Baicalein Relieves Ferroptosis-Mediated Phagocytosis Inhibition of Macrophages in Ovarian Endometriosis.

Iron overload and oxidative stress have been reported to contribute to ferroptosis in endometriotic lesions. However, the possible roles of iron overload on macrophages in endometriosis (EMs) remain unknown. Based on recent reports by single-cell sequencing data of endometriosis, here we found significant upregulations of ferroptosis-associated genes in the macrophage of the endometriotic lesion. Additionally, there was an elevated expression of HMOX1, FTH1, and FTL in macrophages of peritoneal fluid in EMs, as well as iron accumulation in the endometriotic lesions. Notably, cyst fluid significantly up-regulated levels of intracellular iron and ferroptosis in Phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 cells. Additionally, high iron-induced ferroptosis obviously reduced PMA-stimulated THP-1 cells' phagocytosis and increased the expression of angiogenic cytokines, such as vascular endothelial growth factor A (VEGFA) and interleukin 8 (IL8). Baicalein, a potential anti-ferroptosis compound, increased GPX4 expression, significantly inhibited ferroptosis, and restored phagocytosis of THP-1 cells in vitro. Collectively, our study reveals that ferroptosis triggered by high iron in cyst fluid promotes the development of EMs by impairing macrophage phagocytosis and producing more angiogenic cytokines (e.g., IL8 and VEGFA). Baicalein displays the potential for the treatment of EMs, especially in patients with high ferroptosis and low phagocytosis of macrophages.

Precision De Novo Peptide Sequencing Using Mirror Proteases of Ac-LysargiNase and Trypsin for Large-scale Proteomics.

De novo peptide sequencing for large-scale proteomics remains challenging because of the lack of full coverage of ion series in tandem mass spectra. We developed a mirror protease of trypsin, acetylated LysargiNase (Ac-LysargiNase), with superior activity and stability. The mirror spectrum pairs derived from the Ac-LysargiNase and trypsin treated samples can generate full b and y ion series, which provide mutual complementarity of each other, and allow us to develop a novel algorithm, pNovoM, for de novo sequencing. Using pNovoM to sequence peptides of purified proteins, the accuracy of the sequence was close to 100%. More importantly, from a large-scale yeast proteome sample digested with trypsin and Ac-LysargiNase individually, 48% of all tandem mass spectra formed mirror spectrum pairs, 97% of which contained full coverage of ion series, resulting in precision de novo sequencing of full-length peptides by pNovoM. This enabled pNovoM to successfully sequence 21,249 peptides from 3,753 proteins and interpreted 44-152% more spectra than pNovo+ and PEAKS at a 5% FDR at the spectrum level. Moreover, the mirror protease strategy had an obvious advantage in sequencing long peptides. We believe that the combination of mirror protease strategy and pNovoM will be an effective approach for precision de novo sequencing on both single proteins and proteome samples.

Pollution Load Capacity Calculation Study Based on Multi-objective System in Trans-boundary Area, Plain River Network.

In this paper, a new method for pollution load control was proposed to solve the transboundary pollution problem in plain river network. The spatial distribution of load capacity, considering both multiple management requirements and local hydrodynamic features, has been studied in three steps: (1) the multiple objectives calculation system featured by multiple constraints has been proposed considering water quality requirements for different kinds of objectives. (2) the corresponding 1-d and 0-d models for load capacity calculation, considering the complex hydrodynamic characteristic, have been established separately. (3) based on the multi-objective calculation system, the load capacity satisfying the multiple objectives in different administrative units could be calculated. The results indicated that pollution load capacity of the whole transboundary area in Taihu basin is 151806 t/y for chemical oxygen demand, 15,099 t/y for ammonia nitrogen and 3394 t/y for total phosphorus, respectively. Then the rationality of the result has been analyzed and the load capacity calculated has been proved reasonable.

Comparative Transcriptomic and Proteomic Analyses Prove that IFN-λ1 is a More Potent Inducer of ISGs than IFN-α against Porcine Epidemic Diarrhea Virus in Porcine Intestinal Epithelial Cells.

Type III interferon (IFN-λ) is currently considered to be largely nonredundant to type I interferon (IFN-α) in antivirus infection, especially in epithelial cells. Previous studies reported that, compared with IFN-α, IFN-λ exhibited stronger induction of interferon-stimulated genes (ISGs) at the transcriptional level in intestinal epithelial cells and stronger inhibition of porcine epidemic diarrhea virus (PEDV). In this study, the different mechanisms of ISG upregulation induced by IFN-α and IFN-λ1 were compared at the mRNA and protein levels in the porcine intestinal epithelial cell model (IPEC-J2). It was proved that IFN-λ1 consistently exhibited stronger stimulation effects at both levels. At the mRNA level, 132 genes were significantly upregulated upon IFN-λ1 stimulation, while 42 genes upon IFN-α stimulation. At the protein level, 47 proteins were significantly upregulated upon IFN-λ1 stimulation, but only 8 proteins were upregulated upon IFN-α stimulation. The shared upregulated genes/proteins by IFN-λ1 in both transcriptional and translational omics, especially the regulation factors of ISG15, were involved in the JAK-STAT signaling pathway. Compared to IFN-α, IFN-λ1 could induce more consistent upregulation of the key ISGs (ISG15, USP18, OASL, and RSAD2) at 3-24 h postinduction as measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation. It was further confirmed through functional analysis that ISG15 and RSAD2 could inhibit PEDV infection in dose-dependent manners. This study provided solid evidence that IFN-λ1 could induce a more unique and higher ISG expression level, which exhibited anti-PEDV effects on porcine intestinal epithelial cells.

Peptidyl-Lys metalloendopeptidase (Lys-N) purified from dry fruit of Grifola frondosa demonstrates "mirror"digestion property with lysyl endopeptidase (Lys-C).

RATIONALE: Lys-N, also known as lysine-specific metalloendopeptidase, functions as the "sister" enzyme of lysyl endopeptidase (Lys-C) in proteomic research. Its digestion specificity at the N-terminal lysine residue makes it a very useful tool in proteomics analysis, especially in mass spectrometry (MS)-based de novo sequencing of proteins. METHODS: Here we present a complete production process of highly purified Lys-N from dry fruit of Grifola frondosa (maitake mushroom). The purification process includes one step of microfiltration plus one step of UF/DF (ultrafiltration used in tandem with a diafiltration method) recovery and four steps of chromatographic purification. RESULTS: The overall yield of the process was approximately 6.7 mg Lys-N protein/kg dry fruit of G. frondosa. The assay data demonstrated that the purified Lys-N exhibited high enzymatic activity and specificity. CONCLUSIONS: The novel production process provides for the first time the extraction of Lys-N from dry fruit of G. frondosa. The process is also stable and scalable, and provides an economic way of producing the enzyme in large quantities for MS-based proteomics and other biological studies.

Recombinant expression, purification and characterization of acetylated LysargiNase from Escherichia coli with high activity and stability.

RATIONALE: LysargiNase is a novel characterized metalloprotease that can cleave the N-terminii of lysine or arginine residues. The peptides generated by LysargiNase are just mirrors to those generated by trypsin. These characteristics of LysargiNase provide a powerful tool for mass spectrometry (MS)-based proteomics research. A highly active and stable LysargiNase produced by an easy and inexpensive method could greatly benefit proteomics research. Here, we report the soluble recombinant expression, purification and acetyl modification of LysargiNase in Escherichia coli. METHODS: The coding sequence of LysargiNase with an enterokinase cleavage site at the N-terminus was inserted into plasmid pGEX-4 T-2 and transformed into E. coli BL21 (DE3). The strain was cultured in a 14-L fermenter with a working volume of 5 L. The protein expression was induced by adding isopropyl-ß-D-thiogalactoside (IPTG) to a final concentration of 1 mM. The recombinant LysargiNase was loaded onto a GSTrap and an on-column digestion was performed to remove the GST tag and was subsequently purified by chromatographic purification. In vitro acetylation of LysargiNase was performed by using acetic anhydride. The digestion efficiency and specificity of recombinant LysargiNase and acetylated LysargiNase were compared with simple protein substrate, human serum albumin (HSA), and a complex proteomic sample, yeast lysate, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). RESULTS: Highly soluble expression of recombinant LysargiNase was achieved by plasmid pGEX-4 T-2 in E. coli BL21 (DE3). In addition, acetylation of purified LysargiNase significantly increased its resistance to autolysis, which resulted in a more complete digestion of proteomics samples and more identified peptides and proteins by LC/MS/MS. CONCLUSIONS: In this study, we constructed a highly soluble expression system for producing recombinant LysargiNase in E. coli, which gave tremendous advantages in the downstream purification process. We also confirmed that acetyl modification can increase the stability and activity of recombinant LysargiNase. The study provided a superior way to produce this powerful tool for proteomics research.

Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs).

Ubiquitination is one of the most common post-translational modifications, regulating protein stability and function. However, the proteome-wide profiling of ubiquitinated proteins remains challenging due to their low abundance in cells. In this study, we systematically evaluated the affinity of ubiquitin-binding domains (UBDs) to different types of ubiquitin chains. By selecting UBDs with high affinity and evaluating various UBD combinations with different lengths and types, we constructed two artificial tandem hybrid UBDs (ThUBDs), including four UBDs made of DSK2p-derived ubiquitin-associated (UBA) and ubiquilin 2-derived UBA (ThUDQ2) and of DSK2p-derived UBA and RABGEF1-derived A20-ZnF (ThUDA20). ThUBD binds to ubiquitinated proteins, with markedly higher affinity than naturally occurring UBDs. Furthermore, it displays almost unbiased high affinity to all seven lysine-linked chains. Using ThUBD-based profiling with mass spectrometry, we identified 1092 and 7487 putative ubiquitinated proteins from yeast and mammalian cells, respectively, of which 362 and 1125 proteins had ubiquitin-modified sites. These results demonstrate that ThUBD is a refined and promising approach for enriching the ubiquitinated proteome while circumventing the need to overexpress tagged ubiquitin variants and use antibodies to recognize ubiquitin remnants, thus providing a readily accessible tool for the protein ubiquitination research community.

Searching Missing Proteins Based on the Optimization of Membrane Protein Enrichment and Digestion Process.

A membrane protein enrichment method composed of ultracentrifugation and detergent-based extraction was first developed based on MCF7 cell line. Then, in-solution digestion with detergents and eFASP (enhanced filter-aided sample preparation) with detergents were compared with the time-consuming in-gel digestion method. Among the in-solution digestion strategies, the eFASP combined with RapiGest identified 1125 membrane proteins. Similarly, the eFASP combined with sodium deoxycholate identified 1069 membrane proteins; however, the in-gel digestion characterized 1091 membrane proteins. Totally, with the five digestion methods, 1390 membrane proteins were identified with ≥1 unique peptides, among which 1345 membrane proteins contain unique peptides ≥2. This is the biggest membrane protein data set for MCF7 cell line and even breast cancer tissue samples. Interestingly, we identified 13 unique peptides belonging to 8 missing proteins (MPs). Finally, eight unique peptides were validated by synthesized peptides. Two proteins were confirmed as MPs, and another two proteins were candidate detections.

iTRAQ-Based Membrane Proteomics Reveals Plasma Membrane Proteins Change During HepaRG Cell Differentiation.

HepaRG cell, a stabilized bipotent liver progenitor cell line, exhibits hepatocyte functions only after differentiation. However, the mechanism of transition from nondifferentiated to differentiated states, accompanied by proliferation migration and differentiation, remains poorly understood, particularly those proteins residing in the plasma membrane. In this study, the membrane protein expression change of HepaRG cell during differentiation were systematically analyzed using an iTRAQ labeled quantitative membrane proteomics approach. A total of 70 membrane proteins were identified to be differentially expressed among 849 quantified membrane proteins. Function and disease clustering analysis proved that 11 of these proteins are involved in proliferation, migration, and differentiation. Two key factors (MMP-14 and OCLN) were validated by qRT-PCR and Western blot. Blockade of MMP-14 further demonstrated its important function during tumor cell migration. The data sets have been uploaded to ProteomeXchange with the identifier PXD004752.

Quantitative Proteomics Reveals Membrane Protein-Mediated Hypersaline Sensitivity and Adaptation in Halophilic Nocardiopsis xinjiangensis.

The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress.

Increased levels of HMGB1 in trophoblastic debris may contribute to preeclampsia.

Preeclampsia is triggered by an as yet unknown toxin from the placenta. Antiphospholipid antibodies (aPL), a strong risk factor for preeclampsia, have been shown to induce the production of toxic trophoblastic debris from the placenta. High mobility group box 1 (HMGB1) is a proinflammatory danger signal, and the expression of it has been reported to be increased in preeclampsia. This study examined whether aPL or preeclamptic sera increase the expression of HMGB1 in the syncytiotrophoblast or trophoblastic debris. Trophoblastic debris from normal placental explants that had been cultured with aPL or preeclamptic sera was exposed to endothelial cells. Endothelial cell activation was quantified by cell-surface ICAM-1 expression and U937 monocyte adhesion. The expression of HMGB1 in placental explants and trophoblastic debris that had been treated with aPL or preeclamptic sera was measured by immunohistochemistry and western blotting. The expression of the receptor for advanced glycation end products (RAGE) in endothelial cells was quantified by western blotting. Compared with controls, the expression of HMGB1 in the cytoplasm of the syncytiotrophoblast and trophoblastic debris was increased by treating placental explants with aPL or preeclamptic sera. The increased levels of HMGB1 contributed to endothelial cell activation, mediated in part by the RAGE. Preeclamptic sera and aPL both induced an increase in the cytoplasmic levels of the danger signal HMGB1 in trophoblastic debris. This increased HMGB1 in trophoblastic debris may be one of the toxic factors released from the placenta in preeclampsia.

Recombinant expression, refolding, purification and characterization of Pseudomonas aeruginosa protease IV in Escherichia coli.

Several protease IV enzymes are widely used in proteomic research. Specifically, protease IV from Pseudomonas aeruginosa has lysyl endopeptidase activity. Here, we report the recombinant expression, refolding, activation, and purification of this protease in Escherichia coli. Proteolytic instability of the activated intermediate, a major obstacle for efficient production, is controlled through ammonium sulfate precipitation. The purified protease IV exhibits superior lysyl endopeptidase activity compared to a commercial product.

Recombinant acetylated trypsin demonstrates superior stability and higher activity than commercial products in quantitative proteomics studies.

RATIONALE: Trypsin is an important digestive enzyme in peptide sample preparation for proteomics. It digests proteins at the C-terminal of Arg or Lys residues. The majority of commercial products are obtained from animal sources. In a previous study, we reported the production process for recombinant trypsin (r-trypsin) and acetylated trypsin (r-Ac-trypsin). In this paper, we want to evaluate whether the r-trypsin and r-Ac-trypsin are suitable for proteomics research. METHODS: The trypsins used in this research were first normalized to the same concentration and used for further evaluation. The stability and buffer compatibility (2M urea, 0.1% SDS and 10% acetonitrile) were compared and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The digestion efficiency and specificity were compared based on a simple protein substrate, human serum albumin (HSA) and a complex proteomic sample, yeast lysate. The acquisition of proteomics data was achieved by ultra-high performance liquid chromatography (UPLC) connected to an LTQ Orbitrap Velos mass spectrometer. RESULTS: r-Ac-trypsin demonstrated similar tolerance to 2 M urea and 10% acetonitrile but weaker 0.1% SDS tolerance than commercial trypsins. Based on simple protein sample HSA, the activity and specificity of r-Ac-trypsin were similar to that of commercial trypsins. However, it demonstrated superior activity and specificity on complicated samples like yeast lysate. More interestingly, the newly developed r-Ac-trypsin was more resistant to autolysis, which enabled more complete digestion of proteomic samples. CONCLUSIONS: The r-Ac-trypsin described here is a recombinant product. In addition it showed similar or superior properties such as stability activity and specificity to commercial products. It can be used in peptide sample preparation in proteomics studies.

Transcriptome differences between fiber-type and seed-type Cannabis sativa variety exposed to salinity.

The industrial hemp varieties 'Yunma 5' and 'Bamahuoma,' which demonstrate growth vigor and environmental adaptability, have been primarily cultivated in Yunnan and Guangxi, China, respectively, for fiber and seeds. The results of physiological measurements showed the phenotypic differences between the two varieties in response to salt stress. RNA-Seq analysis was first performed on leaves of both varieties sampled at four time intervals (0, 2, 4, 6 days) after treatment with salt (500 mM NaCl) We identified 220 co-up-regulated differentially expressed genes (DEGs) in the two varieties, while 26 up-regulated DEGs and 24 down-regulated DEGs were identified exclusively in the single varieties after 2 days of salt stress. Among the 220 DEGs, we identified 22 transcription factors, including key transcription factors involved in salt stress, such as MYB, NAC, GATA, and HSF. We applied gene expression profile analysis and found that 'Yunma 5' and 'Bamahuoma' have variety-specific pathways for resisting salt stress. The DEGs of 'Yunma 5' were enriched in spliceosome and amino acid metabolism genes, while the DEGs of 'Bamahuoma' were enriched in fatty acid metabolism, amino acid metabolism, and endoplasmic reticulum protein processing pathway. Although there were common DEGs, such as genes encoding cysteine protease and alpha/beta-hydrolase superfamily, the two varieties' responses to salt stress impacted different metabolic pathways. The DEGs that were co-expressed in both varieties under stress may provide useful insights into the tolerance of cultivated hemp and other bast fiber crops to saline soil conditions. These transcriptomes also represent reference sequences for industrial hemp.

Special Enrichment Strategies Greatly Increase the Efficiency of Missing Proteins Identification from Regular Proteome Samples.

As part of the Chromosome-Centric Human Proteome Project (C-HPP) mission, laboratories all over the world have tried to map the entire missing proteins (MPs) since 2012. On the basis of the first and second Chinese Chromosome Proteome Database (CCPD 1.0 and 2.0) studies, we developed systematic enrichment strategies to identify MPs that fell into four classes: (1) low molecular weight (LMW) proteins, (2) membrane proteins, (3) proteins that contained various post-translational modifications (PTMs), and (4) nucleic acid-associated proteins. Of 8845 proteins identified in 7 data sets, 79 proteins were classified as MPs. Among data sets derived from different enrichment strategies, data sets for LMW and PTM yielded the most novel MPs. In addition, we found that some MPs were identified in multiple-data sets, which implied that tandem enrichments methods might improve the ability to identify MPs. Moreover, low expression at the transcription level was the major cause of the "missing" of these MPs; however, MPs with higher expression level also evaded identification, most likely due to other characteristics such as LMW, high hydrophobicity and PTM. By combining a stringent manual check of the MS2 spectra with peptides synthesis verification, we confirmed 30 MPs (neXtProt PE2 ∼ PE4) and 6 potential MPs (neXtProt PE5) with authentic MS evidence. By integrating our large-scale data sets of CCPD 2.0, the number of identified proteins has increased considerably beyond simulation saturation. Here, we show that special enrichment strategies can break through the data saturation bottleneck, which could increase the efficiency of MP identification in future C-HPP studies. All 7 data sets have been uploaded to ProteomeXchange with the identifier PXD002255.

Development of a rapid high-efficiency scalable process for acetylated Sus scrofa cationic trypsin production from Escherichia coli inclusion bodies.

Trypsin is one of the most important enzymatic tools in proteomics and biopharmaceutical studies. Here, we describe the complete recombinant expression and purification from a trypsinogen expression vector construct. The Sus scrofa cationic trypsin gene with a propeptide sequence was optimized according to Escherichia coli codon-usage bias and chemically synthesized. The gene was inserted into pET-11c plasmid to yield an expression vector. Using high-density E. coli fed-batch fermentation, trypsinogen was expressed in inclusion bodies at 1.47 g/L. The inclusion body was refolded with a high yield of 36%. The purified trypsinogen was then activated to produce trypsin. To address stability problems, the trypsin thus produced was acetylated. The final product was generated upon gel filtration. The final yield of acetylated trypsin was 182 mg/L from a 5-L fermenter. Our acetylated trypsin product demonstrated higher BAEE activity (30,100 BAEE unit/mg) than a commercial product (9500 BAEE unit/mg, Promega). It also demonstrated resistance to autolysis. This is the first report of production of acetylated recombinant trypsin that is stable and suitable for scale-up.

Two new species of the genus Eophileurus Arrow, 1908 (Coleoptera: Scarabaeidae: Dynastinae) from the Philippines and Vietnam, with first description of E. quadratifovealis Yang & Pathomwattananurak, 2022 female.

Two new species, Eophileurus varipunctatus Yang, Pathomwattananurak & Zhao, new species from Mindoro and Palawan, the Philippines and Eophileurus vietnamensis Yang, Pathomwattananurak & Zhao, new species from Vietnam are described and illustrated. Eophileurus iwasei Muramoto, 1995 and E. chinensis (Faldermann, 1935) are illustrated and compared to each new species, respectively. The female of E. quadratifovealis Yang & Pathomwattananurak, 2022 is described for the first time.

New species of the tribe Sericini Kirby, 1837 from China, with further updates on their taxonomy and distribution (Coleoptera: Scarabaeidae: Sericinae).

Fifteen new species of Sericini are described from China, including Gastroserica (s. str.) mayunshui Zhao & Ahrens, new species, Pachyserica albopunctata Zhao & Ahrens, new species, P. dongnanensis Zhao & Ahrens, new species, P. jianfengensis Zhao & Ahrens, new species, Serica (s. l.) caiyiyiae Zhao & Ahrens, new species, S. (s. l.) babaoshanensis Zhao & Ahrens, new species, S. (s. l.) jicaiyanae Zhao & Ahrens, new species, S. (s. l.) zhangyaonani Zhao & Ahrens, new species, S. (s. l.) longidentata Zhao & Ahrens, new species, S. (s. l.) callosericoides Zhao & Ahrens, new species, S. (Taiwanoserica) liboyani Zhao & Ahrens, new species, S. (T.) yangzaichuni Zhao & Ahrens, new species, Maladera zhanchaoi Zhao & Ahrens, new species M. parabikouensis Zhao & Ahrens, new species and M. shikengkongensis Zhao & Ahrens, new species. Maladera juxianensis Ahrens, Fabrizi & Liu, 2021 was recognized as a junior synonym of M. aureola (Murayama, 1938). Additional collecting data for 32 other sericine species is presented.

Characterization of permanent deformation properties of densely-compacted unbound granular materials subjected to cyclic loading.

To investigate the long-term deformation properties of unbound granular materials (UGM) that are ordinarily adopted to construct subgrade for high-speed railway, a series of medium-sized cyclic triaxial tests were performed to obtain the relationship between permanent strain and loading cycles under different cyclic stress levels. Moreover, DEM analysis was conducted for the samples to reveal the deformation mechanism and verify the strain developing tendency. It is found that the UGM samples present different long-term deformation properties under different cyclic stress levels. As cyclic stress increases, the permanent strain of UGM sample transfers from rapid stabilization to tardy stabilization, then to tardy failure and finally to rapid failure. Furthermore, the exponent in a power law function was selected as the critical indicator of deformation developing tendency. With the exponent obtained precisely in accordance with the strain rate, the deformation tendency can be analyzed quantitatively. Finally, the characteristics of interparticle force chains induced by different cyclic stress levels were obtained by DEM analysis, which provided evidences for the classification of long-term deformation properties of UGM samples. The achievements have guiding significance for the design of subgrade of both ballasted and unballasted high-speed railway.

A New Approach for Classifying the Permanent Deformation Properties of Granular Materials under Cyclic Loading.

A series of medium-sized cyclic triaxial tests were performed to investigate the permanent deformation properties of granular materials. The strain rate was then plotted against loading cycles to classify the permanent deformation properties of granular materials under different cyclic stress ratios (CSRs). It was found that (1) the permanent strain rate dεp/dN was linearly correlated with loading cycles N using a double-log coordinate on the condition of CSR < 60%; (2) the deformation tendency factor ß, which was extracted from the linear relationship between dεp/dN and N, significantly varied with CSR and, thus, can be adopted to identify the deformation states; (3) ß > 1 implying that permanent strain accumulation ceases in limited cycles and corresponds to the plastic shakedown range, while 0 < ß ≤ 1 indicates the temporary steady state, corresponding to the plastic creep range; (4) sluggish decrease or remarkable increase in dεp/dN appeared as CSR ≥ 60%, leading to soil collapsed in limited loading cycles and resulting in an incremental collapse range. The new approach was validated by the crushed tuff aggregates and subgrade materials reported previously. It is expected that the new approach will have wider applicability than the traditional one and can provide technical guidance for the design and construction of substructures in roadway and railway engineering.