Dead Cell Counts Research Articles

Environmental RNA metabarcoding for ballast water microbial diversity: Minimizing false positives

While maritime transport boosts global trade by shipping bulk goods, it raises concerns about the spread of harmful bacteria via ballast water. Moreover, the dark and cold environments of ballast tanks often harbor extracellular DNA from dead organisms, leading to false positives in traditional environmental DNA (eDNA) metabarcoding analyses. Here, we alternatively employed environmental RNA (eRNA) metabarcoding to assess its potential for reducing false positive in ballast water monitoring. We collected eDNA and eRNA in parallel from ballast water before and after disinfection in three vessels. High-throughput sequencing of the 16S rRNA V4-V5 regions and cDNA counterparts was conducted to compare bacterial community composition. Our findings showed that over 80 % of the top 150 abundant amplicon sequence variants (ASVs) were detected by both eRNA and eDNA metabarcoding. Samples sequenced separately using DNA and RNA consistently clustered together, indicating similar community recovery efficacy. However, 42 % of ASVs were detected exclusively in DNA, resulting in significantly higher bacterial diversity compared to RNA, which suggests false positives in the DNA method. In treated samples with higher dead cell counts, the RNA method showed significantly lower bacterial diversity, indicating its effectiveness in detecting live bacteria. In summary, eRNA metabarcoding offers comparable recovery efficiency while maintaining a lower false-positive rate than eDNA metabarcoding.

Investigation of Cytotoxic Effects of Tragopogon reticulatus Against HepG2 and MCF-7 Cell Lines and Antioxidant, Antimicrobial Effects Against Some Microorganisms

Plants have been used for various purposes throughout history. This subject has received increasing attention in the field of traditional medicine in recent years. Tragopogon reticulatus, as a forage grass plant in turkey, is a species that stands out with rich vitamin and mineral content. The aim of this study was to investigate the antimicrobial, antioxidant, and cytotoxic activities of T. reticulatus. Two separate extracts were prepared at different concentrations using hexane and methanol solvents obtained from the T. reticulatus plant, and cytotoxic activity was evaluated by the MTT Assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method on human liver cancer (HepG2) and human breast cancer (MCF7) cell lines. The antioxidant activity of the extracts obtained from methanol and hexane was determined by the DPPH (2,2-diphenyl 1-picrylhydrazyl) radical scavenging capacity method. Antimicrobial activity was evaluated by the disk diffusion method, and bacteria such as Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Bacillus megaterium, and Candida albicans fungus were used in the study. The hexane extract obtained from the T. reticulatus plant showed a high cytotoxic activity of 44% in the MCF7 cell line, while it showed a 51% dead cell count in the HepG2 cell line. When the antioxidant results were evaluated, it was determined that the DPPH radical scavenging effect of the hexane extract exhibited the most effective activity at 19%. When the antimicrobial results were evaluated, it was determined that the concentrations prepared with hexane exhibited the most effective activity with a zone diameter of 16 mm against B. megaterium. These findings suggest that T. reticulatus plant extracts can be considered as a potential source. These results may shed light on future research into the biological effects of the plant and contribute to the development of new treatment methods.

Experimental Study on Compatibility of Human Bronchial Epithelial Cells in Collagen-Alginate Bioink for 3D Printing.

This paper reports an experimental study on the compatibility of human bronchial epithelial (HBE) cells in a collagen-alginate bioink. The compatibility was assessed using the culture well method with three bioink compositions prepared from a 10% alginate solution and neutralized TeloCol-10 mg/mL collagen stock solution. Cell viability, quantified by (live cell count-dead cell count)/live cell count within the HBE cell-laden hydrogel, was evaluated using the live/dead assay method from Day 0 to Day 6. Experimental results demonstrated that the collagen-alginate 4:1 bioink composition exhibited the highest cell viability on Day 6 (85%), outperforming the collagen-alginate 1:4 bioink composition and the alginate bioink composition, which showed cell viability of 75% and 45%, respectively. Additionally, the live cell count was highest for the collagen-alginate 4:1 bioink composition on Day 0, a trend that persisted through Days 1 to 6, underscoring its superior performance in maintaining cell viability and promoting cell proliferation. These findings show that the compatibility of HBE cells with the collagen-alginate 4:1 bioink composition was higher compared with the other two bioink compositions.

Unveiling the microbial diversity of biofilms on titanium surfaces in full-scale water-cooling plants using metagenomics approach.

Microbial colonization on the titanium condenser material (TCM) used in the cooling system leads to biofouling and corrosion and influences the water supply. The primary investigation of the titanium condenser was infrequently studied on characterizing biofilm-forming bacterial communities. Different treatment methods like electropotential charge, ultrasonication, and copper coating of titanium condenser material may influence the microbial population over the surface of the titanium condensers. The present study aimed to catalog the primary colonizers and the effect of different treatment methods on the microbial community. CFU (1.7 × 109CFU/mL) and ATP count (< 5000 × 10-7 relative luminescence units) showed a minimal microbial population in copper-coated surface biofilm as compared with the other treatments. Live and dead cell result also showed consistency with colony count. The biofilm sample on the copper-coated surface showed an increased dead cell count and decreased live cells. In the metagenomic approach, the microbiome coverage was 10.06Mb in samples derived from copper-coated TCM than in other treated samples (electropotential charge-17.94Mb; ultrasonication-20.01Mb), including control (10.18Mb). Firmicutes preponderate the communities in the biofilm samples, and Proteobacteria stand next in the population in all the treated condenser materials. At the genus level, Lactobacillaceae and Azospirillaceae dominated the biofilm community. The metagenome data suggested that the attached community is different from those biofilm samples based on the environment that influences the bacterial community. The outcome of the present study depicts that copper coating was effective against biofouling and corrosion resistance of titanium condenser material for designing long-term durability.

Abstract 4722: Enhanced phenotypic screening: A kinetic, multiplexed approach to analyzing drug effects using Incucyte® Live-Cell Analysis System

Abstract Phenotypic screening is a valuable tool for analysis of the complex mix of effects that drug treatment has on cells. Traditionally image-based screening uses fixed cells stained with reagents at a pre-chosen end point, however our technique combines kinetic, label-free readouts with non-perturbing fluorescence reporters to enable information-rich quantification of compound effects. A549 lung cancer cells were treated with a library of over 800 FDA-approved drugs while images were acquired in the Incucyte® Live-Cell Analysis System every two hours for four days. Cell growth and viability were measured using label-free readouts of confluence and dead cell count while cell cycle, cell death and Akt pathways were investigated using multiplexed fluorescent readouts. Cytotoxicity was measured using label-free Incucyte® AI Cell Health Analysis Software Module which uses two neural networks to segment individual cells and classify them as live or dead. Assay robustness was quantified using the time course of positive and negative controls (camptothecin and vehicle, respectively). Z’ reached a maximal value &amp;gt;0.9 between 48h and 72h; at 72h 4.4% of the compounds within the library induced &amp;gt;50% cell death. Correlation between % Live cells and % Confluence indicated 3 types of effect: cytotoxic (low viability, low confluence), cytostatic (high viability, low confluence), and none (high viability, high confluence). Morphological changes were observed in a multitude of wells with 29 compounds inducing cell enlargement &amp;gt;1800 µm2, suggesting induction of senescence. Hits from fluorescent readouts were identified as the compounds which perturbed the quantified readout more than 3 standard deviations away from the mean value of the vehicle. Mechanisms of cell death were examined by correlative analysis of the total dead cell population (label-free cell death identification), caspase-active (caspase 3/7-reagent positive) and Annexin V positive cells. This enabled rapid identification of compounds which induced apoptosis by caspase-independent pathways, including the serotonin-selective reuptake inhibitor Sertraline. Kinetic plots of cell cycle stage (% S|G2|M or G1) revealed contrasting mechanisms. Mycophenolic acid induced cell cycle arrest, observed as a consistent increase in the percentage of cells in G1 over time. However flumazenil displayed repeated, transient peaks in the percentage of cells in S|G2|M over the same 72h period, indicating synchronisation behavior. Incucyte® Live-Cell Analysis System as a phenotypic screening platform provides valuable kinetic data from cells within the physiologically relevant environment of a standard incubator. Label-free readouts yield direct cellular information and can be combined with fluorescent data to provide vastly more information than a single endpoint readout. Citation Format: Kirsty McBain, Gillian Lovell, Jasmine Trigg, Nicola Bevan, Daniel Appledorn. Enhanced phenotypic screening: A kinetic, multiplexed approach to analyzing drug effects using Incucyte® Live-Cell Analysis System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4722.

Abstract 225: Integration of patient-derived tumor organoids and patient clinical multimodal data to investigate the role of organoids in predicting treatment response

Abstract Introduction: Patient-derived organoids (PDOs) enable the ex vivo study of tumor heterogeneity and its effect on response to treatment. Our previous work demonstrated that our robust pan-cancer organoid platform shows genetic and transcriptomic concordance with clinical tumor samples and can be deployed for high-throughput drug response screening. Here, we ran a standard of care (SOC) panel on PDOs with paired de-identified clinically curated data to enable a comparison of patient response to the same drugs ex vivo and exploration of potentially more effective treatment alternatives. Methods: Patient tumor samples were cultivated into tumor PDOs in extracellular matrix + chemically defined media. PDOs underwent pathological review to ensure development of malignant tissue. PDOs were identified by clusters of Hoechst-positive cells and live and dead cell counts for each PDO were determined using fluorescent stains. The median number of cells per PDO in the well was calculated, excluding PDOs with &amp;lt;3 cells and the top 1% of PDOs by area. Drug screens were run with 6 doses of the compound and the inverse area under the curve of ToPro3 live cell measurements was calculated to quantify response. Tempus xT DNA-seq and xR whole-transcriptome assays were used to perform NGS on organoids and patient samples where available. Data was processed through our standard pipeline to identify targetable mutations, neoantigens, copy number variants (CNVs) and fusions. Results: We analyzed 38 PDOs with paired patient data across 9 different cancer types comprising mostly lung (n=14) and colorectal (n=12); 17 PDOs had responses associated with treatment given close to the biopsy collection date. Across the full PDO cohort, we observed a range of responses to SOC compounds, providing a platform to better understand biomarkers and mechanisms of response. Treatment response in PDOs correlated with patient response in many cases. In the 3 patients with progression-free survival &amp;gt;1 year and outcomes of either complete response or stable disease, all showed strong responses to the corresponding drugs in the SOC panel. For most patients with progressive disease recorded after treatment the corresponding PDO cluster showed limited response to treatment. The SOC panel includes several targeted therapies that provide insight into how patients respond to treatment beyond chemotherapy. For two patients with limited response to treatment in both patient and PDO, we identified targeted therapies relevant to their clinico-genomic landscape. One patient showed improved clinical outcomes to a later line of therapy with a similar mechanism of action to the drug that showed response in the organoid. Conclusion: These results suggest that PDOs may serve as a powerful tool for predicting patient response to treatment and aid the development of new therapies. Citation Format: Kathleen A. Burke, Brian Larsen, Yi-Hung Carol Tan, Jessica Barbeau, Curtis Brinkman, Elle Moore, Nick Callamaras, Jonathan R. Dry, Iker Huerga, Kate Sasser, Jeffrey A. Borgia. Integration of patient-derived tumor organoids and patient clinical multimodal data to investigate the role of organoids in predicting treatment response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 225.

Abstract 5332: Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development

Abstract Cell viability is one of the critical quality attributes (CQAs) for cell and gene therapy products. Characterization of cell viability, in addition to cell identity, cell count and other CQAs, are essential to ensure the quality, safety and efficacy throughout various developmental stages of cell and gene therapies. To determine cell viability, a variety of viability detection methods have been used, such as trypan blue (TB) exclusion assay, and fluorescence-based dyes including acridine orange/propidium iodide (AO/PI), acridine orange/4′,6-diamidino-2-phenylindole (AO/DAPI), all of which have been employed on flow or image-based cytometry systems. In this work, we employed the Cellaca MX high-throughput cell counter to characterize and compare AO/PI and AO/DAPI viability detection methods. First, we measured the viabilities of different ratios of viable and non-viable Jurkat cells created via two cell dying processes. Next, viabilities of the stained cells were measured multiple times within a 30 min staining time for each method in order to monitor the trend of viability changes. The viability results showed that the AO/DAPI method were generally higher than AO/PI method with differences ranging from 0% to 24%. We also observed the discrepancy of the measured viability differences between naturally-dying and heat-killed Jurkat cells, suggesting that the viability difference might be dependent on the cell dying process. In the time-dependent viability characterization, results from AO/DAPI method showed increasing dead cell counts leading to decreasing viability, revealing an on-going staining process. In contrast, results from AO/PI method showed more consistent cell count and viability measurements, indicating a more instantaneous staining process. Lastly, we conducted a cell proliferation assay as an orthogonal method to confirm replicability of the low viability cell samples. The proliferation assay results showed no significant increase in cell concentrations within the 4-day time frame. The viability results before the culture were approximately 0% for both naturally-dying and heat-killed Jurkat cells, using the AO/PI staining method, and 12% and 9% using the AO/DAPI respectively. Furthermore, spiked samples of natural-dying and heat-killed cells with healthy cells (&amp;gt;90% viability) were generated and measured, which showed exponential growth. The viability results of these spiked samples before the culture were 11% and 9% by AO/PI and 17% and 18% by AO/DAPI, respectively. Taken together, these results suggest that cell viability methods need to be optimized for different fluorescent stains in order to generate reliable, robust, and fit-for-purpose results for the downstream assays for cell and gene therapy products.. Citation Format: Yongyang Huang, Samir Patel, Mackenzie Pierce, Carolina Franco Nitta, Bo Lin, Leo Chan. Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5332.

Optimal Intravenous Administration Procedure for Efficient Delivery of Canine Adipose-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) are currently being investigated for their therapeutic applications in a wide range of diseases. Although many studies examined peripheral venous administration of MSC, few have investigated the detailed intravenous administration procedures of MSC from their preparation until they enter the body. The current study therefore aimed to explore the most efficient infusion procedure for MSC delivery by preparing and infusing them under various conditions. Canine adipose-derived mesenchymal stem cells (cADSC) were infused using different infusion apparatuses, suspension solutions, allogenic serum supplementation, infusion time and rates, and cell densities, respectively. Live and dead cell counts were then assessed by manual measurements and flow cytometry. Efficiency of live- and dead-cell infusion and cell viability were calculated from the measured cell counts and compared under each condition. Efficiency of live-cell infusion differed significantly according to the infusion apparatus, infusion rate, and combination of cell density and serum supplementation. Cell viability after infusion differed significantly between the infusion apparatuses. The optimal infusion procedure resulting in the highest cell delivery and viability involved suspending cADSC in normal saline supplemented with 5% allogenic serum at a density of 5 × 105 cells/mL, and infusing them using an automatic infusion device for 15 min. This procedure is therefore recommended as the standard procedure for the intravenous administration of ADSC in terms of cell-delivery efficiency.

Neuroprotection of Rodent and Human Retinal Ganglion Cells In Vitro/Ex Vivo by the Hybrid Small Molecule SA-2.

The mechanisms underlying the neuroprotective effects of the hybrid antioxidant-nitric oxide donating compound SA-2 in retinal ganglion cell (RGC) degeneration models were evaluated. The in vitro trophic factor (TF) deprivation model in primary rat RGCs and ex vivo human retinal explants were used to mimic glaucomatous neurodegeneration. Cell survival was assessed after treatment with vehicle or SA-2. In separate experiments, tert-Butyl hydroperoxide (TBHP) and endothelin-3 (ET-3) were used in ex vivo rat retinal explants and primary rat RGCs, respectively, to induce oxidative damage. Mitochondrial and intracellular reactive oxygen species (ROS) were assessed following treatments. In the TF deprivation model, SA-2 treatment produced a significant decrease in apoptotic and dead cell counts in primary RGCs and a significant increase in RGC survival in ex vivo human retinal explants. In the oxidative stress-induced models, a significant decrease in the production of ROS was observed in the SA-2-treated group compared to the vehicle-treated group. Compound SA-2 was neuroprotective against various glaucomatous insults in the rat and human RGCs by reducing apoptosis and decreasing ROS levels. Amelioration of mitochondrial and cellular oxidative stress by SA-2 may be a potential therapeutic strategy for preventing neurodegeneration in glaucomatous RGCs.

Bactericidal Efficacy of Nanostructured Surfaces Increases under Flow Conditions.

Bacterial colonization on solid surfaces creates enormous problems across various industries causing billions of dollars' worth of economic damages and costing human lives. Biomimicking nanostructured surfaces have demonstrated a promising future in mitigating bacterial colonization and related issues. The importance of this non-chemical method has been elevated due to bacterial evolvement into antibiotic and antiseptic-resistant strains. However, bacterial attachment and viability on nanostructured surfaces under fluid flow conditions has not been investigated thoroughly. In this study, attachment and viability of Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) on a model nanostructured surface were studied under fluid flow conditions. A wide range of flow rates resulting in a broad spectrum of fluid wall shear stress on a nanostructured surface representing various application conditions were experimentally investigated. The bacterial suspension was pumped through a custom-designed microfluidic device (MFD) that contains a sterile Ti-6Al-4V substrate. The surface of the titanium substrate was modified using a hydrothermal synthesis process to fabricate the nanowire structure on the surface. The results of the current study show that the fluid flow significantly reduces bacterial adhesion onto nanostructured surfaces and significantly reduces the viability of adherent cells. Interestingly, the bactericidal efficacy of the nanostructured surface was increased under the flow by ∼1.5-fold against P. aeruginosa and ∼3-fold against S. aureus under static conditions. The bactericidal efficacy had no dependency on the fluid wall shear stress level. However, trends in the dead-cell count with the fluid wall shear were slightly different between the two species. These findings will be highly useful in developing and optimizing nanostructures in the laboratory as well as translating them into successful industrial applications. These findings may be used to develop antibacterial surfaces on biomedical equipment such as catheters and vascular stents or industrial applications such as ship hulls and pipelines where bacterial colonization is a great challenge.

Vitexin alters Staphylococcus aureus surface hydrophobicity to obstruct biofilm formation

Cell Surface hydrophobicity is one of the determinant biophysical parameters of bacterial aggregation for being networked to form a biofilm. Phytoconstituent, like vitexin, has long been in use for their antibacterial effect. The present work demonstrates the role of vitexin in modulating Staphylococcus aureus surface hydrophobicity while aggregating to form biofilm and pathogenesis in a host. In planktonic form, vitexin shows minimum inhibitory concentration at 252 µg/ml against S. aureus. Sub-MIC doses of vitexin and antibiotics (26 µg/ml of vitexin, 55 µg/ml of azithromycin, and 2.5 µg/ml of gentamicin) were selected to treat S. aureus. Dead cell counts after treatment were studied through flow cytometry. As dead cell counts were minimal (<5 %), these doses were considered for all subsequent experiments. While studying aggregating cells, it was observed that vitexin reduces S. aureus surface hydrophobicity and membrane permeability at the sub-MIC dose of 26 µg/ml. The in silico binding analysis showed a higher binding affinity of vitexin with surface proteins (IcaA, DltA, and SasG) of S. aureus. Down-regulation of dltA and icaAB expression, along with the reduction in membrane potential with a sub-MIC dose of vitexin, explains reduced S. aureus surface hydrophobicity. Vitexin was found to interfere with S. aureus biofilm-associated protein biomass, EPS production, and swarming movement. Subsequently, the suppression of proteases production and down-regulation of icaAB and agrAC gene expression with a sub-MIC dose of vitexin explained the inhibition of S. aureus virulence in vitro. Besides, vitexin was also found to potentiate the antibiofilm activity of sub-MIC doses of gentamicin and azithromycin. Treatment with vitexin exhibits a protective response in S. aureus infected macrophages through modulation of expression of cytokines like IL-10 and IL-12p40 at protein and mRNA levels. Furthermore, CFU count and histological examination of infected mouse tissue (liver and spleen) justify the in vivo protective effect of vitexin from S. aureus biofilm-associated infection. From this study, it can be inferred that vitexin can reduce S. aureus surface hydrophobicity, leading to interference with aggregation at the time of biofilm formation and subsequent pathogenesis in a host.

Fresh Versus Frozen Stool for Fecal Microbiota Transplantation-Assessment by Multimethod Approach Combining Culturing, Flow Cytometry, and Next-Generation Sequencing.

The objective of this work was to compare the quality of FMT preparations made from fresh feces with those made from feces frozen at –30°C without any pre-processing or cryopreservation additives. The research hypothesis was that such preservation protocol (frozen whole stool, then thawed and processed) is equipotent to classical fresh FMT preparation. For that, three complementary methods were applied, including: (i) culturing in aerobic and anaerobic conditions, (ii) measuring viability by flow cytometry, and (iii) next-generation sequencing. Flow cytometry with cell staining showed that the applied freezing protocol causes significant changes in all of the observed bacterial fractions. Alive cell counts dropped four times, from around 70% to 15%, while the other two fractions, dead and unknown cell counts quadrupled and doubled, with the unknown fraction becoming the dominant one, with an average contribution of 57.47% per sample. It will be very interesting to uncover what this unknown fraction is (e.g., bacterial spores), as this may change our conclusions (if these are spores, the viability could be even higher after freezing). Freezing had a huge impact on the structure of cultivable bacterial communities. The biggest drop after freezing in the number of cultivable species was observed for Actinobacteria and Bacilli. In most cases, selected biodiversity indices were slightly lower for frozen samples. PCoA visualization built using weighted UniFrac index showed no donor-wise clusters, but a clear split between fresh and frozen samples. This split can be in part attributed to the changes in the relative abundance of Bacteroidales and Clostridiales orders. Our results clearly show that whole stool freezing without any cryoprotectants has a great impact on the cultivability and biodiversity of the bacterial community, and possibly also on the viability of bacterial cells.

Protein-conjugated microbubbles for the selective targeting of S. aureus biofilms

Staphylococcus aureus (S. aureus) is an important human pathogen and a common cause of bloodstream infection. The ability of S. aureus to form biofilms, particularly on medical devices, makes treatment difficult, as does its tendency to spread within the body and cause secondary foci of infection. Prolonged courses of intravenous antimicrobial treatment are usually required for serious S. aureus infections. This work investigates the in vitro attachment of microbubbles to S. aureus biofilms via a novel Affimer protein, AClfA1, which targets the clumping factor A (ClfA) virulence factor – a cell-wall anchored protein associated with surface attachment. Microbubbles (MBs) are micron-sized gas-filled bubbles encapsulated by a lipid, polymer, or protein monolayer or other surfactant-based material. Affimers are small (∼12 kDa) heat-stable binding proteins developed as replacements for antibodies. The binding kinetics of AClfA1 against S. aureus ClfA showed strong binding affinity (KD = 62 ± 3 nM). AClfA1 was then shown to bind S. aureus biofilms under flow conditions both as a free ligand and when bound to microparticles (polymer beads or microbubbles). Microbubbles functionalized with AClfA1 demonstrated an 8-fold increase in binding compared to microbubbles functionalized with an identical Affimer scaffold but lacking the recognition groups. Bound MBs were able to withstand flow rates of 250 μL/min. Finally, ultrasound was applied to burst the biofilm bound MBs to determine whether this would lead to biofilm biomass loss or cell death. Application of a 2.25 MHz ultrasound profile (with a peak negative pressure of 0.8 MPa and consisting of a 22-cycle sine wave, at a pulse repetition rate of 10 kHz) for 2 s to a biofilm decorated with targeted MBs, led to a 25% increase in biomass loss and a concomitant 8% increase in dead cell count. The results of this work show that Affimers can be developed to target S. aureus biofilms and that such Affimers can be attached to contrast agents such as microbubbles or polymer beads and offer potential, with some optimization, for drug-free biofilm treatment.

Development and in vitro validation of flexible intraretinal probes

The efforts to improve the treatment efficacy in blind patients with retinal degenerative diseases would greatly benefit from retinal activity feedback, which is lacking in current retinal implants. While the door for a bidirectional communication device that stimulates and records intraretinally has been opened by the recent use of silicon-based penetrating probes, the biological impact induced by the insertion of such rigid devices is still unknown. Here, we developed for the first time, flexible intraretinal probes and validated in vitro the acute biological insertion impact in mouse retinae compared to standard silicon-based probes. Our results show that probes based on flexible materials, such as polyimide and parylene-C, in combination with a narrow shank design 50 µm wide and 7 µm thick, and the use of insertion speeds as high as 187.5 µm/s will successfully penetrate the retina, reduce the footprint of the insertion to roughly 2 times the cross-section of the probe, and induce low dead cell counts, while keeping the vitality of the tissue and recording the neural activity at different depths.

Antimicrobial Capacity and Surface Alterations Using Photodynamic Therapy and Light Activated Disinfection on Polymer-Infiltrated Ceramic Material Contaminated with Periodontal Bacteria.

This study determined the antimicrobial efficiency of light-activated disinfection (LAD) and photodynamic therapy (PDT) on polymer-infiltrated ceramic network (PICN) material contaminated with three periodontal bacteria and explored if PDT and LAD cause PICN surface alterations. Sixty PICN discs were contaminated with Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola and randomly divided into five groups (n = 12 samples/each) according to the treatment groups: Group PDT—PDT (630 ± 10 nm diode laser) with methylene blue; Group DL—808 nm diode laser in contact mode without photosensitizer; Group MB–methylene blue without light application; Group CHX—0.12% chlorhexidine digluconate solution and; Group NT—no treatment. Each disc was then placed in tubes containing phosphate buffered saline (PBS) and vortexed for 30 s to remove the remaining bacteria from the discs. A total of 10× serial dilutions were performed followed by plating of 30 μL of suspension on Brucella agar plates. The colony forming units (CFU) were calculated after 72 h. PICN discs with the attached biofilms were used for confocal microscopy investigation for live/dead bacterial viability. A random single sample from each group was selected to study the bacterial adherence and topographical alterations on PICN discs under scanning electron microscope (SEM). The PDT group showed higher reduction for each bacterial species and total counts of bacteria assessed followed by the DL group (p < 0.05). When compared with MB group, the two laser groups were significantly superior (p < 0.05). The MB group did not show significant differences for any bacteria when compared to NT. The bacteria with the CHX group and DL groups appeared dead with few areas of surviving green stained bacteria. The PDT group showed the highest dead cell count (p < 0.05). PDT and DL groups indicate no significant changes on the surface compared to the sterile PICN discs on visual assessment. Photodynamic therapy produced superior periodontal bacteria reduction over the surface of PICN surface. PDT group showed higher reduction for each bacterial species and total counts of bacteria assessed followed by the DL group. Both PDT and DL treatment strategies are effective without producing surface alterations on PICN.

Curcumin induces chemosensitization to doxorubicin in Duke's type B coloadenocarcinoma cell line.

Cancer cells require higher levels of ATP for their sustained growth, proliferation, and chemoresistance. Mitochondrial matrix protein, C1qbp is upregulated in colon cancer cell lines. It protects the mitochondria from oxidative stress, by inhibiting the Membrane Permeability Transition (MPT) pore and providing uninterrupted synthesis of ATP. This intracellular interaction of C1qbp could be involved in chemoresistance development. Natural chemosensitizing agent, curcumin has been used in the treatment of multiple cancers. In this current study, we elucidate the role of C1qbp during curcumin induced chemosensitization to doxorubicin resistant colon cancercells. The possible interaction between C1qbp and curcumin was determined using bioinformatics tools-AutoDock, SYBYL, and PyMol. Intracellular doxorubicin accumulation by fluorimetry and dead cell count was carried out to determine development of chemoresistance. Effect of curcumin treatment and cytotoxicity was measured by MTT and lactate dehydrogenase release. Morphological analysis by phase contrast microscopy and colony forming ability by colonogenic assay were alsoperformed. Inaddition,Cox-2 could mediate P-glycoprotein upregulation via phosphorylation of c-Jun.Thus, the gene level expression of P-glycoprotein and Cox-2 was alsoinvestigated using PCR. Through molecular docking we identified possible interaction between curcumin and C1qbp. We observed development of chemoresistance upon 6th day treatment. Concentration dependent alleviation of chemoresistance development by curcumin was confirmed and wasfound to reducegene level expression ofP-glycoprotein and Cox-2. Hence, curcumin could interact directly with C1qbp protein and this interaction could contribute to the chemosensiting effect to doxorubicin in colon cancer cells.

Biological Effects of Anodic Oxidation on Titanium Miniscrews: An In Vitro Study on Human Cells.

This controlled in vitro study compared the effects of varying the thickness of a TiO2 layer on cellular activity using commercially available miniscrew samples with identical surface features to derive information with direct clinical impact. Titanium grade V plates with four different thicknesses of TiO2 layer/color were used: absent/gray (Control group), 40–50 nm/pink (Pink group), 130 nm/gold (Gold group) and 140 nm/rosé (Rosé group). In vitro experiments used Saos-2 cells and included cell growth analysis, phospho-Histone H3 and procollagen I staining, cell viability analysis, and a cell migration assay at 12, 24, 40 and to 48 h. Few differences were seen among the groups, with no clear behavior of cellular activity according to the TiO2 thickness. The Control group showed a greater cell count. Phospho-Histone H3 staining was similar among the groups and procollagen I staining was greater in the Rosé group. Cell viability analysis showed a significant difference for live cell counts (greater in the Rosé group) and no difference for the dead cell counts. The cell migration assay showed a delay for the Rosé group up to 40 h, where full repopulation of cell-free areas was obtained at 48 h. The results suggest that the TiO2 layers of the commercial miniscrews have minimal biological effects, including cytotoxicity, with possibly negligible or minimal clinical implications.

Investigation of apoptotic effect of juglone on CCL-228-SW 480 colon cancer cell line.

Colon cancer is a major cause of morbidity and mortality in the world. Juglone is a natural compound which has been isolated from Juglans mandshurica Maxim, and it has various pharmacological effects such as antiviral, antibacterial, and anticancer. In our study, we aimed to investigate the effect of juglone on CCL-228-SW 480 colon carcinoma cell line in monolayer and spheroid culture medium. The CCL-228-SW 480 cell lines were cultured in both monolayer and spheroid cultures. Cells were treated with juglone at 24, 48, and 72 h of incubation. ID50 inhibition was determined on the dose for juglone and after it was found 20 μM was applied to the cells to examine the effect of juglone on CCL-228-SW 480 colon carcinoma cell line. After Juglone was applied the BrdU marking index, Transferase dUTP Nick ends Labeling (TUNEL) assay, active caspase-3 assay, apoptosis-inducing factor (AIF) assay were determined by immunohistochemistry in both the monolayer and spheroid cultures. The control group had a healthy pattern of S-phase fraction, and many of the CCL-228-SW 480 cells nuclei were observed to be positive for BrdU. Terminal Deoxynucleotidyl TUNEL-positive cells, active Caspase-3, and AIF were detected after treatment with juglone in both the monolayer and spheroid cultures. The dead cell count was higher in the CCL-228-SW 480 cell lines with juglone applied than in the controls. Juglone significantly inhibits the proliferation and induces the apoptosis of CCL-228-SW 480 cells in vitro.

Expansion capacity of human muscle progenitor cells differs by age, sex, and metabolic fuel preference.

Activation of satellite cells and expansion of the muscle progenitor cell (MPC) population are essential to generate a sufficient number of cells to repair damaged skeletal muscle. Proliferating MPCs have high energetic and biosynthetic material requirements, and the ability to utilize oxidative phosphorylation (OXPHOS) and/or glycolysis may affect expansion capacity of MPCs. In the present study, we investigated the effect of donor age and sex on human (h)MPC expansion capacity and metabolic fuel preference. hMPCs from young and old male and female donors were grown for 408 h (17 days). Percent confluence, live nuclei count, and dead cell count were measured every 24 h. Metabolic phenotype was assessed by glucose uptake, expression of genes related to glycolysis and OXPHOS, and the Seahorse XF24 Phenotype Test Kit during the exponential phase of growth. hMPCs from old male donors had impaired expansion capacity secondary to heightened cell death early in expansion compared with hMPCs from young male donors, an effect not observed in female hMPCs. Age-related differences in metabolism were also sex dependent; markers of OXPHOS were altered in old (vs. young) male hMPCs, whereas markers of metabolism were largely unaffected by age in female hMPCs. For the first time, we identify sex-specific differences in cell death and OXPHOS that contribute to impaired expansion capacity of hMPC cell populations with age.

Determination of Apoptotic Effect of Juglone on Human Bladder Cancer TCC-SUP and RT-4 Cells: An In Vitro Study.

This study aimed to investigate the effects of juglone on the human bladder carcinoma cell lines TCC-SUB and RT-4 in monolayer and spheroid cultures. Cells were treated with juglone at 24, 48, and 72 h of incubation. The activity of caspase-3 was detected in vitro using a caspase-3 colorimetric assay kit according to the manufacturer's instructions. The bromodeoxyuridine (BrdU) labeling index was used to determine the cells of the synthesis phase. The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay was used to determine the death of cells in both the monolayer and spheroid cultures. The control group had a large S-phase fraction and many of the TCC-SUB and RT-4 cells nuclei were observed to be positive for BrdU. The dead cell count was higher in the TCC-SUB and RT-4 cell lines with juglone applied than in the controls. We conclude that juglone significantly inhibits the proliferation and induces the apoptosis of TCC-SUB and RT-4 cells in vitro.