Inhibition of human immunodeficiency virus (HIV-1) infectivity by expression of poorly or broadly neutralizing antibodies against Env in virus-producing cells.

The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein precursor (gp160) trimerizes, is modified by high-mannose glycans in the endoplasmic reticulum, and is transported via Golgi and non-Golgi secretory pathways to the infected cell surface. In the Golgi, gp160 is partially modified by complex carbohydrates and proteolytically cleaved to produce the mature functional Env trimer, which is preferentially incorporated into virions. Broadly neutralizing antibodies (bNAbs) generally recognize the cleaved Env trimer, whereas poorly neutralizing antibodies (pNAbs) bind the conformationally flexible gp160. We found that expression of bNAbs, pNAbs, or soluble/membrane forms of the receptor, CD4, in cells producing HIV-1 all decreased viral infectivity. Four patterns of co-expressed ligand:Env were observed: (i) ligands (CD4, soluble CD4-Ig, and some pNAbs) that specifically recognize the CD4-bound Env conformation resulted in uncleaved Envs lacking complex glycans that were not incorporated into virions; (ii) other pNAbs produced Envs with some complex carbohydrates and severe defects in cleavage, which were relieved by brefeldin A treatment; (iii) bNAbs that recognize gp160 as well as mature Envs resulted in Envs with some complex carbohydrates and moderate decreases in virion Env cleavage; and (iv) bNAbs that preferentially recognize mature Envs produced cleaved Envs with complex glycans in cells and on virions. The low infectivity observed upon co-expression of pNAbs or CD4 could be explained by disruption of Env trafficking, reducing the level of Env and/or increasing the fraction of uncleaved Env on virions. In addition to bNAb effects on virion Env cleavage, the secreted bNAbs neutralized the co-expressed viruses.IMPORTANCEThe Env trimers on the HIV-1 mediate virus entry into host cells. Env is synthesized in infected cells, modified by complex sugars, and cleaved to form a mature, functional Env, which is incorporated into virus particles. Env elicits antibodies in infected individuals, some of which can neutralize the virus. We found that antibodies co-expressed in the virus-producing cell can disrupt Env transit to the proper compartment for cleavage and sugar modification and, in some cases, block incorporation into viruses. These studies provide insights into the processes by which Env becomes functional in the virus-producing cell and may assist attempts to interfere with these events to inhibit HIV-1 infection.

Conformations of membrane human immunodeficiency virus (HIV-1) envelope glycoproteins solubilized in Amphipol A18 lipid-nanodiscs.

Upon binding to the host cell receptor, CD4, the pretriggered (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer undergoes transitions to downstream conformations important for virus entry. State 1 is targeted by most broadly neutralizing antibodies (bNAbs), whereas downstream conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. Extraction of Env from the membranes of viruses or Env-expressing cells disrupts the metastable State-1 Env conformation, even when detergent-free approaches like styrene-maleic acid lipid nanoparticles (SMALPs) are used. Here, we combine three strategies to solubilize and purify mature membrane Envs that are antigenically native (i.e., recognized by bNAbs and not pNAbs): (1) solubilization of Env with a novel amphipathic copolymer, Amphipol A18; (2) use of stabilized pretriggered Env mutants; and (3) addition of the State-1-stabilizing entry inhibitor, BMS-806. Amphipol A18 was superior to the other amphipathic copolymers tested (SMA and AASTY 11-50) for preserving a native Env conformation. A native antigenic profile of A18 Env-lipid-nanodiscs was maintained for at least 7 days at 4°C and 2 days at 37°C in the presence of BMS-806 and was also maintained for at least 1 h at 37°C in a variety of adjuvants. The damaging effects of a single cycle of freeze-thawing on the antigenic profile of the A18 Env-lipid-nanodiscs could be prevented by the addition of 10% sucrose or 10% glycerol. These results underscore the importance of the membrane environment to the maintenance of a pretriggered (State-1) Env conformation and provide strategies for the preparation of lipid-nanodiscs containing native membrane Envs.IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins like Env rely on purification procedures that maintain their natural conformation. In this study, we show that an amphipathic copolymer A18 can directly extract HIV-1 Env from a membrane without the use of detergents. A18 promotes the formation of nanodiscs that contain Env and membrane lipids. Env in A18-lipid nanodiscs largely preserves features recognized by broadly neutralizing antibodies (bNAbs) and conceals features potentially recognized by poorly neutralizing antibodies (pNAbs). Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful for future studies of HIV-1 Env structure, interaction with receptors and antibodies, and immunogenicity.

Peripheral nerve-derived Sema3A promotes osteogenic differentiation of mesenchymal stem cells through the Wnt/ß-catenin/Nrp1 positive feedback loop.

Sensory nerves play a crucial role in maintaining bone homeostasis by releasing Semaphorin 3A (Sema3A). However, the specific mechanism of Sema3A in regulation of bone marrow mesenchymal stem cells (BMMSCs) during bone remodelling remains unclear. The tibial denervation model was used and the denervated tibia exhibited significantly lower mass as compared to sham operated bones. In vitro, BMMSCs cocultured with dorsal root ganglion cells (DRGs) or stimulated by Sema3A could promote osteogenic differentiation through the Wnt/ß-catenin/Nrp1 positive feedback loop, and the enhancement of osteogenic activity could be inhibited by SM345431 (Sema3A-specific inhibitor). In addition, Sema3A-stimulated BMMSCs or intravenous injection of Sema3A could promote new bone formation in vivo. To sum up, the coregulation of bone remodelling is due to the ageing of BMMSCs and increased osteoclast activity. Furthermore, the sensory neurotransmitter Sema3A promotes osteogenic differentiation of BMMSCs via Wnt/ß-catenin/Nrp1 positive feedback loop, thus promoting osteogenesis in vivo and in vitro.

V3 tip determinants of susceptibility to inhibition by CD4-mimetic compounds in natural clade A human immunodeficiency virus (HIV-1) envelope glycoproteins.

IMPORTANCE: CD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants.

Conformations of Human Immunodeficiency Virus Envelope Glycoproteins in Detergents and Styrene-Maleic Acid Lipid Particles.

The mature human immunodeficiency virus (HIV) envelope glycoprotein (Env) trimer, which consists of noncovalently associated gp120 exterior and gp41 transmembrane subunits, mediates virus entry into cells. The pretriggered (State-1) Env conformation is the major target for broadly neutralizing antibodies (bNAbs), whereas receptor-induced downstream Env conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. To examine the contribution of membrane anchorage to the maintenance of the metastable pretriggered Env conformation, we compared wild-type and State-1-stabilized Envs solubilized in detergents or in styrene-maleic acid (SMA) copolymers. SMA directly incorporates membrane lipids and resident membrane proteins into lipid nanoparticles (styrene-maleic acid lipid particles [SMALPs]). The integrity of the Env trimer in SMALPs was maintained at both 4°C and room temperature. In contrast, Envs solubilized in Cymal-5, a nonionic detergent, were unstable at room temperature, although their stability was improved at 4°C and/or after incubation with the entry inhibitor BMS-806. Envs solubilized in ionic detergents were relatively unstable at either temperature. Comparison of Envs solubilized in Cymal-5 and SMA at 4°C revealed subtle differences in bNAb binding to the gp41 membrane-proximal external region, consistent with these distinct modes of Env solubilization. Otherwise, the antigenicity of the Cymal-5- and SMA-solubilized Envs was remarkably similar, both in the absence and in the presence of BMS-806. However, both solubilized Envs were recognized differently from the mature membrane Env by specific bNAbs and pNAbs. Thus, detergent-based and detergent-free solubilization at 4°C alters the pretriggered membrane Env conformation in consistent ways, suggesting that Env assumes default conformations when its association with the membrane is disrupted. IMPORTANCE The human immunodeficiency virus (HIV) envelope glycoproteins (Envs) in the viral membrane mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins rely on purification procedures that allow the proteins to maintain their natural conformation. In this study, we show that a styrene-maleic acid (SMA) copolymer can extract HIV-1 Env from a membrane without the use of detergents. The Env in SMA is more stable at room temperature than Env in detergents. The purified Env in SMA maintains many but not all of the characteristics expected of the natural membrane Env. Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful tools for future studies of HIV-1 Env structure and its interaction with receptors and antibodies.

Analysis of Glycosylation and Disulfide Bonding of Wild-Type SARS-CoV-2 Spike Glycoprotein.

The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S, M, E, and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to purify the Golgi-modified fraction of a wild-type SARS-CoV-2 S glycoprotein trimer and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on other characterized S trimer preparations, is predominantly modified in the Golgi compartment by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions. IMPORTANCE The SARS-CoV-2 coronavirus, which causes COVID-19, uses its spike glycoprotein to enter host cells. The viral spike glycoprotein is the main target of host neutralizing antibodies that help to control SARS-CoV-2 infection and are important for the protection provided by vaccines. The SARS-CoV-2 spike glycoprotein consists of a trimer of two subunits covered with a coat of carbohydrates (sugars). Here, we describe the disulfide bonds that assist the SARS-CoV-2 spike glycoprotein to assume the correct shape and the composition of the sugar moieties on the glycoprotein surface. We also evaluate the consequences of natural virus variation in O-linked sugar addition and in the cysteine residues involved in disulfide bond formation. This information can expedite the improvement of vaccines and therapies for COVID-19.

Functional and Highly Cross-Linkable HIV-1 Envelope Glycoproteins Enriched in a Pretriggered Conformation.

Binding to the receptor, CD4, drives the pretriggered, "closed" (state-1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer into more "open" conformations (states 2 and 3). Broadly neutralizing antibodies, which are elicited inefficiently, mostly recognize the state-1 Env conformation, whereas the more commonly elicited poorly neutralizing antibodies recognize states 2/3. HIV-1 Env metastability has created challenges for defining the state-1 structure and developing immunogens mimicking this labile conformation. The availability of functional state-1 Envs that can be efficiently cross-linked at lysine and/or acidic amino acid residues might assist these endeavors. To that end, we modified HIV-1AD8 Env, which exhibits an intermediate level of triggerability by CD4. We introduced lysine/acidic residues at positions that exhibit such polymorphisms in natural HIV-1 strains. Env changes that were tolerated with respect to gp120-gp41 processing, subunit association, and virus entry were further combined. Two common polymorphisms, Q114E and Q567K, as well as a known variant, A582T, additively rendered pseudoviruses resistant to cold, soluble CD4, and a CD4-mimetic compound, phenotypes indicative of stabilization of the pretriggered state-1 Env conformation. Combining these changes resulted in two lysine-rich HIV-1AD8 Env variants (E.2 and AE.2) with neutralization- and cold-resistant phenotypes comparable to those of natural, less triggerable tier 2/3 HIV-1 isolates. Compared with these and the parental Envs, the E.2 and AE.2 Envs were cleaved more efficiently and exhibited stronger gp120-trimer association in detergent lysates. These highly cross-linkable Envs enriched in a pretriggered conformation should assist characterization of the structure and immunogenicity of this labile state. IMPORTANCE The development of an efficient vaccine is critical for combating HIV-1 infection worldwide. However, the instability of the pretriggered shape (state 1) of the viral envelope glycoprotein (Env) makes it difficult to raise neutralizing antibodies against HIV-1. Here, by introducing multiple changes in Env, we derived two HIV-1 Env variants that are enriched in state 1 and can be efficiently cross-linked to maintain this shape. These Env complexes are more stable in detergent, assisting their purification. Thus, our study provides a path to a better characterization of the native pretriggered Env, which should assist vaccine development.

Basic demographic characteristics and prevalence of comorbidities in acute mesenteric ischemia: a systematic review and proportional meta-analysis.

PURPOSE: The aim of this systematic review and meta-analysis was to examine and assess the basic demographic characteristics and prevalence of comorbidities in acute mesenteric ischemia (AMI) and its various subtypes. PATIENTS AND METHODS: A literature search was conducted by using the databases PubMed, EMBASE, and Google Scholar (to June 1, 2022). Random-effects or fixed-effects models were selected to pool means and proportions and their corresponding 95% confidence intervals (CI), based on heterogeneity between studies. The results of meta-analyses for basic demographic characteristics and prevalence (proportions) of each specific comorbidities of acute mesenteric ischemia (AMI) and its various subtypes were described. RESULTS: Ninety-nine studies were included in the meta-analysis, including 17,103 patients with AMI. Furthermore, 7941 patients with subclass diagnoses of AMI were identified, including 3,239 patients with arterial occlusive mesenteric ischemia (AOMI), 2,977 patients with nonocclusive mesenteric ischemia (NOMI), and 1,725 patients with mesenteric venous thrombosis (MVT). As a surgical emergency, AMI is associated with older patients and a high likelihood of multisystem comorbidities. Comorbidities of AMI involved multiple systemic diseases, including cardiovascular disease, endocrine and metabolic diseases, kidney diseases, digestive diseases, respiratory diseases, cerebrovascular diseases, vascular diseases, and cancer. CONCLUSION: The basic demographic characteristics and the prevalence of comorbidities of different subtypes of AMI are different. The management of comorbidities should be an essential part of improving the prognosis of AMI patients and may contribute to precise prevention of AMI.

Variability of fuel consumption and CO2 emissions of a gasoline passenger car under multiple in-laboratory and on-road testing conditions.

An increasing divergence regarding fuel consumption (and/or CO2 emissions) between real-world and type-approval values for light-duty gasoline vehicles (LDGVs) has posed severe challenges to mitigating greenhouse gases (GHGs) and achieving carbon emissions peak and neutrality. To address this divergence issue, laboratory test cycles with more real-featured and transient traffic patterns have been developed recently, for example, the China Light-duty Vehicle Test Cycle for Passenger cars (CLTC-P). We collected fuel consumption and CO2 emissions data of a LDGV under various conditions based on laboratory chassis dynamometer and on-road tests. Laboratory results showed that both standard test cycles and setting methods of road load affected fuel consumption slightly, with variations of less than 4%. Compared to the type-approval value, laboratory and on-road fuel consumption of the tested LDGV over the CLTC-P increased by 9% and 34% under the reference condition (i.e., air conditioning off, automatic stop and start (STT) on and two passengers). On-road measurement results indicated that fuel consumption under the low-speed phase of the CLTC-P increased by 12% due to the STT off, although only a 4% increase on average over the entire cycle. More fuel consumption increases (52%) were attributed to air conditioning usage and full passenger capacity. Strong correlations (R2 > 0.9) between relative fuel consumption and average speed were also identified. Under traffic congestion (average speed below 25 km/hr), fuel consumption was highly sensitive to changes in vehicle speed. Thus, we suggest that real-world driving conditions cannot be ignored when evaluating the fuel economy and GHGs reduction of LDGVs.

Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects.

SARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects (syncytium formation). We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced syncytium-forming ability. Notably, the D614G change found in globally predominant SARS-CoV-2 strains increased infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, these two features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both host proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent.IMPORTANCE The development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, a process that contributes to the destruction of the virus-infected cell.

Asymmetric Structures and Conformational Plasticity of the Uncleaved Full-Length Human Immunodeficiency Virus Envelope Glycoprotein Trimer.

The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41)3] is produced by cleavage of a conformationally flexible gp160 precursor. gp160 cleavage or the binding of BMS-806, an entry inhibitor, stabilizes the pretriggered, "closed" (state 1) conformation recognized by rarely elicited broadly neutralizing antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers during natural infection recognize more "open" Env conformations (states 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and cross-linking decreased the exposure of pNAb epitopes on cell surface gp160; however, after detergent solubilization, cross-linked and BMS-806-treated gp160 sampled non-state-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume state-2-like conformations. Asymmetry of the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a state-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs. IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage for a persistent virus like HIV-1.

Dual Pathways of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trafficking Modulate the Selective Exclusion of Uncleaved Oligomers from Virions.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer is transported through the secretory pathway to the infected cell surface and onto virion particles. In the Golgi, the gp160 Env precursor is modified by complex sugars and proteolytically cleaved to produce the mature functional Env trimer, which resists antibody neutralization. We observed mostly uncleaved gp160 and smaller amounts of cleaved gp120 and gp41 Envs on the surface of HIV-1-infected or Env-expressing cells; however, cleaved Envs were relatively enriched in virions and virus-like particles (VLPs). This relative enrichment of cleaved Env in VLPs was observed for wild-type Envs, for Envs lacking the cytoplasmic tail, and for CD4-independent, conformationally flexible Envs. On the cell surface, we identified three distinct populations of Envs: (i) the cleaved Env was transported through the Golgi, was modified by complex glycans, formed trimers that cross-linked efficiently, and was recognized by broadly neutralizing antibodies; (ii) a small fraction of Env modified by complex carbohydrates escaped cleavage in the Golgi; and (iii) the larger population of uncleaved Env lacked complex carbohydrates, cross-linked into diverse oligomeric forms, and was recognized by poorly neutralizing antibodies. This last group of more "open" Env oligomers reached the cell surface in the presence of brefeldin A, apparently bypassing the Golgi apparatus. Relative to Envs transported through the Golgi, these uncleaved Envs were counterselected for virion incorporation. By employing two pathways for Env transport to the surface of infected cells, HIV-1 can misdirect host antibody responses toward conformationally flexible, uncleaved Env without compromising virus infectivity.IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus type 1 (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The cleaved, functional Env is incorporated into virus particles from the surface of the infected cell. We found that an uncleaved form of Env is transported to the cell surface by an unconventional route, but this nonfunctional Env is mostly excluded from the virus. Thus, only one of the pathways by which Env is transported to the surface of infected cells results in efficient incorporation into virus particles, potentially allowing the uncleaved Env to act as a decoy to the host immune system without compromising virus infectivity.

Long-Acting BMS-378806 Analogues Stabilize the State-1 Conformation of the Human Immunodeficiency Virus Type 1 Envelope Glycoproteins.

During human immunodeficiency virus type 1 (HIV-1) entry into cells, the viral envelope glycoprotein (Env) trimer [(gp120/gp41)3] binds the receptors CD4 and CCR5 and fuses the viral and cell membranes. CD4 binding changes Env from a pretriggered (state-1) conformation to more open downstream conformations. BMS-378806 (here called BMS-806) blocks CD4-induced conformational changes in Env important for entry and is hypothesized to stabilize a state-1-like Env conformation, a key vaccine target. Here, we evaluated the effects of BMS-806 on the conformation of Env on the surface of cells and virus-like particles. BMS-806 strengthened the labile, noncovalent interaction of gp120 with the Env trimer, enhanced or maintained the binding of most broadly neutralizing antibodies, and decreased the binding of poorly neutralizing antibodies. Thus, in the presence of BMS-806, the cleaved Env on the surface of cells and virus-like particles exhibits an antigenic profile consistent with a state-1 conformation. We designed novel BMS-806 analogues that stabilized the Env conformation for several weeks after a single application. These long-acting BMS-806 analogues may facilitate enrichment of the metastable state-1 Env conformation for structural characterization and presentation to the immune system.IMPORTANCE The envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) mediates the entry of the virus into host cells and is also the target for antibodies. During virus entry, Env needs to change shape. Env flexibility also contributes to the ability of HIV-1 to evade the host immune response; many shapes of Env raise antibodies that cannot recognize the functional Env and therefore do not block virus infection. We found that an HIV-1 entry inhibitor, BMS-806, stabilizes the functional shape of Env. We developed new variants of BMS-806 that stabilize Env in its natural state for long periods of time. The availability of such long-acting stabilizers of Env shape will allow the natural Env conformation to be characterized and tested for efficacy as a vaccine.

The opportunity cost of automated glycopeptide analysis: case study profiling the SARS-CoV-2 S glycoprotein.

Glycosylation analysis of viral glycoproteins contributes significantly to vaccine design and development. Among other benefits, glycosylation analysis allows vaccine developers to assess the impact of construct design or producer cell line choices for vaccine production, and it is a key measure by which glycoproteins that are produced for use in vaccination can be compared to their native viral forms. Because many viral glycoproteins are multiply glycosylated, glycopeptide analysis is a preferrable approach for mapping the glycans, yet the analysis of glycopeptide data can be cumbersome and requires the expertise of an experienced analyst. In recent years, a commercial software product, Byonic, has been implemented in several instances to facilitate glycopeptide analysis on viral glycoproteins and other glycoproteomics data sets, and the purpose of the study herein is to determine the strengths and limitations of using this software, particularly in cases relevant to vaccine development. The glycopeptides from a recombinantly expressed trimeric S glycoprotein of the SARS-CoV-2 virus were first analyzed using an expert-based analysis strategy; subsequently, analysis of the same data set was completed using Byonic. Careful assessment of instances where the two methods produced different results revealed that the glycopeptide assignments from Byonic contained more false positives than true positives, even when the data were assessed using a 1% false discovery rate. The work herein provides a roadmap for removing the spurious assignments that Byonic generates, and it provides an assessment of the opportunity cost for relying on automated assignments for glycopeptide data sets from viral glycoproteins.

Strain-Dependent Activation and Inhibition of Human Immunodeficiency Virus Entry by a Specific PF-68742 Stereoisomer.

Human immunodeficiency virus (HIV-1) entry into cells is mediated by the viral envelope glycoprotein (Env) trimer, which consists of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. When gp120 binds sequentially to the receptors CD4 and CCR5 on the target cell, the metastable Env trimer is triggered to undergo entry-related conformational changes. PF-68742 is a small molecule that inhibits the infection of a subset of HIV-1 strains by interfering with an Env function other than receptor binding. Determinants of HIV-1 resistance to PF-68742 map to the disulfide loop and fusion peptide of gp41. Of the four possible PF-68742 stereoisomers, only one, MF275, inhibited the infection of CD4-positive CCR5-positive cells by some HIV-1 strains. MF275 inhibition of these HIV-1 strains occurred after CD4 binding but before the formation of the gp41 six-helix bundle. Unexpectedly, MF275 activated the infection of CD4-negative CCR5-positive cells by several HIV-1 strains resistant to the inhibitory effects of the compound in CD4-positive target cells. In contrast to CD4 complementation by CD4-mimetic compounds, activation of CD4-independent infection by MF275 did not depend upon the availability of the gp120 Phe 43 cavity. Sensitivity to inhibitors indicates that MF275-activated virus entry requires formation/exposure of the gp41 heptad repeat (HR1) as well as CCR5 binding. MF275 apparently activates a virus entry pathway parallel to that triggered by CD4 and CD4-mimetic compounds. Strain-dependent divergence in Env conformational transitions allows different outcomes, inhibition or activation, in response to MF275. Understanding the mechanisms of MF275 activity should assist efforts to optimize its utility.IMPORTANCE Envelope glycoprotein (Env) spikes on the surface of human immunodeficiency virus (HIV-1) bind target cell receptors, triggering changes in the shape of Env. We studied a small molecule, MF275, that also induced shape changes in Env. The consequences of MF275 interaction with Env depended on the HIV-1 strain, with infection by some viruses inhibited and infection by other viruses enhanced. These studies reveal the strain-dependent diversity of HIV-1 Envs as they undergo shape changes in proceeding down the entry pathway. Appreciation of this diversity will assist attempts to develop broadly active inhibitors of HIV-1 entry.

Drain fluid amylase as a predictor of postoperative salivary fistula in cases with benign parotid tumours.

BACKGROUND: Late diagnosis of a salivary fistula increases the risk of wound infection and scarring. The purpose of the present study was to identify a quantitative predictor of postoperative salivary fistula for cases treated with surgery. METHODS: Demographic, intraoperative and postoperative parameters for 57 cases that received surgery for benign parotid tumours were recorded from June 2017 to June 2018, of which 18 cases developed salivary fistulas. These data were analysed using univariate and binary logistic regression analyses as well as receiver operating curve analysis. RESULTS: Drain fluid amylase concentration was positively correlated with salivary fistula development (p <  0.001), with an odds ratio of 1.14 for a 1 KU/L increase in concentration and an optimal receiver operating curve cut-off value of 51,100 U/L for predicting salivary fistula development. Cases wherein the parotid-masseteric fascia remained intact were associated with a lower risk of salivary fistula development (p = 0.006). CONCLUSION: Drain fluid amylase concentration may be a valuable predictor of postoperative salivary fistula in cases with benign parotid tumours.

Sensory Nerves Affect Bone Regeneration in Rabbit Mandibular Distraction Osteogenesis.

Objectives: To investigate the effects of inferior alveolar nerve on new bone formation in rabbit mandibular distraction osteogenesis. Methods: 20 New Zealand White rabbits underwent bilateral distraction osteogenesis with a rate of 1 mm/day. The inferior alveolar nerve of one side was resected under the surgical microscope, with the inferior alveolar vascular intact. The contralateral side received sham operation. The rabbits were sacrificed at consolidation time of 28 days. The regenerate callus underwent radiograph examination, dual-energy X-ray absorptiometry, haematoxylin and eosin staining and histomorphometric analysis. A paired t-test was performed using SPSS 16.0 software package. Results: The BMD of the new bone in the distraction gap on the denervation side of mandibular was significantly lower (P<0.05) than on the control side. The histological investigation showed that the bone trabeculae were dis-arrayed containing dispersed cartilage cells on the denervation side, whereas the bone trabeculae were orderly with rich blood vessels and no cartilage cell on the control side. Both new bone volume and the thickness of new trabeculae were significantly lower on the denervation side than on the control side (P < 0.05). Conclusion: The loss of the sensory nerves could result in a decrease of the new bone quality during the mandibular distraction osteogenesis.

Osseointegration effect of biomimetic intrafibrillarly mineralized collagen applied simultaneously with titanium implant: A pilot in vivo study.

OBJECTIVES: To investigate the promoting effects of biomimetic intrafibrillarly mineralized collagen (IMC) bone scaffold material on the osseointegration of a titanium implant simultaneously grafted into a critical-sized bone defect as well as the underlying mechanisms involved. MATERIALS AND METHODS: A critical-sized bone defect was created in the rat femur, and a titanium (Ti) implant surrounded by IMC or extrafibrillarly mineralized collagen (EMC) bone scaffold material was placed in the defect. A blank group and a natural bone group were included as controls. Osseointegration was assessed by micro-computed tomographic, histological, and biochemical evaluations at 12 weeks postoperatively. Microarray technology was applied for transcriptional profile analysis at days 7 and 14 postoperatively. RESULTS: Significant bone regeneration and osseointegration were observed in the IMC and EMC groups according to µ-CT and histological analyses. The bone volume (BV)/total volume (TV) fraction, bone-to-implant contact percentage, and bone area percentage as well as ultimate shear strength and maximal pull-out force were all significantly higher in the IMC group than in the EMC group (all p < 0.05). Transcriptional analysis revealed overexpression of genes mainly associated with cell proliferation, immuno-inflammatory response, skeletogenesis, angiogenesis, neurogenesis, and skeletogenesis-related pathways during the early process of osseointegration in the IMC group. CONCLUSION: Our data suggest that IMC placed simultaneously with a Ti implant may be a promising strategy in jawbone defect reconstruction. Several candidate genes that were found to be differentially expressed in the IMC group may be responsible for the superior osseointegration effects in this model.

Restoring Skeletal Marker Points for Severe Maxillary and Mandibular Jaw Defects Using a Linear Regression Approach.

PURPOSE: For severe mandibular or maxillary defects across the midline, doctors often lack data on the shape of the jaws when designing virtual surgery. This study sought to repair the personalized 3-dimensional shape of the jaw, particularly when the jaw is severely damaged. MATERIALS AND METHODS: Two linear regression methods, denoted method I and method II, were used to reconstruct key points of the severely damaged maxilla or mandible based on the remaining jaw. The predictor variable was the position of key points. Outcome variables were the position of key points and the error between the predicted and actual positions. Another variable was the average error. In the final data analysis, the effect of the method was judged based on the mean error and error probability distribution. RESULTS: Computed tomographic data of jaws from 44 normal adults in East China were collected over 2 years by the Shanghai Jiao Tong University School of Medicine (Shanghai, China). Sixteen 16 key points were extracted for each jaw. Method I showed that 2-dimensional regression can yield the best overall result and that the position error of most points can be decreased to smaller than 5 mm. The result of method II was similar to that of method I but showed cumulative errors. CONCLUSIONS: Linear regression can be used to locate key points. Two-dimensional regression has the best effect, which can be used as a reference to develop a surgical plan and perform surgery.

Isoform Evolution in Primates through Independent Combination of Alternative RNA Processing Events.

Recent RNA-seq technology revealed thousands of splicing events that are under rapid evolution in primates, whereas the reliability of these events, as well as their combination on the isoform level, have not been adequately addressed due to its limited sequencing length. Here, we performed comparative transcriptome analyses in human and rhesus macaque cerebellum using single molecule long-read sequencing (Iso-seq) and matched RNA-seq. Besides 359 million RNA-seq reads, 4,165,527 Iso-seq reads were generated with a mean length of 14,875 bp, covering 11,466 human genes, and 10,159 macaque genes. With Iso-seq data, we substantially expanded the repertoire of alternative RNA processing events in primates, and found that intron retention and alternative polyadenylation are surprisingly more prevalent in primates than previously estimated. We then investigated the combinatorial mode of these alternative events at the whole-transcript level, and found that the combination of these events is largely independent along the transcript, leading to thousands of novel isoforms missed by current annotations. Notably, these novel isoforms are selectively constrained in general, and 1,119 isoforms have even higher expression than the previously annotated major isoforms in human, indicating that the complexity of the human transcriptome is still significantly underestimated. Comparative transcriptome analysis further revealed 502 genes encoding selectively constrained, lineage-specific isoforms in human but not in rhesus macaque, linking them to some lineage-specific functions. Overall, we propose that the independent combination of alternative RNA processing events has contributed to complex isoform evolution in primates, which provides a new foundation for the study of phenotypic difference among primates.