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Influenza viruses have an error-prone polymerase complex that facilitates a mutagenic environment. Antigenic mutants swiftly arise from this environment with the capacity to persist in both humans and economically important livestock even in the face of vaccination. Furthermore, influenza viruses can adjust the antigenicity of the haemagglutinin (HA) protein, the primary influenza immunogen, using one of four molecular mechanisms. Two prominent mechanisms are: (1) enhancing binding avidity of HA toward cellular receptors to outcompete antibody binding and (2) amino acid substitutions that introduce an N-linked glycan on HA that sterically block antibody binding. In this study we investigate the impact that adsorptive mutation and N-linked glycosylation have on receptor-binding, viral fitness, and antigenicity. We utilize the H9N2 A/chicken/Pakistan/SKP-827/16 virus which naturally contains HA residue T180 that we have previously shown to be an adsorptive mutant relative to virus with T180A. We find that the addition of N-linked glycans can be beneficial or deleterious to virus replication depending on the background receptor binding avidity. We also find that in some cases, an N-linked glycan can trump the effect of an avidity enhancing substitution with respect to antigenicity. Taken together these data shed light on a potential route to the generation of a virus which is “fit” and able to overcome vaccine pressure.

Determination of the mechanisms of interspecies transmission is of great significance for the prevention of epidemic diseases caused by emerging coronaviruses (CoVs). Recently, porcine deltacoronavirus (PDCoV) was shown to exhibit broad host cell range mediated by surface expression of aminopeptidase N (APN), and humans have been reported to be at risk of PDCoV infection. In the present study, we first demonstrated overexpression of APN orthologues from various species, including mice and felines, in the APN-deficient swine small intestine epithelial cells permitted PDCoV infection, confirming that APN broadly facilitates PDCoV cellular entry and perhaps subsequent interspecies transmission. PDCoV was able to limitedly infect mice in vivo, distributing mainly in enteric and lymphoid tissues, suggesting that mice may serve as a susceptible reservoir of PDCoV. Furthermore, elements (two glycosylation sites and four aromatic amino acids) on the surface of domain B (S1B) of the PDCoV spike glycoprotein S1 subunit were identified to be critical for cellular surface binding of APN orthologues. However, both domain A (S1A) and domain B (S1B) were able to elicit potent neutralizing antibodies against PDCoV infection. The antibodies against S1A inhibited the hemagglutination activity of PDCoV using erythrocytes from various species, which might account for the neutralizing capacity of S1A antibodies partially through a blockage of sialic acid binding. The study reveals the tremendous potential of PDCoV for interspecies transmission and the role of two major PDCoV S1 domains in receptor binding and neutralization, providing a theoretical basis for development of intervention strategies. IMPORTANCE Coronaviruses exhibit a tendency for recombination and mutation, which enables them to quickly adapt to various novel hosts. Previously, orthologues of aminopeptidase N (APN) from mammalian and avian species were found to be associated with porcine deltacoronavirus (PDCoV) cellular entry in vitro. Here, we provide in vivo evidence that mice are susceptible to PDCoV limited infection. We also show that two major domains (S1A and S1B) of the PDCoV spike glycoprotein involved in APN receptor binding can elicit neutralizing antibodies, identifying two glycosylation sites and four aromatic amino acids on the surface of the S1B domain critical for APN binding and demonstrating that the neutralization activity of S1A antibodies is partially attributed to blockage of sugar binding activity. Our findings further implicate PDCoV’s great potential for interspecies transmission, and the data of receptor binding and neutralization may provide a basis for development of future intervention strategies.

Human metapneumovirus (hMPV) is an important cause of acute viral respiratory infection. As the only target of neutralizing antibodies, the hMPV fusion (F) protein has been a major focus for vaccine development and targeting by drugs and monoclonal antibodies (mAbs). While X-ray structures of trimeric pre-fusion and post-fusion hMPV F proteins from genotype A, and monomeric pre-fusion hMPV F protein from genotype B have been determined, structural data for the post-fusion conformation for genotype B is lacking. We determined the crystal structure of this protein and compared the structural differences of post-fusion hMPV F between hMPV A and B genotypes. We also assessed the receptor binding properties of the hMPV F protein to heparin and heparan sulfate (HS). A library of HS oligomers was used to verify the HS binding activity of hMPV F, and several compounds showed binding to predominantly pre-fusion hMPV F, but had limited binding to post-fusion hMPV F. Furthermore, mAbs to antigenic sites III and the 66-87 intratrimeric epitope block heparin binding. In addition, we evaluated the efficacy of post-fusion hMPV B2 F protein as a vaccine candidate in BALB/c mice. Mice immunized with hMPV B2 post-fusion F protein showed a balanced Th1/Th2 immune response and generated neutralizing antibodies against both subgroup A2 and B2 hMPV strains, which protected the mice from hMPV challenge. Antibody competition analysis revealed the antibodies generated by immunization target two known antigenic sites (III and IV) on the hMPV F protein. Overall, this study provides new characteristics of the hMPV F protein, which may be informative for vaccine and therapy development. Importance Human metapneumovirus (hMPV) is an important cause of viral respiratory disease. In this paper, we report the X-ray crystal structure of the hMPV fusion (F) protein in the post-fusion conformation from genotype B. We also assessed binding of the hMPV F protein to heparin and heparan sulfate, a previously reported receptor for the hMPV F protein. Furthermore, we determined the immunogenicity and protective efficacy of post-fusion hMPV B2 F protein, which is the first study using a homogenous conformation of the protein. Antibodies generated in response to vaccination give a balanced Th1/Th2 response and target two previously discovered neutralizing epitopes.

The Bunyavirales order accommodates related viruses (bunyaviruses) with segmented, linear, single-stranded, negative- or ambi-sense RNA genomes. Their glycoproteins form capsomeric projections or spikes on the virion surface and play a crucial role in virus entry, assembly, morphogenesis. Bunyavirus glycoproteins are encoded by a single RNA segment as a polyprotein precursor that is co- and post-translationally cleaved by host cell enzymes to yield two mature glycoproteins, Gn and Gc (or GP1 and GP2 in arenaviruses). These glycoproteins undergo extensive N-linked glycosylation and despite their cleavage, remain associated to the virion to form an integral transmembrane glycoprotein complex. This review summarizes recent advances in our understanding of the molecular biology of bunyavirus glycoproteins, including their processing, structure, and known interactions with host factors that facilitate cell entry.

The brominated flame retardants (BFRs), 1,2-dibromo-4-(1,2 dibromoethyl)cyclohexane (TBECH) and 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE) bind to the androgen receptor (AR). in vitro bioassays have shown that TBECH is a potent androgen agonist while DPTE is a potent AR antagonist. Both TBECH and DPTE alter gene expression associated with AR regulation. However, it remains to be determined if TBECH and DPTE can affect the prostate. For this reason, we exposed CD1 mice to a 1:1 mixture of TBECH diastereomers alpha and beta, a 1:1 mixture of gamma and delta, and to DPTE, and tested their effects on prostate growth, histology and gene expression profiles. Castrated mice were used to study the androgenic effects of TBECH alpha beta and TBECH gamma delta while the antagonistic effects of DPTE were studied in non-castrated mice. We observed that testosterone and TBECH gamma delta increased body and prostate weights while TBECH alpha beta affected neither of them; and that DPTE had no effect on body weight but reduced prostate weight drastically. Histomorphometric analysis of the prostate revealed epithelial and glandular alterations in the TBECH gamma delta group comparable to those in testosterone group while alterations in the TBECH alpha beta group were less pronounced. DPTE displayed androgen antagonist activity reminiscent of castration. The transcription profile of the prostate was altered by castration and exposure to testosterone and to TBECH gamma delta reversed several of these changes. Testosterone and TBECH gamma delta also regulated the expression of several androgen responsive genes implicated in prostate growth and cancer. While DPTE resulted in a drastic reduction in prostate weight, it only affected a small number of genes. The results indicate that TBECH gamma delta and DPTE are of high human health concern as they may contribute to changes in prostate growth, histology and function

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of the Coronavirus disease (COVID-19) pandemic, has so far resulted in more than 1.1 M deaths and 40 M cases worldwide with no confirmed remedy yet available. Since the first outbreak in Wuhan, China in December 2019, researchers across the globe have been in a race to develop therapies and vaccines against the disease. SARS-CoV-2, similar to other previously identified Coronaviridae family members, encodes several structural proteins, such as spike, envelope, membrane, and nucleocapsid, that are responsible for host penetration, binding, recycling, and pathogenesis. Structural biology has been a key player in understanding the viral infection mechanism and in developing intervention strategies against the new coronavirus. The spike glycoprotein has drawn considerable attention as a means to block viral entry owing to its interactions with the human angiotensin-converting enzyme 2 (ACE2), which acts as a receptor. Here, we review the current knowledge of SARS-CoV-2 and its interactions with ACE2 and antibodies. Structural information of SARS-CoV-2 spike glycoprotein and its complexes with ACE2 and antibodies can provide key input for the development of therapies and vaccines against the new coronavirus.

Tetrazole derivatives are a prime class of heterocycles, very important to medicinal chemistry and drug design due to not only their bioisosterism to carboxylic acid and amide moieties but also to their metabolic stability and other beneficial physicochemical properties. Although more than 20 FDA-approved drugs contain 1 H- or 2 H-tetrazole substituents, their exact binding mode, structural biology, 3D conformations, and in general their chemical behavior is not fully understood. Importantly, multicomponent reaction (MCR) chemistry offers convergent access to multiple tetrazole scaffolds providing the three important elements of novelty, diversity, and complexity, yet MCR pathways to tetrazoles are far from completely explored. Here, we review the use of multicomponent reactions for the preparation of substituted tetrazole derivatives. We highlight specific applications and general trends holding therein and discuss synthetic approaches and their value by analyzing scope and limitations, and also enlighten their receptor binding mode. Finally, we estimated the prospects of further research in this field.

Background The paramyxovirus haemagglutinin-neuraminidase (HN) is a multifunctional protein that is responsible for attachment to receptors, removal of receptors from infected cells to prevent viral self-aggregation (neuraminidase, NA) and fusion promotion. It is commonly accepted that there are two receptor binding sites in the globular head of HN, and the second receptor binding site is only involved in the function of receptor binding and fusion promotion. Methods 10 conserved residues in the second receptor binding site of Newcastle disease virus (NDV) HN were chosen and substituted to alanine (A). The desired mutants were examined to detect the functional change in hemadsorption (HAD) ability, NA activity and fusion promotion ability. Results The HAD and fusion promotion ability of mutants C172A, R174A, C196A, D198A, Y526A and E547A were abolished. Compared with wild-type (wt) HN, the HAD of mutants T167A, S202A and R516A decreased to 55.81, 44.53, 69.02%, respectively, and the fusion promotion ability of these three mutants decreased to 54.74, 49.46, 65.26%, respectively; however, mutant G171A still maintained fusion promotion ability comparable with wt HN but had impaired HAD ability. All the site-directed mutations altered the NA activity of NDV HN without affecting protein cell surface expression. Conclusions The data suggest that mutants C172A, R174A, C196A, D198A, Y526A and E547A do not allow the conformational change that is required for fusion promotion ability and HAD activity, while the other mutants only affect the conformational change to a limited extent, except mutant G171A with intact fusion promotion ability. Overall, the conserved amino acids in the second receptor binding site, especially residues C172, R174, C196, D198, Y526 and E547, are crucial to normal NDV HN protein function.

Influenza subtype H3N2 viruses have circulated in humans for over 50 years, continuing to cause annual epidemics. Such viruses have undergone antigenic drift in response to immune pressure, reducing the protective effects of preexisting immunity to previously circulating H3N2 strains. The changes in hemagglutinin (HA) affiliated with drift have implications for the receptor binding properties of these viruses, affecting virus replication in the culture systems commonly used to generate and amplify vaccine strains. Therefore, the antigenic properties of the vaccines may not directly reflect those of the circulating strains from which they were derived, compromising vaccine efficacy. In order to reproducibly provide effective vaccines, it will be critical to understand the interrelationships between binding, antigenicity, and replication properties in different growth substrates., H3N2 strains of influenza A virus emerged in humans in 1968 and have continued to circulate, evolving in response to human immune pressure. During this process of “antigenic drift,” viruses have progressively lost the ability to agglutinate erythrocytes of various species and to replicate efficiently under the established conditions for amplifying clinical isolates and generating vaccine candidates. We have determined the glycome profiles of chicken and guinea pig erythrocytes to gain insights into reduced agglutination properties displayed by drifted strains and show that both chicken and guinea pig erythrocytes contain complex sialylated N-glycans but that they differ with respect to the extent of branching, core fucosylation, and the abundance of poly-N-acetyllactosamine (PL) [-3Galβ1-4GlcNAcβ1-]n structures. We also examined binding of the H3N2 viruses using three different glycan microarrays: the synthetic Consortium for Functional Glycomics array; the defined N-glycan array designed to reveal contributions to binding based on sialic acid linkage type, branched structures, and core modifications; and the human lung shotgun glycan microarray. The results demonstrate that H3N2 viruses have progressively lost their capacity to bind nearly all canonical sialylated receptors other than a selection of biantennary structures and PL structures with or without sialic acid. Significantly, all viruses displayed robust binding to nonsialylated high-mannose phosphorylated glycans, even as the recognition of sialylated structures is decreased through antigenic drift. IMPORTANCE Influenza subtype H3N2 viruses have circulated in humans for over 50 years, continuing to cause annual epidemics. Such viruses have undergone antigenic drift in response to immune pressure, reducing the protective effects of preexisting immunity to previously circulating H3N2 strains. The changes in hemagglutinin (HA) affiliated with drift have implications for the receptor binding properties of these viruses, affecting virus replication in the culture systems commonly used to generate and amplify vaccine strains. Therefore, the antigenic properties of the vaccines may not directly reflect those of the circulating strains from which they were derived, compromising vaccine efficacy. In order to reproducibly provide effective vaccines, it will be critical to understand the interrelationships between binding, antigenicity, and replication properties in different growth substrates.

Bacillus thuringiensis took advantage of important insect cellular proteins, such as chaperones, involved in maintaining protein homeostasis, to enhance its insecticidal activity. This constitutes a positive loop where the concentrations of Hsp90 and Hsp70 in the gut lumen are likely to increase as midgut cells burst due to Cry1A pore formation action. Hsp90 protects Cry1A protoxin from degradation and enhances receptor binding, resulting in increased toxicity. The effect of insect chaperones on Cry toxicity could have important biotechnological applications to enhance the toxicity of Cry proteins to insect pests, especially those that show low susceptibility to these toxins., Bacillus thuringiensis Cry proteins are pore-forming insecticidal toxins with specificity against different crop pests and insect vectors of human diseases. Previous work suggested that the insect host Hsp90 chaperone could be involved in Cry toxin action. Here, we show that the interaction of Cry toxins with insect Hsp90 constitutes a positive loop to enhance the performance of these toxins. Plutella xylostella Hsp90 (PxHsp90) greatly enhanced Cry1Ab or Cry1Ac toxicity when fed together to P. xylostella larvae and also in the less susceptible Spodoptera frugiperda larvae. PxHsp90 bound Cry1Ab and Cry1Ac protoxins in an ATP- and chaperone activity-dependent interaction. The chaperone Hsp90 participates in the correct folding of proteins and may suppress mutations of some client proteins, and we show here that PxHsp90 recovered the toxicity of the Cry1AbG439D protoxin affected in receptor binding, in contrast to the Cry1AbR99E or Cry1AbE129K mutant, affected in oligomerization or membrane insertion, respectively, which showed a slight toxicity improvement. Specifically, PxHsp90 enhanced the binding of Cry1AbG439D protoxin to the cadherin receptor. Furthermore, PxHsp90 protected Cry1A protoxins from degradation by insect midgut proteases. Our data show that PxHsp90 assists Cry1A proteins by enhancing their binding to the receptor and by protecting Cry protoxin from gut protease degradation. Finally, we show that the insect cochaperone protein PxHsp70 also increases the toxicity of Cry1Ac in P. xylostella larvae, in contrast to a bacterial GroEL chaperone, which had a marginal effect, indicating that the use of insect chaperones along with Cry toxins could have important biotechnological applications for the improvement of Cry insecticidal activity, resulting in effective control of insect pests.

Background and Objectives: Previously we have shown that synthetic lunasin, a 43 amino acid residue-containing peptide, after its central (intracisternal) administration in mice demonstrated antagonism against dopaminergic drug behavioural effects, indicating a putative antipsychotic/anti-schizophrenic profile of lunasin. The aims of the present studies were: to test whether lunasin would show an influence on the dopaminergic system after intranasal administration, and to examine the effect(s) of lunasin on serotonin and glutamatergic systems, which could play an essential role in antipsychotic action. Materials and Methods: Lunasin was administered intra-nasally at doses 0.1 and 1 nmol/mouse in ICR mice (n = 7&ndash, 8) and tested in an open field on hyperlocomotion caused by amphetamine, serotonin 5-HT 2A/2C receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)- 2-aminopropane (DOI), and glutamate NMDA receptor antagonist phencyclidine. Following behavioural testing, the contents of neurotransmitters and their metabolites in brain hemispheres (n = 6&ndash, 8) were assessed by ultra-high-performance liquid chromatography-time of flight mas-spectrometry (UHPLC-TOF-MS) method. Also, lunasin binding to serotonin receptors was assessed. Results: Lunasin intra-nasally fully normalized hyper-locomotion and brain monoamine levels in amphetamine- and DOI-treated mice brains. Phencyclidine behavioural effects were not influenced. In vitro receptor binding data demonstrated a low affinity of lunasin (at &micro, M concentrations) compared with DOI (nM concentrations) for the 5-HT2A and 5-HT2C receptors. Conclusions: These results demonstrated, for the first time, that the intranasal administration of oligopeptide lunasin normalized mice behaviour and brain monoamine levels in experimental psychosis mice models. Its neuro-regulatory effects indicated a usefulness of this peptide molecule for the design of novel psychotropic agents.

Background Two new subclades of influenza A(H3N2) viruses became prominent during the 2014-2015 Northern Hemisphere influenza season. The HA glycoproteins of these viruses showed sequence changes previously associated with alterations in receptor-binding properties. To address how these changes influence virus propagation, viruses were isolated and propagated in conventional MDCK cells and MDCK-SIAT1 cells, cells with enhanced expression of the human receptor for the virus, and analysed at each passage. Methods Gene sequence analysis was undertaken as virus was passaged in conventional MDCK cells and MDCK-SIAT1 cells. Alterations in receptor recognition associated with passage of virus were examined by haemagglutination assays using red blood cells from guinea pigs, turkeys and humans. Microneutralisation assays were performed to determine how passage-acquired amino acid substitutions and polymorphisms affected virus antigenicity. Results Viruses were able to infect MDCK-SIAT1 cells more efficiently than conventional MDCK cells. Viruses of both the 3C.2a and 3C.3a subclades showed greater sequence change on passage in conventional MDCK cells than in MDCK-SIAT1 cells, with amino acid substitutions being seen in both HA and NA glycoproteins. However, virus passage in MDCK-SIAT1 cells at low inoculum dilutions showed reducing infectivity on continued passage. Conclusions Current H3N2 viruses should be cultured in the MDCK-SIAT1 cell line to maintain faithful replication of the virus, and at an appropriate multiplicity of infection to retain infectivity.

Background Influenza viruses are undergoing continuous and rapid evolution. The fatal influenza A/H7N9 has drawn attention since the first wave of infections in March 2013, and raised more grave concerns with its increased potential to spread among humans. Experimental studies have revealed several host and virulence markers, indicating differential host binding preferences which can help estimate the potential of causing a pandemic. Here we systematically investigate the sequence pattern and structural characteristics of novel influenza A/H7N9 using computational approaches. Results The sequence analysis highlighted mutations in protein functional domains of influenza viruses. Molecular docking and molecular dynamics simulation revealed that the hemagglutinin (HA) of A/Taiwan/1/2017(H7N9) strain enhanced the binding with both avian and human receptor analogs, compared with the previous A/Shanghai/02/2013(H7N9) strain. The Molecular Mechanics - Poisson Boltzmann Surface Area (MM-PBSA) calculation revealed the change of residue-ligand interaction energy and detected the residues with conspicuous binding preference. Conclusion The results are novel and specific to the emerging influenza A/Taiwan/1/2017(H7N9) strain compared with A/Shanghai/02/2013(H7N9). Its enhanced ability to bind human receptor analogs, which are abundant in the human upper respiratory tract, may be responsible for the recent outbreak. Residues showing binding preference were detected, which could facilitate monitoring the circulating influenza viruses. Electronic supplementary material The online version of this article (10.1186/s12864-018-4461-z) contains supplementary material, which is available to authorized users.

The surface glycoproteins of coronaviruses play an important role in receptor binding and cell entry. Different coronaviruses interact with their specific receptors to enter host cells. Lentiviruses pseudotyped with their spike proteins (S) were compared to analyze the entry efficiency of various coronaviruses. Our results indicated that S proteins from different coronaviruses displayed varied abilities to mediate pseudotyped virus infection. Furthermore, the cell tropisms of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been characterized by live and pseudotyped viruses. Both live and pseudoviruses could infected Vero- CCL-81 (monkey kidney), Huh-7 (human liver), and PK-15 (pig kidney) cells efficiently. CCL94 (cat kidney) cells could be infected efficiently by TGEV but not PEDV. Overall, our study provides new insights into the mechanisms of viral entry and forms a basis for antiviral drug screening.

Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemagglutinin (HA) and Neuraminidase (NA) of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF: receptor binding, esterase activity and membrane fusion. Electronic supplementary material The online version of this article (doi:10.1007/s13238-015-0193-x) contains supplementary material, which is available to authorized users.

Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA) of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering) in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L) to enhance replication in mammals and retained replication efficiency in the original avian host.

Influenza A(H7N9) viruses have caused a large number of zoonotic infections since their emergence in 2013. They remain a public health concern due to the repeated high levels of infection with these viruses and their perceived pandemic potential. A major factor that determines influenza A virus fitness and therefore transmissibility is the interaction of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) with the cell surface receptor sialic acid. Typically, the HA is responsible for binding to the sialic acid to allow virus internalization and the NA is a sialidase responsible for cleaving sialic acid to aid virus spread and release. N9 NA has previously been shown to have receptor binding properties mediated by a sialic acid binding site, termed the hemadsorption (Hb) site, which is discrete from the enzymatically active sialidase site. This study investigated the N9 NA from a zoonotic H7N9 virus strain in order to determine its possible role in virus receptor binding. We demonstrate that this N9 NA has an active Hb site which binds to sialic acid, which enhances overall virus binding to sialic acid receptor analogues. We also show that the N9 NA can also contribute to receptor binding due to unusual kinetic characteristics of the sialidase site which specifically enhance binding to human-like α2,6-linked sialic acid receptors. IMPORTANCE The interaction of influenza A virus glycoproteins with cell surface receptors is a major determinant of infectivity and therefore transmissibility. Understanding these interactions is important for understanding which factors are necessary to determine pandemic potential. Influenza A viruses generally mediate binding to cell surface sialic acid receptors via the hemagglutinin (HA) glycoprotein, with the neuraminidase (NA) glycoprotein being responsible for cleaving the receptor to allow virus release. Previous studies showed that the NA proteins of the N9 subtype can bind sialic acid via a separate binding site distinct from the sialidase active site. This study demonstrates for purified protein and virus that the NA of the zoonotic H7N9 viruses has a binding capacity via both the secondary binding site and unusual kinetic properties of the sialidase site which promote receptor binding via this site and which enhance binding to human-like receptors. This could have implications for understanding human-to-human transmission of these viruses.

Citrus aurantium L. (bitter orange) extracts that contain p‐synephrine as the primary protoalkaloid are widely used for weight loss/weight management, sports performance, appetite control, energy, and mental focus and cognition. Questions have been raised about the safety of p‐synephrine because it has some structural similarity to ephedrine. This review focuses on current human, animal, in vitro, and mechanistic studies that address the safety, efficacy, and mechanisms of action of bitter orange extracts and p‐synephrine. Numerous studies have been conducted with respect to p‐synephrine and bitter orange extract because ephedra and ephedrine were banned from use in dietary supplements in 2004. Approximately 30 human studies indicate that p‐synephrine and bitter orange extracts do not result in cardiovascular effects and do not act as stimulants at commonly used doses. Mechanistic studies suggest that p‐synephrine exerts its effects through multiple actions, which are discussed. Because p‐synephrine exhibits greater adrenergic receptor binding in rodents than humans, data from animals cannot be directly extrapolated to humans. This review, as well as several other assessments published in recent years, has concluded that bitter orange extract and p‐synephrine are safe for use in dietary supplements and foods at the commonly used doses. Copyright © 2017 The Authors Phytotherapy Research Published by John Wiley & Sons Ltd.

Unlike the well-established picture for the entry of enveloped viruses, the mechanism of cellular entry of non-enveloped eukaryotic viruses remains largely mysterious. Picornaviruses are representative models for such viruses, and initiate this entry process by their functional receptors. Here we present the structural and functional studies of SCARB2, a functional receptor of the important human enterovirus 71 (EV71). SCARB2 is responsible for attachment as well as uncoating of EV71. Differences in the structures of SCARB2 under neutral and acidic conditions reveal that SCARB2 undergoes a pivotal pH-dependent conformational change which opens a lipid-transfer tunnel to mediate the expulsion of a hydrophobic pocket factor from the virion, a pre-requisite for uncoating. We have also identified the key residues essential for attachment to SCARB2, identifying the canyon region of EV71 as mediating the receptor interaction. Together these results provide a clear understanding of cellular attachment and initiation of uncoating for enteroviruses. Electronic supplementary material The online version of this article (doi:10.1007/s13238-014-0087-3) contains supplementary material, which is available to authorized users.

Clostridium difficile induces antibiotic-associated diarrhea due to the release of toxin A (TcdA) and toxin B (TcdB), the latter being its main virulence factor. The epidemic strain NAP1/027 has an increased virulence attributed to different factors. We compared cellular intoxication by TcdBNAP1 with that by the reference strain VPI 10463 (TcdBVPI). In a mouse ligated intestinal loop model, TcdBNAP1 induced higher neutrophil recruitment, cytokine release, and epithelial damage than TcdBVPI. Both toxins modified the same panel of small GTPases and exhibited similar in vitro autoprocessing kinetics. On the basis of sequence variations in the frizzled-binding domain (FBD), we reasoned that TcdBVPI and TcdBNAP1 might have different receptor specificities. To test this possibility, we used a TcdB from a NAP1 variant strain (TcdBNAP1v) unable to glucosylate RhoA but with the same receptor-binding domains as TcdBNAP1. Cells were preincubated with TcdBNAP1v to block cellular receptors, prior to intoxication with either TcdBVPI or TcdBNAP1. Preincubation with TcdBNAP1v blocked RhoA glucosylation by TcdBNAP1 but not by TcdBVPI, indicating that the toxins use different host factors for cell entry. This crucial difference might explain the increased biological activity of TcdBNAP1 in the intestine, representing a contributing factor for the increased virulence of the NAP1/027 strain. Universidad de Costa Rica/[803-B8-117]/UCR/Costa Rica Universidad de Costa Rica/[803-B7-183]/UCR/Costa Rica Universidad de Costa Rica/[803-B7-158]/UCR/Costa Rica Universidad de Costa Rica/[803-B6-657]/UCR/Costa Rica UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET) UCR::Vicerrectoría de Docencia::Salud::Facultad de Farmacia UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Laboratorio de Ensayos Biológicos (LEBI) UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicina

Prophylaxis against influenza could be improved by the development of a stable, easy to deliver, potent mucosal vaccine. In this study, we spray-freeze-dried (SFD) whole inactivated virus influenza vaccine (WIV) alone or supplemented with monophosphoryl lipid A (MPLA) using inulin as a lyoprotectant. Physical characterization revealed that the SFD powder consisted of highly porous particles with a size distribution suitable for pulmonary administration. The receptor-binding properties of WIV and the immunostimulatory properties of MPLA were preserved after spray-freeze-drying as indicated by unchanged hemagglutination titers and a retained ability of the vaccine to activate NFkB after incubation with a reporter cell line, respectively. Pulmonary vaccination of mice with MPLA-adjuvanted liquid or powder WIV resulted in induction of higher mucosal and systemic antibody concentrations than vaccination with non-adjuvanted formulations. When exposed to influenza virus, mice immunized with MPLA-adjuvanted pulmonary vaccine showed similar protection in terms of reduction in lung virus titers and prevention ofweight loss as mice immunized intramuscularly with subunit vaccine. Characterization of the antibody response revealed a balanced IgG2a-to-IgG1 profile along with induction of both memory IgA- and IgG-producing B cells in mice immunized with MPLA-adjuvanted vaccine. These studies suggest that themucosal and systemic immune responses to pulmonary delivered influenza vaccines can be significantly enhanced by using MPLA as adjuvant. MPLA-adjuvanted SFD vaccine was particularly effective implying that delivery of adjuvanted vaccine powder to the lungs can be an attractive way of immunization against influenza. (C) 2013 Elsevier B.V. All rights reserved.

Mutant H5N1 influenza viruses have been isolated from humans that have increased human receptor avidity. We have compared the receptor binding properties of these mutants with those of wild-type viruses, and determined the structures of their haemagglutinins in complex with receptor analogues. Mutants from Vietnam bind tighter to human receptor by acquiring basic residues near the receptor binding site. They bind more weakly to avian receptor because they lack specific interactions between Asn-186 and Gln-226. In contrast, a double mutant, Δ133/Ile155Thr, isolated in Egypt has greater avidity for human receptor while retaining wild-type avidity for avian receptor. Despite these increases in human receptor binding, none of the mutants prefers human receptor, unlike aerosol transmissible H5N1 viruses. Nevertheless, mutants with high avidity for both human and avian receptors may be intermediates in the evolution of H5N1 viruses that could infect both humans and poultry., Highlights • H5N1 influenza virus binding. • Haemagglutinin receptor specificity using biolayer interferometry. • Haemagglutinin receptor complex crystal structure determination.

Sialic acid linked to glycoproteins and gangliosides is used by many viruses as a receptor for cell entry. These viruses include important human and animal pathogens, such as influenza, parainfluenza, mumps, corona, noro, rota, and DNA tumor viruses. Attachment to sialic acid is mediated by receptor binding proteins that are constituents of viral envelopes or exposed at the surface of non-enveloped viruses. Some of these viruses are also equipped with a neuraminidase or a sialyl-O-acetyl-esterase. These receptor-destroying enzymes promote virus release from infected cells and neutralize sialic acid-containing soluble proteins interfering with cell surface binding of the virus. Variations in the receptor specificity are important determinants for host range, tissue tropism, pathogenicity, and transmissibility of these viruses.

Evodiamine, a naturally occurring indole alkaloid, is one of the main bioactive ingredients of Evodiae fructus. With respect to the pharmacological actions of evodiamine, more attention has been paid to beneficial effects in insults involving cancer, obesity, nociception, inflammation, cardiovascular diseases, Alzheimer's disease, infectious diseases and themoregulative effects. Evodiamine has evolved a superior ability to bind various proteins, so we also argue that it is good starting point for multi-target drugs. This review is primarily addressed to the description of the recent advances in the biological activity studies of evodiamine, with a focus on pharmacological mechanism. The present review also includes the pharmacokinetics and the detailed exploration of target-binding properties of evodiamine in an attempt to provide a direction for further multi-target drug design.

H9N2 avian influenza viruses are primarily a disease of poultry; however, they occasionally infect humans and are considered a potential pandemic threat. Little work has been performed to assess the intrinsic biochemical properties related to zoonotic potential of H9N2 viruses. The objective of this study, therefore, was to investigate H9N2 haemagglutinins (HAs) using two well-known correlates for human adaption: receptor-binding avidity and pH of fusion. Receptor binding was characterized using bio-layer interferometry to measure virus binding to human and avian-like receptor analogues and the pH of fusion was assayed by syncytium formation in virus-infected cells at different pHs. We characterized contemporary H9N2 viruses of the zoonotic G1 lineage, as well as representative viruses of the zoonotic BJ94 lineage. We found that most contemporary H9N2 viruses show a preference for sulphated avian-like receptor analogues. However, the 'Eastern' G1 H9N2 viruses displayed a consistent preference in binding to a human-like receptor analogue. We demonstrate that the presence of leucine at position 226 of the HA receptor-binding site correlated poorly with the ability to bind a human-like sialic acid receptor. H9N2 HAs also display variability in their pH of fusion, ranging between pH 5.4 and 5.85 which is similar to that of the first wave of human H1N1pdm09 viruses but lower than the pH of fusion seen in zoonotic H5N1 and H7N9 viruses. Our results suggest possible molecular mechanisms that may underlie the relatively high prevalence of human zoonotic infection by particular H9N2 virus lineages.

Emergence and intercontinental spread of highly pathogenic avian influenza A(H5Nx) virus clade 2.3.4.4 is unprecedented. H5N8 and H5N2 viruses have caused major economic losses in the poultry industry in Europe and North America, and lethal human infections with H5N6 virus have occurred in Asia. Knowledge of the evolution of receptor-binding specificity of these viruses, which might affect host range, is urgently needed. We report that emergence of these viruses is accompanied by a change in receptor-binding specificity. In contrast to ancestral clade 2.3.4 H5 proteins, novel clade 2.3.4.4 H5 proteins bind to fucosylated sialosides because of substitutions K222Q and S227R, which are unique for highly pathogenic influenza virus H5 proteins. North American clade 2.3.4.4 virus isolates have retained only the K222Q substitution but still bind fucosylated sialosides. Altered receptor-binding specificity of virus clade 2.3.4.4 H5 proteins might have contributed to emergence and spread of H5Nx viruses.

Subtype H6 influenza A viruses (IAVs) are commonly detected in wild birds and domestic poultry and can infect humans. In 2010, a H6N6 virus emerged in southern China, and since then, it has caused sporadic infections among swine. We show that this virus binds to α2,6-linked and α2,3-linked sialic acids. Mutations at residues 222 (alanine to valine) and 228 (glycine to serine) of the virus hemagglutinin (HA) affected its receptor-binding properties. Experiments showed that the virus has limited transmissibility between ferrets through direct contact or through inhalation of infectious aerosolized droplets. The internal genes of the influenza A(H1N1)pdm09 virus, which is prevalent in swine worldwide, increases the replication efficiency of H6N6 IAV in the lower respiratory tract of ferrets but not its transmissibility between ferrets. These findings suggest H6N6 swine IAV (SIV) currently poses a moderate risk to public health, but its evolution and spread should be closely monitored.

As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process., Highlights • Changes in receptor binding of HA during H5N1 human infection were identified. • Single A134V and L151F substitutions caused reduced affinity for avian receptors. • Glycan array analyses were used to identify changes in receptor binding specificity. • Structural basis for altered receptor binding was examined by X-ray crystallography.

BACKGROUND: Human noroviruses (NoVs) are the main cause of gastroenteritis worldwide. The most commonly detected NoV strains belong to the genetically diverse GII.4 genotype, with new pandemic variants emerging periodically. Despite extensive efforts, NoV investigation has been hampered by the lack of an effective in vitro cell culture system. However, NoV-derived recombinant virus-like particles (VLPs) resembling empty capsids are good surrogates for analysing NoV antigenicity and virus-ligand interactions. NoV VLPs have been reported to bind to histo-blood group antigens (HBGAs). We have analysed the ability of NoV VLPs derived from GI.1 genotype and from three GII.4 genotype variants, GII.4-1999, GII.4-2004 and GII.4-2006b, to bind to porcine gastric mucin (PGM), human saliva and differentiated human intestinal Caco-2 cells (D-Caco-2 cells). RESULTS: Distinct patterns of saliva binding with the NoV GII.4 variant VLPs were observed, although they bound to D-Caco-2 cells independently of the expression of HBGAs. Monoclonal antibodies against Lewis antigens were able to block the binding of NoV VLPs to saliva, but not to D-Caco-2 cells. Blocking HBGAs on the surface of D-Caco-2 cells with specific monoclonal antibodies did not affect NoV VLP binding to cellular membranes. Co-localisation of Lewis y (Le(y)) and H-type 2 antigens with NoV VLPs was not observed by immunofluorescence assays. CONCLUSION: Although the binding of NoV VLPs of GII.4 genotype variants to human saliva samples occur with distinct HBGA binding patterns and can be blocked by antibodies against Lewis antigens, their attachment to D-Caco-2 cells can be mediated by other receptors, which still need further investigation.

Objective: We developed an assay that detects autoantibodies against the main immunogenic region (MIR) located at the extracellular end of the nicotinic acetylcholine receptor (AChR) a subunit, and investigated its clinical relevance in myasthenia gravis (MG). Methods: In this retrospective cohort study, we measured MIR antibody (Ab) titres in sera obtained before treatment and analysed their associations with clinical parameters in 102 MG patients from two neurological centres. MIR Ab titres were determined using a modified competition immunoprecipitation assay in the presence or absence of monoclonal antibody 35. Results: 11 of 23 (47.8%) ocular type and 66 of 72 (91.7%) generalised type MG patients were positive for the presence of MIR Abs, defined as a titre >16.8% (3 SDs above the mean for 70 healthy controls). A significantly higher MIR Ab titre (p, Journal of Neurology, Neurosurgery & Psychiatry, 83(9), pp.935-940; 2012

Recent studies indicate that membrane cholesterol can associate with G protein-coupled receptors (GPCRs) and affect their function. Previously, we reported that manipulation of membrane cholesterol affects ligand binding and signal transduction of the type 1 cholecystokinin receptor (CCK1R), a Class A GPCR. We now demonstrate that the closely related type 2 cholecystokinin receptor (CCK2R) does not share this cholesterol sensitivity. The sequences of both receptors reveal almost identical cholesterol interaction motifs in analogous locations in transmembrane segments two, three, four, and five. The disparity in cholesterol sensitivity between these receptors, despite their close structural relationship, provides a unique opportunity to define the possible structural basis of cholesterol sensitivity of CCK1R. To evaluate the relative contributions of different regions of CCK1R to cholesterol sensitivity, we performed ligand binding studies and biological activity assays of wild-type and CCK2R/CCK1R chimeric receptor-bearing Chinese hamster ovary cells after manipulation of membrane cholesterol. We also extended these studies to site-directed mutations within the cholesterol interaction motifs. The results contribute to a better understanding of the structural requirements for cholesterol sensitivity in CCK1R and provides insight into the function of other cholesterol-sensitive Class A GPCRs.

Summary Egg-based seasonal influenza vaccines are the major preventive countermeasure against influenza virus. However, their effectiveness can be compromised when antigenic changes arise from egg-adaptive mutations on influenza hemagglutinin (HA). The L194P mutation is commonly observed in egg-based H3N2 vaccine seed strains and significantly alters HA antigenicity. An approach to prevent L194P would therefore be beneficial. We show that emergence of L194P during egg passaging can be impeded by preexistence of a G186V mutation, revealing strong incompatibility between these mutations. X-ray structures illustrate that individual G186V and L194P mutations have opposing effects on the HA receptor-binding site (RBS), and when both G186V and L194P are present, the RBS is severely disrupted. Importantly, wild-type HA antigenicity is maintained with G186V, but not L194P. Our results demonstrate that these epistatic interactions can be used to prevent the emergence of mutations that adversely alter antigenicity during egg adaptation., Graphical Abstract, Highlights • Most H3N2 egg isolates carry hemagglutinin mutation G186V or L194P, but not both • Hemagglutinin double mutation G186V/L194P is highly deleterious to the virus • Hemagglutinin double mutation G186V/L194P disrupts the receptor-binding site • Wild-type hemagglutinin antigenicity is maintained in G186V, but not in L194P, Wu et al. characterize the incompatibility between two major egg-adaptive mutations in human H3N2 virus hemagglutinin, G186V and L194P, which confer either minimal or significant antigenic change, respectively. This study suggests that the antigenically disruptive mutation L194P that occurs during egg-based influenza vaccine production can be prevented by mutational incompatibility.

Infectious salmon anaemia virus (ISAV) is a piscine orthomyxovirus causing a serious disease in farmed Atlantic salmon ( Salmo salar L.). The virus surface glycoprotein hemagglutinin-esterase (HE) is responsible for both viral attachment and release. Similarity to bovine and porcine torovirus hemagglutinin-esterase (BToV HE, PToV HE), bovine coronavirus HE (BCoV HE) and influenza C hemagglutinin-esterase-fusion (InfC HEF) proteins were exploited in a computational homology-based structure analysis of ISAV HE. The analysis resolved structural aspects of the protein and identified important features of relevance to ISAV HE activity. By recombinant expression and purification of secretory HE (recHE) proteins, receptor-binding and quantitative analyses of enzymatic activities displayed by ISAV HE molecules are presented for the first time. Three different recHE molecules were constructed: one representing a high virulent isolate, one a low virulent, while in the third a Ser 32 to Ala 32 amino acid substitution was introduced in the enzymatic catalytic site as inferred from the model. The three amino acid differences between the high and low virulent variants, of which two localized to the putative receptor-binding domain and one in the esterase domain, had no impact on receptor-binding or -release activities. In contrast, the Ser 32 amino acid substitution totally abolished enzymatic activity while receptor binding increased, as observed by agglutination of Atlantic salmon red blood cells. This demonstrates the essential role of a serine in the enzyme's catalytic site. In conclusion, structural analysis of ISAV HE in combination with selected recHE proteins gave insights into structure–function relationships and opens up for further studies aiming at dissecting molecular determinants of ISAV virulence.

Selection of high-growth virus variants of strain B/Victoria/504/2000 by serial passage in eggs resulted in three amino acid substitutions, G141E, R162M, and D196Y, in the vicinity of the receptor-binding pocket of viral hemagglutinin. Virus variants containing the identified amino acid substitutions, individually or in various combinations, were constructed using reverse genetics and analyzed for their receptor-binding properties using glycan microarray platform. Three different patterns of virus binding were revealed. A low-growth virus variant, corresponding to the original egg-derived virus B/Victoria/504/2000 prior to acquisition of amino acid changes G141E, R162M, and D196Y, had a clear preference for the oligosaccharide chains terminated with alpha2-6-linked sialic acid with very weak binding of the glycans terminated with alpha2-3-linked sialic acid. Amino acid substitutions R162M and D196Y had similar effects, resulting in viruses that bound with high efficiency almost all terminally sialylated glycans represented on the array regardless of the type of glycosidic linkage. In contrast, substitution of G141E alone, or in combinations with the other two amino acid substitutions, significantly restricted virus glycan-binding capabilities. All virus variants possessing this substitution lost the ability to bind glycans with alpha2-6 glycosidic linkage as well as most of the glycans with alpha2-3 glycosidic linkage. Linear penta- and heptasaccharide chains represented at the non-reducing end by alpha2-3 sialylated Type-II motif (LacNAc) were the only structures bound with high affinity by the virus variants with G141E substitution. In all cases when the effects on virus binding of individual amino acid substitutions differed, the effect of R162M was subordinate to the effect of either G141E or D196Y.

Triggering receptor expressed in myeloid (TREM) cells 2, a receptor expressed by myeloid cells, osteoclasts and microglia, is known to play a protective role in bones and brain. Mutations of the receptor (or of its coupling protein, DAP12) sustain in fact a genetic disease affecting the two organs, the polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy (PLOSL or Nasu-Hakola disease). So far, specific agonist(s) of TREM2 have not been identified and its (their) transduction mechanisms are largely unknown. Heat shock protein 60 (Hsp60) is a mitochondrial chaperone that can also be harboured at the cell surface. By using constructs including the extracellular domain of TREM2 and the Fc domain of IgGs we have identified Hsp60 as the only TREM2-binding protein exposed at the surface of neuroblastoma N2A cells and astrocytes, and lacking in U373 astrocytoma. Treatment with Hsp60 was found to stimulate the best known TREM2-dependent process, phagocytosis, however, only in the microglial N9 cells rich in the receptor. Upon TREM2 down-regulation, the Hsp60-induced stimulation of N9 phagocytosis was greatly attenuated. Hsp60 is also released by many cell types, segregated within exosomes or shedding vesicles which might then undergo dissolution. However, the affinity of its binding (Kd = 3.8 μM) might be too low for the soluble chaperone released from the vesicles to the extracellular space to induce a significant activation of TREM2. It might in contrast be appropriate for the binding of TREM2 to Hsp60 exposed at the surface of cells closely interacting with microglia. The ensuing stimulation of phagocytosis could play protective effects on the brain. © 2009 International Society for Neurochemistry.

The development of functional brain nuclear medicine techniques and their application in the investigation of neuropsychiatric disorders, have contributed significantly in the illumination of the underlying pathophysiological processes of these disorders. Furthermore, Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) brain studies provide information in early diagnosis, differential diagnosis, development of new drugs, and monitoring the response to therapeutic management. SPECT and PET brain imaging require the use of radiopharmaceuticals that cross the intact Blood Brain Barrier (BBB). Such radiotracers have been used in regional Cerebral Blood Flow (rCBF) SPECT and PET imaging and brain metabolism imaging with PET; these are well established methods in the diagnosis and management of various cerebral vascular diseases (e.g. stroke, dementia, epilepsy). Advances in radiotracer chemistry have resulted in the development of molecular imaging which represents the molecular and cellular processes of neurochychiatric diseases. SPECT and PET molecular imaging has become available for the study of acetylcholinergic, dopaminergic and serotonergic systems, as well as for benzodiazepine and opioid receptors, with promising results. More studies are needed to validate the role of molecular imaging in the clinical practice of neuropsychiatric disorders. © 2008 Bentham Science Publishers Ltd. 3 2 99 107

The special issue “Enterotoxins: Microbial Proteins and Host Cell Dysregulation” is comprised of research articles and reviews covering a diverse group of toxins that affect the gut and dysregulate host immune response in mechanistically different ways. [...]

The study reports the synthesis and biological evaluation of two opioid analogs, a monomer and a dimer, obtained as products of the solid-phase, side-chain to side-chain cyclization of the pentapeptide Tyr-d-Lys-Phe-Phe-AspNH2 . The binding affinities to the mu, delta, and kappa opioid receptors, as well as results obtained in a calcium mobilization functional assay are reported. Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 1 was a potent and selective full agonist of mu with sub-nanomolar affinity, while the dimer (Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 )2 2 showed a significant mixed mu/kappa affinity, acting as an agonist at the mu. Molecular docking computations were utilized to explain the ability of the dimeric cyclopeptide 2 to interact with the receptor. Interestingly, in spite of the increased ring size, the higher flexibility allowed 2 to fold and fit into the mu receptor binding pocket. Both cyclopeptides were shown to elicit strong antinociceptive activity after intraventricular injection but only cyclomonomer 1 was able to cross the blood-brain barrier. However, the cyclodimer 2 displayed a potent peripheral antinociceptive activity in a mouse model of visceral inflammatory pain. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 309-317, 2016.

The glycoprotein envelope of alphaviruses consists of two proteins, E1 and E2. E1 is responsible for fusion and E2 is responsible for receptor binding. An atomic structure is available for E1, but one for E2 has not been reported. In this study, transposon linker-insertion mutagenesis was used to probe the function of different domains of E2. A library of mutants, containing 19 amino acid insertions in the E2 glycoprotein sequence of the prototype alphavirus, Sindbis virus (SINV), was generated. Fifty-seven independent E2 insertions were characterized, of which more than half (67%) gave rise to viable virus. The wild-type-like mutants identify regions that accommodate insertions without perturbing virus production and can be used to insert targeting moieties to direct SINV to specific receptors. The defective and lethal mutants give insight into regions of E2 important for protein stability, transport to the cell membrane, E1–E2 contacts, and receptor binding.

International audience; In mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play a key role in inhibiting pluripotency-maintaining genes and activating some major actors of cell differentiation. To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with RAR/RXR genomic redistribution during the differentiation process. In-depth analysis of RAR/RXR binding sites occupancy dynamics and composition show that in undifferentiated cells, RAR/RXR interact with genomic regions characterized by binding of pluripotency-associated factors and high prevalence of the non-canonical DR0-containing RA response element. By contrast, in differentiated cells, RAR/RXR bound regions are enriched in functional Sox17 binding sites and are characterized with a higher frequency of the canonical DR5 motif. Our data offer an unprecedentedly detailed view on the action of RA in triggering pluripotent cell differentiation and demonstrate that RAR/RXR action is mediated via two different sets of regulatory regions tightly associated with cell differentiation status.

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Bacillus thuringiensis subs israelensis produces Cry toxins active against mosquitoes. Receptor binding is a key determinant for specificity of Cry toxins composed of three domains. We found that exposed loop alpha-8 of Cry11Aa toxin, located in domain II, is an important epitope involved in receptor interaction. Synthetic peptides corresponding to exposed regions in domain II (loop alpha-8, beta-4 and loop 3) competed binding of Cry11Aa to membrane vesicles from Aedes aegypti midgut microvilli. The role of loop alpha-8 of Cry11A in receptor interaction was demonstrated by phage display and site-directed mutagenesis. We isolated a peptide-displaying phage (P5.tox), that recognizes loop alpha-8 in Cry11Aa, interferes interaction with the midgut receptor and attenuates toxicity in bioassay. Loop alpha-8 mutants affected in toxicity and receptor binding were characterized.

Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been controversial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that several of these specifically recognize EPOR by Western blot, immunoprecipitation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment.

Interspecies differences in PRL-receptor binding and their relationship with bioactivity deserve investigation since cross-reactivity is relevant to the design of many experiments. We have previously shown that the lower affinity of rabbit prolactin (rbPRL) binding to its homologous receptor is due to its faster and more complete dissociation compared with that of ovine PRL (oPRL). In order to obtain sufficient amounts of rbPRL to study the functional consequences of its low affinity homologous interaction, rbPRL was expressed recombinantly in Escherichia coli (rec rbPRL) as insoluble inclusion bodies, refolded and purified to homogeneity, yielding electrophoretically pure, over 98% monomeric rec rbPRL. Proper renaturation of rec rbPRL was evidenced by comparison of its CD spectra, binding parameters and bioactivity with those determined for the rbPRL. The binding potency of rec rbPRL to its receptor, expressed either endogenously in the mammary gland or recombinantly in mammalian cells is one log unit lower than that to the receptor expressed recombinantly in insect cells. This difference is probably related to differences in cell-dependent receptor densities. The proliferation potency of rbPRL or rec rbPRL was one log unit lower than that of oPRL, consistent with its lower binding affinity, but the differentiation potencies of these PRLs were similar. Thus, the proliferation activity is sensitive to PRL-receptor affinity and dissociation kinetics, whereas the differentiation response is marginally modulated. (C) 2014 Elsevier Inc. All rights reserved.

Retroviral envelope proteins are heavily glycosylated. In some cases, glycosylation has been shown to be important for folding, protein stability, immune evasion, or receptor usage. The receptor-binding subunit (SU or gp85) of the envelope protein (EnvA) of the avian sarcoma/leukosis virus, subtype A (ASLV-A), contains 11 potential N-linked glycosylation sites (NXS/T). To address the importance of N-linked glycosylation for the function of EnvA, we prepared a series of EnvA proteins lacking one or more of these carbohydrate addition sites. Using site-directed mutagenesis, we mutated the S or T in each NXS/T glycosylation sequon to A. We also prepared EnvAs bearing selected double and triple mutations. We examined each mutant EnvA for its ability to be expressed at the cell surface, proteolytically processed into gp85 and gp37, incorporated into MLV pseudotyped virions, and to support infection of cells expressing the ASLV-A receptor, Tva. Eight single mutations were well tolerated, and, in general, EnvA was able to tolerate double mutations of these glycosylation sites. Triple mutations were more variable in their effects. Of the three glycosylation sites important for EnvA function, two are important for folding (EnvA production and processing were severely impaired). For the third, although EnvA processing was impaired, significant amounts of processed EnvA were expressed at the cell surface and incorporated into virions. Nonetheless, this mutant EnvA, EnvADeltaNg10, was unable to support infection. Further examination of EnvADeltaNg10 revealed that it was unable to bind Tva and was severely impaired for binding to a monoclonal antibody which inhibits receptor binding. This work has therefore identified a single N-linked glycosylation site in the SU domain of EnvA that is critical for binding between EnvA and its receptor, Tva.

The Protoparvovirus (PtPV) genus of the Parvoviridae family of viruses includes important animal pathogens and reference molecular models for the entire family. Some virus members of the PtPV genus have arisen as promising tools to treat tumoral processes, as they exhibit marked oncotropism and oncolytic activities while being nonpathogenic for humans. The PtPVs invade and replicate within the nucleus making extensive use of the transport, transcription and replication machineries of the host cells. In order to reach the nucleus, PtPVs need to cross over several intracellular barriers and traffic through different cell compartments, which limit their infection efficiency. In this review we summarize molecular interactions, capsid structural transitions and hijacking of cellular processes, by which the PtPVs enter and deliver their single-stranded DNA genome into the host cell nucleus. Understanding mechanisms that govern the complex PtPV entry will be instrumental in developing approaches to boost their anticancer therapeutic potential and improving their safety profile., Spanish Ministerio de Economía y Competitividad, S2013/ABI-2906-FEDER (Comunidad de Madrid); and by an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa (CSIC-UAM)

We found that the sequences YPLDL and YPLDLF in the large subunit of spinach D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) met the structure YP-aliphatic amino acid which might have opioid activity. We then synthesized these peptides to test their opioid activity. The IC(50) of these peptides in mouse vas deferens assay were 51.0 microM and 24.4 microM, respectively, and those in delta receptor binding assay using [(3)H]deltorphin II as radioligand were 2.09 microM and 0.93 microM, respectively. Both peptides were selective for delta receptor. We named them rubiscolin-5 and -6, respectively. Rubiscolin-5 and -6 have antinociceptive activity in mice after i.c.v. or oral administration. The enzymatic conditions to release rubiscolin were investigated using both spinach Rubisco and synthetic fragment peptides. This is the first example of bioactive peptides derived from plant Rubisco.

We analyzed the tissue distribution of apelin mRNA in rats by a quantitative reverse transcription-polymerase chain reaction and that of immunoreactive apelin (ir-apelin) by an enzyme immunoassay (EIA) using a monoclonal antibody. The expression levels of apelin mRNA and ir-apelin seemed to be consistent among tissues: they were highly expressed in the lung and mammary gland. By the combination of gel filtration and EIA, we found that the molecular forms of apelin differ among respective tissues: apelin molecules with sizes close to apelin-36 (long forms) were major components in the lung, testis, and uterus, but both long and short (whose sizes were close to [