Assessing in vitro dermal absorption of dry residues of agrochemical sprays using human skin within OECD TG 428.

We describe a novel experimental method that mimics exposure to dried agrochemical residues on contact surfaces during re-entry into crops. It includes the creation of dry dislodgeable residues and subsequent transfer to human skin for in vitro measurement of dermal absorption within a standard Organisation for Economic Co-operation and Development test guideline (OECD TG) 428 study. A pre-determined volume of spray containing 14C-labelled active substance is transferred onto a polytetrafluorethylene-coated septum and air-dried. The septum is then gently placed onto the pre-wetted skin mounted in a flow-through Franz diffusion chamber. The septum is gently rotated thrice to transfer the dose. Preliminary tests determined transfer efficiency to ensure the appropriate test concentration on the skin. Then, a standard dermal absorption study is performed according to OECD TG 428. Results from 10 compounds indicate that the methodology can be robustly incorporated into a standard TG study. These data show that the dermal absorption from a dry dislodgeable residue is lower than that from the equivalent dose of the aqueous spray, regardless of formulation type or active substance. Studies following the scenario described above can be a suitable tool to better estimate dermal absorption from dry residues in re-entry worker and resident exposure assessment for agrochemicals.

Towards single egg toxicity screening using microcoil NMR.

Planar microcoils with diameter ranging from 20 to 1000 µm I.D. (130-1130 µm O.D.) are evaluated for their applications in NMR spectroscopy. The coils are first overfilled with a standard sucrose solution and compared against each other. Coils with smaller I.D. (≤100 µm) perform extremely well. One hypothesis is that as the coils get smaller the volume occupied by the copper turns increases relative to the open I.D.; as such a large proportion of the sample is brought in close proximity to the coil turns and likely gives rise to strong sample-coil magnetic coupling, which increases the signal. The applications of the planar microcoils are demonstrated on Cypselurus poecilopterus (fish) and Daphnia magna (water flea) eggs. A single D. magna egg on a 50 µm coil yielded at least 3000 times the mass sensitivity (∼9,000,000 time saving) when compared to a 5 mm probe. This value could be at least 4 times higher if the B1 homogeneity of the coils could be improved. With the current design, 80% of the signal is lost in multiple pulse experiments that rely on phase inversion and signal cancellation between scans. The data were extrapolated to predict that biological samples as small as ∼4 µm may become accessible via planar microcoil designs. To fulfill their potential for in situ metabolic screening, specialized magnetic susceptibility matched sample holders that restrict the sample to the homogeneous B1 field region (i.e. within the 90% RF field) of the coil and advanced experiments that narrow spectral lines, suppress lipids and disperse signals into multiple dimensions will be required.

Evidence-based Update of Pediatric Dental Restorative Procedures: Preventive Strategies.

BACKGROUND: There has been significant advances in the understanding of preventive restorative procedures regarding the advantages and disadvantages for restorative procedures; the evidence for conservative techniques for deep carious lesions; the effectiveness of pit and fissure sealants; and the evidence for use of resin infiltration techniques. AIM: The intent of this review is to help practitioners use evidence to make decisions regarding preventive restorative dentistry in children and young adolescents. STUDY DESIGN: This evidence-based review appraises the literature, primarily between the years 1995-2013, on preventive restorative strategies. The evidence was graded as to strong evidence, evidence in favor, or expert opinion by consensus of authors Results: The preventive strategy for dental caries includes individualized assessment of disease progression and management with appropriate preventive and restorative therapy. There is strong evidence that restoration of teeth with incomplete caries excavation results in fewer signs and symptoms of pulpal disease than complete excavation. There is strong evidence that sealants should be placed on pit and fissure surfaces judged to be at risk for dental caries, and surfaces that already exhibit incipient, non-cavitated carious lesions. There is evidence in favor for resin infiltration to improve the clinical appearance of white spot lesions. CONCLUSIONS: Substantial evidence exists in the literature regarding the value of preventive dental restorative procedures.

Overhauser effects in insulating solids.

We report magic angle spinning, dynamic nuclear polarization (DNP) experiments at magnetic fields of 9.4 T, 14.1 T, and 18.8 T using the narrow line polarizing agents 1,3-bisdiphenylene-2-phenylallyl (BDPA) dispersed in polystyrene, and sulfonated-BDPA (SA-BDPA) and trityl OX063 in glassy glycerol/water matrices. The (1)H DNP enhancement field profiles of the BDPA radicals exhibit a significant DNP Overhauser effect (OE) as well as a solid effect (SE) despite the fact that these samples are insulating solids. In contrast, trityl exhibits only a SE enhancement. Data suggest that the appearance of the OE is due to rather strong electron-nuclear hyperfine couplings present in BDPA and SA-BDPA, which are absent in trityl and perdeuterated BDPA (d21-BDPA). In addition, and in contrast to other DNP mechanisms such as the solid effect or cross effect, the experimental data suggest that the OE in non-conducting solids scales favorably with magnetic field, increasing in magnitude in going from 5 T, to 9.4 T, to 14.1 T, and to 18.8 T. Simulations using a model two spin system consisting of an electron hyperfine coupled to a (1)H reproduce the essential features of the field profiles and indicate that the OE in these samples originates from the zero and double quantum cross relaxation induced by fluctuating hyperfine interactions between the intramolecular delocalized unpaired electrons and their neighboring nuclei, and that the size of these hyperfine couplings is crucial to the magnitude of the enhancements. Microwave power dependent studies show that the OE saturates at considerably lower power levels than the solid effect in the same samples. Our results provide new insights into the mechanism of the Overhauser effect, and also provide a new approach to perform DNP experiments in chemical, biophysical, and physical systems at high magnetic fields.

Regional differences in predictive biomarker testing rates for patients with metastatic NSCLC in the Netherlands.

BACKGROUND: Predictive biomarker testing has a key role in the treatment decision-making for patients with non-small cell lung cancer (NSCLC) and is mandated by (inter)national guidelines. The aim of this study was to establish guideline-adherent biomarker testing rates in the Netherlands in 2019 and to examine associations of demographical, clinical, and environmental factors with guideline-adherent testing. METHODS: This study involved the integration of clinical data of the Netherlands Cancer Registry with pathology reports of the Dutch Nationwide Pathology Databank. Data extracted from these reports included sample type, diagnosis, and molecular testing status of predictive biomarkers. The study population comprised all patients diagnosed with metastatic non-squamous NSCLC in the Netherlands in 2019. RESULTS: In the cohort of 3877 patients with metastatic non-squamous NSCLC under investigation, overall molecular testing rates for non-fusion predictive biomarkers (EGFR, KRAS, BRAF, ERBB2, MET) ranged from 73.9 to 89.0 %, while molecular testing for fusion-drivers (ALK, ROS1, RET, NTRK) ranged from 12.6 % to 63.9 %. Guideline-adherent testing of EGFR, KRAS, and ALK was performed in 85.2 % of patients, with regional rates spanning from 76.0 % to 90.8 %. Demographical and clinical factors associated with guideline-adherent biomarker testing included lower age (OR = 1.05 per one year decrease; p < 0.001), female sex (OR = 1.36; p = 0.002), diagnosis of adenocarcinoma (OR = 2.48; p < 0.001), availability of histological tumor material (OR = 2.46; p < 0.001), and clinical stage of metastatic disease (p = 0.002). Other factors associated with guideline-adherent biomarker testing included diagnosis at academic center (OR = 1.87; p = 0.002) and patient's region of residence (p < 0∙001). CONCLUSION: Optimization of the chain-of-care of predictive biomarker testing in patients with NSCLC in the Netherlands is needed to provide adequate care for these patients.

Naturally occurring plasmid carrying genes for enterotoxin production and drug resistance.

Escherichia coli strain 86, isolated from a piglet with diarrhea, carries plasmid-linked genes for resistance to tetracycline, streptomycin, and sulfonamides and for production of heat-labile and heat-stable enterotoxin. Results of (i) genetic experiments involving conjugal transfer and phage P1-mediated transduction and (ii) physical experiments involving electron microscopic examination of plasmid DNA and heteroduplex analysis show that a single conjugative plasmid carries the genes for drug resistance and production of enterotoxin.

Water Fluoridation and Dental Caries in U.S. Children and Adolescents.

Fluoridation of America's drinking water was among the great public health achievements of the 20th century. Yet there is a paucity of studies from the past 3 decades investigating its dental health benefits in the U.S. POPULATION: This cross-sectional study sought to evaluate associations between availability of community water fluoridation (CWF) and dental caries experience in the U.S. child and adolescent population. County-level estimates of the percentage of population served by CWF (% CWF) from the Centers for Disease Control and Prevention's Water Fluoridation Reporting System were merged with dental examination data from 10 y of National Health and Nutrition Examination Surveys (1999 to 2004 and 2011 to 2014). Dental caries experience in the primary dentition (decayed and filled tooth surfaces [dfs]) was calculated for 7,000 children aged 2 to 8 y and in the permanent dentition (decayed, missing, and filled tooth surfaces [DMFS]) for 12,604 children and adolescents aged 6 to 17 y. Linear regression models estimated associations between % CWF and dental caries experience with adjustment for sociodemographic characteristics: age, sex, race/ethnicity, rural-urban location, head-of-household education, and period since last dental visit. Sensitivity analysis excluded counties fluoridated after 1998. In unadjusted analysis, caries experience in the primary dentition was lower in counties with ≥75% CWF (mean dfs = 3.3; 95% confidence limit [CL] = 2.8, 3.7) than in counties with <75% CWF (mean dfs = 4.6; 95% CL = 3.9, 5.4), a prevented fraction of 30% (95% CL = 11, 48). The difference was also statistically significant, although less pronounced, in the permanent dentition: mean DMFS (95% CL) was 2.2 (2.0, 2.4) and 1.9 (1.8, 2.1), respectively, representing a prevented fraction of 12% (95% CL = 1, 23). Statistically significant associations likewise were seen when % CWF was modeled as a continuum, and differences tended to increase in covariate-adjusted analysis and in sensitivity analysis. These findings confirm a substantial caries-preventive benefit of CWF for U.S. children and that the benefit is most pronounced in primary teeth.

A sandwich-cultured rat hepatocyte system with increased metabolic competence evaluated by gene expression profiling.

A rapid decline of cytochrome P450 (CYP450) enzyme activities remains a drawback of rat hepatocyte-based in vitro cultures. Consequently, judgment of the toxic potential of compounds that need bioactivation by CYP450s may not be adequate using this model. In the present study, an improved hepatocyte-based in vitro system was developed with special focus on metabolic competence. Therefore, a mixture of CYP450 inducers, phenobarbital, dexamethasone and beta-naphthoflavone, was added to culture medium of sandwich-cultured rat hepatocytes. The resulting modified model was evaluated by comparing its genome-wide expression profiles with liver and a standard model without the inducer mixture. Metabolic capacity for CYP450 enzymes showed that the modified model resembled more closely the in vivo situation. Gene expression results revealed large differences between in vivo and both in vitro models. The slight differences between the two sandwich models were predominantly represented by gene expression changes in CYP450s. Importantly, in the modified model, expression ratios of the phase I and the majority of phase II genes more closely resembled liver in vivo. The CYP450 enzyme activities corresponded with gene expression data. In conclusion, for toxicological applications using sandwich-cultured hepatocytes, the modified model may be preferred.

Binding of the arginine repressor of Escherichia coli K12 to its operator sites.

In the arginine regulon of Escherichia coli K12 each of the eight operator sites consists of two 18-base-pair-long palindromic sequences called ARG boxes. In the operator sites for the structural genes of the regulon the two ARG boxes are separated by three base-pairs, in the regulatory gene argR they are separated by two base-pairs. The hexameric arginine repressor, the product of argR, binds to the two ARG boxes in an operator in the presence of L-arginine. From the results of various kinds of in vitro footprinting experiments with the ARG boxes of argF and argR (DNase I protection, hydroxyl radical, ethylation and methylation interference, methylation protection) it can be concluded that: (1) the repressor binds simultaneously to two adjacent ARG boxes; (2) that it binds on one face of the double helix; and (3) that it forms contacts with the major and minor grooves of each ARG box, but not with the central three base-pairs. The repressor can bind also to a single ARG box, but its affinity is about 100-fold lower than for two ARG boxes. From gel retardation experiments with 3H-labeled repressor and 32P-labeled argF operator DNA, it is concluded that the retarded DNA-protein complex contains no more than one repressor molecule per operator site and that most likely one hexamer binds to two ARG boxes. The bound repressor was shown to induce bending of argF operator DNA. The bending angle calculated from the results of gel retardation experiments is about 70 degrees and the bending center was located within the region encompassing the ARG boxes. The main features that distinguish the arginine repressor from other repressors studied in E. coli are its hexameric nature and the simultaneous binding of one hexameric molecule to two palindromic ARG boxes that are close to each other.

A superrepressor mutant of the arginine repressor with a correctly predicted alteration of ligand binding specificity.

Arginine biosynthesis in Escherichia coli is negatively regulated by the hexameric repressor protein ArgR and the corepressor L-arginine. L-Arginine binds to ArgR in the C-terminal domain of the repressor. Binding to operator DNA occurs in the N-terminal domain. The molecular structures of both domains have recently been elucidated. The known stereochemistry of the arginine binding pocket was used for the rational design of a mutant ArgR with altered ligand specificity. Our prediction was that a replacement of Asp128 by asparagine would preferentially lead to the binding of L-citrulline, rather than L-arginine. The D128N mutant was constructed and was shown to fulfill our expectation by several experimental approaches. By isothermal titration calorimetry it was found to bind L-citrulline much more strongly than L-arginine, in contrast to wild-type ArgR. Exchange between the mutant trimers of the hexamer was inhibited by L-citrulline, as it is by L-arginine in the wild-type. The mutant protein was precipitated by L-citrulline but not by L-arginine, whereas the reverse is true for the wild-type protein. Demonstration of a corepressor action was, however, precluded by the superrepressor effect of the D128N mutation by itself. The mutant protein, in the absence of L-citrulline or L-arginine is as strong a repressor as the wild-type protein in the presence of L-arginine. We discuss two possible mechanisms, in terms of the known domain structures that could explain our observations.

Explanation for different types of regulation of arginine biosynthesis in Escherichia coli B and Escherichia coli K12 caused by a difference between their arginine repressors.

In Escherichia coli K12, formation of the enzymes of arginine biosynthesis are controlled by arginine, with complete repression during growth with added arginine, severe repression (about 95%) during growth without added arginine and complete derepression during arginine-limited growth. In E. coli B, the degree of repression is not correlated with arginine concentrations. Under all conditions of growth enzyme formation is repressed, with repression being somewhat less in a medium with arginine than in a medium without arginine. These differences in repressibility between the two strains have been shown previously to be due to the presence of different alleles of argR, the gene for the arginine repressor. Here we have compared the binding of the two repressors to the operator sites of argF (ARG boxes). In DNase I footprinting and gel retardation experiments with argF ARG boxes we have shown that the arginine repressor of E. coli K12 bound to arginine (ArgRK-arg) has a greater affinity than the arginine repressor of E. coli B bound to arginine (ArgRB-arg), whereas free ArgRB (ArgRBf) has a much stronger affinity than free ArgRK (ArgRKf). The stronger binding of ArgRBf can explain the repression seen in E. coli B during arginine-limited growth and indicates that ArgRBf, but not ArgRKf, is able to repress enzyme synthesis under physiological conditions. The weaker repression of E. coli B than of E. coli K12 seen in the presence of arginine can be explained by the lower affinity of ArgRB-arg for operator sites as compared to ArgRK-arg. Another contributing cause for the weaker repression is the reduction of ArgRBf concentration due to autoregulation of the gene for the repressor. Thus the combined effects of repression by ArgRBf, but not ArgRKf, with the weaker repression by ArgRB-arg as compared to ArgRK-arg, convert the arginine dependent regulation in E. coli K12 to arginine independent regulation in E. coli B.

Structure of the oligomerization and L-arginine binding domain of the arginine repressor of Escherichia coli.

The structure of the oligomerization and L-arginine binding domain of the Escherichia coli arginine repressor (ArgR) has been determined using X-ray diffraction methods at 2.2 A resolution with bound arginine and at 2.8 A in the unliganded form. The oligomeric core is a 3-fold rotationally symmetric hexamer formed from six identical subunits corresponding to the 77 C-terminal residues (80 to 156) of ArgR. Each subunit has an alpha/beta fold containing a four-stranded antiparallel beta-sheet and two antiparallel alpha-helices. The hexamer is formed from two trimers, each with tightly packed hydrophobic cores. In the absence of arginine, the trimers stack back-to-back through a dyad-symmetric, sparsely packed hydrophobic interface. Six molecules of arginine bind at the trimer-trimer interface, each making ten hydrogen bonds to the protein including a direct ion pair that crosslinks the two protein trimers. Solution experiments with wild-type ArgR and oligomerization domain indicate that the hexameric form is greatly stabilized upon arginine binding. The crystal structures and solution experiments together suggest possible mechanisms of how arginine activates ArgR to bind to its DNA targets and provides a stereochemical basis for interpreting the results of mutagenesis and biochemical experiments with ArgR.

The DNA-binding domain of the hexameric arginine repressor.

The arginine repressor of Escherichia coli is a classical feedback regulator, signalling the availability of L-arginine inside the cell. It differs from most other bacterial repressors in functioning as a hexamer, but structural details have been lacking and its shares no clear sequence homologies with other transcriptional regulators. Analysis of the amino acid residue sequence and proteolytic cleavage pattern of the repressor was used to identify a region predicted to house the DNA-binding function. When this protein fragment is overexpressed from a clone of the corresponding gene fragment, it represses ornithine transcarbamylase levels in vivo, and binds to the operator DNA in vitro, both in an arginine-independent manner. Sedimentation equilibrium and gel filtration indicate that the purified protein fragment is a monomer in solution. The results thus define the domain organization of the repressor at low resolution, suggesting that the N and C-terminal portions of the polypeptide chain are separated by a structural and functional border that decouples hexamerization and arginine binding from DNA binding.

Nuclear magnetic resonance imaging of solid rocket propellants at 14.1 T.

Proton NMR images of solid propellant materials, consisting of a polybutadiene binder material filled with 82% solid particles, have been obtained at a magnetic field strength of 14.1 T and at a resolution of 8.5 x 8.5 micron. The images are the first of elastomeric materials obtained at a proton frequency of 600 MHz and have the highest spatial resolution yet reported. The images display a high contrast and are rich in information content. They reveal the distribution of individual filler particles in the polymer matrix as well as a thin polymer film of about 10-30 micron which is found to surround some of the larger filler particles.

Magnetic field gradients in solid state magic angle spinning NMR.

Magnetic field gradients have proven useful in NMR for coherence pathway selection, diffusion studies, and imaging. Recently they have been combined with magic angle spinning to permit high-resolution measurements of semi-solids, where magic angle spinning averages any residual dipolar couplings and local variations in the bulk magnetic susceptibility. Here we show the first examples of coherence pathway selection by gradients in dipolar coupled solids. When the gradient evolution competes with dipolar evolution the experiment design must take into account both the strength of the dipolar couplings and the means to refocus it. Examples of both homonuclear and heteronuclear experiments are shown in which gradients have been used to eliminate the need for phase cycling.

Use of dental care by HIV-infected medical patients.

Although increasing attention has been paid to the use of dental care by HIV patients, the existing studies do not use probability samples, and no accurate population estimates of use can be made from this work. The intent of the present study was to establish accurate population estimates of the use of dental services by patients under medical care. The study, part of the HIV Cost and Services Utilization Study (HCSUS), created a representative national probability sample, the first of its kind, of HIV-infected adults in medical care. Both bivariate and logistic regressions were conducted, with use of dental care in the preceding 6 months as the dependent variable and demographic, social, behavioral, and disease characteristics as independent variables. Forty-two percent of the sample had seen a dental health professional in the preceding 6 months. The bivariate logits for use of dental care show that African-Americans, those whose exposure to HIV was caused by hemophilia or blood transfusions, persons with less education, and those who were employed were less likely to use dental care (p < 0.05). Sixty-five percent of those with a usual source of care had used dental care in the preceding 6 months. Use was greatest among those obtaining dental care from an AIDS clinic (74%) and lowest among those without a usual source of dental care (12%). We conclude that, in spite of the high rate of oral disease in persons with HIV, many do not use dental care regularly, and that use varies by patient characteristics and availability of a regular source of dental care.

EPR spectral determination of electronic substituent effects on the D values of hydrocarbon polyradicals (Quintet and septet spin states) composed of localized 1,3-cyclopentanediyl spin-carrying units linked by 1,3-Di- and 1,3,5-trimethylenebenzene ferromagnetic couplers

The parent and p-nitrophenyl-substituted diradicals D-3a,b (triplets), tetraradicals T-3a,b (quintets), and hexaradicals H-3a,b (septets) were photochemically generated in matrix-isolated form (toluene, 77 K) by successive denitrogenation of the trisazoalkanes 3a,b and EPR spectrally characterized. In these high-spin polyradicals the spin-spin interaction within the localized spin-carrying 1,3-cyclopentanediyl diradical unit is much stronger than within the cross-conjugated ferromagnetic coupling unit. Accordingly, a change of the electronic properties in the cyclopentanediyl unit affects decisively the D value of the whole polyradical. Therefore, the spin-accepting p-nitro group reduces the D value of the tetra- and hexaradical in the same amount as that of the diradical. Thus, irrespective of the spin multiplicity, the substituent stabilizes electronically the triplet (D-3a,b), quintet (T-3a,b), and septet (H-3a,b) species with equal efficacy.

Cryopreservation of precision-cut rat liver slices using a computer-controlled freezer.

Precision-cut liver slices are frequently used to study hepatic toxicity and metabolism of xenobiotics in vitro. Successful cryopreservation techniques will enhance an efficient and economic use of scarcely available (human) liver tissue. For primary hepatocytes, slow freezing has been accepted as the best approach towards successful cryopreservation. For slices, however, no agreement exists on the optimal way of cryopreservation and both slow and fast freezing techniques have been reported. The aim of the present study was to determine the applicability of a computer-controlled slow freezing technique for the cryopreservation of (rat) liver slices. Thus far, this technique has not been described in detail. Our studies confirmed that slow freezing was most successful in the cryopreservation of primary rat hepatocytes. Based on this observation, the slow freezing technique was applied to the cryopreservation of rat liver slices. Directly after thawing, slice viability was between 60 and 100% of fresh values, depending on the parameter determined. However, after additional culturing, slice viability was reduced. This decrease in slice viability was more pronounced in comparison to primary hepatocytes. In conclusion, the slow freezing technique was confirmed to be a successful approach for the cryopreservation of primary rat hepatocytes, and was found to be of limited use for the cryopreservation of rat liver slices.

1985 NHIS findings on public knowledge and attitudes about oral diseases and preventive measures.

Two objectives for the nation for 1990 set goals related to the need for schoolchildren and adults to understand the causes of oral diseases and methods of prevention. Five questions related to these objectives were included in the 1985 National Health Interview Survey. Survey responses of adults ages 18 years and older indicated that while the public is generally aware of the importance of a number of factors in the prevention of tooth decay, only 18 percent had both heard of, and knew the purpose of, dental sealants. At the same time, the public fails to discriminate between effective disease preventive factors related to periodontal diseases as opposed to those related to dental decay. Knowledge of oral disease prevention modalities generally varies across educational, income, age, and racial categories. However, there appears to be little variation in knowledge by gender. Additional information from upcoming surveys may shed more light on the relationships between knowledge of oral diseases and their prevention and personal preventive practices.