Stability of enveloped and nonenveloped viruses in hydrolyzed gelatin liquid formulation.

BACKGROUND: The thermal stability of viruses in gelatin liquid formulations for medical research and application is poorly understood and this study aimed to examine the thermal stability of 4 enveloped and nonenveloped DNA and RNA viruses in hydrolyzed gelatin liquid formulations. METHODS: Bovine herpesvirus (BHV) was used as a model virus to examine the molecular weight (MW), concentration and gelatin type and to optimize virus stability in liquid formulations at 25 °C and 4 °C. Using the model virus liquid formulation, the stability of multiple enveloped and nonenveloped RNA and DNA viruses, including parainfluenza virus, reovirus (RV), BHV, and adenovirus (AdV), was monitored over up to a 30-week storage period. RESULTS: The BHV model virus was considered stable after 3 weeks in hydrolyzed gelatin (MW: 4000) with a 0.8 LRV (log10 reduction value) at 25 °C or a 0.2 LRV at 4 °C, compared to the stabilities observed in higher MW gelatin (60,000 and 160,000) with an LRV above 1. Based on the gelatin type, BHV in alkaline-treated hydrolyzed gelatin samples were unexpectantly more stable than in acid-treated hydrolyzed gelatin sample. All four viruses exhibited stability at 4 °C for at least 8 weeks, BHV or AdV remained stable for over 30 weeks of storage, and at 25 °C, AdV and RV remained stable for 8 weeks. CONCLUSION: The results demonstrated that 5% of 4000 MW hydrolyzed gelatin formulation can act as a relevant stabilizer for the thermal stability of viruses in medical research and application.

Structure of the dopamine D2 receptor in complex with the antipsychotic drug spiperone.

In addition to the serotonin 5-HT2A receptor (5-HT2AR), the dopamine D2 receptor (D2R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D2R have been described in complex with the inverse agonists risperidone (D2Rris) and haloperidol (D2Rhal). Here we describe the structure of human D2R in complex with spiperone (D2Rspi). In D2Rspi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D2Rris and D2Rhal, demonstrating that ECL2 in D2R is highly dynamic. Moreover, D2Rspi exhibited an extended binding pocket to accommodate spiperone's phenyl ring, which probably contributes to the selectivity of spiperone to D2R and 5-HT2AR. Together with D2Rris and D2Rhal, the structural information of D2Rspi should be of value for designing novel antipsychotics with improved safety and efficacy.

Low response in eliciting neuraminidase inhibition activity of sera among recipients of a split, monovalent pandemic influenza vaccine during the 2009 pandemic.

Antibodies against influenza virus neuraminidase (NA) protein prevent releasing of the virus from host cells and spreading of infection foci and are considered the 'second line of defence' against influenza. Haemagglutinin inhibition antibody-low responders (HI-LRs) are present among influenza split vaccine recipients. The NA inhibition (NAI) antibody response in vaccinees is worth exploring, especially those in the HI-LRs population. We collected pre- and post-vaccination sera from 61 recipients of an inactivated, monovalent, split vaccine against A/H1N1pdm09 and acute and convalescent sera from 49 unvaccinated patients naturally infected with the A/H1N1pdm09 virus during the 2009 influenza pandemic. All samples were subjected to haemagglutinin inhibition (HI), NAI and neutralisation assays. Most paired sera from naturally infected patients exhibited marked elevation in the NAI activity, and seroconversion rates (SCR) among HI-LRs and HI-responders (HI-Rs) were 60% and 87%, respectively; however, those from vaccinees displayed low increase in the NAI activity, and the SCR among HI-LRs and HI-Rs were 0% and 12%, respectively. In both HI-LRs and HI-Rs, vaccination with the inactivated, monovalent, split vaccine failed to elicit the NAI activity efficiently in the sera of the naive population, compared with the natural infection. Hence, the improvement of influenza vaccines is warranted to elicit not only HI but also NAI antibodies.

Cryo-EM structure of the human PAC1 receptor coupled to an engineered heterotrimeric G protein.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide hormone. The PACAP receptor PAC1R, which belongs to the class B G-protein-coupled receptors (GPCRs), is a drug target for mental disorders and dry eye syndrome. Here, we present a cryo-EM structure of human PAC1R bound to PACAP and an engineered Gs heterotrimer. The structure revealed that transmembrane helix TM1 plays an essential role in PACAP recognition. The extracellular domain (ECD) of PAC1R tilts by ~40° compared with that of the glucagon-like peptide-1 receptor (GLP-1R) and thus does not cover the peptide ligand. A functional analysis demonstrated that the PAC1R ECD functions as an affinity trap and is not required for receptor activation, whereas the GLP-1R ECD plays an indispensable role in receptor activation, illuminating the functional diversity of the ECDs in class B GPCRs. Our structural information will facilitate the design and improvement of better PAC1R agonists for clinical applications.

Fluctuations in Antibody Titers against Enterovirus D68 in Pediatric Sera Collected in a Community before, during, and after a Possible Outbreak.

We previously reported a hospital-based epidemiological study on enterovirus (EV)-D68 infection among children during the autumn of 2015, which indirectly inferred an outbreak in Sendai, Japan. In this study, stocked sera of children (aged 0-6 years; without symptoms of infectious diseases) in the Sendai community collected during 4 periods (1 year before, 6 months before, immediately after, and 1 year after the possible outbreak period) were analyzed using the neutralization antibody titer assay to determine community children's immunity levels against EV-D68 infection. The immunity levels were confirmed to have increased during the possible outbreak period and to have gradually waned over 1 year without another outbreak. These results provide background information supporting the results of our previous hospital-based surveillance study.

Crystal structure of human endothelin ETB receptor in complex with peptide inverse agonist IRL2500.

Endothelin receptors (ETA and ETB) are G-protein-coupled receptors activated by endothelin-1 and are involved in blood pressure regulation. IRL2500 is a peptide-mimetic of the C-terminal tripeptide of endothelin-1, and has been characterized as a potent ETB-selective antagonist, which has preventive effects against brain edema. Here, we report the crystal structure of the human ETB receptor in complex with IRL2500 at 2.7 Å-resolution. The structure revealed the different binding modes between IRL2500 and endothelin-1, and provides structural insights into its ETB-selectivity. Notably, the biphenyl group of IRL2500 penetrates into the transmembrane core proximal to D2.50, thus stabilizing the inactive conformation. Using the newly-established constitutively active mutant, we clearly demonstrate that IRL2500 functions as an inverse agonist for the ETB receptor. The current findings will expand the chemical space of ETR antagonists and facilitate the design of inverse agonists for other class A GPCRs.

Conformational transitions of a neurotensin receptor 1-Gi1 complex.

Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-Gi/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.

Illuminating G-Protein-Coupling Selectivity of GPCRs.

Heterotrimetic G proteins consist of four subfamilies (Gs, Gi/o, Gq/11, and G12/13) that mediate signaling via G-protein-coupled receptors (GPCRs), principally by receptors binding Gα C termini. G-protein-coupling profiles govern GPCR-induced cellular responses, yet receptor sequence selectivity determinants remain elusive. Here, we systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini. For each receptor, we probed chimeric Gα subunit activation via a transforming growth factor-α (TGF-α) shedding response in HEK293 cells lacking endogenous Gq/11 and G12/13 proteins, and complemented G-protein-coupling profiles through a NanoBiT-G-protein dissociation assay. Interrogation of the dataset identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which we used to develop a coupling predictor that outperforms previous methods. We used the predictor to engineer designer GPCRs selectively coupled to G12. This dataset of fine-tuned signaling mechanisms for diverse GPCRs is a valuable resource for research in GPCR signaling.

Structures of the 5-HT2A receptor in complex with the antipsychotics risperidone and zotepine.

Many drugs target the serotonin 2A receptor (5-HT2AR), including second-generation antipsychotics that also target the dopamine D2 receptor (D2R). These drugs often produce severe side effects due to non-selective binding to other aminergic receptors. Here, we report the structures of human 5-HT2AR in complex with the second-generation antipsychotics risperidone and zotepine. These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket, the movements of which are important for receptor activation. 5-HT2AR is structurally similar to 5-HT2CR but possesses a unique side-extended cavity near the orthosteric binding site. A docking study and mutagenic studies suggest that a highly 5-HT2AR-selective antagonist binds the side-extended cavity. The conformation of the ligand-binding pocket in 5-HT2AR significantly differs around extracellular loops 1 and 2 from that in D2R. These findings are beneficial for the rational design of safer antipsychotics and 5-HT2AR-selective drugs.

Crystal structures of human ETB receptor provide mechanistic insight into receptor activation and partial activation.

Endothelin receptors (ETA and ETB) are class A GPCRs activated by vasoactive peptide endothelins, and are involved in blood pressure regulation. ETB-selective signalling induces vasorelaxation, and thus selective ETB agonists are expected to be utilized for improved anti-tumour drug delivery and neuroprotection. Here, we report the crystal structures of human ETB receptor in complex with ETB-selective agonist, endothelin-3 and an ETB-selective endothelin analogue IRL1620. The structure of the endothelin-3-bound receptor reveals that the disruption of water-mediated interactions between W6.48 and D2.50 is critical for receptor activation, while these hydrogen-bonding interactions are partially preserved in the IRL1620-bound structure. Consistently, functional analysis reveals the partial agonistic effect of IRL1620. The current findings clarify the detailed molecular mechanism for the coupling between the orthosteric pocket and the G-protein binding, and the partial agonistic effect of IRL1620, thus paving the way for the design of improved agonistic drugs targeting ETB.

Case study-based time-course analysis of symptoms of respiratory syncytial virus infections followed by acute sinusitis in otherwise-healthy adults.

Reports on respiratory syncytial virus (RSV) infections are abundant in pediatric and geriatric populations but not many in healthy adults, and particularly, those which demonstrated the illness throughout its time course are rare. We report two otherwise-healthy adult cases, showing a number of evidence essential for confirmation of exclusive infections with RSV, and document their clinical features from the onset of the disease to recovery, including secondary sinusitis with magnetic resonance (MR) and computed tomography (CT) images. The infection was proven by isolating RSV belonging to subgroup B and by observing elevated anti-RSV antibody titer in the paired sera. Possible contribution of other pathogens including almost all respiratory viruses and representative bacteria, was excluded by negative results in multiplex PCR examination. In the first case, illness initiated with pharyngeal pain, followed by symptoms of sneezing, severe rhinorrhea and coughing, which peaked at approximately 5-7 days and persisted for 12 days. The patient experienced a slight chill, but the body temperature did not exceed 37 °C during illness. The patient showed no significant finding but only a slight increase in serum C-reactive protein level in the routine clinical laboratory examinations. On the 9th day of illness, a dull headache started persisting for at least a week after which it gradually waned. Sinusitis was found by chance on MR images of maxillary sinus 8 days after the headache started, and the finding disappeared on CT images taken after 6 months. In the second case, the symptoms included severe rhinorrhea and dull facial pain around the upper nose; the pain also occurred on the 9th day of illness and the symptom was clinically diagnosed to be acute sinusitis during a visit to a physician.

X-ray structures of endothelin ETB receptor bound to clinical antagonist bosentan and its analog.

Endothelin receptors (ETRs) have crucial roles in vascular control and are targets for drugs designed to treat circulatory-system diseases and cancer progression. The nonpeptide dual-ETR antagonist bosentan is the first oral drug approved to treat pulmonary arterial hypertension. Here we report crystal structures of human endothelin ETB receptor bound to bosentan and to the ETB-selective analog K-8794, at 3.6-Å and 2.2-Å resolution, respectively. The K-8794-bound structure reveals the detailed water-mediated hydrogen-bonding network at the transmembrane core, which could account for the weak negative allosteric modulation of ETB by Na+ ions. The bosentan-bound structure reveals detailed interactions with ETB, which are probably conserved in the ETA receptor. A comparison of the two structures shows unexpected similarity between antagonist and agonist binding. Despite this similarity, bosentan sterically prevents the inward movement of transmembrane helix 6 (TM6), and thus exerts its antagonistic activity. These structural insights will facilitate the rational design of new ETR-targeting drugs.

Differences in viral load among human respiratory syncytial virus genotypes in hospitalized children with severe acute respiratory infections in the Philippines.

BACKGROUND: Human respiratory syncytial virus (HRSV) is a leading viral etiologic agent of pediatric lower respiratory infections, including bronchiolitis and pneumonia. Two antigenic subgroups, HRSV-A and B, each contain several genotypes. While viral load may vary among HRSV genotypes and affect the clinical course of disease, data are scarce regarding the actual differences among genotypes. Therefore, this study estimated and compared viral load among NA1 and ON1 genotypes of HRSV-A and BA9 of HRSV-B. ON1 is a newly emerged genotype with a 72-nucleotide duplication in the G gene as observed previously with BA genotypes in HRSV-B. FINDINGS: Children <5 years of age with an initial diagnosis of severe or very severe pneumonia at a hospital in the Philippines from September 2012 to December 2013 were enrolled. HRSV genotypes were determined and the viral load measured from nasopharyngeal swabs (NPS). The viral load of HRSV genotype NA1 were significantly higher than those of ON1 and BA9. Regression analysis showed that both genotype NA1 and younger age were significantly associated with high HRSV viral load. CONCLUSIONS: The viral load of NA1 was higher than that of ON1 and BA9 in NPS samples. HRSV genotypes may be associated with HRSV viral load. The reasons and clinical impacts of these differences in viral load among HRSV genotypes require further evaluation.