Simultaneous single-cell three-dimensional genome and gene expression profiling uncovers dynamic enhancer connectivity underlying olfactory receptor choice.

The simultaneous measurement of three-dimensional (3D) genome structure and gene expression of individual cells is critical for understanding a genome's structure-function relationship, yet this is challenging for existing methods. Here we present 'Linking mRNA to Chromatin Architecture (LiMCA)', which jointly profiles the 3D genome and transcriptome with exceptional sensitivity and from low-input materials. Combining LiMCA and our high-resolution scATAC-seq assay, METATAC, we successfully characterized chromatin accessibility, as well as paired 3D genome structures and gene expression information, of individual developing olfactory sensory neurons. We expanded the repertoire of known olfactory receptor (OR) enhancers and discovered unexpected rules of their dynamics: OR genes and their enhancers are most accessible during early differentiation. Furthermore, we revealed the dynamic spatial relationship between ORs and enhancers behind stepwise OR expression. These findings offer valuable insights into how 3D connectivity of ORs and enhancers dynamically orchestrate the 'one neuron-one receptor' selection process.

Phase-matching of high-order harmonic generation in the extreme ultraviolet region with orbital angular momentum.

High-order harmonics can generate vortex beams with orbital angular momentum (OAM) in the extreme ultraviolet region. However, experimental research on their phase-matching (PM) characteristics is limited. In this study, vortex high-order harmonic generation (HHG) in the extreme ultraviolet region was generated with Ar gas. Phase-matched HHG with OAM was obtained by optimizing the focus position, laser energy, and gas pressure. The dependence of the PM characteristics on these parameters was analyzed. In addition, we conducted an experimental analysis of the dimensional properties of vortex harmonics under PM conditions. This study is a contribution towards the intense vortex high-order harmonic light sources and their applications.

Single-cell bisulfite-free 5mC and 5hmC sequencing with high sensitivity and scalability.

Existing single-cell bisulfite-based DNA methylation analysis is limited by low DNA recovery, and the measurement of 5hmC at single-base resolution remains challenging. Here, we present a bisulfite-free single-cell whole-genome 5mC and 5hmC profiling technique, named Cabernet, which can characterize 5mC and 5hmC at single-base resolution with high genomic coverage. Cabernet utilizes Tn5 transposome for DNA fragmentation, which enables the discrimination between different alleles for measuring hemi-methylation status. Using Cabernet, we revealed the 5mC, hemi-5mC and 5hmC dynamics during early mouse embryo development, uncovering genomic regions exclusively governed by active or passive demethylation. We show that hemi-methylation status can be used to distinguish between pre- and post-replication cells, enabling more efficient cell grouping when integrated with 5mC profiles. The property of Tn5 naturally enables Cabernet to achieve high-throughput single-cell methylome profiling, where we probed mouse cortical neurons and embryonic day 7.5 (E7.5) embryos, and constructed the library for thousands of single cells at high efficiency, demonstrating its potential for analyzing complex tissues at substantially low cost. Together, we present a way of high-throughput methylome and hydroxymethylome detection at single-cell resolution, enabling efficient analysis of the epigenetic status of biological systems with complicated nature such as neurons and cancer cells.

Correlated Protein Modules Revealing Functional Coordination of Interacting Proteins Are Detected by Single-Cell Proteomics.

Single-cell proteomics has attracted a lot of attention in recent years because it offers more functional relevance than single-cell transcriptomics. However, most work to date has focused on cell typing, which has been widely accomplished by single-cell transcriptomics. Here we report the use of single-cell proteomics to measure the correlation between the translational levels of a pair of proteins in a single mammalian cell. In measuring pairwise correlations among ∼1000 proteins in a population of homogeneous K562 cells under a steady-state condition, we observed multiple correlated protein modules (CPMs), each containing a group of highly positively correlated proteins that are functionally interacting and collectively involved in certain biological functions, such as protein synthesis and oxidative phosphorylation. Some CPMs are shared across different cell types while others are cell-type specific. Widely studied in omics analyses, pairwise correlations are often measured by introducing perturbations into bulk samples. However, some correlations of gene or protein expression under the steady-state condition would be masked by perturbation. The single-cell correlations probed in our experiment reflect intrinsic steady-state fluctuations in the absence of perturbation. We note that observed correlations between proteins are experimentally more distinct and functionally more relevant than those between corresponding mRNAs measured in single-cell transcriptomics. By virtue of single-cell proteomics, functional coordination of proteins is manifested through CPMs.

Excessive consumption of mucin by over-colonized Akkermansia muciniphila promotes intestinal barrier damage during malignant intestinal environment.

Gut microbiota disorders damage the intestinal barrier, which causes intestinal disease. Thus, we screened the microbiota with significant changes using an in situ malignant colorectal cancer (CRC) model. Among the colonies with increased abundance, Akkermansia muciniphila (A. muciniphila) is known for its characteristic of breaking down mucin, which is an essential component of the intestinal barrier. The role of A. muciniphila remains controversial. To investigate the effect of excess A. muciniphila on the intestinal barrier, we established an over-colonized A. muciniphila mouse model by administering a live bacterial suspension after disrupting the original gut microbiome with antibiotics. The results showed that over-colonization of A. muciniphila decreased intestinal mucin content. The mRNA and protein expression levels of tight junction proteins also decreased significantly in the over-colonized A. muciniphila mouse model. Our findings reveal that excess colonization by A. muciniphila breaks the dynamic balance between mucin secretion and degradation, reduces the thickness of the intestinal mucus layer, and damages the intestinal barrier, which would eventually aggravate the development of colitis and CRC. These results will raise awareness about the safety of A. muciniphila serving as a probiotic.

Corrigendum: Lycium barbarum Glycopeptide prevents the development and progression of acute colitis by regulating the composition and diversity of the gut microbiota in mice.

[This corrects the article DOI: 10.3389/fcimb.2022.921075.].

Highly sensitive single-cell chromatin accessibility assay and transcriptome coassay with METATAC.

Recent advances in single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) and its coassays have transformed the field of single-cell epigenomics and transcriptomics. However, the low detection efficiency of current methods has limited our understanding of the true complexity of chromatin accessibility and its relationship with gene expression in single cells. Here, we report a high-sensitivity scATAC-seq method, termed multiplexed end-tagging amplification of transposase accessible chromatin (METATAC), which detects a large number of accessible sites per cell and is compatible with automation. Our high detectability and statistical framework allowed precise linking of enhancers to promoters without merging single cells. We systematically investigated allele-specific accessibility in the mouse cerebral cortex, revealing allele-specific accessibility of promotors of certain imprinted genes but biallelic accessibility of their enhancers. Finally, we combined METATAC with our high-sensitivity single-cell RNA sequencing (scRNA-seq) method, multiple annealing and looping based amplification cycles for digital transcriptomics (MALBAC-DT), to develop a joint ATAC-RNA assay, termed METATAC and MALBAC-DT coassay by sequencing (M2C-seq). M2C-seq achieved significant improvements for both ATAC and RNA compared with previous methods, with consistent performance across cell lines and early mouse embryos.

Lycium barbarum Glycopeptide prevents the development and progression of acute colitis by regulating the composition and diversity of the gut microbiota in mice.

In most cases, recurrent chronic colitis is caused by the recurrence of acute colitis after incomplete recovery and re-exposure to irritating factors, and the gut microbiome, which is the largest micro-ecosystem in the human body, plays a crucial role in the development of colitis. Plant polysaccharides have always been reported to have the ability for anti-inflammation, and they are closely related to the gut microbiome. Lycium barbarum Glycopeptide (LbGP), the most potent component obtained by further isolation and purification from Lycium barbarum fruit, has been shown to inhibit inflammation in animal models. However, its therapeutic efficacy in colitis and its mechanism in gut microbiota regulation have not been fully studied. In our study, the dextran sulfate sodium (DSS)-induced mouse model was used to dynamically evaluate the effect of LbGP in the treatment of acute colitis and the mechanism from the perspective of the gut microbiome through the 16S rDNA sequence. The results showed that LbGP treatment significantly alleviated acute colitis and improved the gut microbiome compared with that in the model group. Harmful bacteria, such as Lachnoclostridium spp. and Parabacteroides_distasonis, were inhibited and probiotics, such as Bacteroides_acidifaciens, Lactobacillus spp., Turicibacter spp., and Alistipes spp., were increased by LbGP treatment. Further, a Random Forest analysis with 10-fold cross-validation identified a family named Muribaculaceae representing colitis development and recovery upon LbGP treatment. In conclusion, our study demonstrated the capability of LbGP to prevent the development of acute colitis by regulating the composition and diversity of the gut microbiota and highlighted the dynamic process of gut microbiota with the colitis progression. Further, it provides evidence to develop LbGP as a functional food supplement and future drug acting on intestinal disease.

Resonance absorption of the inner shell during high-order harmonic generation.

In this work, we report the observation of resonance absorption of the inner shell during the high-order harmonic generation (HHG) from xenon (Xe) and krypton (Kr). The absorption peaks show a periodic variation with the change of carrier-envelope phase of driving laser pulses and the delay of two-color laser field, which indicates the absorption peaks come from the collective multielectron effects during the HHG. With the increase of gas pressure, the depth of absorption peak will continue to increase, while due to the phase matching effect, there will be an optimal pressure for the intensity of harmonic signal. Our experimental results pave the way to uncover the physical mechanism of the collective multielectron effects involving inner-shell electrons in the HHG process.

Controlling of the harmonic generation induced by the Berry curvature.

High-order harmonic generation in solid state has attracted a lot of attentions. The Berry curvature (BC), a geometrical property of the Bloch energy band, plays an important role for the harmonic generation in crystal. As we all know, the influence of BC on the harmonic emission has been investigated before and BC is simplified as a 1D structure. However, many other materials including MoS2 are 2D materials. In this work, we extend the investigation for BC to 2D structure and get a generalized equation, which not only gives a new method to control the harmonic emission with BC, but also gives a deeper understanding for the influence of the BC. We show the ability to control the harmonic emission related to the BC using the orthogonal two-color (OTC) laser field. By tuning the delay of OTC laser field, one can steer the trajectory of electrons and modulate the emission of harmonics. This study can provide us a deeper insight into the role of the BC which is difficult to be measured experimentally.

Changes in genome architecture and transcriptional dynamics progress independently of sensory experience during post-natal brain development.

Both transcription and three-dimensional (3D) architecture of the mammalian genome play critical roles in neurodevelopment and its disorders. However, 3D genome structures of single brain cells have not been solved; little is known about the dynamics of single-cell transcriptome and 3D genome after birth. Here, we generated a transcriptome (3,517 cells) and 3D genome (3,646 cells) atlas of the developing mouse cortex and hippocampus by using our high-resolution multiple annealing and looping-based amplification cycles for digital transcriptomics (MALBAC-DT) and diploid chromatin conformation capture (Dip-C) methods and developing multi-omic analysis pipelines. In adults, 3D genome "structure types" delineate all major cell types, with high correlation between chromatin A/B compartments and gene expression. During development, both transcriptome and 3D genome are extensively transformed in the first post-natal month. In neurons, 3D genome is rewired across scales, correlated with gene expression modules, and independent of sensory experience. Finally, we examine allele-specific structure of imprinted genes, revealing local and chromosome (chr)-wide differences. These findings uncover an unknown dimension of neurodevelopment.

Structurally Resolved SARS-CoV-2 Antibody Shows High Efficacy in Severely Infected Hamsters and Provides a Potent Cocktail Pairing Strategy.

Understanding how potent neutralizing antibodies (NAbs) inhibit SARS-CoV-2 is critical for effective therapeutic development. We previously described BD-368-2, a SARS-CoV-2 NAb with high potency; however, its neutralization mechanism is largely unknown. Here, we report the 3.5-Å cryo-EM structure of BD-368-2/trimeric-spike complex, revealing that BD-368-2 fully blocks ACE2 recognition by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their "up" or "down" conformations. Also, BD-368-2 treats infected adult hamsters at low dosages and at various administering windows, in contrast to placebo hamsters that manifested severe interstitial pneumonia. Moreover, BD-368-2's epitope completely avoids the common binding site of VH3-53/VH3-66 recurrent NAbs, evidenced by tripartite co-crystal structures with RBDs. Pairing BD-368-2 with a potent recurrent NAb neutralizes SARS-CoV-2 pseudovirus at pM level and rescues mutation-induced neutralization escapes. Together, our results rationalized a new RBD epitope that leads to high neutralization potency and demonstrated BD-368-2's therapeutic potential in treating COVID-19.

Laser-induced inner-shell excitations through direct electron re-collision versus indirect collision.

The dynamics and the decay processes of inner-shell excited atoms are of great interest in physics, chemistry, biology, and technology. The highly excited state decays very quickly through different channels, both radiative and non-radiative. It is therefore a long-standing goal to study such dynamics directly in the time domain. Using few-cycle infrared laser pulses, we investigated the excitation and ionization of inner-shell electrons through laser-induced electron re-collision with the original parent ions and measured the dependence of the emitted x-ray spectra on the intensity and ellipticity of the driving laser. These directly re-colliding electrons can be used as the initiating pump step in pump/probe experiments for studying core-hole dynamics at their natural temporal scale. In our experiment we found that the dependence of the x-ray emission spectrum on the laser intensity and polarization state varies distinctly for the two kinds of atomic systems. Relying on our data and numerical simulations, we explain this behavior by the presence of different excitation mechanisms that are contributing in different ratios to the respective overall x-ray emission yields. Direct re-collision excitation competes with indirect collisions with neighboring atoms by electrons having "drifted away" from the original parent ion.

Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients' B Cells.

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 Å cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.

Infrared-laser-induced ultrafast modulation on the spectrum of an extreme-ultraviolet attosecond pulse.

We present a theoretical investigation of the isolated extreme-ultraviolet (XUV) attosecond pulse propagating in the ionizing gas induced by a synchronized strong infrared (IR) laser, with the numerical solution of the nonadiabatic one-dimensional propagation model. Upon scanning the relative delay between the XUV and the IR pulse, it is found that the delay-dependent XUV transmission spectrogram exhibits the unique pattern that is controllable by the chirp of the XUV pulse. Furthermore, we demonstrate that this kind of spectrum modulation can be attributed to the term of the light energy loss involved in the propagation equation. The characteristics of the spectrum modulation dependent on the XUV chirp might provide an all-optical way for the reconstruction of the XUV spectral phase.

Molecular targeting of VEGF/VEGFR signaling by the anti-VEGF monoclonal antibody BD0801 inhibits the growth and induces apoptosis of human hepatocellular carcinoma cells in vitro and in vivo.

Hepatocellular carcinoma (HCC) is the third-leading cause of cancer-related deaths with 750,000 newly diagnosed cases each year. Surgery, radiotherapy, and chemotherapy constitute the main treatment modalities for HCC, but liver cirrhosis and damage often occur. Molecular targeted drugs have been recently developed to treat HCC. Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) autocrine signaling is closely related to the growth, progression, and metastasis of HCC, making the VEGF/VEGFR axis an ideal target for the development of molecular targeted agents. Here, we report the effects of the novel anti-VEGF humanized monoclonal antibody BD0801 on the growth of HCC cells in vitro and in vivo as well as the underlying mechanisms. BD0801 significantly inhibited the proliferation of HepG2, SMMC-7721, and Bel7402 cells in vitro, accompanied with an induction of apoptosis and cell cycle arrest at the G1 phase. BD0801 potently suppressed AKT, Erk1/2, and retinoblastoma (Rb) phosphorylation, while increasing p21 and decreasing cyclin D1 protein levels. BD0801 significantly inhibited growth in mouse tumor xenografts and induced cell apoptosis of HepG2 and SMMC-7721 tumor xenografts. Furthermore, BD0801 effectively reduced the vascular density and tumor tissue microvessel density (MVD). Similarly, BD0801 decreased AKT, Erk1/2, and Rb phosphorylation and cyclin D1 expression whereas it increased p21 protein expression in mouse HCC tumor xenografts. Importantly, BD0801 showed a better effect than Bevacizumab (Bev) on the inhibition of cell growth and induction of apoptosis in HCC cells in vitro and in vivo. These findings suggest that BD0801 is a potent anti-VEGF monoclonal antibody for the treatment of HCC.

Attosecond chirp effect on the transient absorption spectrum of laser-dressed helium atom.

We theoretically investigate the attosecond transient absorption spectrum of helium atom in the presence of an infrared-dressed laser pulse upon scanning their relative delay, with the particular emphasis on the chirp effect of the attosecond pulse. By numerically solving the fully three-dimensional time-dependent Schrödinger equation, we identify the attoscecond chirp can induce the temporal shift of the absorption spectrogram along the delay axis. Additionally, it is found that the extent of the temporal shift is dependent on both the position of the absorption line and the infrared pulse wavelength, which is well confirmed and reproduced by a three-level model. Moreover, we demonstrate that the observed features can be quantitatively explained in terms of the indirect two-photon absorption processes through some virtual states. This effect might provide a way to measure the chirp of attosecond pulse in an all-optical way.

Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos.

CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing.

Efficacy and safety of the oxaliplatin-based chemotherapy in the treatment of advanced primary hepatocellular carcinoma: A meta-analysis of prospective studies.

BACKGROUND: Many clinical studies have demonstrated the survival benefits of oxaliplatin-based chemotherapy for advanced hepatocellular carcinoma patients. Therefore, we aim to evaluate the efficacy and safety of oxaliplatin-based chemotherapy in patients with advanced hepatocellular carcinoma by conducting a meta-analysis of prospective studies. METHODS: A comprehensive literature search was performed using the PubMed, Cochrane Library, EMBASE, and Web of Science databases from their inception to June 2016. Only prospective studies evaluating oxaliplatin-based chemotherapy in patients with advanced hepatocellular carcinoma were selected. The main outcomes included objective response rate (ORR), progression-free survival (PFS), overall survival (OS), and main adverse events. RESULTS: Ten prospective studies involving 525 patients were included. The pooled ORR, 1-year PFS, and OS were 14.4% (95% confidence interval [CI] 9.2-19.6%), 9.3% (95%CI 10-28%), and 35.7% (95%CI 27-44%), respectively, for oxaliplatin-based chemotherapy. The median PFS and OS were 4.7 and 9.4 months, respectively. The incidences of grade 3/4 toxicities of neutropenia, thrombopenia, anemia, neurotoxicity, diarrhea, and nausea/vomiting were 17.2%, 9.2%, 6.0%, 4.8%, 3.1%, and 1.8%, respectively. Subgroup analysis revealed that the pooled ORR was 13.9% (95%CI 6.8-21%) in Asian patients and 12.8% (95%CI 6.8-18.7%) in Western patients. For Asian patients, the median PFS and OS were 4.2 and 9.2 months, and the 1-year PFS and OS were 12.5% and 30.5%, respectively. For Western patients, the median PFS and OS were 4.7 and 9.5 months, and the 1-year PFS and OS were 19.6% and 42.4%, respectively. There were no significant differences in the ORR, 1-year PFS, and OS (P > 0.05) between Asian and Western patients. CONCLUSIONS: Oxaliplatin-based chemotherapy appears to be effective and safe for the treatment of advanced hepatocellular carcinoma.