Flint corn silage management: influence of maturity stage, inoculation with Lentilactobacillus buchneri, and storage time on fermentation pattern, aerobic stability, and nutritional characteristics.

Introduction: High quality corn silage depends on factors such as corn type, stage of crop development at harvest time, fermentation time, in addition to use or not of inoculants. This study aimed to investigate the impact of maturity stage, bacterial inoculation, and storage time on fermentation, aerobic stability, and nutritional characteristics of flint corn silage and their implications for corn silage management. Methods: A flint corn hybrid was harvested very early, early, and medium (at 250, 300 and 350 g dry matter (DM)/kg as fed, respectively) and ensiled in mini-silos without (control) or with Lentilactobacillus buchneri CNCM I-4323 at 1 × 105 cfu/g for 120, 240 and 360 d to investigate how these factors interact with each other. Results and discussion: There was only a small increase (7 g/kg starch; p = 0.003) in starch digestibility (starch-D) in the silages stored for 360 d when compared to that stored for 240 d, but with no difference for 120 d. Despite the reduced starch-D (526 vs. 694 g/kg starch; p < 0.001), silages produced from medium harvest had higher (p < 0.001) starch content (317 vs. 137 g/kg DM) and higher amount of digestible starch (169 vs. 98.5 g/kg DM; p < 0.001) compared to very early harvest. The 2-way interactions (inoculation × storage time and maturity × storage time) showed that inoculation of corn silage with L. buchneri increased (p < 0.001) the aerobic stability, and that more mature crop silage had higher aerobic stability (140 h; p = 0.036) than the others (118 and 48.5 h for those silages from very early and early harvest). Conclusion: The storage for a longer time (>120 d) with the goal of increasing silage digestibility did not occur. Harvesting whole-crop flint corn with 300 to 350 g/kg DM is desirable to have higher DM yield and starch accumulation. Inoculation with L. buchneri is recommended to preserve the silage against aerobic deterioration. This study has shown the importance of harvesting flint corn at the right time, and the need for inoculation with L. buchneri to ensure greater yield, starch accumulation, and silage preservation, if 120 days of storage are not exceeded.

Conformational preferences induced by cyclization in orbitides: a vibrational CD study.

Orbitides are bioactive head-to-tail natural cyclic peptides from plant species. Their bioactivity is intrinsically related to the main conformations adopted in solution, whose correct characterization represents an important bottleneck for medicinal chemistry applications. To date, NMR spectrosocopy has been the most frequently used technique to assess the secondary structure of orbitides. Despite the amount of structural information commonly available from NMR, its time scale frequently results in a limited conformational ensemble with a single mean structure, which may not represent the bioactive conformation. Additionally, problems with inter-residue NOE/ROE signals can reduce the accuracy and confidence of the 3D assignments. Vibrational circular dichroism (VCD), on the other hand, has been demostrated as a powerful tool to probe the stereostructure of chiral molecules, including peptides and proteins, with enhanced sensitivity to individual conformations in the condensed phase. Herein, we present the first VCD stereochemical investigation of orbitides. By combining IR/VCD experiments in ACN-d3 and ACN-d3/D2O mixtures with DFT calculations in different levels of theory we were able to determine the solution-state conformational behavior, as well as the main structural restraints induced by cyclization, of the seven-residue orbitide pohlianin A and its linear precursor. VCD results indicated inverse γ-turns as the most prevalent structural motif for the linear precursor in partially aqueous solution, while type I and type VI ß-turns were induced by the cyclization process, along with some classic γ-turns. In addition to the conformation of the cyclic peptide already described from NMR data, two previously unidentified conformations with distinct secondary structures were found to significantly populate the sample. This conformational discriminatory power of VCD for both linear and cyclic peptides in different solvent systems may lead to more accurate structural characterization of turn-rich peptidic natural products and help the design of conformationally-tailored peptides for more precise structure-activity relationship results.

Antimicrobial peptides as a feed additive alternative to animal production, food safety and public health implications: An overview.

In the last few years, feed additives have been used in animal nutrition to improve nutrient utilization, health parameters and animal performance. However, the use of antibiotics as feed additives has allowed the occurrence of antimicrobial resistance (AMR), which can bring as a consequence, an increase in the morbidity and mortality of diseases that were previously treatable with antibiotics. In this context, antimicrobial peptides (AMP) have appeared as a promising strategy because they have multiple biological activities and represent a powerful strategy to prevent the development of resistant microorganisms. Despite the small number of studies applied in vivo, AMP appear as a potent alternative to the use of antibiotics in animal nutrition, due to an increase in feed efficiency and the prevention/treatment of some animal diseases. This review discusses the problems associated with antimicrobial resistance and the use of AMP as a strong candidate to replace conventional antibiotics, mainly in the animal industry.

Active-electrode biosensor of SnO2 nanowire for cyclodextrin detection from microbial enzyme.

Cyclodextrin (CD) is a conical compound used in food and pharmaceutical industry to complexation of hydrophobic substances. It is a product of microbial enzymes which converts starch into CD during their activity. We aim to detect CD using active-electrode biosensor of SnO2. They were grown on active electrode by the VLS method. The CD consists of several glucose units which have hydroxyl groups which tend to bind to interface states present in nanowires changing their conductivity. Experimental results of electrical conductivity at different CD concentrations are presented. A model that describes the influence of adsorbed glucose on nanowires and its electrical properties is also presented. Some general observations are performed on the applicability of the CD adsorption method by the nanowire-based biosensor.

Dissecting the mechanism of action of actinoporins. Role of the N-terminal amphipathic α-helix in membrane binding and pore activity of sticholysins I and II.

Actinoporins sticholysin I and sticholysin II (St I, St II) are proposed to lyse model and biomembranes via toroidal pore formation by their N-terminal domain. Based on the hypothesis that peptide fragments can reproduce the structure and function of this domain, the behavior of peptides containing St I residues 12-31 (StI12-31), St II residues 11-30 (StII11-30), and its TOAC-labeled analogue (N-TOAC-StII11-30) was examined. Molecular modeling showed a good match with experimental structures, indicating amphipathic α-helices in the same regions as in the toxins. CD spectra revealed that the peptides were essentially unstructured in aqueous solution, acquiring α-helical conformation upon interaction with micelles and large unilamellar vesicles (LUV) of variable lipid composition. Fluorescence quenching studies with NBD-containing lipids indicated that N-TOAC-StII11-30's nitroxide moiety is located in the membranes polar head group region. Pyrene-labeled phospholipid inter-leaflet redistribution suggested that the peptides form toroidal pores, according to the mechanism of action proposed for the toxins. Binding occurred only to negatively charged LUV, indicating the importance of electrostatic interactions; in contrast the peptides bound to both negatively charged and zwitterionic micelles, pointing to a lesser influence of these interactions. In addition, differences between bilayers and micelles in head group packing and in curvature led to differences in peptide-membrane interaction. We propose that the peptides topography in micelles resembles that of the toxins in the toroidal pore. The peptides mimicked the toxins permeabilizing activity, St II peptides being more effective than StI12-31. To our knowledge, this is the first demonstration that differences in the toxins N-terminal amphipathic α-helix play a role in the difference between St I and St II activities.

HsDHODH Microdomain-Membrane Interactions Influenced by the Lipid Composition.

Human dihydroorotate dehydrogenase (HsDHODH) enzyme has been studied as selective target for inhibitors to block the enzyme activity, intending to prevent proliferative diseases. The N-terminal microdomain seems to play an important role in the enzyme function. However, the molecular mechanism of action and dynamics of this region are not totally understood yet. This study analyzes the interaction and conformation in model membranes of HsDHODH microdomain using peptide analogues containing the paramagnetic amino acid TOAC at strategic positions. In buffer solution, the analogues presented a disordered conformation, but acquired a high content of α-helical structure in membrane mimetics, which was found to be lipid dependent. The microdomain peptide structure in micelles showed a very different peptide conformation when compared to the reported crystal structure, displaying a conformational flexibility of its helices, promoted by the connecting loop, which might be functionally relevant. Electron spin resonance in membrane compositions containing POPC, POPE, and cardiolipin showed that interaction of the analogues was enhanced by the presence of cardiolipin, indicating that the microdomain preferentially interacts with cardiolipin-containing membranes. Therefore, the great flexibility of the microdomain and the cardiolipin affinity should be considered in further studies aimed at finding new inhibitory compounds to fight proliferative diseases.

SARS-CoV fusion peptides induce membrane surface ordering and curvature.

Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed.

Randomized controlled trial comparing hyaluronic acid, platelet-rich plasma and the combination of both in the treatment of mild and moderate osteoarthritis of the knee.

Objective: This study aims at evaluating the clinical effects of Platelet Rich Plasma (PRP) and Hyaluronic Acid (HA) as individual treatments for mild to moderate Osteoarthritis (OA) and it also examines the potential synergistic effects of PRP in combination with HA. Research continues to emerge examining the potential therapeutic efficacy of HA and PRP as autologous injectable treatments for joint arthritis. However, there is a paucity of research investigating the effects of combining HA and PRP on pain and functional status in patients with OA. Design: In this multi-center, randomized, controlled, double blind, prospective trial, 105 patients with mild to moderate knee osteoarthritis, who met the study criteria, were randomly allocated to one of three interventions: HA (n=36), PRP (n=36), or HA+PRP (n=33). Each patient received 3 intra-articular knee injections of their assigned substance, with 2 week intervals between each injection. Clinical outcomes were evaluated using the Western Ontario and McMaster Universities Arthritis Index (WOMAC) and Visual Analogue Scale (VAS) questionnaire at baseline and after 1,3,6 and 12 months. Results: The study showed that the PRP group have significant reduction in VAS scores at 1 (p= 0.003), 3 (p= 0.0001), 6 (p= 0.0001) and 12 (p= 0.000) months when compared to HA. In addition, the PRP group illustrated greater improvement in WOMAC physical activity scale at 12 months (p= 0.008) when compared to the HA group. Combining HA and PRP resulted in a significant decreases in pain (p=0.0001) and functional limitation (p=0.0001) when compared to HA alone at 1 year post treatment; and significantly increased physical function at 1 (p=0.0004) and 3 (p=.011) months when compared to PRP alone. Conclusion: The findings of the study support the use of autologous PRP as an effective treatment of mild to moderate knee osteoarthritis. It also shows that the combination of HA and PRP resulted to better outcomes than HA alone up to 1 year and PRP alone up to 3 months. Furthermore, the results suggest that combination of PRP and HA could potentially provide better functional outcomes in the first 30 days after treatment with both PRP and HA alone.

Conformational changes of the HsDHODH N-terminal Microdomain via DEER Spectroscopy.

The human enzyme dihydroorotate dehydrogenase (HsDHODH) has been studied for being a target for development of new antineoplasic and antiproliferative drugs. The synthetic peptide N-t(DH) represents the N-terminal microdomain of this enzyme, responsible for anchoring it to the inner mitochondrial membrane. Also, it is known to harbor quinones that are essential for enzyme catalysis. Here we report structural features of the peptide/membrane interactions obtained by using CD and DEER spectroscopic techniques, both in micelles and in lipid vesicles. The data revealed different peptide conformational states in micelles and liposomes, which could suggest that this microdomain acts in specific regions or areas of the mitochondria, which can be related with the control of the quinone access to the HsDHODH active site. This is the first study to report on conformational changes of the HsDHODH N-terminal microdomain through a combination of CD and DEER spectroscopic techniques.

N-terminal microdomain peptide from human dihydroorotate dehydrogenase: structure and model membrane interactions.

The N-terminus of the human dihydroorotate dehydrogenase (HsDHODH) has been described as important for the enzyme attachment in the inner mitochondrial membrane and possibly to regulate enzymatic activity. In this study, we synthesized the peptide acetyl-GDERFYAEHLMPTLQGLLDPESAHRL AVRFTSLGamide, comprising the residues 33-66 of HsDHODH N-terminal conserved microdomain. Langmuir monolayers and circular dichroism (CD) were employed to investigate the interactions between the peptide and membrane model, as micelles and monolayers of the lipids phosphatidylcholine (PC), 3-phosphatidylethanolamine (PE) and cardiolipin (CL). These lipids represent the major constituents of inner mitochondrial membranes. According to CD data, the peptide adopted a random structure in water, whereas it acquired α-helical structures in the presence of micelles. The π-A isotherms and polarization- modulated infrared reflection-absorption spectroscopy on monolayers showed that the peptide interacted with all lipids, but in different ways. In DPPC monolayers, the peptide penetrated into the hydrophobic region. The strongest initial interaction occurred with DPPE, but the peptide was expelled from this monolayer at high surface pressures. In CL, the peptide could induce a partial dissolution of the monolayer, leading to shorter areas at the monolayer collapse. These results corroborate the literature, where the HsDHODH microdomain is anchored into the inner mitochondrial membrane. Moreover, the existence of distinct conformations and interactions with the different membrane lipids indicates that the access to the enzyme active site may be controlled not only by conformational changes occurring at the microdomain of the protein, but also by some lipid-protein synergetic mechanism, where the HsDHODH peptide would be able to recognize lipid domains in the membrane.

Dynamics and conformational studies of TOAC spin labeled analogues of Ctx(Ile(21))-Ha peptide from Hypsiboas albopunctatus.

Antimicrobial peptides (AMPs) isolated from several organisms have been receiving much attention due to some specific features that allow them to interact with, bind to, and disrupt cell membranes. The aim of this paper was to study the interactions between a membrane mimetic and the cationic AMP Ctx(Ile(21))-Ha as well as analogues containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) incorporated at residue positions n = 0, 2, and 13. Circular dichroism studies showed that the peptides, except for [TOAC(13)]Ctx(Ile(21))-Ha, are unstructured in aqueous solution but acquire different amounts of α-helical secondary structure in the presence of trifluorethanol and lysophosphocholine micelles. Fluorescence experiments indicated that all peptides were able to interact with LPC micelles. In addition, Ctx(Ile(21))-Ha and [TOAC(13)]Ctx(Ile(21))-Ha peptides presented similar water accessibility for the Trp residue located near the N-terminal sequence. Electron spin resonance experiments showed two spectral components for [TOAC(0)]Ctx(Ile(21))-Ha, which are most likely due to two membrane-bound peptide conformations. In contrast, TOAC(2) and TOAC(13) derivatives presented a single spectral component corresponding to a strong immobilization of the probe. Thus, our findings allowed the description of the peptide topology in the membrane mimetic, where the N-terminal region is in dynamic equilibrium between an ordered, membrane-bound conformation and a disordered, mobile conformation; position 2 is most likely situated in the lipid polar head group region, and residue 13 is fully inserted into the hydrophobic core of the membrane.

Solid-phase peptide synthesis in highly loaded conditions.

The use of very highly substituted resins has been avoided for peptide synthesis due to the aggravation of chain-chain interactions within beads. To better evaluate this problem, a combined solvation-peptide synthesis approach was herein developed taking as models, several peptide-resins and with peptide contents values increasing up to near 85%. Influence of peptide sequence and loading to solvation characteristics of these compounds was observed. Moreover, chain-chain distance and chain concentration within the bead were also calculated in different loaded conditions. Of note, a severe shrinking of beads occurred during the α-amine deprotonation step only when in heavily loaded resins, thus suggesting the need for the modification of the solvent system at this step. Finally, the yields of different syntheses in low and heavily loaded conditions were comparable, thus indicating the feasibility of applying this latter "prohibitive" chemical synthesis protocol. We thought these results might be basically credited to the possibility, without the need of increasing molar excess of reactants, of carrying out the coupling reaction in higher concentration of reactants - near three to seven folds - favored by the use of smaller amount of resin. Additionally, the alteration in the solvent system at the α-amine deprotonation step might be also improving the peptide synthesis when in heavily loaded experimental protocol.