Dosing metric in cellular experiments: The mol/cell metric has its limitations.

It has been argued that the mol/cell metric is more universal than concentration of the toxic agent since in many cases the effect of dose expressed as mol/cell is independent of ex-perimental setup. We confirmed it for hemolysis of erythrocytes in phosphate-buffered saline induced by hypochlorite where the amount of femtomoles/cell of hypochlorite needed for 50% hemolysis was independent of erythrocyte concentration. However, in the presence of blood plasma this metric became dependent on cell concentration. Similarly, the effect of 3-bromopyruvic acid (3-BP) on PEO1 cells as a function of mol/cell ratio depended on the volume of the 3-BP containing medium, due to the reaction of 3-BP with components of the medium. Hemolytic amounts of sodium dodecyl sulfate and Triton X-100 expressed as mol/cell decreased with increasing cell concentration while the effect of DMSO on the viability of a constant number of fibroblasts was independent of the volume of DMSO-containing medium. These results demonstrate that the mol/cell metric is still dependent on experimental conditions when the toxic agent interacts with components of the medium or when its physical state is modified by the target cells, and the effect is independent of the mol/per cell ratio for high excess of a cell damaging agent.

Olfaction alters spatial memory strategy of scatter-hoarding animals.

Although it has been suggested that olfaction is closely interconnected with hippocampal systems, whether olfaction regulates spatial memory strategy remains never known. Furthermore, no study has examined how olfaction mediates spatial memory established on the external objects, for example, caches made by scatter-hoarding animals. Here, we experimentally induced nondestructive and reversible olfaction loss of a scatter-hoarding animal Leopoldamys edwardsi, to test whether and how olfaction regulates spatial memory to mediate cache recovery and pilferage. Our results showed that the normal L. edwardsi preferred to pilfer caches of others rather than to recover their own using accurate spatial memory (35.7% vs. 18.6%). Anosmic L. edwardsi preferred to recover the caches they made prior to olfaction loss rather than to pilfer from others relied on spatial memory (54.2% vs. 36.0%). However, L. edwardsi with anosmia showed no preference either to the caches they established after olfaction loss or caches made by others (25.8% vs. 29.1%). These collectively indicate that olfaction loss has a potential to affect new memory formation but not previously established spatial memory on caches. Our study first showed that olfaction modified spatial memory strategy in cache recovery and pilferage behaviors of scatter-hoarding animals. We suggest that future studies pay more attention to the evolution of olfaction and its relationship with spatial memory strategy.

Inhibition of Notch signaling pathway using γ-secretase inhibitor delivered by a low dose of Triton-X100 in cultured oral cancer cells.

How to effectively delivering therapeutic agents, including γ-secretase inhibitors (GSIs), into live cells, remains a significant challenge. This study assessed the effect of Notch signaling inhibition by examining levels of the Notch1 intracellular domain (N1ICD) in cultured oral cancer cells analyzed with random stitched images (2D) and 3D visualizations using confocal microscopy and quantitative gene analysis. Substantially, we have developed a novel method to assist the delivery of γ-secretase inhibitor, DAPT, into live cells in the presence of an effective minimum concentration of Triton-X100 (0.001%) without damaging cell activity and membrane integrity assessed with cell proliferation assays. The images obtained in this study showed that DAPT alone could not block the γ-secretase inhibitor despite inhibiting cell growth. Further analysis of quantitative gene expressions of Notch signaling canonical pathway to verify the effectiveness of the novel method for delivering inhibitor into live cells, displayed deregulation of Notch1, Delta-like ligand 1 (DLL1) and hairy and enhancer of split 1 (Hes1). Our data suggest that Notch1/Hes1 signaling pathway is deactivated using DAPT with a low dose of Triton-X100 in this cancer cells. And the finding also suggests that Notch1 could be engaged by DLL1 to promote differentiation in oral cancer cells. Using this approach, we demonstrate that Triton-X100 is a promising and effective permeabilization agent to deliver γ-secretase inhibitor DAPT into live oral epithelial cells. This strategy has the potential to implicate in the treatment of cancer diseases.

Influence of Mixed Additives on the Physicochemical Properties of a 5.25% Sodium Hypochlorite Solution: An Unsupervised Multivariate Statistical Approach.

INTRODUCTION: This article reports for the first time the effects of multiple additives (polyethylene glycol 400, Triton X-100, benzalkonium chloride, and ethyl formate) on the surface tension, pH, and viscosity of 5.25% sodium hypochlorite (NaOCl) irrigant solution. Advanced statistical approaches based on unsupervised multivariate analysis (cluster analysis and principal component analysis) were used to quantify the variability of the physicochemical properties of the modified NaOCl solution for the first time in dentistry. METHODS: Solutions of 5.25% NaOCl were modified with multiple additives in various concentrations, physicochemical parameters were measured at 22°C and 37°C, and the results were statistically analyzed to group the solutions and reveal the effects of additives. RESULTS: Cluster analysis and principal component analysis revealed that pH and surface tension were the significant parameters (P < .05) for grouping the modified solutions. Four principal components, accounting for 90.6% of the total variance, were associated with flow characteristics (37.3%) determined by polyethylene glycol; the wetting property (22.5% and 10.5%), which was dependent on cationic and nonionic surfactant; and the antimicrobial effect (20.3%) influenced by ethyl formate. Varimax rotation of the principal components showed that the cationic surfactant (benzalkonium chloride) had significantly decreased surface tension compared with the nonionic surfactant (Triton-X). Although ethyl formate was introduced as an odor modifier, it had a significant effect on pH decrease and the occurrence of effervescence with O2 and hypochlorous acid release. CONCLUSIONS: The statistical results revealed that the 5.25% NaOCl irrigant solution should be modified with a mixture of 0.1% benzalkonium chloride, 1% ethyl formate, and 7% polyethylene glycol for obtaining a low pH and low surface tension.

Building a better mouse embryo assay: effects of mouse strain and in vitro maturation on sensitivity to contaminants of the culture environment.

PURPOSE: The aim of this study is to compare the sensitivity of the standard one-cell mouse embryo assay (MEA) to that using in vitro-matured oocytes from hybrid and outbred mice. METHODS: The study was done by culturing embryos in the presence or absence of two concentrations (0.0005 or 0.001 % v/v) of Triton X-100 (TX100). Embryonic development, blastocyst cell numbers (total and allocation to the trophectoderm [TE] and inner cell mass [ICM]), and blastocyst gene expression were evaluated. RESULTS: Neither concentration of TX100 affected (P > 0.05) cleavage, blastocyst development, or hatching in one-cell embryos from BDF1 mice. However, all cell number endpoints were reduced (P < 0.05) by the high concentration of TX100 and the number of ICM cells was reduced (P < 0.05) by the low concentration of TX100. Inhibitory (P < 0.05) effects of the high concentration of TX100 were observed in in vitro maturation (IVM) embryos from BDF1, CF1, and SW, but not ICR, mice. Cell number and allocation were negatively affected by the high concentration of TX100 in CF1 and SW embryos, but not in BDF1 or ICR embryos. The only developmental endpoints affected by the low concentration of TX100 were cleavage of BDF1 oocytes, blastocyst development of SW embryos, and cell numbers (total and inner cell mass (ICM)) of SW blastocysts. CONCLUSIONS: The sensitivity of the MEA to TX100 is improved by using embryos from in vitro-matured oocytes, using oocytes from some outbred (SW or CF1, not ICR) strains of mice, and evaluating blastocyst cell number and allocation.

A novel technique for simultaneous whole-body and multi-organ decellularization: umbilical artery catheterization as a perfusion-based method in a sheep foetus model.

The aim of this study was to develop a method to generate multi-organ acellular matrices. Using a foetal sheep model have developed a method of systemic pulsatile perfusion via the umbilical artery which allows for simultaneous multi-organ decellularization. Twenty sheep foetuses were systemically perfused with Triton X-100 and sodium dodecyl sulphate. Following completion of the whole-body decellularization, multiple biopsy samples were taken from different parts of 21 organs to ascertain complete cell component removal in the preserved extracellular matrices. Both the natural and decellularized organs were subjected to several examinations. The samples were obtained from the skin, eye, ear, nose, throat, cardiovascular, respiratory, gastrointestinal, urinary, musculoskeletal, central nervous and peripheral nervous systems. The histological results depicted well-preserved extracellular matrix (ECM) integrity and intact vascular structures, without any evidence of residual cellular materials, in all decellularized bioscaffolds. Scanning electron microscope (SEM) and biochemical properties remained intact, similar to their age-matched native counterparts. Preservation of the collagen structure was evaluated by a hydroxyproline assay. Dense organs such as bone and muscle were also completely decellularized, with a preserved ECM structure. Thus, as shown in this study, several organs and different tissues were decellularized using a perfusion-based method, which has not been previously accomplished. Given the technical challenges that exist for the efficient generation of biological scaffolds, the current results may pave the way for obtaining a variety of decellularized scaffolds from a single donor. In this study, there have been unique responses to the single acellularization protocol in foetuses, which may reflect the homogeneity of tissues and organs in the developing foetal body.

The contribution of vascular smooth muscle, elastin and collagen on the passive mechanics of porcine carotid arteries.

The main components responsible for the mechanical behavior of the arterial wall are collagen, elastin, and smooth muscle cells (SMCs) in the medial layer. We determined the structural and mechanical changes in porcine carotid arteries after administration of Triton® X-100, elastase, and collagenase using the inflation-deflation test. The arteries were intraluminarly pressurized from 0 to 200 mmHg, and the outer diameter of the artery was measured. The pressure-strain elastic modulus was determined based on the pressure/diameter ratio. The intima-media thickness, wall thickness, thickness of the tunica adventitia layer, and the area fractions of SMCs, elastin, and collagen within the arterial wall (A(A)(SMC/elastin/collagen, wall)) were measured using stereological methods. The relative changes in the relevant components of the treated samples were as follows: the decrease in A(A)(SMC, wall) after administration of Triton® X-100 was 11% ± 7%, the decrease in A(A)(elastin, wall) after administration of elastase was 40% ± 22%, and the decrease in A(A)(collagen, wall) after the application of collagenase was 51% ± 22%. The Triton® X-100 treatment led to a decrease in the SMC content that was associated with enlargement of the arterial wall (outer diameter) for pressures up to 120 mmHg, and with mechanical stiffening of the arterial wall at higher pressures. Elastase led to a decrease in the elastin content that was associated with enlargement of the arterial wall, but not with stiffening or softening. Collagenase led to a decrease in collagen content that was associated with a change in the stiffness of the arterial wall, although the exact contribution of mechanical loading and the duration of treatment (enlargement) could not be quantified.

Olfactory bulb recovery following reversible deafferentation with repeated detergent application in the adult zebrafish.

The neuroplasticity and regenerative properties of the olfactory system make it a useful model for studying the ability of the nervous system to recover from damage. We have developed a novel method for examining the effects of long-term deafferentation and regeneration of the olfactory organ and resulting influence on the olfactory bulb in adult zebrafish. To test the hypothesis that repeated damage to the olfactory epithelium causes reduced olfactory bulb afferent input and cessation of treatment allows recovery, we chronically ablated the olfactory organ every 2-3 days for 3 weeks with the detergent Triton X-100 while another group was allowed 3 weeks of recovery following treatment. Animals receiving chronic treatment showed severe morphological disruption of the olfactory organ, although small pockets of epithelium remained. These pockets were labeled by anti-calretinin, indicating the presence of mature olfactory sensory neurons (OSNs). Following a recovery period, the epithelium was more extensive and neuronal labeling increased, with three different morphologies of sensory neurons observed. Repeated peripheral exposure to Triton X-100 also affected the olfactory bulb. Bulb volumes and anti-tyrosine hydroxylase-like immunoreactivity, which is an indicator of afferent activity, were diminished in the olfactory bulb of the chronically treated group compared to the control side. In the recovery group, there was little difference in bulb volume or antibody staining. These results suggest that repeated, long-term nasal irrigation with Triton X-100 eliminates a substantial number of mature OSNs and reduces afferent input to the olfactory bulb. It also appears that these effects are reversible and regeneration will occur in both the peripheral olfactory organ and the olfactory bulb when given time to recover following cessation of treatment. We report here a new method that allows observation not only of the effects of deafferentation on the olfactory bulb but also the effects of reinnervation.

Kinetics of solvent-free lipase-catalyzed glycerolysis of olive oil in surfactant system.

This work reports experimental data and kinetic modeling of solvent-free glycerolysis of olive oil using a commercial immobilized lipase (Novozym 435) in the presence of Triton X-100 surfactant for the production of monoacylglycerols (MAG) and diacylglycerols (DAG). The experiments were performed in batch mode evaluating the effects of temperature (30-70 degrees C), enzyme concentration (2.5-18 wt %), Triton X-100 concentration (10-20 wt %), and glycerol to oil molar ratio (3:1, 6:1, and 9:1). Experimental results showed that lipase-catalyzed solvent-free glycerolysis with the addition of Triton X-100 might be a potential alternative route to conventional organic solvent methods, as good conversions were obtained with relatively low enzyme concentrations (9 wt %) in short reaction times (240 min). The glycerolysis and hydrolysis parallel reactions were considered with rate constants estimated by minimizing a maximum likelihood function. A very satisfactory agreement between experimental data and model results was obtained, thus allowing a better understanding of the reaction kinetics.

Absorption enhancement of poorly absorbed hydrophilic compounds from various mucosal sites by combination of mucolytic agent and non-ionic surfactant.

Absorption enhancement of poorly absorbed hydrophilic compounds from various mucosal sites by co-administration of a mucolytic agent and a non-ionic surfactant was examined in rats. Fluorescein isothiocyanate-labeled dextran with average molecular weight of ca. 4.4kDa (FD-4), and salmon calcitonin (SCT) were used as model compounds. N-acetylcysteine (NAC) and p-t-octyl phenol polyoxyethylene-9.5 (Triton X-100, TX-100) were selected as a mucolytic agent and a non-ionic surfactant, respectively. Dosing solutions containing these agents were administered into various mucosal sites including the nose, the lung and the large intestine, and the bioavailabilities were determined. The combination of 5% NAC and 5% TX-100 significantly enhanced the nasal, the pulmonary and the large intestinal absorption of FD-4 compared to the control, and the enhancement ratios relative to the control were 7.2-, 2.8- and 4.5-fold, respectively. The different enhancement ratio among the administration sites explored indicates that the absorption enhancing effect of the combination of NAC and TX-100 is site-dependent. This combination also improved the nasal and the pulmonary absorption of SCT, and the enhancement ratios relative to the control were 6.1- and 8.1-fold, respectively. All these results suggest that the combination strategy of a mucolytic agent and a non-ionic surfactant may be widely applicable to various mucosal deliveries of poorly absorbed hydrophilic compounds.

Beta vulgaris L. suspension cultures permeabilized with triton X-100 retain cell viability and betacyanines production ability: a digital image analysis study.

The influence of Triton X-100 on Beta vulgaris L. permeabilized cell culture viability, regrowth, and ability to produce betacyanines was evaluated in this study. A non-destructive method based on the analysis of images in the RGB (red, green, blue) system was developed to estimate betacyanines content. A treatment for 15 min with 0.7 mM Triton X-100 induced the release of 30% of betacyanines without loss of cell viability (>or=70%). After this permeabilization treatment, B. vulgaris cultures regrew normally, reaching a maximum biomass concentration of 48% higher than non-permeabilized cultures after 14 days of culture. Also, maximum betacyanines concentration was only 25% lower than that of non-permeabilized cultures.

Solvent/detergent plasma for prevention of bleeding in recessively inherited coagulation disorders: dosing, pharmacokinetics and clinical efficacy.

BACKGROUND AND OBJECTIVES: This open-label multicenter trial of solvent/detergent (SD) plasma involving 17 patients with recessively inherited coagulation disorders (one afibrinogenemia, four FV, six combined FV and FVIII, one FX and five FXI deficiencies) evaluated the pharmacokinetics of the deficient factors and hemostatic efficacy. DESIGN AND METHODS: In vivo recovery (IVR) of the deficient coagulation factor was determined in a non-bleeding state in all patients and the mean values for FV, FVIII, FX, FXI and fibrinogen were 1.3, 1.2, 1.5, 1.3 and 1.5 dL/Kg, respectively. The mean plasma half-life of FV, FVIII and FX was 18, 43 and 33 hours, respectively. All patients underwent replacement therapy for elective procedures at risk of bleeding (surgery in 14 cases and vaginal delivery in two patients), except one treated for a central nervous system surgical emergency. RESULTS: Treatment courses with SD plasma were judged fully effective in 13/16 cases (81%). In the remaining three cases, mild bleeding occurred after major surgery in a FV deficient patient with a factor level of 43% and in a FXI deficient patient when factor levels were between 20% and 41%; and after minor surgery in a patient with FV and FVIII deficiency when factor levels were 41% and 18%, respectively. Bleeding was controlled by continuing or increasing treatment with SD plasma. INTERPRETATION AND CONCLUSIONS: These results suggest that, even though the current absolute risk of blood-borne infections associated with fresh-frozen plasma is relatively small, SD plasma should be preferred in patients with recessively inherited coagulation disorders who need replacement therapy when virus-inactivated single-factor concentrates are not available.

Effects of temperature on the wall strength and compliance of frog mesenteric microvessels.

In single perfused mesenteric microvessels of pithed frogs, we assessed wall strength from the critical pressure, PB, which has to be applied within the vessel in order to induce openings in the walls through which fluid and cells can extravasate. PB was determined in capillaries and venules of tissues at 12-20 The P(B) (mean +/- S.E.M.) in 22 vessels between 12 and 20 degrees C, P(B) was 92.0 +/- 7.40 cm H2O which was significantly higher than at room temperatures (P<0.001). The compliance of the vessel wall was estimated using both the red cell method and the oil meniscus technique. There was no measurable effect of temperature on wall compliance. The compliance of vessels from which the cells had been removed by previous perfusion with detergent solutions was very similar to that of intact vessels between 12 and 20 degrees C and between 0 and 5 degrees C. The negligible effects of temperature upon compliance suggest that microvessel walls have to be distended to a greater extent in cold tissue before P(B) is reached. This, together with their rapid closure, is consistent with the hypothesis that pressure-induced openings in microvascular walls are dependent on an active response of the endothelium rather than being the result of stress failure of the basement membrane.

Erythrocyte hemolysis by detergents.

The numbers of Triton X-100 and sodium dodecyl sulfate molecules required to form respective pores were estimated from the relationship between the detergent concentrations and the rates of fast and slow hemolysis components. It has been found that the slow hemolysis component evoked by Triton X-100 is related to the existence of two different pores. It is shown that the fast hemolysis component induced by sodium dodecyl sulfate is associated with the modification of phosphatidylcholine which determines the break in the Arrhenius plots of the hemolysis rate within the range of 20 degrees C. The shape of hemolysis kinetic curves and the dependence of hemolytic parameters on the detergent concentration and temperature are discussed based on the concept of hemolysis caused by the formation of pores in various membrane lipid regions and by releasing vesicles from erythrocytes.

Transurethral enzymatic ablation of the prostate: canine model.

OBJECTIVES: The purpose of this study was to investigate the toxicity and potential usefulness of transurethral prostatic injection of a collagenase-based solution in treating benign prostatic hyperplasia in dogs. METHODS: The injected solution contained collagenase, hyaluronidase, Triton X-100, and gentamicin. Twenty-one dogs were randomly divided into three groups for transurethral prostatic needle injection: two treatment groups were observed for 6 and 12 weeks and a control group was observed for 12 weeks. Laboratory studies, clinical monitoring, and complete postmortem examination were performed in all animals. RESULTS: Gross hematuria was noted in all dogs for a mean of 4 days after injection. No significant postoperative morbidity was noted. There were no significant differences in the values of laboratory tests among the three groups except for a mean increase in serum level of aspartate transaminase for treatment groups on postoperative day 1; this resolved by postoperative day 7. Histologically, all treated prostates had stromal atrophy and cystic acinar dilation involving about 30% of the gland without extraprostatic extension of these changes. The urethra, bladder, rectum, testicles, kidney, liver, and lungs were normal and intact in all animals. CONCLUSIONS: Transurethral injection of this enzyme solution creates a predictable, favorable histologic response in the canine prostate. The procedure appears safe and warrants further investigation for treatment of human benign prostatic hyperplasia.

A polymer-Triton X-100 conjugate capable of PH-dependent red blood cell lysis: a model system illustrating the possibility of drug delivery within acidic intracellular compartments.

Poly(amidoamines) are soluble polymers containing tertiary amino and amido groups regularly arranged along the macromolecular chain, and their net average charge alters considerably as pH changes from neutral to acidic leading to a change in conformation. This property provides the possibility to design polymer-drug conjugates that are, following intravenous administration, relatively compacted and thus protect a drug payload in the circulation, but following pinocytic internalisation into acidic intracellular compartments unfold permitting pH-triggered intracellular drug delivery. To study the feasibility of this approach, a covalent conjugate of a poly(amidoamine) (MBI) was prepared to contain the membrane lytic non-ionic detergent Triton X-100 (as a model), and its ability to lyse red blood cells in vitro was used as an indicator of conjugate conformation at at different pHs. Although Triton X-100 was highly lytic at pH 5.5, 7.4 and 8.0, and the parent polymer MBI was not lytic under any conditions, the conjugate only showed concentration-dependent red blood cell lysis at pH 5.5. Moreover, incubation of human leukaemic cells (CCRF) with these substrates showed conjugate to be more toxic than MBI (IC50 values of 100 micrograms/ml and 650 micrograms/ml respectively) and less toxic than Triton X-100 (IC50 of 1 microgram/ml).

Low concentration of Triton X-100 inhibits diacylglycerol acyltransferase without measurable effect on phosphatidate phosphohydrolase in the human primordial placenta.

Agents causing accumulation of endogenous diacylglycerols (DAG) may be helpful in studies on the intracellular regulatory effects and on the mechanisms of attenuation of this putative second messenger. In a previous study (Tóth et al., BBA 921 (1987) 417-425) we have shown a marked stimulatory effect of low micellar concentration (0.05%, v/v) of Triton X-100 on the labelling of phosphatidic acid with (32P)phosphate in minced human primordial placenta. The present results demonstrate that 0.05% Triton X-100 inhibits nearly completely the conversion of (3H)diacylglycerols into (3H)triacylglycerols in placenta fragments incubated with (3H)glucose. However, this concentration of the detergent does not have any effect on the appearance of label in the sum of acylglycerols (comprising mono-, di- and triacylglycerols) and phosphatidylcholine, indicating a lack of effect on phosphatidate phosphohydrolase. The about 5-fold elevation of (3H)diacylglycerols was attended by an approximately 3-fold rise in (3H)phosphatidic acid and a 1.3-fold increase in the labelling of phosphatidylcholine. These findings provide supportive evidence that 1,2-DAG was formed due to inhibition of DAG-acyltransferase and suggest that some of the DAG was transformed into phosphatidic acid by diacylglycerol-kinase.