Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C

The aim of the present work was to study the effects of aflatoxin (AF) on sperm parameters in rams, and to determine the protective efficiency of esterified glucomannan (EG) co-administered with AF up to 96 h of the liquid storage of ram semen at 5 °C. Thirty-two Merino rams (12–14 months old) were used. The animals were examined for their general health status. To ensure their adaptation to the environment and the new feeding regimen, a 15-day acclimatization programme was applied to the animals, prior to the start of the study. Experimental feeding was continued for ninety-two days. The experimental design consisted of four dietary treatments. The control group (C) was fed with commercial feed. The AF group was fed with commercial feed plus 250 μg/day of total AF. The EG group received commercial feed plus 2 g/day of EG. The AF + EG group was given commercial feed plus 250 μg/day of total AF and 2 g/day of EG. In the study, ejaculates were obtained from rams twice a week for 12 weeks, using an electro-ejaculator. After collected, the ejaculates were diluted with a skimmed milk extender, and stored at 5 °C. Sperm motility and rates of abnormal and nonviable spermatozoa were determined for the different treatment groups at 5 °C at 0, 24, 48, 72 and 96 h of liquid storage.     

A Novel Receptor-induced Activation Site in the Nipah Virus Attachment Glycoprotein (G) Involved in Triggering the Fusion Glycoprotein (F)

Cellular entry of paramyxoviruses requires the coordinated action of both the attachment (G/H/HN) and fusion (F) glycoproteins, but how receptor binding activates G to trigger F-mediated fusion during viral entry is not known. Here, we identify a receptor (ephrinB2)-induced allosteric activation site in Nipah virus (NiV) G involved in triggering F-mediated fusion. We first generated a conformational monoclonal antibody (monoclonal antibody 45 (Mab45)) whose binding to NiV-G was enhanced upon NiV-G-ephrinB2 binding. However, Mab45 also inhibited viral entry, and its receptor binding-enhanced (RBE) epitope was temperature-dependent, suggesting that the Mab45 RBE epitope on G may be involved in triggering F. The Mab45 RBE epitope was mapped to the base of the globular domain (β6S4/β1H1). Alanine scan mutants within this region that did not exhibit this RBE epitope were also non-fusogenic despite their ability to bind ephrinB2, oligomerize, and associate with F at wild-type (WT) levels. Although circular dichroism revealed conformational changes in the soluble ectodomain of WT NiV-G upon ephrinB2 addition, no such changes were detected with soluble RBE epitope mutants or short-stalk G mutants. Additionally, WT G, but not a RBE epitope mutant, could dissociate from F upon ephrinB2 engagement. Finally, using a biotinylated HR2 peptide to detect pre-hairpin intermediate formation, a cardinal feature of F-triggering, we showed that ephrinB2 binding to WT G, but not the RBE-epitope mutants, could trigger F. In sum, we implicate the coordinated interaction between the base of NiV-G globular head domain and the stalk domain in mediating receptor-induced F triggering during viral entry.The paramyxoviruses comprise a group of important human pathogens, such as measles, mumps, human parainfluenza viruses, and the highly pathogenic Nipah (NiV)⁴ and Hendra (HeV) viruses. NiV infections have a mortality rate in humans of up to 75%, and NiV is classified as a BSL4 pathogen because of its bio- or agro-terrorism potential (1). The efficacy of entry inhibitors targeted against HIV suggests that a better understanding of Paramyxovirus entry and fusion will facilitate similarly efficacious antiviral therapeutics.Although past studies have identified regions in either the fusion (F) or attachment (G/H/HN) glycoproteins that are important for membrane fusion or F-G/H/HN association (2–10), the region(s) in G important for receptor-activated triggering of F-mediated fusion remains unknown. Current models of Paramyxovirus membrane fusion posit that receptor binding to the attachment glycoprotein (G, H, or HN) triggers a conformational cascade in the fusion protein (F). Such F-triggering results in fusion peptide (FP) exposure, which involves formation of a pre-hairpin intermediate and subsequent six-helix bundle formation (11). The energy released upon refolding into the stable six-helix bundle ground state is what drives the fusion of the viral and host-cell membranes. These are common functional and structural features responsible for membrane fusion for all enveloped viruses regardless of whether the fusion protein has predominantly trimeric α-helical coiled-coil (Class I), β (Class II), or a combination of α and β (Class III) core structures (12). Important human pathogens such as the HIV, influenza, and various paramyxoviruses have Class I fusion proteins, and their similar structural features point to similar membrane fusion mechanisms (11, 12). Besides sharing trimeric coiled-coil structures, they are synthesized as precursors that are cleaved into a metastable conformation; cleavage generates a new hydrophobic N terminus FP that gets released and inserted into the target cell membrane upon triggering (11, 12). Class I fusion proteins have two heptad repeat regions, HR1 and HR2, at their N and C termini, respectively, that fold up onto each other during six-helix bundle formation to bring about merging of target cell and viral membranes (12). For Paramyxovirus F proteins, the C-terminal HR2 region is generally thought to be pre-formed, but the N-terminal HR1 region is formed only upon F-triggering and FP insertion (11, 13). The formation of this trimeric HR1 core just before six-helix bundle formation, is known as the pre-hairpin intermediate.Despite their common features, viral fusion proteins vary in their detailed structures, triggering factors, and number of viral surface proteins involved. For paramyxoviruses, receptor binding and fusion functions are carried out by two distinct transmembrane proteins (attachment (G, H, or HN) and fusion (F) proteins, respectively), and with few exceptions both are required for membrane fusion. The underlying mechanism of fusion triggering by the attachment protein may vary depending on their use of protein versus carbohydrate receptors (14). For example, we and others have observed an inverse correlation between fusogenicity of the F protein and the avidity of the F/G or F/H interactions for NiV and measles virus (2, 3, 5, 15, 16), both of which use protein-based receptors; however, for Newcastle disease virus, a glycan-using Paramyxovirus, there seems to be a direct correlation between fusogenicity and the avidity of F/HN interactions (8).For the paramyxoviruses, the early steps in the fusion cascade, particularly how H/HN “triggers” F, are not well understood, and the region(s) in H/HN responsible for F triggering remains unclear, although the stalk domain of H/HN appears to be important for F triggering or for interaction with F (5–8). For NiV, the G attachment glycoprotein binds either the ephrinB2 (B2) or ephrinB3 (B3) protein receptors (17–19), but it is not known how receptor engagement induces G to undergo the allosteric changes involved in triggering F. However, by homology to H or HN, it is likely that the stalk domain in NiV-G is also involved in F-triggering (20). Here we analyze the early steps in the fusion cascade for NiV and identify a specific region in NiV-G distinct from the receptor binding site that is involved in 1) B2-induced changes that trigger FP exposure in F, 2) modulating the avidity of F/G interactions resulting in displacement of F from G, and 3) transducing receptor-induced membrane fusion. Our results offer testable hypotheses as to whether this model of fusion cascade holds true for other paramyxoviruses that use protein-based receptors.     

A Mechanistic Paradigm for Broad-Spectrum Antivirals that Target Virus-Cell Fusion

LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC₅₀≤0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001''s specific inhibition of virus-cell fusion.The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen (¹O₂) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated ¹O₂. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. ¹O₂-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced ¹O₂-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides.Based on our understanding of LJ001''s mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001''s limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC₅₀<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of ¹O₂ generation), and (4) 10–100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion.     

Analysis of the association of the G-7A polymorphism of the matrix Gla protein gene with ischemic atherothrombotic stroke in humans with its different risk factors

The G-7A polymorphism (rs1800801) of the matrix Gla protein (MGP) gene was determined in 170 patients with ischemic atherothrombotic stroke (IATS) and 124 control persons. It was found that the distribution of the major allele homozygotes, heterozygotes, and minor allele homozygotes was 35.9, 48.8, and 15.3% in patients with IATS (in the control, 43.5, 50.0, and 6.5%; P = 0.051 by the χ² test). Significant differences in the distribution of genotypes were revealed only in women (P = 0.022). The odds ratio (OR) for minor allele homozygotes (A/A) vs. major allele carriers (G/A + G/G) was 2.618 (P = 0.023), while in women it was equal to 6.645 (P = 0.015). In patients with the A/A genotype, the values of the blood coagulation parameters (prothrombin time) indicated increased their predisposition to hypercoagulability. The results obtained prove that the A/A variant of the MGP gene is associated with an increased risk of IATS in females of the Ukrainian population and may be related to blood hypercoagulability and formation of thrombi.     

Effects of Chronic Boron Exposure on Semen Profile

The possible changes in semen quality were studied in men living in a boron mining area. The subjects in the boron group had exposure to boron at an average level of 6.5 mg/day, as determined by urinary analysis. The results obtained by the boron group were compared to those obtained for the control group whose subjects were living in the same geographical area but away from the boron region; average exposure level was 1.4 mg/day for this group. The semen samples were analyzed according to the recommendations of the World Health Organization. Boron levels were established in the water samples obtained from various locations in the study region. In the boron mining fields where the subjects in the boron group live, water samples contained boron in the range of 1.4–6.5 mg/L, while the values were <0.01 mg/L for the water samples obtained from the region where the subjects of the control group reside. No negative effects were found in the sperm samples obtained from the subjects of the boron group.     

Small molecule diffusion into swelling iota-carrageenan gels: a fluorescence study

Small molecule diffusion into Iota-Carrageenan gel was studied by using steady-state fluorescence (SSF) technique. Pyranine, dissolved in water was used as fluorescence probe. Fluorescence emission intensity, I(p), and scattered light intensity, I(sc), were monitored to study diffusion and swelling processes at various temperatures respectively. Fickian and Li-Tanaka models were elaborated to produce diffusion, D, and collective diffusion, D(0), coefficients. Diffusion and swelling activation energies were also obtained and found to be 20.5 kj mol(-1) and 28.2 kj mol(-1), respectively.     

The NMDA receptor NR1 C1 region bound to calmodulin: structural insights into functional differences between homologous domains

Calmodulin (CaM) regulates tetrameric N-methyl-D-aspartate receptors (NMDARs) by binding tightly to the C0 and C1 regions of its NR1 subunit. A crystal structure (2HQW; 1.96 A) of calcium-saturated CaM bound to NR1C1 (peptide spanning 875-898) showed that NR1 S890, whose phosphorylation regulates membrane localization, was solvent protected, whereas the endoplasmic reticulum retention motif was solvent exposed. NR1 F880 filled the CaM C-domain pocket, whereas T886 was closest to the N-domain pocket. This 1-7 pattern was most similar to that in the CaM-MARCKS complex. Comparison of CaM-ligand wrap-around conformations identified a core tetrad of CaM C-domain residues (FLMM(C)) that contacted all ligands consistently. An identical tetrad of N-domain residues (FLMM(N)) made variable sets of contacts with ligands. This CaM-NR1C1 structure provides a foundation for designing mutants to test the role of CaM in NR1 trafficking as well as insights into how the homologous CaM domains have different roles in molecular recognition.