluorescence in FFPE vascularized tissues and can be broadly used in fluorescence microscopy analyses.

Abstract A16: Evaluation of the genotoxicity of ruthenium complex, trans-[Ru(ThySMet)(PPh3)2(bipy)]PF6, by comet assay in vitro on breast cells and in vivo

Abstract Cancer remains a major public health problem, accounting for one in six deaths worldwide. One of the most common types of cancer in women is breast cancer, which is extremely heterogeneous and composed of different subtypes. The majority of drugs currently used in cancer treatment show no selectivity for tumor cells, provoking undesired side effects. Cisplatin, discovered more than 40 years ago, and successive generations of platinum-based antitumor drugs have demonstrated that metal complexes may play an important role in the cancer treatment. Metals have unique characteristics that can be exploited by the chemistry coordination for the design of new drugs with fewer side effects. In the last two decades, ruthenium complexes have been widely studied and gained prominence for cancer treatment due to their unique characteristics, such as high rate of ligand exchange, accessible oxidation states, and the ability to mimic the iron to bind biologic molecules. This study aimed to evaluate the genotoxicity effects of the ruthenium complex trans-[Ru(ThySMet)(PPh3)2(bipy)]PF6 in vitro and in vivo by the comet assay, which can be used to measure DNA damage in individual eukaryotic cells. Briefly, for in vitro assays, MDA-MB-231 malignant breast cells and MCF-10A nonmalignant breast cells were seeded (1x105/1000µL) into sterile 12-well plates. Cells were allowed to grow at 37°C in 5% CO2 for 24h and then treated or not (control) with different concentrations (2, 4 or 8µM) of the ruthenium complex or positive control cisplatin (8µM) for 1h. After this period, cells were harvested, centrifuged and resuspended in 500μL of culture medium. For in vivo assays, the animals, swiss mice, were kept in a climate-controlled environment (22±2°C) with a 12/12h light/dark cycle and food and water ad libitum. The Animal Bioethics Committee (CEUA/UNESP FAMEMA, Marília, São Paulo, Brazil) approved this study (protocol no. 828/2016). Mice were divided into five groups with five animals in each group. The ruthenium complex (12.5, 25 or 50mg/kg), positive control doxorubicin (20mg/kg), or negative control (vehicle - DMSO) were administered intraperitoneally for 3 days at 24h interval. On the third day, peripheral blood from the tail vein was collected after 4h after the administration of the last treatment. Then both, cell suspension or blood, were mixed with 120µL of 0.5% low-melting point agarose and dropped on slides precoated with 1.5% normal melting point agarose and taken to a lysis solution for 1h. After denaturation (20min) and alkaline electrophoresis (25V, 300mA, 20min) the slides were neutralized and fixed. The slide staining was performed with GelRed® (Uniscience) and comet analysis of at least 150 random cells per group was done visually according to comet tail size using a fluorescence microscope (Olympus BX61-TRF5). In vitro results of genotoxic evaluation of trans-[Ru(ThySMet)(PPh3)2(bipy)]PF6 demonstrate that this complex was able to induce significant DNA damage in MDA-MB-231 cells at the highest concentrations (4 and 8μM), as well as the positive control, cisplatin (8μM). However, the complex did not cause DNA damage in MCF-10A cells; only cisplatin induced damage to these cells. The results of genotoxic evaluation of in vivo assays indicated that only the group of animals treated with the highest dose of the complex (50mg/kg) and the animals treated with doxorubicin presented DNA damage. These results demonstrate that this complex causes DNA damages in vitro with more specificity for tumor cells and that only in the highest dose it causes DNA damages in vivo. Therefore, more studies should be performed to evaluate its potential for breast cancer treatment. Citation Format: Amanda Blanque Becceneri, Cecília Patrícia Popolin, Ana Maria Plutin, Edson Luis Maistro, Alzir Azevedo Batista, Márcia Regina Cominetti. Evaluation of the genotoxicity of ruthenium complex, trans-[Ru(ThySMet)(PPh3)2(bipy)]PF6, by comet assay in vitro on breast cells and in vivo [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A16.