Cocktail Mixtures Research Articles

Wavelength-Selective Xanthene-Based Monochromophoric Photoremovable Protecting Groups for Tuning Soft Matter Material Properties.

Photocontrolled deprotection of specific functional groups has garnered significant interest over the past two decades. Notably, the selective deprotection of distinct groups based on wavelength has emerged as a prominent focus in recent research. The achievement of this objective has primarily involved the utilization of linker-based bichromophoric systems and diverse cocktail mixtures of photoresponsive protecting groups (PRPGs), each responsive to varying wavelengths of light. Herein, we present the first wavelength-selective monochromophoric system based on a hydroxanthene moiety, enabling the wavelength-selective release of two distinct functionalities under 450 and 600 nm light, respectively. The mechanism of the wavelength-selective photodegradation was thoroughly investigated by 1H NMR, UV-vis, and fluorescence spectroscopy, suggesting a proton-coupled electron transfer mechanism in the first photorelease step and electron transfer based arylmethyl type of photorelease in the second step. The utility of the xanthene-based wavelength-selective PRPGs was demonstrated in the multistep degradation of microparticles and dual-color tuning of polymer chain architecture, thus opening an avenue to design advanced photoreactive wavelength-controlled systems for applications in soft matter materials.

Coassembled Multicomponent Protein Nanoparticles Elicit Enhanced Antibacterial Activity.

The waning pipeline of the useful antibacterial arsenal has necessitated the urgent development of more effective antibacterial strategies with distinct mechanisms to rival the continuing emergence of resistant pathogens, particularly Gram-negative bacteria, due to their explicit drug-impermeable, two-membrane-sandwiched cell wall envelope. Herein, we have developed multicomponent coassembled nanoparticles with strong bactericidal activity and simultaneous bacterial cell envelope targeting using a peptide coassembly strategy. Compared to the single-component self-assembled nanoparticle counterparts or cocktail mixtures of these at a similar concentration, coassembled multicomponent nanoparticles showed higher bacterial killing efficiency against Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli by several orders of magnitude (about 100-1,000,000-fold increase). Comprehensive confocal and electron microscopy suggest that the superior antibacterial activity of the coassembled nanoparticles proceeds via multiple complementary mechanisms of action, including membrane destabilization, disruption, and cell wall hydrolysis, actions that were not observed with the single nanoparticle counterparts. To understand the fundamental working mechanisms behind the improved performance of coassembled nanoparticles, we utilized a "dilution effect" system where the antibacterial components are intermolecularly mixed and coassembled with a non-antibacterial protein in the nanoparticles. We suggest that coassembled nanoparticles mediate enhanced bacterial killing activity by attributes such as optimized local concentration, high avidity, cooperativity, and synergy. The nanoparticles showed no cytotoxic or hemolytic activity against tested eukaryotic cells and erythrocytes. Collectively, these findings reveal potential strategies for disrupting the impermeable barrier that Gram-negative pathogens leverage to restrict antibacterial access and may serve as a platform technology for potential nano-antibacterial design to strengthen the declining antibiotic arsenal.

Acute toxicity of piggery effluent and veterinary pharmaceutical cocktail on freshwater organisms.

Intensive livestock farming has increased the use of veterinary pharmaceuticals in many developing countries, and this is considered a significant concern to the freshwater ecosystem. However, the information on the potential acute toxicity of piggery effluent waste and the veterinary pharmaceutical effluent discharged into the aquatic environment is limited. This study assessed the adverse effect of a piggery effluent and the cocktail mixtures of high- and low-level doses of three frequently occurring veterinary pharmaceuticals (tetracycline (TETR), ivermectin (IVER), and salicylic acid (SALA)) on freshwater organisms using three representative freshwater biotests organisms: Pseudokirchneriella subcapitata (P. subcapitata), Daphnia magna (D. magna), and Tetrahymena thermophila (T. thermophila). The freshwater organism test results showed that the 24-h and 48-h EC50 algal toxicity to P. subcapitata exposed to 10% unfiltered piggery effluent were 25.6 and 49.3% respectively while the 24-h LC50 value to Cladocera, D. magna exposed to unfiltered piggery effluent was 23.2 (17.7-30.4)%. The 24-h EC50 protozoan toxicity to T. thermophila exposed to 1% HLD veterinary pharmaceuticals was 0.014μg/L. Thus, the study established the different sensitivities of freshwater organisms to various percentage levels of piggery effluent and high- and low-level doses of veterinary pharmaceutical. The piggery effluent and the pharmaceutical cocktail mixtures have potential toxicological effects on the freshwater ecosystem.

Effect of yogurt-based marinade combined with essential oils on the behavior of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella spp. in camel meat chunks during storage

The present study evaluated the effect of yogurt-based marinade combined with active essential oil components (EOs) namely: thymol (TH), carvacrol (CA), and cinnamaldehyde (CI) on Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella spp. in camel meat (CM) chunks during storage at 4 and 10 °C. Fresh cocktail mixtures of L.monocytogenes, E. coli O157:H7 and Salmonella spp. strains were inoculated on CM samples. Subsequently, a yogurt-based marinade, with or without 1% or 2% of the added EOs, was mixed with the CM chunks. After treatment, marinated camel samples were stored at 4 and 10 °C for 0, 1, 4 and 7 days. Adding yogurt-based marinade to the CM samples did not cause any significant changes in L.monocytogenes, E. coli O157:H7 and Salmonella spp. numbers at 4 °C, but at 10 °C resulted in a significant decrease in numbers on day 4 and 7 of storage by 1.4–1.5; 2.7–2.1 and 2.5–2.8 log CFU/g, respectively, compared to untreated CM samples. The incorporation of EOs into the CM with marination (CMM) further enhanced the microbial reduction of E. coli O157:H7 and Salmonella. At 10 °C, the synergistic effect of EOs with marinade was greater than at 4 °C. Increasing the concentration of the EOs used in this study from 1% to 2%, enhanced E. coli O157:H7 and Salmonella spp. reduction during storage at 4 and 10 °C while L.monocytogenes numbers were not affected. Increasing active EO component concentrations to 2% caused further significant reductions in Salmonella spp. in the CMM samples during storage by 1.0–2.7 log CFU/g (P ˂ 0.05) at 4 and 10 °C. At 10 °C, increasing the concentration of CI and TH to 2% caused a further reduction (P ˂ 0.05) of E. coli O157:H7 numbers by days 4 and 7 in the range of 3.6–4.4 log CFU/g. Among all tested EOs, 2% TH and 2% CI had the greatest effect against E. coli O157:H7 and Salmonella spp. in CMM during storage at 4 and 10 °C. In comparison to CMM, the highest scores of all examined sensory attributes were found in CMM samples with 1% and 2% CI added. Results indicate that the EO component CI can be used as an effective tool to decrease populations of E. coli O157:H7 and Salmonella spp. in CM with minor sensory changes.

A new synergistic relationship between xylan-active LPMO and xylobiohydrolase to tackle recalcitrant xylan

BackgroundHemicellulose accounts for a significant part of plant biomass, and still poses a barrier to the efficient saccharification of lignocellulose. The recalcitrant part of hemicellulose is a serious impediment to the action of cellulases, despite the use of xylanases in the cellulolytic cocktail mixtures. However, the complexity and variety of hemicelluloses in different plant materials require the use of highly specific enzymes for a complete breakdown. Over the last few years, new fungal enzymes with novel activities on hemicelluloses have emerged. In the present study, we explored the synergistic relationships of the xylan-active AA14 lytic polysaccharide monooxygenase (LPMO), PcAA14B, with the recently discovered glucuronoxylan-specific xylanase TtXyn30A, of the (sub)family GH30_7, displaying xylobiohydrolase activity, and with commercial cellobiohydrolases, on pretreated natural lignocellulosic substrates.ResultsPcAA14B and TtXyn30A showed a strong synergistic interaction on the degradation of the recalcitrant part of xylan. PcAA14B was able to increase the release of xylobiose from TtXyn30A, showing a degree of synergism (DS) of 3.8 on birchwood cellulosic fibers, and up to 5.7 on pretreated beechwood substrates. The increase in activity was dose- and time- dependent. A screening study on beechwood materials pretreated with different methods showed that the effect of the PcAA14B–TtXyn30A synergism was more prominent on substrates with low hemicellulose content, indicating that PcAA14B is mainly active on the recalcitrant part of xylan, which is in close proximity to the underlying cellulose fibers. Simultaneous addition of both enzymes resulted in higher DS than sequential addition. Moreover, PcAA14B was found to enhance cellobiose release from cellobiohydrolases during hydrolysis of pretreated lignocellulosic substrates, as well as microcrystalline cellulose.ConclusionsThe results of the present study revealed a new synergistic relationship not only among two recently discovered xylan-active enzymes, the LPMO PcAA14B, and the GH30_7 glucuronoxylan-active xylobiohydrolase TtXyn30A, but also among PcAA14B and cellobiohydrolases. We hypothesize that PcAA14B creates free ends in the xylan polymer, which can be used as targets for the action of TtXyn30A. The results are of special importance for the design of next-generation enzymatic cocktails, able to efficiently remove hemicelluloses, allowing complete saccharification of cellulose in plant biomass.

Surface pH of Fresh Beef as a Parameter To Validate Effectiveness of Lactic Acid Treatment against Escherichia coli O157:H7 and Salmonella

The U.S. beef industry must provide documentation to the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) that the antimicrobial interventions implemented or any subsequent changes in the process are effective under the actual conditions that apply in its operation. The main objective of this study was to determine whether surface pH after application of diluted lactic acid solution on surfaces of fresh meat can be used as a control measure indicator for the reduction of E. coli O157:H7 and Salmonella. Samples (240 each) of lean and adipose beef tissues were inoculated with cocktail mixtures of Escherichia coli O157:H7 and Salmonella. Application parameters were varied such that lean and adipose tissues were spray treated with either 2 or 4.5% lactic acid solution at either 38 or 60°C for 1 to 10 s. Lean and adipose tissues were collected before and after spray treatments for enumeration of the pathogens. Based on the conditions of this study, there was no difference between spray treatments at 38 or 60°C, but 4.5% lactic acid solution reduced pathogens more effectively ( P ≤ 0.05) than did 2% lactic acid solution. Spray treatment with lactic acid solution for 1 to 10 s reduced surface pH values of lean tissues (3 to 3.8) and adipose tissues (2.75 to 3.65). At surface pH values of 3.0 and 2.75, lactic acid reduced E. coli O157:H7 on surfaces of lean and adipose tissues by approximately 1.60 and 1.54 log CFU/cm2, respectively. At surface pH values of 3.8 and 3.65, lactic acid reduced E. coli O157:H7 on lean and adipose tissues by approximately 0.3 and 0.42 log CFU/cm2, respectively. The surface pH values after lactic acid treatment and the reductions of both pathogens showed a strong linear relationship; this indicates that a surface pH of 3.1 would provide at least 1-log reduction of E. coli O157:H7 and Salmonella, regardless of lactic acid application parameters. Therefore, surface pH after spray treatment with lactic acid could be used to validate pathogen reduction.

Hazard screening of photo-transformation products from pharmaceuticals: Application to selective β1-blockers atenolol and metoprolol.

The identification of toxic components in cocktail mixtures of pollutants, their metabolites and transformation products (TPs) generated from environmental and treatment processes remains an arduous task. This study expanded in this area by applying a combination of chemical analytics, a battery of in vitro bioassays and an in silico "testing battery" to UV photolysis mixtures of active pharmaceutical ingredients. The objectives were to understand the toxic nature of the mixtures and to prioritize photo-TPs for risk analysis. The selective β1-blockers Atenolol (ATL) and Metoprolol (MTL) that are ubiquitous in the aquatic environment were used as an example. The photolysis mixtures were cytotoxic to Vibrio fischeri and mammalian cells but not mutagenic in the Ames test or genotoxic in the in vitro micronucleus and umu tests. Potentially hazardous TPs were proposed by relating the observed effects to the kinetics of TP occurrence and applying in silico toxicity predictions for individual photo-TPs. This model study was done to identify principal mechanisms rather than accurately simulating environmental transformation processes. Several photo-TPs were proposed to present a greater hazard than the selected β-blockers and therefore fate and toxicity assessments may be required to determine their environmental relevance.

Precise Ratiometric Control of Dual Drugs through a Single Macromolecule for Combination Therapy.

A major challenge of combinatorial therapy is the unification of the pharmacokinetics and cellular uptake of various drug molecules with precise control of the dosage thereby maximizing the combined effects. To realize ratiometric delivery and synchronized release of different drugs from a single carrier, a novel approach was designed in this study to load dual drugs onto the macromolecular carrier with different molar ratio by covalently preconjugating dual drugs through peptide linkers to form drug conjugates. In contrast to loading individual types of drugs separately, these drug conjugates enable the loading of dual drugs onto the same carrier in a precisely controllable manner to reverse multidrug resistance (MDR) of human hepatoma (HepG2) cells. As a proof of concept, the synthesis and characterization of xyloglucan-mitomycin C/doxorubicin (XG-MMC/DOX) conjugates were demonstrated. This approach enabled MMC and DOX to be conjugated to the same polymeric carrier with precise control of drug dosage. The cytotoxicity and combinatorial effects were significantly improved compared to the cocktail mixtures of XG-MMC and XG-DOX as well as the individual conjugate of the mixture. Moreover, the results also showed that there was an optimum ratio of dual drugs showing the best cytotoxicity effect and greatest synergy among other tested polymeric conjugate formulations.

Efficacy of Antimicrobial Compounds on Surface Decontamination of Seven Shiga Toxin-Producing Escherichia coli and Salmonella Inoculated onto Fresh Beef

Several antimicrobial compounds have been used in commercial meat processing plants for decontamination of pathogens on beef carcasses, but there are many commercially available, novel antimicrobial compounds that may be more effective and suitable for use in beef processing pathogen-reduction programs. Sixty-four prerigor beef flanks (cutaneous trunci) were used in a study to determine whether hypobromous acid, neutral acidified sodium chlorite, and two citric acid-based antimicrobial compounds effectively reduce seven Shiga toxin-producing Escherichia coli (STEC) serogroups and Salmonella on the surface of fresh beef. Two cocktail mixtures were inoculated onto prerigor beef flank surfaces. Cocktail mixture 1 was composed of STEC serogroups O26, O103, O111, O145, and O157; and cocktail mixture 2 was composed of STEC serogroups O45, O121, and O157 and Salmonella. The inoculated fresh beef flanks were subjected to spray treatments with four antimicrobial compounds. Following antimicrobial treatments, both control and treated fresh beef samples were either enumerated immediately or were stored for 48 h at 4°C before enumeration. All four antimicrobial compounds caused 0.7- to 2.0-log reductions of STEC, Salmonella, aerobic plate counts, and Enterobacteriaceae. Results also indicated that the four antimicrobial compounds were as effective at reducing the six non-O157 STEC strains as they were at reducing E. coli O157:H7 on the surfaces of fresh beef. The recovery of all seven STEC strains and Salmonella in a low-inoculation study indicated that none of the four antimicrobial compounds eliminated all of the tested pathogens.

New insights into enzymatic hydrolysis of heterogeneous cellulose by using carbohydrate-binding module 3 containing GFP and carbohydrate-binding module 17 containing CFP

BackgroundThe in-depth understanding of the enzymatic hydrolysis of cellulose with heterogeneous morphology (that is, crystalline versus amorphous) may help develop better cellulase cocktail mixtures and biomass pretreatment, wherein cost-effective release of soluble sugars from solid cellulosic materials remains the largest obstacle to the economic viability of second generation biorefineries.ResultsIn addition to the previously developed non-hydrolytic fusion protein, GC3, containing a green fluorescent protein (GFP) and a family 3 carbohydrate-binding module (CBM3) that can bind both surfaces of amorphous and crystalline celluloses, we developed a new protein probe, CC17, which contained a mono-cherry fluorescent protein (CFP) and a family 17 carbohydrate-binding module (CBM17) that can bind only amorphous cellulose surfaces. Via these two probes, the surface accessibilities of amorphous and crystalline celluloses were determined quantitatively. Our results for the enzymatic hydrolysis of microcrystalline cellulose (Avicel) suggested that: 1) easily accessible amorphous cellulose on the surface of Avicel is preferentially hydrolyzed at the very early period of hydrolysis (that is, several minutes with a cellulose conversion of 2.8%); 2) further hydrolysis of Avicel is a typical layer-by-layer mechanism, that is, amorphous and crystalline cellulose regions were hydrolyzed simultaneously; and 3) most amorphous cellulose within the interior of the Avicel particles cannot be accessed by cellulase.ConclusionsThe crystallinity index (CrI), reflecting a mass-average (three-dimensional) cellulose characteristic, did not represent the key substrate surface (two-dimensional) characteristic related to enzymatic hydrolysis.

Polymeric nanoparticles with precise ratiometric control over drug loading for combination therapy.

We report a novel approach for nanoparticle-based combination chemotherapy by concurrently incorporating two different types of drugs into a single polymeric nanoparticle with ratiometric control over the loading of the two drugs. By adapting metal alkoxide chemistry, we synthesize highly hydrophobic drug-poly-L-lactide (drug-PLA) conjugates, of which the polymer has the same chain length while the drug may differ. These drug-polymer conjugates are then encapsulated into lipid-coated polymeric nanoparticles through a single-step nanoprecipitation method. Using doxorubicin (DOX) and camptothecin (CPT) as two model chemotherapy drugs, various ratios of DOX-PLA and CPT-PLA conjugates are loaded into the nanoparticles with over 90% loading efficiency. The resulting nanoparticles are uniform in size, size distribution and surface charge. The loading yield of DOX and CPT in the particles can be precisely controlled by simply adjusting the DOX-PLA:CPT-PLA molar ratio. Cellular cytotoxicity results show that the dual-drug loaded nanoparticles are superior to the corresponding cocktail mixtures of single-drug loaded nanoparticles. This dual-drug delivery approach offers a solution to the long-standing challenge in ratiometric control over the loading of different types of drugs onto the same drug delivery vehicle. We expect that this approach can be exploited for many types of chemotherapeutic agents containing hydroxyl groups and thus enable codelivery of various drug combinations for combinatorial treatments of diseases.

Improved measurements of Na + fluxes in plants using calixarene-based microelectrodes

Ion-selective microelectrodes are a powerful tool in studying adaptive responses of plant cells and tissues to various abiotic stresses. However, application of this technique in Na + flux measurements was limited due to poor selectivity for Na + ions of commercially available Na + cocktails. Often, these cocktails cannot discriminate between Na + and other interfering ions such as K + and Ca 2+, leading to inaccurate measurements of Na + concentration and, consequently, inaccurate Na + flux calculations. To overcome this problem, three Na +-selective cocktail mixtures were prepared using tetramethoxyethyl ester derivative of p-t-butyl calix[4]arene. These cocktail mixtures were compared with commercially available ETH 227-based Na + cocktail for selectivity for Na + ions over other ions (particularly K + and Ca 2+). Among the three calixarene-based Na + cocktails tested, cocktail 2 [in % w/w: Na + ionophore (4- tert-butylcalix[4]arene-tetra acetic acid tetraethyl ester) 3.5, the plasticizer (2-nitrophenyl octyl ether) 95.9 and lipophilic anion (potassium tetrakis (4-chlorophenyl) borate) 0.6] showed the best selectivity for Na + ions over K + and Ca 2+ ions and was highly stable over time (up to 10 h). Na + flux measurements under a wide range of NaCl concentrations (25–150 mM) using Na + cocktail 2 established a clear dose–response relationship between severity of salt stress and magnitude of Na + influx at the distal elongation and mature zones of Arabidopsis thaliana roots. Furthermore, Na + cocktail 2 was compared with commercially available ETH 227-based Na + cocktail by measuring Na + fluxes at the two Arabidopsis root zones in response to 100 mM NaCl treatment. With calixarene-based Na + cocktail 2, a large decreasing Na + influx (0–15 min) followed by small Na + influx (15–45 min) was measured, whereas with ETH-based Na + cocktail Na + influx was short-lived (1–3 min) and was followed by Na + efflux (3–45 min) that might have been due to K + and Ca 2+ efflux measured together with Na + influx. In conclusion, Na +-selective calixarene-based microelectrodes have excellent potential to be used in real-time Na + flux measurements in plants.

Detection of glucocorticoid bioactivity in bovine urine samples using a reporter gene assay

The illegal use of anabolic substances in the meat producing industry is an ongoing problem due to the continual production of new synthetic compounds and/or the practice of low-level cocktail administration to avoid detection by the surveillance schemes of EU member states National Plan surveillance systems. We present a highly sensitive reporter gene assay and sample extraction procedure based on a two step solid phase extraction and high performance liquid chromatography, developed for the detection of glucocorticoid abuse in bovine urine. The assay is capable of detecting compounds with glucocorticoid bioactivity and is extremely sensitive with an EC 50 of 0.79 ng mL −1 for dexamethasone. New or unknown compounds with glucocorticoid bioactivity and low-level cocktail mixtures are detectable by this assay. Cross-reactivity data for a range of 11β-hydroxyglucocorticoids has been provided. This assay shows low interference from the 11-keto prohormones and other steroidal hormones. The assay may be suitable for application in other matrices such as hair. In conclusion this screening assay offers advantages over existing analytical techniques.

Development and Validation of a Most-Probable-Number–Immunomagnetic Separation Methodology of Enumerating Escherichia coli O157 in Cattle Feces

A method to validate enumeration of Escherichia coli O157 in fecal samples from feedlot cattle was developed in these studies. Due to background flora, bovine fecal sample enumeration cannot be performed by simple direct plating techniques. Known quantities of E. coli O157:H7 were inoculated into feces, and populations were determined by direct plating of the cocktail (studies 1, 2, and 3) and manure and cocktail (studies 4 and 5) mixtures and compared with a most-probable-number (MPN)–immunomagnetic separation (IMS) method. The three-tube MPN combined preenrichment in gram-negative broth with confirmation using IMS. Five separate enumeration studies (study 1, sterile feces inoculated with 102E. coli O157:H7 per g; study 2, nonsterile feces inoculated with 103E. coli O157:H7 per g; study 3, nonsterile feces inoculated with 101E. coli O157:H7 per g; study 4, sterile feces inoculated with 104 streptomycin-resistant E. coli O157:H7 per g; and study 5, sterile feces inoculated with 102 streptomycin-resistant E. coli O157:H7 per g) were conducted. These studies were performed to determine the precision, accuracy, and specificity at low and high levels of pathogen contamination in feces, using direct plating compared with the MPN-IMS methodology tested. There was an overall difference (P < 0.01) between direct plating and MPN-IMS methodologies, but this difference was biologically negligible due to the difference in least-squares means (0.29 ± 0.10) being so low. The direct plating and MPN-IMS methods were correlated (r = 0.93). These results suggest that using the MPN-IMS procedures is an effective method of estimating E. coli O157 populations in naturally infected bovine fecal samples.

Gross alpha/beta analyses in water by liquid scintillation counting

The standard procedure for analyzing gross alpha and gross beta in water is evaporation of the sample and radioactivity determination of the resultant solids by proportional counting. This technique lacks precision, and lacks sensitivity for samples with high total dissolved solids. Additionally, the analytical results are dependent on the choice of radionuclide calibration standard and the sample matrix. Direct analysis by liquid scintillation counting has the advantages of high counting efficiencies and minimal sample preparation time. However, due to the small sample aliquants used for analysis, long count times are necessary to reach required detection limits. The procedure proposed consists of evaporating a sample aliquant to dryness, dissolving the resultant solids in a small volume of dilute acid, followed by liquid scintillation counting to determine radioactivity. This procedure can handle sample aliquants containing up to 500 mg of dissolved solids. Various acids, scintillation cocktail mixtures, instrument discriminator settings, and regions of interest (ROI) were evaluated to determine optimum counting conditions. Precision is improved and matrix effects are reduced as compared to proportional counting. Tests indicate that this is a viable alternative to proportional counting for gross alpha and gross beta analyses of water samples.

Cocktail modulator mixtures for overcoming multidrug resistance in renal cell carcinoma

Objectives. To determine the effect of various modulators on intracellular drug accumulation in renal cell carcinoma (RCC) tumor cells and the modulation of cytotoxicity of various chemotherapeutic drugs on the native RCC cell line and acquired intrinsic multidrug resistance (MDR) sublines because MDR is a major obstacle to effective chemotherapy of RCC. Methods. The cytotoxity of adriamycin to RCC 8701 and its MDR subline was analyzed. Fourteen MDR modulators, including calcium antagonists, protein kinase C inhibitor, glutathione transferase inhibitor, protein/peptide synthesis inhibitors, respiratory chain inhibitors, uncoupling reagent, adenosine triphosphate synthesis inhibitor, and ionophores, were examined for their MDR-reverse activity using the microplate tetrazolium test. Results. The intracellular adriamycin concentration significantly increased and reached maximum 4 hours after simultaneous treatment with calcium antagonists, tamoxifen, and oligomycin. The result demonstrated that verapamil, quinidine, tamoxifen, and oligomycin had an additive effect on the cytotoxicity of adriamycin and vinblastine against RCC8701 and RCC8701/ADR800 tumor cells. RCC8701/ADR800 tumor cells were more sensitive to modulator enhancement than native cells. The enhancement was related to the dosage and treatment duration of the modulators. Further trials on simultaneous additions to cocktail mixtures of the above four modulators showed no additive or synergistic effect on cytotoxicity against RCC8701/ADR800 tumor cells. Conclusions. Calcium antagonists and tamoxifen and oligomycin can individually be an effective chemotherapy adjunct for overcoming the native drug resistance or acquired MDR in RCC. Combination regimens, however, need more study regarding timing of administration, dosage, and frequency of modulators.

NITROUS OXIDE—OXYGEN SEDATION FOR MINOR SURGERY?

Nitrous oxide-oxygen inhalation sedation is a helpful method of calming children and adults when suturing and other painful minor surgical procedures are needed. It is within the scope of family physicians, pediatricians and emergency department physicians, and other medical doctors and our data1 indicate that it is quite safe when proper equipment and a thorough understanding of the technique are used.... In our opinion, of the different modalities available for sedation, nitrous oxideoxygen inhalation sedation is one of the safest, when properly used. This is especially so because its effects can be reversed rapidly with a few deep breaths. Certainly, it is much safer than the frequently used injectible cocktail mixtures of a sedative, tranquilizer and narcotic. Editorial Comment.2 Griffin and colleagues ... report no recognizable problem. Their report is understandably enthusiastic; they admit to no "controls," and they do not attempt to quantify the resultant relief achieved....Will their committment to careful selection in carrying out their technique be replicated by others? ... Regardless of these concerns, the work of Griffin and colleagues cannot be disregarded out of hand; a comparable technique has been used in dentistry and while it is found objectionable by many anesthesiologists, largely on a hypothetical basis, its use has been deemed beneficial. Certainly replication of the current study should proceed: the outcome remains "not proved."