Quantitative catalogue of mammalian mitotic chromosome-associated RNAs.

The faithful transmission of a cell's identity and functionality to its daughters during mitosis requires the proper assembly of mitotic chromosomes from interphase chromatin in a process that involves significant changes in the genome-bound material, including the RNA. However, our understanding of the RNA that is associated with the mitotic chromosome is presently limited. Here, we present complete and quantitative characterizations of the full-length mitotic chromosome-associated RNAs (mCARs) for 3 human cell lines, a monkey cell line, and a mouse cell line derived from high-depth RNA sequencing (3 replicates, 47 M mapped read pairs for each replicate). Overall, we identify, on average, more than 20,400 mCAR species per cell-type (including isoforms), more than 5,200 of which are enriched on the chromosome. Notably, overall, more than 2,700 of these mCARs were previously unknown, which thus also expands the annotated genome of these species. We anticipate that these datasets will provide an essential resource for future studies to better understand the functioning of mCARs on the mitotic chromosome and in the cell.

Evaluation of Spike Protein Epitopes by Assessing the Dynamics of Humoral Immune Responses in Moderate COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike protein (S) of SARS-CoV-2 is a major target for diagnosis and vaccine development because of its essential role in viral infection and host immunity. Currently, time-dependent responses of humoral immune system against various S protein epitopes are poorly understood. In this study, enzyme-linked immunosorbent assay (ELISA), peptide microarray, and antibody binding epitope mapping (AbMap) techniques were used to systematically analyze the dynamic changes of humoral immune responses against the S protein in a small cohort of moderate COVID-19 patients who were hospitalized for approximately two months after symptom onset. Recombinant truncated S proteins, target S peptides, and random peptides were used as antigens in the analyses. The assays demonstrated the dynamic IgM- and IgG recognition and reactivity against various S protein epitopes with patient-dependent patterns. Comprehensive analysis of epitope distribution along the spike gene sequence and spatial structure of the homotrimer S protein demonstrated that most IgM- and IgG-reactive peptides were clustered into similar genomic regions and were located at accessible domains. Seven S peptides were generally recognized by IgG antibodies derived from serum samples of all COVID-19 patients. The dynamic immune recognition signals from these seven S peptides were comparable to those of the entire S protein or truncated S1 protein. This suggested that the humoral immune system recognized few conserved S protein epitopes in most COVID-19 patients during the entire duration of humoral immune response after symptom onset. Furthermore, in this cohort, individual patients demonstrated stable immune recognition to certain S protein epitopes throughout their hospitalization period. Therefore, the dynamic characteristics of humoral immune responses to S protein have provided valuable information for accurate diagnosis and immunotherapy of COVID-19 patients.

Robust Acquisition of Spatial Transcriptional Programs in Tissues With Immunofluorescence-Guided Laser Capture Microdissection.

The functioning of tissues is fundamentally dependent upon not only the phenotypes of the constituent cells but also their spatial organization in the tissue, as local interactions precipitate intra-cellular events that often lead to changes in expression. However, our understanding of these processes in tissues, whether healthy or diseased, is limited at present owing to the difficulty in acquiring comprehensive transcriptional programs of spatially- and phenotypically-defined cells in situ. Here we present a robust method based on immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq) to acquire finely resolved transcriptional programs with as few as tens of cells from snap-frozen or RNAlater-treated clinical tissues sufficient to resolve even isoforms. The protocol is optimized to protect the RNA with a small molecule inhibitor, the ribonucleoside vanadyl complex (RVC), which thereby enables the typical time-consuming immunostaining and laser capture steps of this procedure during which RNA is usually severely degraded in existing approaches. The efficacy of this approach is exemplified by the characterization of differentially expressed genes between the mouse small intestine lacteal cells at the tip versus the main capillary body, including those that function in sensing and responding to local environmental cues to stimulate intra-cellular signalling. With the extensive repertoire of specific antibodies that are presently available, our method provides an unprecedented capability for the analysis of transcriptional networks and signalling pathways during development, pathogenesis, and aging of specific cell types within native tissues.

Single-Molecule Micromanipulation and Super-Resolution Imaging Resolve Nanodomains Underlying Chromatin Folding in Mitotic Chromosomes.

The folding of interphase chromatin into highly compact mitotic chromosomes is one of the most recognizable changes during the cell cycle. However, the structural organization underlying this drastic compaction remains elusive. Here, we combine several super resolution methods, including structured illumination microscopy (SIM), binding-activated localization microscopy (BALM), and atomic force microscopy (AFM), to examine the structural details of the DNA within the mitotic chromosome, both in the native state and after up to 30-fold extension using single-molecule micromanipulation. Images of native chromosomes reveal widespread ∼125 nm compact granules (CGs) throughout the metaphase chromosome. However, at maximal extensions, we find exclusively ∼90 nm domains (mitotic nanodomains, MNDs) that are unexpectedly resistant to extensive forces of tens of nanonewtons. The DNA content of the MNDs is estimated to be predominantly ∼80 kb, which is comparable to the size of the inner loops predicted by a recent nested loop model of the mitotic chromosome. With this DNA content, the total volume expected of the human genome assuming closely packed MNDs is nearly identical to what is observed. Thus, altogether, these results suggest that these mechanically stable MNDs, and their higher-order assembly into CGs, are the dominant higher-level structures that underlie the compaction of chromatin from interphase to metaphase.

Epithelial Cells in 2D and 3D Cultures Exhibit Large Differences in Higher-order Genomic Interactions.

Recent studies have characterized the genomic structures of many eukaryotic cells, often focusing on their relation to gene expression. However, these studies have largely investigated cells grown in 2D cultures, although the transcriptomes of 3D-cultured cells are generally closer to their in vivo phenotypes. To examine the effects of spatial constraints on chromosome conformation, we investigated the genomic architecture of mouse hepatocytes grown in 2D and 3D cultures using in situ Hi-C. Our results reveal significant differences in higher-order genomic interactions, notably in compartment identity and strength as well as in topologically associating domain (TAD)-TAD interactions, but only minor differences are found at the TAD level. Our RNA-seq analysis reveals an up-regulated expression of genes involved in physiological hepatocyte functions in the 3D-cultured cells. These genes are associated with a subset of structural changes, suggesting that differences in genomic structure are critically important for transcriptional regulation. However, there are also many structural differences that are not directly associated with changes in gene expression, whose cause remains to be determined. Overall, our results indicate that growth in 3D significantly alters higher-order genomic interactions, which may be consequential for a subset of genes that are important for the physiological functioning of the cell.

The binding epitope of sintilimab on PD-1 revealed by AbMap.

PD-1 plays an important role as an immune checkpoint. Sintilimab is a newly approved PD-1 antibody for cancer immunotherapy with an unknown binding epitope on PD-1. In this study, to elucidate the molecular mechanism by which sintilimab blocks PD-1 activation, we applied Antibody binding epitope Mapping (AbMap) to identify the binding epitope of sintilimab. An epitope was successfully identified, i.e. SLAPKA, aa 127-132. By constructing a series of point mutations, the dominant residues S127, L128, A129, P130, and A132 of PD-1 were further validated by western blot analysis, biolayer interferometry, and flow cytometry. Structural analysis showed that the epitope is partially within the binding interface of PD-1 and PD-L1, and this epitope also partially overlaps with that of nivolumab and pembrolizumab. These results demonstrate that sintilimab can attenuate PD-1 activation by directly competing with the interaction between PD-1 and PD-L1 through binding with the key residues of the FG loop on PD-1. This study also demonstrates the high efficiency and accuracy of AbMap for determining the binding epitope of therapeutic antibodies.

Anomalous self-optimization of sulfate ions for boosted oxygen evolution reaction.

Broadly, the oxygen evolution reaction (OER) has been deeply understood as a significant part of energy conversion and storage. Nevertheless, the anions in the OER catalysts have been neglected for various reasons such as inactive sites, dissolution, and oxidation, amongst others. Herein, we applied a model catalyst s-Ni(OH)2 to track the anionic behavior in the catalyst during the electrochemical process to fill this gap. The advanced operando synchrotron radiation Fourier transform infrared (SR-FTIR) spectroscopy, synchrotron radiation photoelectron spectroscopy (SRPES) depth detection and differential X-ray absorption fine structure (Δ-XAFS) spectrum jointly point out that some oxidized sulfur species (SO42-) will self-optimize new Ni-S bonds during OER process. Such amazing anionic self-optimization (ASO) behavior has never been observed in the OER process. Subsequently, the optimization-derived component shows a significantly improved electrocatalytic performance (activity, stability, etc.) compared to reference catalyst Ni(OH)2. Theoretical calculation further suggests that the ASO process indeed derives a thermodynamically stable structure of the OER catalyst, and then gives its superb catalytic performance by optimizing the thermodynamic and kinetic processes in the OER, respectively. This work demonstrates the vital role of anions in the electrochemical process, which will open up new perspectives for understanding OER and provide some new ideas in related fields (especially catalysis and chemistry).

Antibody Binding Epitope Mapping (AbMap) of Hundred Antibodies in a Single Run.

Antibodies play essential roles in both diagnostics and therapeutics. Epitope mapping is essential to understand how an antibody works and to protect intellectual property. Given the millions of antibodies for which epitope information is lacking, there is a need for high-throughput epitope mapping. To address this, we developed a strategy, Antibody binding epitope Mapping (AbMap), by combining a phage displayed peptide library with next-generation sequencing. Using AbMap, profiles of the peptides bound by 202 antibodies were determined in a single test, and linear epitopes were identified for >50% of the antibodies. Using spike protein (S1 and S2)-enriched antibodies from the convalescent serum of one COVID-19 patient as the input, both linear and potentially conformational epitopes of spike protein specific antibodies were identified. We defined peptide-binding profile of an antibody as the binding capacity (BiC). Conceptually, the BiC could serve as a systematic and functional descriptor of any antibody. Requiring at least one order of magnitude less time and money to map linear epitopes than traditional technologies, AbMap allows for high-throughput epitope mapping and creates many possibilities.

Evidence for heightened genetic instability in precancerous spasmolytic polypeptide expressing gastric glands.

BACKGROUND: Spasmolytic polypeptide-expressing metaplasia (SPEM) is present in more than 90% of resected gastric cancer tissues. However, although widely regarded as a pre-cancerous tissue, its genetic characteristics have not been well studied. METHODS: Immunohistochemistry using Trefoil factor 2 (TFF2) antibodies was used to identify TFF2-positive SPEM cells within SPEM glands in the stomach of Helicobacter felis (H. felis) -infected mice and human clinical samples. Laser microdissection was used to isolate specific cells from both the infected mice and the human samples. The genetic instability in these cells was examined by measuring the lengths of microsatellite (MS) markers using capillary electrophoresis. Also, genome-wide genetic variations in the SPEM cells from the clinical sample was examined using deep whole-exome sequencing. RESULTS: SPEM cells indeed exhibit not only heightened MS instability (MSI), but also genetic instabilities that extend genome-wide. Furthermore, surprisingly, we found that morphologically normal, TFF2-negative cells also contain a comparable degree of genomic instabilities as the co-resident SPEM cells within the SPEM glands. CONCLUSION: These results, for the first time, clearly establish elevated genetic instability as a critical property of SPEM glands, which may provide a greater possibility for malignant clone selection. More importantly, these results indicate that SPEM cells may not be the sole origin of carcinogenesis in the stomach and strongly suggest the common progenitor of these cells, the stem cells, as the source of these genetic instabilities, and thus, potential key players in carcinogenesis.

Ultra-deep sequencing of ribosome-associated poly-adenylated RNA in early Drosophila embryos reveals hundreds of conserved translated sORFs.

There is growing recognition that small open reading frames (sORFs) encoding peptides shorter than 100 amino acids are an important class of functional elements in the eukaryotic genome, with several already identified to play critical roles in growth, development, and disease. However, our understanding of their biological importance has been hindered owing to the significant technical challenges limiting their annotation. Here we combined ultra-deep sequencing of ribosome-associated poly-adenylated RNAs with rigorous conservation analysis to identify a comprehensive population of translated sORFs during early Drosophila embryogenesis. In total, we identify 399 sORFs, including those previously annotated but without evidence of translational capacity, those found within transcripts previously classified as non-coding, and those not previously known to be transcribed. Further, we find, for the first time, evidence for translation of many sORFs with different isoforms, suggesting their regulation is as complex as longer ORFs. Furthermore, many sORFs are found not associated with ribosomes in late-stage Drosophila S2 cells, suggesting that many of the translated sORFs may have stage-specific functions during embryogenesis. These results thus provide the first comprehensive annotation of the sORFs present during early Drosophila embryogenesis, a necessary basis for a detailed delineation of their function in embryogenesis and other biological processes.

Complex clonal mosaicism within microdissected intestinal metaplastic glands without concurrent gastric cancer.

BACKGROUND: The transformation of healthy gastric tissue into intestinal metaplasia (IM) is thought to be a critical premalignant step in the development of intestinal-type gastric adenocarcinoma (GA). How such premalignancies contribute to the development of GA is, however, poorly understood. METHODS: In this study, the extent and clonal complexity in IM tissue from patients without gastric cancer were analysed by measuring variations of multiple microsatellite (MS) markers. RESULTS: Even though these tissues are generally regarded as clinically benign, we found extensive MS length heterogeneity between and within individual IM glands, indicating that complex genome diversity is already pervasive in these tissues. Based on a clonal relationship analysis, we found that there exist multiple clones within individual IM glands and that MS alterations can accumulate in these clones. Moreover, we found spatially distant IM glands with the same MS phenotype, suggesting that these MS alterations were progressed by gland fission. CONCLUSIONS: These results provide evidence that genetic instability is an early event, present within metaplastic tissues of otherwise non-cancer patients, and such frequent genetic alterations can be part of the pathophysiological rationale for the requirement of this phase during gastric carcinogenesis.

The non-coding RNA composition of the mitotic chromosome by 5'-tag sequencing.

Mitotic chromosomes are one of the most commonly recognized sub-cellular structures in eukaryotic cells. Yet basic information necessary to understand their structure and assembly, such as their composition, is still lacking. Recent proteomic studies have begun to fill this void, identifying hundreds of RNA-binding proteins bound to mitotic chromosomes. However, by contrast, there are only two RNA species (U3 snRNA and rRNA) that are known to be associated with the mitotic chromosome, suggesting that there are many mitotic chromosome-associated RNAs (mCARs) not yet identified. Here, using a targeted protocol based on 5'-tag sequencing to profile the mammalian mCAR population, we report the identification of 1279 mCARs, the majority of which are ncRNAs, including lncRNAs that exhibit greater conservation across 60 vertebrate species than the entire population of lncRNAs. There is also a significant enrichment of snoRNAs and specific SINE RNAs. Finally, ∼40% of the mCARs are presently unannotated, many of which are as abundant as the annotated mCARs, suggesting that there are also many novel ncRNAs in the mCARs. Overall, the mCARs identified here, together with the previous proteomic and genomic data, constitute the first comprehensive catalogue of the molecular composition of the eukaryotic mitotic chromosomes.

Superior Mechanical Properties of Epoxy Composites Reinforced by 3D Interconnected Graphene Skeleton.

Epoxy-based composites reinforced by three-dimensional graphene skeleton (3DGS) were fabricated in resin transfer molding method with respect to the difficulty in good dispersion and arrangement of graphene sheets in composites by directly mixing graphene and epoxy. 3DGS was synthesized in the process of self-assembly and reduction with poly(amidoamine) dendrimers. In the formation of 3DGS, graphene sheets were in good dispersion and ordered state, which resulted in exceptional mechanical properties and thermal stability for epoxy composites. For 3DGS/epoxy composites, the tensile and compressive strengths significantly increased by 120.9% and 148.3%, respectively, as well as the glass transition temperature, which increased by a notable 19 °C, unlike the thermal exfoliation graphene/epoxy composites via direct-mixing route, which increased by only 0.20 wt % content of fillers. Relative to the graphene/epoxy composites in direct-mixing method mentioned in literature, the increase in tensile and compressive strengths of 3DGS/epoxy composites was at least twofold and sevenfold, respectively. It can be expected that 3DGS, which comes from preforming graphene sheets orderly and dispersedly, would replace graphene nanosheets in polymer nanocomposite reinforcement and endow composites with unique structure and some unexpected performance.

Genome-wide analysis of core promoter structures in Schizosaccharomyces pombe with DeepCAGE.

The core promoter, which immediately flanks the transcription start site (TSS), plays a critical role in transcriptional regulation of eukaryotes. Recent studies on higher eukaryotes have revealed an unprecedented complexity of core promoter structures that underscores diverse regulatory mechanisms of gene expression. For unicellular eukaryotes, however, the structures of core promoters have not been investigated in detail. As an important model organism, Schizosaccharomyces pombe still lacks the precise annotation for TSSs, thus hampering the analysis of core promoter structures and their relationship to higher eukaryotes. Here we used a deep sequencing-based approach (DeepCAGE) to generate 16 million uniquely mapped tags, corresponding to 93,736 positions in the S. pombe genome. The high-resolution TSS landscape enabled identification of over 8,000 core promoters, characterization of 4 promoter classes and observation of widespread alternative promoters. The landscape also allowed precise determination of the representative TSSs within core promoters, thus redefining the 5' UTR for 82.8% of S. pombe genes. We further identified the consensus initiator (Inr) sequence--PyPyPuN(A/C)(C/A), the TATA-enriched region (between position -25 and -37) and an Inr immediate downstream motif--CC(T/A)(T/C)(T/C/A)(A/G)CCA(A/T/C), all of which were associated with highly expressed promoters. In conclusion, the detailed analysis of core promoters not only significantly improves the genome annotation of S. pombe, but also reveals that this unicellular eukaryote shares a highly similar organization in the core promoters with higher eukaryotes. These findings lend additional evidence for the power of this model system in delineating complex regulatory processes in multicellular organisms, despite its perceived simplicity.