Framework for validation and implementation of in vitro toxicity tests

Summary The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community—academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.     

Prevalence and trends of common mental disorders from 2007-2009 to 2019-2022: results from the Netherlands Mental Health Survey and Incidence Studies (NEMESIS), including comparison of prevalence rates before vs. during the COVID-19 pandemic

Up-to-date information on the prevalence and trends of common mental disorders is relevant to health care policy and planning, owing to the high burden associated with these disorders. In the first wave of the third Netherlands Mental Health Survey and Incidence Study (NEMESIS-3), a nationally representative sample was interviewed face-to-face from November 2019 to March 2022 (6,194 subjects; 1,576 interviewed before and 4,618 during the COVID-19 pandemic; age range: 18-75 years). A slightly modified version of the Composite International Diagnostic Interview 3.0 was used to assess DSM-IV and DSM-5 diagnoses. Trends in 12-month prevalence rates of DSM-IV mental disorders were examined by comparing these rates between NEMESIS-3 and NEMESIS-2 (6,646 subjects; age range: 18-64 years; interviewed from November 2007 to July 2009). Lifetime DSM-5 prevalence estimates in NEMESIS-3 were 28.6% for anxiety disorders, 27.6% for mood disorders, 16.7% for substance use disorders, and 3.6% for attention-deficit/hyperactivity disorder. Over the last 12 months, prevalence rates were 15.2%, 9.8%, 7.1%, and 3.2%, respectively. No differences in 12-month prevalence rates before vs. during the COVID-19 pandemic were found (26.7% pre-pandemic vs. 25.7% during the pandemic), even after controlling for differences in socio-demographic characteristics of the respondents interviewed in these two periods. This was the case for all four disorder categories. From 2007-2009 to 2019-2022, the 12-month prevalence rate of any DSM-IV disorder significantly increased from 17.4% to 26.1%. A stronger increase in prevalence was found for students, younger adults (18-34 years) and city dwellers. These data suggest that the prevalence of mental disorders has increased in the past decade, but this is not explained by the COVID-19 pandemic. The already high mental disorder risk of young adults has particularly further increased in recent years.     

Holocene vegetation and water level history in two bogs of the Cordillera de Talamanca, Costa Rica

Pollen records of Holocene sediment cores from the Costa Rican Cordillera de Talamanca (La Chonta bog, 2310 m and La Trinidad bog, 2700 m) show the postglacial development of the montane oak forest zone from ca. 9500 to 1500 yr BP. During the early Holocene (ca. 9500–700 yr BP), alder vegetation covered the La Chonta and La Trinidad bogs and their adjacent hills. The upper forest line is inferred to be at 2800–3000 m elevation. A Podocarpus-Quercus forest characterised the middle Holocene (ca. 7000–4500 yr BP). The upper forest line is located at >3000 m reaching the present-day altitudinal distribution. A Quercus forest characterised the late Holocene (ca. 4500–1500 yr BP). Compared to modern conditions, the early Holocene has similar average temperatures, but the moisture level was probably higher. Pollen evidence for the late Holocene indicates drier environmental conditions than today. In order to improve the paleoecological interpretation, we described the local vegetation and used moss samples as pollen traps at both montane bogs along strong soil moisture gradients.The Netherlands Centre for Geo-ecological Research, ICG     

Limits to Catalysis by Ribonuclease A

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of the P-O(5') bond in RNA. Although this enzyme has been the object of much landmark work in bioorganic chemistry, the nature of its rate-limiting transition state and its catalytic rate enhancement had been unknown. Here, the value of k(cat)/K(m) for the cleavage of UpA by wild-type RNase A was found to be inversely related to the concentration of added glycerol. In contrast, the values of k(cat)/K(m) for the cleavage of UpA by a sluggish mutant of RNase A and the cleavage of the poor substrate UpOC(6)H(4)-p-NO(2) by wild-type RNase A were found to be independent of glycerol concentration. Yet, UpA cleavage by the wild-type and mutant enzymes was found to have the same dependence on sucrose concentration, indicating that catalysis of UpA cleavage by RNase A is limited by desolvation. The rate of UpA cleavage by RNase A is maximal at pH 6.0, where k(cat) = 1.4 × 10(3) s(-1) and k(cat)/K(m) = 2.3 × 10(6) M(-1)s(-1) at 25°C. At pH 6.0 and 25°C, the uncatalyzed rate of [5,6-(3)H]Up[3,5,8-(3)H]A cleavage was found to be k(uncat) = 5 × 10(-9) s(-1) (t(1/2) = 4 years). Thus, RNase A enhances the rate of UpA cleavage by 3 × 10(11)-fold by binding to the transition state for P-O(5') bond cleavage with a dissociation constant of <2 × 10(-15) M.     

Ventilatory response to helium-oxygen breathing during exercise: effect of airway anesthesia

Krishnan, Bharath S., Ron E. Clemens, Trevor A. Zintel,Martin J. Stockwell, and Charles G. Gallagher. Ventilatory response to helium-oxygen breathing during exercise: effect of airwayanesthesia. J. Appl. Physiol. 83(1):82-88, 1997.The substitution of a normoxic helium mixture(HeO₂) for room air (Air) during exercise results in a sustained hyperventilation, which is present evenin the first breath. We hypothesized that this response is dependent onintact airway afferents; if so, airway anesthesia (Anesthesia) shouldaffect this response. Anesthesia was administered to the upper airwaysby topical application and to lower central airways by aerosolinhalation and was confirmed to be effective for over 15 min. Subjectsperformed constant work-rate exercise (CWE) at 69 ± 2 (SE) % maximal work rate on a cycle ergometer on three separate days: twiceafter saline inhalation (days 1 and3) and once after Anesthesia(day 2). CWE commenced after a briefwarm-up, with subjects breathing Air for the first 5 min (Air-1),HeO₂ for the next 3 min, and Airagain until the end of CWE (Air-2). The resistance of the breathingcircuit was matched for Air andHeO₂. BreathingHeO₂ resulted in a small butsignificant increase in minute ventilation(I) anddecrease in alveolar PCO₂ in both theSaline (average of 2 saline tests; not significant) and Anesthesiatests. Although Anesthesia had no effect on the sustainedhyperventilatory response to HeO₂breathing, theI transientswithin the first six breaths ofHeO₂ were significantly attenuatedwith Anesthesia. We conclude that theI response to HeO₂ is not simply due to areduction in external tubing resistance and that, in humans, airwayafferents mediate the transient but not the sustained hyperventilatoryresponse to HeO₂ breathing duringexercise.

     

RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) AND PARSIMONY METHODS

Abstract — Random amplified polymorphic DNA (RAPD) data possess a number of undesirable features for parsimony analysis. These features include their non-codominant inheritance, their anonymous nature, their different (a)symmetrical transformation probabilities, and their possible GC priming bias. As a consequence, no single parsimony method seems appropriate for RAPD data. Moreover, the presence/absence coding of RAPDs is equivalent to the invalid independent allele model for allozymes. These issues are discussed and the way in which parsimony analysis of RAPDs can be misleading is illustrated.     

Role of the conserved C-repeat region of the M protein of Streptococcus pyogenes

The surface-located M protein functions to protect Streptococcus pyogenes (the group A streptococcus) from phagocytosis by polymorphonuclear leukocytes. It has been suggested that this protection results from the ability of M protein to bind factor H, a serum protein that can inhibit the activation of complement. Among different serological variants of M protein, the C-repeat domain is highly conserved and is exposed on the bacterial surface. This domain has been implicated in binding to complement factor H and in M-protein-mediated adherence of streptococci to human keratinocytes in the cutaneous epithelium. In this study, we constructed an S. pyogenes mutant strain which expresses an M6 protein from which the entire C-repeat domain was deleted. As predicted, this mutant did not adhere well to human keratinocytes and was unable to bind to factor H. Unexpectedly, the mutant was able to survive and multiply in human blood. Therefore, while the binding of factor H and the facilitation of adherence to keratinocytes appear to involve recognition of the C-repeat domain, a region of the M-protein molecule distinct from the C-repeat domain confers upon S. pyogenes its ability to resist phagocytosis.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Discrimination of flash-induced fast and slow electric potential components

This work aimed at the resolution of the multi-component electric potential changes induced by single-turnover flash illumination of Photosystem-I-enriched subchloroplast vesicles. If supplemented with ferredoxin and under carefully adjusted redox poising, these vesicles show a pronounced slow-rising and -decaying electric potential component, as monitored by endogenous and exogenous field-sensitive probes, carotenoids and oxonol VI, respectively. The fast and slow potential components can be easily discriminated without the need for computer-assisted deconvolution after selective presaturation of the slow component by preillumination or a transmembrane ΔpH, after selective suppression of the slow component by low valinomycin or uncoupler concentrations or in the absence of ferredoxin. The slow electric potential component, as compared to the fast one, is relatively sensitive to low concentrations of ionophores and uncouplers, detergent, ageing and lower temperatures (4–12°C), is associated with electrogenic proton displacements and is interpreted to respond to a field that is more located on the membrane-bulk interface. Temperature effects show transition temperatures around 20°C for both the rise and decay of the slow potential component. The results provide further evidence that the carotenoids and oxonol VI sense the same (slow) electric field, but may be differently located in the thylakoid membrane.