Hyperthermia along with hydrocortisone (HC) are proven teratogens that can negatively influence embryo development during early pregnancy. Proliferation of cells is one of the main developmental processes during the early embryogenesis. This study was focused on testing the effect of elevated temperature and HC addition on proliferation of cells in in vitro cultures. The V79-4 cell line was treated with HC and cultured in vitro at 37 °C or 39 °C, respectively. To reveal the effect of both factors, the proliferation of cells cultured under different conditions was evaluated using various approaches (colony formation assay, generation of growth curves, computation of doubling times, and mitotic index estimation). Our results indicate that a short-term exposure to elevated temperature slightly stimulates and a long-term exposure suppresses cell proliferation. However, HC (0.1 mg/ml) acts as a stimulator of cell proliferation. Interestingly, the interaction of HC and long-term elevated temperature (39 °C) exposure results in at least partial compensation of the negative impact of elevated temperature by HC addition and in higher proliferation if compared with cells cultured at 39 °C without addition of HC.
Leishmania, the dixenous trypanosomatid parasites, are the causative agents of leishmaniasis currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania, and the recently described Mundinia, consisting of six species distributed sporadically all over the world infecting humans and/or animals. These parasites infect various mammalian species and also cause serious human diseases, but their reservoirs are unknown. Thus, adequate laboratory models are needed to enable proper research of Mundinia parasites. In this complex study, we compared experimental infections of five Mundinia species (L. enriettii, L. macropodum, L. chancei, L. orientalis, and four strains of L. martiniquensis) in three rodent species: BALB/c mouse, Chinese hamster (Cricetulus griseus) and steppe lemming (Lagurus lagurus). Culture-derived parasites were inoculated intradermally into the ear pinnae and progress of infection was monitored for 20 weeks, when the tissues and organs of animals were screened for the presence and quantity of Leishmania. Xenodiagnoses with Phlebotomus duboscqi were performed at weeks 5, 10, 15 and 20 post-infection to test the infectiousness of the animals throughout the experiment. BALB/c mice showed no signs of infection and were not infectious to sand flies, while Chinese hamsters and steppe lemmings proved susceptible to all five species of Mundinia tested, showing a wide spectrum of disease signs ranging from asymptomatic to visceral. Mundinia induced significantly higher infection rates in steppe lemmings compared to Chinese hamsters, and consequently steppe lemmings were more infectious to sand flies: In all groups tested, they were infectious from the 5th to the 20th week post infection. In conclusion, we identified two rodent species, Chinese hamster (Cricetulus griseus) and steppe lemming (Lagurus lagurus), as candidates for laboratory models for Mundinia allowing detailed studies of these enigmatic parasites. Furthermore, the long-term survival of all Mundinia species in steppe lemmings and their infectiousness to vectors support the hypothesis that some rodents have the potential to serve as reservoir hosts for Mundinia.
- MeSH
- Arvicolinae * parazitologie MeSH
- Cricetulus MeSH
- křečci praví MeSH
- Leishmania * klasifikace MeSH
- leishmanióza * parazitologie MeSH
- modely nemocí na zvířatech * MeSH
- myši inbrední BALB C * MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Selective activation of individual subtypes of muscarinic receptors is a promising way to safely alleviate a wide range of pathological conditions in the central nervous system and the periphery as well. The flexible G-protein interface of muscarinic receptors allows them to interact with several G-proteins with various efficacy, potency, and kinetics. Agonists biased to the particular G-protein mediated pathway may result in selectivity among muscarinic subtypes and, due to the non-uniform expression of individual G-protein alpha subunits, possibly achieve tissue specificity. Here, we demonstrate that novel tetrahydropyridine-based agonists exert specific signalling profiles in coupling with individual G-protein α subunits. These signalling profiles profoundly differ from the reference agonist carbachol. Moreover, coupling with individual Gα induced by these novel agonists varies among subtypes of muscarinic receptors which may lead to subtype selectivity. Thus, the novel tetrahydropyridine-based agonist can contribute to the elucidation of the mechanism of pathway-specific activation of muscarinic receptors and serve as a starting point for the development of desired selective muscarinic agonists.
- MeSH
- agonisté muskarinových receptorů * farmakologie MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- karbachol farmakologie MeSH
- lidé MeSH
- proteiny vázající GTP - alfa-podjednotky metabolismus genetika MeSH
- proteiny vázající GTP metabolismus MeSH
- pyridiny farmakologie MeSH
- receptory muskarinové * metabolismus MeSH
- signální transdukce účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca2+ concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions.
- MeSH
- analgetika farmakologie terapeutické užití MeSH
- beta-cyklodextriny * farmakologie MeSH
- bolest chemicky indukované farmakoterapie metabolismus MeSH
- CHO buňky MeSH
- cholesterol metabolismus MeSH
- Cricetulus MeSH
- HEK293 buňky MeSH
- kationtové kanály TRPM * metabolismus genetika MeSH
- lidé MeSH
- membránové mikrodomény metabolismus účinky léků MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- pregnenolon farmakologie MeSH
- pyrimidinony farmakologie MeSH
- sfingomyelinfosfodiesterasa * metabolismus farmakologie MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In late 2023, several SARS-CoV-2 XBB descendants, notably EG.5.1, were predominant worldwide. However, a distinct SARS-CoV-2 lineage, the BA.2.86 variant, also emerged. BA.2.86 is phylogenetically distinct from other Omicron sublineages, accumulating over 30 amino acid mutations in its spike protein. Here, we examined the virological characteristics of the BA.2.86 variant. Our epidemic dynamics modeling suggested that the relative reproduction number of BA.2.86 is significantly higher than that of EG.5.1. Additionally, four clinically available antivirals were effective against BA.2.86. Although the fusogenicity of BA.2.86 spike is similar to that of the parental BA.2 spike, the intrinsic pathogenicity of BA.2.86 in hamsters was significantly lower than that of BA.2. Since the growth kinetics of BA.2.86 are significantly lower than those of BA.2 both in vitro and in vivo, the attenuated pathogenicity of BA.2.86 is likely due to its decreased replication capacity. These findings uncover the features of BA.2.86, providing insights for control and treatment.
- MeSH
- aminokyseliny MeSH
- COVID-19 * MeSH
- kinetika MeSH
- křečci praví MeSH
- mutace MeSH
- SARS-CoV-2 genetika MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In late 2022, SARS-CoV-2 Omicron subvariants have become highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged through the recombination of two cocirculating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022. XBB.1 is the variant most profoundly resistant to BA.2/5 breakthrough infection sera to date and is more fusogenic than BA.2.75. The recombination breakpoint is located in the receptor-binding domain of spike, and each region of the recombinant spike confers immune evasion and increases fusogenicity. We further provide the structural basis for the interaction between XBB.1 spike and human ACE2. Finally, the intrinsic pathogenicity of XBB.1 in male hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provides evidence suggesting that XBB is the first observed SARS-CoV-2 variant to increase its fitness through recombination rather than substitutions.
- MeSH
- COVID-19 * MeSH
- fylogeneze MeSH
- glykoprotein S, koronavirus genetika MeSH
- křečci praví MeSH
- lidé MeSH
- rekombinace genetická MeSH
- SARS-CoV-2 genetika MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In late 2022, various Omicron subvariants emerged and cocirculated worldwide. These variants convergently acquired amino acid substitutions at critical residues in the spike protein, including residues R346, K444, L452, N460, and F486. Here, we characterize the convergent evolution of Omicron subvariants and the properties of one recent lineage of concern, BQ.1.1. Our phylogenetic analysis suggests that these five substitutions are recurrently acquired, particularly in younger Omicron lineages. Epidemic dynamics modelling suggests that the five substitutions increase viral fitness, and a large proportion of the fitness variation within Omicron lineages can be explained by these substitutions. Compared to BA.5, BQ.1.1 evades breakthrough BA.2 and BA.5 infection sera more efficiently, as demonstrated by neutralization assays. The pathogenicity of BQ.1.1 in hamsters is lower than that of BA.5. Our multiscale investigations illuminate the evolutionary rules governing the convergent evolution for known Omicron lineages as of 2022.
The problem of the efficient treatment of acute organophosphorus (OP) poisoning needs more efforts in the development of a versatile antidote, applicable for treatment of the injuries of both peripheral and central nervous systems. A series of N-H, N-methyl, N-butyl, and N-phenyl derivatives of benzhydroxamic (1a-1d), 3-methoxybenzhydroxamic (2a-2d), 4-methoxybenzhydroxamic (3a-3d) acids, and corresponding salycilhydroxamates (4a-4d) was prepared. Their predicted hydrophobicity (log P) was evaluated as regards to ВВВ score by the open access cheminformatics tools; prediction of the passive transport across the BBB was found by means on the parallel artificial membrane permeability assay (PAMPA). The data on reactivation capacity of human acetylcholinesterase (HssAChE) inhibited by GB, VX, and paraoxon was supported by molecular docking study on binding to the active site of the AChE, viability study against mammalian cells (Chinese hamster ovary CHO-K1), and biodegradability (Closed Bottle test OECD 301D). Among the studied compounds, N-butyl derivatives have better balanced combination of properties; among them, N-butylsalicylhydroxamic acid is most promising. The studied compounds demonstrate modest reactivation capacity; change of N-H by N-Me ensures the reactivation capacity in studied concentrations on all studied OP substrates; among N-butyl derivatives, the N-butylsalicylhydroxamic acid demonstrates most promising results within the series. The found regularities may lead to selection of perspective structures to complement current formulations for medical countermeasures against poisoning by organophosphorus toxicants.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- antidota farmakologie MeSH
- CHO buňky MeSH
- cholinesterasové inhibitory chemie farmakologie MeSH
- Cricetulus MeSH
- křečci praví MeSH
- lidé MeSH
- otrava organofosfáty * MeSH
- oximy chemie MeSH
- reaktivátory cholinesterázy * chemie farmakologie MeSH
- simulace molekulového dockingu MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
STUDY QUESTION: Does addition of choriogonadotropin beta (recombinant CG beta) to follitropin delta increase the number of good-quality blastocysts following ovarian stimulation in a long GnRH agonist protocol? SUMMARY ANSWER: At the doses investigated, the addition of CG beta reduced the number of intermediate follicles and related down-stream parameters including the number of oocytes and blastocysts. WHAT IS KNOWN ALREADY: CG beta is a novel recombinant hCG (rhCG) molecule expressed by a human cell line (PER.C6®) and has a different glycosylation profile compared to urinary hCG or rhCG derived from a Chinese Hamster Ovary (CHO) cell line. In the first-in-human trial, the CG beta pharmacokinetics were similar between men and women. In women, the AUC and the peak serum concentration (Cmax) increased approximately dose proportionally following single and multiple daily doses. In men, a single dose of CG beta provided higher exposure with a longer half-life and proportionately higher testosterone production than CHO cell-derived rhCG. STUDY DESIGN, SIZE, DURATION: This placebo-controlled, double-blind, randomized trial (RAINBOW) was conducted in five European countries to explore the efficacy and safety of CG beta as add-on treatment to follitropin delta in women undergoing ovarian stimulation in a long GnRH agonist protocol. Randomization was stratified by centre and age (30-37 and 38-42 years). The primary endpoint was the number of good-quality blastocysts (Grade 3 BB or higher). Subjects were randomized to receive either placebo or 1, 2, 4, 8 or 12 μg CG beta added to the daily individualized follitropin delta dose during ovarian stimulation. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 620 women (30-42 years) with anti-Müllerian hormone (AMH) levels between 5 and 35 pmol/l were randomized in equal proportions to the six treatment groups and 619 subjects started treatment. All 619 subjects were treated with an individualized dose of follitropin delta determined based on AMH (Elecsys AMH Plus Immunoassay) and body weight. Triggering with rhCG was performed when 3 follicles were ≥17 mm but no more than 25 follicles ≥12 mm were reached. MAIN RESULTS AND THE ROLE OF CHANCE: The demographic characteristics were comparable between the six treatment groups and the overall mean age, body weight and AMH were 35.6 ± 3.3 years, 65.3 ± 10.7 kg and 15.3 ± 7.0 pmol/l, respectively. The incidence of cycle cancellation (range 0-2.9%), total follitropin delta dose (mean 112 μg) and duration of stimulation (mean 10 days) were similar across the groups. At stimulation Day 6, the number and size of follicles was similar between the treatment groups, whereas at the end-of-stimulation dose-related decrease of the intermediate follicles between 12 and 17 mm was observed in comparison to the placebo group. In contrast, the number of follicles ≥17 mm was similar between the CG beta dose groups and the placebo group. A reduced number of intermediate follicles (12 to 17 mm) and fewer oocytes (mean range 9.7 to 11.2) were observed for all doses of CG beta compared to the follitropin delta only group (mean 12.5). The mean number of good-quality blastocysts was 3.3 in the follitropin delta group and ranged between 2.1 and 3.0 across the CG beta groups. The incidence of transfer cancellation was higher in the 4, 8 and 12 μg group, mostly as no blastocyst was available for transfer. In the group receiving only follitropin delta, the ongoing pregnancy rate (10-11 weeks after transfer) was 43% per started cycle versus 28-39% in CG beta groups and 49% per transfer versus 38-50% in the CG beta groups. There was no apparent effect of CG beta on the incidence of adverse events, which was 48.1% in the placebo group and 39.6-52.3% in the CG beta dose groups. In line with the number of collected oocytes, the overall ovarian hyperstimulation syndrome incidence remained lower following follitropin delta with CG beta (2.0-10.3%) compared with follitropin delta only treatment (11.5%). Regardless of the dose, CG beta was safe and well-tolerated with low risk of immunogenicity. LIMITATIONS, REASONS FOR CAUTION: The effect of the unique glycosylation of CG beta and its associated potency implications in women were not known prior to this trial. Further studies will be needed to evaluate optimal doses of CG beta for this and/or different indications. WIDER IMPLICATIONS OF THE FINDINGS: The high ongoing pregnancy rate in the follitropin delta group supports the use of individualized follitropin delta dosing in a long GnRH agonist protocol. The addition of CG beta reduced the presence of intermediate follicles with the investigated doses and negatively affected all down-stream parameters. Further clinical research will be needed to assess the optimal dose of CG beta in the optimal ratio to follitropin delta to develop this novel combination product containing both FSH and LH activity for ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by Ferring Pharmaceuticals, Copenhagen, Denmark. B.M. and P.L. are employees of Ferring Pharmaceuticals. M.F.S., H.V., C.Y.A., M.F., C.B., A.P. and Y.K. have received institutional clinical trial fees from Ferring Pharmaceuticals. C.B. has received payments for lectures from Organon, Ferring Pharmaceuticals, Merck A/S and Abbott. M.F.S. has received payment for lectures from Ferring Pharmaceuticals. Y.K. has received payment for lectures from Merck and travel support from Gedeon Richter. H.V. has received consulting fees from Oxo and Obseva and travel support from Gedeon Richter, Ferring Pharmaceuticals and Merck. C.Y.A. has received payment for lectures from IBSA, Switzerland. M.F and C.Y.A. were reimbursed as members of the Data Monitoring Board in this trial. M.F. has an issued patent about unitary combination of FSH and hCG (EP1633389). TRIAL REGISTRATION NUMBER: 2017-003810-13 (EudraCT Number). TRIAL REGISTRATION DATE: 21 May 2018. DATE OF FIRST PATIENT’S ENROLMENT: 13 June 2018.
- MeSH
- antimülleriánský hormon MeSH
- CHO buňky MeSH
- choriogonadotropin MeSH
- Cricetulus MeSH
- fertilizace in vitro metody MeSH
- folikuly stimulující hormon lidský * MeSH
- hormon uvolňující gonadotropiny MeSH
- indukce ovulace * metody MeSH
- křečci praví MeSH
- léčivé přípravky MeSH
- lidé MeSH
- lidský choriogonadotropin, beta podjednotka MeSH
- randomizované kontrolované studie jako téma MeSH
- rekombinantní proteiny MeSH
- těhotenství MeSH
- tělesná hmotnost MeSH
- úhrn těhotenství na počet žen v reprodukčním věku MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- protokol klinické studie MeSH
The flavivirus, tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and may cause severe and potentially lethal neurological tick-borne encephalitis (TBE) in humans. Studying TBEV requires the use of secondary methodologies to detect the virus in infected cells. To overcome this problem, we rationally designed and constructed a recombinant reporter TBEV that stably expressed the mCherry reporter protein. The resulting TBEV reporter virus (named mCherry-TBEV) and wild-type parental TBEV exhibited similar growth kinetics in cultured cells; however, the mCherry-TBEV virus produced smaller plaques. The magnitude of mCherry expression correlated well with progeny virus production but remained stable over <4 passages in cell culture. Using well-characterized antiviral compounds known to inhibit TBEV, 2'-C-methyladenosine and 2'-deoxy-2'-β-hydroxy-4'-azidocytidine (RO-9187), we demonstrated that mCherry-TBEV is suitable for high-throughput screening of antiviral drugs. Serum samples from a TBEV-vaccinated human and a TBEV-infected dog were used to evaluate the mCherry-based neutralization test. Collectively, recombinant mCherry-TBEV reporter virus described here provides a powerful tool to facilitate the identification of potential antiviral agents, and to measure levels of neutralizing antibodies in human and animal sera.
- MeSH
- antivirové látky izolace a purifikace MeSH
- buněčné linie MeSH
- klíšťová encefalitida imunologie virologie MeSH
- křečci praví MeSH
- ledviny cytologie MeSH
- lidé MeSH
- luminescentní proteiny genetika MeSH
- neutralizační testy * MeSH
- neutralizující protilátky krev MeSH
- protilátky virové krev MeSH
- rychlé screeningové testy metody MeSH
- viry klíšťové encefalitidy genetika růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
