Pre-sliding of the femoral neck system to prevent postoperative shortening of femoral neck fractures.

Objective: The purpose of this study is to evaluate the effect of pre-sliding of the femoral neck system (FNS) in the prevention of postoperative femoral neck shortening in femoral neck fractures. Method: This study was designed to retrospectively analyze data from 109 patients with femoral neck fractures who were admitted to a Level I trauma center between April 2020 and June 2022. Of these patients, 90 were followed up for more than 12 months. The study included 52 males and 38 females, with 35 cases of Garden I and II fractures and 55 cases of Garden III and IV fractures. The Harris Hip Score at 12 months postoperatively were recorded. The patients were divided into two groups based on their surgical records and postoperative radiography: the Pre-sliding group and the No-pre-sliding group. The purpose of this study is to analyze the role of pre-sliding in preventing femoral neck shortening, fracture healing time, degree of postoperative shortening, complications, and Harris Hip Score, and to make a comparison between the two groups. Results: All 90 patients were followed up for over one year after surgery. A statistically significant difference was observed in the preoperative Garden classification (P < 0.05). At 1 year after the operation, the shortening distance was 6.5 ± 6.4 mm in the No-pre-sliding group and 3.9 ± 3.4 mm in the Pre-sliding group. The Harris Hip Score were 88.7 (79.8, 93.5) in the No-pre-sliding group and 94.8 (87.7, 96.9) in the Pre-sliding group, with a statistically significant difference between the two groups (P < 0.05). Shortening was concentrated at 3 months postoperatively and reached a stable state within 6 months, with less persistent shortening occurring after 6 months. There was no statistically significant difference in the preoperative baseline data. Conclusion: Pre-sliding of the FNS prevents postoperative shortening of the femoral neck and improves hip function as measured by the Harris Hip Score.

LncCMRR Plays an Important Role in Cardiac Differentiation by Regulating the Purb/Flk1 Axis.

As crucial epigenetic regulators, long noncoding RNAs (lncRNAs) play critical functions in development processes and various diseases. However, the regulatory mechanism of lncRNAs in early heart development is still limited. In this study, we identified cardiac mesoderm-related lncRNA (LncCMRR). Knockout (KO) of LncCMRR decreased the formation potential of cardiac mesoderm and cardiomyocytes during embryoid body differentiation of mouse embryonic stem (ES) cells. Mechanistic analyses showed that LncCMRR functionally interacted with the transcription suppressor PURB and inhibited its binding potential at the promoter region of Flk1, which safeguarded the transcription of Flk1 during cardiac mesoderm formation. We also carried out gene ontology term and signaling pathway enrichment analyses for the differentially expressed genes after KO of LncCMRR, and found significant correlation of LncCMRR with cardiac muscle contraction, dilated cardiomyopathy, and hypertrophic cardiomyopathy. Consistently, the expression level of Flk1 at E7.75 and the thickness of myocardium at E17.5 were significantly decreased after KO of LncCMRR, and the survival rate and heart function index of LncCMRR-KO mice were also significantly decreased as compared with the wild-type group. These findings indicated that the defects in early heart development led to functional abnormalities in adulthood heart of LncCMRR-KO mice. Conclusively, our findings elucidate the main function and regulatory mechanism of LncCMRR in cardiac mesoderm formation, and provide new insights into lncRNA-mediated regulatory network of mouse ES cell differentiation.

Discovery of novel bis-evodiamine derivatives with potent antitumor activity.

Inspired by antitumor natural product evodiamine, a series of novel bis-evodiamine derivatives were designed and synthesized, which showed potent antitumor activity. In particular, compound 13b effectively inhibited the proliferation and migration of HCT116 cells. Further mechanism studies revealed that compound 13b acted by inducing HCT116 cell apoptosis and arresting the cell cycle at the G2/M phase. Thus, compound 13b represents a promising lead compound for the discovery of novel antitumor agents.

Design, Synthesis, and Structure-Activity relationships of Evodiamine-Based topoisomerase (Top)/Histone deacetylase (HDAC) dual inhibitors.

On the basis of synergistic effect between topoisomerase (Top) and histone deacetylase (HDAC) inhibitors, a series of novel evodiamine-based Top/HDAC dual inhibitors were designed and synthesized. Systematic structure-activity relationship (SAR) studies led to the discovery of compounds 29b and 45b, which simultaneously inhibited Top and HDAC and exhibited potent antitumor activities against the HCT116 cell line. Compounds 29b and 45b efficiently induced apoptosis with G2 cell cycle arrest and significantly inhibited cellular HDACs in HCT116 cells with good in vitro metabolic stabilities. Collectively, this work provides valuable SAR information and lead compounds for evodiamine-based Top/HDAC dual inhibitors.

Sin3a drives mesenchymal-to-epithelial transition through cooperating with Tet1 in somatic cell reprogramming.

BACKGROUND: Identifying novel regulatory factors and uncovered mechanisms of somatic cell reprogramming will be helpful for basic research and clinical application of induced pluripotent stem cells (iPSCs). Sin3a, a multifunctional transcription regulator, has been proven to be involved in the maintenance of pluripotency in embryonic stem cells (ESCs), but the role of Sin3a in somatic cell reprogramming remains unclear. METHODS: RNA interference of Sin3a during somatic cell reprogramming was realized by short hairpin RNAs. Reprogramming efficiency was evaluated by the number of alkaline phosphatase (AP)-positive colonies and Oct4-GFP-positive colonies. RNA sequencing was performed to identify the influenced biological processes after Sin3a knockdown and further confirmed by quantitative RT-PCR (qRT-PCR), western blotting and flow cytometry. The interaction between Sin3a and Tet1 was detected by coimmunoprecipitation. The enrichment of Sin3a and Tet1 at the epithelial gene promoters was measured by chromatin immunoprecipitation. Furthermore, DNA methylation patterns at the gene loci were investigated by hydroxymethylated DNA immunoprecipitation. Finally, Sin3a mutants that disrupt the interaction of Sin3a and Tet1 were also introduced to assess the importance of the Sin3a-Tet1 interaction during the mesenchymal-to-epithelial transition (MET) process. RESULTS: We found that Sin3a was gradually increased during OSKM-induced reprogramming and that knockdown of Sin3a significantly impaired MET at the early stage of reprogramming and iPSC generation. Mechanistic studies showed that Sin3a recruited Tet1 to facilitate the hydroxymethylation of epithelial gene promoters. Moreover, disrupting the interaction of Sin3a and Tet1 significantly blocked MET and iPSC generation. CONCLUSIONS: Our studies revealed that Sin3a was a novel mediator of MET during early reprogramming, where Sin3a functioned as an epigenetic coactivator, cooperating with Tet1 to activate the epithelial program and promote the initiation of somatic cell reprogramming. These findings highlight the importance of Sin3a in the MET process and deepen our understanding of the epigenetic regulatory network of early reprogramming.

Human-gained heart enhancers are associated with species-specific cardiac attributes.

The heart, a vital organ which is first to develop, has adapted its size, structure and function in order to accommodate the circulatory demands for a broad range of animals. Although heart development is controlled by a relatively conserved network of transcriptional/chromatin regulators, how the human heart has evolved species-specific features to maintain adequate cardiac output and function remains to be defined. Here, we show through comparative epigenomic analysis the identification of enhancers and promoters that have gained activity in humans during cardiogenesis. These cis-regulatory elements (CREs) are associated with genes involved in heart development and function, and may account for species-specific differences between human and mouse hearts. Supporting these findings, genetic variants that are associated with human cardiac phenotypic/disease traits, particularly those differing between human and mouse, are enriched in human-gained CREs. During early stages of human cardiogenesis, these CREs are also gained within genomic loci of transcriptional regulators, potentially expanding their role in human heart development. In particular, we discovered that gained enhancers in the locus of the early human developmental regulator ZIC3 are selectively accessible within a subpopulation of mesoderm cells which exhibits cardiogenic potential, thus possibly extending the function of ZIC3 beyond its conserved left-right asymmetry role. Genetic deletion of these enhancers identified a human gained enhancer that was required for not only ZIC3 and early cardiac gene expression at the mesoderm stage but also cardiomyocyte differentiation. Overall, our results illuminate how human gained CREs may contribute to human-specific cardiac attributes, and provide insight into how transcriptional regulators may gain cardiac developmental roles through the evolutionary acquisition of enhancers.

Unveiling Complexity and Multipotentiality of Early Heart Fields.

[Figure: see text].

Cardiac cell type-specific gene regulatory programs and disease risk association.

Misregulated gene expression in human hearts can result in cardiovascular diseases that are leading causes of mortality worldwide. However, the limited information on the genomic location of candidate cis-regulatory elements (cCREs) such as enhancers and promoters in distinct cardiac cell types has restricted the understanding of these diseases. Here, we defined >287,000 cCREs in the four chambers of the human heart at single-cell resolution, which revealed cCREs and candidate transcription factors associated with cardiac cell types in a region-dependent manner and during heart failure. We further found cardiovascular disease-associated genetic variants enriched within these cCREs including 38 candidate causal atrial fibrillation variants localized to cardiomyocyte cCREs. Additional functional studies revealed that two of these variants affect a cCRE controlling KCNH2/HERG expression and action potential repolarization. Overall, this atlas of human cardiac cCREs provides the foundation for illuminating cell type-specific gene regulation in human hearts during health and disease.

Structural simplification of evodiamine: Discovery of novel tetrahydro-ß-carboline derivatives as potent antitumor agents.

Natural products (NPs) have played a crucial role in the discovery and development of antitumor drugs. However, the high structural complexity of NPs generally results in unfavorable physicochemical profiles and poor drug-likeness. A powerful strategy to tackle this obstacle is the structural simplification of NPs by truncating nonessential structures. Herein, a series of tetrahydro-ß-carboline derivatives were designed by elimination of the D ring of NP evodiamine. Structure-activity relationship studies led to the discovery of compound 45, which displayed highly potent antitumor activity against all the tested cancer cell lines and excellent in vivo antitumor activity in the HCT116 xenograft model with low toxicity. Further mechanistic research indicated that compound 45 acted by dual Top1/2 inhibition and induced caspase-dependent cell apoptosis coupled with G2/M cell cycle arrest. This proof-of-concept study validated the effectiveness of structural simplification in NP-based drug development, discovered compound 45 as a potent antitumor lead compound and enriched the structure-activity relationships of evodiamine.

Multigene editing reveals that MtCEP1/2/12 redundantly control lateral root and nodule number in Medicago truncatula.

The multimember CEP (C-terminally Encoded Peptide) gene family is a complex group that is involved in various physiological activities in plants. Previous studies demonstrated that MtCEP1 and MtCEP7 control lateral root formation or nodulation, but these studies were based only on gain of function or artificial miRNA (amiRNA)/RNAi approaches, never knockout mutants. Moreover, an efficient multigene editing toolkit is not currently available for Medicago truncatula. Our quantitative reverse transcription-PCR data showed that MtCEP1, 2, 4, 5, 6, 7, 8, 9, 12, and 13 were up-regulated under nitrogen starvation conditions and that MtCEP1, 2, 7, 9, and 12 were induced by rhizobial inoculation. Treatment with synthetic MtCEP peptides of MtCEP1, 2, 4, 5, 6, 8, and 12 repressed lateral root emergence and promoted nodulation in the R108 wild type but not in the cra2 mutant. We optimized CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9] genome editing system for M. truncatula, and thus created single mutants of MtCEP1, 2, 4, 6, and 12 and the double mutants Mtcep1/2C and Mtcep5/8C; however, these mutants did not exhibit significant differences from R108. Furthermore, a triple mutant Mtcep1/2/12C and a quintuple mutant Mtcep1/2/5/8/12C were generated and exhibited more lateral roots and fewer nodules than R108. Overall, MtCEP1, 2, and 12 were confirmed to be redundantly important in the control of lateral root number and nodulation. Moreover, the CRISPR/Cas9-based multigene editing protocol provides an additional tool for research on the model legume M. truncatula, which is highly efficient at multigene mutant generation.

The Use of Autologous Iliotibial Band With Gerdy's Tubercle for Irreparable Rotator Cuff Tears.

OBJECTIVE: To determine the radiographic and functional outcomes after autologous iliotibial band with Gerdy's tubercle (ITB-GT) interposition in patients with irreparable rotator cuff tears (IRCTs). METHODS: From December 2015 to March 2017, a total of 16 patients who underwent autologous ITB-GT interposition for IRCTs were identified, including 4 males and 12 females. The mean age was 56.1 ± 10.3 years (range, 44-67 years). Functional assessment consisted of active range of motion (ROM), Visual Analogue scale (VAS), American Shoulder and Elbow Surgeons (ASES) score, Constant-Murley score, and patient satisfaction rate. The tendon integrity and acromiohumeral interval (AHI) were assessed by postoperative magnetic resonance imaging (MRI). RESULTS: The mean duration of follow-up was 25.3 ± 3.5 months. At final follow-up, the patients exhibited significantly improved forward elevation, external rotation, and internal rotation (75.00° ± 13.16° to 159.37° ± 8.51°, t = 26.71, P = 0.001; 17.81° ± 11.54° to 35.31° ± 8.26°, t = 6.57, P = 0.001; 2 to 11, t = 13.10, P = 0.001). Other functional outcomes as measured by VAS score, ASES score, and Constant-Murley score also improved significantly (6.50 ± 1.41 to 1.06 ± 0.93, t = 11.68, P = 0.001; 38.50 ± 8.68 to 81.75 ± 6.80, t = 15.42, P = 0.001; 32.50 ± 8.53 to 77.12 ± 6.72, t = 17.28, P = 0.001). The overall satisfaction rate was 87.5%. The postoperative MRI showed that the tendon integrity was fully intact in 14 patients and partially intact in two patients. The AHI improved significantly from 3.63 ± 1.25 mm preoperatively to 8.37 ± 1.02 mm postoperatively (t = 11.78, P = 0.001). CONCLUSION: Autologous ITB-GT interposition was a useful treatment option for patients with IRCTs, which resulted in significant improvements in active ROM, subjective functional outcomes, and AHI with excellent tendon integrity at a minimum 2-year follow-up.

A CEP Peptide Receptor-Like Kinase Regulates Auxin Biosynthesis and Ethylene Signaling to Coordinate Root Growth and Symbiotic Nodulation in Medicago truncatula.

Because of the large amount of energy consumed during symbiotic nitrogen fixation, legumes must balance growth and symbiotic nodulation. Both lateral roots and nodules form on the root system, and the developmental coordination of these organs under conditions of reduced nitrogen (N) availability remains elusive. We show that the Medicago truncatula COMPACT ROOT ARCHITECTURE2 (MtCRA2) receptor-like kinase is essential to promote the initiation of early symbiotic nodulation and to inhibit root growth in response to low N. C-TERMINALLY ENCODED PEPTIDE (MtCEP1) peptides can activate MtCRA2 under N-starvation conditions, leading to a repression of YUCCA2 (MtYUC2) auxin biosynthesis gene expression, and therefore of auxin root responses. Accordingly, the compact root architecture phenotype of cra2 can be mimicked by an auxin treatment or by overexpressing MtYUC2, and conversely, a treatment with YUC inhibitors or an MtYUC2 knockout rescues the cra2 root phenotype. The MtCEP1-activated CRA2 can additionally interact with and phosphorylate the MtEIN2 ethylene signaling component at Ser643 and Ser924, preventing its cleavage and thereby repressing ethylene responses, thus locally promoting the root susceptibility to rhizobia. In agreement with this interaction, the cra2 low nodulation phenotype is rescued by an ein2 mutation. Overall, by reducing auxin biosynthesis and inhibiting ethylene signaling, the MtCEP1/MtCRA2 pathway balances root and nodule development under low-N conditions.

The Chromosome-Level Genome Sequence of the Autotetraploid Alfalfa and Resequencing of Core Germplasms Provide Genomic Resources for Alfalfa Research.

Alfalfa (Medicago sativa) is one of the most important forage crops in the world; however, its molecular genetics and breeding research are hindered due to the lack of a high-quality reference genome. Here, we report a de novo assembled 816-Mb high-quality, chromosome-level haploid genome sequence for 'Zhongmu No.1' alfalfa, a heterozygous autotetraploid. The contig N50 is 3.92 Mb, and 49 165 genes are annotated in the genome. The alfalfa genome is estimated to have diverged from M. truncatula approximately 8 million years ago. Genomic population analysis of 162 alfalfa accessions revealed high genetic diversity, weak population structure, and extensive gene flow from wild to cultivated alfalfa. Genome-wide association studies identified many candidate genes associated with important agronomic traits. Furthermore, we showed that MsFTa2, a Flowering Locus T homolog, whose expression is upregulated in salt-resistant germplasms, may be associated with fall dormancy and salt resistance. Taken together, these genomic resources will facilitate alfalfa genetic research and agronomic improvement.

A CEP Peptide Receptor-like Kinase Regulates Auxin Biosynthesis and Ethylene Signaling to Coordinate Root Growth and Symbiotic Nodulation in Medicago truncatula.

Because of the high energy consumed during symbiotic nitrogen fixation, legumes must balance growth and symbiotic nodulation. Both lateral roots and nodules form on the root system and the developmental coordination of these organs according to reduced nitrogen (N) availability remains elusive. We show that the Compact Root Architecture 2 (MtCRA2) receptor-like kinase is essential to promote the initiation of early symbiotic nodulation and to inhibit root growth in response to low-N. MtCEP1 peptides can activate MtCRA2 under N-starvation conditions, leading to a repression of MtYUC2 auxin biosynthesis gene expression, and therefore of auxin root responses. Accordingly, the compact root architecture phenotype of cra2 can be mimicked by an auxin treatment or by over-expressing MtYUC2, and conversely, a treatment with YUC inhibitors or a MtYUC2 knock-out rescues the cra2 root phenotype. The MtCEP1-activated CRA2 can additionally interact with and phosphorylate the MtEIN2 ethylene signaling component at Ser643 and Ser924, preventing its cleavage and therefore repressing ethylene responses, thus locally promoting the root susceptibility to rhizobia. In agreement, the cra2 low nodulation phenotype is rescued by an ein2 mutation. Overall, by reducing auxin biosynthesis and inhibiting ethylene signaling, the MtCEP1/MtCRA2 pathway balances root and nodule development under low-N conditions.

Arthroscopic side-to-side repair for complete radial posterior lateral meniscus root tears.

BACKGROUND: The aim of this study was to determine the radiographic, second-look, and functional outcomes after arthroscopic side-to-side repair for complete radial posterior lateral meniscus root tears (PLMRTs). METHODS: Patients who underwent arthroscopic side-to-side repair for complete radial PLMRTs were identified. Clinical assessment consisted of symptoms (locking, catching, giving way and effusion), examinations of joint-line tenderness and McMurray test, and subjective scores of International Knee Documentation Committee (IKDC), Lysholm, and Tegner. In addition, postoperative MRI scan and second-look arthroscopy were performed to assess the healing status of the repaired meniscus. RESULTS: Twenty-nine patients met the inclusion criteria. The mean age was 25.41 years. The mean follow-up period was 26.68 months. During the follow-up, none of the patients had symptoms of meniscal retear, lateral joint-line tenderness or a positive McMurray test. The postoperative subjective scores of IKDC, Lysholm, and Tegner improved significantly compared to the preoperative values (P = 0.01). Postoperative MRI scan showed that 28/29 (96.6%) patients achieved meniscus healing. Twenty-two patients underwent second-look arthroscopy, among whom 19 (86.4%) patients showed complete meniscus healing and 3 (13.6%) patients showed partial healing. CONCLUSION: Arthroscopic side-to-side repair was a valuable surgical repair technique for complete radial PLMRTs, which leaded to significant improvements in both objective and subjective functional outcomes with a high rate of meniscus healing. LEVEL OF EVIDENCE: Level IV, case series.

Osteochondral allograft transplantation for large Hill-Sachs lesions: a retrospective case series with a minimum 2-year follow-up.

PURPOSE: To investigate the clinical outcomes after osteochondral allograft transplantation for large Hill-Sachs lesions. METHODS: Patients who underwent osteochondral allograft transplantation for large Hill-Sachs lesions were identified. Clinical assessment consisted of active range of motion (ROM), American Shoulder and Elbow Surgeons score (ASES), Constant-Murley score, Rowe score, and patient satisfaction rate. Radiographic assessment was performed with CT scan. RESULTS: Nineteen patients met the inclusion criteria. The mean age was 21.7 years. The mean preoperative size of the Hill-Sachs lesion was 35.70 ± 3.02%. The mean follow-up was 27.8 months. All grafts achieved union at an average of 3.47 months after surgery. At the final follow-up, graft resorption was observed in 43.1% of patients. The average size of residual humeral head articular arc loss was 12.31 ± 2.79%. Significant improvements (P < .001) were observed for the active ROM, ASES score, Constant-Murley score, and Rowe score. The overall satisfaction rate was 94.7%. No significant difference was found between the resorption group and the nonresorption group in postoperative clinical outcomes. CONCLUSION: Osteochondral allograft transplantation is a useful treatment option for patients with large Hill-Sachs lesions. Although the incidence of graft resorption may be relatively high, the clinical outcomes at a minimum 2-year follow-up are favorable. LEVEL OF EVIDENCE: Level IV, case series.

Transcription Factor bHLH2 Represses CYSTEINE PROTEASE77 to Negatively Regulate Nodule Senescence.

Legume-rhizobia symbiosis is a time-limited process due to the onset of senescence, which results in the degradation of host plant cells and symbiosomes. A number of transcription factors, proteases, and functional genes have been associated with nodule senescence; however, whether other proteases or transcription factors are involved in nodule senescence remains poorly understood. In this study, we identified an early nodule senescence mutant in Medicago truncatula, denoted basic helix-loop-helix transcription factor2 (bhlh2), that exhibits decreased nitrogenase activity, acceleration of plant programmed cell death (PCD), and accumulation of reactive oxygen species (ROS). The results suggest that MtbHLH2 plays a negative role in nodule senescence. Nodules of wild-type and bhlh2-TALEN mutant plants at 28 d postinoculation were used for transcriptome sequencing. The transcriptome data analysis identified a papain-like Cys protease gene, denoted MtCP77, that could serve as a potential target of MtbHLH2. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis demonstrated that MtbHLH2 directly binds to the promoter of MtCP77 to inhibit its expression. MtCP77 positively regulates nodule senescence by accelerating plant PCD and ROS accumulation. In addition, the expression of MtbHLH2 in the nodules gradually decreased from the meristematic zone to the nitrogen fixation zone, whereas the expression of MtCP77 showed enhancement. These results indicate that MtbHLH2 and MtCP77 have opposite functions in the regulation of nodule senescence. These results reveal significant roles for MtbHLH2 and MtCP77 in plant PCD, ROS accumulation, and nodule senescence, and improve our understanding of the regulation of the nodule senescence process.

Collaborative ISL1/GATA3 interaction in controlling neuroblastoma oncogenic pathways overlapping with but distinct from MYCN.

Background: Transcription factor ISL1 plays a critical role in sympathetic neurogenesis. Expression of ISL1 has been associated with neuroblastoma, a pediatric tumor derived from sympatho-adrenal progenitors, however the role of ISL1 in neuroblastoma remains unexplored. Method: Here, we knocked down ISL1 (KD) in SH-SY5Y neuroblastoma cells and performed RNA-seq and ISL1 ChIP-seq analyses. Results: Analyses of these data revealed that ISL1 acts upstream of multiple oncogenic genes and pathways essential for neuroblastoma proliferation and differentiation, including LMO1 and LIN28B. ISL1 promotes expression of a number of cell cycle associated genes, but represses differentiation associated genes including RA receptors and the downstream target genes EPAS1 and CDKN1A. Consequently, Knockdown of ISL1 inhibits neuroblastoma cell proliferation and migration in vitro and impedes tumor growth in vivo, and enhances neuronal differentiation by RA treatment. Furthermore, genome-wide mapping revealed a substantial co-occupancy of binding regions by ISL1 and GATA3, and ISL1 physically interacts with GATA3, and together they synergistically regulate the aforementioned oncogenic pathways. In addition, analyses of the roles of ISL1 and MYCN in MYCN-amplified and MYCN non-amplified neuroblastoma cells revealed an epistatic relationship between ISL1 and MYCN. ISL1 and MYCN function in parallel to regulate common yet distinct oncogenic pathways in neuroblastoma. Conclusion: Our study has demonstrated that ISL1 plays an essential role in neuroblastoma regulatory networks and may serve as a potential therapeutic target in neuroblastoma.

Medicago falcata MfSTMIR, an E3 ligase of endoplasmic reticulum-associated degradation, is involved in salt stress response.

Recent studies on E3 of endoplasmic reticulum (ER)-associated degradation (ERAD) in plants have revealed homologs in yeast and animals. However, it remains unknown whether the plant ERAD system contains a plant-specific E3 ligase. Here, we report that MfSTMIR, which encodes an ER-membrane-localized RING E3 ligase that is highly conserved in leguminous plants, plays essential roles in the response of ER and salt stress in Medicago. MfSTMIR expression was induced by salt and tunicamycin (Tm). mtstmir loss-of-function mutants displayed impaired induction of the ER stress-responsive genes BiP1/2 and BiP3 under Tm treatment and sensitivity to salt stress. MfSTMIR promoted the degradation of a known ERAD substrate, CPY*. MfSTMIR interacted with the ERAD-associated ubiquitin-conjugating enzyme MtUBC32 and Sec61-translocon subunit MtSec61γ. MfSTMIR did not affect MtSec61γ protein stability. Our results suggest that the plant-specific E3 ligase MfSTMIR participates in the ERAD pathway by interacting with MtUBC32 and MtSec61γ to relieve ER stress during salt stress.

[γ-Al2O3-graphene oxide absorbent material for detection of nucleosides in urine].

γ-Al2O3 nanoparticle-bonded graphene oxide (γ-Al2O3-GO) was prepared for use as a solid phase extraction absorbent and for preconcentration of four nucleosides in human urine samples for investigation by high-performance liquid chromatography (HPLC). Transmission electron microscopy (TEM), thermal gravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FT-IR) were employed to characterize the absorbent. Several parameters (eluent species, absorbent amount, sample volume, and sample pH) were optimized, and the absorption efficiency was determined based on equilibrium absorbent amounts. Under optimal conditions, the γ-Al2O3-GO absorbent displayed enhanced absorbent ability for nucleosides. The calibration curve showed good linearities in the range of 0.10-10 mg/L for cytidine, inosine, and guanosine, and 0.05-10 mg/L for uridine; the correlation coefficient was observed to be in the range of 0.9967-0.9973. The limits of detection for the four nucleosides were in the range of 0.010-0.021 mg/L. Intra-day and inter-day relative standard deviations were 0.1%-0.8% and 1.0%-3.1%, respectively. The recoveries of the four nucleosides in urine samples ranged from 71.3% to 107.4%, and the relative standard deviations (n=3) were less than 4.8%. The results demonstrated that the proposed method is faster, more sensitive, and more efficient. Therefore, solid phase extraction (SPE)-based γ-Al2O3-GO can be considered more suitable for the determination and enrichment of nucleosides in urine samples.