Considerations for the development of guidance on dose level selection for developmental and reproductive toxicity studies.

In 2022, the European Chemicals Agency issued advice on the selection of high dose levels for developmental and reproductive toxicity (DART) studies indicating that the highest dose tested should aim to induce clear evidence of reproductive toxicity without excessive toxicity and severe suffering in parental animals. In addition, a recent publication advocated that a 10% decrease in body weight gain should be replaced with a 10% decrease in bodyweight as a criterion for dose adequacy. Experts from the European Centre for Ecotoxicology and Toxicology of Chemicals evaluated these recent developments and their potential impact on study outcomes and interpretation and identified that the advice was not aligned with OECD test guidelines or with humane endpoints guidance. Furthermore, data analysis from DART studies indicated that a 10% decrease in maternal body weight during gestation equates to a 25% decrease in body weight gain, which differs from the consensus of experts at a 2010 ILSI/HESI workshop. Dose selection should be based on a biological approach that considers a range of other factors. Excessive dose levels that cause frank toxicity and overwhelm homeostasis should be avoided as they can give rise to effects that are not relevant to human health assessments.

[Flow cytometry evaluation of the rhesus monkey cellular immunity following the Zaire ebolavirus (Filoviridae; Ebolavirus: Zaire ebolavirus) experimental infection].

INTRODUCTION: The outbreaks of the Zaire ebolavirus (ZE) disease (ZED) that have arisen in the last decade determine the need to study the infection pathogenesis, the formation of specific immunity forming as well as the development of effective preventive and therapeutic means. All stages of fight against the ZED spread require the experimental infection in sensitive laboratory animals, which are rhesus monkeys in case of this disease .The aim of the study is to evaluate the rhesus monkey cellular immunity following the ZE experimental infection by the means of flow cytometry (cytofluorimetry). MATERIAL AND METHODS: Male rhesus monkeys were intramuscularly infected by the dose of 15 LD50 (dose of the pathogen that causes 50% mortality of infected animals) of the ZE, the Zaire strain (ZEBOV). Levels of 18 peripheral blood lymphocyte populations of the animals before the ZE experimental infection and at the terminal stage of the disease were assessed using flow cytometry. RESULTS AND DISCUSSION: The certain changes in the levels of the lymphocyte populations were observed following infection, indicating simultaneous activation and suppression of the immune system during ZED. The increase in content was observed for T-lymphocytes, T-helper and cytotoxic T-lymphocytes expressing the corresponding markers of early activation. The decrease was recorded for T-lymphocytes and double-positive T-lymphocytes expressing corresponding markers of late activation, as well as natural killer cells expressing CD8 (p < 0.05). CONCLUSION: For the first time in the Russian Federation, the rhesus monkey cellular immunity before and after the ZE experimental infection was assessed using flow cytometry.

Safety and immunogenicity of GamEvac-Combi, a heterologous VSV- and Ad5-vectored Ebola vaccine: An open phase I/II trial in healthy adults in Russia.

Ebola hemorrhagic fever, also known as Ebola virus disease or EVD, is one of the most dangerous viral diseases in humans and animals. In this open-label, dose-escalation clinical trial, we assessed the safety, side effects, and immunogenicity of a novel, heterologous prime-boost vaccine against Ebola, which was administered in 2 doses to 84 healthy adults of both sexes between 18 and 55 years. The vaccine consists of live-attenuated recombinant vesicular stomatitis virus (VSV) and adenovirus serotype-5 (Ad5) expressing Ebola envelope glycoprotein. The most common adverse event was pain at the injection site, although no serious adverse events were reported. The vaccine did not significantly impact blood, urine, and immune indices. Seroconversion rate was 100 %. Antigen-specific IgG geometric mean titer at day 42 was 3,277 (95 % confidence interval 2,401-4,473) in volunteers immunized at full dose. Neutralizing antibodies were detected in 93.1 % of volunteers immunized at full dose, with geometric mean titer 20. Antigen-specific response in peripheral blood mononuclear cells was also detected in 100 % of participants, as well as in CD4+ and CD8+ T cells in 82.8 % and 58.6 % of participants vaccinated at full dose, respectively. The data indicate that the vaccine is safe and induces strong humoral and cellular immune response in up to 100 % of healthy adult volunteers, and provide a rationale for testing efficacy in Phase III trials. Indeed, the strong immune response to the vaccine may elicit long-term protection. This trial was registered with grls.rosminzdrav.ru (No. 495*), and with zakupki.gov.ru (No. 0373100043215000055).

Cholinesterase inhibition and toxicokinetics in immature and adult rats after acute or repeated exposures to chlorpyrifos or chlorpyrifos-oxon.

The effect of age or dose regimen on cholinesterase inhibition (ChEI) from chlorpyrifos (CPF) or CPF-oxon (CPFO) was studied in Crl:CD(SD) rats. Rats were exposed to CPF by gavage in corn oil, rat milk (pups), or in the diet (adults) or to CPFO by gavage in corn oil. Blood CPF/CPFO levels were measured. With acute exposure, ChEI NOELs were 2 mg/kg CPF for brain and 0.5 mg/kg CPF for red blood cells (RBCs) in both age groups. In pups, ChEI and blood CPF levels were similar using either milk or corn oil vehicles. Compared to gavage, adults given dietary CPF (12 h exposure) had greater RBC ChEI, but lower brain ChEI at corresponding CPF doses, indicating an effect of dose rate. With repeated CPF exposures, ChEI NOELs were the same across ages (0.5 and 0.1 mg/kg/day for brain and RBCs, respectively). With CPFO dosing, the ChEI NOELs were 0.1 mg/kg (acute) and 0.01 mg/kg/day (repeated doses) for RBCs with no ChEI in brain at CPFO doses up to 0.5 (pup) or 10 mg/kg (adult) for acute dosing or 0.5 mg/kg/day for both ages with repeat dosing. Thus, there were no age-dependent differences in CPF ChEI via acute or repeated exposures. Pups had less ChEI than adults at comparable blood CPF levels. Oral CPFO resulted in substantial RBC ChEI, but no brain ChEI, indicating no CPFO systemic bioavailability to peripheral tissues.

Acute toxicologic and neurotoxic effects of inhaled 1,2-dichloroethane in adult Fischer 344 rats.

Acute toxicologic and neurotoxic effects were evaluated in Fischer 344 rats exposed to 0, 50, 200, 600, or 2000 ppm 1,2-dichloroethane (ethylene dichloride; EDC) for 4 h or 0, 50, 100 or 150 ppm for 8 h. Neurobehavioral and neuropathologic effects were assessed using a functional observational battery (FOB; baseline, days 1, 8, and 15), and by light microscopy, respectively. Acute toxicologic effects were assessed by bronchoalveolar lavage (BAL) and histopathology of the respiratory tract and selected target organs. Neurobehavioral effects consistent with central nervous system (CNS) depression were present at concentrations >200 ppm and were restricted to day 1. There were no neuropathologic changes in the CNS, however, olfactory epithelial regeneration 15 days after exposure to > or = 200 ppm was observed. The no-observed-effect concentration (NOEC) for behavioral neurotoxicity was 200 ppm EDC for 4 h. There were no effects on BAL parameters in any exposure group. Exposure to 2000 ppm EDC altered adrenal gland, kidney, and liver weights, and resulted in morphologic alterations in the kidney and liver. Degeneration/necrosis of the olfactory epithelium was observed at > or = 200 ppm for 4 h and > or = 100 ppm for 8 h. Based on olfactory epithelial degeneration/necrosis, the most sensitive indicator of toxicity in this study, the overall NOEC was 50 ppm EDC for up to 8 h in rats.

Evaluation of the safety and nutritional equivalence of a genetically modified cottonseed meal in a 90-day dietary toxicity study in rats.

Meal prepared from Cry1F/Cry1Ac transgenic/genetically modified cottonseed (WIDESTRIKE Insect Protection, hereafter referred to as WIDESTRIKE) was compared to cottonseed meal prepared from four conventionally bred lines of cotton (three commercial non-transgenic line controls (PHY72, PHY78 and 98M-2983), and a near isoline non-transgenic control (PSC355) in a 90-day dietary study to evaluate safety and nutritional equivalence. Diets were formulated with 10% WIDESTRIKE cottonseed meal equivalent to 7,235 mg/kg/day for males and 7,935 mg/kg/day for females. Animals were evaluated by cage-side and hand-held detailed clinical observations, body weight, and feed consumption. Functional tests, motor activity and ophthalmic examinations were conducted pre-exposure and prior to study termination. Standard hematology, clinical chemistry, prothrombin time and urinalysis parameters were evaluated. All rats had a complete necropsy and selected organs were weighed. Histopathologic examinations were performed on all rats fed the diets containing the near isoline non-transgenic control or WIDESTRIKE. Following 90 days of feeding, no adverse effects were observed during the conduct of clinical observations or in any of the parameters measured in this study. This study demonstrated that rodent diets prepared with 10% cottonseed meal from WIDESTRIKE cottonseeds do not produce any untoward effects and are nutritionally equivalent to cottonseed meals prepared from other, non-transgenic cottonseeds.

Double-hadron leptoproduction in the nuclear medium.

The first measurements of double-hadron production in deep-inelastic scattering within the nuclear medium were made with the HERMES spectrometer at DESY HERA using a 27.6 GeV positron beam. By comparing data for deuterium, nitrogen, krypton, and xenon nuclei, the influence of the nuclear medium on the ratio of double-hadron to single-hadron yields was investigated. Nuclear effects on the additional hadron are clearly observed, but with little or no difference among nitrogen, krypton, or xenon, and with smaller magnitude than effects seen on previously measured single-hadron multiplicities. The data are compared with models based on partonic energy loss or prehadronic scattering and with a model based on a purely absorptive treatment of the final-state interactions. Thus, the double-hadron ratio provides an additional tool for studying modifications of hadronization in nuclear matter.

Measurement of the tensor structure function b1 of the deuteron.

The Hermes experiment has investigated the tensor spin structure of the deuteron using the 27.6 GeV/c positron beam of DESY HERA. The use of a tensor-polarized deuteron gas target with only a negligible residual vector polarization enabled the first measurement of the tensor asymmetry A(d)zz and the tensor structure function b(d)1 for average values of the Bjorken variable 0.01< <0.45 and of the negative of the squared four-momentum transfer 0.5 GeV2 < <5 GeV2. The quantities A(d)zz and b(d)1 are found to be nonzero. The rise of b(d)1 for decreasing values of x can be interpreted to originate from the same mechanism that leads to nuclear shadowing in unpolarized scattering.

Single-spin asymmetries in semi-inclusive deep-inelastic scattering on a transversely polarized hydrogen target.

Single-spin asymmetries for semi-inclusive electroproduction of charged pions in deep-inelastic scattering of positrons are measured for the first time with transverse target polarization. The asymmetry depends on the azimuthal angles of both the pion (phi) and the target spin axis (phi(S)) about the virtual-photon direction and relative to the lepton scattering plane. The extracted Fourier component sin((phi+phi(S))(pi)(UT) is a signal of the previously unmeasured quark transversity distribution, in conjunction with the Collins fragmentation function, also unknown. The component sin((phi-phi(S)(pi)(UT) arises from a correlation between the transverse polarization of the target nucleon and the intrinsic transverse momentum of quarks, as represented by the previously unmeasured Sivers distribution function. Evidence for both signals is observed, but the Sivers asymmetry may be affected by exclusive vector meson production.

Flavor decomposition of the sea-quark helicity distributions in the nucleon from semiinclusive deep inelastic scattering.

Double-spin asymmetries of semiinclusive cross sections for the production of identified pions and kaons have been measured in deep inelastic scattering of polarized positrons on a polarized deuterium target. Five helicity distributions including those for three sea quark flavors were extracted from these data together with reanalyzed previous data for identified pions from a hydrogen target. These distributions are consistent with zero for all three sea flavors. A recently predicted flavor asymmetry in the polarization of the light quark sea appears to be disfavored by the data.

Q2 dependence of nuclear transparency for exclusive rho0 production.

Exclusive coherent and incoherent electroproduction of the rho(0) meson from 1H and 14N targets has been studied at the HERMES experiment as a function of coherence length (l(c)), corresponding to the lifetime of hadronic fluctuations of the virtual photon, and squared four-momentum of the virtual photon (-Q2). The ratio of 14N to 1H cross sections per nucleon, called nuclear transparency, was found to increase (decrease) with increasing l(c) for coherent (incoherent) rho(0) electroproduction. For fixed l(c), a rise of nuclear transparency with Q2 is observed for both coherent and incoherent rho(0) production, which is in agreement with theoretical calculations of color transparency.

Presentación atípica de tuberculosis pulmonaren la infancia / Atipical presentation of tuberculosis in children

Introducción: En lactantes y niños, la tuberculosis pulmonar primaria suele ser asintomática y con escasas manifestaciones radiológicas. Una forma de presentación poco frecuente es la de masa pulmonar. El diagnostico diferencial en estos casos es difícil e incluye malformaciones pulmonares y tumores propios de la infancia. Pacientes: Presentamos los casos de dos niñas menores de un año en las que se realizó una lobectomía por una tuberculosis pulmonar. En ambas, el diagnóstico inicial era erróneo a pesar de que las exploraciones realizadas fueron las apropiadas en el estudio de una masa pulmonar. Discusión y conclusiones: La TBC pulmonar debe considerarse en el diagnóstico diferencial en niños que presentan masa pulmonar. La confusión más frecuente ocurre con malformación adenomatoide quística y tumores mediastínicos. Los antecedentes y la clínica pueden ser de poca ayuda en estos casos. La TAC permite una buena definición de la lesión aunque no son raros los errores diagnósticos. El tratamiento mediante lobectomía está indicado cuando existe destrucción tisular irreversible. Las principales complicaciones son el empiema y la fístula broncopleural (AU)

Overview of purification and analysis of synthetic nucleic acids.

Synthetic nucleic acids are produced routinely for a wide variety of applications, including biological and chemical research, and diagnostic or therapeutic applications. To ensure an adequate level of quality and purity, rapid and convenient analytical methods are necessary. This unit discusses basic principles to guide in the selection of appropriate purification and analysis protocols.

Polyacrylamide gel electrophoresis (PAGE) of synthetic nucleic acids.

Protocols are given for analysis of oligonucleotides by PAGE, using either methylene blue staining or radiolabeling to mark the oligonucleotide. In addition, a separate protocol is provided for purification by PAGE.

Analysis and purification of synthetic nucleic acids using HPLC.

HPLC is a powerful and popular method for analyzing and purifying biomolecules. Reversed-phase HPLC allows a high-capacity method for purification, and uses volatile buffer systems that simplify product recovery. Anion-exchange HPLC provides better resolution and a more predictable elution pattern. This unit presents protocols that are optimized for HPLC of oligonucleotides. Because of the resolution limits of both reversed-phase and anion-exchange HPLC, it can be used for oligonucleotides of up to approximately 50 nt in length.

Base composition analysis of nucleosides using HPLC.

In this protocol, nuclease digestion of an oligonucleotide is followed by dephosphorylation and HPLC analysis of the monomers on a reversed-phase C18 column. This method can be used to detect and quantitate a wide variety of nucleobase modifications in oligonucleotides. Integrated areas of the nucleoside chromatogram give precise quantitation of nucleoside composition when the relative extinction coefficient cofactors are applied to the sum of the areas of the four bases. The protocol is also useful for analysis of oligonucleotides containing conjugated moieties and carbohydrate modifications.

Cartridge methods for oligonucleotide purification.

Protocols are given for purification of oligonucleotides by dimethoxytrityl-sensitive and affinity desalting methods. The protocols are applicable for many of the convenient disposable products available for rapid oligonucleotide purification, clean-up by selective adsorption, and elution on solid-phase media. Many of these products are prepackaged, single-use cartridges or columns filled with affinity or size-exclusion media.

Signature of superfluid density in the single-particle excitation spectrum of Bi(2)Sr(2)CaCu(2)O(8+delta)

We report that the doping and temperature dependence of photoemission spectra near the Brillouin zone boundary of Bi(2)Sr(2)CaCu(2)O(8+delta)exhibit unexpected sensitivity to the superfluid density. In the superconducting state, the photoemission peak intensity as a function of doping scales with the superfluid density and the condensation energy. As a function of temperature, the peak intensity shows an abrupt behavior near the superconducting phase transition temperature where phase coherence sets in, rather than near the temperature where the gap opens. This anomalous manifestation of collective effects in single-particle spectroscopy raises important questions concerning the mechanism of high-temperature superconductivity.

Efficient synthesis of double dye-labeled oligodeoxyribonucleotide probes and their application in a real time PCR assay.

A fast cleaving non-nucleosidic tetramethylrhodamine dye-labeled support has been developed for automated synthesis of double dye-labeled oligodeoxyribonucleotides in high yield. A mixture (1:1:2) of t-butylamine:methanol:water is used for cleavage and deprotection of dye-labeled oligodeoxyribonucleotides without any degradation or modification of dyes and nucleobases. The cleavage rate of oligodeoxyribonucleotides is significantly increased by using a diglycolate ester linkage instead of the commonly used succinate linkage. These double dye-labeled probes are used in PCR for real time detection of a specific PCR product. Using a 5'-exonuclease assay, detected on the ABI PRISM 7700 Sequence Detection System, there was no distinguishable difference in performance of probes synthesized using the dye-labeled support compared with traditional post-synthetic attachment of rhodamine.

Determination of interactions between structured nucleic acids by fluorescence resonance energy transfer (FRET): selection of target sites for functional nucleic acids.

We previously developed a method for monitoring the integrity of oligonucleotides in vitro and in vivo by quantitating fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. As an extension of this analysis, we examined changes in the extent of FRET in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In this system FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. We used a single-stranded oligodeoxyribonucleotide labeled at its 5'-end and its 3'-end with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. Moreover, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. The decrease in the extent of FRET depended on the length of the stem region of the hairpin structure and also on the higher-ordered structure of the probe. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. This conclusion based on changes in FRET was confirmed by gel-shift assays.