Hypervariable loops of HIV-1 Env protein gp120 are speculated to play roles in the conformational transition of Env to the receptor binding-induced metastable state. Structural analysis of full-length Env-based immunogens, containing the entire V2 loop, displayed tighter association between gp120 subunits, resulting in a smaller trimeric diameter than constructs lacking V2. A prominent basal quaternary location of V2 and V3' that challenges previous reports would facilitate gp41-independent gp120-gp120 interactions and suggests a quaternary mechanism of epitope occlusion facilitated by hypervariable loops. Deletion of V2 resulted in dramatic exposure of basal, membrane-proximal gp41 epitopes, consistent with its predicted basal location. The structural features of HIV-1 Env characterized here provide grounds for a paradigm shift in loop exposure and epitope occlusion, while providing substantive rationale for epitope display required for elicitation of broadly neutralizing antibodies, as well as substantiating previous pertinent literature disregarded in recent reports.

Helicases contribute to diverse biological processes including replication, transcription and translation. Recent reports suggest that unwinding of some helicases display repetitive activity, yet the functional role of the repetitiveness requires further investigation. Using single-molecule fluorescence assays, we elucidated a unique unwinding mechanism of RNA helicase A (RHA) that entails discrete substeps consisting of binding, activation, unwinding, stalling and reactivation stages. This multi-step process is repeated many times by a single RHA molecule without dissociation, resulting in repetitive unwinding/rewinding cycles. Our kinetic and mutational analysis indicates that the two double stand RNA binding domains at the N-terminus of RHA are responsible for such repetitive unwinding behavior in addition to providing an increased binding affinity to RNA. Further, the repetitive unwinding induces an efficient annealing of a complementary RNA by making the unwound strand more accessible. The complex and unusual mechanism displayed by RHA may help in explaining how the repetitive unwinding of helicases contributes to their biological functions.

Despite decades of intensive research and a number of demonstrable successes, quantitative structure-activity relationship (QSAR) models still fail to yield predictions with reasonable accuracy in some circumstances, especially when the QSAR paradox occurs. In this study, to avoid the QSAR paradox, we proposed a novel integrated approach to improve the model performance through using both structural and biological information from compounds. As a proof-of-concept, the integrated models were built on a toxicological dataset to predict non-genotoxic carcinogenicity of compounds, using not only the conventional molecular descriptors but also expression profiles of significant genes selected from microarray data. For test set data, our results demonstrated that the prediction accuracy of QSAR model was dramatically increased from 0.57 to 0.67 with incorporation of expression data of just one selected signature gene. Our successful integration of biological information into classic QSAR model provided a new insight and methodology for building predictive models especially when QSAR paradox occurred.

The increase in recent outbreaks and unpredictable changes of highly pathogenic avian influenza (HPAI) H5N1 in birds and humans highlights the urgent need to develop a cross-protective H5N1 vaccine. We here report our development of a multiple-clade H5N1 influenza vaccine tested for immunogenicity and efficacy to confer cross-protection in an animal model.

Hepatitis E Virus-like particles self-assemble in to noninfectious nanocapsids that are resistant to proteolytic/acidic mucosal delivery conditions. Previously, the nanocapsid was engineered to specifically bind and enter breast cancer cells, where successful tumor targeting was demonstrated in animal models. In the present study, the nanocapsid surface was modified with a solvent-exposed cysteine to conjugate monolayer protected gold nanoclusters (AuNC). Unlike commercially available gold nanoparticles, AuNCs monodisperse in water and are composed of a discrete number of gold atoms, forming a crystalline gold core. Au102 pMBA44 (Au102) was an ideal conjugate given its small 2.5 nm size and detectability in cryoEM. Au102 was bound directly to nanocapsid surface cysteines via direct ligand exchange. In addition, Au102 was functionalized with a maleimide linker (Au102_C6MI) for maleimide-thiol conjugation to nanocapsid cysteines. The AuNC-bound nanocapsid constructs were conjugated in various conditions. We found Au102_C6MI to bind nanocapsid more efficiently, while Au102 remained more soluble over time. Nanocapsids conjugated to Au102_C6MI were imaged in cryoEM for single particle reconstruction to localize AuNC position on the nanocapsid surface. We resolved five unique high intensity volumes that formed a ring-shaped density at the 5-fold symmetry center. This finding was further supported by independent rigid modeling.

Clostridium difficile disease is mediated primarily by toxins A and B (TcdA and TcdB, respectively). The receptor binding domains (RBD) of TcdA and TcdB are immunogenic, and anti-RBD antibodies are protective. Since these toxins act locally, an optimal C. difficile vaccine would generate both systemic and mucosal responses. We have repurposed an attenuated Salmonella enterica serovar Typhimurium strain (YS1646) to produce such a vaccine. Plasmid-based candidates expressing either the TcdA or TcdB RBD were screened. Different vaccine routes and schedules were tested to achieve detectable serum and mucosal antibody titers in C57BL/6J mice. When given in a multimodality schedule over 1 week (intramuscularly and orally [p.o.] on day 0 and p.o. on days 2 and 4), several candidates provided 100% protection against lethal challenge. Substantial protection (82%) was achieved with combined p.o. TcdA and TcdB vaccination alone (days 0, 2, and 4). These data demonstrate the potential of the YS1646-based vaccines for C. difficile and strongly support their further development.

Purpose: Studies involving mouse models and human uterine smooth muscle cells have shown that phenylephrine inhibits uterine contractions in non-pregnant mice and human in vitro cell via cyclic adenosine monophosphate (cAMP) signaling. However, there has been no limited exploration to date of the effect of phenylephrine on uterine contractions in clinical practice. This study aimed to compare the dose requirement of oxytocin with or without the infusion of prophylactic phenylephrine to prevent post spinal hypotension during cesarean delivery under combined spinal and epidural anesthesia. Methods: This was a double-blinded, single-center, randomized, control study. One hundred and sixty pregnant patients provided informed consent and were randomly allocated to the phenylephrine (phenylephrine infusion) and control (saline infusion) groups. Patients randomized to the phenylephrine group received an intravenous prophylactic phenylephrine infusion at a fixed rate of 0.5 μg/kg/min. The control group received a saline placebo at the same rate and used the same apparatus for delivery. After neonatal delivery and clamping of the umbilical cord, patients received a standard institutional oxytocin protocol. The primary outcome measure was the total dose of oxytocin administered during CD. Secondary outcomes including the proportion (%) of patients requiring a secondary uterotonic agent and estimated blood loss (EBL) in the first 24 h after surgery. Results: The median oxytocin dose administered was significantly higher in the phenylephrine group than in the control group [6.9 ± 2.5 international standardized units (IU) vs. 5.4 ± 2.4 IU, p = 0.0004]. The number of patients that required a secondary uterotonic agent was significantly higher in the phenylephrine group than in the control group (24.2% vs. 9.1%; p = 0.034). The EBL in the first 24-h postoperatively was similar between the two groups (467 ± 47 ml vs. 392 ± 38 ml; p = 0.22). Conclusions: Prophylactic infusion of phenylephrine used to prevent post-spinal hypotension during CD was associated with a higher dose of oxytocin. This has important clinical implications, as the suboptimal use of oxytocin is associated with an increased risk of postpartum hemorrhage and increased maternal morbidity and mortality. Further studies are now needed to confirm these findings.

Accurate detection of SARS-CoV-2 neutralizing antibody (nAb) is critical for assessing the immunity levels after virus infection or vaccination. As fast, cost-effective alternatives to viral infection-based assays, competitive binding (CB) assays were developed to quantitate nAb by monitoring the ability of sera to inhibit the binding of viral spike (S) protein to the angiotensin converting enzyme 2 (ACE2) receptor. Herein, we established a bead-based flow cytometric CB assay and tested the detection performance of six combination models, i.e. immobilized ACE2 and soluble Fc-tagged S1 subunit of S protein (iACE2/S1-Fc), immobilized ACE2 and soluble Fc-tagged receptor binding domain (RBD) of S protein (iACE2/RBD-Fc), immobilized S1 and soluble Fc-tagged ACE2 (iS1/ACE2-Fc), immobilized S1 and soluble His-tagged ACE2 (iS1/ACE2-His), immobilized RBD and soluble Fc-tagged ACE2 (iRBD/ACE2-Fc), and immobilized RBD and soluble His-tagged ACE2 (iRBD/ACE2-His). Using SARS-CoV-2 monoclonal antibodies and sera of convalescent COVID-19 patients and vaccinated subjects, the combination models iACE2/RBD-Fc, iACE2/S1-Fc and iS1/ACE2-His were identified to be able to specifically detect SARS-CoV-2 nAb, among which iACE2/RBD-Fc model showed the highest sensitivity, superior to a commercial SARS-CoV-2 surrogate virus neutralization test (sVNT) ELISA kit. Further studies demonstrated that the sensitivity and specificity of CB assays were affected by the tag of ACE2, type of spike and method of measuring binding rate between ACE2 and spike. Moreover, the iACE2/RBD-Fc model showed good performance in detecting kinetic development of nAb against both the prototype SARS-CoV-2 strain and an omicron variant of SARS-CoV-2 in people immunized by an inactivated SARS-CoV-2 vaccine, and the results of iACE2/RBD-Fc model are correlated well with those of live virus-based and pseudovirus-based neutralization tests, demonstrating the potential to be developed into a highly sensitive, specific, versatile and high-throughput method for detecting SARS-CoV-2 nAb in clinical practice.

The porcine epidemic diarrhea virus (PEDV) causes a highly contagious disease in pigs, which is one of the most devastating viral diseases of swine in the world. In China, PEDV was first confirmed in 1984 and PEDV infections occurred sporadically from 1984 to early 2010. From late 2010 until present, PEDV infections have swept every province or region in China. In this study, we analyzed a total of 186 full-length spike genes and deduced proteins of all available complete genomes of PEDVs isolated in China during 2007-2019. A total of 28 potential recombination events were identified in the spike genes of PEDVs in China. Spike gene recombination not only expanded the genetic diversity of PEDVs in the GII genogroup, but also resulted in the emergence of a new evolutional branch GI-c during 2016-2018. In addition, comparative analysis of spike proteins between GI-a prototype virulent CV777 and GII strain AJ1102 reveals that the amino acid variations could affect 20 potential linear B cell epitopes, demonstrating a dramatic antigen drift in the spike protein. These results provide a thorough view of the information about the genetic and antigenic diversity of PEDVs circulating in China and therefore could benefit the development of suitable strategies for disease control.

Macrophages and monocytes orchestrate inflammatory processes in the lungs. However, their role in the pathogenesis of chronic obstructive pulmonary disease (COPD), an inflammatory condition, is not well known. Here, we determined the characteristics of these cells in lungs of COPD patients and identified novel therapeutic targets.

In early spring 2013, the emergence of the influenza A (H7N9) virus in humans in Eastern China raised concerns of a new influenza pandemic. Development of a safe and effective H7N9 influenza vaccine is urgently needed. To this end, we first synthesized the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A (H7N9) virus A/AnHui/1/2013. Using reverse genetics, we rescued a reassortant virus (H7N9/PR8) that contained the HA and NA genes from wild-type H7N9 and six genes encoding internal proteins from the A/Puerto Rico/8/34 (PR8) virus. Next, the pathogenicity of the reassortant virus was evaluated both in vivo and in vitro. We found that the virus was non-pathogenic in mice and was stable after serial passaging in eggs. Furthermore, we found that a monovalent influenza A (H7N9) split vaccine prepared from the virus was immunogenic in mice and ferrets. When given intramuscularly, the vaccine (two doses of at least 15-µg) completely protected mice from normally lethal wild-type H7N9 virus challenge. In summary, our H7N9 vaccine, developed over a short time, is a potential candidate for further clinical evaluation and human use.

Studies have shown that stem cells exert their therapeutic effects on acute kidney injury (AKI) through paracrine/endocrine actions. If the protective effect is mediated in an endocrine manner, the injection of the factors that these cells secrete could be effective, but the effect of conditioned medium (CM) remains controversial.

In the early stage of sepsis, M1 macrophages result in the production of inflammatory mediators and AKI. Heparin-binding protein (HBP) have been shown to play important roles in sepsis-induced AKI. In this study, we investigate the association of HBP with M1 macrophages in sepsis-induced AKI.

The continued spread of a highly pathogenic avian influenza (HPAI) H5N1 virus among poultry and wild birds has posed a potential threat to human public health. An influenza pandemic happens, when a new subtype that has not previously circulated in humans emerges. Almost all of the influenza pandemics in history have originated from avian influenza viruses (AIV). Birds are significant reservoirs of influenza viruses. In the present study, we performed a survey of avian influenza virus in ostriches and H5N1 virus (A/Ostrich/SuZhou/097/03, China097) was isolated. This H5N1 virus is highly pathogenic to both chickens and mice. It is also able to replicate in the lungs of, and to cause death in, BALB/c mice following intranasal administration. It forms plaques in chicken embryo fibroblast (CEF) cells in the absence of trypsin. The hemagglutinin (HA) gene of the virus is genetically similar to A/Goose/Guangdong/1/96(H5N1) and belongs to clade 0. The HA sequence contains multiple basic amino acids adjacent to the cleavage site, a motif associated with HPAI viruses. More importantly, the existence of H5N1 isolates in ostriches highlights the potential threat of wild bird infections to veterinary and public health.

Coxsackievirus B (CVB) enteroviruses are common pathogens that can cause acute and chronic myocarditis, dilated cardiomyopathy, aseptic meningitis, and they are hypothesized to be a causal factor in type 1 diabetes. The licensed enterovirus vaccines and those currently in clinical development are traditional inactivated or live attenuated vaccines. Even though these vaccines work well in the prevention of enterovirus diseases, new vaccine technologies, like virus-like particles (VLPs), can offer important advantages in the manufacturing and epitope engineering. We have previously produced VLPs for CVB3 and CVB1 in insect cells. Here, we describe the production of CVB3-VLPs with enhanced production yield and purity using an improved purification method consisting of tangential flow filtration and ion exchange chromatography, which is compatible with industrial scale production. We also resolved the CVB3-VLP structure by Cryo-Electron Microscopy imaging and single particle reconstruction. The VLP diameter is 30.9 nm on average, and it is similar to Coxsackievirus A VLPs and the expanded enterovirus cell-entry intermediate (the 135s particle), which is ~2 nm larger than the mature virion. High neutralizing and total IgG antibody levels, the latter being a predominantly Th2 type (IgG1) phenotype, were detected in C57BL/6J mice immunized with non-adjuvanted CVB3-VLP vaccine. The structural and immunogenic data presented here indicate the potential of this improved methodology to produce highly immunogenic enterovirus VLP-vaccines in the future.

Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy in autoimmune diseases, but no small-molecule IL-17A antagonists have yet progressed into clinical trials. Investigation of a series of linear peptide ligands to IL-17A and characterization of their binding site has enabled the design of novel macrocyclic ligands that are themselves potent IL-17A antagonists.

We report the identification of three structurally diverse compounds - compound 4, GC376, and MAC-5576 - as inhibitors of the SARS-CoV-2 3CL protease. Structures of each of these compounds in complex with the protease revealed strategies for further development, as well as general principles for designing SARS-CoV-2 3CL protease inhibitors. These compounds may therefore serve as leads for the basis of building effective SARS-CoV-2 3CL protease inhibitors.

The receptor-binding domains (RBDs) located in toxin A and toxin B of Clostridium difficile are known to be nontoxic and immunogenic. We need to develop a new type vaccine based on RBDs. In this study, we expressed and purified recombinant proteins (named RBD-TcdA and RBD-TcdB) as vaccine candidates containing the RBDs of toxin A and toxin B, respectively, from the C. difficile reference strain VPI10463. The immunogenicity and protection of the vaccine candidates RBD-TcdA, RBD-TcdB, and RBD-TcdA/B was evaluated by ELISA and survival assays. The data indicated that mice immunized with all vaccine candidates displayed potent levels of RBD-specific serum IgG. Following intramuscular immunization of mice with RBD-TcdA and/or RBD-TcdB, these vaccine candidates triggered immune responses that protected mice compared to mice immunized with aluminum hydroxide alone. Taken together, the results of this study reveal that recombinant proteins containing RBDs of C. difficile toxins can be used for vaccine development. Additionally, we found that an RBD-TcdA/B vaccine can elicit a stronger humoral immune response and provide better immunoprotection than the univalent vaccines. This RBD vaccine candidate conferred significant protection against disease symptoms and death caused by toxins from a wild-type C. difficile strain.

Swine acute diarrhea syndrome (SADS) is a highly contagious infectious disease characterized by acute vomiting and watery diarrhea in neonatal piglets. The causative agent for SADS is the swine acute diarrhea syndrome coronavirus (SADS-CoV), an alphacoronavirus in the family Coronaviridae. Currently, SADS-CoV was identified only in Guangdong and Fujian provinces of China, not in any other regions or countries in the world. To explore the genetic diversity of SADS-CoV isolates, herein we comparatively analyzed 44 full-length genomes of viruses isolated in Guangdong and Fujian provinces during 2017-2019. The spike glycoprotein gene of SADS-CoV strain CH/FJWT/2018 isolated in Fujian province is distinct from that of other viral isolates in either spike glycoprotein gene-based phylogenetic analysis or whole genome-based gene similarity analysis. Moreover, at least 7 predicted linear B cell epitopes in the spike glycoprotein of CH/FJWT/2018 would be affected by amino acid variations when compared with a representative virus isolated in Guangdong province. The spike glycoprotein of coronaviruses determines viral host range and tissue tropism during virus infection via specific interactions with the cellular receptor and also plays critical roles in eliciting the production of neutralizing antibodies. Since SADS-CoVs have a broad cell tropism, the results in this report further emphasize that the spike glycoprotein gene is a pivotal target in the surveillance of SADS-CoV.

The purpose of this study was to evaluate the associations between blood glucose and hemoglobin A1c (HbA1c) levels with the degree of stress urinary incontinence (SUI) and urgency urinary incontinence (UUI) in women.