Galectin-10 in serum extracellular vesicles reflects asthma pathophysiology.

BACKGROUND: Novel biomarkers (BMs) are urgently needed for bronchial asthma (BA) with various phenotypes and endotypes. OBJECTIVE: We sought to identify novel BMs reflecting tissue pathology from serum extracellular vesicles (EVs). METHODS: We performed data-independent acquisition of serum EVs from 4 healthy controls, 4 noneosinophilic asthma (NEA) patients, and 4 eosinophilic asthma (EA) patients to identify novel BMs for BA. We confirmed EA-specific BMs via data-independent acquisition validation in 61 BA patients and 23 controls. To further validate these findings, we performed data-independent acquisition for 6 patients with chronic rhinosinusitis without nasal polyps and 7 patients with chronic rhinosinusitis with nasal polyps. RESULTS: We identified 3032 proteins, 23 of which exhibited differential expression in EA. Ingenuity pathway analysis revealed that protein signatures from each phenotype reflected disease characteristics. Validation revealed 5 EA-specific BMs, including galectin-10 (Gal10), eosinophil peroxidase, major basic protein, eosinophil-derived neurotoxin, and arachidonate 15-lipoxygenase. The potential of Gal10 in EVs was superior to that of eosinophils in terms of diagnostic capability and detection of airway obstruction. In rhinosinusitis patients, 1752 and 8413 proteins were identified from EVs and tissues, respectively. Among 11 BMs identified in EVs and tissues from patients with chronic rhinosinusitis with nasal polyps, 5 (including Gal10 and eosinophil peroxidase) showed significant correlations between EVs and tissues. Gal10 release from EVs was implicated in eosinophil extracellular trapped cell death in vitro and in vivo. CONCLUSION: Novel BMs such as Gal10 from serum EVs reflect disease pathophysiology in BA and may represent a new target for liquid biopsy approaches.

Sphingomyelin Deacylase, the Enzyme Involved in the Pathogenesis of Atopic Dermatitis, Is Identical to the ß-Subunit of Acid Ceramidase.

A ceramide deficiency in the stratum corneum (SC) is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). Previously, we reported that sphingomyelin (SM) deacylase, which hydrolyzes SM and glucosylceramide at the acyl site to yield their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine, respectively, instead of ceramide and/or acylceramide, is over-expressed in AD skin and results in a ceramide deficiency. Although the enzymatic properties of SM deacylase have been clarified, the enzyme itself remains unidentified. In this study, we purified and characterized SM deacylase from rat skin. The activities of SM deacylase and acid ceramidase (aCDase) were measured using SM and ceramide as substrates by tandem mass spectrometry by monitoring the production of SPC and sphingosine, respectively. Levels of SM deacylase activity from various rat organs were higher in the order of skin > lung > heart. By successive chromatography using Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and Shodex RP18-415, SM deacylase was purified to homogeneity with a single band of an apparent molecular mass of 43 kDa with an enrichment of > 14,000-fold. Analysis by MALDI-TOF MS/MS using a protein spot with SM deacylase activity separated by 2D-SDS-PAGE allowed its amino acid sequence to be determined and identified as the ß-subunit of aCDase, which consists of α- and ß-subunits linked by amino bonds and a single S-S bond. Western blotting of samples treated with 2-mercaptoethanol revealed that, whereas recombinant human aCDase was recognized by antibodies to the α-subunit at ~56 kDa and ~13 kDa and the ß-subunit at ~43 kDa, the purified SM deacylase was detectable only by the antibody to the ß-subunit at ~43 kDa. Breaking the S-S bond of recombinant human aCDase with dithiothreitol elicited the activity of SM deacylase with ~40 kDa upon gel chromatography. These results provide new insights into the essential role of SM deacylase expressed as an aCDase-degrading ß-subunit that evokes the ceramide deficiency in AD skin.

Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish.

BACKGROUND: Fear conditioning is a form of learning essential for animal survival and used as a behavioral paradigm to study the mechanisms of learning and memory. In mammals, the amygdala plays a crucial role in fear conditioning. In teleost, the medial zone of the dorsal telencephalon (Dm) has been postulated to be a homolog of the mammalian amygdala by anatomical and ablation studies, showing a role in conditioned avoidance response. However, the neuronal populations required for a conditioned avoidance response via the Dm have not been functionally or genetically defined. RESULTS: We aimed to identify the neuronal population essential for fear conditioning through a genetic approach in zebrafish. First, we performed large-scale gene trap and enhancer trap screens, and created transgenic fish lines that expressed Gal4FF, an engineered version of the Gal4 transcription activator, in specific regions in the brain. We then crossed these Gal4FF-expressing fish with the effector line carrying the botulinum neurotoxin gene downstream of the Gal4 binding sequence UAS, and analyzed the double transgenic fish for active avoidance fear conditioning. We identified 16 transgenic lines with Gal4FF expression in various brain areas showing reduced performance in avoidance responses. Two of them had Gal4 expression in populations of neurons located in subregions of the Dm, which we named 120A-Dm neurons. Inhibition of the 120A-Dm neurons also caused reduced performance in Pavlovian fear conditioning. The 120A-Dm neurons were mostly glutamatergic and had projections to other brain regions, including the hypothalamus and ventral telencephalon. CONCLUSIONS: Herein, we identified a subpopulation of neurons in the zebrafish Dm essential for fear conditioning. We propose that these are functional equivalents of neurons in the mammalian pallial amygdala, mediating the conditioned stimulus-unconditioned stimulus association. Thus, the study establishes a basis for understanding the evolutionary conservation and diversification of functional neural circuits mediating fear conditioning in vertebrates.

Hypoxia decreases glucagon-like peptide-1 secretion from the GLUTag cell line.

Glucagon-like peptide-1 (GLP-1), an incretin hormone, is secreted from L cells located in the intestinal epithelium. It is known that intestinal oxygen tension is decreased postprandially. In addition, we found that the expression of hypoxia-inducible factor-1α (HIF-1α), which accumulates in cells under hypoxic conditions, was significantly increased in the colons of mice with food intake, indicating that the oxygen concentration is likely reduced in the colon after eating. Therefore, we hypothesized that GLP-1 secretion is affected by oxygen tension. We found that forskolin-stimulated GLP-1 secretion from GLUTag cells, a model of intestinal L cells, is suppressed in hypoxia (1% O2). Forskolin-stimulated elevations of preproglucagon (ppGCG) and proprotein convertase 1/3 (PC1/3) mRNA expression were decreased under hypoxic conditions. The finding that H89, a protein kinase A (PKA) inhibitor, inhibited the forskolin-stimulated increase of ppGCG and PC1/3 indicated that the cAMP-PKA pathway is involved in the hypoxia-induced suppression of the genes. Hypoxia decreased hexokinase 2 mRNA and protein expression and increased lactate dehydrogenase A mRNA and protein expression. Concomitantly, lactate production was increased and ATP production was decreased. Together, the results indicate that hypoxia decreases glucose utilization for ATP production, which probably causes a decrease in cAMP production and in subsequent GLP-1 production. Our findings suggest that the postprandial decrease in oxygen tension in the intestine attenuates GLP-1 secretion.

Idiopathic pulmonary fibrosis-specific Bayesian network integrating extracellular vesicle proteome and clinical information.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by severe lung fibrosis and a poor prognosis. Although the biomolecules related to IPF have been extensively studied, molecular mechanisms of the pathogenesis and their association with serum biomarkers and clinical findings have not been fully elucidated. We constructed a Bayesian network using multimodal data consisting of a proteome dataset from serum extracellular vesicles, laboratory examinations, and clinical findings from 206 patients with IPF and 36 controls. Differential protein expression analysis was also performed by edgeR and incorporated into the constructed network. We have successfully visualized the relationship between biomolecules and clinical findings with this approach. The IPF-specific network included modules associated with TGF-ß signaling (TGFB1 and LRC32), fibrosis-related (A2MG and PZP), myofibroblast and inflammation (LRP1 and ITIH4), complement-related (SAA1 and SAA2), as well as serum markers, and clinical symptoms (KL-6, SP-D and fine crackles). Notably, it identified SAA2 associated with lymphocyte counts and PSPB connected with the serum markers KL-6 and SP-D, along with fine crackles as clinical manifestations. These results contribute to the elucidation of the pathogenesis of IPF and potential therapeutic targets.

SFTPB in serum extracellular vesicles as a biomarker of progressive pulmonary fibrosis.

Progressive pulmonary fibrosis (PPF), defined as the worsening of various interstitial lung diseases (ILDs), currently lacks useful biomarkers. To identify novel biomarkers for early detection of patients at risk of PPF, we performed a proteomic analysis of serum extracellular vesicles (EVs). Notably, the identified candidate biomarkers were enriched for lung-derived proteins participating in fibrosis-related pathways. Among them, pulmonary surfactant-associated protein B (SFTPB) in serum EVs could predict ILD progression better than the known biomarkers, serum KL-6 and SP-D, and it was identified as an independent prognostic factor from ILD-gender-age-physiology index. Subsequently, the utility of SFTPB for predicting ILD progression was evaluated further in 2 cohorts using serum EVs and serum, respectively, suggesting that SFTPB in serum EVs but not in serum was helpful. Among SFTPB forms, pro-SFTPB levels were increased in both serum EVs and lungs of patients with PPF compared with those of the control. Consistently, in a mouse model, the levels of pro-SFTPB, primarily originating from alveolar epithelial type 2 cells, were increased similarly in serum EVs and lungs, reflecting pro-fibrotic changes in the lungs, as supported by single-cell RNA sequencing. SFTPB, especially its pro-form, in serum EVs could serve as a biomarker for predicting ILD progression.

Evolutionarily conserved replication timing profiles predict long-range chromatin interactions and distinguish closely related cell types.

To identify evolutionarily conserved features of replication timing and their relationship to epigenetic properties, we profiled replication timing genome-wide in four human embryonic stem cell (hESC) lines, hESC-derived neural precursor cells (NPCs), lymphoblastoid cells, and two human induced pluripotent stem cell lines (hiPSCs), and compared them with related mouse cell types. Results confirm the conservation of coordinately replicated megabase-sized "replication domains" punctuated by origin-suppressed regions. Differentiation-induced replication timing changes in both species occur in 400- to 800-kb units and are similarly coordinated with transcription changes. A surprising degree of cell-type-specific conservation in replication timing was observed across regions of conserved synteny, despite considerable species variation in the alignment of replication timing to isochore GC/LINE-1 content. Notably, hESC replication timing profiles were significantly more aligned to mouse epiblast-derived stem cells (mEpiSCs) than to mouse ESCs. Comparison with epigenetic marks revealed a signature of chromatin modifications at the boundaries of early replicating domains and a remarkably strong link between replication timing and spatial proximity of chromatin as measured by Hi-C analysis. Thus, early and late initiation of replication occurs in spatially separate nuclear compartments, but rarely within the intervening chromatin. Moreover, cell-type-specific conservation of the replication program implies conserved developmental changes in spatial organization of chromatin. Together, our results reveal evolutionarily conserved aspects of developmentally regulated replication programs in mammals, demonstrate the power of replication profiling to distinguish closely related cell types, and strongly support the hypothesis that replication timing domains are spatially compartmentalized structural and functional units of three-dimensional chromosomal architecture.

Genome-wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis.

Differentiation of mouse embryonic stem cells (mESCs) is accompanied by changes in replication timing. To explore the relationship between replication timing and cell fate transitions, we constructed genome-wide replication-timing profiles of 22 independent mouse cell lines representing 10 stages of early mouse development, and transcription profiles for seven of these stages. Replication profiles were cell-type specific, with 45% of the genome exhibiting significant changes at some point during development that were generally coordinated with changes in transcription. Comparison of early and late epiblast cell culture models revealed a set of early-to-late replication switches completed at a stage equivalent to the post-implantation epiblast, prior to germ layer specification and down-regulation of key pluripotency transcription factors [POU5F1 (also known as OCT4)/NANOG/SOX2] and coinciding with the emergence of compact chromatin near the nuclear periphery. These changes were maintained in all subsequent lineages (lineage-independent) and involved a group of irreversibly down-regulated genes, at least some of which were repositioned closer to the nuclear periphery. Importantly, many genomic regions of partially reprogrammed induced pluripotent stem cells (piPSCs) failed to re-establish ESC-specific replication-timing and transcription programs. These regions were enriched for lineage-independent early-to-late changes, which in female cells included the inactive X chromosome. Together, these results constitute a comprehensive "fate map" of replication-timing changes during early mouse development. Moreover, they support a model in which a distinct set of replication domains undergoes a form of "autosomal Lyonization" in the epiblast that is difficult to reprogram and coincides with an epigenetic commitment to differentiation prior to germ layer specification.

CD4+ T-cell dysfunctions through the impaired lipid rafts ameliorate concanavalin A-induced hepatitis in sphingomyelin synthase 1-knockout mice.

Membrane microdomains consisting of sphingomyelin (SM) and cholesterol appear to be important for signal transduction in T-cell activation. The present study was designed to elucidate the role of membrane SM in vivo and in vitro using sphingomyelin synthase 1 (SMS1) knock out (SMS1(-/-)) mice and Concanavalin A (ConA)-induced hepatitis. After establishing SMS1(-/-) mice, we investigated CD4+ T-cell functions including proliferation, cytokine production and signal transduction in vivo. We also examined severity of hepatitis, cytokine production in serum and liver after ConA injection at a dose of 20 mg kg(-1). CD4+ T cells from SMS1(-/-) mice showed severe deficiency of membrane SM and several profound defects compared with wild-type controls as follows: (i) cellular proliferation and production of IL-2 and IFN-γ by co-cross-linking of CD3 and CD4; (ii) tyrosine phosphorylation of LAT and its association with ZAP-70; (iii) clustering and co-localization of TCR with lipid rafts. Consistent with these impaired CD4+ T-cell functions in vitro, SMS1(-/-) mice showed decreased serum levels of IL-6 and IFN-γ by ConA injection, which renders SMS1(-/-) mice less sensitive to ConA-induced hepatitis. These results indicated that the deficiency of membrane SM caused the CD4+ T-cell dysfunction through impaired lipid raft function contributed to protection of ConA-induced liver injury, suggesting that the membrane SM is critical for full T-cell activation both in vitro and in vivo.

Palytoxin causes nonoxidative necrotic damage to PC12 cells in culture.

Palytoxin (PTX) is a potent marine toxin that causies serious damage to various tissues and organs. It has been reported to affect the transport of cations across the plasma membranes, which is commonly recognized as being the principal mechanism of its highly toxic action on mammals, including humans. However, although some marine toxins have been shown to cause toxic effects on the nervous system by interfering with the transmission of nerve impulses, the effect of PTX on neuronal cells has not yet been fully elucidated. Therefore, the toxic action of PTX on PC12 cells was examined as an in vitro model experiment to elucidate the neurotoxic properties of this toxin, and PTX was shown to reduce the viability of PC12 cells in a concentration-dependent manner. The cytotoxic action of PTX was not significantly altered by the presence of the antioxidant N-acetylcysteine and reduced-form glutathione in the cultures. Fluorescence staining of the cells and the electrophoretic analysis of genomic DNA showed that PTX failed to cause chromatin condensation and DNA fragmentation within the cells. On the other hand, the exposure to PTX caused positive staining of the cytoplasmic space of the cells with propidium iodide and the release of lactate dehydrogenase into the culture medium. Based on these observations, PTX is considered to cause cell death as a consequence of disrupting the plasma membranes, thus causing nonoxidative necrotic damage to PC12 cells.

A novel corpus of molecular to higher-order events that facilitates the understanding of the pathogenic mechanisms of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a severe and progressive chronic fibrosing interstitial lung disease with causes that have remained unclear to date. Development of effective treatments will require elucidation of the detailed pathogenetic mechanisms of IPF at both the molecular and cellular levels. With a biomedical corpus that includes IPF-related entities and events, text-mining systems can efficiently extract such mechanism-related information from huge amounts of literature on the disease. A novel corpus consisting of 150 abstracts with 9297 entities intended for training a text-mining system was constructed to clarify IPF-related pathogenetic mechanisms. For this corpus, entity information was annotated, as were relation and event information. To construct IPF-related networks, we also conducted entity normalization with IDs assigned to entities. Thereby, we extracted the same entities, which are expressed differently. Moreover, IPF-related events have been defined in this corpus, in contrast to existing corpora. This corpus will be useful to extract IPF-related information from scientific texts. Because many entities and events are related to lung diseases, this freely available corpus can also be used to extract information related to other lung diseases such as lung cancer and interstitial pneumonia caused by COVID-19.

Latent inter-organ mechanism of idiopathic pulmonary fibrosis unveiled by a generative computational approach.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease characterized by complex lung pathogenesis affecting approximately three million people worldwide. While the molecular and cellular details of the IPF mechanism is emerging, our current understanding is centered around the lung itself. On the other hand, many human diseases are the products of complex multi-organ interactions. Hence, we postulate that a dysfunctional crosstalk of the lung with other organs plays a causative role in the onset, progression and/or complications of IPF. In this study, we employed a generative computational approach to identify such inter-organ mechanism of IPF. This approach found unexpected molecular relatedness of IPF to neoplasm, diabetes, Alzheimer's disease, obesity, atherosclerosis, and arteriosclerosis. Furthermore, as a potential mechanism underlying this relatedness, we uncovered a putative molecular crosstalk system across the lung and the liver. In this inter-organ system, a secreted protein, kininogen 1, from hepatocytes in the liver interacts with its receptor, bradykinin receptor B1 in the lung. This ligand-receptor interaction across the liver and the lung leads to the activation of calmodulin pathways in the lung, leading to the activation of interleukin 6 and phosphoenolpyruvate carboxykinase 1 pathway across these organs. Importantly, we retrospectively identified several pre-clinical and clinical evidence supporting this inter-organ mechanism of IPF. In conclusion, such feedforward and feedback loop system across the lung and the liver provides a unique opportunity for the development of the treatment and/or diagnosis of IPF. Furthermore, the result illustrates a generative computational framework for machine-mediated synthesis of mechanisms that facilitates and complements the traditional experimental approaches in biomedical sciences.

The keratin-related Ouroboros proteins function as immune antigens mediating tail regression in Xenopus metamorphosis.

Tail resorption during amphibian metamorphosis has been thought to be controlled mainly by a cell-autonomous mechanism of programmed cell death triggered by thyroid hormone. However, we have proposed a role for the immune response in metamorphosis, based on the finding that syngeneic grafts of tadpole tail skin into adult Xenopus animals are rejected by T cells. To test this, we identified two tail antigen genes called ouro1 and ouro2 that encode keratin-related proteins. Recombinant Ouro1 and Ouro2 proteins generated proliferative responses in vitro in T cells isolated from naive adult Xenopus animals. These genes were expressed specifically in the tail skin at the climax of metamorphosis. Overexpression of ouro1 and ouro2 induced T-cell accumulation and precocious tail degeneration after full differentiation of adult-type T cells when overexpressed in the tail region. When the expression of ouro1 and ouro2 were knocked down, tail skin tissue remained even after metamorphosis was complete. Our findings indicate that Ouro proteins participate in the process of tail regression as immune antigens and highlight the possibility that the acquired immune system contributes not only to self-defense but also to remodeling processes in vertebrate morphogenesis.

In vitro cytotoxic effect of ethanol extract prepared from sporophyll of Undaria pinnatifida on human colorectal cancer cells.

Brown seaweed Undaria pinnatifida (Harvey) Suringar is popular as a foodstuff, and used for medical care in East Asian countries. The major components of this seaweed are shown to benefit hypertension and hyperlipidemia, and considered to reduce the risks of infarction and ischemic diseases. Furthermore, the intake of dietary fiber of seaweeds is considered to prevent the production and proliferation of cancer in the gastrointestinal tract. The direct effect of an ethanol extract prepared from Undaria pinnatifida sporophyll (mekabu) on HCT116 human colorectal cancer cells was examined, and the mekabu extract was shown to induce the non-oxidative apoptotic damage to the cells, thus resulting in the reduction of their viabilities in a concentration-dependent manner. Moreover, the cytotoxic effects of carcinostatic drugs, such as 5-fluorouracil (5-FU) and irinotecan (CPT-11), were observed only in the medium containing sera, while the mekabu extract could effectively reduce the cell viabilities even in the serum-free medium. These findings suggest that the mekabu extract may contain a potential active substance inducing the non-oxidative apoptotic cell death probably through a mechanism different from those of 5-FU and CPT-11, and hence mekabu is possibly useful as an auxiliary drug to the chemotherapy of colorectal cancer.

Extract of fermented brown rice induces apoptosis of human colorectal tumor cells by activating mitochondrial pathway.

Brown rice fermented with Aspergillus oryzae, designated as FBRA, is a dietary fiber-rich food, and fully appreciated as one of the prebiotics, which are generally considered to be beneficial to the health of the body, because of stimulating the growth and/or the activity of bacteria in the digestive system. To assess the effectiveness of FBRA as a functional food, the direct effect of FBRA extract on human colorectal tumor cells was examined. The exposure of HCT116 cells to FBRA extract reduced their viabilities in a concentration-dependent manner, and the reduction of the cell viability might be attributed to the induction of apoptosis probably through the oxidative damage to the cells. Further studies showed that FBRA extract caused a significant elevation of Bax protein and a slight reduction of Bcl2 protein levels, and furthermore caused the activation of caspase-3 in the cells. Thus, it seems reasonable to conclude that FBRA extract can exert oxidative damage to the cells, resulting in apoptotic cell death by activating the mitochondrial pathway in human colorectal tumor cells. Therefore, daily intake of FBRA can be expected to be beneficial for preventing carcinogenesis and/or suppressing tumor growth in the digestive tract.

Mouse tissue glycome atlas 2022 highlights inter-organ variation in major N-glycan profiles.

This study presents "mouse tissue glycome atlas" representing the profiles of major N-glycans of mouse glycoproteins that may define their essential functions in the surface glycocalyx of mouse organs/tissues and serum-derived extracellular vesicles (exosomes). Cell surface glycocalyx composed of a variety of N-glycans attached covalently to the membrane proteins, notably characteristic "N-glycosylation patterns" of the glycocalyx, plays a critical role for the regulation of cell differentiation, cell adhesion, homeostatic immune response, and biodistribution of secreted exosomes. Given that the integrity of cell surface glycocalyx correlates significantly with maintenance of the cellular morphology and homeostatic immune functions, dynamic alterations of N-glycosylation patterns in the normal glycocalyx caused by cellular abnormalities may serve as highly sensitive and promising biomarkers. Although it is believed that inter-organs variations in N-glycosylation patterns exist, information of the glycan diversity in mouse organs/tissues remains to be elusive. Here we communicate for the first-time N-glycosylation patterns of 16 mouse organs/tissues, serum, and serum-derived exosomes of Slc:ddY mice using an established solid-phase glycoblotting platform for the rapid, easy, and high throughput MALDI-TOFMS-based quantitative glycomics. The present results elicited occurrence of the organ/tissue-characteristic N-glycosylation patterns that can be discriminated to each other. Basic machine learning analysis using this N-glycome dataset enabled classification between 16 mouse organs/tissues with the highest F1 score (69.7-100%) when neural network algorithm was used. A preliminary examination demonstrated that machine learning analysis of mouse lung N-glycome dataset by random forest algorithm allows for the discrimination of lungs among the different mouse strains such as the outbred mouse Slc:ddY, inbred mouse DBA/2Crslc, and systemic lupus erythematosus model mouse MRL-lpr/lpr with the highest F1 score (74.5-83.8%). Our results strongly implicate importance of "human organ/tissue glycome atlas" for understanding the crucial and diversified roles of glycocalyx determined by the organ/tissue-characteristic N-glycosylation patterns and the discovery research for N-glycome-based disease-specific biomarkers and therapeutic targets.

Integrative Network Modeling Highlights the Crucial Roles of Rho-GDI Signaling Pathway in the Progression of non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and a leading cause of cancer-related deaths worldwide. Using an integrative approach, we analyzed a publicly available merged NSCLC transcriptome dataset using machine learning, protein-protein interaction (PPI) networks and bayesian modeling to pinpoint key cellular factors and pathways likely to be involved with the onset and progression of NSCLC. First, we generated multiple prediction models using various machine learning classifiers to classify NSCLC and healthy cohorts. Our models achieved prediction accuracies ranging from 0.83 to 1.0, with XGBoost emerging as the best performer. Next, using functional enrichment analysis (and gene co-expression network analysis with WGCNA) of the machine learning feature-selected genes, we determined that genes involved in Rho GTPase signaling that modulate actin stability and cytoskeleton were likely to be crucial in NSCLC. We further assembled a PPI network for the feature-selected genes that was partitioned using Markov clustering to detect protein complexes functionally relevant to NSCLC. Finally, we modeled the perturbations in RhoGDI signaling using a bayesian network; our simulations suggest that aberrations in ARHGEF19 and/or RAC2 gene activities contributed to impaired MAPK signaling and disrupted actin and cytoskeleton organization and were arguably key contributors to the onset of tumorigenesis in NSCLC. We hypothesize that targeted measures to restore aberrant ARHGEF19 and/or RAC2 functions could conceivably rescue the cancerous phenotype in NSCLC. Our findings offer promising avenues for early predictive biomarker discovery, targeted therapeutic intervention and improved clinical outcomes in NSCLC.

Next-generation proteomics of serum extracellular vesicles combined with single-cell RNA sequencing identifies MACROH2A1 associated with refractory COVID-19.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is widespread; however, accurate predictors of refractory cases have not yet been established. Circulating extracellular vesicles, involved in many pathological processes, are ideal resources for biomarker exploration. METHODS: To identify potential serum biomarkers and examine the proteins associated with the pathogenesis of refractory COVID-19, we conducted high-coverage proteomics on serum extracellular vesicles collected from 12 patients with COVID-19 at different disease severity levels and 4 healthy controls. Furthermore, single-cell RNA sequencing of peripheral blood mononuclear cells collected from 10 patients with COVID-19 and 5 healthy controls was performed. RESULTS: Among the 3046 extracellular vesicle proteins that were identified, expression of MACROH2A1 was significantly elevated in refractory cases compared to non-refractory cases; moreover, its expression was increased according to disease severity. In single-cell RNA sequencing of peripheral blood mononuclear cells, the expression of MACROH2A1 was localized to monocytes and elevated in critical cases. Consistently, single-nucleus RNA sequencing of lung tissues revealed that MACROH2A1 was highly expressed in monocytes and macrophages and was significantly elevated in fatal COVID-19. Moreover, molecular network analysis showed that pathways such as "estrogen signaling pathway," "p160 steroid receptor coactivator (SRC) signaling pathway," and "transcriptional regulation by STAT" were enriched in the transcriptome of monocytes in the peripheral blood mononuclear cells and lungs, and they were also commonly enriched in extracellular vesicle proteomics. CONCLUSIONS: Our findings highlight that MACROH2A1 in extracellular vesicles is a potential biomarker of refractory COVID-19 and may reflect the pathogenesis of COVID-19 in monocytes.

Global reorganization of replication domains during embryonic stem cell differentiation.

DNA replication in mammals is regulated via the coordinate firing of clusters of replicons that duplicate megabase-sized chromosome segments at specific times during S-phase. Cytogenetic studies show that these "replicon clusters" coalesce as subchromosomal units that persist through multiple cell generations, but the molecular boundaries of such units have remained elusive. Moreover, the extent to which changes in replication timing occur during differentiation and their relationship to transcription changes has not been rigorously investigated. We have constructed high-resolution replication-timing profiles in mouse embryonic stem cells (mESCs) before and after differentiation to neural precursor cells. We demonstrate that chromosomes can be segmented into multimegabase domains of coordinate replication, which we call "replication domains," separated by transition regions whose replication kinetics are consistent with large originless segments. The molecular boundaries of replication domains are remarkably well conserved between distantly related ESC lines and induced pluripotent stem cells. Unexpectedly, ESC differentiation was accompanied by the consolidation of smaller differentially replicating domains into larger coordinately replicated units whose replication time was more aligned to isochore GC content and the density of LINE-1 transposable elements, but not gene density. Replication-timing changes were coordinated with transcription changes for weak promoters more than strong promoters, and were accompanied by rearrangements in subnuclear position. We conclude that replication profiles are cell-type specific, and changes in these profiles reveal chromosome segments that undergo large changes in organization during differentiation. Moreover, smaller replication domains and a higher density of timing transition regions that interrupt isochore replication timing define a novel characteristic of the pluripotent state.

[Synchronous double cancer of the gallbladder and rectum successfully treated with S-1 as second-line chemotherapy- a case report].

A 66-year-old man was referred to our hospital with obstructive jaundice. Computed tomography(CT)scan showed thickening of the gallbladder wall, invasion into the liver bed, and thickening of the rectal wall. Colonoscopy revealed a type 2 rectal cancer, in which adenocarcinoma was identified by endoscopic biopsy. He was diagnosed with double-cancer of the gallbladder and rectum. Because his gallbladder cancer was more life threatening than his rectal cancer, gemcitabine was administered at 1, 000 mg/m2 on days 1, 8, and 15 of a 28-day course. After 3 courses of gemcitabine, the CT scan showed that the lymph nodes in the hepatoduodenal ligament had been enlarged, and duodenal stenosis had occurred as a result of gallbladder cancer invasion. S-1 was administered orally at doses of 120 mg/day twice daily on days 1-28 of a 42-day course. Partial response was confirmed by CT scan. After 8 courses of S-1, the gallbladder cancer had progressed and liver metastases had appeared. He subsequently died of disease progression. He survived for 17 months after the first course of chemotherapy, and the progression-free survival with S-1 was 10 months. Therefore, S-1 could be an effective agent for synchronous double cancer of the gallbladder and rectum.