Ratchet, swivel, tilt and roll: a complete description of subunit rotation in the ribosome.

Protein synthesis by the ribosome requires large-scale rearrangements of the 'small' subunit (SSU; ∼1 MDa), including inter- and intra-subunit rotational motions. However, with nearly 2000 structures of ribosomes and ribosomal subunits now publicly available, it is exceedingly difficult to design experiments based on analysis of all known rotation states. To overcome this, we developed an approach where the orientation of each SSU head and body is described in terms of three angular coordinates (rotation, tilt and tilt direction) and a single translation. By considering the entire RCSB PDB database, we describe 1208 fully-assembled ribosome complexes and 334 isolated small subunits, which span >50 species. This reveals aspects of subunit rearrangements that are universal, and others that are organism/domain-specific. For example, we show that tilt-like rearrangements of the SSU body (i.e. 'rolling') are pervasive in both prokaryotic and eukaryotic (cytosolic and mitochondrial) ribosomes. As another example, domain orientations associated with frameshifting in bacteria are similar to those found in eukaryotic ribosomes. Together, this study establishes a common foundation with which structural, simulation, single-molecule and biochemical efforts can more precisely interrogate the dynamics of this prototypical molecular machine.

Effect of slow-release urea on intake, ingestive behavior, digestibility, nitrogen metabolism, microbial protein production, blood and ruminal parameters of sheep.

We conducted two experiments. The first aimed to obtain and characterize microparticles of slow-release urea (SRU) using calcium alginate as the encapsulating agent. The second experiment evaluated their inclusion in sheep diets. In the first experiment, four treatments from a completely randomized design were employed to develop an SRU through the ionic gelification technique testing two drying methods (oven and lyophilizer) and addition or no of sulfur (S): SRU oven-dried with sulfur (MUSO) and without sulfur (MUO), SRU freeze-dried/lyophilized with (MUSL), and without sulfur (MUL). MUO exhibited better yield and encapsulation efficiency among these formulations than the others. Therefore, the second experiment was conducted to compare free urea (U) as control and three proportions (1%, 1.5%, and 2% of total dry matter) of MUO in the diet of sheep. Twenty-four non-castrated male Santa Ines lambs, with an average body weight of 22 ± 3.0 kg, were used and distributed in a completely randomized design with four treatments and six replications. The inclusion of 1% alginate-encapsulated urea (MUO1%) resulted in higher dry matter (DM) intake than free urea (p ≤ 0.05). MUO2% inclusion promoted higher NDF digestibility than U and MUO1%. MUO1% showed higher DM than MUO2% and higher NFC digestibility than U and MUO2% (p ≤ 0.05). Sheep fed MUO1.5% and MUO2% exhibited similar nutrient intake and digestibility. Sheep receiving MUO1% had higher N-intake, N-urinary, N-excretion total, N-digested, and N-retained compared to U. Sheep fed MUO1% showed greater N-retained (as % ingested and digested), microbial protein production, and efficiency when compared to other treatments (p ≤ 0.05). MUO2% addition (SRU) promoted the lowest microbial protein production and efficiency in sheep. MUO dietary inclusion increased feeding time and reduced idleness time compared to U, regardless of the MUO level (p ≤ 0.05). Adding MUO1% improved the intake efficiency of DM and NDF and resulted in more feed boli than the other MUO levels (p ≤ 0.05). Sheep receiving U had (4 h after fending) higher NH3-N, pH, and blood urea nitrogen (BUN) and lower TGL serum compared to sheep fed MUO (p ≤ 0.05), without significant difference among MUO levels (p > 0.05), except NH3-N was higher in MUO1.5% and MUO2% compared to MUO1.0%. The external ionic gelation technique proved suitable for urea microencapsulation in calcium alginate (3%), demonstrating high quality, efficiency, and yield. MUO represents a promising slow-release urea for ruminants and is recommended for sheep diets at an inclusion level of 1.0%. This inclusion level improves intake efficiency and nutrient digestibility, increases rumen nitrogen retention, and reduces BUN without compromising sheep health.

Production, characterization, and dietary supplementation effect of rumen-protected fat on ruminal function and blood parameters of sheep.

Rumen-protected fat (RPF) was produced in the 1st experimental stage through melt-emulsification technique using buriti oil (BO) as core, at concentrations of 10% (BO10), 20% (BO20), and 30% (BO30) (w/w), and carnauba wax (CW) as encapsulant material. After obtention and characterization, protected fat microspheres were tested in a 2nd experimental stage on the sheep' diet using six castrated 2-year-old male Santa Ines with initial weight 48.9 ± 5.23 kg, fistulated in rumen and distributed in a double Latin square design with 3 treatments × 3 periods, to evaluate rumen pH, temperature, protozoal count, and blood parameters. There was no difference (P > 0.05) among RPF microspheres for microencapsulation yield. However, microencapsulation efficiency increased (P < 0.05) with BO addition ranging from 36 to 61.3% for BO10 and BO30, respectively. The inclusion of BO10 in the sheep's diet did not affect the ruminal dry matter degradability (DMD) of BO over time (P > 0.05); however, BO20 and BO30 had higher (P < 0.05) DMD values than BO10. No significant differences were observed among RPF for rumen pH and temperature (P > 0.05). There was an increase (P < 0.05) in the protozoal population in the rumen environment due to the microencapsulated BO30 inclusion. There was also increase (P < 0.05) in serum albumin, cholesterol, aspartate aminotransferase (ALT), and gamma-glutamyltransferase (GGT), and a reduction (P < 0.05) in serum triglycerides of the sheep when RPF microspheres increased in the diet. Melt-emulsification proved to be a good technique for microencapsulation of buriti oil into the carnauba wax matrix. RPF from buriti oil protected into carnauba wax is recommended for sheep diet because it increases energy density, without adverse effects on the protozoal populations and blood serum metabolites from the bypass effect in the rumen.

Cholinesterase inhibitors assessment of aporphine alkaloids from Annona crassiflora and molecular docking studies.

Annona crassiflora Mart. is an endemic plant from Brazilian Cerrado (savanna) biome, commonly employed in traditional medicine to treat wounds, diarrhea, and scalp infections. The pulp of the fruits is edible and has a characteristic taste, being employed to prepare sweets like jam, cakes, and ice cream by the people who live in the region of the Cerrado, although the peels are discarded. In this way, the A. crassiflora fruit peels ethanol extract was prepared and subjected to liquid-liquid extraction, which yielded the alkaloidal fraction (CH2Cl2). Subjected to high-performance liquid chromatography separations, this fraction allowed the purification of the aporphine alkaloids stephalagine (1), liriodenine (2), and atherospermidine (3), that were structurally characterized by high-resolution mass spectrometry with electrospray ionization, and nuclear magnetic resonance spectroscopy analyses. Aporphine alkaloids are recognized for their acetylcholinesterase (AChE) inhibitory activity, an important target in Alzheimer's disease therapy. Thus, the ethanol extract, alkaloidal fraction, and compounds1,2,and3were evaluated for acetyl- and butyrylcholinesterase (BChE) inhibitory activities. Compound1(IC50 104.2 µmol L-1) exhibited better BChE inhibitory activity than the standard compound galanthamine (IC50 162.7 µmol L-1), while2had a comparable result(and IC50 167.3 µmol L-1). Furthermore, molecular docking was performed to predict the compound's binding mode to the human AChE at a molecular level. Semiempirical calculation results show that the enthalpy interaction energy (ΔHint) between AChE and BChE active sites and all ligands were favorable for both enzymes, with the ligands interacting even more strongly with AChE, corroborating with IC50 results.

Biotin-painted proteins have thermodynamic stability switched by kinetic folding routes.

Biotin-labeled proteins are widely used as tools to study protein-protein interactions and proximity in living cells. Proteomic methods broadly employ proximity-labeling technologies based on protein biotinylation in order to investigate the transient encounters of biomolecules in subcellular compartments. Biotinylation is a post-translation modification in which the biotin molecule is attached to lysine or tyrosine residues. So far, biotin-based technologies proved to be effective instruments as affinity and proximity tags. However, the influence of biotinylation on aspects such as folding, binding, mobility, thermodynamic stability, and kinetics needs to be investigated. Here, we selected two proteins [biotin carboxyl carrier protein (BCCP) and FKBP3] to test the influence of biotinylation on thermodynamic and kinetic properties. Apo (without biotin) and holo (biotinylated) protein structures were used separately to generate all-atom structure-based model simulations in a wide range of temperatures. Holo BCCP contains one biotinylation site, and FKBP3 was modeled with up to 23 biotinylated lysines. The two proteins had their estimated thermodynamic stability changed by altering their energy landscape. In all cases, after comparison between the apo and holo simulations, differences were observed on the free-energy profiles and folding routes. Energetic barriers were altered with the density of states clearly showing changes in the transition state. This study suggests that analysis of large-scale datasets of biotinylation-based proximity experiments might consider possible alterations in thermostability and folding mechanisms imposed by the attached biotins.

Antiparasitary and antiproliferative activities in vitro of a 1,2,4-oxadiazole derivative on Trypanosoma cruzi.

Chagas disease (CD) is a neglected disease, prevalent and endemic in Latin America, but also present in Europe and North America. The main treatment used for this disease is benznidazole, but its efficacy is variable in the chronic phase and presents high toxicity. So, there is a need for the development of new therapeutic agents. The five-membered heterocyclic 1,2,4-oxadiazole ring has received attention for its unique properties and a broad spectrum of biological activities and is therefore a potential candidate for the development of new drugs. Thus, the aim of this study was to evaluate the activity of the N-cyclohexyl-3-(3-methylphenyl)-1,2,4-oxadiazol-5-amine (2) on the evolutionary forms of Trypanosoma cruzi strain Y, as well as its mechanisms of action and in silico theoretical approach. The results by computational method showed an interaction of the 1,2,4-oxadiazole (2) with TcGAPDH, cruzain, and trypanothione reductase, showing good charge distribution and affinity in those three targets. Furthermore, cytotoxicity in LLC-MK2 cells was performed by the MTT method. In the assays with different parasite forms, the tested compound showed similar time-dependent concentration effect. The evaluation of the antiamastigote effect between the two concentrations tested showed a reduction in the number of infected cells and also in the number of amastigotes per infected cell. By flow cytometry, the compound (2) displayed alterations suggestive of necrotic events. Finally, in scanning electron microscopy structural alterations were present, characteristic of necrosisin the epimastigote forms. Overall, the 1,2,4-oxadiazole derivative (2) here evaluated opens perspectives to the development of new antichagasic agents.

Antihyperlipidemic Activity of Glycoconjugated Phthalimides in Mice Submitted to a Model of Dyslipidemia and Insulin Resistance.

Alterations in lipid and lipoprotein metabolism are factors that trigger several negative metabolic complications. Hyperlipidemia is the starting point for the development of comorbidities of the cardiovascular system, such as atherosclerosis. The search for compounds that reduce high levels of total cholesterol and triglycerides has been widely reported in several publications in the literature. Phthalimide derivatives have been extensively researched with various biological actions. In this study we evaluated the antihyperlipidemic ability of three phthalimide derivatives (FGT-2, FGT-3 and FGT-4) on a model of obesity and insulin resistance in mice. The animals were submitted to a hyperlipid diet for 60 days. On the thirtieth day they were treated with phthalimides (20 mg/kg). The positive control group was treated with Simvastatin (20 mg/kg) and the negative control received only the carboxymethylcellulose vehicle. Biochemical and histological analyzes of all groups were analyzed. The animals treated with phthalimidic derivatives had a reduction in total cholesterol, low density and very low density lipoproteins (LDL-c and VLDL-c), triglycerides and fasting glycemia when compared to the negative control group. The treated animals also showed good results when analyzing the atherogenic indexes Castelli i and II and the ratio Triglycerides/HDL-c. In the oral glucose tolerance test and in the insulin tolerance test, animals treated with phthalimides were more sensitive to the action of the hormone regulating carbohydrate uptake. In the evaluation of the transaminases (AST/ALT), the animals of the group treated with phthalimides presented a lower elevation than the other groups of the experiment, the same observed with the uric acid evaluation. Histological analyzes were performed on liver, kidney, heart and pancreas samples. The groups treated with the compounds FGT-2 and FGT3 presented discrete alterations in the liver and kidney. FGT-4 did not present histological alterations for both tissues and the three phthalimide derivatives did not cause alterations in the other organs. These results suggest that the phthalimides tested can act as antihyperlipidemic agents and have a pleiotropic action, by acting also reducing glycemia in insulin resistance model mimicking diabetes mellitus type 2. These compounds may appear as a new approach in the treatment of obesity and complications, which are multifaceted.

Effect of replacing soybean meal by a blend of ground corn and urea-ammonium sulphate on milk production and composition, digestibility and N balance of dairy Murrah buffaloes.

This study evaluated the effect of replacing soybean meal by a blend of ground corn and urea-ammonium sulphate (GCU-S) in the diet of lactating buffaloes on milk production and composition, digestibility, N balance and blood metabolites. Twelve multiparous dairy Murrah buffaloes (Bubalus bubalis), at 100 ± 4 d in milk and yielding 10 ± 2.5 kg/d, were randomly distributed in a triple 4 × 4 Latin square, with four different inclusions of GCU-S at U-S levels: 0 (control), 8.0, 16.4 and 24.1 g/kg dry matter (DM) total. Replacing soybean meal with GC-US had a significant depressing effect on absolute DM intake, which was still numerically evident but no longer significant when expressed on a body weight-related basis. Intakes of crude protein (CP) and N as well as N-urinary excretion were also significantly depressed. Digestibility of dry matter was improved and, as a proportion of DMI, intake of total digestible nutrients increased significantly. Perhaps as a result, feed efficiency (kg DMI required per kg 6% fat-corrected milk) was significantly improved. Efficiency of protein use for milk production was significantly improved, but not when expressed as milk protein output. N transfer into milk, as a proportion of total N intake, also increased significantly. However, GC-US inclusion had no significant effect on milk production or milk composition, nor did it affect serum metabolites, digestibilities (apart from dry matter) or N measures of balance apart from those mentioned above. Inclusion of this blend of ground corn with urea and ammonium suplate can be recommended for dairy buffalo because, although it decreases DMI and N-excretion, it improves DM digestibility and feeding efficiency whilst maintaining milk production and composition.

Physicochemical composition, fatty acid profile and sensory attributes of meat (longissimus lumborum muscle) from Nellore and Nellore-cross bulls.

This study aimed to compare the physicochemical characteristics, fatty acid composition, and sensory attributes of the meat from three genetic groups: Nellore (Nell), ½ Nellore × ½ Angus (NeAn), and » Nellore × » Angus × ½ Senepol (NASe). Longissimus lumborum muscle from 30 slaughtered bulls with a body weight of 549 ± 32.5 kg was used. The water holding capacity was greater for the Nell and NeAn groups than for the NASe group. Meat samples from the NASe group exhibited a higher L* index than those from the NeAn group and lower a* and b* color indexes than those from the Nell group. The meat fatty acid profiles showed that the Nell group had higher concentrations of 12:0, 14:0, 18:1 t11, 14:1 c9, 16:1 c9, 18:1 c9, 18:1 c11, 18:2 c9, t11 (conjugated linoleic acid (CLA)), and 20:3 n-6 polyunsaturated fatty acid (PUFA) than the NeAn and NASe groups. The total saturated (ΣSFA), unsaturated (ΣUFA), and monounsaturated (ΣMUFA) fatty acid concentrations were higher and the ΣPUFA:ΣSFA ratio was lower in the Nell group than in the NeAn group. The Δ9-desaturase C16 activity was significantly higher in the Nell and NASe groups than in the NeAn group. The atherogenicity index (AI) tended to be lower in the crossbreeds than in the Nell breed. The NASe group presented meat with better tenderness, juiciness, and overall acceptance than the Nell and NeAn groups and was therefore the best genetic group for beef production of the tested groups.

Shedding Light on the Inhibitory Mechanisms of SARS-CoV-1/CoV-2 Spike Proteins by ACE2-Designed Peptides.

Angiotensin-converting enzyme 2 (ACE2) is the host cellular receptor that locks onto the surface spike protein of the 2002 SARS coronavirus (SARS-CoV-1) and of the novel, highly transmissible and deadly 2019 SARS-CoV-2, responsible for the COVID-19 pandemic. One strategy to avoid the virus infection is to design peptides by extracting the human ACE2 peptidase domain α1-helix, which would bind to the coronavirus surface protein, preventing the virus entry into the host cells. The natural α1-helix peptide has a stronger affinity to SARS-CoV-2 than to SARS-CoV-1. Another peptide was designed by joining α1 with the second portion of ACE2 that is far in the peptidase sequence yet grafted in the spike protein interface with ACE2. Previous studies have shown that, among several α1-based peptides, the hybrid peptidic scaffold is the one with the highest/strongest affinity for SARS-CoV-1, which is comparable to the full-length ACE2 affinity. In this work, binding and folding dynamics of the natural and designed ACE2-based peptides were simulated by the well-known coarse-grained structure-based model, with the computed thermodynamic quantities correlating with the experimental binding affinity data. Furthermore, theoretical kinetic analysis of native contact formation revealed the distinction between these processes in the presence of the different binding partners SARS-CoV-1 and SARS-CoV-2 spike domains. Additionally, our results indicate the existence of a two-state folding mechanism for the designed peptide en route to bind to the spike proteins, in contrast to a downhill mechanism for the natural α1-helix peptides. The presented low-cost simulation protocol demonstrated its efficiency in evaluating binding affinities and identifying the mechanisms involved in the neutralization of spike-ACE2 interaction by designed peptides. Finally, the protocol can be used as a computer-based screening of more potent designed peptides by experimentalists searching for new therapeutics against COVID-19.

Searching Anti-Zika Virus Activity in 1H-1,2,3-Triazole Based Compounds.

Zika virus (ZIKV) is a mosquito-borne virus belonging to the Flaviviridae family and is responsible for an exanthematous disease and severe neurological manifestations, such as microcephaly and Guillain-Barré syndrome. ZIKV has a single strand positive-sense RNA genome that is translated into structural and non-structural (NS) proteins. Although it has become endemic in most parts of the tropical world, Zika still does not have a specific treatment. Thus, in this work we evaluate the cytotoxicity and antiviral activities of 14 hybrid compounds formed by 1H-1,2,3-triazole, naphthoquinone and phthalimide groups. Most compounds showed low cytotoxicity to epithelial cells, specially the 3b compound. After screening with all compounds, 4b was the most active against ZIKV in the post-infection test, obtaining a 50% inhibition concentration (IC50) of 146.0 µM and SI of 2.3. There were no significant results for the pre-treatment test. According to the molecular docking compound, 4b was suggested with significant binding affinity for the NS5 RdRp protein target, which was further corroborated by molecular dynamic simulation studies.

Effect of Edible Onion (Allium cepa L.) Film on Quality, Sensory Properties and Shelf Life of Beef Burger Patties.

The production of edible film from onion (Allium cepa L.) to be applied as packaging is attractive, due to its chemical properties and biodegradable characteristics. Thus, we tested the hypothesis that edible onion film can positively influence the sensory properties, quality and increasing shelf life of beef burgers patties. The experiment was designed in a 4 × 2 factorial scheme, with two treatments (beef burgers patties with or without edible onion film) at an interval of four storage times (0, 3, 6 and 9 days) at 4 °C. The uncoated burger patties (control) suffered the most intense color modifications during the storage (p < 0.05). The luminosity index was higher (p < 0.05) in the control at all storage times, except at day 6, and redness, yellowness and chrome were higher (p < 0.05) in the edible onion film patties at all storage times. The pH of the beef burger patties was lower (p < 0.05) at all storage times when the edible onion film was applied. For the texture profile, only the chewiness was affected, as the inclusion of the edible onion film improved the chewing of the beef burgers patties over the storage time (p < 0.05). Additionally, there was an inhibition of the microbial growth of mesophiles and psychrophiles with the application of the edible onion film in beef burgers patties. The use of edible onion film improved the perception of panelists for the variables texture, color, flavor, odor and overall appearance, and increased the preference of panelists. The edible onion film is recommended for preserving beef burgers patties, as it delays the proliferation of unwanted microorganisms, stabilizes and improves the color parameters and sensory attributes, and increases the overall acceptance of the consumer.

Days in milk alters the milk fatty acid profile of grazing donkeys: A preliminary study.

The aim of this study was to characterize and determine how days in milk (DIM) affect the milk fatty acid (FA) profile of grazing donkeys. Donkey milk is very similar to human milk, containing bioactive molecules such as FA and proteins. However, there is a lack of scientific and technical information on the changes in the FA profile of asinine milk in pasture-feeding systems. Seven multiparous Pega donkeys, maintained in an exclusively extensive system, were used. Milking was undertaken twice a day, once a week for 16 weeks. Milk samples were composed according to the average lactating days as follows: 55, 110, 165, 220 and 275 DIM. A descriptive analysis of the milk fat composition was performed, and the linear and quadratic effects of DIM on the milk FA profile were tested. The milk FA profile of grazing donkeys is influenced by lactation days and is characterized by high concentrations of polyunsaturated FA (PUFA) with a low n-6 to n-3 ratio (0.66 g/100 g), mainly due to the high level of linolenic acid (16.8 g/100 g). Most of the individual FAs did not change during lactation, but stearic and oleic acid linearly decreased (p < .05), and total polyunsaturated FA (PUFA) and n-3 FA increased (p < .05) with DIM. The milk profile of grazing donkeys is influenced by the day of lactation and is characterized by a high concentration of n-3 PUFA (mainly α-linolenic acid) and a lower n-6 to n-3 ratio. As the number of days of lactation increased, the concentration of n-3 PUFA also increased. Therefore, higher milk quality may be associated with higher days in milk.

Effect of different blend levels of spineless cactus and Mombasa hay as roughage on intake, digestibility, ingestive behavior, and performance of lambs.

This study aimed to determine the best level of the blend of spineless cactus (Nopalea cochenillifera) and Mombasa (Panicum maximum) hay as roughage in the diet of lambs based on intake, digestibility, ingestive behavior, and lamb performance. Forty-eight uncastrated crossbred Santa Ines breed lambs with an average age of 4 months old and an average initial BW of 20.5 kg ± 2.8 kg were distributed in two experiments using a completely randomized design: experiment 1: 16 lambs (four treatments and four replicates) for in vivo digestibility trial in metabolic cage; experiment 2: 32 lambs (four treatments and eight replicates) in feedlot to determine intake, ingestive behavior, and performance. As the amount of cactus in the roughage increased, there was a linear reduction (P <0.001) on intake (g/day) of dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), non-fibrous carbohydrates (NFC) digestibility, time spent ruminating, feeding efficiency of NDF (P = 0.0153), NDF rumination efficiency rate (P = 0.032), final BW, TWG, and ADG of lamb. However, there was a linear increase (P <0.001) on the intake of the NFC and TDN, digestibility coefficient of DM, CP, total carbohydrates (TC) and NDF gross energy, metabolizable energy, and time spent idling when spineless cactus increased in the roughage blend. The least amount of cactus in the roughage (112-637 g/kg DM) promoted higher intake of DM and CP, improving lamb performance. However, the blend up to 450-300 g/kg DM improved digestibility and energy production as well as it did not affect the feed efficiency.

The Role of Electrostatics and Folding Kinetics on the Thermostability of Homologous Cold Shock Proteins.

Understanding which aspects contribute to the thermostability of proteins is a challenge that has persisted for decades, and it is of great relevance for protein engineering. Several types of interactions can influence the thermostability of a protein. Among them, the electrostatic interactions have been a target of particular attention. Aiming to explore how this type of interaction can affect protein thermostability, this paper investigated four homologous cold shock proteins from psychrophilic, mesophilic, thermophilic, and hyperthermophilic organisms using a set of theoretical methodologies. It is well-known that electrostatics as well as hydrophobicity are key-elements for the stabilization of these proteins. Therefore, both interactions were initially analyzed in the native structure of each protein. Electrostatic interactions present in the native structures were calculated with the Tanford-Kirkwood model with solvent accessibility, and the amount of hydrophobic surface area buried upon folding was estimated by measuring both folded and extended structures. On the basis of Energy Landscape Theory, the local frustration and the simplified alpha-carbon structure-based model were modeled with a Debye-Hückel potential to take into account the electrostatics and the effects of an implicit solvent. Thermodynamic data for the structure-based model simulations were collected and analyzed using the Weighted Histogram Analysis and Stochastic Diffusion methods. Kinetic quantities including folding times, transition path times, folding routes, and Φ values were also obtained. As a result, we found that the methods are able to qualitatively infer that electrostatic interactions play an important role on the stabilization of the most stable thermophilic cold shock proteins, showing agreement with the experimental data.

Antileishmanial activity of 4-phenyl-1-[2-(phthalimido-2-yl)ethyl]-1H-1,2,3-triazole (PT4) derivative on Leishmania amazonensis and Leishmania braziliensis: In silico ADMET, in vitro activity, docking and molecular dynamic simulations.

Organic compounds obtained by click chemistry reactions have demonstrated a broad spectrum of biological activities being widely applied for the development of molecules against pathogens of medical and veterinary importance. Cutaneous leishmaniasis (CL), caused by intracellular protozoa parasite of genus Leishmania, comprises a complex of clinical manifestations that affect the skin and mucous membranes. The available drugs for the treatment are toxic and costly, with long periods of treatment, and the emergence of resistant strains has been reported. In this study we investigated the in vitro effects of a phthalimide-1,2,3-triazole derivative, the 4-Phenyl-1-[2-(phthalimido-2-yl)ethyl]-1H-1,2,3-triazole (PT4) obtained by click chemistry, on mammalian cells and on L. amazonensis and L. braziliensis, the causative agents of CL in Brazil. In silico ADMET evaluation of PT4 showed that this molecule has good pharmacokinetic properties with no violation of Lipinski's rules. The in vitro assays showed that PT4 was more selective for both Leishmania species than to mammalian cells. This compound also presented low cytotoxicity to mammalian cells with CC50 > 500 µM. Treatment of promastigote forms with different concentrations of PT4 resulted in ultrastructural alterations, such as plasma membrane wrinkling, shortening of cell body, increased cell volume and cell rupture. The molecular dynamic simulations showed that PT4 interacts with Lanosterol 14 α-demethylase from Leishmania, an essential enzyme of lipid synthesis pathway in this parasite. Our results demonstrated PT4 was effective against both species of Leishmania. PT4 caused a decrease of mitochondrial membrane potential and increased production of reactive oxygen species, which may lead to parasite death. Taken together, our results pointed PT4 as promissing therapeutic agent against CL.

Fatty acid, chemical, and tissue composition of meat comparing Santa Inês breed sheep and Boer crossbreed goats submitted to different supplementation strategies.

To compare the fatty acid (FA) composition, and chemical and tissue composition of meat, 24 uncastrated males, 12 sheep of the Santa Inês breed, and 12 crossbred goats (F1 Boer × undefined breed) with the weight of 24.3 ± 2.38 kg. The animals were distributed in a randomized block design and treatments were arranged in a 2 × 2 factorial design, two small ruminant species/breed (Boer crossbreed goat × Santa Inês breed sheep), and two supplementation strategies (energy × protein energy). There was not an interaction between the small ruminant species and the supplementation on average daily gain, tissue composition, chemical composition, or FA profile of the longissimus lumborum muscle. Sheep presented greater weight and yield of fat in the subcutaneous, intermuscular, and total depots than goats, which results in better finishing of the sheep for slaughter. Goat meat presented a lower lipid concentration than sheep meat. The protein-energy supplementation increased oleic acid, ΣUFA, ΣMUFA, hypocholesterolemic/ Hypercholesterolemic index, and enzymatic activity Δ9-desaturase C18 and decreased SFA capric acid in the muscle when compared with energetic supplementation. In Caatinga biome conditions, Boer crossbreed goats meat has a lower concentration of lipids and a healthier FA composition compared with Santa Inês breed sheep because it has a lower SFA and greater PUFA content, which are sources of n-3 and n-6, which may contribute to the reduction of blood cholesterol (LDL). In addition, protein-energy supplementation also improved the quality of animal fat compared with supplementation only with the energetic concentrate, regardless of species.

A Dynamic Hydrophobic Core and Surface Salt Bridges Thermostabilize a Designed Three-Helix Bundle.

Thermostable proteins are advantageous in industrial applications, as pharmaceuticals or biosensors, and as templates for directed evolution. As protein-design methodologies improve, bioengineers are able to design proteins to perform a desired function. Although many rationally designed proteins end up being thermostable, how to intentionally design de novo, thermostable proteins is less clear. UVF is a de novo-designed protein based on the backbone structure of the Engrailed homeodomain (EnHD) and is highly thermostable (Tm > 99°C vs. 52°C for EnHD). Although most proteins generally have polar amino acids on their surfaces and hydrophobic amino acids buried in their cores, protein engineers followed this rule exactly when designing UVF. To investigate the contributions of the fully hydrophobic core versus the fully polar surface to UVF's thermostability, we built two hybrid, chimeric proteins combining the sets of buried and surface residues from UVF and EnHD. Here, we determined a structural, dynamic, and thermodynamic explanation for UVF's thermostability by performing 4 µs of all-atom, explicit-solvent molecular dynamics simulations at 25 and 100°C, Tanford-Kirkwood solvent accessibility Monte Carlo electrostatic calculations, and a thermodynamic analysis of 40 temperature runs by the weighted-histogram analysis method of heavy-atom, structure-based models of UVF, EnHD, and both chimeric proteins. Our models showed that UVF was highly dynamic because of its fully hydrophobic core, leading to a smaller loss of entropy upon folding. The charged residues on its surface made favorable electrostatic interactions that contributed enthalpically to its thermostability. In the chimeric proteins, both the hydrophobic core and charged surface independently imparted thermostability.

Importance of the ß5-ß6 Loop for the Structure, Catalytic Efficiency, and Stability of Carbapenem-Hydrolyzing Class D ß-Lactamase Subfamily OXA-143.

The class D ß-lactamase OXA-143 has been described as an efficient penicillinase, oxacillinase, and carbapenemase. The D224A variant, known as OXA-231, was described in 2012 as exhibiting less activity toward imipenem and increased oxacillinase activity. Additionally, the P227S mutation was reported as a case of convergent evolution for homologous enzymes. To investigate the impact of both mutations (D224A and P227S), we describe in this paper a deep investigation of the enzymatic activities of these three homologues. OXA-143(P227S) presented enhanced catalytic activity against ampicillin, oxacillins, aztreonam, and carbapenems. In addition, OXA-143(P227S) was the only member capable of hydrolyzing ceftazidime. These enhanced activities were due to a combination of a higher affinity (lower Km) and a higher turnover number (higher kcat). We also determined the crystal structure of apo OXA-231. As expected, the structure of this variant is very similar to the published OXA-143 structure, except for the two M223 conformations and the absence of electron density for three solvent-exposed loop segments. Molecular dynamics calculations showed that both mutants experience higher flexibility compared to that of the wild-type form. Therefore, our results illustrate that D224A and P227S act as deleterious and positive mutations, respectively, within the evolutionary path of the OXA-143 subfamily toward a more efficient carbapenemase.

Drift-diffusion (DrDiff) framework determines kinetics and thermodynamics of two-state folding trajectory and tunes diffusion models.

The stochastic drift-diffusion (DrDiff) theory is an approach used to characterize the dynamical properties of simulation data. With new features in transition times analyses, the framework characterized the thermodynamic free-energy profile [F(Q)], the folding time (τf), and transition path time (τTP) by determining the coordinate-dependent drift-velocity [v(Q)] and diffusion [D(Q)] coefficients from trajectory time traces. In order to explore the DrDiff approach and to tune it with two other methods (Bayesian analysis and fep1D algorithm), a numerical integration of the Langevin equation with known D(Q) and F(Q) was performed and the inputted coefficients were recovered with success by the diffusion models. DrDiff was also applied to investigate the prion protein (PrP) kinetics and thermodynamics by analyzing folding/unfolding simulations. The protein structure-based model, the well-known Go¯-model, was employed in a coarse-grained Cα level to generate long constant-temperature time series. PrP was chosen due to recent experimental single-molecule studies in D and τTP that stressed the importance and the difficulty of probing these quantities and the rare transition state events related to prion misfolding and aggregation. The PrP thermodynamic double-well F(Q) profile, the "X" shape of τf(T), and the linear shape of τTP(T) were predicted with v(Q) and D(Q) obtained by the DrDiff algorithm. With the advance of single-molecule techniques, the DrDiff framework might be a useful ally for determining kinetic and thermodynamic properties by analyzing time observables of biomolecular systems. The code is freely available at https://github.com/ronaldolab/DrDiff.