Research progress on the types, functions, biosynthesis, and metabolic regulation of ginkgo terpenoids.

Ginkgo biloba L. is a relict plant endemic to China that is commonly considered a "living fossil". It contains unique medicinal compounds that play important roles in its response to various stresses and help maintain human health. Ginkgo terpenoids are known to be important active ingredients but have received less attention than flavonoids. Hence, this review focuses on recent progress in research on the pharmacological effects of ginkgo terpenoid and the bioactivities of different terpenoid monomers. Many key structural genes, enzyme-encoding genes, transcription factors, and noncoding RNAs involved in the ginkgo terpenoid pathway were identified. Finally, many external factors (ecological factors, hormones, etc.) that regulate the biosynthesis and metabolism of terpenoids were proposed. All these findings improve the understanding of the biosynthesis, accumulation, and medicinal functions of terpenoids. Finally, this review includes an in-depth discussion regarding the limitations of terpenoid-related studies and potential future research directions.

A Flexible and Mechanical Robust All-Solid-State Polymer Electrolyte with Microphase Separated Structure for Lithium-Ion Battery.

Polyethylene oxide (PEO)-based electrolytes are the most widely used solid polymer electrolyte (SPE) due to their high safety, excellent ability to dissociate lithium salts, low cost, and ease of preparation. However, low ionic conductivity and narrow electrochemical stability window limit their potential for further development. "Polymer-in-salt" electrolytes exhibit superior electrochemical performance; however, the high lithium salt concentration makes the SPE mechanically fragile when facing lithium dendrites. Therefore, preparing an SPE that can withstand a high concentration of lithium salt while still maintaining good mechanical properties has become a valuable challenge. In this study, a macroscopically homogeneous but nanoscopically phase-separated polymer matrix was designed as an electrolyte that can withstand a high concentration of lithium salt while retaining good mechanical properties, and this study investigated changes in the Li+ solvation structure within the electrolyte and analyzed the reasons for the simultaneous achievement of good ionic conductivity (1.02 × 10-3 S cm-1 at 60 °C) and mechanical properties (7 MPa at room temperature). The formation of large ion clusters at the phase interface and selective enrichment of lithium salt in specific regions are found to play crucial roles, and the critical current density (CCD) can reach a value of 2.2 mA cm-2. This work demonstrates a promising design approach for polymer electrolytes that achieves an optimal balance between SPE conductivity and mechanical properties through microstructure control.

Duloxetine protected indomethacin-induced gastric mucosal injury by increasing serotonin-dependent RANTES expression and activating PI3K-AKT-VEGF pathway.

Antidepressant duloxetine has been shown protective effect on indomethacin-induced gastric ulcer, which was escorted by inflammation in the gastric mucosa. Cytokines are the principal mediators of inflammation. Thus, by screening the differential expression of cytokines in the gastric mucosa using cytokine array at 3 h after indomethacin exposure, when the gastric ulcer began to format, we found that indomethacin increased cytokines which promoted inflammation responses, whereas duloxetine decreased pro-inflammatory cytokines increased by indomethacin and increased RANTES expression. RANTES was consistently increased by pretreated with both 5 mg/kg and 20 mg/kg duloxetine at 3 h and 6 h after indomethacin exposure in male rats. Selective blockade of RANTES-CCR5 axis by a functional antagonist Met-RANTES or a CCR5 antagonist maraviroc suppressed the protection of duloxetine. Considering the pharmacologic action of duloxetine on reuptake of monoamine neurotransmitters, we examined the serotonin (5-HT), norepinephrine and dopamine contents in the blood and discovered 20 mg/kg duloxetine increased 5-HT levels in platelet-poor plasma, while treatment with 5-HT promoted expression of RANTES in the gastric mucosa and alleviated the indomethacin-induced gastric injury. Furthermore, duloxetine activated PI3K-AKT-VEGF signaling pathway, which was regulated by RANTES-CCR5, and selective inhibitor of VEGF receptor axitinib blocked the prophylactic effect of duloxetine. Furthermore, duloxetine also protected gastric mucosa from indomethacin in female rats, and RANTES was increased by duloxetine after 6 h after indomethacin exposure too. Together, our results identified the role of cytokines, particularly RANTES, and the underlying mechanisms in gastroprotective effect of duloxetine against indomethacin, which advanced our understanding in inflammatory modulation by monoamine-based antidepressants.

Albizzia chinensis (Osbeck) Merr extract YS ameliorates ethanol-induced acute gastric ulcer injury in rats by regulating NRF2 signaling pathway.

BACKGROUND: Around the world, there is a high incidence of gastric ulcers. YS, an extract from the Chinese herb Albizzia chinensis (Osbeck) Merr, has potential therapeutic applications for gastrointestinal diseases. Here we elucidated the protective effect and underlying mechanism of action of YS on gastric ulcer in rats injured by ethanol. METHODS: The ethanol-induced gastric ulcer rat model was used to assess the protective effect of YS. A pathological examination of gastric tissue was performed by H&E staining. GES-1 cells damaged by hydrogen peroxide were used to simulate oxidative damage in gastric mucosal epithelial cells. Endogenous NRF2 was knocked down using small interfering RNA. Immunoprecipitation was used to detect ubiquitination of NRF2. Co-immunoprecipitation was used to detect the NRF2-Keap1 interaction. RESULTS: YS (10 and 30 mg/kg, i.g.) significantly reduced the ulcer index, decreased MDA level, and increased SOD and GSH levels in gastric tissues damaged by ethanol. YS promoted NRF2 translocation from cytoplasm to nucleus and enhanced the NQO1 and HO-1 expression levels in injured rat gastric tissue. In addition, YS regulated NQO1 and HO-1 via NRF2 in H2O2-induced oxidative injured GES-1 cells. Further studies on the underlying mechanism indicated that YS reduced the interaction between NRF2 and Keap1 and decreased ubiquitylation of NRF2, thereby increasing its stability and expression of downstream factors. NRF2 knockdown abolished the effect of YS on MDA and SOD in GES-1 cells treated with H2O2. CONCLUSION: YS reduced the NRF2-Keap1 interaction, promoting NRF2 translocation into the nucleus, which increasing the transcription and translation of NQO1 and HO-1 and improved the antioxidant capacity of rat stomach.

CRL2APPBP2-mediated TSPYL2 degradation counteracts human mesenchymal stem cell senescence.

Cullin-RING E3 ubiquitin ligases (CRLs), the largest family of multi-subunit E3 ubiquitin ligases in eukaryotic cells, represent core cellular machinery for executing protein degradation and maintaining proteostasis. Here, we asked what roles Cullin proteins play in human mesenchymal stem cell (hMSC) homeostasis and senescence. To this end, we conducted a comparative aging phenotype analysis by individually knocking down Cullin members in three senescence models: replicative senescent hMSCs, Hutchinson-Gilford Progeria Syndrome hMSCs, and Werner syndrome hMSCs. Among all family members, we found that CUL2 deficiency rendered hMSCs the most susceptible to senescence. To investigate CUL2-specific underlying mechanisms, we then applied CRISPR/Cas9-mediated gene editing technology to generate CUL2-deficient human embryonic stem cells (hESCs). When we differentiated these into hMSCs, we found that CUL2 deletion markedly accelerates hMSC senescence. Importantly, we identified that CUL2 targets and promotes ubiquitin proteasome-mediated degradation of TSPYL2 (a known negative regulator of proliferation) through the substrate receptor protein APPBP2, which in turn down-regulates one of the canonical aging marker-P21waf1/cip1, and thereby delays senescence. Our work provides important insights into how CRL2APPBP2-mediated TSPYL2 degradation counteracts hMSC senescence, providing a molecular basis for directing intervention strategies against aging and aging-related diseases.

Inferring the Regulatory Network of miRNAs on Terpene Trilactone Biosynthesis Affected by Environmental Conditions.

As a medicinal tree species, ginkgo (Ginkgo biloba L.) and terpene trilactones (TTLs) extracted from its leaves are the main pharmacologic activity constituents and important economic indicators of its value. The accumulation of TTLs is known to be affected by environmental stress, while the regulatory mechanism of environmental response mediated by microRNAs (miRNAs) at the post-transcriptional levels remains unclear. Here, we focused on grafted ginkgo grown in northwestern, southwestern, and eastern-central China and integrally analyzed RNA-seq and small RNA-seq high-throughput sequencing data as well as metabolomics data from leaf samples of ginkgo clones grown in natural environments. The content of bilobalide was highest among detected TTLs, and there was more than a twofold variation in the accumulation of bilobalide between growth conditions. Meanwhile, transcriptome analysis found significant differences in the expression of 19 TTL-related genes among ginkgo leaves from different environments. Small RNA sequencing and analysis showed that 62 of the 521 miRNAs identified were differentially expressed among different samples, especially the expression of miRN50, miR169h/i, and miR169e was susceptible to environmental changes. Further, we found that transcription factors (ERF, MYB, C3H, HD-ZIP, HSF, and NAC) and miRNAs (miR319e/f, miRN2, miRN54, miR157, miR185, and miRN188) could activate or inhibit the expression of TTL-related genes to participate in the regulation of terpene trilactones biosynthesis in ginkgo leaves by weighted gene co-regulatory network analysis. Our findings provide new insights into the understanding of the regulatory mechanism of TTL biosynthesis but also lay the foundation for ginkgo leaves' medicinal value improvement under global change.

A Polytetrafluoroethylene-Based Solvent-Free Procedure for the Manufacturing of Lithium-Ion Batteries.

Lithium-ion batteries (LIBs) have recently become popular for energy storage due to their high energy density, storage capacity, and long-term cycle life. Although binders make up only a small proportion of LIBs, they have become the key to promoting the transformation of the battery preparation process. Along with the development of binders, the battery manufacturing process has evolved from the conventional slurry-casting (SC) process to a more attractive solvent-free (SF) method. Compared with traditional LIBs manufacturing method, the SF method could dramatically reduce and increase the energy density due to the reduced preparation steps and enhanced electrode loading. Polytetrafluoroethylene (PTFE), as a typical binder, has played an important role in fabricating high-performance LIBs, particularly in regards to the SF technique. In this paper, the development history and application status of PTFE binder was introduced, and then its contributions and the inherent problems involved in the SF process were described and analyzed. Finally, the viewpoints concerning the future trends for PTFE-based SF manufacturing methods were also discussed. We hope this work can inspire future research concerning high-quality SF binders and assist in promoting the evolution of the SF manufacturing technology in regards to LIBs.

Dynamic Cerebral Autoregulation After Carotid Endarterectomy.

OBJECTIVE: Studies have shown that dynamic cerebral autoregulation (dCA) is impaired in patients with severe internal carotid artery (ICA) stenosis and that carotid endarterectomy (CEA) may improve dCA in these patients. However, the time course of dCA changes in patients after CEA remains unclear. Therefore, this study aimed to investigate the effects of CEA on the dCA in patients with carotid artery stenosis at different time points. METHODS: This prospective study enrolled 44 patients (19 symptomatic stenosis patients and 25 asymptomatic stenosis patients) who underwent CEA and 44 age- and sex-matched controls. In the CEA group, the patients underwent dCA measurements at baseline, within 3 days, and 1 month after CEA. Transfer function parameters, phase difference (PD), and gain were used to quantify dCA. Changes in dCA before and after CEA were analyzed in detail. RESULTS: The bilateral PD of the patients before CEA was significantly lower than that of the control group. This damage did not improve within 3 days after surgery. One month after surgery, the PD on the affected side of the patients significantly improved compared with before surgery and reached the level of the control group. The PD of affected side across time points in symptomatic and asymptomatic stenosis patients is consistent with that in all patients. CONCLUSIONS: The dCA level did not improve immediately after CEA but significantly improved 1 month after surgery. This suggests that the occurrence of stroke should be considered in the acute period after CEA surgery, and its preventive effect on stroke may be effective after 1 month. CLINICAL IMPACT: We found the dCA level did not improve immediately after CEA but significantly improved 1 month after surgury. This suggests that the occuttencce of stroke and surgical complications (such as cerebral hyperperfusion syndrome) associated with impaired dCA in the acute phase after CEA surgery should be of particular concern.

Overview and Recent Progress on the Biosynthesis and Regulation of Flavonoids in Ginkgo biloba L.

Flavonoids and their derivatives play important roles in plants, such as exerting protective activity against biotic and abiotic stresses, functioning in visual signaling to attract pollinators, and regulating phytohormone activity. They are also important secondary metabolites that are beneficial to humans. Ginkgo biloba L. is a well-known relict plant considered to be a "living fossil". Flavonoids present in ginkgo leaves have antioxidant and anti-aging capacities and show good therapeutic effects on a variety of neurological diseases. To date, studies on flavonoids have mainly focused on their extraction, pharmacological effects, and component analysis and on the expression levels of the key genes involved. However, a systematic review summarizing the biosynthesis and regulatory mechanisms of ginkgo flavonoids is still lacking. Thus, this review was conducted to comprehensively introduce the biological characteristics, value, and utilization status of ginkgo; summarize the effects, biosynthetic pathways, and transcriptional regulation of flavonoids; and finally, discuss the factors (ecological factors, hormones, etc.) that regulate the biosynthesis of flavonoids in ginkgo. This review will provide a reference basis for future research on the biosynthesis and efficient utilization of flavonoids in ginkgo.

Mitochondrial complex I inhibition by homoharringtonine: A novel strategy for suppression of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a hematologic malignancy predominantly driven by the BCR-ABL fusion gene. One of the significant challenges in treating CML lies in the emergence of resistance to tyrosine kinase inhibitors (TKIs), especially those associated with the T315I mutation. Homoharringtonine (HHT) is an FDA-approved, naturally-derived drug with known anti-leukemic properties, but its precise mechanisms of action remain incompletely understood. In this study, we rigorously evaluated the anti-CML activity of HHT through both in vitro and in vivo assays, observing substantial anti-CML effects. To elucidate the molecular mechanisms underpinning these effects, we performed proteomic analysis on BCR-ABL T315I mutation-bearing cells treated with HHT. Comprehensive pathway enrichment analysis identified oxidative phosphorylation (OXPHOS) as the most significantly disrupted, suggesting a key role in the mechanism of action of HHT. Further bioinformatics exploration revealed a substantial downregulation of proteins localized within mitochondrial complex I (MCI), a critical OXPHOS component. These results were validated through Western blot analysis and were supplemented by marked reductions in MCI activity, ATP level, and oxygen consumption rate (OCR) upon HHT exposure. Collectively, our results shed light on the potent anti-CML properties of HHT, particularly its effectiveness against T315I mutant cells through MCI inhibition. Our study underscores a novel therapeutic strategy to overcome BCR-ABL T315I mutation resistance, illuminating a previously uncharted mechanism of action for HHT.

Sensitive N-Glycopeptide Profiling of Single and Rare Cells Using an Isobaric Labeling Strategy without Enrichment.

Single-cell omics is critical in revealing population heterogeneity, discovering unique features of individual cells, and identifying minority subpopulations of interest. As one of the major post-translational modifications, protein N-glycosylation plays crucial roles in various important biological processes. Elucidation of the variation in N-glycosylation patterns at single-cell resolution may largely facilitate the understanding of their key roles in the tumor microenvironment and immune therapy. However, comprehensive N-glycoproteome profiling for single cells has not been achieved due to the extremely limited sample amount and incompatibility with the available enrichment strategies. Here, we have developed an isobaric labeling-based carrier strategy for highly sensitive intact N-glycopeptide profiling for single cells or a small number of rare cells without enrichment. Isobaric labeling has unique multiplexing properties, by which the "total" signal from all channels triggers MS/MS fragmentation for N-glycopeptide identification, while the reporter ions provide quantitative information. In our strategy, a carrier channel using N-glycopeptides obtained from bulk-cell samples significantly improved the "total" signal of N-glycopeptides and, therefore, promoted the first quantitative analysis of averagely 260 N-glycopeptides from single HeLa cells. We further applied this strategy to study the regional heterogeneity of N-glycosylation of microglia in mouse brain and discovered region-specific N-glycoproteome patterns and cell subtypes. In conclusion, the glycocarrier strategy provides an attractive solution for sensitive and quantitative N-glycopeptide profiling of single/rare cells that cannot be enriched by traditional workflows.

Proteomics Identifies Circulating TIMP-1 as a Prognostic Biomarker for Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, although disease stratification using in-depth plasma proteomics has not been performed to date. By measuring more than 1000 proteins in the plasma of 147 DLBCL patients using data-independent acquisition mass spectrometry and antibody array, DLBCL patients were classified into four proteomic subtypes (PS-I-IV). Patients with the PS-IV subtype and worst prognosis had increased levels of proteins involved in inflammation, including a high expression of metalloproteinase inhibitor-1 (TIMP-1) that was associated with poor survival across two validation cohorts (n = 180). Notably, the combination of TIMP-1 with the international prognostic index (IPI) identified 64.00% to 88.24% of relapsed and 65.00% to 80.49% of deceased patients in the discovery and two validation cohorts, which represents a 24.00% to 41.67% and 20.00% to 31.70% improvement compared to the IPI score alone, respectively. Taken together, we demonstrate that DLBCL heterogeneity is reflected in the plasma proteome and that TIMP-1, together with the IPI, could improve the prognostic stratification of patients.

Molecular Characterization and Genetic Diversity of Ginkgo (Ginkgo biloba L.) Based on Insertions and Deletions (InDel) Markers.

As a "living fossil", ginkgo (Ginkgo biloba L.) has significant ornamental, medicinal, and timber value. However, the breeding improvement of ginkgo was limited by the lack of enough excellent germplasms and suitable molecular markers. Here, we characterized numerous polymorphic insertion/deletion (InDel) markers using RAD-seq in 12 different ginkgo cultivars. The total of 279,534 InDels identified were unequally distributed across 12 chromosomes in the ginkgo genome. Of these, 52.56% (146,919) and 47.44% (132,615) were attributed to insertions and deletions, respectively. After random selection and validation, 26 pairs of polymorphic primers were used for molecular diversity analysis in 87 ginkgo cultivars and clones. The average values of observed heterozygosity and polymorphism information were 0.625 and 0.517, respectively. The results of population structure analyses were similar to those of neighbor-joining and principal component analyses, which divided all germplasms into two distinct groups. Moreover, 11 ginkgo core collections accounted for approximately 12.64% of the total ginkgo germplasms obtained, representing well the allelic diversity of all original germplasms. Therefore, these InDels can be used for germplasm management and genetic diversity analyses in ginkgo and the core collections will be used effectively for ginkgo genetic improvement.

The Metasequoia genome and evolutionary relationships among redwoods.

Redwood trees (Sequoioideae), including Metasequoia glyptostroboides (dawn redwood), Sequoiadendron giganteum (giant sequoia), and Sequoia sempervirens (coast redwood), are threatened and widely recognized iconic tree species. Genomic resources for redwood trees could provide clues to their evolutionary relationships. Here, we report the 8-Gb reference genome of M. glyptostroboides and a comparative analysis with two related species. More than 62% of the M. glyptostroboides genome is composed of repetitive sequences. Clade-specific bursts of long terminal repeat retrotransposons may have contributed to genomic differentiation in the three species. The chromosomal synteny between M. glyptostroboides and S. giganteum is extremely high, whereas there has been significant chromosome reorganization in S. sempervirens. Phylogenetic analysis of marker genes indicates that S. sempervirens is an autopolyploid, and more than 48% of the gene trees are incongruent with the species tree. Results of multiple analyses suggest that incomplete lineage sorting (ILS) rather than hybridization explains the inconsistent phylogeny, indicating that genetic variation among redwoods may be due to random retention of polymorphisms in ancestral populations. Functional analysis of ortholog groups indicates that gene families of ion channels, tannin biosynthesis enzymes, and transcription factors for meristem maintenance have expanded in S. giganteum and S. sempervirens, which is consistent with their extreme height. As a wetland-tolerant species, M. glyptostroboides shows a transcriptional response to flooding stress that is conserved with that of analyzed angiosperm species. Our study offers insights into redwood evolution and adaptation and provides genomic resources to aid in their conservation and management.

In-Depth Serum Proteomics Reveals the Trajectory of Hallmarks of Cancer in Hepatitis B Virus-Related Liver Diseases.

Hepatocellular carcinoma (HCC) is a prevalent cancer in China, with chronic hepatitis B (CHB) and liver cirrhosis (LC) being high-risk factors for developing HCC. Here, we determined the serum proteomes (762 proteins) of 125 healthy controls and Hepatitis B virus-infected CHB, LC, and HCC patients and constructed the first cancerous trajectory of liver diseases. The results not only reveal that the majority of altered biological processes were involved in the hallmarks of cancer (inflammation, metastasis, metabolism, vasculature, and coagulation) but also identify potential therapeutic targets in cancerous pathways (i.e., IL17 signaling pathway). Notably, the biomarker panels for detecting HCC in CHB and LC high-risk populations were further developed using machine learning in two cohorts comprised of 200 samples (discovery cohort = 125 and validation cohort = 75). The protein signatures significantly improved the area under the receiver operating characteristic curve of HCC (CHB discovery and validation cohort = 0.953 and 0.891, respectively; LC discovery and validation cohort = 0.966 and 0.818, respectively) compared to using the traditional biomarker, alpha-fetoprotein, alone. Finally, selected biomarkers were validated with parallel reaction monitoring mass spectrometry in an additional cohort (n = 120). Altogether, our results provide fundamental insights into the continuous changes of cancer biology processes in liver diseases and identify candidate protein targets for early detection and intervention.

GbFLSa overexpression negatively regulates proanthocyanin biosynthesis.

Flavonoids are important secondary metabolites with extensive pharmacological functions. Ginkgo biloba L. (ginkgo) has attracted extensive attention because of its high flavonoid medicinal value. However, little is understood about ginkgo flavonol biosynthesis. Herein, we cloned the full-length gingko GbFLSa gene (1314 bp), which encodes a 363 amino acid protein that has a typical 2-oxoglutarate (2OG)-Fe(II) oxygenase region. Recombinant GbFLSa protein with a molecular mass of 41 kDa was expressed in Escherichia coli BL21(DE3). The protein was localized to the cytoplasm. Moreover, proanthocyanins, including catechin, epicatechin, epigallocatechin and gallocatechin, were significantly less abundant in transgenic poplar than in nontransgenic (CK) plants. In addition, dihydroflavonol 4-reductase, anthocyanidin synthase and leucoanthocyanidin reductase expression levels were significantly lower than those of their CK counterparts. GbFLSa thus encodes a functional protein that might negatively regulate proanthocyanin biosynthesis. This study helps elucidate the role of GbFLSa in plant metabolism and the potential molecular mechanism of flavonoid biosynthesis.

Quantitative Proteomics Reveal the Inherent Antibiotic Resistance Mechanism against Norfloxacin Resistance in Aeromonas hydrophila.

Recently, the prevalence of Aeromonas hydrophila antibiotic-resistant strains has been reported in aquaculture, but its intrinsic antibiotic resistance mechanisms are largely unknown. In the present study, a label-free proteomics technology was used to compare the differential protein abundances in response to norfloxacin (NOR) stress in A. hydrophila. The results showed that there were 186 proteins decreasing and 220 proteins increasing abundances in response to NOR stress. Bioinformatics analysis showed that the differentially expressed proteins were enriched in several biological processes, such as sulfur metabolism and homologous recombination. Furthermore, the antibiotic sensitivity assays showed that the deletion of AHA_0904, cirA, and cysI significantly decreased the resistance against NOR, whereas ΔAHA_1239, ΔcysA, ΔcysD, and ΔcysN significantly increased the resistance against NOR. Our results provide insights into NOR resistance mechanisms and indicate that AHA_0904, cirA, AHA_1239, and sulfur metabolism may play important roles in NOR resistance in A. hydrophila.

Genome-Wide Identification of Expansin Gene Family and Their Response under Hormone Exposure in Ginkgo biloba L.

Expansins are pH-dependent enzymatic proteins that irreversibly and continuously facilitate cell-wall loosening and extension. The identification and comprehensive analysis of Ginkgo biloba expansins (GbEXPs) are still lacking. Here, we identified and investigated 46 GbEXPs in Ginkgo biloba. All GbEXPs were grouped into four subgroups based on phylogeny. GbEXPA31 was cloned and subjected to a subcellular localization assay to verify our identification. The conserved motifs, gene organization, cis-elements, and Gene Ontology (GO) annotation were predicted to better understand the functional characteristics of GbEXPs. The collinearity test indicated segmental duplication dominated the expansion of the GbEXPA subgroup, and seven paralogous pairs underwent strong positive selection during expansion. A majority of GbEXPAs were mainly expressed in developing Ginkgo kernels or fruits in transcriptome and real-time quantitative PCR (qRT-PCR). Furthermore, GbEXLA4, GbEXLA5, GbEXPA5, GbEXPA6, GbEXPA8, and GbEXPA24 were inhibited under the exposure of abiotic stresses (UV-B and drought) and plant hormones (ABA, SA, and BR). In general, this study expanded our understanding for expansins in Ginkgo tissues' growth and development and provided a new basis for studying GbEXPs in response to exogenous phytohormones.

A rapid and sensitive single-cell proteomic method based on fast liquid-chromatography separation, retention time prediction and MS1-only acquisition.

Single-cell analysis has received much attention in recent years for elucidating the widely existing cellular heterogeneity in biological systems. However, the ability to measure the proteome in single cells is still far behind that of transcriptomics due to the lack of sensitive and high-throughput mass spectrometry methods. Herein, we report an integrated strategy termed "SCP-MS1" that combines fast liquid chromatography (LC) separation, deep learning-based retention time (RT) prediction and MS1-only acquisition for rapid and sensitive single-cell proteome analysis. In SCP-MS1, the peptides were identified via four-dimensional MS1 feature (m/z, RT, charge and FAIMS CV) matching, therefore relieving MS acquisition from the time consuming and information losing MS2 step and making this method particularly compatible with fast LC separation. By completely omitting the MS2 step, all the MS analysis time was utilized for MS1 acquisition in SCP-MS1 and therefore led to 65%-138% increased MS1 feature collection. Unlike "match between run" methods that still needed MS2 information for RT alignment, SCP-MS1 used deep learning-based RT prediction to transfer the measured RTs in long gradient bulk analyses to short gradient single cell analyses, which was the key step to enhance both identification scale and matching accuracy. Using this strategy, more than 2000 proteins were obtained from 0.2 ng of peptides with a 14-min active gradient at a false discovery rate (FDR) of 0.8%. Comparing with the DDA method, improved quantitative performance was also observed for SCP-MS1 with approximately 50% decreased median coefficient of variation of quantified proteins. For single-cell analysis, 1715 ± 204 and 1604 ± 224 proteins were quantified in single 293T and HeLa cells, respectively. Finally, SCP-MS1 was applied to single-cell proteome analysis of sorafenib resistant and non-resistant HepG2 cells and revealed clear cellular heterogeneity in the resistant population that may be masked in bulk studies.

A three-stage search strategy combining database reduction and retention time filtering to improve the sensitivity of low-input and single-cell proteomic analysis.

When performing proteome profiling of low-input and single-cell samples, achieving deep protein coverage is very challenging due to the sensitivity limitation of current proteomic methods. Herein, we introduce a three-stage search strategy that combines the advantages of database reduction and Δ retention time (ΔRT) filtering. The strategy improves peptide/protein identification and reproducibility by retaining more correct identifications and filtering out incorrect identifications. The raw data were first merged and searched against a Uniprot database with a relaxed false discovery rate (FDR) of 40% to identify the possible detectable proteins. The identified proteins were then used as a new database to search the raw data against with a tighter FDR of 10%. After this, the results were filtered using ΔRT (the difference between the measured and predicted RT) to reduce the incorrect identifications and maintain the FDR below 1%. This strategy resulted in over 30% improvement in proteome coverage for single-cells and samples of similar size. The reproducibility of identification and quantification was also enhanced for the low-input samples. Moreover, the 50% higher number of differential proteins found in the two types of single neurons indicates the application potential of this strategy.