Shedding Light on Dark Chemical Matter: The Discovery of a SARS-CoV-2 Mpro Main Protease Inhibitor through Intensive Virtual Screening and In Vitro Evaluation.

The development of specific antiviral therapies targeting SARS-CoV-2 remains fundamental because of the continued high incidence of COVID-19 and limited accessibility to antivirals in some countries. In this context, dark chemical matter (DCM), a set of drug-like compounds with outstanding selectivity profiles that have never shown bioactivity despite being extensively assayed, appears to be an excellent starting point for drug development. Accordingly, in this study, we performed a high-throughput screening to identify inhibitors of the SARS-CoV-2 main protease (Mpro) using DCM compounds as ligands. Multiple receptors and two different docking scoring functions were employed to identify the best molecular docking poses. The selected structures were subjected to extensive conventional and Gaussian accelerated molecular dynamics. From the results, four compounds with the best molecular behavior and binding energy were selected for experimental testing, one of which presented inhibitory activity with a Ki value of 48 ± 5 µM. Through virtual screening, we identified a significant starting point for drug development, shedding new light on DCM compounds.

Negative effects of the spatial clumping of thermal resources on lizard thermoregulation in a fragmented habitat.

In ecosystems threatened by the expansion of croplands, habitat fragmentation and climate change, two of the main extinction drivers, may have thermoregulation-mediated interacting effects on demographic trends of terrestrial ectotherms. We studied the thermal biology of a metapopulation of the widespread Mediterranean lacertid Psammodromus algirus in ten fragments of evergreen or deciduous oak forests interspersed among cereal fields. We obtained thermoregulation statistics (selected temperature range, body and operative temperatures, thermal quality of the habitat, and precision, accuracy, and effectiveness of thermoregulation) that could be compared among fragments and with conspecific populations living in unfragmented habitat. We also measured the selection (use vs. availability) and spatial distribution of sunlit and shaded patches used for behavioral thermoregulation in fragments, and we estimated operative temperatures and thermal habitat quality in the agricultural matrix surrounding the fragments. Variation of the thermal environment was much larger within fragments than among them, and thermoregulation was accurate, precise, and efficient throughout the fragmented landscape; its effectiveness was similar to that of previously studied unfragmented populations. The average distance between sunlit and shaded patches was shorter in deciduous than in evergreen fragments, producing a more clumped distribution of the mosaic of thermal resources. Consequently, in evergreen habitat the cost of thermoregulation was higher, because lizards were more selective in their choice of sunlit sites (i.e. they used sunlit patches closer to shade and refuge than expected at random, and the extent of such selection was larger than at deciduous habitat). Temperatures available in croplands were too high to allow lizard dispersal, at least in the post-breeding season. This result confirms the role of croplands as a thermal barrier that promotes inbreeding and associated fitness losses in isolated fragments, and it forecasts a dark future for populations of forest lizards in agricultural landscapes under the combined effects of habitat fragmentation and global warming.

Reliability of the Coimbra Reactive Agility Soccer Test (CRAST).

Agility is a fitness-skill-related component that should be a part of the standard physiological testing for soccer players and one of the key performance indicators in soccer. The present study aimed to assess the reliability of the CRAST as a research tool in the study of soccer skills. Twenty-one university soccer players (chronological age: 19.3 ± 1.4 years; body mass: 69.6 ± 8.2 kg; stature: 173.5 ± 6.5 cm; federated training experience: 9.7 ± 3.6 years) volunteered for the testing protocol. The CRAST requires players to complete random courses six times as quickly as possible. In addition, the CRAST requires players to control and dribble the markers (four different colors: green, yellow, blue, and red). The soccer players completed three trials, each separated by one week. The first trial accounted for familiarization; the second and third were considered for analysis. The correlation for overall performance was very strong. The reliability of the CRAST was slightly better for total time than that for the penalty score (0.95 vs. 0.93). The TEM and the associated CV range of 7.04%-7.54% were for the penalty score and the total time, respectively. For both measurements, the ICC values also represent excellent reliability, as both values were over 0.900. The CRAST is a reliable protocol for assessing agility in soccer players.

Fragment dissolved molecular dynamics: a systematic and efficient method to locate binding sites.

Diverse computational methods to support fragment-based drug discovery (FBDD) are available in the literature. Despite their demonstrated efficacy in supporting FBDD campaigns, they exhibit some drawbacks such as protein denaturation or ligand aggregation that have not yet been clearly overcome in the framework of biomolecular simulations. In the present work, we discuss a systematic semi-automatic novel computational procedure, designed to surpass these difficulties. The method, named fragment dissolved Molecular Dynamics (fdMD), utilizes simulation boxes of solvated small fragments, adding a repulsive Lennard-Jones potential term to avoid aggregation, which can be easily used to solvate the targets of interest. This method has the advantage of solvating the target with a low number of ligands, thus preventing the denaturation of the target, while simultaneously generating a database of ligand-solvated boxes that can be used in further studies. A number of scripts are made available to analyze the results and obtain the descriptors proposed as a means to trustfully discard spurious binding sites. To test our method, four test cases of different complexity have been solvated with ligand boxes and four molecular dynamics runs of 200 ns length have been run for each system, which have been extended up to 1 µs when needed. The reported results point out that the selected number of replicas are enough to identify the correct binding sites irrespective of the initial structure, even in the case of proteins having several close binding sites for the same ligand. We also propose a set of descriptors to analyze the results, among which the average MMGBSA and the average KDEEP energies have emerged as the most robust ones.

Regulation of the inositol transporter Itr1p by hydrogen peroxide in Saccharomyces cerevisiae.

Myo-inositol is a precursor of several membrane phospholipids and sphingolipids and plays a key role in gene regulation in Saccharomyces cerevisiae (S. cerevisiae). Here, we tested whether H2O2 was affecting the levels of the inositol transporters and thus inositol uptake. In S. cerevisiae cells adapted to H2O2 Itr1-GFPp accumulated in the plasma membrane until 20 min, concomitantly with an inhibition of its internalization. Exposure to H2O2 did not alter Itr2-GFPp cellular levels and induced only an 8% decrease at 10 min in the plasma membrane. Therefore, decreased inositol intracellular levels are not caused by decreased levels of inositol transporters in the plasma membrane. However, results show that H2O2 adaptation affects Itr1p turnover and, consequently, H2O2-adapted yeast cells display an inositol transporter phenotype comparable to cells grown in the absence of inositol in growth medium, i.e. accumulation in the plasma membrane and decreased degradation.

A multistep docking and scoring protocol for congeneric series: Implementation on kinase DFG-out type II inhibitors.

AIM: Rescoring of docking-binding poses can significantly improve molecular docking results. Our aim was to evaluate postprocessing docking protocols in order to determine the most suitable methodology for the study of the binding of congeneric compounds to protein kinases. MATERIALS & METHODS: Diverse ligand-receptor poses generated after docking were submitted to different relaxation protocols. The Molecular Mechanics Poisson-Boltzmann (Generalized Born) Surface Area approach was applied for the evaluation of the binding affinity of complexes obtained. The performance of various Molecular Mechanics Poisson-Boltzmann (Generalized Born) Surface Area methodologies was compared. RESULTS: The inclusion of a postprocessing protocol after docking enhances the quality of the results, although the best methodology is system dependent. CONCLUSION: An examination of the interactions established has allowed us to suggest useful modifications for the design of new type II inhibitors.

Does day length affect winter bird distribution? Testing the role of an elusive variable.

Differences in day length may act as a critical factor in bird biology by introducing time constraints in energy acquisition during winter. Thus, differences in day length might operate as a main determinant of bird abundance along latitudinal gradients. This work examines the influence of day length on the abundance of wintering crested tits (Lophophanes cristatus) in 26 localities of Spanish juniper (Juniperus thurifera) dwarf woodlands (average height of 5 m) located along a latitudinal gradient in the Spanish highlands, while controlling for the influence of food availability, minimum night temperature, habitat structure and landscape characteristics. Top regression models in the AIC framework explained 56% of variance in bird numbers. All models incorporated day length as the variable with the highest magnitude effect. Food availability also played an important role, although only the crop of ripe juniper fruits, but not arthropods, positively affected crested tit abundance. Differences in vegetation structure across localities had also a strong positive effect (average tree height and juniper tree density). Geographical variation in night temperature had no influence on crested tit distribution, despite the low winter temperatures reached in these dwarf forests. This paper demonstrates for the first time that winter bird abundance increases with day length after controlling for the effect of other environmental variables. Winter average difference in day length was only 10.5 minutes per day along the 1°47' latitudinal interval (190 km) included in this study. This amount of time, which reaches 13.5 h accumulated throughout the winter season, appears to be large enough to affect the long-term energy budget of small passerines during winter and to shape the distribution of winter bird abundance under restrictive environmental conditions.

[Measurement of blood pressure in consultation and automated mesurement (BPTru(®)) to evaluate the white coat effect]. / Medida de la presión arterial en consulta y automatizada (BPTru®) para evaluar el efecto de bata blanca.

BACKGROUND AND OBJECTIVE: White coat effect (WCE) is one of the main bias that can affect office blood pressure (BP) measurement. Therefore, it is a factor must be considered in hypertensives to avoid mistakes in diagnosis and/or treatment. Employment of automated office BP (AOBP) devices could diminish that effect. METHODS: Two studies were designed with the objective of evaluating differences between routinely office and AOBP measurements. WCE was also assessed. First, the TRUE-ESP study included normotensive and hypertensive patients attending specialized consultations at Cardiology, Nephrology, Internal Medicine, Endocrinology and Family Practice. Second, the TRUE-HTA study included hypertensives attending a protocoled Hypertension Unit, with a trained staff. RESULTS: TRUE-ESP study included 300 patients, 76% being hypertensives. A significant difference between office BP and AOBP measurement (SBP/DBP 9.8±11.6/3.4±7.9 mmHg, P<.001) was observed. Percentage of patients gathering WCE criteria was 27.7%. TRUE-HTA study included 101 hypertensive patients. A significant difference between office BP and AOBP measurement (SBP/DBP 5.7±9.3/2.1±5.3 mmHg, P<.001) and activity period-ABPM (SBP/DBP 8.5±6.7/3.5±2.5 mmHg, P<.001) was observed. Percentage of WCE patients was 32.1%. CONCLUSIONS: Use of AOBP devices can contribute to decrease WCE and to improve accuracy of office BP measurement.

Involvement of regional lymph nodes after penetration of Schistosoma mansoni cercariae in Naive and infected mice

The parotid lymph nodes of naive and previously infected Balb/c mice were studied after, respectively, infection and re-infection with cercariae of Schistosoma mansoni via the ears. Schistosomula were able to pass through the lymph node by following the lymph flow or by penetrating the veins of the medullary cords. The number of nodal mast cells was higher from day 2 to 6 of primary infection; and from day 5 to 11 of re-infection. The amount of degranulating mast cells was significantly higher at day 4 of infection and at day 1 of re-infection. Eosinophils characterized the nodal inflammatory processes observed after day 5 in both primarily-infected and re-infected mice. However, only in the latter the eosinophils were able to adhere to the larval surface. In primarily-infected mice, no intranodal larva presented signs of degeneration. In contrast, in re-infected animals, some degenerating larvae were found inside eosinophilic infiltrates. The eosinophils reached the nodal tissue by migrating through the high endothelial venules and their collecting veins.

Schistosoma mansoni: resistência cutânea em camundongos portadores de infecçäo primária / Schistosoma mansoni: cutaneous resistance in mice with primary infection

No presente trabalho avaliou-se a resistência cutânea de camundongos ao Schistosoma mansoni, usando-se a orelha como sítio de infecçäo e de recuperaçäo de esquistossômulos através da incubaçäo de seus fragmentos em recipiente posto em contacto com Elac tamponado com Hepes. Essa técnica mostrou-se eficiente na discriminaçäo do número de esquistossômulos recuperados de camundongos normais e de camundongos previamente infectados (camundongos imunes), quando comparada à técnica de recuperaçäo de parasitas através da digestäo da pele em meio contendo colagenase. Através dessa técnica, verificou-se que camundongos imunes reduzem o parasitismo do primeiro ao sétimo dia após a reinfecçäo (42 a 46%). Essa resistência foi observada portadores de infecçäo bissexuada (6ª a 15ª semanas) e unissexuada (33ª e 34ª semanas) e em linhagens isogênicas (C57 BL/10, CBA e F1 do cruzamento CBA x DBA/2 e näo isogênica (Swiss). Revelando-se apropriadas ao estudo da resistência anti-esquistossomótica que se manifesta ao nível da pele, sugere-se que orelhas possam ser utilizadas como via de infecçäo em experimentos que visem analisar os fatores que participam da imunidade de camundongos ao S. mansoni

Schistosoma Mansoni: infecçäo experimental de camundongos através da orelha e quantificaçäo do parasitismo na pele / Schistosoma mansoni: experimental infection in mice through the ear and quantification of the parasitism in the skin

Desenvolveu-se método de infecçäo de camundongos através da orelha e de recuperaçäo de esquistossômulos resultantes dessas infecçöes. Cerca de 80% das cercárias postas em contacto com orelhas de camundongos penetraram. Destas, 30% foram recuperadas como vermes adultos, do sistema porta. Da pele (das orelhas) as maiores recuperaçöes de esquistossômulos ocorreram nos dois primeiros dias após a infecçäo. Os parasitas permaneceram nesse sítio por dois dias. No terceiro dia, os parasitas foram recuperados tanto da pele como dos pulmöes. A partir do 4§ dia, foi predominante a recuperaçäo de esquistossômulos ao nível dos pulmöes. Do total de parasitas que potencialmente atingiram o sistema porta, proporçäo elevada (73-80%) pode ser recuperada da pele, no segundo dia após a infecçäo, como esquistossômulos. Revelando-se apropriadas ao acesso, à migraçäo no hospedeiro e às técnicas de recuperaçäo de parasitas, sugere-se que orelhas de camundongos podem ser utilizadas como sítio de infecçäo para estudos que visem a análise parasitológica dos eventos iniciais da infeççäo em animais normais ou imunes