Biosensor capability of the endometrium is mediated in part, by altered miRNA cargo from conceptus-derived extracellular vesicles.

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin, in vivo (high developmental potential (IV)), in vitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus-conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria. There were substantial differences in the endometrial response to in vivo versus in vitro and in vivo versus cloned conceptuses (1153 and 334DEGs respectively) with limited differences between in vitro Vs cloned conceptuses (36 DEGs). The miRNA cargo contained in conceptus-derived EVs was similar between all three groups (426 miRNA in common). Only 8 miRNAs were different between in vivo and cloned conceptuses, while only 6 miRNAs were different between in vivo and in vitro-derived conceptuses. Treatment of endometrial epithelial cells with mimic or inhibitors for miR-128 and miR-1298 changed the proteomic content of target cells (96 and 85, respectively) of which mRNAs are altered in the endometrium in vivo (PLXDC2, COPG1, HSPA12A, MCM5, TBL1XR1, and TTF). In conclusion, we have determined that the biosensor capability of the endometrium is mediated in part, by its response to different EVs miRNA cargo produced by the conceptus during the peri-implantation period of pregnancy.

Refining details of the structural and electronic properties of the CuB site in pMMO enzyme through sequential molecular dynamics/CPKS-EPR calculations.

This work investigated the structural and electronic properties of the copper mononuclear site of the PmoB part of the pMMO enzyme at the molecular level. We propose that the CuB catalytic site in the soluble portion of pMMO at room temperature and under physiological conditions is a mononuclear copper complex in a distorted octahedral arrangement with the residues His33, His137, and His139 on the equatorial base and two water molecules on the axial axis. Our view was based on the molecular dynamics results and DFT calculations of the electronic paramagnetic resonance parameters and comparisons with experimental EPR data. This new proposed model for the CuB site brings additional support concerning the recent experimental evidence, which pointed out that a saturated coordination sphere of the copper ion in the CuB center is an essential factor that makes it less efficient than the CuC site in the methane oxidation. Therefore, according to the CuB site model proposed here, an additional step involving a displacement of at least one water molecule of the copper coordination sphere by the O2 molecule prior to its activation must be necessary. This scenario is less likely to occur in the CuC center once this one is buried in the alpha-helices, which are part of the pMMO structure bound to the membrane wall, and consequently located in a less solvent-exposed region. In addition, we also present a simple and efficient sequential S-MD/CPKS protocol to compute EPR parameters that can, in principle, be expanded for the study of other copper-containing proteins.

Aerobic exercise with blood flow restriction: Energy expenditure, excess postexercise oxygen consumption, and respiratory exchange ratio.

We compared the effects of aerobic exercise with and without blood flow restriction (BFR) to high-intensity aerobic exercise on energy expenditure (EE), excess Postexercise oxygen consumption (EPOC), and respiratory exchange ratio (RER) during and after exercise. Twenty-two recreationally active males randomly completed the following experimental conditions: AE-aerobic exercise without BFR, AE + BFR-aerobic exercise with BFR, HIAE-high-intensity aerobic exercise, CON-non-exercise control condition. EE was significantly (p < 0.05) greater during exercise for HIAE compared to all conditions, and for AE + BFR compared to AE and CON during and postexercise exercise. There were no significant (p > 0.05) differences in EPOC between HIAE and AE + BFR at any time point, however, both conditions were significantly (p < 0.05) greater than the AE (d = 1.50 and d = 1.03, respectively) and CON at the first 10 min postexercise. RER during exercise for HIAE was significantly (p < 0.05) greater than AE + BFR at the first 6 min of exercise (p = 0.003, d = 0.88), however, no significant differences were observed from 9 min up to the end of the exercise. HIAE was also significantly (p < 0.05) greater than AE and CON at all time points during exercise, whereas, AE + BFR was significantly (p < 0.05) greater than CON at all time points but not significantly (p < 0.05) different than AE (p < 0.05); although the overall session RER was significantly (p < 0.05) greater during AE + BFR than AE. Altogether, continuous AE + BFR results in greater EE compared to volume matched AE, as well as a similar EPOC compared to HIAE.

Effect of extreme mechanical densification on the electrical properties of carbon nanotube micro-yarns.

We have explored the effect of high pressure post-treatment in optimizing the properties of carbon nanotube yarns and found that the application of dry hydrostatic pressure reduces porosity and enhances electrical properties. The CNT yarns were prepared by the dry-spinning method directly from CNT arrays made by the hot filament chemical vapour deposition (HF-CVD) process. Mechanical hydrostatic pressure up to 360 MPa induces a decrease in yarn resistivity between 3% and 35%, associated with the sample's permanent densification, with CNT yarn diameter reduction of 10%-25%. However, when increasing the pressure in the 1-3 GPa domain in non-hydrostatic conditions, the recovered samples show lower electrical conductivity. This might be due to concomitant macroscopic effects such as increased twists and damage to the yarn shown by SEM imaging (caused by strong shear stresses and friction) or by the collapse of the CNTs indicated byin situhigh pressure Raman spectroscopy data.

A "turn-off" fluorescent sensor based on electrospun polycaprolactone nanofibers and fluorene(bisthiophene) derivative for nitroaromatic explosive detection.

The preparation of fluorene(bisthiophene)-based fluorescent nanofibers for nitroaromatic explosive detection provides a convenient rapid and low-cost strategy aiming at forensic applications. Polycaprolactone (PCL) and fluorene(bisthiophene) derivative (FBT) nanofibers were obtained by electrospinning technique as a free-standing mat and characterized by SEM, FTIR, thermal analysis and fluorescence spectroscopy. The PCL/FBT nanofibers presented high sensitivity towards 2,4,6-trinitrotoluene (TNT) and picric acid (PA), with fluorescence quenching (turn-off mechanism), and selectivity to another kind of explosives. The free-standing mats were used as a cloth strip that was swiped on surfaces contaminated with TNT traces allowing its visual detection under UV light source. These findings are particularly important for the development of a facile and promising strategy to assembly portable optical devices for nitroaromatic explosive detection.

Small extracellular vesicles derived from in vivo- or in vitro-produced bovine blastocysts have different miRNAs profiles-Implications for embryo-maternal recognition.

In vivo- and in vitro-produced bovine embryos have different metabolic profiles and differences in gene transcription patterns. These embryos also have a distinct ability to establish and sustain early pregnancies. Small extracellular vesicles (sEVs) are secreted by embryos and carry bioactive molecules, such as miRNAs. We hypothesize that in vivo or in vitro-produced bovine hatched blastocysts on Day 9 and the sEVs secreted by them have different miRNA profiles. To address this hypothesis, embryos of both groups were placed in in vitro culture on Day 7. After 48 h, hatched embryos and hatched embryo-conditioned media (eCM) of both groups were collected. A total of 210 miRNAs were detected in embryos of both groups, of these 6 miRNAs were downregulated, while 7 miRNAs were upregulated in vitro group when compared to in vivo group. sEVs were isolated from eCM to determine miRNA profile. A total of 106 miRNAs were detected in both groups, including 14 miRNAs upregulated in sEVs from in vivo-eCM, and 2 miRNAs upregulated in sEVs from in vitro-eCM. These miRNAs express in embryos and sEVs secreted by them regulate early embryonic developmental and endometrial pathways, which can modify embryo-maternal communication during early pregnancy and consequently affect pregnancy establishment.

Effects of supplemental fat concentration on feeding logistics, animal performance, and nutrient losses of heifers fed finishing diets based on steam-flaked corn and sorghum-based distiller's grains.

The use of distiller's grains (DG) in beef cattle finishing diets is a common practice. However, the effects of supplemental fat on performance and nutrient losses of cattle fed diets containing DG are not known. Therefore, we fed 398 crossbred yearling heifers (initial BW = 373.5 kg) for 106 d to determine the effects of dietary fat concentration and sorghum-based wet distiller's grains with solubles (SWDGS) on performance, carcass characteristics, and nutrient losses of finishing cattle. Treatments included two 92% concentrate, steam-flaked corn (SFC)-based diets with 0% or 3% added fat from yellow grease and 3 SFC-based diets with 15% SWDGS (DM basis) that contained either 0%, 1.5%, or 3% added fat (8 pens per treatment) in a randomized block design. Overall DMI and ADG were 5% to 6% greater (P < 0.01) for heifers fed 15% SWDGS than for those fed 0% SWDGS. Among heifers fed 15% SWDGS, DMI was greatest (P = 0.04; quadratic effect) and ADG tended (P = 0.12; quadratic effect) to be greatest for heifers fed 1.5% fat. The ADG:DMI did not differ between 0% SWDGS with 0% or 3% fat, and was not altered by replacing a portion of SFC with SWDGS (P > 0.36). However, ADG:DMI tended to increase as more fat was added to diets with 15% SWDGS (P = 0.06). Average hot carcass weight (HCW) was 5 kg greater (P = 0.05) when SWDGS was fed, but HCW tended to be greatest for heifers fed 15% SWDGS with 1.5% fat (P = 0.09, quadratic effect). Heifers fed 0% SWDGS with 0% fat tended to have a lower marbling score, less rib fat, lower average yield grade (P < 0.08), and more (P < 0.01) yield grade 1 carcasses than heifers fed 0% SWDGS with 3% fat. Averaged across fat levels, heifers fed 15% SWDGS had more rib fat and a higher yield grade (P < 0.03) than heifers fed 0% SWDGS. Feeding 15% SWDGS did not alter carcass quality grade distribution compared to feeding 0% SWDGS, but 15% SWDGS produced fewer yield grade 3 carcasses (P = 0.03) than 0% SWDGS. The calculated NEg of SWDGS (1.36 Mcal/kg) was 91% of the tabular value for dry rolled corn (1.50 Mcal/kg) and 84% of the tabular value for SFC (1.62 Mcal/kg). Nitrogen intake, and N excretion were greater (P < 0.05) in heifers fed 15% SWDGS than in heifers fed the 0% SWDGS diets, but N loss as a % of N intake was less (P < 0.05). Our results suggest adding 1.5% fat to diets containing 15% SWDGS may improve beef cattle performance; however, feeding logistics need to be considered when pricing wet DG.

Effects of wet corn distiller's grains with solubles and nonprotein nitrogen on feeding efficiency, growth performance, carcass characteristics, and nutrient losses of yearling steers12.

Wet distiller's grains with solubles (WDGS) are a common by-product feedstuff generated by the grain-ethanol industry, and it is used extensively by the cattle feeding industry. Distillers grains are typically high in protein; however, the protein in WDGS has a low ruminal degradability, and thus may result in a deficiency of RDP in the diet even when dietary CP concentrations are high. Assessment of the RDP needs in diets containing WDGS is needed to aid the cattle feeding industry in managing feed costs and potential environmental issues. To that end, we conducted 2 feeding studies to evaluate the supplemental RDP requirements of beef cattle fed steam-flaked corn-based finishing diets. In Exp. 1, 525 yearling steers (initial body weight = 373 ± 13 kg) received treatments in a 2 × 3 + 1 factorial. Dietary factors included WDGS (15 or 30% of DM) and nonprotein N (NPN; 0, 1.5, or 3.0% of DM) from urea (0, 0.52, and 1.06%). The control diet without WDGS contained 3.0% NPN (1.06% urea) and cottonseed meal. Diets were formulated to have equal crude fat concentrations. Overall gain efficiency among steers fed 15% WDGS was greatest for 1.5% NPN and least for 0% NPN (P = 0.07, quadratic), whereas gain efficiency decreased linearly (P < 0.09) as NPN increased in the 30% WDGS diets. Dressing percent was greater (P < 0.01) for the Control diet than for 15 or 30% WDGS. In Exp. 2, 296 steer calves (initial BW = 344 ± 12 kg) were fed 1 of 4 experimental diets that included a Control diet without WDGS (contained 3% NPN from urea, and cottonseed meal) and 15% WDGS diets with either 1.50, 2.25, or 3.00% NPN (0.52, 0.78, and 1.04% urea, respectively, on a DM basis). Overall gain efficiency on either a live or carcass-adjusted basis was not different among treatments (P > 0.15). Dietary NPN concentration did not influence growth performance (P > 0.21). Increasing dietary WDGS concentration resulted in decreasing (P < 0.05) diet digestibility (determined with an internal marker) and increasing (P < 0.05) N volatilization losses (determined by diet and manure N:P ratio); however, the effects of NPN level on digestibility and N losses were somewhat inconsistent across experiments. Results suggest that optimum performance for cattle fed 15% WDGS occurred when the diet contained between 1.5 and 2.25% NPN. However, no supplemental NPN was needed to support optimum performance in diets containing 30% WDGS.

Impact of the Molecular Environment on Thiol-Ene Coupling For Biofunctionalization and Conjugation.

Thiol-ene radical coupling is increasingly used for the biofunctionalization of biomaterials and the formation of 3D hydrogels enabling cell encapsulation. Indeed, thiol-ene chemistry presents interesting features that are particularly attractive for platforms requiring specific reactions of peptides or proteins, in particular, in situ, during cell culture or encapsulation. Despite such interest, little is known about the factors impacting thiol-ene chemistry in situ, under biologically relevant conditions. Here we explore some of the molecular parameters controlling photoinitiated thiol-ene couplings with a series of alkenes and thiols, including peptides, in buffered conditions. (1)H NMR and HPLC were used to quantify the efficiency of couplings and the impact of the pH of the buffer, as well as the molecular structure and local microenvironment close to alkenes and thiols to be coupled. Some of these observations are supported by molecular dynamics and quantum mechanics calculations. An important finding of our work is that the pKa of thiols (and its variation upon changes in molecular structure) have a striking impact on coupling efficiencies. Similarly, positively charged and aromatic amino acids are found to have some impact on thiol-ene couplings. Hence, our study demonstrates that molecular design should be carefully selected in order to achieve high biofunctionalization levels in biomaterials with peptides or promote the efficient formation of peptide-based hydrogels.

Phosphorane lifetime and stereo-electronic effects along the alkaline hydrolysis of phosphate esters.

Hybrid quantum mechanical/effective fragment potential (QM/EFP) calculations, in conjunction with the quantum theory of atoms in molecules (QTAIM) and energy decomposition analysis (EDA), were employed to investigate the reaction mechanism and stereo-electronic effects along the alkaline hydrolysis of the monoethyl phosphate dianion (MEP) and the diethylphosphate monoanion (DEP). Reactions proceed through a synchronous bimolecular ANDN mechanism for MEP and a stepwise (AN + DN) mechanism for DEP, with the formation of a phosphorane intermediate, having an overall reaction free energy and barrier of 11.5 and 43.0 kcal mol(-1), respectively. In addition, ab initio molecular dynamics simulations were performed to investigate the stability of the phosphorane pentacoordinate intermediate observed in the reaction of the phosphate diester. The phosphorane intermediate has a lifetime of ∼1 ps after which it decomposes into the corresponding alcohol and phosphate monoester dianion. Electrostatics governs the interaction between the nucleophile and the phosphate ester. However, some degree of covalence in the interaction starts to appear at distances shorter than 2.45 Šfor MEP and 2.63 Šfor DEP. For the monoester, the electrostatic repulsive terms are the dominant contributions for the formation of the transition state. On the other hand, for the phosphate diester, the formation of the P-OH bond is dominated by associative terms of electrostatic nature.

Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase.

The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a water molecule. A computational analysis of this pathway in the [FeFe]-hydrogenase from Clostridium pasteurianum revealed that the solvent-exposed residue of the pathway (Glu282) forms hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implying that these residues could function in regulating proton transfer. In this study, we show that substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in an ∼3-fold enhancement of H2 production activity when methyl viologen is used as an electron donor, suggesting that Arg286 may help control the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate ∼5-fold, implying that it either acts as a member of the pathway or influences Glu282 to permit proton transfer. Interestingly, quantum mechanics/molecular mechanics and molecular dynamics calculations indicate that Ser320 does not play a structural role or indirectly influence the barrier for proton movement at the entrance of the channel. Rather, it may act as an additional proton acceptor for the pathway or serve in a regulatory role. While further studies are needed to elucidate the role of Ser320, collectively these data provide insights into the complex proton transport process.

A radical rebound mechanism for the methane oxidation reaction promoted by the dicopper center of a pMMO enzyme: a computational perspective.

In this article, we investigated the hydroxylation of methane catalyzed by the binuclear copper site of a pMMO enzyme, through a radical rebound mechanism. All intermediates and transition states along the reaction coordinate were located and the energies involved in the mechanism calculated using the B3LYP functional including dispersion effects. Our B3LYP-D2 results show that the singlet state of the (µ-1,2-peroxo)Cu(II)2 complex plays an important role as the lowest energy species prior to C-H bond activation. A crossing between the singlet and triplet PES is suggested to occur before the cleavage of the C-H bond of methane, where the triplet (bis-µ-oxo)Cu(III)2 is very reactive towards activation of the strong C-H bond of methane. The C-H bond activation is the rate-determining step of the reaction, with an activation energy of 18.6 kcal mol(-1) relative to the singlet (µ-1,2-peroxo)Cu(II)2 species. Comparison with previous theoretical results for a non-synchronous concerted mechanism suggests the radical rebound mechanism as a possible alternative pathway.

A combined experimental and computational study of novel nanocage-based metal-organic frameworks for drug delivery.

Three new metal organic frameworks (MOFs) with chemical formulae [(CH3)2NH2] [Sm3(L1)2(HCOO)2(DMF)2(H2O)]·2DMF·18H2O (1), [Cu2(L2)(H2O)2]·2.22DMA (2) and [Zn2(L1)(DMA)]·1.75DMA were synthesized and structurally characterized. 1 and 2 show a classical NbO-like topology and have two types of interconnected cages. 3 exhibits an uncommon zzz topology and has two types of interconnected cages. These MOFs can adsorb large amounts of the drug 5-fluorouracil (5-FU) and release it in a progressive way. 5-FU was incorporated into desolvated 1, 2 and 3 with loadings of 0.40, 0.42, and 0.45 g g(-1), respectively. The drug release rates were 72%, 96% and 79% of the drug after 96 hours in 1, 120 hours in 2 and 96 hours in 3, respectively. Grand Canonical Monte Carlo (GCMC) simulations were performed to investigate the molecular interactions during 5-FU adsorption to the three novel materials. The GCMC simulations reproduced the experimental trend with respect to the drug loading capacity of each material. They also provided a structural description of drug packing within the frameworks, helping to explain the load capacity and controlled release characteristics of the materials. 5-FU binding preferences to 1, 2 and 3 reflect the diversity in pore types, chemistry and sizes. The calculated drug load is more related to the molecular properties of accessible volume Vacc than to the pore size.

Assessment of the 3 D Pore Structure and Individual Components of Preshaped Catalyst Bodies by X-Ray Imaging.

Porosity in catalyst particles is essential because it enables reactants to reach the active sites and it enables products to leave the catalyst. The engineering of composite-particle catalysts through the tuning of pore-size distribution and connectivity is hampered by the inability to visualize structure and porosity at critical-length scales. Herein, it is shown that the combination of phase-contrast X-ray microtomography and high-resolution ptychographic X-ray tomography allows the visualization and characterization of the interparticle pores at micro- and nanometer-length scales. Furthermore, individual components in preshaped catalyst bodies used in fluid catalytic cracking, one of the most used catalysts, could be visualized and identified. The distribution of pore sizes, as well as enclosed pores, which cannot be probed by traditional methods, such as nitrogen physisorption and isotherm analysis, were determined.

Mass density and water content of saturated never-dried calcium silicate hydrates.

Calcium silicate hydrates (C-S-H) are the most abundant hydration products in ordinary Portland cement paste. Yet, despite the critical role they play in determining mechanical and transport properties, there is still a debate about their density and exact composition. Here, the site-specific mass density and composition of C-S-H in hydrated cement paste are determined with nanoscale resolution in a nondestructive approach. We used ptychographic X-ray computed tomography in order to determine spatially resolved mass density and water content of the C-S-H within the microstructure of the cement paste. Our findings indicate that the C-S-H at the border of hydrated alite particles possibly have a higher density than the apparent inner-product C-S-H, which is contrary to the common expectations from previous works on hydrated cement paste.

C-H bond activation of methane in aqueous solution: a hybrid quantum mechanical/effective fragment potential study.

In this study, we investigated the C-H bond activation of methane catalyzed by the complex [PtCl(4)](2-), using the hybrid quantum mechanical/effective fragment potential (EFP) approach. We analyzed the structures, energetic properties, and reaction mechanism involved in the elementary steps that compose the catalytic cycle of the Shilov reaction. Our B3LYP/SBKJC/cc-pVDZ/EFP results show that the methane activation may proceed through two pathways: (i) electrophilic addition or (ii) direct oxidative addition of the C-H bond of the alkane. The electrophilic addition pathway proceeds in two steps with formation of a σ-methane complex, with a Gibbs free energy barrier of 24.6 kcal mol(-1), followed by the cleavage of the C-H bond, with an energy barrier of 4.3 kcal mol(-1) . The activation Gibbs free energy, calculated for the methane uptake step was 24.6 kcal mol(-1), which is in good agreement with experimental value of 23.1 kcal mol(-1) obtained for a related system. The results shows that the activation of the C-H bond promoted by the [PtCl(4)](2-) catalyst in aqueous solution occurs through a direct oxidative addition of the C-H bond, in a single step, with an activation free energy of 25.2 kcal mol(-1), as the electrophilic addition pathway leads to the formation of a σ-methane intermediate that rapidly undergoes decomposition. The inclusion of long-range solvent effects with polarizable continuum model does not change the activation energies computed at the B3LYP/SBKJC/cc-pVDZ/EFP level of theory significantly, indicating that the large EFP water cluster used, obtained from Monte Carlo simulations and analysis of the center-of-mass radial pair distribution function, captures the most important solvent effects.

DFT study of the full catalytic cycle for the propene hydroformylation catalyzed by a heterobimetallic HPt(SnCl3)(PH3)2 model catalyst.

DFT calculations were carried out to study the full catalytic cycle for the hydroformylation of propene, catalyzed by the heterobimetallic model catalyst trans-Pt(H)(PH(3))(2)(SnCl(3)). Before the study of the full catalytic cycle, the performance of six pure GGA, one GGA with inclusion of dispersion corrections, four hybrid-GGA, and three meta-GGA exchange correlation functional to describe a model reaction promoted by Pt-Sn catalyst were assessed. It is shown that the BP86 and GPW91 functionals, using extended basis set, provides reliable energetic results when compared with the CCSD(T) calculations. All intermediates and transition states along the elementary steps of the entire catalytic cycle were located and the energies involved in the catalytic cycle calculated using BP86 functional. The solvent effects along the entire catalytic cycle were evaluated using the polarizable continuum model. In contrast with the rhodium catalysts, the regioselectivity of the hydroformylation is set at the carbonylation step. The hydrogenolysis is the rate determining step of the entire cycle, with the activation energy of approximately 21 kcal mol(-1) in agreement with the experimental value of approximately 25 kcal mol(-1). The trans effect of the SnCl(3)(-) ligand seems to be pronounced only in the first step of the catalytic cycle, facilitating the insertion of the olefin into the Pt-H bond trans to it. The analysis of the stationary points obtained along each elementary step of the catalytic cycle is carried out separately and discussed. The BP86/cc-pVTZ/SBKJC results shows that the pathway leading to the linear aldehyde is preferred, being in agreement with the experimental findings.