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Summary: The structural and characteristic features of HIV-1 broadly neutralizing antibodies (bnAbs) from chronically infected pediatric donors are currently unknown. Herein, we characterized a heavy chain matured HIV-1 bnAb 44m, identified from a pediatric elite-neutralizer. Interestingly, in comparison to its wild-type AIIMS-P01 bnAb, 44m exhibited moderately higher level of somatic hypermutations of 15.2%. The 44m neutralized 79% of HIV-1 heterologous viruses (n = 58) tested, with a geometric mean IC50 titer of 0.36 μg/mL. The cryo-EM structure of 44m Fab in complex with fully cleaved glycosylated native-like BG505.SOSIP.664.T332N gp140 envelope trimer at 4.4 Å resolution revealed that 44m targets the V3-glycan N332-supersite and GDIR motif to neutralize HIV-1 with improved potency and breadth, plausibly attributed by a matured heavy chain as compared to that of wild-type AIIMS-P01. This study further improves our understanding on pediatric HIV-1 bnAbs and structural basis of broad HIV-1 neutralization by 44m may be useful blueprint for vaccine design in future.

HOPS 87 is a Class 0 protostellar core known to harbor an extremely young bipolar outflow and a hot corino. We report the discovery of localized, chemically rich regions near the bases of the two-lobe bipolar molecular outflow in HOPS 87 containing molecules such as H _2 CO, ^13 CS, H _2 S, OCS, and CH _3 OH, the simplest complex organic molecule (COM). The locations and kinematics suggest that these localized features are due to jet-driven shocks rather than being part of the hot-corino region encasing the protostar. The COM compositions of the molecular gas in these jet-localized regions are relatively simpler than those in the hot-corino zone. We speculate that this simplicity is due to either the liberation of ice with a less complex chemical history or the effects of shock chemistry. Our study highlights the dynamic interplay between the protostellar bipolar outflow, disk, inner-core environment, and the surrounding medium, contributing to our understanding of molecular complexity in solar-like young stellar objects.

We conducted a Ka -band (26.1–35 GHz) line survey toward Orion KL using the TianMa 65 m Radio Telescope (TMRT). It is the first blind line survey in the Ka band and achieves a sensitivity at the mK level (1–3 mK at a spectral resolution of ∼1 km s ^−1 ). In total, 592 Gaussian features are extracted. Among them, 257 radio recombination lines (RRLs) are identified. The maximum Δ n of RRLs of H, He, and C are 20, 15, and 5, respectively. Through stacking, we have detected the β lines of ion RRLs (RRLs of C ^+ with the possible contribution of other ions like O ^+ ) for the first time, and a tentative signal of the γ lines of ion RRLs can also be seen on the stacked spectrum. Besides this, 318 other line features were assigned to 37 molecular species, and 10 of these species were not detected in the Q -band survey of TMRT. The vibrationally excited states of nine species were also detected. The emission of most species can be modeled under LTE. A number of transitions of E-CH3OH ( J _2 − J _1 ) display maser effects, which are confirmed by our modeling, and besides the bumping peak at J ∼ 6, there is another peak at J ∼ 13. Methylcyanoacetylene (CH _3 C _3 N) is detected in Orion KL for the first time. This work emphasizes that the Ka band, which was long ignored for spectral line surveys, is very useful for surveying RRLs and molecular lines simultaneously.

Protostellar outflows and jets are almost ubiquitous characteristics during the mass accretion phase and encode the history of stellar accretion, complex organic molecule (COM) formation, and planet formation. Episodic jets are likely connected to episodic accretion through the disk. Despite the importance, studies on episodic accretion and ejection links have not been done yet in a systematic fashion using high-sensitivity and high-resolution observations. To explore episodic accretion mechanisms and the chronologies of episodic events, we investigated 39 fields containing protostars with Atacama Large Millimeter/submillimeter Array observations of CO, SiO, and 1.3 mm continuum emission. We detected SiO emission in 19 fields, where 17 sources are driving molecular jets. Jet velocities, mass-loss rates, mass accretion rates, and periods of accretion events appear to have some dependence on the driving forces of the jet (e.g., bolometric luminosity, envelope mass). Next, velocities and mass-loss rates appear to be somewhat correlated with the surrounding envelope mass, suggesting that the presence of high mass around protostars increases the ejection–accretion activity. We determine mean periods of ejection events of 20–175 yr for our sample, which could be associated with perturbation zones of ∼2−25 au extent around the protostars. In addition, mean ejection periods show an apparent anticorrelation with the envelope mass, where high accretion rates may trigger more frequent ejection events. The observed periods of outburst/ejection are much shorter than the freezeout timescale of the simplest COMs like CH _3 OH, suggesting that episodic events could affect the ice–gas balance inside and around the snowline.

The internal structure of the prestellar core G208.68-19.02-N2 (G208-N2) in the Orion Molecular Cloud 3 (OMC-3) region has been studied with the Atacama Large Millimeter/submillimeter Array. The dust continuum emission revealed a filamentary structure with a length of ∼5000 au and an average H _2 volume density of ∼6 × 10 ^7 cm ^−3 . At the tip of this filamentary structure, there is a compact object, which we call a nucleus , with a radius of ∼150–200 au and a mass of ∼0.1 M _⊙ . The nucleus has a central density of ∼2 × 10 ^9 cm ^−3 with a radial density profile of r ^−1.87±0.11 . The density scaling of the nucleus is ∼3.7 times higher than that of the singular isothermal sphere (SIS). This as well as the very low virial parameter of 0.39 suggests that the gravity is dominant over the pressure everywhere in the nucleus. However, there is no sign of CO outflow localized to this nucleus. The filamentary structure is traced by the N _2 D ^+ 3–2 emission, but not by the C ^18 O 2–1 emission, implying the significant CO depletion due to high density and cold temperature. Toward the nucleus, the N _2 D ^+ also shows the signature of depletion. This could imply either the depletion of the parent molecule, N _2 , or the presence of the embedded very-low luminosity central source that could sublimate the CO in the very small area. The nucleus in G208-N2 is considered to be a prestellar core on the verge of first hydrostatic core (FHSC) formation or a candidate for the FHSC.

One of the most poorly understood aspects of low-mass star formation is how multiple-star systems are formed. Here we present the results of Atacama Large Millimeter/submillimeter Array (ALMA) Band 6 observations toward a forming quadruple protostellar system, G206.93-16.61E2, in the Orion B molecular cloud. ALMA 1.3 mm continuum emission reveals four compact objects, of which two are Class I young stellar objects and the other two are likely in prestellar phase. The 1.3 mm continuum emission also shows three asymmetric ribbon-like structures that are connected to the four objects, with lengths ranging from ∼500 to ∼2200 au. By comparing our data with magnetohydrodynamic simulations, we suggest that these ribbons trace accretion flows and also function as gas bridges connecting the member protostars. Additionally, ALMA CO J = 2−1 line emission reveals a complicated molecular outflow associated with G206.93-16.61E2, with arc-like structures suggestive of an outflow cavity viewed pole-on.

Abstract The structures of a cyanophage small heat shock protein (sHSP) were determined as octahedrons of 24-mers and 48-mers and as icosahedrons of 60-mers. An N-terminal deletion construct of an 18 kDa sHSP of Synechococcus sp. phage S-ShM2 crystallized as a 24-mer and its structure was determined at a resolution of 7 Å. The negative stain electron microscopy (EM) images showed that the full-length protein is a mixture of a major population of larger and a minor population of smaller cage-like particles. Their structures have been determined by electron cryomicroscopy 3D image reconstruction at a resolution of 8 Å. The larger particles are 60-mers with icosahedral symmetry and the smaller ones are 48-mers with octahedral symmetry. These structures are the first of the viral/phage origin and the 60-mer is the largest and the first icosahedral assembly to be reported for sHSPs.

Current influenza vaccines need to be updated annually due to mutations in the globular head of the viral surface protein, hemagglutinin (HA). To address this, vaccine candidates have been designed based on the relatively conserved HA stem domain and have shown protective efficacy in animal models. Oligomerization of the antigens either by fusion to oligomerization motifs or display on self-assembling nanoparticle scaffolds, can induce more potent immune responses compared to the corresponding monomeric antigen due to multivalent engagement of B-cells. Since nanoparticle display can increase manufacturing complexity, and often involves one or more mammalian cell expressed components, it is important to characterize and compare various display and oligomerization scaffolds. Using a structure guided approach, we successfully displayed multiple copies of a previously designed soluble, trimeric influenza stem domain immunogen, pH1HA10, on the ferritin like protein, MsDps2 (12 copies), Ferritin (24 copies) and Encapsulin (180 copies). All proteins were expressed in Escherichia coli. The nanoparticle fusion immunogens were found to be well folded and bound to the influenza stem directed broadly neutralizing antibodies with high affinity. An 8.5 Å Cryo-EM map of Msdps2-pH1HA10 confirmed the successful design of the nanoparticle fusion immunogen. Mice immunization studies with the soluble trimeric stem and nanoparticle fusion constructs revealed that all of them were immunogenic, and protected mice against homologous (A/Belgium/145-MA/2009) and heterologous (A/Puerto Rico/8/1934) challenge with 10MLD50 mouse adapted virus. Although nanoparticle display conferred a small but statistically significant improvement in protection relative to the soluble trimer in a homologous challenge, heterologous protection was similar in both nanoparticle-stem immunized and trimeric stem immunized groups. Such rapidly producible, bacterially expressed antigens and nanoparticle scaffolds are useful modalities to tackle future influenza pandemics.

Stabilization of the metastable envelope glycoprotein (Env) of HIV-1 is hypothesized to improve induction of broadly neutralizing antibodies. We improved the expression yield and stability of the HIV-1 envelope glycoprotein BG505SOSIP.664 gp140 by means of a previously described automated sequence and structure-guided computational thermostabilization approach, PROSS. This combines sequence conservation information with computational assessment of mutant stabilization, thus taking advantage of the extensive natural sequence variation present in HIV-1 Env. PROSS is used to design three gp140 variants with 17–45 mutations relative to the parental construct. One of the designs is experimentally observed to have a fourfold improvement in yield and a 4 °C increment in thermostability. In addition, the designed immunogens have similar antigenicity profiles to the native flexible linker version of wild type, BG505SOSIP.664 gp140 (NFL Wt) to major epitopes targeted by broadly neutralizing antibodies. PROSS eliminates the laborious process of screening many variants for stability and functionality, providing a proof of principle of the method for stabilization and improvement of yield without compromising antigenicity for next generation complex, highly glycosylated vaccine candidates.

Hot corinos are of great interest due to their richness in interstellar complex organic molecules (COMs) and the consequent potential prebiotic connection to solar-like planetary systems. Recent surveys have reported an increasing number of detected hot corinos in Class 0/I protostars; however, the relationships between their physical properties and the hot-corino signatures remain elusive. In this study, our objective is to establish a general picture of the detectability of hot corinos by identifying the origins of the hot-corino signatures in the sample of young stellar objects (YSOs) obtained from the Atacama Large Millimeter/submillimeter Array Survey of Orion Planck Galactic Cold Clumps project. We apply spectral energy distribution modeling to our sample and identify the physical parameters of the modeled YSOs directly, linking the detection of hot-corino signatures to the envelope properties of the YSOs. Imaging simulations of the methanol emission further support this scenario. We therefore posit that the observed COM emission originates from the warm inner envelopes of the sample YSOs, based on both the warm region size and the envelope density profile. The former is governed by the source luminosity and is additionally affected by the disk and cavity properties, while the latter is related to the evolutionary stages. This scenario provides a framework for detecting hot-corino signatures toward luminous Class 0 YSOs, with fewer detections being observed toward similarly luminous Class I sources.

Starless cores represent the initial stage of evolution toward (proto)star formation, and a subset of them, known as prestellar cores, with high density (∼ 10 ^6 cm ^−3 or higher) and being centrally concentrated are expected to be embryos of (proto)stars. Determining the density profile of prestellar cores therefore provides an important opportunity to gauge the initial conditions of star formation. In this work, we perform rigorous modeling to estimate the density profiles of three nearly spherical prestellar cores among a sample of five highly dense cores detected by our recent observations. We employed multiscale observational data of the (sub)millimeter dust continuum emission, including those obtained by SCUBA-2 on the James Clerk Maxwell Telescope with a resolution of ∼ 5600 au and by multiple Atacama Large Millimeter/submillimeter Array observations with a resolution as high as ∼ 480 au. We are able to consistently reproduce the observed multiscale dust continuum images of the cores with a simple prescribed density profile, which bears an inner region of flat density and an r ^−2 profile toward the outer region. By utilizing the peak density and the size of the inner flat region as a proxy for the dynamical stage of the cores, we find that the three modeled cores are most likely unstable and prone to collapse. The sizes of the inner flat regions, as compact as ∼ 500 au, signify them as being the highly evolved prestellar cores rarely found to date.

Rapid emergence of the SARS-CoV-2 variants has dampened the protective efficacy of existing authorized vaccines. Nanoparticle platforms offer a means to improve vaccine immunogenicity by presenting multiple copies of desired antigens in a repetitive manner which closely mimics natural infection. We have applied nanoparticle display combined with the SpyTag–SpyCatcher system to design encapsulin–mRBD, a nanoparticle vaccine displaying 180 copies of the monomeric SARS-CoV-2 spike receptor-binding domain (RBD). Here we show that encapsulin–mRBD is strongly antigenic and thermotolerant for long durations. After two immunizations, squalene-in-water emulsion (SWE)-adjuvanted encapsulin–mRBD in mice induces potent and comparable neutralizing antibody titers of 105 against wild-type (B.1), alpha, beta, and delta variants of concern. Sera also neutralizes the recent Omicron with appreciable neutralization titers, and significant neutralization is observed even after a single immunization.

In current seasonal influenza vaccines, neutralizing antibody titers directed against the hemagglutinin surface protein are the primary correlate of protection. These vaccines are, therefore, quantitated in terms of their hemagglutinin content. Adding other influenza surface proteins, such as neuraminidase and M2e, to current quadrivalent influenza vaccines would likely enhance vaccine efficacy. However, this would come with increased manufacturing complexity and cost. To address this issue, as a proof of principle, we have designed genetic fusions of hemagglutinin ectodomains from H3 and H1 influenza A subtypes. These recombinant H1-H3 hemagglutinin ectodomain fusions could be transiently expressed at high yield in mammalian cell culture using Expi293F suspension cells. Fusions were trimeric, and as stable in solution as their individual trimeric counterparts. Furthermore, the H1-H3 fusion constructs were antigenically intact based on their reactivity with a set of conformation-specific monoclonal antibodies. H1-H3 hemagglutinin ectodomain fusion immunogens, when formulated with the MF59 equivalent adjuvant squalene-in-water emulsion (SWE), induced H1 and H3-specific humoral immune responses equivalent to those induced with an equimolar mixture of individually expressed H1 and H3 ectodomains. Mice immunized with these ectodomain fusions were protected against challenge with heterologous H1N1 (Bel/09) and H3N2 (X-31) mouse-adapted viruses with higher neutralizing antibody titers against the H1N1 virus. Use of such ectodomain-fused immunogens would reduce the number of components in a vaccine formulation and allow for the inclusion of other protective antigens to increase influenza vaccine efficacy.

Summary: Desensitization, signaling, and trafficking of G-protein-coupled receptors (GPCRs) are critically regulated by multifunctional adaptor proteins, β-arrestins (βarrs). The two isoforms of βarrs (βarr1 and 2) share a high degree of sequence and structural similarity; still, however, they often mediate distinct functional outcomes in the context of GPCR signaling and regulation. A mechanistic basis for such a functional divergence of βarr isoforms is still lacking. By using a set of complementary approaches, including antibody-fragment-based conformational sensors, we discover structural differences between βarr1 and 2 upon their interaction with activated and phosphorylated receptors. Interestingly, domain-swapped chimeras of βarrs display robust complementation in functional assays, thereby linking the structural differences between receptor-bound βarr1 and 2 with their divergent functional outcomes. Our findings reveal important insights into the ability of βarr isoforms to drive distinct functional outcomes and underscore the importance of integrating this aspect in the current framework of biased agonism. : Ghosh et al. discover structural differences between β-arrestin isoforms (β-arrestin 1 and 2), which are universal regulators of signaling and trafficking of G-protein-coupled receptors (GPCRs). These findings have direct implications for understanding the regulatory and signaling paradigms of GPCRs and designing novel therapeutics targeting this important class of receptors. Keywords: GPCRs, β-arrestins, cellular signaling, antibody fragments, biosensors, biased agonism, desensitization, negative staining, electron microscopy

In this manuscript, we report the application of graphene oxide (GO) in the preparation of cryo-electron microscopy (cryo-EM) and transmission electron microscopy (TEM) grids. We treated GO with water and organic solvents, such as, methanol, ethanol and isopropanol separately to isolate significantly large GO monolayer flake to fabricate the grids for cryo-EM and TEM study. We implemented a simplified approach to isolate flakes of GO monolayer for constructing the TEM grids, independent of expensive heavy equipment (Langmuir–Blodgett trough, glow-discharge system, carbon-evaporator or plasma-cleaner or peristaltic pumps). We employed confocal microscopy, SEM and TEM to characterize the flake size, stability and transparency of the GO monolayer and atomic force microscopy (AFM) to probe the depth of GO coated grids. Additionally, GO grids are visualized at cryogenic condition for suitability of GO monolayer for cryo-EM study. In addition, GO-Met-H2O grids reduce the effect of preferred orientation of biological macromolecules within the amorphous ice. The power-spectrum and contrast-transfer-function unequivocally suggest that GO-Met-H2O fabricated holey grids have excellent potential for application in high-resolution structural characterization of biomolecules. Furthermore, only 200 movies and ~8000 70S ribosome particles are selected on GO-coated grids for cryo-EM reconstruction to achieve high-resolution structure.

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein.