A gp41 MPER-specific Llama VHH Requires a Hydrophobic CDR3 for Neutralization but not for Antigen Recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10

New Noncovalent Inhibitors of Penicillin-Binding Proteins from Penicillin-Resistant Bacteria

BACKGROUND: Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs beta-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for beta-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs. METHODOLOGY/PRINCIPAL FINDINGS: Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains. CONCLUSIONS: We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.Eur-Intafa

In vitro rumen fermentation of diets with different types of condensed tannins derived from sainfoin (Onobrychis viciifolia Scop.) pellets and hazelnut (Corylus avellana L.) pericarps

The aim of this study was to evaluate the in vitro rumen fermentation parameters of diets including pellets of sainfoin pellets and/or hazelnut pericarps, which are two plant resources that containing different types of condensed tannins (CT) with contrasted structures, using a batch culture system during for 24 h. The treatments were a basal diet (control), the basal diet + pellets of dehydrated sainfoin (PS), the basal diet + freeze-dried hazelnut pericarps (HP), and the basal diet + PS + HP. The diets were adjusted to be isotannic (20 g/kg dry matter (DM), except for the control) and isoproteic (132 g/kg DM). Total gas and methane (CH4) productions were measured after 3.5 h and 24 h of incubation. At the end of incubation, pH, in vitro DM degradability (IVDMD) and the concentration of fermentation end-products in the medium were also measured. The CT structures of CT infrom PS and HP were very different: as PS showed a PD-dominant profilehad mostly prodelphinidins and HP showed a PC-dominant profile mostly procyanidins. After 24 h of incubation, the total gas and methane productions and IVDMD were greater for the basal diet than for the diet + HP and the diet + PS (P<0.05). The CH4 production increased significantly with the diet + HP in the presence of PEG, a compound CT-inactivating CTcompound (P<0.001), and tended to increase for the diet + PS (P<0.1). The volatile fatty acids (VFA) net productions were globally similar among treatments, while the NH3 concentration was lower for the diet + PS (with a significant PEG effect) than for the diets including HP, and was the highest for the basal diet. It was concluded that the inclusion of PS and HP in a basal diet results in lower rumen fermentability and that but their CT decreased CH4 production and protein degradability;, PS being were more efficient effective than HP for the latterreducing protein degradability

Molecular Dynamics Simulation of the Complex PBP-2x with Drug Cefuroxime to Explore the Drug Resistance Mechanism of Streptococcus suis R61

Drug resistance of Streptococcus suis strains is a worldwide problem for both humans and pigs. Previous studies have noted that penicillin-binding protein (PBPs) mutation is one important cause of β-lactam antibiotic resistance. In this study, we used the molecular dynamics (MD) method to study the interaction differences between cefuroxime (CES) and PBP2x within two newly sequenced Streptococcus suis: drug-sensitive strain A7, and drug-resistant strain R61. The MM-PBSA results proved that the drug bound much more tightly to PBP2x in A7 (PBP2x-A7) than to PBP2x in R61 (PBP2x-R61). This is consistent with the evidently different resistances of the two strains to cefuroxime. Hydrogen bond analysis indicated that PBP2x-A7 preferred to bind to cefuroxime rather than to PBP2x-R61. Three stable hydrogen bonds were formed by the drug and PBP2x-A7, while only one unstable bond existed between the drug and PBP2x-R61. Further, we found that the Gln569, Tyr594, and Gly596 residues were the key mutant residues contributing directly to the different binding by pair wise energy decomposition comparison. By investigating the binding mode of the drug, we found that mutant residues Ala320, Gln553, and Thr595 indirectly affected the final phenomenon by topological conformation alteration. Above all, our results revealed some details about the specific interaction between the two PBP2x proteins and the drug cefuroxime. To some degree, this explained the drug resistance mechanism of Streptococcus suis and as a result could be helpful for further drug design or improvement

Comparison of five models used to describe gas accumulation profiles in the gas test method with horse caecal fluid as inoculum

The in vivo organic matter digestibility (OMD) in ruminants can be predicted with the gas test method by the amount of gas produced after 24-h fermentation (Menke et al., 1979). Mathematical models can also be used and can provide useful information concerning the kinetics of fermentation. In ruminants, various models have been used to fit fermentation profiles. The early models are based on first-order kinetics with a constant fractional rate of fermentation (Ørskov and McDonald, 1979; Khazaal et al., 1993). Recently, more sophisticated models have been used to describe gas production kinetics obtained from new automated gas production equipment (Theodorou et al., 1995; Cone et al., 1996). The aim of this study was to compare the relevance and the accuracy of five models for describing gas production kinetics and for predicting the organic matter digestibility (OMD) of forages in horses.</jats:p

Unexpected tricovalent binding mode of boronic acids within the active site of a penicillin binding protein.

Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and mimicking the transition state of the enzymatic reaction. We have solved the structures of complexes of a penicillin-binding protein, the DD-peptidase from Actinomadura sp. R39, with four amidomethylboronic acids (2,6 dimethoxybenzamidomethylboronic acid, phenylacetamidomethylboronic acid, 2-chlorobenzamidomethylboronic acid, and 2-nitrobenzamidomethylboronic acid) and the pinacol ester derived from phenylacetamidomethylboronic acid. We found that, in each case, the boron forms a tricovalent adduct with Ogamma of Ser49, Ser298, and the terminal amine group of Lys410, three key residues involved in the catalytic mechanism of penicillin-binding proteins. This represents the first tricovalent enzyme-inhibitor adducts observed by crystallography. In two of the five R39-boronate structures, the boronic acid is found as a tricovalent adduct in two monomers of the asymmetric unit and as a monocovalent adduct with the active serine in the two remaining monomers of the asymmetric unit. Formation of the tricovalent complex from a classical monocovalent complex may involve rotation around the Ser49 Calpha-Cbeta bond to place the boron in a position to interact with Ser298 and Lys410, and a twisting of the side chain amide such that its carbonyl oxygen is able to hydrogen bond to the oxyanion hole NH of Thr413. Biphasic kinetics were observed in three of the five cases and details of the reaction between R39 and 2,6-dimethoxybenzamidomethylboronic acid were studied. Observation of biphasic kinetics was not, however, thought to be correlated to formation of tricovalent complexes, assuming that the latter do form in solution. Based on the crystallographic and kinetic results, a reaction scheme for this unexpected inhibition by boronic acids is proposed

Comparison of in vitro digestion of hays with horse caecal and sheep rumen fluids

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Utilization of the gas test method and horse faeces as a source of inoculum

The gas test is commonly described as a simple, rapid and cheap technique for predicting the energy value of forages but it requires animals fitted with a canula. In horses, this method can be implemented with caecal fluid as an inoculum to predict the organic matter digestibility (OMD) of forages (Macheboeuf et al., 1998). In the present work, as bacterial content of faeces was known to be very high (Meyer et al., 1993), the use of faeces as a source of inoculum for predicting OMD of forages in horses was studied.</jats:p