Stem cell function is regulated by intrinsic as well as microenvironmental factors, including chemical and mechanical signals. Conducting polymer-based cell culture substrates provide a powerful tool ...to control both chemical and physical stimuli sensed by stem cells. Here we show that polypyrrole (PPy), a commonly used conducting polymer, can be tailored to modulate survival and maintenance of rat fetal neural stem cells (NSCs). NSCs cultured on PPy substrates containing different counter ions, dodecylbenzenesulfonate (DBS), tosylate (TsO), perchlorate (ClO(4)) and chloride (Cl), showed a distinct correlation between PPy counter ion and cell viability. Specifically, NSC viability was high on PPy(DBS) but low on PPy containing TsO, ClO(4) and Cl. On PPy(DBS), NSC proliferation and differentiation was comparable to standard NSC culture on tissue culture polystyrene. Electrical reduction of PPy(DBS) created a switch for neural stem cell viability, with widespread cell death upon polymer reduction. Coating the PPy(DBS) films with a gel layer composed of a basement membrane matrix efficiently prevented loss of cell viability upon polymer reduction. Here we have defined conditions for the biocompatibility of PPy substrates with NSC culture, critical for the development of devices based on conducting polymers interfacing with NSCs.
•Intake and rumen capacity play an important role in the reticulorumen kp of growing goats.•Intake level appears to summarize the combined effect of intake and rumen capacity on the kp in goats.•Diet ...composition is more relevant to reticulorumen kp of particles than on solutes.•Reticulorumen kp of particles seems to be dependent on kp of solutes.•Reticulorumen kp of solutes can be predicted using only DM intake level (g/kg BW).
The reticulorumen (RR) fractional passage rate (kp; /h) of particles and solutes plays an important role in fiber digestion, methane production, and microbial yield. However, none of the available models for predicting RR kp consider individuals' characteristics of growing goats. The objective was to develop empirical models for predicting the RR kp of particles and solutes in growing goats. Our database involved 175 individual records of castrated males (n = 61), females (n = 57), and intact males (n = 57) growing Saanen goats fed ad libitum, 75% or 50% of ad libitum. Goats were slaughtered around 15, 22, 30, 37, or 45 kg BW. We used Akaike’s information criterion to select the best prediction models. We evaluated the predictive ability of these models using Lin’s concordance correlation coefficient (CCC) and RMSE of prediction (RMSPE) in a 4-fold cross-evaluation. The DM intake (DMI; kg/day), potentially digestible NDF intake (pdNDFI) level (g/kg BW), and RR wet pool size (kg) demonstrated similar importance in predicting RR kp of solutes (CCC = 0.59; RMSPE = 0.050 /h or 34.43%). However, when RR wet pool size was not included in the model, RR kp of solutes could still be precisely and accurately predicted using only DMI level (g/kg BW) (CCC = 0.47; RMSPE = 0.053 /h or 36.58%). The RR wet tissues and wet pool size (kg), NDF intake (NDFI) (kg/day), and indigestible NDFI (iNDFI):NDFI ratio were important predictors of RR kp of particles (CCC = 0.51; RMSPE = 0.0064 /h or 25.43 %). However, when RR wet tissues and wet pool size were not included in the model, iNDFI:NDFI ratio, NDFI level (g/kg BW), and RR kp of solutes presented greater importance in predicting RR kp of particles (CCC = 0.20; RMSPE = 0.0074 /h or 29.55%). Sex was not a significant predictor variable for the selected models. In summary, the RR kp of solutes was more dependent on feed intake level while the RR kp of particles was more dependent on diet composition and RR kp of solutes. Our models were precise and accurate for predicting RR kp of solutes (CCC = 0.57 and 0.47; RMSPE = 0.051 and 0.054 /h) and particles (CCC = 0.48 and 0.17; RMSPE = 0.0066 and 0.0076 /h) after cross-evaluation. This suggests that our models can be integrated into feeding systems with mechanistic approaches that simulate other reticulorumen functions, such as digestion, microbial growth, and methane emission.
Essential AA (EAA), particularly leucine, isoleucine, methionine, and histidine, possess signaling properties for promoting cellular anabolic metabolism, whereas methionine, lysine, and histidine are ...considered also to be substrate limiting AA. The objective of this study was to evaluate production responses to supplementation of 2 AA groups in a 2 × 2 factorial design. Eight cows (99 ± 18 days in milk) were assigned to 4 jugular infusion treatments consisting of saline (CON), methionine plus lysine plus histidine (MKH), isoleucine plus leucine (IL), or MKH plus IL, in a replicated 4 × 4 Latin square design. Periods were 18 d in length, comprising 8 d of rest followed by 10 d of jugular infusion. Daily infusion amounts were 21 g of methionine, 38 g of lysine, 20 g of histidine, 50 g of leucine, and 22 g of isoleucine. Cows were ad libitum fed a common diet consisting of 15.2% crude protein and 1.61 Mcal/kg NEL on a dry matter basis that was predicted to meet rumen degradable protein requirements but was 15% deficient in metabolizable protein. Milk and energy-corrected milk yields increased by 2.3 kg/d and 1.9 kg/d, respectively, with infused IL, and no change was observed for MKH. Milk protein concentration increased by 0.13 percentage units for MKH, whereas milk protein yield increased for both MKH and IL by 84 g/d and 64 g/d, respectively. The milk protein yield increase for MKH+IL was 145 g/d versus CON. Gross feed efficiency tended to increase with IL infusion, and N efficiency tended to increase with MKH infusion. Aggregate arterial EAA concentrations less Met, Lys, and His declined by 7.2% in response to MKH infusion. Arterial EAA less Ile and Leu also declined by 6.2% in response to IL infusion. Net total AA (TAA) and EAA uptake by the udder tended to increase in response to MKH infusion, whereas mammary blood flow increased in response to IL infusion, but TAA and EAA net uptakes were unaffected. Apparent udder affinity increased for TAA and EAA less Met, Lys, and His in response to MKH infusion, whereas affinity for EAA less Ile and Leu increased for IL infusion. Venous Met and Leu concentrations increased by 192% and 35% from the MKH and IL infusions, respectively, compared with CON, which indicates that intracellular concentration of these EAA changed substantially. Increases in milk protein yield were observed from 2 groups of amino acids independently and additively, which contradicts the single limiting amino acid theory that a single EAA will limit milk protein yield.
The use of DNA as a nanoscale construction material has been a rapidly developing field since the 1980s, in particular since the introduction of scaffolded DNA origami in 2006. Although software is ...available for DNA origami design, the user is generally limited to architectures where finding the scaffold path through the object is trivial. Herein, we demonstrate the automated conversion of arbitrary two‐dimensional sheets in the form of digital meshes into scaffolded DNA nanostructures. We investigate the properties of DNA meshes based on three different internal frameworks in standard folding buffer and physiological salt buffers. We then employ the triangulated internal framework and produce four 2D structures with complex outlines and internal features. We demonstrate that this highly automated technique is capable of producing complex DNA nanostructures that fold with high yield to their programmed configurations, covering around 70 % more surface area than classic origami flat sheets.
Flat‐sheet DNA nanostructures: Using algorithmic tools, DNA nanostructures were designed from 2D meshes with varying internal geometries. Using this method, structures with complex internal and external features were prepared that self‐assemble under physiological salt concentrations and have larger surface areas compared to classic DNA origami flat‐sheet designs.
We report a Monte Carlo (MC) simulation study of a model discotic liquid crystal (DLC) confined between hybrid walls with controllable penetrability. The model consists of oblate hard Gaussian ...overlap (HGO) particles. Particle–substrate interactions are modeled as follows: each substrate sees a particle as a disc of zero thickness and diameter D less than or equal to that of the actual particle, σ0, embedded inside the particle and located halfway along, and perpendicular to, its minor axis. This allows us to control the anchoring properties of the substrates, from planar (edge-on) for D ≈ 0 to homeotropic (face-on) for D ≈ σ0, which can be done independently at either substrate. Depending on the values of Ds ≡ D/σ0 at the top (D s t ) and bottom (D s b ) substrates, we find domains in (D s b , D s t ) space in which particle alignment is uniform planar (UP), is uniform homeotropic (UH), or varies linearly from planar at one substrate to homeotropic at the other (Lin). These domains are separated by regions of bistability (P–Lin and H–Lin), which appear to be wider than for prolate HGOs, and there may be also a small tristable (P–H–Lin) region. Results are compared with the predictions of density functional theory, implemented at the level of Onsager’s second-virial approximation with Parsons-Lee rescaling. As in the case of symmetric confinement studied previously, the agreement between theory and simulation is substantially less good than for prolate HGOs: in particular, for the investigated substrate separation L = 6σ0, the Lin configuration is never predicted. These discrepancies are likely a consequence of the fact that Onsager’s theory is less accurate for discs than for rods.
The Notch signaling pathway has fundamental roles in embryonic development and in the nervous system. The current model of receptor activation involves initiation via a force-induced conformational ...change. Here, we define conditions that reveal pulling force-independent Notch activation using soluble multivalent constructs. We treat neuroepithelial stem-like cells with molecularly precise ligand nanopatterns displayed from solution using DNA origami. Notch signaling follows with clusters of Jag1, and with chimeric structures where most Jag1 proteins are replaced by other binders not targeting Notch. Our data rule out several confounding factors and suggest a model where Jag1 activates Notch upon prolonged binding without appearing to need a pulling force. These findings reveal a distinct mode of activation of Notch and lay the foundation for the development of soluble agonists.
This study aimed to examine the effects of feeding corn silage inoculated without or with either Lactobacillus buchneri (LB) alone or a combination of LB and Lactobacillus plantarum (LBLP) on the ...apparent digestibility, ruminal fermentation, microbial protein synthesis, and growth performance of lambs. Thirty Santa Inês×Dorper crossbred intact males lambs weighing 20.4±3.8 kg were blocked by weight into 10 groups. Lambs in each group were randomly assigned to 1 of the following 3 dietary treatments: untreated (Control), LB, and LBLP silage. Lambs were fed experimental diets for 61 d. The apparent digestibility was indirectly estimated from indigestible NDF measured on d 57 to 59. Spot urine samples were collected from all animals on d 59 to estimate microbial protein synthesis. Lambs were slaughtered for carcass evaluation on d 61 when they weighed 32.4±5.2 kg. Six additional ruminally cannulated Santa Inês×Dorper crossbred wethers weighing 40.5±1.8 kg were used to examine dietary effects on ruminal fermentation. Average daily gain was increased when lambs were fed LBLP silage (P<0.05) but not LB silage. The LBLP silage had the highest (P<0.05) lactic acid concentration and both inoculated silages had greater acetic acid concentrations than the Control silage (P<0.05). Inoculation of corn silage increased intakes of DM, OM, CP, NDF, total carbohydrate (CHO), and GE by the lambs but decreased digestibility of DM, OM, CP, total and nonstructural carbohydrates, and concentration of GE and ME. (P<0.05). Nevertheless, lambs fed inoculated silages had greater microbial N supply than those on the Control treatment (P<0.05). The acetate to propionate ratio was lower in ruminal fluid of wethers in LBLP treatment than LB and Control treatment (P<0.05) and ruminal pH tended to be greater in LB lambs than in LBLP and Control wethers (P<0.10). Finally, the inoculation with both bacteria combined enhanced the silage fermentation. The intakes of DM, OM, CP, NDF, and GE were improved in the lambs fed corn silage inoculated with L. buchneri alone or combined with L. plantarum. The microbial N supply was enhanced in the lambs fed corn silage inoculated with L. buchneri. The inoculation of L. buchneri combined with L. plantarum reduced the acetate to propionate ratio in ruminal fluid and improved the ADG of lambs.
•The RSM model yields the optimal non-toxic extraction conditions.•Temperature and organic solvent percentage are the most critical factors.•White/red varieties’ grape stems required differential ...extraction conditions.
A Box–Behnken design of Response Surface Methodology (RSM) was conducted to analyse the effect of time (10–30min), temperature (25–95°C), and solvents concentration (5–90%) on the extraction of total phenolics, flavonoids, ortho-diphenols, and anthocyanins as well as to assess the ABTS+ scavenging capacity, which were considered as response variables. Values coefficients of determination (R2), ranging from 0.903 to 0.996, fitted for describing efficient extraction of bioactive (poly)phenols and antioxidant activity. The recorded data allowed to establish the optimal extraction conditions at 23.0min, 95.0°C, and 57.9% of food-quality ethanol/water for Vitis vinifera L. var. ‘Viosinho’ (white variety), and 23.4min, 84.2°C, and 63.8% for var. ‘Touriga Nacional’ (red variety). The achievement of optimal extraction conditions of phenolics from grape stems using solvents compatible with further uses in food/pharma industries demonstrated that RSM constitutes a powerful tool for deriving optimal conditions for extraction of antioxidant phenolic compounds from grape stems.
Skeletal muscle cells contain hundreds of myonuclei within a shared cytoplasm, presenting unique challenges for regulating gene expression. Certain transcriptional programs (e.g., postsynaptic ...machinery) are segregated to specialized domains, while others (e.g., contractile proteins) do not show spatial confinement. Furthermore, local stimuli, such as denervation, can induce transcriptional responses that are propagated along the muscle cells. Regulated transport of nuclear proteins (e.g., transcription factors) between myonuclei represents a potential mechanism for coordinating gene expression. However, the principles underlying the transport of nuclear proteins within multinucleated cells remain poorly defined. Here we used a mosaic transfection model to create myotubes that contained exactly one myonucleus expressing a fluorescent nuclear reporter and monitored its distribution among all myonuclei. We found that the transport properties of these model nuclear proteins in myotubes depended on molecular weight and nuclear import rate, as well as on myotube width. Interestingly, muscle hypertrophy increased the transport of high molecular weight nuclear proteins, while atrophy restricted the transport of smaller nuclear proteins. We have developed a mathematical model of nuclear protein transport within a myotube that recapitulates the results of our in vitro experiments. To test the relevance to nuclear proteins expressed in skeletal muscle, we studied the transport of two transcription factors—aryl hydrocarbon receptor nuclear translocator and sine oculis homeobox 1—and found that their distributions were similar to the reporter proteins with corresponding molecular weights. Together, these results define a set of variables that can be used to predict the spatial distributions of nuclear proteins within a myotube.
As DNA origami applications in biomedicine are expanding, more knowledge is needed to assess these structures’ interaction with biological systems. Here, uptake and penetration in cell and cell ...spheroid tissue models (CSTMs) are studied to elucidate whether differences in internal structure can be a factor in the efficacy of DNA‐origami‐based delivery. Two structures bearing largely similar features in terms of both geometry and molecular weight, but with different internal designs—being either compact, lattice‐based origami or following an open, wireframe design—are designed. In CSTMs, wireframe rods are able to penetrate deeper than close‐packed rods. Moreover, doxorubicin‐loaded wireframe rods show a higher cytotoxicity in CSTMs. These results can be explained by differences in structural mechanics, local deformability, local material density, and accessibility to cell receptors between these two DNA origami design paradigms. In particular, it is suggested that the main reason for the difference in penetration dynamic arises from differences in interaction with scavenger receptors where lattice‐based structures appear to be internalized to a higher degree than polygonal structures of the same size and shape. It is thus argued that the choice of structural design method constitutes a crucial parameter for the application of DNA origami in drug delivery.
To probe the potential for DNA‐origami‐based delivery devices, the effect of the folding arrangement of the DNA, on passive uptake and penetration, is studied. Despite almost identical composition and shape of the structures, wireframe‐style origami penetrates deeper in tumor models than classical origami and this is likely an effect due to differences in scavenger receptor mediated uptake.
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