Live Cell Counts Research Articles

Influence of Dental Pulp Harvesting Method on the Viability and Differentiation Capacity of Adult Dental Pulp-Derived Mesenchymal Stem Cells.

Objective To compare two pulp harvesting methods for stem cell expansion, namely, conservative pulpotomy and pulpectomy from exodontia. Method Ten freshly extracted sound third molars from five patients were selected. Five were used in the control group, where pulp harvesting was performed by exodontia and the remaining teeth were used in the test group, where the pulp was harvested by conservative pulpotomy (preserving the tooth). This was a split-mouth design study, where a third molar from one side was randomly allocated into the test group and the contralateral tooth in the control group. After pulp harvesting, the following evaluations were performed: cell morphology, sterility test, immunophenotyping, differentiation assays, first pass live cell counts, time to cryopreservation, and total number of expanded cells at the end of the fourth pass. Results Regarding morphology, the cells from both groups presented a fibroblastic phenotype. All samples were sterile. Immunophenotyping demonstrated a positive expression for CD105, CD90, and CD73 and negative expression for CD45 in both groups. Differentiation assays were positive for osteogenic and chondrogenic differentiation in both groups. Regarding live cell counts in the first passage, the control group had 95.8% live cells in the total count and the test group 91.2% (p < 0.05). The time required for cryopreservation was equivalent in both groups 51.6 days and 52.6 days, respectively (p > 0.05). The total number of cells at the end of the fourth passage was 5,286,782 and 5,736,862, respectively (p > 0.05). Conclusion These results suggest that adult stem cell harvesting from conservative pulpotomy is as effective as the traditional exodontia-based method.

Effect of Low Nutrition and T-2 Toxin on C28/I2 Chondrocytes Cell Line and Chondroitin Sulfate-Modifying Sulfotransferases.

To investigate the effects of low nutrition and trichothecenes-2 toxin (T-2) on human chondrocytes cell line C28/I2 and the gene expression levels of some chondroitin sulfate (CS)-modifying sulfotransferases. The chondrocytes were divided into 4 intervention groups: (a) control group (Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 [DMEM/F-12] with fetal bovine serum [FBS]), (b) low-nutrition group (DMEM/F-12 without FBS), (c) T-2 group (DMEM/F-12 with FBS plus 20 ng/mL T-2), and (d) combined group (DMEM/F-12 without FBS plus 20 ng/mL T-2). Twenty-four hours postintervention, ultrastructural changes in the chondrocytes were observed by transmission electron microscopy (TEM). Live cell staining and methyl thiazolyl tetrazolium (MTT) assay were performed to observe cell viability. The expression of CS-modifying sulfotransferases, including carbohydrate sulfotransferase 3, 12, 13, 15 (CHST-3, CHST-12, CHST-13, and CHST-15, respectively), and uronyl 2-O-sulfotransferase (UST) were examined by quantitative real-time polymerase chain reaction (RT-qPCR) analysis. The cells in the T-2 group and combined group had significantly lower live cell counts and relative survival rates than the control group. TEM pictures revealed decreased electron density of mitochondria in the low-nutrition group. The T-2 group and combined group both caused mitochondrial swelling, damage, and reduction in mitochondrial number. RT-qPCR showed a trend of altered expression of CHST and increased expression of UST genes under low-nutrition, T-2 toxin and combined interventions. These results show early-stage Kashin-Beck disease chondrocyte pathophysiology, consisting of chondrocyte cell damage and compensatory upregulation of CHST and UST genes.

P060 A2B adenosine receptor deficiency ameliorates chronic murine Crohn’s-like-ileitis

Abstract Background The A2B adenosine receptor (Adora2b) has been demonstrated to play an immunomodulatory and epithelial barrier protective role in acute murine colitis. A2B agonist treatment during acute murine colitis has been demonstrated to decrease colonic inflammation and damage suggesting targeting the A2B adenosine receptor may protect the intestinal barrier in acute intestinal inflammation. Crohn’s disease (CD) is associated with chronic inflammation and damage in the small intestine, most often the ileum. To date the role, if any, of the A2B adenosine receptor in the small intestine and in CD is unknown. Methods The TNFΔARE model of Crohn’s-like ileitis was used to investigate a possible role for A2B in small intestinal inflammation. TNFΔARE mice develop discontinuous, transmural inflammation in the distal ileum that progresses with age and is representative of the chronic inflammation observed in CD. Whole body A2B receptor deficient (Adora2b -/-) mice were crossed on to the TNFΔARE background to generate TNFΔARE mice deficient in the A2B adenosine receptor (Adora2b -/-/TNFΔARE+/-). Whole ileum was harvested from Adora2b -/-/TNFΔARE+/- and wildtype TNFΔARE mice for histological analysis during early (6–8 weeks), active (10–12 weeks) and chronic disease (&amp;gt;20 weeks). Histological scoring was performed by a board-certified pathologist blinded to the study. Whole tissue was also harvested for digestion to perform live cell counts and cytokine analysis. Results Histological analysis revealed no significant difference between wildtype and Adora2b deficient TNFΔARE mice during early or active disease. However, during chronic disease Adora2b -/-/TNFΔARE+/- histological scores for active inflammation, chronic inflammation and degree of tissue damage were significantly reduced (approximately 25%) compared to wildtype TNFΔARE mice. This dramatic improvement in histological tissue health in mice with chronic disease was accompanied by an approximately 50% reduction in live cell counts in the ileum of Adora2b -/-/TNFΔARE+/- mice compared to wildtype TNFΔARE mice with a corresponding reduction in pro-inflammatory mediators CXCL1 and IL-1β Conclusion Our findings demonstrate that sustained loss of A2B receptor expression improves the outcome of chronic small intestinal inflammation. Previous studies demonstrate that A2B receptor expression promotes pro-fibrotic responses in chronic lung fibrosis. Our future studies will address the possible functional role of A2B in pro-fibrotic responses associated with chronic intestinal inflammation.

The “Burn”: water quality and microbiological impacts related to limited free chlorine disinfection periods in a chloramine system

To control microbial proliferation and nitrification within distribution systems, utilities practicing secondary disinfection chloramination often discontinue their ammonia feed and provide a short, free chlorine disinfection period (FClP), commonly referred to as a “chlorine burn”. However, the success and practicality of this approach is often criticized because of the return to nitrification; yet, previous studies conducted in full-scale distribution systems do not contain the sampling frequency to determine how quickly nitrification can return. In this research, a total of 15 hydrants distributed across hydraulically modeled water ages were sampled for 21 sampling events over a period spanning two annual FClPs (2018 and 2019) to investigate the water quality, planktonic community, and, using a new sampling technique, established biofilm community impacts within a single, distribution system pressure zone. Hydrants measured elevated nitrite only 10 weeks after the end of the FClP and live cell counts in the bulk and scour samples statistically significantly increased within two weeks after the FClP ended and chloramine disinfection resumed, indicating limited impacts from a FClP. Furthermore, the FClP significantly increased iron concentrations during the period of free chlorine disinfection creating a consumer water quality concern. Microbial fingerprint analysis using flow cytometry revealed that beta diversity did not significantly change for sampling locations that experienced even periodic low total chlorine concentrations. Only locations that maintained high chlorine residuals throughout both chloramine and free chlorine disinfection periods demonstrated significant changes in bulk water microbial community. Even for these locations, microbial communities of the scoured biofilms remained similar over the course of the study.

Stability of Bacillus coagulans IBRC-M 10807 and Lactobacillus plantarum PTCC 1058 in Milk Proteins Concentrate (MPC)-Based Edible Film

Controlled release of probiotic bacteria via edible films as a vehicle is a novel approach. Therefore, in this study, the stability of two probiotic microorganisms, Bacillus coagulans IBRC-M 10807 and Lactobacillus plantarum PTCC 1058, were investigated in milk protein concentrate (MPC)-based edible films under different temperatures. For this purpose, effect of four factors, the probiotics (B. coagulans IBRC-M 10807 and L. plantarum PTCC 1058), glycerol concentration (6, 7.5, and 9%w/w), storage temperature (− 16, 4.5, and 25 °C) and storage time (3, 5, and 7 days) was investigated in edible films prepared using 10% w/w of milk protein concentrate containing 7 Log10 CFU/g of probiotics. Then the viability of each probiotic bacterium as well as important characteristics of probiotic edible films: pH, moisture content, solubility, water vapor permeability, and whiteness index were evaluated for optimization. Probiotic type and glycerol concentration had a significant effect on live-cell count (p < 0.05). The survival of B. coagulans spores was higher than that of L. plantarum. The optimum conditions included 9% glycerol concentration, − 16 °C and 7 day storage, in which survivability of B. coagulans and L. plantarum was 6.67 and 6.53 Log10 CFU/g, respectively. The overall results showed that MPC-based edible film could be a suitable carrier for probiotic bacteria to pack food stored at freezing temperatures.

Magnesium ions regulate mesenchymal stem cells population and osteogenic differentiation: A fuzzy agent-based modeling approach

Mesenchymal stem cells (MSCs) are proliferative and multipotent cells that play a key role in the bone regeneration process. Empirical data have repeatedly shown the bioregulatory importance of magnesium (Mg) ions in MSC growth and osteogenesis. In this study, we propose an agent-based model to predict the spatiotemporal dynamics of the MSC population and osteogenic differentiation in response to Mg2+ ions. A fuzzy-logic controller was designed to govern the decision-making process of cells by predicting four cellular processes of proliferation, differentiation, migration, and mortality in response to several important bioregulatory factors such as Mg2+ ions, pH, BMP2, and TGF-β1. The model was calibrated using the empirical data obtained from three sets of cell culture experiments. The model successfully reproduced the empirical observations regarding live cell count, viability, DNA content, and the differentiation-related markers of alkaline phosphate (ALP) and osteocalcin (OC). The simulation results, in agreement with the empirical data, showed that Mg2+ ions within 3–6 mM concentration have the highest stimulation effect on cell population growth. The model also correctly reproduced the stimulatory effect of Mg2+ ions on ALP and its inhibitory effect on OC as the early and late differentiation markers, respectively. Besides, the numerical simulation shed light on the innate cellular differences of the cells cultured in different experiments in terms of the proliferative capacity as well as sensitivity to Mg2+ ions. The proposed model can be adopted in the study of the osteogenesis around Mg-based implants where ions released due to degradation interact with local cells and regulate bone regeneration.

Effect of Hydrogen Peroxide and Phosphate Addition on Biopolymers Formation and Changes in Head Loss in Biological Activated Carbon Process

Objectives:The purpose of this study was to suggest a more efficient operation condition for the BAC(biological activated carbon) process by evaluating the change in the concentration of biopolymers in the effluent of the BAC process and the head loss of the BAC filter layer according to phosphate (PO4-P) and hydrogen peroxide (H2O2) input.Methods:During the experiment period (Feb. to Aug. 2020), the O3 dosage was fixed at 1 mg･O3/mg･DOC. Four columns with an inner diameter of 20 cm and a height of 250 cm were prepared. Empty bed contact time (EBCT) was fixed at 20 minutes and backwash was performed once a week. The four BAC columns are conventional BAC(control-BAC), enhanced BAC with hydrogen peroxide (H2O2+BAC), enhanced BAC with phosphate (PO4-P+BAC), and enhanced BAC with phosphate and hydrogen peroxide together (PO4-P+H2O2+BAC). In the case of enhanced BAC with PO4-P added, PO4-P was added with a concentration of 0.010 mg/L in the influent, and in BAC with H2O2, H2O2 was added with a concentration of 1 mg/L to the influent.Results and Discussion:According to the change of water temperature, the average head loss in control-BAC was 4.4 (25~28℃)~7.7 cm(8~12℃). In addition, PO4-P+BAC, H2O2+BAC and PO4-P+H2O2+BAC were 3.9~5.8 cm, 2.5~3.5 cm, and 2.6~3.5 cm, respectively. The head loss was reduced by the input of PO4-P and H2O2. During the low water temperature period, in control-BAC, the effluent biopolymers (BP) concentration was higher than the influent concentration, indicating that a large amount of EPS (extracellular polymeric substances) was produced and released from the attached biofilm. In PO4-P+BAC, H2O2+BAC and PO4-P+H2O2+BAC processes, the BP concentration ratio (Cout/Cin) was about 36~57% lower than that of the control-BAC during the low water temperature period. The BP concentration ratio was high when the water temperature (8~12℃) was low, and the BP concentration ratio gradually decreased as the water temperature increased. These results were very similar to those of the head loss change in the control-BAC process and the enhanced BAC process, and the BP concentration ratio and the head loss showed a very high correlation (r2=0.82~0.87). To evaluate the stability of the biofilm during the operation period, the total cell counts (TCC) in BAC treated waters were investigated. In control-BAC, PO4-P+BAC, H2O2+BAC and PO4-P+H2O2+BAC process, the average TCC was 46.8×106 cells, 30.3×106 cells, 21.8×106 cells, and 18.8×106 cells, respectively. Compared to the control-BAC, it was found to be 35~60% lower in the enhanced BAC processes. In addition, live cell count (LCC) ratio (LCC/TCC) was 0.84~0.89 in the enhanced BAC processes compared to 0.53 in the control-BAC. These results indicate that the biofilm stability of the enhanced BAC processes is higher than that of control-BAC.Conclusions:During the experiment, compared to the conventional BAC process, the enhanced BAC processes in which PO4-P and H2O2 were added showed a clear effect of reducing the head loss. In particular, the effect of reducing the head loss was higher when H2O2 was added than when PO4-P was added. A rapid head loss increase occurred in the conventional BAC process compared to the enhanced BAC processes in the low water temperature season is the result of the production of large amounts of EPS in the attached biofilm. The input of PO4-P or H2O2 reduces the head loss by improving the stability of the attached biofilm and reducing EPS production.

Extrusion-based 3D printed biodegradable porous iron

Extrusion-based 3D printing followed by debinding and sintering is a powerful approach that allows for the fabrication of porous scaffolds from materials (or material combinations) that are otherwise very challenging to process using other additive manufacturing techniques. Iron is one of the materials that have been recently shown to be amenable to processing using this approach. Indeed, a fully interconnected porous design has the potential of resolving the fundamental issue regarding bulk iron, namely a very low rate of biodegradation. However, no extensive evaluation of the biodegradation behavior and properties of porous iron scaffolds made by extrusion-based 3D printing has been reported. Therefore, the in vitro biodegradation behavior, electrochemical response, evolution of mechanical properties along with biodegradation, and responses of an osteoblastic cell line to the 3D printed iron scaffolds were studied. An ink formulation, as well as matching 3D printing, debinding and sintering conditions, was developed to create iron scaffolds with a porosity of 67%, a pore interconnectivity of 96%, and a strut density of 89% after sintering. X-ray diffracometry confirmed the presence of the α-iron phase in the scaffolds without any residuals from the rest of the ink. Owing to the presence of geometrically designed macropores and random micropores in the struts, the in vitro corrosion rate of the scaffolds was much improved as compared to the bulk counterpart, with 7% mass loss after 28 days. The mechanical properties of the scaffolds remained in the range of those of trabecular bone despite 28 days of in vitro biodegradation. The direct culture of MC3T3-E1 preosteoblasts on the scaffolds led to a substantial reduction in living cell count, caused by a high concentration of iron ions, as revealed by the indirect assays. On the other hand, the ability of the cells to spread and form filopodia indicated the cytocompatibility of the corrosion products. Taken together, this study shows the great potential of extrusion-based 3D printed porous iron to be further developed as a biodegradable bone substituting biomaterial.

Chebulinic acid inhibits MDA-MB-231 breast cancer metastasis and promotes cell death through down regulation of SOD1 and induction of autophagy.

Triple-negative breast cancers (TNBC) are highly aggressive and drug resistant accounting for majority of cases with poor outcome. Purified natural compounds display substantial anticancer activity with reduced cytotoxicity providing a new avenue to combat TNBC. Chebulinic acid (CA), a polyphenol derived from the fruits of various medicinal plants has potent anticancer activity. Here, we demonstrate that CA shows significant cytotoxicity against triple negative MDA-MB-231 cells. CA exhibited cytotoxicity to MDA-MB-231 cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Further, CA mitigated MDA-MB-231 cells viability and proliferation as shown by reduced live cell count, crystal violet staining, colony formation assay, soft agar assay and cell cycle analysis. Wound healing assay and trans-well migration assay demonstrated that CA significantly inhibited migration of MDA-MB-231 cells. Also reduced MMP9 expression was observed in CA-treated cells by gelatin zymography. CA negatively regulated mesenchymal characteristics of MDA-MB-231 cells demonstrated by F-actin staining and reduced expression of N-cadherin by confocal microscopy and western blot analysis. Annexin V/propidium iodide (PI) and active caspase-3 staining showed that CA was able to induce apoptosis in MDA-MB-231 cells but did not activate caspase-3. Two-dimensional gel electrophoresis based proteomic analysis demonstrated that CA regulated proteins belonging to the oxidative stress pathway, apoptotic pathway and proteins with antiproliferative activity. Western blot analysis analysis revealed that CA negatively regulated superoxide dismutase 1 (SOD1) and enhanced oxidative stress in MDA-MB-231 cells. SOD1 in-gel activity assay also showed reduced SOD1 activity upon CA treatment. Overexpression studies with GFP-LC3 and tandem tagged RFP-GFP-LC-3 also demonstrated enhanced autophagy upon CA treatment.

Abstract 1727: Challenging resistance to temozolomide in glioblastoma by drug encapsulation in apoferritin

Abstract Background: Temozolomide (TMZ) is a DNA alkylating prodrug used for the treatment of malignant, glioblastoma multiforme (GBM) tumors. Yet, TMZ therapy is blighted by resistance systems in GBM that thwart eradication of all cancer cells remaining after surgery and radiotherapy. Resistance mechanisms include drug efflux by P-glycoprotein 1 (Pgp), present on the blood brain barrier (BBB) and on GBM cells, overexpression of O6-methylguanine DNA-methyltransferase (MGMT), which removes cytotoxic O6-methylated guanine (O6-MeG) lesions caused by TMZ, and deficiency in mismatch repair (MMR), which leads to tolerance of O6-MeG lesions. Nanoscale delivery systems are proposed as a strategy to enhance drug targeting and accumulation under systemically tolerable conditions. Apoferritin (AFt), a hollow protein capsule, has been used to deliver anticancer agents. Transferrin receptor 1 (TfR1) binding sites on the AFt cage has reportedly been shown to allow AFt to cross the BBB and accumulate in GBM cells, as both BBB endothelial and GBM (but not glial) cells express TfR1. Thus, we use AFt for targeted delivery, specific uptake and controlled release of TMZ in GBM. Methods: TMZ was encapsulated into AFt via molecular diffusion through the channels in the AFt cage. Encapsulation efficiency and stability for this formulation were assessed by dynamic light scattering (DLS) and UV-vis spectroscopy. In vitro assays were conducted (MTT, live cell count, clonogenic, flow cytometry and ELISA) against isogenic GBM cell lines, U373V (vector control) and U373M (MGMT overexpressing), as well as MMR deficient and Pgp overexpressing HCT116 (colorectal carcinoma) and healthy MRC-5 (lung fibroblasts) cells. Environmental scanning electron microscopy (ESEM) and confocal microscopy were further employed to assess morphological changes to the cells after treatment. Results: Encapsulation of &amp;gt; 500 molecules of TMZ per AFt cage was achieved, with encapsulation efficiency &amp;gt; 70%. MTT assays demonstrated significantly increased activity of AFt-TMZ compared to TMZ alone in all cancer cell lines, with 50% growth inhibition (GI50) in resistant cancer cell lines at &amp;lt; 15 μM, compared to &amp;gt; 200 μM for TMZ alone. Clonogenic and flow cytometric cell cycle and DNA damage analyses further corroborate these findings. Additionally, ELISA quantification demonstrated significantly (P &amp;lt; 0.001) elevated O6-MeG levels following treatment of U373V and U373M cells with AFt-TMZ compared to naked TMZ, correlating with enhanced γ-H2AX burden in cells following exposure to AFt-TMZ. Against MRC-5, lower activity was observed, with GI50 for AFt-TMZ &amp;gt; 70 μM. Furthermore, cell shrinkage and blebbing were observed in GBM cells following 24 h treatment with AFt-TMZ. Conclusions: TMZ delivered by AFt demonstrated enhanced potency. Overcoming resistance by enhancing TMZ intracellular accumulation, may prove to be a turning point in enhancing drug efficacy in GBM. Citation Format: Kaouthar Bouzinab, Neil R. Thomas, Lyudmila Turyanska, Pavel Gershkovich, Nicola Weston, Marianne B. Ashford, Tracey D. Bradshaw. Challenging resistance to temozolomide in glioblastoma by drug encapsulation in apoferritin [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1727.

Evaluation of mtr cluster expression in Shewanella RCRI7 during uranium removal.

In recent years, bioremediation is considered as an efficient method to remove the pollutants from the industrial wastewater. In this study, quantitative gene expressions (Real-time RT-PCR) of mtr gene cluster (mtrA, mtrB, mtrC, mtrD, mtrE, mtrF and omcA) in five different uranium concentrations (0.1, 0.25, 0.5, 1 and 2mM) were performed with ICP and microscopic live cell counting analysis under anaerobic condition, by Shewanella RCRI7 as a native bacterium. The results indicated that the amount of uranium removal and live-cell counting were decreased in the higher uranium concentrations (1 and 2mM), due to the uranium toxicity, suggesting 0.5mM as the optimum uranium concentration for Shewanella RCRI7 resistance. The expression of mtrCED and omcA genes presented increasing trend in the lower uranium concentrations (0.1, 0.25 and 0.5mM) and a decreasing trend in 1 and 2mM, while mtrABF, presented an inverse pattern, proving the alternative role of mtrF for mtrC and omcA, as the substantial multiheme cytochromes in Extracellular Electron Transfer (EET) pathway. These data are a proof of these gene vital roles in the EET pathway, proposing them for genetic engineering toward EET optimization, as the certain pathway in heavy metal bioremediation process.

Using direct epifluorescent microscopic count for rapid enumeration of viable yeast and bacteria in injured conditions

Traditional method for assessing cell count requires an incubation period of 2-3 days. The direct microscopic count (DMC) method gives rapid enumeration of yeast and bacteria. However, the application is limited, since it is not possible to distinguish accurately between viable and nonviable cell. In this report, several nucleic acid dyes from Molecular Probe Inc. were used to stain the cells for enumeration of live and dead yeast in injured conditions, in which the dye can stain the DNA of nucleus and mitochondria, which then fluoresce yellow under fluorescence microscope, as well as stain the cytoplasm of actively growing cells to fluoresce green, while the cytoplasm of inactive cells fluoresced orange. The direct microscopic count multiplied by the live cell count ratio obtained by this direct epifluoroscent microscopic count (DEMC) method gives the estimated viable yeast count. According to our results, the correlation coefficient (R2) between the cell count obtained by the DEMC method and that by the standard plate count was 0.91 for yeast and 0.96 for bacteria in frozen and heating conditions, respectively. Requiring only 30 min, this method can also be used for the rapid enumeration of viable bacteria in injured conditions.

Cardiovascular Effects of Polychlorinated Biphenyls and Their Major Metabolites.

Background:Xenobiotic metabolism is complex, and accounting for bioactivation and detoxification processes of chemicals remains among the most challenging aspects for decision making with in vitro new approach methods data.Objectives:Considering the physiological relevance of human organotypic culture models and their utility for high-throughput screening, we hypothesized that multidimensional chemical-biological profiling of chemicals and their major metabolites is a sensible alternative for the toxicological characterization of parent molecules vs. metabolites in vitro.Methods:In this study, we tested 25 polychlorinated biphenyls (PCBs) [PCB 3, 11, 52, 126, 136, and 153 and their relevant metabolites (hydroxylated, methoxylated, sulfated, and quinone)] in concentration–response () for effects in human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) and endothelial cells (ECs) (iPSC-derived and HUVECs). Functional phenotypic end points included effects on beating parameters and intracellular flux in CMs and inhibition of tubulogenesis in ECs. High-content imaging was used to evaluate cytotoxicity, mitochondrial integrity, and oxidative stress.Results:Data integration of a total of 19 physicochemical descriptors and 36 in vitro phenotypes revealed that chlorination status and metabolite class are strong predictors of the in vitro cardiovascular effects of PCBs. Oxidation of PCBs, especially to di-hydroxylated and quinone metabolites, was associated with the most pronounced effects, whereas sulfation and methoxylation of PCBs resulted in diminished bioactivity.Discussion:Risk characterization analysis showed that although in vitro derived effective concentrations exceeded the levels measured in the general population, risks cannot be ruled out due to the potential for population variability in susceptibility and the need to fill data gaps using read-across approaches. This study demonstrated a strategy for how in vitro data can be used to characterize human health risks from PCBs and their metabolites. https://doi.org/10.1289/EHP7030

A novel egg shell-based bio formulation for remediation of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) contaminated soil

Environmental contamination by secondary explosive has been posing threat to human health and the ecosystem. We investigated the potential of a novel bioformulation developed from poultry waste for the bioremediation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) contaminated soils. Eggshells and additives immobilized with an indigenous explosive degrading microbe Janibacter cremeus were utilized for the development of the wettable powder bioformulation. Treatments carried out under unsaturated and saturated soil conditions resulted in 62 and 73 % removal of RDX respectively in 35 days meeting the soil clean up goals. The saturated treatment sets exhibited better microbial growth during the study in terms of live cell count and total enzyme activity. The bacteria, J. cremeus was observed to exhibit significant release of nitrite under both unsaturated as well as saturated conditions. Mass spectrometric studies showed that, both the conditions lead to the formation of nitroso-derivatives of RDX. But under saturated condition, an intermediate, 5-hydroxy-4-nitro-2,4-diazapentanal was observed which is a precursor to 4-nitro-2,4-diazabuatnal ultimately leading to mineralization. An accessible bio resource from poultry waste when used as a carrier for explosive degrading microbe has proven effective for in situ remediation of explosive contaminated soils.

Tapioca Starch Modulates Cellular Events in Oral Probiotic Streptococcus salivarius Strains.

Considering the implications of microbiota in health, scientists are in search of microbiota-oriented strategies for the effective prevention and/or treatment of a wide variety of serious diseases. A microbiota comprises diverse microorganisms with either probiotic or pathogenic properties. The fermentation of prebiotic carbohydrates by probiotic bacteria can affect host metabolism. Therefore, understanding the prebiotic-mediated metabolic modulations in probiotics is crucial to develop functional foods for the improvement of disturbed microbiota. Studies have emphasized the importance of prebiotics in probiotic therapies for mucosal diseases and highlighted the need for extensive research on oral bacteria. In the present study, the cellular events have been studied in batch cultures of probiotic Streptococcus salivarius exposed to the natural prebiotic, tapioca starch (TS). TS modulated the keystone metabolic events in Streptococcus salivarius in a dose-dependent manner. Besides increasing the live cell counts and altering the colony morphologies, TS affected the protein metabolism in terms of cellular expression and conformational changes in protein secondary structures. After treatment with TS, the nucleic acid synthesis increased and B-DNA was more than A- and Z-DNA, together with the diminished fatty acids and increased polysaccharide synthesis. The study results can be considered for the assessment of functional foods and probiotics in oral health.

Bacterial ecotoxicity and shifts in bacterial communities associated with the removal of ibuprofen, diclofenac and triclosan in biopurification systems

The proliferation and possible adverse effects of emerging contaminants such as pharmaceutical and personal care products (PPCPs) in waters and the environment is a cause for increasing concern. We investigated the dissipation of three PPCPs: ibuprofen (IBP), diclofenac (DCF) and triclosan (TCS), separately and in mixtures, in the ppm range in biopurification system (BPS) microcosms, paying special attention to their effect on bacterial ecotoxicity, as well as bacterial community structure and composition. The results reveal that BPS microcosms efficiently dissipate IBP and DCF with 90% removed after 45 and 84 days of incubation, respectively. However, removal of TCS required a longer incubation period of 127 days for 90% removal. Furthermore, dissipation of the PPCPs was slower when a mixture of all three was applied to BPS microcosms. TCS had an initial negative effect on bacterial viability by a decrease of 34–43% as measured by live bacterial cell counts using LIVE/DEAD® microscopy; however, this effect was mitigated when the three PPCPs were present simultaneously. The bacterial communities in BPS microcosms were more affected by incubation time than by the PPCPs used. Nonetheless, the PPCPs differentially affected the composition and relative abundance of bacterial taxa. IBP and DCF initially increased bacterial diversity and richness, while exposure to TCS generally provoked an opposite effect without full recovery at the end of the incubation period. TCS, which negatively affected the relative abundance of Acidobacteria, Methylophilales, and Legionellales, had the largest impact on bacterial groups. Biomarker OTUs were identified in the BPS microcosms which were constrained to higher concentrations of the PPCPs and thus are likely to harbour degradation and/or detoxification mechanisms. This study reveals for the first time the effect of PPCPs on bacterial ecotoxicity and diversity in biopurification system microcosms and also facilitates the design of further applications of biomixtures to eliminate PPCPs.

Impact of activated carbon block point‐of‐use filters on chloraminated water quality

AbstractWashington, District of Columbia, installed activated carbon‐based lead mitigation filters on all water fountains/sinks in break rooms, classrooms, and health suites located in public elementary schools. To investigate the impact of these point‐of‐use filters, 12 water fountains/taps with various filter ages, with and without sediment prefilters, spread across four schools were monitored weekly from July to December 2017, along with two unfiltered sources per school (n = 8). Within the first 100 days postinstallation, filtrate from filters in schools with high influent monochloramine and no prefilter temporarily increased nitrite concentrations, with concentrations of some filters exceeding 0.4 mg/L N. Subsequent decreases in filtrate nitrite for these filters coincided with increases in live cell counts. Microbial community “fingerprints” were determined with flow cytometry. Filtered and unfiltered water fingerprints differed, indicating selective microbial growth on, and release from, the filters into the filtrate.

Comparison of Cell and Organoid-Level Analysis of Patient-Derived 3D Organoids to Evaluate Tumor Cell Growth Dynamics and Drug Response.

3D cell culture models have been developed to better mimic the physiological environments that exist in human diseases. As such, these models are advantageous over traditional 2D cultures for screening drug compounds. However, the practicalities of transitioning from 2D to 3D drug treatment studies pose challenges with respect to analysis methods. Patient-derived tumor organoids (PDTOs) possess unique features given their heterogeneity in size, shape, and growth patterns. A detailed assessment of the length scale at which PDTOs should be evaluated (i.e., individual cell or organoid-level analysis) has not been done to our knowledge. Therefore, using dynamic confocal live cell imaging and data analysis methods we examined tumor cell growth rates and drug response behaviors in colorectal cancer (CRC) PDTOs. High-resolution imaging of H2B-GFP-labeled organoids with DRAQ7 vital dye permitted tracking of cellular changes, such as cell birth and death events, in individual organoids. From these same images, we measured morphological features of the 3D objects, including volume, sphericity, and ellipticity. Sphericity and ellipticity were used to evaluate intra- and interpatient tumor organoid heterogeneity. We found a strong correlation between organoid live cell number and volume. Linear growth rate calculations based on volume or live cell counts were used to determine differential responses to therapeutic interventions. We showed that this approach can detect different types of drug effects (cytotoxic vs cytostatic) in PDTO cultures. Overall, our imaging-based quantification workflow results in multiple parameters that can provide patient- and drug-specific information for screening applications.

Kappa opioid receptors internalization is protective against oxygen-glucose deprivation through β-arrestin activation and Akt-mediated signaling pathway

Hypoxia induces reversible κ-opioid receptor (KOR) internalization similar to the internalization that is induced by KOR agonists. In the current study, we demonstrate that this KOR internalization is a protective mechanism via the β-arrestin specific pathway in an oxygen-glucose deprivation (OGD) model. Mouse neuroblastoma Neuro2A cells were stably transfected with mouse KOR–tdTomato fusion protein (N2A-mKOR-tdT cells). Various concentrations of salvinorin A (SA), a highly selective KOR agonist, were given in the presence and absence of norbinaltorphimine (norBNI), which is a KOR antagonist, or Dyngo-4a (internalization inhibitor) or API-2 (Akt/Protein kinase B signaling inhibitor-2). Various concentrations of SA and RB-64 (22-thiocyanatosalvinorin A, selective for the G protein signaling pathway) were administered both in normoxic and hypoxic conditions. Autophagosomes and ultrastructural components of cells were observed using transmission electron microscopy (TEM). Cell viability, severity of cell injury, and levels of proteins related to the Akt signaling pathway were evaluated using live cell counting (by Cell Counting Kit-8), the lactic acid dehydrogenase (LDH) release rate, and Western blot analysis, respectively. SA promoted cell survival and attenuated OGD-induced cell injury. The Akt signaling pathway is activated by SA. KOR internalization, when blocked by norBNI or Dyngo-4a, increased LDH release and decreased cell viability under OGD. Treatment with SA significantly inhibited autophagy, and the effects of SA on autophagy were reversed by API-2 pretreatment. RB-64 in a low concentration without β-arrestin recruitment did not reduce LDH release and increase cell viability as observed with SA. KOR internalization through β-arrestin activation is a protective mechanism against OGD. The Akt pathway might play a critical role in modulating these protective effects by inhibiting autophagy.

Enhanced Microbial Decontamination Using Non-thermal Low Pressure Argon Plasma Jet.

Atmospheric pressure plasma jet (APPJ) gained great interest due to its effectiveness as selective non-lethal technique with low operational costs. In this study, argon APPJ system was designed and the generated cold plasma was applied in disinfection of microbial cells. Argon APPJ was generated by blowing argon through capillary metallic tube inserted in alumina and powered by 8-25 kHz sinusoidal voltage waveform. The plasma applied in inactivation of microbes by direct exposure of cell suspension, approximately 10 mm below jet nozzle, for different intervals. Interference of organics in exposure medium, on lethal activity of plasma was investigated. APPJ jet induced high levels of reactive oxygen (ROS) and nitrogen species (RNS). Jet length increased with applied voltage and flow rate in laminar mode, but decreased with flow rate in turbulent mode. Percent reduction in living cell count was 98.3 and 94.1%, for E. coli and S. aureus suspended in water after 30s of exposure, respectively, with 2.7- and 2-folds increase in plasma lethal activity, as compared with LB broth medium. D-values (Decimal Reduction Time) were increased from 34-333, 37-476 and 139-385 s for E. coli, S. aureus and C. albicans in water and complex liquid organic media, respectively. Designed argon APPJ system can be used in disinfection of different microbes. Plasma antimicrobial activity drastically decreased in presence of organic matter. The generated plasma can be promising approach for treatment of diseases, especially caused by antibiotic-resistant pathogens.