Effect of feeding Bengal gram residual forage-based pelleted total mixed ration on growth performance, nutrient availability, carcass traits and composition in finisher Barbari kids.

The study aimed to assess the impact of feeding Bengal gram residual forage-based pelleted Total Mixed Ration (TMR) with varying concentrate (C) to roughage (R) ratios on feed intake, nutrient utilization, growth, and carcass characteristics in Barbari kids. Sixteen weaned male Barbari kids (av. age, 233 ± 11 days; weight, 13.86 ± 0.76 kg) were divided into two groups (T1 and T2), each receiving different pelleted diets (TMR) with distinct concentrate to roughage ratios (T1 with 60:40; T2 with 40:60). The kids were fed for 133 days, and a digestion trial was conducted at the end of the study. After completion, all kids were slaughtered. Although, kids under T1 consumed higher (P < 0.001) amount of dry matter, and crude protein compared to T2, which was due to a higher concentrate to roughage ratio in T1. But, the average daily body weight gain (ADG) of finisher kids was 88.53, and 79.83 g/d/kid in T1 and T2, respectively; however, the difference was non-significant. Digestibility of organic matter, crude protein, and total carbohydrate was also greater in T1 compared to T2. Total digestible nutrients intake was higher (P < 0.001) in T1; similarly intake of digestible energy, and metabolizable energy were significantly increased (P < 0.01) in T1 compared to T2. Concentrations of volatile fatty acids and NH3-nitrogen were also enhanced (P < 0.05) in T1 compared to T2. We observed similar carcass weight, and dressing percentage in both groups, and carcass composition remained unaffected. The pelleted diet containing greater ratio of concentrate: roughage (60:40) had no additional benefits in terms of ADG, and carcass traits in finisher kids. Therefore, it may be concluded that the Bengal gram residual forage-based pelleted TMR diet containing C40: R60 (TDN 57.13%, DCP 7.64%, ME 9.11MJ/kg feed) is suitable for optimizing growth performance with desirable carcass traits, and meat composition in finisher Barbari kids reared under the intensive system.

A Marcus-Type Inverted Region in the Translocation Kinetics of a Knotted Protein.

Knotted proteins are rare but important species, yet how their complex topologies affect their physical properties is not fully understood. Here we combine single molecule nanopore experiments and all-atom MD simulations to study the electric-field-driven unfolding during the translocation through a model pore of individual protein knots important for methylating tRNA. One of these knots shows an unusual behavior that resembles the behavior of electrons hopping between two potential surfaces: as the electric potential driving the translocation reaction is increased, the rate eventually plateaus or slows back down in the "Marcus inverted regime". Our results shed light on the influence of topology in knotted proteins on their forced translocation through a pore connecting two electrostatic potential wells.

Threading single proteins through pores to compare their energy landscapes.

Translocation of proteins is correlated with structural fluctuations that access conformational states higher in free energy than the folded state. We use electric fields at the solid-state nanopore to control the relative free energy and occupancy of different protein conformational states at the single-molecule level. The change in occupancy of different protein conformations as a function of electric field gives rise to shifts in the measured distributions of ionic current blockades and residence times. We probe the statistics of the ionic current blockades and residence times for three mutants of the [Formula: see text]-repressor family in order to determine the number of accessible conformational states of each mutant and evaluate the ruggedness of their free energy landscapes. Translocation becomes faster at higher electric fields when additional flexible conformations are available for threading through the pore. At the same time, folding rates are not correlated with ease of translocation; a slow-folding mutant with a low-lying intermediate state translocates faster than a faster-folding two-state mutant. Such behavior allows us to distinguish among protein mutants by selecting for the degree of current blockade and residence time at the pore. Based on these findings, we present a simple free energy model that explains the complementary relationship between folding equilibrium constants and translocation rates.

Direct Observation of Single-Protein Transition State Passage by Nanopore Ionic Current Jumps.

Conformational transitions of proteins are governed by chemical kinetics, often toggled by passage through an activated state separating two conformational ensembles. The passage time of a protein through the activated state can be too fast to be detected by single-molecule experiments without the aid of viscogenic agents. Here, we use high-bandwidth nanopore measurements to resolve microsecond-duration transitions that occur between conformational states of individual protein molecules partly blocking pore current. We measure the transition state passage time between folded and unfolded states of a two-state λ6-85 mutant and between metastable intermediates and the unfolded state of the multistate folder cytochrome c. Consistent with the principle of microscopic reversibility, the transition state passage time is the same for the forward and backward reactions. A passage time distribution whose tail is broader than a single exponential observed in cytochrome c suggests a multidimensional energy landscape for this protein.

Discrimination of RNA fiber structures using solid-state nanopores.

RNA fibers are a class of biomaterials that can be assembled using HIV-like kissing loop interactions. Because of the programmability of molecular design and low immunorecognition, these structures present an interesting opportunity to solve problems in nanobiotechnology and synthetic biology. However, the experimental tools to fully characterize and discriminate among different fiber structures in solution are limited. Herein, we utilize solid-state nanopore experiments and Brownian dynamics simulations to characterize and distinguish several RNA fiber structures that differ in their degrees of branching. We found that, regardless of the electrolyte type and concentration, fiber structures that have more branches produce longer and deeper ionic current blockades in comparison to the unbranched fibers. Experiments carried out at temperatures ranging from 20-60 °C revealed almost identical distributions of current blockade amplitudes, suggesting that the kissing loop interactions in fibers are resistant to heating within this range.

Electrical unfolding of cytochrome c during translocation through a nanopore constriction.

Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome c (cyt c) through ultrathin silicon nitride (SiNx) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of ∼30 to 100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field-induced deformability.

Rosette Nanotube Porins as Ion Selective Transporters and Single-Molecule Sensors.

Rosette nanotubes (RNTs) are a class of materials formed by molecular self-assembly of a fused guanine-cytosine base (G∧C base). An important feature of these self-assembled nanotubes is their precise atomic structure, intriguing for rational design and optimization as synthetic transmembrane porins. Here, we present experimental observations of ion transport across 1.1 nm inner diameter RNT porins (RNTPs) of various lengths in the range 5-200 nm. In a typical experiment, custom lipophilic RNTPs were first inserted into lipid vesicles; the vesicles then spontaneously fused with a planar lipid bilayer, which produced stepwise increases of ion current across the bilayer. Our measurements in 1 M KCl solution indicate ion transport rates of ∼50 ions s-1 V-1 m, which for short channels amounts to conductance values of ∼1 nS, commensurate with naturally occurring toxin channels such as α-hemolysin. Measurements of interaction times of α-cyclodextrin with RNTPs reveal two distinct unbinding time scales, which suggest that interactions of either face of α-cyclodextrin with the RNTP face are differentiable, backed with all-atom molecular dynamics simulations. Our results highlight the potential of RNTPs as self-assembled nonproteinaceous single-molecule sensors and selective nanofilters with tunable functionality through chemistry.

More protected vibrational states at the dissociation limit of SCCl2.

Local vibrational coupling models predict that intramolecular vibrational energy redistribution (IVR) is not completely statistical even at the dissociation limit of polyatomic molecules. Thus states protected from IVR and from rapid dissociation form regular progressions and can be assigned vibrational quantum numbers. We previously observed such regular progressions of states in vibrational spectra of the molecule SCCl2, but a discrepancy in the density of such states remained between theory and experiment. Here we show that the gap can be closed by observing and assigning additional vibrational transitions above the dissociation limit of SCCl2, and by carefully analyzing the theoretically expected density of protected states. The newly observed transitions originate from recently assigned and more highly excited vibrational levels in the B̃ electronic state, connecting to the X̃ ground state by different Franck-Condon factors. Based on our analysis of Franck-Condon activity, we conclude that theory and experiment agree within measurement uncertainty. Consistency between theory and experiment implies that even more protected states should be observed for larger molecules, leading to nonstatistical dissociation reactions.