Efficacy of ensitrelvir against SARS-CoV-2 in a delayed-treatment mouse model.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19) and a devastating worldwide health concern. Development of safe and effective treatments is not only important for interventions during the current pandemic, but also for providing general treatment options moving forward. We have developed ensitrelvir, an antiviral compound that targets the 3C-like protease of SARS-CoV-2. In this study, a delayed-treatment mouse model was used to clarify the potential in vivo efficacy of ensitrelvir. METHODS: Female BALB/cAJcl mice of different ages were infected with the SARS-CoV-2 gamma strain (hCoV-19/Japan/TY7-501/2021) or mouse-adapted SARS-CoV-2 MA-P10 and then 24 h post-infection orally administered various doses of ensitrelvir or vehicle. Viral titres and RNA levels in the lungs were quantified using VeroE6/TMPRSS2 cells and RT-qPCR, respectively. Body weight loss, survival, lung weight, cytokine/chemokine production, nucleocapsid protein expression and lung pathology were evaluated to investigate the in vivo efficacy of ensitrelvir. RESULTS: Based on infectious viral titres and viral RNA levels in the lungs of infected mice, ensitrelvir reduced viral loads in a dose-dependent manner. The antiviral efficacy correlated with increased survival, reduced body weight loss, reduced pulmonary lesions and suppression of inflammatory cytokine/chemokine levels. CONCLUSIONS: This was the first evaluation of the in vivo anti-SARS-CoV-2 efficacy of ensitrelvir in a delayed-treatment mouse model. In this model, ensitrelvir demonstrated high antiviral potential and suppressed lung inflammation and lethality caused by SARS-CoV-2 infection. The findings support the continued clinical development of ensitrelvir as an antiviral agent to treat patients with COVID-19.

Castor bean cake in top-dressing application as a source of nitrogen on the production and quality of zucchini organic seeds / Torta de mamona em cobertura como fonte de nitrogênio na produção e qualidade de sementes orgânicas de abobrinha-de-moita

Studies in vegetables show that fertilization influences seed production positively, however, when the quality of the seeds is analyzed, the results are mostly inconsistent. The objective of this study was to evaluate the effect of castor bean cake (CBC) dose splitting in top-dressing application on the production and quality of zucchini organic seeds, as well as its effect on the macronutrient content of fruits and seeds. The experimental design was a randomized block design and four replications. There were 13 treatments with four doses of CBC in top-dressing (1.7, 3.4, 5.1 and 6.8 t ha-1) per three applications (33.3-33.3-33.3%, 50-50% and 33-50-17%) and the control treatment without any kind of top-dressing fertilization. The study evaluated the number of ripe fruits per plant, the production (number and mass) of seeds per fruit and per plant, the mass of one hundred seeds, seed germination, first germination count, and macronutrient content in the diagnosis leaf, in mature fruits (without seeds) and seeds. The number of ripe fruits per plant was not affected by the CBC doses in top-dressing, neither by the splitting of the applications. It was observed that the number of seeds per fruit, mass of seeds per fruit and mass of seeds per plant showed quadratic behavior. Regarding seed quality, only the 1.7; 3.4 and 5.1 t ha-1 doses produced seed with superior quality than the other doses. In conclusion, the CBC doses increased the number of seeds per fruit, mass of seeds per fruit and per plant up to the 4.5t ha-1 dose, approximately.

Trabalhos em horticultura já realizados mostram que a adubação influencia positivamente na produção de sementes, porém, ao se analisar a qualidade das sementes, os resultados na maioria das vezes não são concordantes. O objetivo do trabalho foi de estudar o efeito de parcelamentos e doses de torta de mamona (TM) em cobertura na produção e qualidade de sementes orgânicas de abobrinha-de-moita, assim como nos teores de macronutrientes nos frutos e sementes. Foi utilizado o delineamento experimental em blocos ao acaso, e quatro repetições. Totalizaram 13 tratamentos, sendo quatro doses de TM em cobertura (1,7; 3,4; 5,1 e 6,8 t ha-1) x três parcelamentos das aplicações (33,3-33,3-33,3%; 50-50% e 33-50-17%) e o controle sem nenhum tipo de adubação em cobertura. Foram avaliados o número de frutos maduros por planta, produção (número e massa) de sementes por fruto e por planta, massa de mil sementes, germinação, primeira contagem de germinação e teores de macronutrientes na folha diagnose, nos frutos maduros (sem as sementes) e nas sementes. O número de frutos maduros por planta não foi influenciado pelas doses de TM em cobertura nem pelos parcelamentos. Observou-se ajuste quadrático para o número de sementes por fruto, massa de sementes por fruto e massa de sementes por planta. Para a qualidade das sementes as doses de 1,7; 3,4; e 5,1 t ha-1 foram superiores às demais doses. Pode-se concluir que as doses de TM aumentaram o número de sementes por fruto, massa de sementes por fruto e massa de sementes por planta até a dose 4,5 t ha-1, aproximadamente.

Single Molecule Fluorescence Imaging Reveals the Stoichiometry of BKγ1 Subunit in Living HEK293 Cell Expression System.

Large conductance Ca2+-activated K+ (BKCa) channels are ubiquitously expressed in plasma membrane of both excitable and non-excitable cells and possess significant physiological functions. A tetrameric complex of α subunit (BKα) forms a functional pore of BKCa channel. The properties of BKCa channel, such as voltage-dependence, Ca2+ sensitivity and pharmacological responses, are extensively modulated by co-expressing accessory ß subunits (BKß), which can associate with BKα in one to one manner. Although the functional significance of newly identified γ subunits (BKγ) has been revealed, the stoichiometry between BKα and BKγ1 remains unclear. In the present study, we utilized a single molecule fluorescence imaging with a total internal reflection fluorescence (TIRF) microscope to directly count the number of green fluorescent protein (GFP)-tagged BKγ1 (BKγ1-GFP) within a single BKCa channel complex in HEK293 cell expression system. BKγ1-GFP significantly enhanced the BK channel activity even when the intracellular Ca2+ concentration was kept lower, i.e., 10 nM, than the physiological resting level. BKγ1-GFP stably formed molecular complexes with BKα-mCherry in the plasma membrane. Counting of GFP bleaching steps revealed that a BKCa channel can contain up to four BKγ1 per channel at the maximum. These results suggest that BKγ1 forms a BKCa channel complex with BKα in a 1 : 1 stoichiometry in a human cell line.

Involvement of the γ1 subunit of the large-conductance Ca2+-activated K+ channel in the proliferation of human somatostatinoma cells.

Pancreatic neuroendocrine tumors (pNETs) occur due to the abnormal growth of pancreatic islet cells and predominantly develop in the duodenal-pancreatic region. Somatostatinoma is one of the pNETs associated with tumors of pancreatic δ cells, which produce and secrete somatostatin. Limited information is currently available on the pathogenic mechanisms of somatostatinoma. The large-conductance Ca2+-activated K+ (BKCa) channel is expressed in several types of cancer cells and regulates cell proliferation, migration, invasion, and metastasis. In the present study, the functional expression of the BKCa channel was examined in a human somatostatinoma QGP-1 cell line. In QGP-1 cells, outward currents were elicited by membrane depolarization at pCa 6.5 (300 nM) in the pipette solution and inhibited by the specific BKCa channel blocker, paxilline. Paxilline-sensitive currents were detected, even at pCa 8.0 (10 nM) in the pipette solution, in QGP-1 cells. In addition to the α and ß2-4 subunits of the BKCa channel, the novel regulatory γ1 subunit (BKCaγ1) was co-localized with the α subunit in QGP-1 cells. Paxilline-sensitive currents at pCa 8.0 in the pipette solution were reduced by the siRNA knockdown of BKCaγ1. Store-operated Ca2+ entry was smaller in BKCaγ1 siRNA-treated QGP-1 cells. The proliferation of QGP-1 cells was attenuated by paxilline or the siRNA knockdown of BKCaγ1. These results strongly suggest that BKCaγ1 facilitates the proliferation of human somatostatinoma cells. Therefore, BKCaγ1 may be a novel therapeutic target for somatostatinoma.

Roles of LRRC26 as an auxiliary γ1-subunit of large-conductance Ca2+-activated K+ channels in bronchial smooth muscle cells.

In visceral smooth muscle cells (SMCs), the large-conductance Ca2+-activated K+ (BK) channel is one of the key elements underlying a negative feedback mechanism that is essential for the regulation of intracellular Ca2+ concentration. Although leucine-rich repeat-containing (LRRC) proteins have been identified as novel auxiliary γ-subunits of the BK channel (BKγ) in several cell types, its physiological roles in SMCs are unclear. The BKγ expression patterns in selected SM tissues were examined using real-time PCR analyses and Western blotting. The functional contribution of BKγ1 to BK channel activity was examined by whole cell patch-clamp in SMCs and heterologous expression systems. BKγ1 expression in mouse bronchial SMCs (mBSMCs) was higher than in other several SMC types. Coimmunoprecipitation and total internal reflection fluorescence imaging analyses revealed molecular interaction between BKα and BKγ1 in mBSMCs. Under voltage-clamp, steady-state activation of BK channel currents at pCa 8.0 in mBSMCs occurred in a voltage range comparable to that of reconstituted BKα/BKγ1 complex. However, this range was much more negative than in mouse aortic SMCs (mASMCs) or in HEK293 cells expressing BKα alone and ß-subunit (BKß1). Mallotoxin, a selective activator of BK channel that lacks BKγ1, dose-dependently activated BK currents in mASMCs but not in mBSMCs. The abundant expression of BKγ1 in mBSMCs extensively facilitates BK channel activity to keep the resting membrane potential at negative values and prevents contraction under physiological conditions.