BCKDK modification enhances the anticancer efficacy of CAR-T cells by reprogramming branched chain amino acid metabolism.

Altered branched chain amino acids (BCAAs), including leucine, isoleucine, and valine, are frequently observed in patients with advanced cancer. We evaluated the efficacy of chimeric antigen receptor (CAR) T cell-mediated cancer cell lysis potential in the immune microenvironment of BCAA supplementation and deletion. BCAA supplementation increased cancer cell killing percentage, while accelerating BCAA catabolism and decreasing BCAA transporter decreased cancer cell lysis efficacy. We thus designed BCKDK engineering CAR T cells for the reprogramming of BCAA metabolism in the tumor microenvironment based on the genotype and phenotype modification. BCKDK overexpression (OE) in CAR-T cells significantly improved cancer cell lysis, while BCKDK knockout (KO) resulted in inferior lysis potential. In an in vivo experiment, BCKDK-OE CAR-T cell treatment significantly prolonged the survival of mice bearing NALM6-GL cancer cells, with the differentiation of central memory cells and an increasing proportion of CAR-T cells in the peripheral circulation. BCKDK-KO CAR-T cell treatment resulted in shorter survival and a decreasing percentage of CAR-T cells in the peripheral circulation. In conclusion, BCKDK-engineered CAR-T cells exert a distinct phenotype for superior anticancer efficiency.

The L-type calcium channel CaV1.3: A potential target for cancer therapy.

Cancer remains a prominent cause to life expectancy, and targeted cancer therapy stands as a pivotal approach in contemporary therapy. Calcium (Ca2+) signalling plays a multifaceted role in cancer progression, such as proliferation, invasion and distant metastasis. Otherwise, it also exerts an important influence on the efficacy of clinical treatment, including cancer therapy resistance. In this review we discuss the role of the L-type calcium channel CaV1.3 (calcium voltage-gated channel subunit alpha1 D) in different types of cancers, highlighting its potential as a therapeutic target for certain cancer types. The development of selective blockers of the CaV1.3 channel has been of great interest and is expected to be a new option for the treatment of cancers such as prostate cancer and endometrial cancer. We present the pharmacological properties of CaV1.3 and the current status of selective blocker development, and analyse the challenges and possible directions for breakthroughs in the development of tailored medicines.

Resolvin and lipoxin metabolism network regulated by Hyssopus Cuspidatus Boriss extract in asthmatic mice.

Resolvin (Rv) and lipoxin (Lx) play important regulative roles in the development of several inflammation-related diseases. The dysregulation of their metabolic network is believed to be closely related to the occurrence and development of asthma. The Hyssopus Cuspidatus Boriss extract (SXCF) has long been used as a treatment for asthma, while the mechanism of anti-inflammatory and anti-asthma action targeting Rv and Lx has not been thoroughly investigated. In this study, we aimed to investigate the effects of SXCF on Rv, Lx in ovalbumin (OVA)-sensitized asthmatic mice. The changes of Rv, Lx before and after drug administration were analyzed based on high sensitivity chromatography-multiple response monitoring (UHPLC-MRM) analysis and multivariate statistics. The pathology exploration included behavioral changes of mice, IgE in serum, cytokines in BALF, and lung tissue sections stained with H&E. It was found that SXCF significantly modulated the metabolic disturbance of Rv, Lx due to asthma. Its modulation effect was significantly better than that of dexamethasone and rosmarinic acid which is the first-line clinical medicine and the main component of Hyssopus Cuspidatus Boriss, respectively. SXCF is demonstrated to be a potential anti-asthmatic drug with significant disease-modifying effects on OVA-induced asthma. The modulation of Rv and Lx is a possible underlying mechanism of the SXCF effects.

A direct electrochemical sensor based on covalent organic frameworks/platinum nanoparticles for the detection of ofloxacin in water.

A direct electrochemical sensor based on covalent organic frameworks (COFs)/platinum nanoparticles (PtNPs) composite was fabricated for the detection of ofloxacin (OFX) in water. Firstly, the COF material was synthesized via the condensation reaction of 1,3,5-tris(4-aminophenyl)benzene (TAPB) with terephthalaldehyde (TPA) and integrated with PtNPs by in situ reduction. Then, TAPB-TPA-COFs/PtNPs composite was loaded onto the surface of the glassy carbon electrode (GCE) by drip coating to construct the working electrode (TAPB-TPA-COFs/PtNPs/GCE). The electrochemical performance of TAPB-TPA-COFs/PtNPs/GCE showed a significant improvement compared with that of TAPB-TPA-COFs/GCE, leading to a 3.2-fold increase in the electrochemical signal for 0.01 mM OFX. Under optimal conditions, the TAPB-TPA-COFs/PtNPs/GCE exhibited a wide linear range of 9.901 × 10-3-1.406 µM and 2.024-15.19 µM with a detection limit of 2.184 × 10-3 µM. The TAPB-TPA-COFs/PtNPs/GCE-based electrochemical sensor with excellent performance provides great potential for the rapid and trace detection of residual OFX.

D2HGDH-mediated D2HG catabolism enhances the anti-tumor activities of CAR-T cells in an immunosuppressive microenvironment.

The effect of immunotherapy is limited by oncometabolite D-2-hydroxyglutarate (D2HG). D2HGDH is an inducible enzyme that converts D2HG into the endogenous metabolite 2-oxoglutarate. We aimed to evaluate the impairment of CD8 T lymphocyte function in the high-D2HG environment and to explore the phenotypic features and anti-tumor effect of D2HGDH-modified CAR-T cells. D2HG treatment inhibited the expansion of human CD8 T lymphocytes and CAR-T cells, increased their glucose uptake, suppressed effector cytokine production, and decreased the central memory cell proportion. D2HGDH-modified CAR-T cells displayed distinct phenotypes, as D2HGDH knock-out (KO) CAR-T cells exhibited a significant decrease in central memory cell differentiation and intracellular cytokine production, while D2HGDH over-expression (OE) CAR-T cells showed predominant killing efficacy against NALM6 cancer cells in high-D2HG medium. In vivo xenograft experiments confirmed that D2HGDH-OE CAR-T cells decreased serum D2HG and improved the overall survival of mice bearing NALM6 cancer cells with mutation IDH1. Our findings demonstrated that the immunosuppressive effect of D2HG and distinct phenotype of D2HGDH modified CAR-T cells. D2HGDH-OE CAR-T cells can take advantage of the catabolism of D2HG to foster T cell expansion, function, and anti-tumor effectiveness.

Molecularly imprinted 3D SERS sensor with inorganic frameworks for specific and recyclable SERS sensing application.

Poor selectivity and reusability of Au/Ag nanostructures are the main challenges for surface-enhanced Raman spectroscopy (SERS) in real sample detection. Herein, a novel specific and reusable three-dimensional (3D) SERS sensor with dual functions of selective trapping and photocatalytic degradation was designed. Firstly, Au-Ag bimetallic nanoparticles decorated silicon nanowires array (SiNWs-AuAg) were prepared as 3D SERS substrate. Then, silicon-based inorganic-framework molecularly imprinted TiO2 (TiO2@SiMIP) was synthesized and immobilized on SiNWs-AuAg by using rhodamine 6G (R6G) as template molecule. Owing to the excellent SERS performance of SiNWs-AuAg and the specific affinity of TiO2@SiMIP to template molecule, the prepared SERS sensor enables sensitive and selective detection of R6G in food samples with a limit of detection (LOD) of 0.27 nM. In addition, due to the photocatalysis of TiO2 and the stability of silicon-based inorganic framework, the residual templates in TiO2@SiMIP can be completely removed by UV irradiation, and the imprinted cavity of regenerated sensors still maintained good selectivity after regeneration by UV irradiation.

Metabolic disorders sensitise endometrial carcinoma through endoplasmic reticulum stress.

BACKGROUND: Metabolic disorder is considered a well-established risk factor for endometrial carcinoma (EC). However, the mechanism remains unclear. Insulin resistance and excessive flux of free fatty acids serve as fundamental pathogenic factors in metabolic disorders, including obesity and type 2 diabetes. The aim of this study was to test the correlation between insulin resistance and dyslipidaemia in EC and to determine the effect of insulin and saturated fatty acids on EC cells. METHODS: A retrospective study on the medical records of patients with EC and RNA-seq from the TCGA database analysed with edgR and Gene Ontology (GO) were used to assess the correlation of dyslipidaemia and diabetes as well as obesity. Crystal violet assays and CCK-8 assays were used to detect the proliferation of EC cells, and Annexin V-PI was used to examine apoptosis. Transient changes in mitochondrial Ca2+ and reactive oxygen species (ROS) were monitored via confocal microscopy. DNA damage was assessed by comet assays. Changes in signalling pathways were detected via phospho-kinase array. western blotting was used to assess the molecular changes in endoplasmic reticulum (ER) stress and DNA damage. RESULTS: We found that glucose metabolism disorders accompanied dyslipidaemia in patients with EC. As a key regulator of glucose metabolism disorders, insulin promoted DNA damage, ROS and Ca2+ homoeostasis imbalance in a panel of established EC cell lines. Interestingly, excessive insulin boosted saturated fatty acid-induced pro-apoptotic effects in EC cells. Furthermore, our data showed that insulin synergised with saturated fatty acids to activate the mechanistic target of rapamycin kinase/70 kDa ribosomal protein S6 kinase (mTOR/p70S6K) pathway and ER stress, resulting in Ca2+ release from ER and unfolded protein response (UPR) activation, which contributed to combined insulin and saturated fatty acid treatment-induced apoptosis and tumour progression. CONCLUSIONS: Our data are the first to illustrate that impaired glucose metabolism accelerates dyslipidaemia-promoted EC progression, which is attributed to hyperinsulinaemia and saturated fatty acid-induced Ca2+ dyshomoeostasis and UPR activation in EC cells via ER stress.

Clinicopathological characteristics of placental chorioangioma: A clinicopathological study of 77 cases.

OBJECTIVE: To explore clinicopathological characteristics, diagnosis, differential diagnoses, treatment, and prognoses of placental chorioangioma (PCA). METHODS: Placenta of 77 cases of PCA firmly diagnosed by pathology from 2009 through 2019 were collected, and clinicopathological characteristics of the patients were retrospectively analyzed. RESULTS: Seventy-seven patients were 20-41 (mean age, 28.8) years old at onset. Thirty patients showed pregnancy comorbidity. In one patient with the largest tumor (diameter, 16 cm), intrauterine fetal demise occurred at 33 weeks of gestation. Tumors were macroscopically manifested. The placental fetal surface showed a raised dark red to a pale pink nodule, quasi-round, with a maximum diameter of 0.2-16 cm. Microscopically, the tumors had a lot of capillaries and some interstitial loose connective tissue. One case was of atypical cellular chorioangioma. Immunophenotypically, CD34 (+) and Ki-67 proliferation indexes were less than 10%. CONCLUSIONS: Large PCA often accompanies pregnancy comorbidity. Atypical cellular chorangioma is rare and may be misdiagnosed as a malignant tumor. Therefore, improvement of understanding of such tumors can provide a basis for clinical diagnosis and intervention.

Simple and sensitive nitric oxide biosensor based on the electrocatalysis of horseradish peroxidase on AuNPs@metal-organic framework composite-modified electrode.

Fe-based metal-organic framework (MIL-101(Fe)) was synthesized through a simple solvothermal synthesis and then used to prepare the AuNPs-decorated MIL-101(Fe) nanocomposite (APPPM(Fe)) by a multi-step layer-by-layer assembly process. Benefited from the porous structure of MIL-101(Fe) and the multilayer assemble process, the loading amount of AuNPs on APPPM(Fe) was enhanced and exhibited a fine biocompatible interface and high conductivity. Through the intense Au-S bond, high loading amount of horseradish peroxidase was immobilized on APPPM(Fe) and the native bioactivity of HRP was kept to realize its direct electrochemistry. From the electrochemical kinetics, the constructed biosensor displayed fast electron transfer and good electrocatalysis activity for the detection of nitric oxide (NO) with wide linear range from 0.033 to 5370 µM and a low detection limit of 0.01 µM (3 σ) as well as fine stability, reproducibility and specificity. According to results of real sample analysis, the proposed electrochemical biosensor offers fast and simple detection of NO in real serum. Therefore, the present strategy definitely provided a potential application prospect in NO clinic detection and disease therapy.

Hierarchical CeO2@N-C Ultrathin Nanosheets for Efficient Selective Oxidation of Benzylic Alcohols in Water.

A monodisperse CeO2@N-C ultrathin nanosheet self-assembled hierarchical structure (USHR) has been prepared by metal-organic framework template methods. The uniform coating of nitrogen-doped carbon (N-C) layers could play an important role in the adsorption and activation of benzylic alcohol. The unique 3D hierarchical structure self-assembled by ultrathin nanosheets provided enough active sites for the catalytic reaction. Therefore, the CeO2@N-C USHR can afford excellent catalytic performance for selective oxidation of benzylic alcohols in water.

Combining N,S-Codoped C and CeO2: A Unique Hinge-like Structure for Efficient Photocatalytic Hydrogen Evolution.

N- and S-doped C-encapsulated CeO2 with a hinge-like nanostructure (CeO2@N,S-C HN) has been obtained by using a Ce-based metal-organic framework (Ce-MOF-808) as a precursor. Due to the synergistic effects of its special structure and N,S-doped C layer, CeO2@N,S-C HN exhibits high activity of photocatalytic hydrogen (H2) evolution. Density functional calculations are used to obtain a fundamental understanding of the high H2 production rate of CeO2@N,S-C HN.

Ratiometric fluorescent detection of Cu2+ based on dual-emission ZIF-8@rhodamine-B nanocomposites.

Zeolitic imidazolate framework-8 (ZIF-8) loading rhodamine-B (ZIF-8@rhodamine-B) nanocomposites was proposed and used as ratiometric fluorescent sensor to detect copper(II) ion (Cu2+ ). Scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray powder diffraction, nitrogen adsorption/desorption isotherms and fluorescence emission spectroscopy were employed to characterize the ZIF-8@rhodamine-B nanocomposites. The results showed the rhodamine-B was successfully assembled on ZIF-8 based on the π-π interaction and the hydrogen bond between the nitrogen atom of ZIF-8 and -COOH of rhodamine-B. The as-obtained ZIF-8@rhodamine-B nanocomposites were octahedron with size about 150-200 nm, had good water dispersion, and exhibited the characteristic fluorescence emission of ZIF-8 at 335 nm and rhodamine-B at 575 nm. The Cu2+ could quench fluorescence of ZIF-8 rather than rhodamine-B. The ZIF-8 not only acted as the template to assemble rhodamine-B, but also was employed as the signal fluorescence together with the fluorescence of rhodamine-B as the reference to construct a novel ratiometric fluorescent sensor to detect Cu2+ . The resulted ZIF-8@rhodamine-B nanocomposite fluorescence probe showed good linear range (68.4 nM to 125 µM) with a low detection limit (22.8 nM) for Cu2+ combined with good sensitivity and selectivity. The work also provides a better way to design ratiometric fluorescent sensors from ZIF-8 and other fluorescent molecules.

Ultrasonic Non-Destructive Testing System of Semi-Enclosed Workpiece with Dual-Robot Testing System.

With the rapid development of material science, more and more workpieces, especially workpieces with complex curved surfaces, are being made of composite materials. Robotic non-destructive testing (NDT) systems for complex curved surface composite material parts are being used more and more. Despite the emergence of such flexible NDT systems, the detection of semi-enclosed parts is also a challenge for robotic NDT systems. In order to overcome the problem, this paper establishes an NDT solution for semi-enclosed workpieces based on a dual-robot system of synchronous motion, in which an extension arm is installed on one of the robots and presents a trajectory planning method that always ensures the extension arm is parallel to the rotary axis of a semi-enclosed workpiece and that the ultrasonic probes are perpendicular to the workpiece surface. Trajectory analysis experiments and ultrasonic NDT experiments utilizing the optimal water path distance determined by simulation result of multi-Gaussian beam model for two types of semi-enclosed workpieces are performed with the dual-robot NDT system. Experimental results prove that the dual-robot NDT scheme functions well and the planned trajectories are correct. All the hole-shaped artificial defects with diameters ≥3 mm are detected by using 2.25 MHz ultrasonic probes through the transmission testing method. Vivid 3D C-scan image of a small diameter cylindrical workpiece based on the testing result is provided for convenience of observation.

Comparative metabolomics reveals the metabolic variations between two endangered Taxus species (T. fuana and T. yunnanensis) in the Himalayas.

BACKGROUND: Plants of the genus Taxus have attracted much attention owing to the natural product taxol, a successful anti-cancer drug. T. fuana and T. yunnanensis are two endangered Taxus species mainly distributed in the Himalayas. In our study, an untargeted metabolomics approach integrated with a targeted UPLC-MS/MS method was applied to examine the metabolic variations between these two Taxus species growing in different environments. RESULTS: The level of taxol in T. yunnanensis is much higher than that in T. fuana, indicating a higher economic value of T. yunnanensis for taxol production. A series of specific metabolites, including precursors, intermediates, competitors of taxol, were identified. All the identified intermediates are predominantly accumulated in T. yunnanensis than T. fuana, giving a reasonable explanation for the higher accumulation of taxol in T. yunnanensis. Taxusin and its analogues are highly accumulated in T. fuana, which may consume limited intermediates and block the metabolic flow towards taxol. The contents of total flavonoids and a majority of tested individual flavonoids are significantly accumulated in T. fuana than T. yunnanensis, indicating a stronger environmental adaptiveness of T. fuana. CONCLUSIONS: Systemic metabolic profiling may provide valuable information for the comprehensive industrial utilization of the germplasm resources of these two endangered Taxus species growing in different environments.

Unexpected link between insect innexins and apoptosis of HeLa cells.

Little is known about how mammalian cells respond to the expression of innexins (Inxs), which are known to mediate cell-to-cell communication that causes apoptosis in the cells of the insect Spodoptera litura. The mammalian expression system, p3xFLAG tag protein, containing the CMV promoter, allowed us to construct two C-terminally elongated innexins (Cte-Inxs), SpliInx2 (Inx2-FLAG), and SpliInx3 (Inx3-FLAG), which were predicted to have the same secondary topological structures as the native SpliInx2 and SpliInx3. Here, we found that only the mRNAs of the two Cte-Inxs were expressed under the control of the CMV promoter in HeLa cells. Unexpectedly, mRNA expression of the two Cte-Inxs enhanced apoptosis of HeLa cells. The two Cte-Inx mRNAs were associated with a significant decrease in Akt phosphorylation in HeLa cells undergoing apoptosis. Furthermore, Inx3-FLAG mRNA expression in nonapoptotic HCT116 cells was also associated with a significant decrease in the levels of phosphorylated Akt. Intriguingly, expression of the mRNAs of the two Cte-Inxs did not activate caspase 3, but it markedly reduced Bid levels in HeLa cells undergoing apoptosis. These results suggest that mRNA expression of the two Cte-Inxs may activate a Bid-dependent apoptotic pathway in HeLa cells. Our study demonstrates that invertebrate gap junction mRNAs can function in vertebrate cancer cells as tumor suppressors.

Association of Genes implicated in primary angle-closure Glaucoma and the ocular biometric parameters of anterior chamber depth and axial length in a northern Chinese population.

BACKGROUND: The membrane frizzled-related protein (MFRP) gene is involved in axial length (AL) regulation and MFRP mutations cause nanophthalmos; also, the hepatocyte growth factor (HGF) gene is reported to result in morphologic changes of the anterior segment and abnormal aqueous regulation that increases the risk of primary angle-closure glaucoma (PACG), while the zinc ring finger 3 (ZNRF3) gene is associated with AL. The present study investigated the association of single nucleotide polymorphisms (SNPs) in ZNRF3, HGF and MFRP with PACG in a northern Chinese population, as well as the association of these SNPs with the ocular biometric parameters of anterior chamber depth (ACD) and AL. METHODS: A total of 500 PACG patients and 720 controls were recruited. All individuals were genotyped for 12 SNPs in three genes (rs7290117, rs2179129, rs4823006 and rs3178915 in ZNRF3; rs5745718, rs12536657, rs12540393, rs17427817 and rs3735520 in HGF, rs2510143, rs36015759 and rs3814762 in MFRP) using an improved multiplex ligation detection reaction (iMLDR) technique. Genotypic distribution was analyzed for Hardy-Weinberg equilibrium. Differences in the allelic and genotypic frequencies were evaluated and adjusted by age and sex. Linkage disequilibrium (LD) patterns were tested and haplotype analysis was conducted by a logistic regression model. Generalized estimation equation (GEE) analysis was conducted using SPSS for primary association testing between genotypes and ocular biometric parameters. Bonferroni corrections for multiple comparisons were performed, and the statistical power was calculated by power and sample size calculations. RESULTS: The rs7290117 SNP in ZNRF3 was significantly associated with the AL, with a p-value of 0.002. We did not observe any significant associations between the SNPs and PACG or ACD. In a stratification analysis by ethnicity, rs12540393 and rs17427817 in HGF showed a nominal association with PACG in the Hui cohort, although significance was lost after correction. CONCLUSIONS: The present study suggests rs7290117 in ZNRF3 may be involved in the regulation of AL, though our results do not support a contribution of the SNPs we tested in ZNRF3, HGF and MFRP to PACG in northern Chinese people. Further studies in a larger population are warranted to confirm this conclusion.

Development of SCoT-Based SCAR Marker for Rapid Authentication of Taxus Media.

Taxus media is an important species in the family Taxaceae with high medicinal and commercial value. Overexploitation and illegal trade have led T. media to a severe threat of extinction. In addition, T. media and other Taxus species have similar morphological traits and are easily misidentified, particularly during the seedling stage. The purpose of this study is to develop a species-specific marker for T. media. Through a screening of 36 start codon targeted (SCoT) polymorphism primers, among 15 individuals of 4 Taxus species (T. media, T. chinensis, T. cuspidate and T. fuana), a clear species-specific DNA fragment (amplified by primer SCoT3) for T. media was identified. After isolation and sequencing, a DNA sequence with 530 bp was obtained. Based on this DNA fragment, a primer pair for the sequence-characterized amplified region marker was designed and named MHSF/MHSR. PCR analysis with primer pair MHSF/MHSR revealed a clear amplified band for all individuals of T. media but not for T. chinensis, T. cuspidate and T. fuana. Therefore, this marker can be used as a quick, efficient and reliable tool to identify T. media among other related Taxus species. The results of this study will lay an important foundation for the protection and management of T. media as a natural resource.

Ratiometric fluorescence detection of superoxide anion based on AuNPs-BSA@Tb/GMP nanoscale coordination polymers.

A novel ratiometric fluorescence nanosensor for superoxide anion (O2•- ) detection was designed with gold nanoparticles-bovine serum albumin (AuNPs-BSA)@terbium/guanosine monophosphate disodium (Tb/GMP) nanoscale coordination polymers (NCPs) (AuNPs-BSA@Tb/GMP NCPs). The abundant hydroxyl and amino groups of AuNPs-BSA acted as binding points for the self-assembly of Tb3+ and GMP to form core-shell AuNPs-BSA@Tb/GMP NCP nanosensors. The obtained probe exhibited the characteristic fluorescence emission of both AuNPs-BSA and Tb/GMP NCPs. The AuNPs-BSA not only acted as a template to accelerate the growth of Tb/GMP NCPs, but also could be used as the reference fluorescence for the detection of O2•- . The resulting AuNPs-BSA@Tb/GMP NCP ratiometric fluorescence nanosensor for the detection of O2•- demonstrated high sensitivity and selectivity with a wide linear response range (14 nM-10 µM) and a low detection limit (4.7 nM).

A mathematical model to characterize the degree of coalification based on the low-angle region of the X-ray diffractogram.

Based on X-ray diffraction (XRD) pattern of coal, an empirical model for judging the coalification degree is used to calculate the ratio of the 002 peak height to the Full width at half maximum (FWHM). However, the existing models are often simpler and more suitable for judgment of the medium and low rank coal, while are not feasible in determination of high rank coal. In order to address this issue, the objective of this study is to establish a new modified mathematical model based on optimization of the existing empirical models. Through the calculation of Bragg equation, it demonstrates that the low-angle region (2θ= 3-10°) in the XRD pattern reflects the information of micropore in coal with a diameter of (0.884-2.94) nm. Accordingly, its diffraction intensity corresponds to the porosity rate in coal. As a result, the modified mathematical model has been established for characterizing the coalification degree by introducing the variation of porosity rate with the coal ranks creatively. The synergistic effects of the change regulation of organic matter peak and the porosity rate with coal rank ensure the accuracy of the model. Furthermore, the good stability and high reliability of new model are verified through the recalculation of a total of 14 coal samples. Study results demonstrated that the new method enabled to determine coal rank more conveniently and accurately in the industrial production.

Succinyl-proteome profiling of Dendrobium officinale, an important traditional Chinese orchid herb, revealed involvement of succinylation in the glycolysis pathway.

BACKGROUND: Lysine succinylation is a ubiquitous and important protein post-translational modification in various eukaryotic and prokaryotic cells. However, its functions in Dendrobium officinale, an important traditional Chinese orchid herb with high polysaccharide contents, are largely unknown. RESULTS: In our study, LC-MS/MS was used to identify the peptides that were enriched by immune-purification with a high-efficiency succinyl-lysine antibody. In total, 314 lysine succinylation sites in 207 proteins were identified. A gene ontology analysis showed that these proteins are associated with a wide range of cellular functions, from metabolic processes to stimuli responses. Moreover, two types of conserved succinylation motifs, '***Ksuc******K**' and '****EKsuc***', were identified. Our data showed that lysine succinylation occurred on five key enzymes in the glycolysis pathway. The numbers of average succinylation sites on these five enzymes in plants were lower than those in bacteria and mammals. Interestingly, two active site amino acids residues, K103 and K225, could be succinylated in fructose-bisphosphate aldolase, indicating a potential function of lysine succinylation in the regulation of glycolytic enzyme activities. Furthermore, the protein-protein interaction network for the succinylated proteins showed that several functional terms, such as glycolysis, TCA cycle, oxidative phosphorylation and ribosome, are consisted. CONCLUSIONS: Our results provide the first comprehensive view of the succinylome of D. officinale and may accelerate future biological investigations of succinylation in the synthesis of polysaccharides, which are major active ingredients.