Antibody Lineages with Vaccine-Induced Antigen-Binding Hotspots Develop Broad HIV Neutralization.

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

Vaccination induces maturation in a mouse model of diverse unmutated VRC01-class precursors to HIV-neutralizing antibodies with >50% breadth.

Vaccine elicitation of broadly neutralizing antibodies (bnAbs) is a key HIV-research goal. The VRC01 class of bnAbs targets the CD4-binding site on the HIV-envelope trimer and requires extensive somatic hypermutation (SHM) to neutralize effectively. Despite substantial progress, vaccine-induced VRC01-class antibodies starting from unmutated precursors have exhibited limited neutralization breadth, particularly against viruses bearing glycan on loop D residue N276 (glycan276), present on most circulating strains. Here, using sequential immunization of immunoglobulin (Ig)-humanized mice expressing diverse unmutated VRC01-class antibody precursors, we elicited serum responses capable of neutralizing viruses bearing glycan276 and isolated multiple lineages of VRC01-class bnAbs, including two with >50% breadth on a 208-strain panel. Crystal structures of representative bnAbs revealed the same mode of recognition as known VRC01-class bnAbs. Structure-function studies further pinpointed key mutations and correlated their induction with specific immunizations. VRC01-class bnAbs can thus be matured by sequential immunization from unmutated ancestors to >50% breadth, and we delineate immunogens and regimens inducing key SHM.

Cell competition constitutes a barrier for interspecies chimerism.

Cell competition involves a conserved fitness-sensing process during which fitter cells eliminate neighbouring less-fit but viable cells1. Cell competition has been proposed as a surveillance mechanism to ensure normal development and tissue homeostasis, and has also been suggested to act as a barrier to interspecies chimerism2. However, cell competition has not been studied in an interspecies context during early development owing to the lack of an in vitro model. Here we developed an interspecies pluripotent stem cell (PSC) co-culture strategy and uncovered a previously unknown mode of cell competition between species. Interspecies competition between PSCs occurred in primed but not naive pluripotent cells, and between evolutionarily distant species. By comparative transcriptome analysis, we found that genes related to the NF-κB signalling pathway, among others, were upregulated in less-fit 'loser' human cells. Genetic inactivation of a core component (P65, also known as RELA) and an upstream regulator (MYD88) of the NF-κB complex in human cells could overcome the competition between human and mouse PSCs, thereby improving the survival and chimerism of human cells in early mouse embryos. These insights into cell competition pave the way for the study of evolutionarily conserved mechanisms that underlie competitive cell interactions during early mammalian development. Suppression of interspecies PSC competition may facilitate the generation of human tissues in animals.

The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations.

Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase-the positive transcription elongation factor complex (P-TEFb)-from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype.

Spatial transcriptomics reveals light-induced chlorenchyma cells involved in promoting shoot regeneration in tomato callus.

Callus is a reprogrammed cell mass involved in plant regeneration and gene transformation in crop engineering. Pluripotent callus cells develop into fertile shoots through shoot regeneration. The molecular basis of the shoot regeneration process in crop callus remains largely elusive. This study pioneers the exploration of the spatial transcriptome of tomato callus during shoot regeneration. The findings reveal the presence of highly heterogeneous cell populations within the callus, including epidermis, vascular tissue, shoot primordia, inner callus, and outgrowth shoots. By characterizing the spatially resolved molecular features of shoot primordia and surrounding cells, specific factors essential for shoot primordia formation are identified. Notably, chlorenchyma cells, enriched in photosynthesis-related processes, play a crucial role in promoting shoot primordia formation and subsequent shoot regeneration. Light is shown to promote shoot regeneration by inducing chlorenchyma cell development and coordinating sugar signaling. These findings significantly advance our understanding of the cellular and molecular aspects of shoot regeneration in tomato callus and demonstrate the immense potential of spatial transcriptomics in plant biology.

Dot Scanner: open-source software for quantitative live-cell imaging in planta.

Confocal microscopy has greatly aided our understanding of the major cellular processes and trafficking pathways responsible for plant growth and development. However, a drawback of these studies is that they often rely on the manual analysis of a vast number of images, which is time-consuming, error-prone, and subject to bias. To overcome these limitations, we developed Dot Scanner, a Python program for analyzing the densities, lifetimes, and displacements of fluorescently tagged particles in an unbiased, automated, and efficient manner. Dot Scanner was validated by performing side-by-side analysis in Fiji-ImageJ of particles involved in cellulose biosynthesis. We found that the particle densities and lifetimes were comparable in both Dot Scanner and Fiji-ImageJ, verifying the accuracy of Dot Scanner. Dot Scanner largely outperforms Fiji-ImageJ, since it suffers far less selection bias when calculating particle lifetimes and is much more efficient at distinguishing between weak signals and background signal caused by bleaching. Not only does Dot Scanner obtain much more robust results, but it is a highly efficient program, since it automates much of the analyses, shortening workflow durations from weeks to minutes. This free and accessible program will be a highly advantageous tool for analyzing live-cell imaging in plants.

The TRAPPIII subunit, Trs85, has a dual role in the trafficking of cellulose synthase complexes in Arabidopsis.

Plant cell walls are essential for defining plant growth and development, providing structural support to the main body and responding to abiotic and biotic cues. Cellulose, the main structural polymer of plant cell walls, is synthesized at the plasma membrane by cellulose synthase complexes (CSCs). The construction and transport of CSCs to and from the plasma membrane is poorly understood but is known to rely on the coordinated activity of cellulose synthase-interactive protein 1 (CSI1), a key regulator of CSC trafficking. In this study, we found that Trs85, a TRAPPIII complex subunit, interacted with CSI1 in vitro. Using functional genetics and live-cell imaging, we have shown that trs85-1 mutants have reduced cellulose content, stimulated CSC delivery, an increased population of static CSCs and deficient clathrin-mediated endocytosis in the primary cell wall. Overall, our findings suggest that Trs85 has a dual role in the trafficking of CSCs, by negatively regulating the exocytosis and clathrin-mediated endocytosis of CSCs.

SAIBR: a simple, platform-independent method for spectral autofluorescence correction.

Biological systems are increasingly viewed through a quantitative lens that demands accurate measures of gene expression and local protein concentrations. CRISPR/Cas9 gene tagging has enabled increased use of fluorescence to monitor proteins at or near endogenous levels under native regulatory control. However, owing to typically lower expression levels, experiments using endogenously tagged genes run into limits imposed by autofluorescence (AF). AF is often a particular challenge in wavelengths occupied by commonly used fluorescent proteins (GFP, mNeonGreen). Stimulated by our work in C. elegans, we describe and validate Spectral Autofluorescence Image Correction By Regression (SAIBR), a simple platform-independent protocol and FIJI plug-in to correct for autofluorescence using standard filter sets and illumination conditions. Validated for use in C. elegans embryos, starfish oocytes and fission yeast, SAIBR is ideal for samples with a single dominant AF source; it achieves accurate quantitation of fluorophore signal, and enables reliable detection and quantification of even weakly expressed proteins. Thus, SAIBR provides a highly accessible low-barrier way to incorporate AF correction as standard for researchers working on a broad variety of cell and developmental systems.

Cell biology of primary cell wall synthesis in plants.

Building a complex structure such as the cell wall, with many individual parts that need to be assembled correctly from distinct sources within the cell, is a well-orchestrated process. Additional complexity is required to mediate dynamic responses to environmental and developmental cues. Enzymes, sugars, and other cell wall components are constantly and actively transported to and from the plasma membrane during diffuse growth. Cell wall components are transported in vesicles on cytoskeletal tracks composed of microtubules and actin filaments. Many of these components, and additional proteins, vesicles, and lipids are trafficked to and from the cell plate during cytokinesis. In this review, we first discuss how the cytoskeleton is initially organized to add new cell wall material or to build a new cell wall, focusing on similarities during these processes. Next, we discuss how polysaccharides and enzymes that build the cell wall are trafficked to the correct location by motor proteins and through other interactions with the cytoskeleton. Finally, we discuss some of the special features of newly formed cell walls generated during cytokinesis.

R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters.

R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed.

Essential role of Alix in regulating cardiomyocyte exosome biogenesis under physiological and stress conditions.

BACKGROUND: Exosomes released by cardiomyocytes are essential mediators of intercellular communications within the heart, and various exosomal proteins and miRNAs are associated with cardiovascular diseases. However, whether the endosomal sorting complex required for transport (ESCRT) and its key component Alix is required for exosome biogenesis within cardiomyocyte remains poorly understood. METHODS: Super-resolution imaging was performed to investigate the subcellular location of Alix and multivesicular body (MVB) in primary cardiomyocytes. Cardiomyocyte-specific Alix-knockout mice were generated using AAV9/CRISPR/Cas9-mediated in vivo gene editing. A stable Alix-knockdown H9c2 cardiomyocyte line was constructed through lentiviral-mediated delivery of short hairpin RNA. In order to determine the role of Alix in controlling exosome biogenesis, exosomes from cardiomyocyte-specific Alix-knockout mice plasma and Alix-knockdown H9c2 culture medium were isolated and examined by western blot, NTA analysis and transmission electron microscopy. Biochemical and immunofluorescence analysis were performed to determine the role of ESCRT machinery in regulating MVB formation. Lastly, transverse aortic constriction (TAC)-induced cardiac pressure overload model was established to further explore the role of Alix-mediated exosome biogenesis under stress conditions. RESULTS: A significant proportion of Alix localized to the MVB membrane within cardiomyocytes. Genetic deletion of Alix in murine heart resulted in a reduction of plasma exosome content without affecting cardiac structure or contractile function. Consistently, the downregulation of Alix in H9c2 cardiomyocyte line also suppressed the biogenesis of exosomes. We found the defective ESCRT machinery and suppressed MVB formation upon Alix depletion caused compromised exosome biogenesis. Remarkably, TAC-induced cardiac pressure overload led to increased Alix, MVB levels, and elevated plasma exosome content, which could be totally abolished by Alix deletion. CONCLUSION: These results establish Alix as an essential and stress-sensitive regulator of cardiac exosome biogenesis and the findings may yield valuable therapeutic implications.

mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells.

Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo.

Critical Residues Involved in the Coassembly of L1 and L2 Capsid Proteins of Human Papillomavirus 16.

Human papillomaviruses (HPV) are small DNA viruses associated with cervical cancer, warts, and other epithelial tumors. Structural studies have shown that the HPV capsid consists of 360 copies of the major capsid protein, L1, arranged as 72 pentamers in a T=7 icosahedral lattice, coassembling with substoichiometric amounts of the minor capsid protein, L2. However, the residues involved in the coassembly of L1 and L2 remain undefined due to the lack of structure information. Here, we investigated the solvent accessibility surfaces (SASs) of the central cavity residues of the HPV16 L1 pentamer in the crystal structure because those internal exposed residues might mediate the association with L2. Twenty residues in L1 protein were selected to be analyzed, with four residues in the lumen of the L1 pentamer identified as important: F256, R315, Q317, and T340. Mutations to these four residues reduced the PsV (pseudovirus) infection capacity in 293FT cells, and mutations to R315, Q317, and T340 substantially perturb L2 from coassembling into L1 capsid. Compared with wild-type (WT) PsVs, these mutant PsVs also have a reduced ability to become internalized into host cells. Finally, we identified a stretch of negatively charged residues on L2 (amino acids [aa] 337 to 340 [EEIE]), mutations to which completely abrogate L2 assembly into L1 capsid and subsequently impair the endocytosis and infectivity of HPV16 PsVs. These findings shed light on the elusive coassembly between HPV L1 and L2. IMPORTANCE Over 200 types of HPV have been isolated, with several high-risk types correlated with the occurrence of cervical cancer. The HPV major capsid protein, L1, assembles into a T=7 icosahedral viral shell, and associates with the minor capsid protein, L2, which plays a critical role in the HPV life cycle. Despite the important role of the L2 protein, its structure and coassembly with L1 remain elusive. In this study, we analyzed the amino acid residues at the proposed interface between L1 and L2. Certain mutations at these sites decreased the amount of L2 protein assembled into the capsid, which, in turn, led to a decrease in viral infectivity. Knowledge about these residues and the coassembly of L1 and L2 could help to expand our understanding of HPV biology and aid in the development of countermeasures against a wide range of HPV types by targeting the L2 protein.

Structural basis for broad neutralization of human antibody against Omicron sublineages and evasion by XBB variant.

IMPORTANCE: The ongoing COVID-19 pandemic has been characterized by the emergence of new SARS-CoV-2 variants including the highly transmissible Omicron XBB sublineages, which have shown significant resistance to neutralizing antibodies (nAbs). This resistance has led to decreased vaccine effectiveness and therefore result in breakthrough infections and reinfections, which continuously threaten public health. To date, almost all available therapeutic nAbs, including those authorized under Emergency Use Authorization nAbs that were previously clinically useful against early strains, have recently been found to be ineffective against newly emerging variants. In this study, we provide a comprehensive structural basis about how the Class 3 nAbs, including 1G11 in this study and noted LY-CoV1404, are evaded by the newly emerged SARS-CoV-2 variants.

Bromine Substitution Improves the Photothermal Performance of π-Conjugated Phototheranostic Molecules.

NIR-II fluorescence imaging-guided photothermal therapy (PTT) has been widely investigated due to its great application potential in tumor theranostics. PTT is an effective and non-invasive tumor treatment method that can adapt to tumor hypoxia; nevertheless, simple and effective strategies are still desired to develop new materials with excellent PTT properties to meet clinical requirements. In this work, we developed a bromine-substitution strategy to enhance the PTT of A-D-A'-D-A π-conjugated molecules. The experimental results reveal that bromine substitution can notably enhance the absorptivity (ϵ) and photothermal conversion efficiency (PCE) of the π-conjugated molecules, resulting in the brominated molecules generating two times more heat (ϵ808 nm ×PCE) than their unsubstituted counterpart. We disclose that the enhanced photothermal properties of bromine-substituted π-conjugated molecules are a combined outcome of the heavy-atom effect, enhanced ICT effect, and more intense bromine-mediate intermolecular π-π stacking. Finally, the NIR-II tumor imaging capability and efficient PTT tumor ablation of the brominated π-conjugated materials demonstrate that bromine substitution is a promising strategy for developing future high-performance NIR-II imaging-guided PTT agents.

Entangled dark state mediated by a dielectric cavity within epsilon-near-zero materials.

Two emitters can be entangled by manipulating them through optical fields within a photonic cavity. However, maintaining entanglement for a long time is challenging due to the decoherence of the entangled qubits, primarily caused by cavity loss and atomic decay. Here, we found the entangled dark state between two emitters mediated by a dielectric cavity within epsilon-near-zero (ENZ) materials, ensuring entanglement maintenance over an extended period. To obtain the entangled dark state, we derived an effective model with degenerate mode modulation. In the dielectric cavities within ENZ materials, the decay rate of emitters can be regarded as 0, which is the key to achieving the entangled dark state. Meanwhile, the dark state immune to cavity loss exists when two emitters are in symmetric positions in the dielectric cavity. Additionally, by adjusting the emitters to specific asymmetric positions, it is possible to achieve transient entanglement with higher concurrence. By overcoming the decoherence of the entangled qubits, this study demonstrates stable, long-term entanglement with ENZ materials, holding significant importance for applications such as nanodevice design for quantum communication and quantum information processing.

Combinatory data-independent acquisition and parallel reaction monitoring method for revealing the lipid metabolism biomarkers of coronary heart disease and its comorbidities.

Disorders of lipid metabolism are a common cause of coronary heart disease (CHD) and its comorbidities. In this study, ultra-performance liquid chromatography-high-resolution mass spectrometry in data-independent acquisition (DIA) mode was applied to collect abundant tandem mass spectrometry data, which provided valuable information for lipid annotation. For the lipid isomers that could not be completely separated by chromatography, parallel reaction monitoring (PRM) mode was used for quantification. A total of 223 plasma lipid metabolites were annotated, and 116 of them were identified for their fatty acyl chain composition and location. In addition, 152 plasma lipids in patients with CHD and its comorbidities were quantitatively analyzed. Multivariate statistical analysis and metabolic pathway analysis demonstrated that glycerophospholipid and sphingolipid metabolism deserved more attention for CHD. This study proposed a method combining DIA and PRM for high-throughput characterization of plasma lipids. The results also improved our understanding of metabolic disorders of CHD and its comorbidities, which can provide valuable suggestions for medical intervention.

Determination of organic acids for predicting sourness intensity of tea beverage by liquid chromatography-tandem mass spectrometry and chemometrics methods.

The contents of organic acids (OAs) in tea beverage and their relationship with taste intensity have not been fully understood. In this work, a rapid (10 min for a single run) and sensitive (limits of quantification: 0.0044-0.4486 µg/mL) method was developed and validated for the simultaneous determination of 17 OAs in four types of tea, based on liquid chromatography-tandem mass spectrometry with multiple reaction monitoring mode. The contents of 17 OAs in 96 tea samples were measured at levels between 0.01 and 11.80 g/kg (dried weight). Quinic acid, citric acid, and malic acid were determined as the major OAs in green, black, and raw pu-erh teas, while oxalic acid and tartaric acid exhibited the highest contents in ripe pu-erh tea. Taking the OAs composition as input features, a partial least squares regression model was proposed to predict the sourness intensity of tea beverages. The model achieved a root-mean-square error of 0.58 and a coefficient of determination of 0.84 for the testing set. The proposed model provides a theoretical way to evaluate the sensory quality of tea infusion based on its chemical composition.

Effects of electronic screen exposure time on hypertensive disorders in pregnancy: a retrospective cohort study.

BACKGROUND: We previously conducted a case-control study and found that exposure to electronic screen before nocturnal sleep was associated with hypertensive disorders in pregnancy (HDP). Hence, we carried out this cohort study aiming to identify the effects of screen exposure time on the incidence rate and severity of HDP. METHODS: A retrospective cohort study was conducted from January 2022 and July 2022 from three hospitals in Wuxi and Changzhou cities. A total of 732 women were recruited and the information included socio-demographic characteristics, screen exposure and outcomes. Generalized estimating equations and binary non-conditional logistic models were applied to multivariate analysis, calculating the odds ratios (ORs) and 95% confidence intervals (CIs) of screen exposure time. RESULTS: The duration order of total screen time was smartphone > computer > television, while the duration order of screen time before nocturnal sleep was smartphone > television > computer. Multivariate analyses showed that the susceptibility of HDP among women who exposed to television before nocturnal sleep was 81.5% percent higher than those not exposed (P = 0.018, OR[95%CI] = 1.815[1.106-2.981]). In addition, total daily exposure time of television in the third trimester of pregnancy significantly increased the severity of HDP (P = 0.021, OR[95%CI] = 3.641[1.213-10.927]). CONCLUSIONS: Based on this preliminary study, we would suggest that pregnant women do not watch television before nocturnal sleep. While in the third trimester of pregnancy, total exposure time of television should be limited. Investigations from other areas and experimental studies should be conducted to verify the conclusion.

Fluorescence sensor based on optimized quantum yield manganese-carbon polymer dots and smartphone-integrated sensing platform for tetracycline detection.

The one-step synthesis of Mn-doped carbon quantum dots (Mn-CPDs) with a high quantum yield (QY = 45%) is reported using the microwave-assisted method. Subsequently, Mn-CPDs were successfully combined with Eu3+ ions to construct an Eu3+@Mn-CPDs fluorescence sensor. The presence of tetracycline (TC) induced a transition of fluorescence emission from blue (434 nm) to red (618 nm), and a robust linear relationship was observed between the ratio of F618 nm / F434 nm and the TC concentration (5 - 50 nmol/L), with a limit of detection (LOD) of 5.76 nmol/L. The underlying mechanism of Eu3+@Mn-CPDs and TC sensing is unveiled as a synergistic effect involving inner filter effect (IFE) and concurrent interactions. Notably, the smartphone-integrated sensing platform based on Eu3+@Mn-CPDs enables rapid and quantitative TC detection within a short time (< 30 s) by monitoring fluorescence color changes, achieving high-detection sensitivities (with a LOD of 6.18 nmol/L). This versatile and efficient sensing platform demonstrates its potential for the determination of TC concentrations in milk, honey, and tap water samples.