Cycloalkane degradation by an uncultivated novel genus of Gammaproteobacteria derived from China's marginal seas.

The consumption of cycloalkanes is prevalent in low-temperature marine environments, likely influenced by psychrophilic microorganisms. Despite their significance, the primary active species responsible for marine cycloalkane degradation remain largely unidentified due to cultivation challenges. In this study, we provide compelling evidence indicating that the uncultured genus C1-B045 of Gammaproteobacteria is a pivotal participant in cycloalkane decomposition within China's marginal seas. Notably, the relative abundance of C1-B045 surged from 15.9% in the methylcyclohexane (MCH)-consuming starter culture to as high as 97.5% in MCH-utilizing extinction cultures following successive dilution-to-extinction and incubation cycles. We used stable isotope probing, Raman-activated gravity-driven encapsulation, and 16 S rRNA gene sequencing to link cycloalkane-metabolizing phenotype to genotype at the single-cell level. By annotating key enzymes (e.g., alkane monooxygenase, cyclohexanone monooxygenase, and 6-hexanolactone hydrolase) involved in MCH metabolism within C1-B045's representative metagenome-assembled genome, we developed a putative MCH degradation pathway.

Optical-based microbubble for on-demand droplet release from static droplet array (SDA) for dispensing one droplet into one tube.

Static droplet array (SDA) is a pivotal tool for high-capacity screening assays, yet extraction and collection the target droplets that contain unique analytes or cells from the SDA remains one major technical bottleneck that limits its broader application. Here we present an optical-based on-demand droplet release (OODR) system by incorporating a 1064 nm laser-responsive indium tin oxide (ITO) layer into a chamber array-based droplet microfluidic chip. By focusing the 1064 nm laser onto the ITO layer, microbubbles can be created via local heating to selectively push-out the droplets from the chamber. Then the released droplet is readily exported in a one-droplet-one-tube (ODOT) manner by the inherent capillary force into pipette tip. Releasing of the droplets containing fluorescein sodium demonstrated ∼100% successful rate (9 out of 6400 droplets were successfully released) and low residual (only ∼5% of the droplet volume remains in the chamber). White or fluorescence image-based releasing of single-cell-droplets directly after cell loading or multi-cells-droplets derived from on-chip single-cell cultivation for both E. coli and yeast cells further demonstrated the wide applicability of OODR. The present system is user-friendly and has the potential to be applied in various high-throughput screening assays, including single molecule/cell analysis, drug screening, and phenotype-based cell sorting.

Corrigendum: Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase.

[This corrects the article DOI: 10.3389/fbioe.2023.1233856.].

Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase.

Single-cell genomic whole genome amplification (WGA) is a crucial step in single-cell sequencing, yet its low amplification efficiency, incomplete and uneven genome amplification still hinder the throughput and efficiency of single-cell sequencing workflows. Here we introduce a process called Improved Single-cell Genome Amplification (iSGA), in which the whole single-cell sequencing cycle is completed in a high-efficient and high-coverage manner, through phi29 DNA polymerase engineering and process engineering. By establishing a disulfide bond of F137C-A377C, the amplification ability of the enzyme was improved to that of single-cell. By further protein engineering and process engineering, a supreme enzyme named HotJa Phi29 DNA Polymerase was developed and showed significantly better coverage (99.75%) at a higher temperature (40°C). High single-cell genome amplification ability and high coverage (93.59%) were also achieved for commercial probiotic samples. iSGA is more efficient and robust than the wild-type phi29 DNA polymerase, and it is 2.03-fold more efficient and 10.89-fold cheaper than the commercial Thermo Scientific EquiPhi29 DNA Polymerase. These advantages promise its broad applications in large-scale single-cell sequencing.

Sequence and expression regulation of the BCL2L2 gene in pigs.

B cell lymphoma-2-like 2 (BCL2L2), an important regulator of apoptosis, plays vital roles in several physiological processes, as revealed by studies in humans and mice. However, reports on pig BCL2L2 are few, and the encoding gene has not been identified experimentally. This study was designed to clone the porcine BCL2L2 gene and its alternative splicing (AS) transcripts using molecular biological techniques and to analyze the regulatory mechanisms underlying transcription and translation. The BCL2L2 cDNA (V1) was 807 bp in length and encoded a polypeptide of 193 aa containing four BCL-2 homology domains. A total of nine AS transcripts were obtained, among which V2 and V3 differed from V1 in the 5' untranslated region (UTR). The core promoter was mapped to a range of -1102 to -759 bp (the first nucleotide of the start codon was designated as +1). There were several functional cis-elements, including one SP1 and two C/EBPα binding sites at around -759 bp. AS in the 5' UTR is involved in the regulation of gene expression, as revealed by dual-luciferase reporter and western blot analysis, and the secondary structure of the 5' UTRs may be the reason for the differential expression of V1-3. At the same time, an upstream open reading frame (ORF) existed in each of the three 5' UTRs, was found to repress the expression of the main ORF. Additionally, the roles of porcine BCL2L2 in cell proliferation and apoptosis were preliminarily analyzed. The results will contribute to further characterizing the role of BCL2L2.

Single-cell rapid identification, in situ viability and vitality profiling, and genome-based source-tracking for probiotics products.

Rapid expansion of the probiotics industry demands fast, sensitive, comprehensive, and low-cost strategies for quality assessment. Here, we introduce a culture-free, one-cell-resolution, phenome-genome-combined strategy called Single-Cell Identification, Viability and Vitality tests, and Source-tracking (SCIVVS). For each cell directly extracted from the product, the fingerprint region of D2O-probed single-cell Raman spectrum (SCRS) enables species-level identification with 93% accuracy, based on a reference SCRS database from 21 statutory probiotic species, whereas the C-D band accurately quantifies viability, metabolic vitality plus their intercellular heterogeneity. For source-tracking, single-cell Raman-activated Cell Sorting and Sequencing can proceed, producing indexed, precisely one-cell-based genome assemblies that can reach ~99.40% genome-wide coverage. Finally, we validated an integrated SCIVVS workflow with automated SCRS acquisition where the whole process except sequencing takes just 5 h. As it is >20-fold faster, >10-time cheaper, vitality-revealing, heterogeneity-resolving, and automation-prone, SCIVVS is a new technological and data framework for quality assessment of live-cell products.

Lignocellulosic xylitol production from corncob using engineered Kluyveromyces marxianus.

Xylitol production from lignocellulose hydrolysate is a sustainable and environment-friendly process. In this study, a systematic process of converting corncob waste into xylitol is described. First, the corncobs are hydrolyzed with acid to a hydrolysate. Second, Kluyveromyces marxianus YZJQ016 derived from K. marxianus YZJ074, constructed by overexpressing ScGAL2-N376F from Saccharomyces cerevisiae, CtXYL1 from Candida tropicalis, and KmZWF1 from K. marxianus, produces xylitol from the hydrolysate. A total of ten xylose reductase genes were evaluated, and CtXYL1 proved best by showing the highest catalytic activity under the control of the KmGAPDH promoter. A 5 L fermenter at 42°C produced 105.22 g/L xylitol using K. marxianus YZJQ016-the highest production reported to date from corncob hydrolysate. Finally, for crystallization of the xylitol, the best conditions were 50% (v/v) methanol as an antisolvent, at 25°C, with purity and yield of 99%-100% and 74%, respectively-the highest yield reported to date.

Diagnostic value of elevated serum carbohydrate antigen 125 level in sarcoidosis.

BACKGROUND: Sarcoidosis is a multisystem disorder with unknown etiology, and it predominantly affects the lungs and intrathoracic lymph nodes. For patients with atypical clinical manifestations, the diagnosis of sarcoidosis is difficult and specific biomarkers may play an important role in assisting diagnosis. Previous research has demonstrated a correlation between sarcoidosis and increased carbohydrate antigen 125 (CA125), but remains a lack of large cohort studies to validate this observation. AIM: To compare serum CA125 levels in sarcoidosis patients and healthy controls, and explore whether CA125 can be used as a biomarker for the diagnosis of sarcoidosis. METHODS: In this study, the serum CA125 levels were measured by enzyme-linked immunosorbent assay in 108 consecutive sarcoidosis patients between June 2016 and December 2020 (31 males, 77 females; age at diagnosis 49.69 ± 9.10 years) and 112 healthy subjects. Data on the C-reactive protein, erythrocyte sedimentation rate, and angiotensin-converting enzyme were also collected. The association of serum CA125 levels with clinical, radiological, and respiratory functional characteristics was analyzed between patient groups with CA125 ≤ 35 U/mL or CA125 > 35 U/mL. RESULTS: We found that serum CA125 levels were higher in sarcoidosis patients compared to healthy controls (median: 44.78 vs 19.11 U/mL, P < 0.001). The area under the receiver operator characteristic was 0.9833 (95%CI: 0.9717-0.9949), and the best cutoff point was 32.33 U/mL. The elevated serum CA125 was notably associated with the percentage of predicted forced vital capacity (FVC%) and neutrophil-to-lymphocyte ratio (P = 0.043 and P = 0.038, respectively) in sarcoidosis patients. Multivariate analysis revealed that FVC% was a statistically notable predictor of elevated serum CA125 (P = 0.029). Also, our research revealed that compared to patients with Stage I of radiology classification, patients with Stage II and III showed a higher concentration of serum CA125 (46.16 ± 8.32 vs 41.00 ± 6.04 U/mL, P = 0.005, and 47.92 ± 10.10 vs 41.00 ± 6.04 U/mL, P = 0.002, respectively). CONCLUSION: Serum CA125 was highly increased in sarcoidosis patients and showed high efficiency for noninvasive diagnosis of the disease. In addition, abnormally elevated serum CA125 was correlated with pulmonary function and radiological Scadding's classification of sarcoidosis.

SENP1 regulates the transformation of lung resident mesenchymal stem cells and is associated with idiopathic pulmonary fibrosis progression.

BACKGROUND: Lung resident mesenchymal stem cells (LR-MSCs) play an important role in idiopathic pulmonary fibrosis (IPF) by transforming into myofibroblasts, thereby losing their repair ability. Evidence suggests that key proteins of multiple signaling pathways are involved in myofibroblast differentiation of LR-MSCs, such as ß-Catenin and GLI family zinc finger 1 (GLI1). These proteins are regulated by SUMO (small ubiquitin-like modifier) modification, which is a post-translational modification that promotes protein degradation, while Sumo specific protein 1 (SENP1)-mediated deSUMOylation produces the opposite biological effects. Therefore, we speculated that SENP1 might be a potential target for treating pulmonary fibrosis by preventing the myofibroblast differentiation of LR-MSCs. METHODS: LR-MSCs were isolated from mice by using immunomagnetic beads. The extracted LR-MSCs were identified by flow cytometric analysis and multilineage differentiation assays. Lentivirus packaged shRNA silenced the expression of SENP1 in vitro and vivo. The silencing efficacy of SENP1 was verified by real-time quantitative PCR. The effect of down-regulated SENP1 on the myofibroblast differentiation of LR-MSCs was assessed by Immunofluorescence and Western blot. Immunoprecipitation was used to clarify that SENP1 was a key target for regulating the activity of multiple signaling pathways in the direction of LR-MSCs differentiation. LR-MSCs resident in the lung was analyzed with in vivo imaging system. HE and Masson staining was used to evaluate the therapeutic effect of LR-MSCs with SENP1 down-regulation on the lung of BLM mice. RESULTS: In this study, we found that the myofibroblast differentiation of LR-MSCs in IPF lung tissue was accompanied by enhanced SENP1-mediated deSUMOylation. The expression of SENP1 increased in LR-MSCs transition of bleomycin (BLM)-induced lung fibrosis. Interfering with expression of SENP1 inhibited the transformation of LR-MSCs into myofibroblasts in vitro and in vivo and restored their therapeutic effect in BLM lung fibrosis. In addition, activation of the WNT/ß-Catenin and Hedgehog/GLI signaling pathways depends on SENP1-mediated deSUMOylation. CONCLUSIONS: SENP1 might be a potential target to restore the repair function of LR-MSCs and treat pulmonary fibrosis. Video Abstract.

Exome Sequencing Reveals Genetic Variability and Identifies Chronic Prognostic Loci in Chinese Sarcoidosis Patients.

Background: Sarcoidosis is an inflammatory disease characterized by non-caseating granuloma formation in various organs, with several recognized genetic and environmental risk factors. Despite substantial progress, the genetic determinants associated with its prognosis remain largely unknown. Objectives: This study aimed to identify the genetic changes involved in sarcoidosis and evaluate their clinical relevance. Methods: We performed whole-exome sequencing (WES) in 116 sporadic sarcoidosis patients (acute sarcoidosis patients, n=58; chronic sarcoidosis patients, n=58). In addition, 208 healthy controls were selected from 1000 G East Asian population data. To identify genes enriched in sarcoidosis, Fisher exact tests were performed. The identified genes were included for further pathway analysis using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Additionally, we used the STRING database to construct a protein network of rare variants and Cytoscape to identify hub genes of signaling pathways. Results: WES and Fisher's exact test identified 1,311 variants in 439 protein-coding genes. A total of 135 single nucleotide polymorphisms (SNPs) on 30 protein-coding genes involved in the immunological process based on the GO and KEGG enrichment analysis. Pathway enrichment analysis showed osteoclast differentiation and cytokine-cytokine receptor interactions. Three missense mutations (rs76740888, rs149664918, and rs78251590) in two genes (PRSS3 and CNN2) of immune-related genes showed significantly different mutation frequencies between the disease group and healthy controls. The correlation of genetic abnormalities with clinical outcomes using multivariate analysis of the clinical features and mutation loci showed that the missense variant (rs76740888, Chr9:33796673 G>A) of PRSS3 [p=0.04, odds ratio (OR) = 2.49] was significantly associated with chronic disease prognosis. Additionally, the top two hub genes were CCL4 and CXCR4 based on protein-protein interaction (PPI) network analysis. Conclusion: Our study provides new insights into the molecular pathogenesis of sarcoidosis and identifies novel genetic alterations in this disease, especially PRSS3, which may be promising targets for future therapeutic strategies for chronic sarcoidosis.

Endoplasmic reticulum stress modulates the fate of lung resident mesenchymal stem cell to myofibroblast via C/EBP homologous protein during pulmonary fibrosis.

BACKGROUND: As a fatal interstitial lung disease, idiopathic pulmonary fibrosis (IPF) was characterized by the insidious proliferation of extracellular matrix (ECM)-producing mesenchymal cells. Recent studies have demonstrated that lung resident mesenchymal/stromal cells (LR-MSC) are the source of myofibroblasts. Endoplasmic reticulum (ER) stress is prominent in IPF lung. This study sought to investigate the effects of ER stress on the behavior of LR-MSC during pulmonary fibrosis. METHODS: ER stress and myofibroblast differentiation of LR-MSC in patients with IPF were evaluated. Primary mouse LR-MSC was harvested and used in vitro for testing the effects of ER stress and C/EBP homologous protein (CHOP) on LR-MSC. Adoptive transplantation of LR-MSC to bleomycin-induced pulmonary fibrosis was done to test the in vivo behavior of LR-MSC and its influence on pulmonary fibrosis. RESULTS: We found that myofibroblast differentiation of LR-MSC is associated with ER stress in IPF and bleomycin-induced mouse fibrotic lung. Tunicamycin-induced ER stress impairs the paracrine, migration, and reparative function of mouse LR-MSC to injured type 2 alveolar epithelial cells MLE-12. Overexpression of the ER stress responder C/EBP homologous protein (CHOP) facilitates the TGFß1-induced myofibroblast transformation of LR-MSC via boosting the TGFß/SMAD signaling pathway. CHOP knockdown facilitates engraftment and inhibits the myofibroblast transformation of LR-MSC during bleomycin-induced pulmonary fibrosis, thus promoting the efficacy of adopted LR-MSC in alleviating pulmonary fibrosis. CONCLUSION: Our work revealed a novel role that ER stress involved in pulmonary fibrosis by influencing the fate of LR-MSC and transformed to "crime factor" myofibroblast, during which CHOP acts as the key modulator. These results indicate that pharmacies targeting CHOP or therapies based on CHOP knockdown LR-MSC may be promising ways to treat pulmonary fibrosis.

C/EBP homologous protein promotes Sonic Hedgehog secretion from type II alveolar epithelial cells and activates Hedgehog signaling pathway of fibroblast in pulmonary fibrosis.

BACKGROUND: Endoplasmic reticulum (ER) stress is involved in the pathological process of pulmonary fibrosis, including IPF. It affects a broad scope of cellular types during pulmonary fibrosis but the role in epithelial-mesenchymal crosstalk has not been fully defined. The present study aimed to investigate the effects of Shh secretion by ER  stress-challenged type II alveolar epithelial cells (AECII) on fibroblast and pulmonary fibrosis. METHODS: Conditioned medium (CM) from tunicamycin (TM)-treated AECII was collected and incubated with fibroblast. Short hairpin RNA (shRNA) was used for RNA interference of C/EBP homologous protein (CHOP). The effects of CHOP and HH signaling were evaluated by TM administration under the background of bleomycin-induced pulmonary fibrosis in mice. RESULTS: Both expression of CHOP and Shh in AECII, and HH signaling in mesenchyme were upregulated in IPF lung. TM-induced Shh secretion from AECII activates HH signaling and promotes pro-fibrotic effects of fibroblast. Interfering CHOP expression reduced ER stress-induced Shh secretion and alleviated pulmonary fibrosis in mice. CONCLUSIONS: Our work identified a novel mechanism by which ER stress is involved in pulmonary fibrosis. Inhibition of ER stress or CHOP in epithelial cells alleviated pulmonary fibrosis by suppressing Shh/HH signaling pathway of fibroblasts.

Revealing CO2-Fixing SAR11 Bacteria in the Ocean by Raman-Based Single-Cell Metabolic Profiling and Genomics.

The majority of marine microbes remain uncultured, which hinders the identification and mining of CO2-fixing genes, pathways, and chassis from the oceans. Here, we investigated CO2-fixing microbes in seawater from the euphotic zone of the Yellow Sea of China by detecting and tracking their 13C-bicarbonate (13C-HCO3-) intake via single-cell Raman spectra (SCRS) analysis. The target cells were then isolated by Raman-activated Gravity-driven Encapsulation (RAGE), and their genomes were amplified and sequenced at one-cell resolution. The single-cell metabolism, phenotype and genome are consistent. We identified a not-yet-cultured Pelagibacter spp., which actively assimilates 13C-HCO3-, and also possesses most of the genes encoding enzymes of the Calvin-Benson cycle for CO2 fixation, a complete gene set for a rhodopsin-based light-harvesting system, and the full genes necessary for carotenoid synthesis. The four proteorhodopsin (PR) genes identified in the Pelagibacter spp. were confirmed by heterologous expression in E. coli. These results suggest that hitherto uncultured Pelagibacter spp. uses light-powered metabolism to contribute to global carbon cycling.

Single-cell Raman-activated sorting and cultivation (scRACS-Culture) for assessing and mining in situ phosphate-solubilizing microbes from nature.

Due to the challenges in detecting in situ activity and cultivating the not-yet-cultured, functional assessment and mining of living microbes from nature has typically followed a 'culture-first' paradigm. Here, employing phosphate-solubilizing microbes (PSM) as model, we introduce a 'screen-first' strategy that is underpinned by a precisely one-cell-resolution, complete workflow of single-cell Raman-activated Sorting and Cultivation (scRACS-Culture). Directly from domestic sewage, individual cells were screened for in-situ organic-phosphate-solubilizing activity via D2O intake rate, sorted by the function via Raman-activated Gravity-driven Encapsulation (RAGE), and then cultivated from precisely one cell. By scRACS-Culture, pure cultures of strong organic PSM including Comamonas spp., Acinetobacter spp., Enterobacter spp. and Citrobacter spp., were derived, whose phosphate-solubilizing activities in situ are 90-200% higher than in pure culture, underscoring the importance of 'screen-first' strategy. Moreover, employing scRACS-Seq for post-RACS cells that remain uncultured, we discovered a previously unknown, low-abundance, strong organic-PSM of Cutibacterium spp. that employs secretary metallophosphoesterase (MPP), cell-wall-anchored 5'-nucleotidase (encoded by ushA) and periplasmic-membrane located PstSCAB-PhoU transporter system for efficient solubilization and scavenging of extracellular phosphate in sewage. Therefore, scRACS-Culture and scRACS-Seq provide an in situ function-based, 'screen-first' approach for assessing and mining microbes directly from the environment.

Ni-Al layered double hydroxides modified sponge skeleton with polydopamine coating for enhanced U(VI) extraction from aqueous solution.

Uranium (VI) (U(VI)) is a major fuel for nuclear power, and the mass of it in seawater is about 4.5 billion tons. However, many current U(VI) adsorbents exist in the form of powder, which hinders the smooth recovery of U(VI) from aqueous solutions and its application in actual environments. Herein, the MP@LDH as an easy-to-recover and 3D macroscopic adsorbent for U(VI) extraction from aqueous solution was successfully prepared. The adsorbent was obtained via anchoring of LDH on the surface of melamine sponge (MS) coated with polydopamine (PDA). The maximum experimental adsorption capacity of MP@LDH sponge for U(VI) was as high as 559.8 mg g-1 (at pH = 8.0, T = 298 K). In the competitive sorption experiments against the competitions of Ba(II), Sr(II), Zn(II) and others ions, MP@LDH still exhibited a particularly excellent selectivity for U(VI) (almost 90% removal rate). Furthermore, the removal rate of MP@LDH towards U(VI) reached 88.18% under simulated seawater (C0 = 3.47 µg L-1). We believe that the MP@LDH with easy retrieval, excellent selectivity and uptake amount provides a convenient way for U(VI) extraction from seawater.

CCAAT/enhancer-binding protein (C/EBP) homologous protein promotes alveolar epithelial cell senescence via the nuclear factor-kappa B pathway in pulmonary fibrosis.

Alveolar epithelial cell senescence is a core event in the development of pulmonary fibrosis. Endoplasmic reticulum stress accelerates cellular senescence significantly; however, whether this stress promotes alveolar epithelial cell senescence in pulmonary fibrosis and its mechanisms are unclear. As a common intersection of endoplasmic reticulum stress signaling pathways, CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) activates the oxidative stress pathway, which in turn accelerates cellular senescence. Therefore, we speculated CHOP pathway activation would affect endoplasmic reticulum stress-induced alveolar epithelial cell senescence in pulmonary fibrosis. In this study, we observed that alveolar epithelial cell senescence was accompanied by CHOP overexpression in idiopathic pulmonary fibrosis lung tissues. Bleomycin and tunicamycin combination models in vivo and in vitro showed that CHOP downregulation rescued alveolar epithelial cell senescence, reduced fibroblast activation mediated by the senescence-associated secretory phenotype, and improved pulmonary fibrosis pathology. Mechanistic studies showed that CHOP accelerated alveolar epithelial cell senescence by promoting reactive oxygen species generation, which activated the nuclear factor-kappa B pathway. Our study suggested that CHOP activates the downstream nuclear factor-kappa B pathway, thus contributing to endoplasmic reticulum stress-induced alveolar epithelial cell senescence and pulmonary fibrosis.

Artificial intelligence-assisted automatic and index-based microbial single-cell sorting system for One-Cell-One-Tube.

Identification, sorting, and sequencing of individual cells directly from in situ samples have great potential for in-depth analysis of the structure and function of microbiomes. In this work, based on an artificial intelligence (AI)-assisted object detection model for cell phenotype screening and a cross-interface contact method for single-cell exporting, we developed an automatic and index-based system called EasySort AUTO, where individual microbial cells are sorted and then packaged in a microdroplet and automatically exported in a precisely indexed, "One-Cell-One-Tube" manner. The target cell is automatically identified based on an AI-assisted object detection model and then mobilized via an optical tweezer for sorting. Then, a cross-interface contact microfluidic printing method that we developed enables the automated transfer of cells from the chip to the tube, which leads to coupling with subsequent single-cell culture or sequencing. The efficiency of the system for single-cell printing is >93%. The throughput of the system for single-cell printing is ~120 cells/h. Moreover, >80% of single cells of both yeast and Escherichia coli are culturable, suggesting the superior preservation of cell viability during sorting. Finally, AI-assisted object detection supports automated sorting of target cells with high accuracy from mixed yeast samples, which was validated by downstream single-cell proliferation assays. The automation, index maintenance, and vitality preservation of EasySort AUTO suggest its excellent application potential for single-cell sorting.

Regulation of the IGF1 signaling pathway is involved in idiopathic pulmonary fibrosis induced by alveolar epithelial cell senescence and core fucosylation.

Idiopathic pulmonary fibrosis (IPF) mainly occurs in elderly people over the age of sixty. IPF pathogenesis is associated with alveolar epithelial cells (AECs) senescence. Activation of PI3K/AKT signaling induced by insulin-like growth factor 1 (IGF1) participates in AEC senescence and IPF by releasing CTGF, TGF-ß1, and MMP9. Our previous study demonstrated that core fucosylation (CF) modification, catalyzed by a specific core fucosyltransferase (FUT8) can regulate the activation of multiple signaling pathways, and inhibiting CF can alleviate pulmonary fibrosis in mice induced by bleomycin. However, whether CF is involved in IGF1-mediated AEC senescence in IPF remains unclear. In this study, we found that the IGF1/PI3K/AKT signaling pathway was activated in IPF lung tissue. Meanwhile, CF was present in senescent AECs. We also showed that IGF1 could induce AECs senescence with enhanced CF in vivo and in vitro. Inhibiting CF alleviated AECs senescence and pulmonary fibrosis induced by IGF1. In addition, activation of IGF1/PI3K/AKT signaling depends on CF. In conclusion, this study confirmed that CF is an important target regulating the IGF1 signaling pathway in AEC senescence and IPF, which might be a candidate target to treat IPF in the future.

One-Cell Metabolic Phenotyping and Sequencing of Soil Microbiome by Raman-Activated Gravity-Driven Encapsulation (RAGE).

Soil harbors arguably the most metabolically and genetically heterogeneous microbiomes on Earth, yet establishing the link between metabolic functions and genome at the precisely one-cell level has been difficult. Here, for mock microbial communities and then for soil microbiota, we established a Raman-activated gravity-driven single-cell encapsulation and sequencing (RAGE-Seq) platform, which identifies, sorts, and sequences precisely one bacterial cell via its anabolic (incorporating D from heavy water) and physiological (carotenoid-containing) functions. We showed that (i) metabolically active cells from numerically rare soil taxa, such as Corynebacterium spp., Clostridium spp., Moraxella spp., Pantoea spp., and Pseudomonas spp., can be readily identified and sorted based on D2O uptake, and their one-cell genome coverage can reach ∼93% to allow high-quality genome-wide metabolic reconstruction; (ii) similarly, carotenoid-containing cells such as Pantoea spp., Legionella spp., Massilia spp., Pseudomonas spp., and Pedobacter spp. were identified and one-cell genomes were generated for tracing the carotenoid-synthetic pathways; and (iii) carotenoid-producing cells can be either metabolically active or inert, suggesting culture-based approaches can miss many such cells. As a Raman-activated cell sorter (RACS) family member that can establish a metabolism-genome link at exactly one-cell resolution from soil, RAGE-Seq can help to precisely pinpoint "who is doing what" in complex ecosystems. IMPORTANCE Soil is home to an enormous and complex microbiome that features arguably the highest genomic diversity and metabolic heterogeneity of cells on Earth. Their in situ metabolic activities drive many natural processes of pivotal ecological significance or underlie industrial production of numerous valuable bioactivities. However, pinpointing "who is doing what" in a soil microbiome, which consists of mainly yet-to-be-cultured species, has remained a major challenge. Here, for soil microbiota, we established a Raman-activated gravity-driven single-cell encapsulation and sequencing (RAGE-Seq) method, which identifies, sorts, and sequences at the resolution of precisely one microbial cell via its catabolic and anabolic functions. As a Raman-activated cell sorter (RACS) family member that can establish a metabolism-genome link at one-cell resolution from soil, RAGE-Seq can help to precisely pinpoint "who is doing what" in complex ecosystems.

Microbial metabolism and necromass mediated fertilization effect on soil organic carbon after long-term community incubation in different climates.

Understanding the effects of changing climate and long-term human activities on soil organic carbon (SOC) and the mediating roles of microorganisms is critical to maintain soil C stability in agricultural ecosystem. Here, we took samples from a long-term soil transplantation experiment, in which large transects of Mollisol soil in a cold temperate region were translocated to warm temperate and mid-subtropical regions to simulate different climate conditions, with a fertilization treatment on top. This study aimed to understand fertilization effect on SOC and the role of soil microorganisms featured after long-term community incubation in warm climates. After 12 years of soil transplantation, fertilization led to less reduction of SOC, in which aromatic C increased and the consumption of O-alkyl C and carbonyl C decreased. Soil live microbes were analyzed using propidium monoazide to remove DNAs from dead cells, and their network modulization explained 60.4% of variations in soil labile C. Single-cell Raman spectroscopy combined with D2O isotope labeling indicated a higher metabolic activity of live microbes to use easily degradable C after soil transplantation. Compared with non-fertilization, there was a significant decrease in soil α- and ß-glucosidase and delay on microbial growth with fertilization in warmer climate. Moreover, fertilization significantly increased microbial necromass as indicated by amino sugar content, and its contribution to soil resistant C reached 22.3%. This study evidentially highlights the substantial contribution of soil microbial metabolism and necromass to refractory C of SOC with addition of nutrients in the long-term.