Nucleic Acid Stain Research Articles

Evidence of genotoxicity and cytotoxicity of X-rays in the oral mucosa epithelium of adults subjected to cone beam computed tomography.

Evidence of genotoxicity and cytotoxicity of X-rays in the oral mucosa epithelium of adults subjected to cone beam computed tomography.

PF523 NOVEL THIENOPYRIDINE DERIVATIVES INDUCE TUBULIN DISRUPTION AND CAUSE CELL CYCLE ARREST IN MALIGNANT B‐CELLS

Background:Use of targeted therapy for B‐cell malignancies has resulted in significant improvements in patient overall‐ and progression‐free‐survival. These treatments have focused on inhibition of components of the B‐cell receptor (BCR) such as BTK and PI3Kδ, targeted by ibrutinib and idelalisib respectively, and are often used in combination with other agents such as Microtubule Targeting Agents (MTA), alkylating agents and glucocorticoids. However, relapse and resistant disease remain a significant challenge. Thienopyridines, commonly used as anti‐thrombotic therapy, have recently been identified as potential anti‐cancer agents, with PLCγ2 (acting downstream of BTK in BCR signalling), Jak2 and Lck suggested as direct targets, while mitotic spindle inhibition via microtubule de‐stabilisation has also been proposed. Theinopyridines may therefore have potential as a future treatment for B‐cell malignancies.Aims:This study aimed to investigate the anti‐proliferative and microtubule‐disrupting properties of ten novel thienopyridine compounds synthesised by our group, in B‐cell Lymphoma cells in‐vitro, to determine whether this class of compound has potential as future targeted therapy for B‐cell malignancies.Methods:DAUDI Burkit Lymphoma cells were cultured in the presence of novel thienopyridines for 24–72 h at a range of concentrations (100 μM to 1 nM). Synthesis of the thienopyridine compounds (DJ0014, DJ0021, DJ0041, DJ0081, DJ0109, DJ0171, DJ0199, DJ0206 and DJ0209[ND1] ) has been previously published. Cellular biochemical activity was assessed using the MTS assay, while apoptosis and necrosis were observed using the Annexin V/Propidium Iodide (AV/PI) flow cytometry assay. Cell cycle arrest was determined by flow cytometric Propidium Iodide cell cycle assay, and alpha‐tubulin expression was visualised by confocal microscopy following staining with α‐Tubulin (DM1A) Mouse mAb (Alexa Fluor® 488 Conjugate) antibody and DAPI nucleic acid stain. The rate of tubulin polymerisation in the presence of the compounds was assessed by Tubulin Polymerisation Assay, with the rate of polymerisation measured using spectrophotometry. Paclitaxel was used as a positive control for tubulin stabilisation.Results:Across all assays at 48 and 72 h, compounds DJ0014, DJ0041, DJ0081, DJ0199 and DJ0206 resulted in significantly reduced cell viability by MTS assay and increased early and late apoptosis by AV/PI, with corresponding reduction in live cell populations and absence of necrosis at 10 μM and 100 μM concentrations. Increased tetraploidy / G2/M arrest was also seen following treatment with the same compounds at 10 μM and 100 μM concentrations. These compounds showed a significant inhibitory effect on tubulin polymerisation at 10 μM dosage and signs of microtubule disruption via confocal microscopy.Summary/Conclusion:The study demonstrates the anti‐mitotic activity of these novel thienopyridine derivatives in malignant B‐cells and their potential for use in the treatment of mature B‐cell malignancies. Future work aims to clarify their effect on PLCγ2 and the mechanism by which this may result in the observed effect on microtubule assembly.

Superbright organosilica nanodots as a universal sensor for fast discrimination and accurate quantification of live/dead cells

Rapid and quantitative discrimination between live and dead cells is highly desirable yet remains a challenging task. Here, we report the use of the ultrabright green-emitting organosilica nanodots (OSiNDs, photoluminescence quantum yield: ˜100%) for fast (1 min in vitro and 5 min in vivo), wash-free, and universal labeling of the dead bacterial, fungal, and mammalian cells, which meets the demand of the green fluorescent probes that can specifically image the dead cells. The photostability of OSiNDs surpasses that of the commercial dyes, such as propidium iodide (PI) and SYTOX Green nucleic acid stain (SYTOX), making OSiNDs more suitable for long-term and stable monitoring of the cell viability. With the assistance of a commercial red fluorescent dye SYTO 60, we develop an in situ method for rapid and accurate quantification of live/dead cells. Motivated by the unique optical advantages and high sensitivity of OSiNDs to the cell viability, the OSiNDs are employed to observe the in vivo cellular changes induced by doxorubicin using zebrafish as a model, demonstrating their great potential for evaluating the toxicity/efficacy of the therapeutic agents in real time. Owing to the excellent biocompatibility and low cost, the OSiNDs can be a universal probe for efficient and accurate differentiation and quantification of the live and dead cells, which may find wide applications in the biomedical field.

The staining efficiency of cyanine dyes for single-stranded DNA is enormously dependent on nucleotide composition.

The staining of nucleic acids with fluorescent dyes is one of the most fundamental technologies in relevant areas of science. For reliable and quantitative analysis, the staining efficiency of the dyes should not be very dependent on the sequences of the specimens. However, this assumption has not necessarily been confirmed by experimental results, especially in the staining of ssDNA (and RNA). In this study, we found that both SYBR Green II and SYBR Gold did not stain either homopyrimidines or ssDNA composed of only adenine (A) and cytosine (C). However, these two dyes emit strong fluorescence when the ssDNA contains both guanine (G) and C (and/or both A and thymine (T)) and form potential Watson-Crick base pairs. Interestingly, SYBR Gold, but not SYBR Green II, strongly stains ssDNA consisting of G and A (or G and T). Additionally, we found that the secondary structure of ssDNA may play an important role in DNA staining. To obtain reliable results for practical applications, sufficient care must be paid to the composition and sequence of ssDNA.

Evaluation of an Alternative Staining Method Using SYTO 13 to Determine Reticulated Platelets.

Reticulated platelets reflect the rate of platelet turnover and represent the youngest circulating platelets in peripheral blood. Reticulated platelets contain residual ribonucleic acid (RNA) from megakaryocytes which is lost in a time-dependent manner and can be transcribed into proteins even in the absence of a nucleus. An increased proportion of reticulated platelets is associated with higher platelet reactivity, cardiovascular events and mortality. At present, a fully automated assay system (SYSMEX haematology analyser) is available for analysis. This method, however, is not suitable for extended laboratory investigations like subsequent cell sorting. Flow cytometry analysis after staining with thiazole orange (TO) is frequently used in such settings despite several limitations. Here, we describe a new assay for determination of reticulated platelets by flow cytometry using the nucleic acid staining dye SYTO 13 and compare it with SYSMEX and TO staining as current standards. A significant correlation between immature platelet fraction (IPF) determined by SYSMEX XE-2100 analyser and results obtained with the SYTO 13-based assay was observed (r = 0.668, p < 0.001) which was stable during a reasonable time period. In contrast, the correlation between TO staining and IPF was weaker (r = 0.478, p = 0.029) and lost after 90 minutes of staining. SYTO 13 staining of platelets enabled sorting of RNAlow and RNArich platelets which was confirmed by RNA quantification of sorted platelets. Except for fixation of platelets, sorting of these platelet sub-populations was stable under various experimental settings. In summary, determination of reticulated platelets with the new SYTO 13 assay offers distinct technical advantages enabling further laboratory processing.

Rapid Recovery of DNA from Dried Blood Spots: Use of a Novel Electrophoretic Method

The demand for rapid, effective point of care diagnostics has created a pressing need for new and improved methods to quantify DNA from patient samples. Dried blood spotting is a well‐established testing methodology that has gained increasing importance in in vitro diagnostics due to its low cost, low sampling requirements, and long‐term sample stability. In recent years, dried blood spots have been increasingly used for targeted genetic and metabolite profiling. Large‐scale genetic screening of neonatal dried blood spots has a great potential to lower patient mortality and morbidity through early diagnosis of primary immunodeficiencies. However, DNA extraction from the surface of the dried blood spot remains the most time consuming, costly and labor‐intensive part of DNA analysis. Thus, the objective of this study was to develop and optimize a simple and rapid methodology to extract DNA from dried blood spots.In the present study, we created a novel methodology to extract DNA from a dried blood spot using only a lysis buffer (10mM KOH/30mM Tris buffer), 50V, and heat. Briefly, during the electric field extraction spots and lysis buffer were placed in an electroporation cuvette and DNA was extracted by applying 25–50 volts across the plates of the cuvette. The spot was then removed and DNA in the supernatant was analyzed using qPCR or fluorescent nucleic acid stains. This methodology was optimized by the addition of a 70°C heating step before the electric field extraction to better retrieve episomal DNA.This electric field DNA extraction is to our knowledge, the first methodology to use an electric field to extract episomal DNA from a dried blood spot. Results of this study show that this methodology produces significant recoveries of both genomic and episomal DNA (Ct (FAM) 33.0 (50V, 60s) to Ct (FAM) 35.0 (heating alone) p &lt;0.01). Varying recoveries of episomal and genomic DNA were also seen at different voltage conditions suggesting that this methodology may be useful for the isolation and purification of episomal DNA (FAM (25V, 300s (no amplification) and 50V, 60s (Ct 33.0)). Furthermore, preliminary qualitative and spectroscopic analyses suggest that protein removal could be achieved using this dual voltage technique. The 25‐minute electric field extraction procedure has one of the lowest times for the extraction of DNA found within the literature. This optimized procedure negates the need for costly treatment and wash steps, simplifying DNA extraction considerably. Most importantly, the electric field extraction reduces the time of proprietary procedures by more than 30 minutes, while retaining 75–80% of the yield. Thus, the results show that this novel method of electric field extraction provides a rapid and effective protocol to remove episomal and genomic DNA from dried blood spots. This optimized electric field device not only simplifies bioanalytical techniques, but provides a significant step towards point of care diagnostics and should be expanded to other sample types.Support or Funding InformationThe authors thank PerkinElmer for providing the funding for this study.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rapid and reasonable molecular identification of bacteria and fungi in microbiological diagnostics using rapid real-time PCR and Sanger sequencing

Analyses of short subunit gene sequences have been established for taxonomic classification and identification of bacteria and fungi. To produce partial bacterial ribosomal 16S rRNA and rpoB and fungal ribosomal ITS/LSU gene sequences for DNA sequencing, real-time PCR assays supplemented with the nucleic acid stain SYBR Green were created. Generation of PCR products was monitored based on amplification and melting curves. The PCR products were subsequently subjected to Sanger sequencing on demand for identification of bacteria and fungi in routine microbiological diagnostics within a period of two days.From a total of 78 bacterial isolates 40 (51%) or 67 (86%) could be identified at species level using only partial 16S rRNA or additionally rpoB gene sequences based on BLASTN (NCBI) database queries, respectively. Using partial 16S rRNA and rpoB gene sequencing unambiguous assignment was not possible for the closely related species of the Bacillus (B.) cereus group, Bordetella (B.) pertussis/ B. parapertussis/ B. bronchiseptica, Brucella spp., Enterobacter cloacae complex, Escherichia/ Shigella spp., Staphylococcus (S.) hyicus/ S. agnetis and Yersinia (Y.) pseudotuberculosis/ Y. pestis. However, partial rpoB gene sequencing succeeded in identifying 27 bacterial isolates at species level in addition to 16S rRNA gene sequencing.Regarding ITS/LSU gene sequencing, best results could be achieved by ITS gene sequencing followed by LSU gene sequencing, resulting in 32 (63%) and 21 (43%) of a total of 51 fungal isolates that could be identified at species level, respectively. Insufficient identification at species level was observed for the genera Apiotrichum, Aspergillus, Cladosporium, Cryptococcus, Microsporum, Nannizziopsis, Penicillium, Trichosporon, and Tolypocladium included in this study.The concept of this procedure is suitable for rapid and reasonable molecular identification of bacteria and fungi within two days and is therefore applicable in routine microbiological diagnostic laboratories.

Abyssinone V, a prenylated flavonoid isolated from the stem bark of Erythrina melanacantha increases oxidative stress and decreases stress resistance in Caenorhabditis elegans.

Recent studies showed that distinct extracts of Erythrina species used in the traditional medicine of sub-Saharan Africa are protective against stress conditions. However, the underlying molecular mechanisms as well as relevant compounds remain unclear. We used the model organism Caenorhabditis elegans to investigate compounds isolated from the stem bark of Erythrina melanacantha (abyssinone V (1), abyssinon-4'O-methylether (2), sigmoidin B-4'O-methylether (3), glabranin (4), 8-prenylnaringenin (5), citflavanone (6), exiguaflavanone (7) and homoeriodictyol (8)). Antioxidative capacity in vitro (trolox equivalent antioxidative capacity assay) and modulation of oxidative stress in vivo (2', 7'-dichlorofluorescein assay) were investigated; stress resistance was analysed using the nucleic acid stain SYTOX green. None of the prenylated flavonoids caused protection against thermal stress; in contrast, most of the compounds (1, 4, 5, 8) decreased stress resistance. None of the compounds decreased the accumulation of reactive oxygen species, but abyssinone V (1) caused an increase in oxidative stress. In line with these results, none of these compounds showed radical-scavenging effects in vitro. The stem bark of E. melanacantha contains various prenylated flavonoids, but no compound protected C. elegans against stress conditions. In contrast, abyssinone V increases oxidative stress and reduces stress resistance in this model organism.

Loop-Mediated Isothermal Amplification in Microchambers on a Cell Microarray Chip for Identification of Plasmodium Species

Malaria is caused by Plasmodium spp., a parasitic protist that infects erythrocytes. A method that can detect the parasite with high sensitivity and that can identify the parasite species is urgently required for the control of malaria. The cell microarray chip was made using polystyrene with 200 cone-shaped frustum microchambers (800-μm top diameter, 636-μm bottom diameter, and 225 μm deep). Approximately 3,000 erythrocytes could be accommodated in each microchamber with monolayer formation, there being 60,000 erythrocytes in total microchambers on a cell microarray. Plasmodium could be quantitatively detected with high sensitivity with the use of cell microarray chips. Plasmodium parasitizing in erythrocytes was labeled with a cell-permeant fluorescent nucleic acid stain (SYTO 21), which could be detected in erythrocytes in the microchambers. Next, we used loop-mediated isothermal amplification (LAMP) in the microchambers (on-chip LAMP) to identify the parasite species detected in the microchambers. LAMP was performed in the microchambers (in a reaction volume of 0.09 μl) using Plasmodium falciparum-infected erythrocytes as the template and specific primers targeting 18S rRNA. To avoid evaporation of the reaction buffer during heat treatment, mineral oil was overlaid on each microchamber and the cell microarray chips were heated at 63 C for 1 hr. The results of on-chip LAMP were assessed using a portable ultraviolet transilluminator. We showed that this method has the potential for detection of parasites in 600,000 erythrocytes and for identification of the parasite species on a cell microarray chip. In conclusion, the parasites can be detected quantitatively with high sensitivity, and the species can be identified with the use of cell microarray chips.

Hibiscus sabdariffa L. extract prolongs lifespan and protects against amyloid-β toxicity in Caenorhabditis elegans: involvement of the FoxO and Nrf2 orthologues DAF-16 and SKN-1.

Hibiscus sabdariffa L. is commonly used as an ingredient for herbal teas and food supplements. Several studies demonstrated the beneficial effects of Hibiscus sabdariffa L. extracts (HSE); however, the bioactive components and their mode of action still remain unclear. Caenorhabditis elegans (C. elegans) was used to study health-related effects and the underlying molecular mechanisms of HSE in this model organism as well as effects of hydroxycitric acid (HCA), a main compound of HSE, and its structural analogue isocitric acid (ICA). Survival and locomotion were detected by touch-provoked movement. Thermotolerance was analysed using the nucleic acid stain SYTOX green, and intracellular ROS accumulation was measured via oxidation of H2DCF. Localisation of the transcription factors DAF-16 and SKN-1 was analysed in transgenic strains (DAF-16::GFP, SKN-1::GFP). The involvement of DAF-16 and SKN-1 was further investigated using loss-of-function strains as well as gene silencing by feeding RNAi-inducing bacteria. Protection against amyloid-β toxicity was analysed using a transgenic strain with an inducible expression of human amyloid-β peptides in body wall muscle cells (paralysis assay). HSE treatment resulted in a prominent extension of lifespan (up to 24%) and a reduction of the age-dependent decline in locomotion. HCA, a main compound of HSE increased lifespan too, but to a lesser extent (6%) while ICA was not effective. HSE and HCA did not modulate resistance against thermal stress conditions and did not exert antioxidative effects: HSE rather increased intracellular ROS levels, suggesting a pro-oxidative effect of the extract in vivo. HSE and HCA increased the nuclear localisation of the pivotal transcription factors DAF-16 and SKN-1 indicating an activation of these factors. Consistent with this result, lifespan prolongation by HSE was dependent on both transcription factors. In addition to the positive effect on lifespan, HSE treatment also elicited a (strong) protection against amyloid-ß induced toxicity in C. elegans in a DAF-16- and SKN-1-dependent manner. Our results demonstrate that HSE increases lifespan and protects against amyloid-β toxicity in the model organism C. elegans. These effects were mediated, at least in parts via modulation of pathways leading to activation/nuclear localisation of DAF-16 and SKN-1. Since HCA, a main component of HSE causes only minor effects, additional bioactive compounds like flavonoids or anthocyanins as well as synergistic effects of these compounds should be investigated.

Stability of lysozyme incorporated into electrospun fibrous mats for wound healing

In this study, we investigated the feasibility of incorporating protein drugs into electrospun fibrous mats (EFMs) for wound healing using lysozyme as a model drug. Lysozyme nanoparticles (Lyso- NPs) were first obtained by electrospray. Lysozyme solutions were prepared with a binary solvent mixture of ethanol (EtOH)-water (H2O) at varied volume ratios. Subsequently, Lyso-NPs were suspended in poly(lactic-co-glycolic acid) (PLGA) solutions using trifluoroethanol (TFE) as a solvent. Lyso-NPs loaded EFMs were obtained by electrospinning of the aforementioned suspensions, and the bioactivity of lysozyme in the EFMs was investigated using fluorescence-based assay kit. The electrosprayed Lyso-NPs were spherical with barely altered bioactivity as compared to the untreated raw material when using EtOH- H2O (30:70, v/v) as the solvent. After the subsequent electrospinning process, more than 90% of the bioactivity of lysozyme was retained compared to the raw material. The cytotoxicity of the produced EFMs was evaluated by thiazolyl blue tetrazolium bromide (MTT) study and the proliferation and distribution of mouse fibroblast cells (L929) growing on EFMs were investigated using 4,6-diamidino-2-phenylindol dihydrochloride (DAPI) for nucleic acid staining. Nearly negligible cytotoxicity of all the EFMs was observed according to the MTT study. Furthermore, it was observed that the L929 cells grew well on the Lyso-EFMs, especially those with the modification of polyethylene glycol (PEG) that was added to improve the hydrophilicity of EFMs. This study demonstrated that the electrospray/electrospinning processes are suitable for loading biomacromolecules to produce functionalized wound dressings to promote wound healing.

Microfluidic Isolation of Circulating Tumor Cells and Cancer Stem-Like Cells from Patients with Pancreatic Ductal Adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) requires multimodal therapeutic approaches and disease monitoring for effective treatment. Liquid biopsy biomarkers, including circulating tumor cells (CTCs) and cancer stem-like cells (CSCs), hold promise for evaluating treatment response promptly and guiding therapeutic modifications.Methods: From 24 patients with metastatic PDAC (stage IV, M1) undergoing active systemic treatment, we collected 78 blood samples at different time points for CTC and CSC isolation using a microfluidic platform functionalized with antibodies against a CTC biomarker, epithelial cell adhesion molecule (EpCAM), or a CSC biomarker, CD133. These isolated cells were further verified, via fluorescent staining and imaging, using cytokeratin (CK), CD45, and nucleic acid stain 4',6-diamidino-2-phenylindole (DAPI).Results: The majority (84.4%) of patient blood samples were positive for CTCs (EpCAM+CK+CD45-DAPI+) and 70.8% of patient blood samples were positive for CSCs (CD133+CK+CD45-DAPI+), using the highest baseline value of healthy samples as threshold. The CTC subtypes (EpCAM+CK+CD45-DAPI+CD133+ and EpCAM+CK+CD45-DAPI+CD133-) and CSC subtypes (CD133+CK+CD45-DAPI+EpCAM+ and CD133+CK+CD45-DAPI+EpCAM-) were also analyzed using immunochemical methods. In several cases, CSCs exhibited cytokeratin expression that did not express EpCAM, indicating that they will not be detected using EpCAM-based isolation.Conclusion: The microfluidic platform enabled the reliable isolation of CTCs and CSCs from PDAC patient samples, as well as their subtypes. Complementary assessment of both CTCs and CSCs appears advantageous to assess the profile of tumor progressing in some cases. This research has important implications for the application and interpretation of approved methods to detect CTCs.

Inhibitory effects of methanolic Olea europaea and acetonic Acacia laeta on growth of Babesia and Theileria

Objective: To evaluate the antipiroplasmic activities of methanolic extract of Olea europaea (MOE) and acetonic extract of Acacia laeta (AAL) against Babesia and Theileria parasites in vitro and evaluate the chemotherapeutic effects of these extracts against Babesia (B.) microti in vivo. Methods: Fluorescence assay using SYBR Green 1 nucleic acid stain was used to detect inhibitory effects of the two extracts as well as the combination effects of the two extracts with diminazene aceturate and atovaquone on four Babesia species and Theileria equi in vitro while for in vivo experiments, 8-weekold female BALB/c mice were injected intraperitoneally with 1 × 107B. microti-iRBCs and treated orally at a dose of 150 mg/kg of both extracts. Results: The half maximal inhibitory concentration (IC50) values of AAL against B. bovis, B. bigemina, B. divergens, B. caballi, and Theileria equi were lower than those of MOE extracts. Toxicity assay on Madin–Darby bovine kidney, mouse embryonic fibroblast (MH/3T3), and human foreskin fibroblast cell lines showed that MOE and AAL affected only the viability of Madin–Darby bovine kidney cell line with half maximal effective concentrations (EC50) of (794.7±41.9) and (873.9±17.5) μg/mL, respectively. The oral treatments of MOE and AAL at 150 mg/kg inhibited the growth of B. microti in mice by 80.4% and 64.4%, respectively. The MOE and diminazene aceturate combination showed a higher chemotherapeutic effect than that of monotherapy. Conclusions: MOE and AAL have the potential to be an alternative remedy for treating piroplasmosis. Furthermore, the combination therapy of MOE + DA was more potent against B. microti infection in mice than their monotherapies.

Aptamer-induced thermofluorimetric protein stabilization and G-quadruplex nucleic acid staining by SYPRO orange dye

Serendipitously discovered nucleic acid staining by SYPRO Orange dye utilized to demonstrate proteins thermal stabilization (increase in melting temperature,Tm) as a function of increased DNA aptamer binding affinity (decrease in dissociation constant,Kd).

Development and viability of biofilms grown on experimental abutments mimicking dental implants: An in vivo model.

BackgroundTo determine whether an experimental abutment mimicking the macro- and microstructure of a dental implant is a suitable method for recovering biofilm, and to describe the features of biofilms formed around such abutments on healthy implants.Material and MethodsExperimental abutments were used in 15 patients without peri-implant diseases. After 14 days’ absence of dental hygiene in this area, the abutments were retrieved and analyzed through confocal laser scanning microscopy and scanning electron microscopy. The biofilm formation on the surface of the first 5 abutments was determined by a fluorescence-staining method using SYTO9 nucleic acid stain. In order to study the biofilm’s coverage and vitality, 10 additional abutments were assessed using live & dead bacterial viability. Descriptive and bivariate analyses of the data were performed.ResultsA global plaque coverage of the abutments was observed in all cases. The submucosal area of the abutment was mostly covered with biofilm (over 21%). Moreover, significant differences between supra- and subgingival locations were detected.ConclusionsThis in vivo experimental model allows detailed observation of the extensive plaque growth found on exposed experimental abutments mimicking dental implants when hygiene measures are absent. The biofilm coverage is significantly higher in the supragingival zone than in the subgingival portion. Key words:Dental implants, biofilm, peri-implant diseases.

Effects of Laser Texture Oxidation and High-Temperature Annealing of TiV Alloy Thin Films on Mechanical and Antibacterial Properties and Cytotoxicity

Titanium dioxide and vanadium oxides have been applied extensively in industrial and medical fields. The objective of this study was to develop various composite structures of titanium and vanadium oxide (Ti-V-O) coatings on pure titanium through high-temperature annealing and laser texturing oxidation, separately; additionally, surface morphologies, tribological and hydrophilic properties, and antibacterial and biocompatibility abilities of these Ti-V-O coatings were evaluated. TiV alloy thin films were deposited on pure titanium and then annealed to form Ti-V-O coatings through thermal oxidation and laser texturing oxidation. Ball-on-disc wear tests and contact angle tests were conducted to evaluate the tribological properties and wettability of the coatings, respectively. The antibacterial activity of the coatings was estimated by SYTO9 nucleic acid staining with Staphylococcus aureus (Gram-positive bacteria). The cell cytotoxicity of the coatings was analyzed following the ISO 10995-5:2009 standard with human skin fibroblast cells. The Ti-V-O coatings, subjected to annealing at 700 °C, demonstrated higher hardness (Hv 1171) and a lower friction coefficient (0.6). The highest hardness (Hv 2711) and the lowest friction coefficient (0.52) were obtained for the Ti-V-O after laser surface texturing oxidation at 100 kHz. The oxide coating obtained from 100 kHz laser texturing oxidation exhibited the lotus effect because of its systematic textured microstructures, and displayed superhydrophobic surface properties. Compared with the unannealed TiV coating, both the samples with high-temperature annealing and laser surface texturing oxidation had excellent antibacterial properties to Staphylococcus aureus. However, the Ti-V-O thin films exhibited notable cell cytotoxicity. Although the cell viability on Ti-V-O coatings were not ideal, this study confirmed improvement in surface hardness, tribology, and antibacterial performance in Ti-V-O coatings, which may have potential for use in biomedical tools, devices, and equipment.

23 Viability staining techniques for cryopreserved spermatozoa in 3 caudata species

Salamanders are the most threatened vertebrate taxa; thus, conservation-based research including spermatozoa cryopreservation and other assisted reproductive technologies is essential to their survival. To determine the effectiveness of sperm cryopreservation, methods for evaluating sperm quality are necessary but underdeveloped in caudate research. Evaluating motility has been the primary analysis for sperm viability but is difficult to perform due to the scythe-like morphology, slow rotating progression, and minute undulations of the tail membrane. Estimating apoptosis is a new approach to evaluating caudate spermatozoa survival through cryostress. Fluorescent dyes, such as SYBR-14, annexin-V, and propidium iodide (PI), are valuable tools for identifying degrees of cell viability, apoptosis, and necrosis. Annexin-V marks the externalization of phosphatidylserine on the cell membrane indicating early steps in the apoptosis signalling cascade. Compromised membranes allow PI, a nucleic acid stain, access to DNA, marking cellular necrosis. The SYBR-14 is a nucleic acid stain that permeatesssss intact membranes, labelling live cells. These fluorescent stains were assessed for marking viability and stages of cell death in post-thaw spermatozoa across 3 caudate species: the Eastern tiger salamander (Ambystoma tigrinum), Kweichow Emperor newt (Tylototriton kweichowensis), and black-spotted newt (Notophthalmus meridionalis). For each species, spermic urine samples were acquired by hormone treatment and frozen based on protocols developed in A. tigrinum, yielding an average of 18.2% relative motility recovered at thaw. Straws were thawed for 5min at 20°C. Viability was tested by staining 5μL 1:1 with a 1:50 dilution of SYBR-14 and 2μL of PI. Stages of cell death were evaluated by staining 10μL with 2μL of annexin-V and 2μL of PI. Cell viability was assessed immediately under a fluorescence microscope. For each of the 3 species, 2 samples were stained with both assays in triplicate. Sperm stained with SYBR-14 alone were considered viable, and sperm stained with any annexin-V or PI were considered not viable. Visible dynamic shifting from SYBR-14 to PI staining was observed in real time, indicating rapid necrosis. Morphological abnormalities, not observed in unstained samples, were prevalent across all species following staining, signifying a possible cytotoxic effect of the dyes. High mortality and abnormality rates suggest that fluorescent dyes have elevated toxicity and permeability in caudate sperm. Caudate spermic urine has a very low osmolality, implying high permeability, which could lead to rapid staining and toxicity effects. Shorter incubation times may be required for accurate staining. Results may also indicate that cryopreservation protocols need to be species specific and do not transfer well across taxa. This is one of the earliest studies to evaluate the use of fluorescent stain protocols on measuring cell viability in caudate sperm and indicates that further refinement is required.

N-Acetylcysteine As a Therapeutic for JAK2V617F Myeloproliferative Neoplasms

Chronic inflammation is common in MPN and drives disease progression and worsens symptom burden. It has been reported that JAK2V617F hematopoietic stem and progenitor cells (HSPCs) exhibit elevated basal oxidative stress compared to wild-type cells. However, JAK2V617F HSCs have a differential response than normal HSPCs upon stimulation with pro-inflammatory cytokines or lipopolysaccharide (LPS) invivo and in vitro. Specifically, JAK2V617F HSPCs do not significantly increase reactive oxygen species, exit quiescence, or increase DNA damage following LPS exposure (Blood 2017 130:4211). Therefore, we hypothesized that oxidative stress is important for differential responses of wild-type and JAK2V617F HSCs to inflammation.To compare cell responses to oxidative stress, we treated bone marrow cells from JAK2V617F knock-in and wild-type mice ex vivo with L-buthionine-S,R-sulfoximine (BSO), which reduces glutathione levels, then measured the impact on myeloid colony formation in methylcellulose. JAK2V617F knock-in bone marrow cells are resistant to oxidative stress-induced reduction in colony formation compared to wild-type bone marrow cells. This data suggests that JAK2V617F myeloid progenitors produce less reactive oxygen species in response to BSO or that these cells are resistant to oxidative stress-induced cell death.Next, we tested the effect of the anti-oxidant n-acetylcysteine (NAC) in a JAK2V617F knock-in mouse model. All hematopoietic cells in this model express JAK2V617F, these mice develop elevated blood counts, splenomegaly, and die suddenly at approximately 2-3 months of age. Surprisingly, addition of NAC (2g/L) in the drinking water extended the lifespan of JAK2V617F-knock in mice (p<0.02, n=6-12). However, aspirin (16mg/L) in drinking water did not extend the lifespan of JAK2V617F-knock in mice. NAC did not alter peripheral blood counts in either JAK2V617F-knock in or wild-type mice, which suggests that NAC's utility in JAK2V617F is not through cytoreduction but is due to its ability to reduce oxidative stress or thrombosis. Additionally, NAC had no effect on thrombin-induced platelet activation, which was assayed by P-selectin expression, phosphatidylserine exposure, and platelet-leukocyte aggregation via flow cytometry.There is a positive correlation between leukocytosis and thrombotic risk in MPN patients. Additionally, MPN patients and mice exhibit elevated neutrophil extracellular trap (NET) formation compared to healthy controls, which contributes to the increased thrombosis in MPN. To test the effect of antioxidants on NET formation in vitro, we treated neutrophils from MPN patients or healthy donors with phorbol myristate acetate concurrently with antioxidants (NAC or ferulic acid) and assayed the presence of extracellular DNA using a SYTOX Green nucleic acid stain. Unstimulated neutrophils from MPN patients exhibited sporadic NET formation while neutrophils from healthy donors did not. Additionally, NAC and ferulic acid reduced DNA release, which is indicative of NET formation.Taken together, these data demonstrate that JAK2V617F progenitors are resistant to oxidative stress-induced cell death. Furthermore, reduction of oxidative stress with n-acetylcysteine in vivo prevented NET-induced thrombosis in JAK2V617F knock-in mice as well as in vitro in normal and MPN neutrophils. These data provide a rationale for investigating the utility of n-acetylcysteine as a therapeutic in myeloproliferative neoplasms. DisclosuresNo relevant conflicts of interest to declare.

Small-scale culture of Plasmodium falciparum using μ-Slide Angiogenesis followed by automatic infection rate counting to assess drug effects

It is very important to reduce the costs involved in malarial drug development by small-scale culture of Plasmodium falciparum, and automation of the assay system for drug efficacy against the parasites for high-throughput screening. In this study, we report that P. falciparum-infected erythrocytes can be stably cultured on μ-Slide Angiogenesis, which is used to investigate angiogenesis in tube formation assays, followed by automatic counting of the infection rate (parasitaemia). After 10 μL of parasite-infected erythrocytes were added to the inner well of μ-Slide Angiogenesis to prevent a multilayer of erythrocytes, 30 μL of silicon oil was overlaid on the culture medium to avoid evaporation of the medium, leading to stable small-scale parasite cultivation. The parasites were stained with a cell-permeant fluorescent nucleic acid stain (SYTO21) followed by cultivation. After taking bright field and fluorescent images using an inverted microscope, the infection rate could be calculated automatically by counting the number of erythrocytes and parasites using MetaMorph Offline software. The effect of anti-malarial drugs on parasite growth could be investigated on μ-Slide Angiogenesis, in which the parasite culture was added to the inner wells containing the drugs followed by their cultivation. Taken together, this method may be useful for image-based screening for anti-malarial drug candidates with automatic counting of parasite infection rates.

Effects of RSC96 Schwann Cell-Derived Exosomes on Proliferation, Senescence, and Apoptosis of Dorsal Root Ganglion Cells In Vitro.

BackgroundStress urinary incontinence is a common condition in women and can be associated with peripheral nerve injury. Exosomes. derived from Schwann cells, can enhance the regeneration of axons of the peripheral nervous system. This study aimed to investigate the effects of RSC96 Schwann cell-derived exosomes in a novel in vitro model of dorsal root ganglion (DRG) cell injury induced by cyclic mechanical strain (CMS).Material/MethodsRSC96 Schwann cells and DRG cells were cultured in vitro. CMS in DRG cells involved mechanical stretch trauma with 5333 μ strain. ExoQuick-TC polymer was used to precipitate exosomes from RSC96 Schwann cell culture medium and identified by nanoparticle tracking analysis, electron microscopy, and Western blot for detection of CD9 and tumor susceptibility gene 101 (Tsg101) protein. Cultured DRG cells were treated with RSC96 Schwann cell-derived exosomes, followed by measurement of cell viability, proliferation, senescence, and apoptosis using the cell counting kit-8 (CCK-8) assay, senescence-associated beta-galactosidase (SA-β-gal) staining, and Hoechst 33258 (blue) fluorescence nucleic acid staining using flow cytometry.ResultsMechanical stretch with 5333 μ strain for 8 hours at 1 Hz decreased the activity of cultured DRG cells. RSC96 Schwann cell-derived exosomes promoted cell proliferation and significantly inhibited apoptosis and senescence of DRG cells following injury induced by CMS.ConclusionsAn in vitro model of DRG cell injury induced by CMS, showed that RSC96 Schwann cell-derived exosomes had a protective effect. The effects of Schwann cell-derived exosomes on peripheral nerve injury, including in stress urinary incontinence, require future in vivo studies.