Mettl14-mediated m6 A modification regulates the abnormal differentiation of small intestinal epithelial stem cells in diabetic state.

Diabetes mellitus (DM) and its related complications are a global epidemic characterized by high morbidity and mortality. However, little is known about diabetic enteropathy (DE) and its the potential underlying mechanism. Intestinal epithelial stem cells (IESCs) were harvested from experimental mice, and the levels of dominant N6-methyladenosine (m6 A)-related enzyme were detected by RT-PCR, Western blotting, immunohistochemistry. The role of Mettl14 in the abnormal differentiation of intestinal epithelial cells (IECs) during DM was confirmed by knockdown experiments. RT-PCR, MeRIP, and bioinformatics analysis were carried out to confirm the downstream target of Mettl14. Through bioinformatics analysis, RT-PCR, and Western blotting, we further analyzed the differentiation-related gene in the IECs from mice with DM. In this study, the levels of Mettl14 and m6 A were higher in db/db mice than that in control mice. And abnormal differentiation of IECs in DM was associated with Mettl14 overexpression. Additionally, Mettl14 is a major determinant of IESCs identity and organoid-forming upon DM state. Mechanistically, we revealed that the candidate binding target of Mettl14 was Fzd2 mRNA and affected Fzd2 stability. Moreover, Mettl14 downregulation was observed to attenuate the abnormal differentiation of IECs through modulating Fzd2 m6A modification in DM state. Together, our results provide definitive evidence for the essential role of Mettl14 in differentiation of IESCs in DM state.

Anterior column realignment via a minimally invasive hybrid approach in adult spinal deformity surgery: a short-term retrospective study.

BACKGROUND: Anterior column realignment (ACR) is a novel surgical method for correcting spinal sagittal balance. meanwhile, oblique lumbar interbody fusion (OLIF) and anterior lumbar interbody fusion (ALIF) are considered minimally invasive surgical methods through natural anatomical space. This study aimed to explore the corrective effects and clinical outcomes of OLIF or ALIF combined with ACR technology in patients with adult spinal deformity (ASD). METHODS: We retrospectively analyzed patients with sagittal imbalance who received OLIF and/or ALIF and ACR treatment from 2018 to 2021. Surgical time and intraoperative bleeding volume are recorded, the corrective effect is determined by the intervertebral space angle (IVA), lumbar lordosis (LL), the sagittal vertical axis (SVA), clinical outcome is evaluated by preoperative and final follow-up visual analog pain score (VAS), Japanese orthopedic association scores (JOA) and complications. RESULTS: Sixty-four patients were enrolled in the study, average age of 65.1(range, 47-82) years. All patients completed 173 fusion segments, for 150 segments of ACR surgery. The operation time of ALIF-ACR was 50.4 ± 22.1 min; The intraoperative bleeding volume was 50.2 ± 23.6 ml. The operation time and intraoperative bleeding volume of single-segment OLIF-ACR was 66.2 ± 19.4 min and 70.2 ± 31.6 ml. At the follow-up of 6 months after surgery, the intervertebral space angle correction for OLIF-ACR and ALIF-ACR is 9.2° and 12.2°, the preoperative and postoperative lumbar lordosis were 16.7° ± 6.4°and 47.1° ± 3.6° (p < 0.001), VAS and JOA scores were improved from 6.8 to 1.8 and 7.8 to 22.1 respectively, statistically significant differences were observed in these parameters. The incidence of surgical related complications is 29.69%, but without serious complications. CONCLUSION: ACR via a minimally invasive hybrid approach for ASD has significant advantages in restoring local intervertebral space angulation and correcting the overall sagittal balance. Simultaneously, it can achieve good clinical outcomes and fewer surgical complications.

Frequency and predictors of implant-related complications after posterior cervical fusion.

STUDY DESIGN: Retrospective, observational study. PURPOSE: To determine the frequency and predictors of implant-related complications in adults after posterior cervical fusion. OVERVIEW OF LITERATURE: Published literature on lumbosacral fusion suggest that implant-related complications are not uncommon. Although posterior cervical fusion is a common operation, data on frequency and predictors of implant-related complications after posterior cervical fusion is still scarce. METHODS: 86 patients (with 740 screws) who underwent posterior cervical fusion were included. Implant-related complications were identified by the presence of: (1) halo sign, (2) screw pull-out/breakage (3) post-operative kyphosis and (4) implant-related complications requiring revision surgery. These were stratified into two groups: (a) minor - isolated halo sign or screw pull-out/breakage (b) major - post-operative kyphosis > 10 degrees, and revision surgery. Demographic, operative and radiological data was collected. Rates of implant-related complications were determined and associated risk factors identified. RESULTS: 33 (38.4%) patients had signs of implant-related complications. Of these, 29 (87.9%) had minor complications and 4 (12.1%) had major complications. Charlson Comorbidity Index (CCI) (p = 0.03179) and pre-op C2-C7 sagittal vertical alignment (SVA) (p = 0.02449) were the only significant risk factors for all-cause implant-related complications during multivariate logistic regression. Other intraoperative parameters (type of screw, length of fusion, levels decompressed, and extension of fusion beyond the levels decompressed) were not significantly associated with implant-related complications. CONCLUSIONS: Implant-related complications are not uncommon but rarely require revision surgery. Higher pre-operative SVA and CCI were significant risk factors; length of construct and extent of decompression were not. These findings may assist clinicians when deciding the extent of fusion and in selecting patients for closer follow-up.

We assessed the frequency and predictors of implant-related complications in adults after posterior cervical fusion. Implant-related complications (halo sign, screw pull-out/breakage, post-operative kyphosis) are not uncommon but rarely require revision surgery. Higher pre-operative SVA and CCI were significant risk factors; length of construct and extent of decompression were not.

[Core connotation of compatibility of Aconiti Lateralis Radix Praeparata-Glycyrrhizae Radix et Rhizoma herb pair under physiological/ pathological conditions based on metabolomics and network pharmacology].

This study aims to explore the core connotation of the compatibility of Aconiti Lateralis Radix Praeparata(Fuzi)-Glycyrrhizae Radix et Rhizoma(Gancao) herb pair under physiological and pathological conditions. The biochemical indicators of serum/myocardial tissue, pathological changes of the myocardial tissue, and serum metabolic profiles of normal rats and heart failure model rats treated with Fuzi Decoction and Fuzi Gancao Decoction were determined. Network pharmacology and metabolomics were employed to establish the metabolite-target-pathway network for Glycyrrhizae Radix et Rhizoma in enhancing the efficacy and reducing the toxicity of Aconiti Lateralis Radix Praeparata, Western blotting was employed to verify the representative pathways in the network. The results showed that both decoctions lowered the levels of creatine kinase and other indicators and mitigate myocardial pathological injury in model rats. However, they caused the abnormal rises in creatine kinase and other indicators and myocardial pathological injury in normal rats. The results indicated that the compatibility reduced the toxicity in normal rats and enhanced the efficacy in model rats. The results of metabolomics showed that Fuzi Gancao Decoction recovered more metabolites in model rats and had weaker effect on interfe-ring with the metabolites in normal rats than Fuzi Decoction. The association analysis showed that the network of Glycyrrhizae Radix et Rhizoma enhancing the efficacy of Aconiti Lateralis Radix Praeparata involved 112 metabolites, 89 targets, and 15 pathways, including calcium and cAMP signaling pathways. The network of Glycyrrhizae Radix et Rhizoma reducing the cardiotoxicity of Aconiti Lateralis Radix Praeparata involved 36 metabolites, 59 targets, and 11 pathways, including adrenergic signaling and tricarboxylic acid cycle in cardiomyocytes. The experimental results of protein expression verified the reliability of the association analysis. This study demonstrated that the core connotation of the herb pair of Aconiti Lateralis Radix Praeparata-Glycyrrhizae Radix et Rhizoma changed under physio-logical and pathological states, and the compatibility results of enhancing efficacy and reducing toxicity were achieved with different metabolic pathways and biological processes.

Metabolomics reveals a correlation between hydroxyeicosatetraenoic acids and allergic asthma: Evidence from three years' immunotherapy.

BACKGROUND: Subcutaneous immunotherapy (SCIT) is an effective, safe, preventative treatment for allergic asthma; however, potential biomarkers for monitoring SCIT have rarely been reported. OBJECTIVE: Metabolomics was utilized for the discovery of new biomarkers and analyzing disease pathophysiology of allergic asthma, and it was also applied to determine the metabolomic profiles of serum samples from children with asthma undergoing SCIT and identify potential biomarkers for allergic asthma and its therapeutic monitoring. METHODS: Untargeted metabolomics using ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry was performed on 15 asthmatic and 15 healthy pediatric sera to profile carboxylic acids. Statistical analysis combined with pathway enrichment analysis was applied to identify potential biomarkers. Then, targeted metabolomics was performed to study longitudinal changes of eicosanoid profiles on sera from 20 participants with asthma who received SCIT at baseline, 6 months, one, two, and three years (ChiCTR-DDT-13003728). RESULTS: Metabolomic analysis revealed that levels of eicosanoids, particularly 12(S)-hydroxyeicosatetraenoic acid (HETE; AUC = 0.94, p < .0001) and 15(S)-HETE (AUC = 0.89, p = .0028), metabolized from arachidonic acid by lipoxygenase and glutathione peroxidase enzymes, were significantly higher in asthma group than in healthy individuals. Furthermore, levels of these important metabolites increased in the first year of SCIT treatment and then decreased from years one to three, being significantly lower after three years of treatment than baseline levels. CONCLUSION: 12(S)- and 15(S)-HETEs are potential biomarkers to participate in the pathogenesis and treatment of allergic asthma. Moreover, these metabolites may be a new target for biological indicators to monitor the therapeutic effect of SCIT, particularly in the setting of allergic asthma.

Genomic analysis of a lignocellulose degrading strain from the underexplored genus Meridianimaribacter.

The genus Meridianimaribacter is one of the least-studied genera within Cytophaga-Flavobacteria. To date, no genomic analysis of Meridianimaribacter has been reported. In this study, Meridianimaribacter sp. strain CL38, a lignocellulose degrading halophile was isolated from mangrove soil. The genome of strain CL38 was sequenced and analyzed. The assembled genome contains 17 contigs with 3.33 Mbp, a GC content of 33.13% and a total of 2982 genes predicted. Lignocellulose degrading enzymes such as cellulases (GH3, 5, 9, 16, 74 and 144), xylanases (GH43 and CE4) and mannanases (GH5, 26, 27 and 130) are encoded in the genome. Furthermore, strain CL38 demonstrated its ability to decompose empty fruit bunch, a lignocellulosic waste residue arising from palm oil industry. The genome information coupled with experimental studies confirmed the ability of strain CL38 to degrade lignocellulosic biomass. Therefore, Meridianimaribacter sp. strain CL38, with its halotolerance, could be useful for seawater based lignocellulosic biorefining.

Draft genome sequence of Parvularcula flava strain NH6-79 T, revealing its role as a cellulolytic enzymes producer.

To date, the genus Parvularcula consists of 6 species and no potential application of this genus was reported. Current study presents the genome sequence of Parvularcula flava strain NH6-79 T and its cellulolytic enzyme analysis. The assembled draft genome of strain NH6-79 T consists of 9 contigs and 7 scaffolds with 3.68 Mbp in size and GC content of 59.87%. From a total of 3,465 genes predicted, 96 of them are annotated as glycoside hydrolases (GHs). Within these GHs, 20 encoded genes are related to cellulosic biomass degradation, including 12 endoglucanases (5 GH10, 4 GH5, and 3 GH51), 2 exoglucanases (GH9) and 6 ß-glucosidases (GH3). In addition, highest relative enzyme activities (endoglucanase, exoglucanase, and ß-glucosidase) were observed at 27th hour when the strain was cultured in the carboxymethyl cellulose/Avicel®-containing medium for 45 h. The combination of genome analysis with experimental studies indicated the ability of strain NH6-79 T to produce extracellular endoglucanase, exoglucanase, and ß-glucosidase. These findings suggest the potential of Parvularcula flava strain NH6-79 T in cellulose-containing biomass degradation and that the strain could be used in cellulosic biorefining process.

Robertkochia solimangrovi sp. nov., isolated from mangrove soil, and emended description of the genus Robertkochia.

To date, there is sparse information for the genus Robertkochia with Robertkochia marina CC-AMO-30DT as the only described member. We report here a new species isolated from mangrove soil collected at Malaysia Tanjung Piai National Park and perform polyphasic characterization to determine its taxonomic position. Strain CL23T is a Gram-negative, yellow-pigmented, strictly aerobic, catalase-positive and oxidase-positive bacterium. The optimal growth conditions were determined to be at pH 7.0, 30-37 °C and in 1-2 % (w/v) NaCl. The major respiratory quinone was menaquinone-6 (MK-6) and the highly abundant polar lipids were four unidentified lipids, a phosphatidylethanolamine and two unidentified aminolipids. The 16S rRNA gene similarity between strain CL23T and R. marina CC-AMO-30DT is 96.67 %. Strain CL23T and R. marina CC-AMO-30DT clustered together and were distinguished from taxa of closely related genera in 16S rRNA gene phylogenetic analysis. Genome sequencing revealed that strain CL23T has a genome size of 4.4 Mbp and a G+C content of 40.72 mol%. Overall genome related indexes including digital DNA-DNA hybridization value and average nucleotide identity are 17.70 % and approximately 70%, below the cutoffs of 70 and 95%, respectively, indicated that strain CL23T is a distinct species from R. marina CC-AMO-30DT. Collectively, based on the phenotypic, chemotaxonomic, phylogenetic and genomic evidences presented here, strain CL23T is proposed to represent a new species with the name Robertkochia solimangrovi sp. nov. (KCTC 72252T=LMG 31418T). An emended description of the genus Robertkochia is also proposed.

miR-155 facilitates calcium oxalate crystal-induced HK-2 cell injury via targeting PI3K associated autophagy.

Nephrolithiasis is one of the most common and highly recurrent diseases worldwide. Accumulating evidence revealed the elevated miR-155 levels both in serum and urine of nephrolithiasis patients. The aim of our research was to explore the role of miR-155 in CaOx-induced apoptosis in HK-2 cells. The expression levels of miR-155 in serum and renal tissues were quantified in 20 patients with nephrolithiasis using qRT-PCR assay. ELISA was performed to determine urinary levels of interleukin (IL)-1ß, IL-6 and tumor necrosis factor-alpha (TNF-α). Renal tubular cell model of CaOx nephrolithiasis was established to investigate the role and molelular mechanism of miR-155. Cell viability and apoptosis were assessed by MTT and flow cytometry, respectively. Immunofluoresent staining of LC3 autophagosome and western blotting were performed to evaluate the autophagic activity. Luciferase reporter assay was employed to verify the interaction between miR-155 and PI3KCA/Rheb. PI3K/Akt/mTOR signaling was further examined by western blotting. Serum and renal levels of miR-155 and inflammatory factors were significantly elevated in nephrolithiasis patients than in controls. CaOx treatment caused up-regulation of miR-155 and induced autophagy in renal tubular epithelial cells, while silencing miR-155 or inhibition of autophagy by 3-metheladenine (3-MA) ameliorated CaOx crystal-induced cell injury. PI3KCA and Rheb was identified as downstream targets of miR-155. Moreover, miR-155 activates autophagy and promotes cell injury through repressing PI3K/Akt/mTOR signaling pathway. Taken together, these findings demonstrated that miR-155 facilitates CaOx crystal-induced renal tubular epithelial cell injury via PI3K/Akt/mTOR-mediated autophagy, providing therapeutic targets for ameliorating cellular damage by CaOx crystals.

Determination of pharmacokinetic parameters of vitamin K1 in rats after an intravenous infusion.

Pharmacokinetic parameters of vitamin K1 have a large range of values in different literature. The aim of this study was to determine the pharmacokinetic parameters of vitamin K1 following post-constant speed intravenous infusion (PCSII) to provide rational pharmacokinetic parameters of vitamin K1 and compare these with results of noncompartmental analysis following intravenous injection (IV). After 15 hours intravenous infusion of vitamin K1 in rats, the logarithmic concentration-time curve of vitamin K1 was fit to a linear equation following PCSII (R2  = 0.9599 ± 0.0096). Then, half-time (T1/2 ), apparent volume of distribution (Vd ), and clearance rate (CL) were estimated successively. T1/2 of vitamin K1 was 4.07 ± 0.41 hour, CL was 89.47 ± 3.60 mL/h, and Vd was 525.38 ± 54.45 mL in rats following PCSII. There was no significant difference in pharmacokinetic parameters of vitamin K1 among different sampling times. For noncompartmental analysis, T1/2 and mean residence time (MRTINF ) for a sampling duration of 6h were shorter than those of 12 hours or 24 hours sampling duration following IV (P < .05, P < .01). In addition, T1/2 of vitamin K1 was obviously different from MRT-equated half-time (T1/2,MRT )(P < .05). Vd and CL of vitamin K1 following PCSII were larger than those following IV based on noncompartmental analysis (P < .01). The results demonstrated that drug distribution in the body was balanced and the Napierian logarithmic concentration-time curve of vitamin K1 fit to a linear equation following PCSII. Vitamin K1 has a long T1/2 and a relatively large Vd following PCSII.

Integrated Proteomics, Biological Functional Assessments, and Metabolomics Reveal Toosendanin-Induced Hepatic Energy Metabolic Disorders.

Toosendanin (TSN), a compound from Melia toosendan, exhibits severe hepatotoxicity, which restricts its clinical application. However, the mechanism is not clear. Our previous research found that covalent modification of TSN for proteins might be a possible reason using human liver microsomes, and the glycolytic enzymes, triosephosphate isomerase 1 (TPIS) and α-enolase (ENOA), were responsible for the hepatotoxicity. In this study, we tried to prove these findings in cell and animal models by integration of proteomics, metabolomics, and biological methods. Proteomics analysis in rats showed that TPIS and ENOA were covalently modified by TSN reactive metabolites. The biological functional assessments revealed that the modifications inhibited the activity of TPIS and induced the activity of ENOA, in vitro and in vivo, followed by an increase in the level of cellular methylglyoxal, advanced glycation end products, and reactive oxygen species/superoxide, and the induction of mitochondrial dysfunction, which further inhibited oxidative phosphorylation and stimulated glycolysis. Furthermore, metabolomics demonstrated the decrease in the level of metabolites in the tricarboxylic acid cycle, fatty acid ß-oxidation, and amino acid metabolism; i.e., TSN induced hepatocyte energy metabolism disorder. In conclusion, these data suggest novel mechanistic insights into TSN-induced liver injury on the upstream level and provide valuable proteins and energy metabolic targets for diagnosis and therapy in the clinic.

Hispidulin induces ER stress-mediated apoptosis in human hepatocellular carcinoma cells in vitro and in vivo by activating AMPK signaling pathway.

Hispidulin (4',5,7-trihydroxy-6-methoxyflavone) is a phenolic flavonoid isolated from the medicinal plant S. involucrata, which exhibits anti-neoplastic activity against several types of cancer. However, the mechanism underlying its anti-cancer activity against hepatocellular carcinoma (HCC) has not been fully elucidated. In this study, we investigated whether and how hispidulin-induced apoptosis of human HCC cells in vitro and in vivo. We showed that hispidulin (10, 20 µmol/L) dose-dependently inhibited cell growth and promoted apoptosis through mitochondrial apoptosis pathway in human HCC SMMC7721 cells and Huh7 cells. More importantly, we revealed that its pro-apoptotic effects depended on endoplasmic reticulum stress (ERS) and unfolded protein response (UPR), as pretreatment with salubrinal, a selective ERS inhibitor, or shRNA targeting a UPR protein CHOP effectively abrogated hispidulin-induced cell apoptosis. Furthermore, we showed that hispidulin-induced apoptosis was mediated by activation of AMPK/mTOR signaling pathway as pretreatment with Compound C, an AMPK inhibitor, or AMPK-targeting siRNA reversed the pro-apoptotic effect of hispidulin. In HCC xenograft nude mice, administration of hispidulin (25, 50 mg/kg every day, ip, for 27 days) dose-dependently suppressed the tumor growth, accompanied by inducing ERS and apoptosis in tumor tissue. Taken together, our results demonstrate that hispidulin induces ERS-mediated apoptosis in HCC cells via activating the AMPK/mTOR pathway. This study provides new insights into the anti-tumor activity of hispidulin in HCC.

In Situ Real-Time Tracing of Organophosphorus Pesticides in Apples by Solid-Phase Microextraction with Developed Sampling-Rate Calibration.

An in situ tracing study based on solid-phase microextraction (SPME) was conducted to investigate the uptake and elimination of organophosphorus pesticides in apples. A matrix-compatible polydimethylsiloxane/poly(styrene-co-divinylbenzene)/polydimethylsiloxane fiber was produced to meet the needs of in situ sampling. The fiber had high extraction ability, good sensitivity and accuracy with respect to the analytes in apple pulp, and could be used 85 times. Although the sampling rate was changing over time, quantification was still achieved by the sampling rate calibration method. Some factors that affect its applicability were studied. The limits of detection were 0.18 ng/g for diazinon and 0.20 ng/g for chlorpyrifos, rather lower than the maximum residue limits of the National Food Safety Standard of China (GB 2763-2016) and the European Commission (Reg.(EU) No 834/2013, 2018/686). The accuracy of in situ SPME quantification was verified by comparing with the results obtained by the traditional liquid-liquid extraction method. In this work, the in situ sampling method is developed using apples, diazinon, and chlorpyrifos as a model system; however, this method can be used for in vivo analysis of fruits and vegetables for nutrition and safety monitoring.

Polarity-Tuning Derivatization-LC-MS Approach for Probing Global Carboxyl-Containing Metabolites in Colorectal Cancer.

Carboxyl-containing metabolites (CCMs) widely exist in living systems and are the essential components for life. Global characteristics of CCMs in biological samples are critical for the understanding of physiological processes and the discovery for the onset of relevant diseases. However, their determination represents a challenge due to enormous polarity differences, structural diversity, high structural similarity, and poor ionization efficiency in mass spectrometry. Herein, 5-(diisopropylamino)amylamine (DIAAA) derivatization coupled with liquid chromatography-mass spectrometry (LC-MS) was developed for mapping the CCMs. With this methodology, the sensitivity was significantly enhanced. More importantly, the hydrophobicity of polar CCMs, amino acids, TCA cycle intermediates, and short-chain fatty acids and the hydrophilicity of low-polar CCMs, long-chain fatty acids, and bile acids were significantly increased, resulting in a remarkable separation efficiency for which 68 CCMs can be simultaneously determined. Furthermore, the polarity-tuning effect was confirmed to be induced by the different impacts of aliphatic chains and nitrogen atom in DIAAA, the latter existing as a cation in the acidic mobile phase, using different derivatization reagents. Finally, this derivatization method was utilized to hunt for the potential biomarkers in colorectal cancer (CRC) patients and 52 CCMs, related with several key metabolic pathways, including amino acids metabolism, TCA cycle, fatty acid metabolism, pyruvate metabolism, and gut flora metabolism were identified. This innovative polarity-tuning derivatization-LC-MS approach was proved to be a valuable tool for probing global metabolome with high separation efficiency and sensitivity in various biological samples.

Different Recovery Processes of Soil Ammonia Oxidizers from Flooding Disturbance.

Understanding how microorganisms respond to environmental disturbance is one of the key focuses in microbial ecology. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are responsible for ammonia oxidation which is a crucial step in the nitrogen cycle. Although the physiology, distribution, and activity of AOA and AOB in soil have been extensively investigated, their recovery from a natural disturbance remains largely unknown. To assess the recovery capacities, including resistance and resilience, of AOA and AOB, soil samples were taken from a reservoir riparian zone which experienced periodically water flooding. The samples were classified into three groups (flooding, recovery, and control) for a high-throughput sequencing and quantitative PCR analysis. We used a relative quantitative index of both the resistance (RS) and resilience (RL) to assess the variation of gene abundance, alpha-diversity, and community composition. The AOA generally demonstrated a better recovery capability after the flooding disturbance compared to AOB. In particular, AOA were more resilient after the flooding disturbance. Taxa within the AOA and AOB showed different RS and RL values, with the most abundant taxa showing in general the highest RS indices. Soil NH4+ and Fe2+/Fe3+ were the main variables controlling the key taxa of AOA and AOB and probably influenced the resistance and resilience properties of AOA and AOB communities. The distinct mechanisms of AOA and AOB in maintaining community stability against the flooding disturbance might be linked to the different life-history strategies: the AOA community was more likely to represent r-strategists in contrast to the AOB community following a K-life strategy. Our results indicated that the AOA may play a vital role in ammonia oxidation in a fluctuating habitat and contribute to the stability of riparian ecosystem.

MicroRNA 494 increases chemosensitivity to doxorubicin in gastric cancer cells by targeting phosphodiesterases 4D.

Acquired drug resistance is one of the main limitations in pharmacological therapy of malignancies including gastric cancer. MicroRNAs (miRNAs) are a class of small noncoding RNAs that suppress their targets by binding to the 3'UTR region of genes. In this study, we explored investigate the target gene of miR-494 and its roles in chemoresistance of gastric cancer. We found that miR-494 was significantly down-regulated in gastric cancer cells lines compared to the normal gastric epithelial cell line. Exogenous overexpression of miR-494 increased the chemosensitivity of gastric cancer cells to doxorubicin. Moreover, miR-494 expression was reduced in a doxorubicin-resistant gastric cancer cells (AGS/dox) compared with the parental cells. MTT assay showed that AGS/dox cells exhibited an elevated viability compared with the parental cells. Enforced expression of miR-494 inhibited AGS/dox cell viability and colony formation ability. In addition, we demonstrated that elevated expression of miR-494 inhibited the mRNA and protein expression of phosphodiesterases 4D (PDE4D) in gastric cancer cell. Luciferase assay showed that miR-494 directly targeted the 3'UTR region of PDE4D. Furthermore, restoration of PDE4D recovered the chemoresistance in miR-494-overexpressed gastric cancer cells. Taken together, this study demonstrated that miR-494 enhanced doxorubicin sensitivity via regulation of PDE4D expression, suggesting a novel therapeutic strategy for anti-chemoresistance in gastric cancer.

Recent development in mass spectrometry and its hyphenated techniques for the analysis of medicinal plants.

INTRODUCTION: Medicinal plants are gaining increasing attention worldwide due to their empirical therapeutic efficacy and being a huge natural compound pool for new drug discovery and development. The efficacy, safety and quality of medicinal plants are the main concerns, which are highly dependent on the comprehensive analysis of chemical components in the medicinal plants. With the advances in mass spectrometry (MS) techniques, comprehensive analysis and fast identification of complex phytochemical components have become feasible, and may meet the needs, for the analysis of medicinal plants. OBJECTIVE: Our aim is to provide an overview on the latest developments in MS and its hyphenated technique and their applications for the comprehensive analysis of medicinal plants. METHODOLOGY: Application of various MS and its hyphenated techniques for the analysis of medicinal plants, including but not limited to one-dimensional chromatography, multiple-dimensional chromatography coupled to MS, ambient ionisation MS, and mass spectral database, have been reviewed and compared in this work. RESULTS: Recent advancs in MS and its hyphenated techniques have made MS one of the most powerful tools for the analysis of complex extracts from medicinal plants due to its excellent separation and identification ability, high sensitivity and resolution, and wide detection dynamic range. CONCLUSION: To achieve high-throughput or multi-dimensional analysis of medicinal plants, the state-of-the-art MS and its hyphenated techniques have played, and will continue to play a great role in being the major platform for their further research in order to obtain insight into both their empirical therapeutic efficacy and quality control.

[Exploring Chinese medicine and Western medicine group modules in acute phase of ischemic stroke disease].

Under the theoretical guidance of "combination of disease and syndrome， correspondence between syndrome and prescription， and dynamic space-time"， 11 135 acute ischemic stroke patients were collected from hospital information system(HIS) of many 3A grade hospitals of traditional Chinese medicine. Complex network analysis was adopted to obtain the core syndrome elements in different periods of acute ischemic stroke patients， and it was found that the core syndrome elements were blood stasis syndrome， phlegm， endogenous wind， Yin deficiency， Qi deficiency， heat， hyperactivity of liver Yang， liver， and kidney of patients in hospital for the first day， and during 8-14 d in hospitalization， the core syndrome elements were blood stasis， phlegm， Yin deficiency， Qi deficiency， endogenous wind， hyperactivity of liver Yang， liver， and kidney. The data with "improved" and "cured" treatment outcomes were adopted for complex network analysis and correlation analysis to identify the Chinese and Western medicine group modules in patients with different disease conditions in different phases after hospitalization. It was found that the Chinese and Western medicine modules within 14 d after hospitalization mainly included "blood-activating and stasis-dissolving module "consisted by "anti-platelet drug + circulation-improving medicine(or anticoagulant drug and anti-fibrinogen drug， et al) + blood-activating and stasis-dissolving drugs"， as well as "stasis-dissolving and phlegm-reducing module" consisted by "anti-platelet drugs + circulation-improving medicine(or anticoagulant drug and anti-fibrinogen drug， et al) + phlegm refreshing drug". The core Chinese and Western medicine modules in patients with urgent and general conditions within 7 d after hospitalization mainly used "blood-activating and stasis-dissolving module" and "stasis-dissolving and phlegm-reducing module". Three or more Chinese medicine and Western medicines module with more than 1% utilization rate was not found in the patients with critical disease condition in admission. The urgent， general and critically ill patients in admission mainly used "blood-activating and stasis-dissolving module" in 8-14 d. From the real world medical big data research， it was found that the combined use of Chinese and Western medicines were consistent with "combination of disease and syndrome， correspondence between syndrome and prescription， and dynamic space-time" theory， and multiple multidimensional dynamic Chinese medicine and Western medicine group modules of "patient-syndrome-drug-time-effective" at the acute ischemic stroke stage were dug out， forming the method of Chinese and Western medicine combination research based on electrical medical big data.

Acid/Salt/pH Gradient Improved Resolution and Sensitivity in Proteomics Study Using 2D SCX-RP LC-MS.

The usage of strong cation exchange (SCX) chromatography in proteomics is limited by its poor resolution and nonspecific hydrophobic interactions with peptides, which lead to peptide overlap across fractions and change of peptide retention, respectively. The application of high concentration of salt (up to 1000 mM) in SCX also restricted its use in online 2D SCX-RP LC. In the present research, we first exploited the chromatographic ability of online 2D SCX-RP LC by combination of acid, salt, and pH gradient, three relatively independent modes of eluting peptides from SCX column. 50% ACN was added to elution buffer for eliminating hydrophobic interactions between SCX matrix and peptides, and the concentration of volatile salt was reduced to 50 mM. Acid/salt/pH gradient showed superior resolution and sensitivity as well as uniform distribution across fractions, consequently leading to significant improvements in peptide and protein identification. 112 191 unique peptides and 7373 proteins were identified by acid/salt/pH fractionation, while 69 870 unique peptides and 4536 proteins were identified by salt elution, that is, 62.5 and 60.6% more proteins and unique peptides, respectively, identified by the former. Fraction overlap was also significantly minimized by acid/salt/pH approach. Furthermore, acid/salt/pH elution showed more identification for acidic peptides and hydrophilic peptides.

Strategy for Hepatotoxicity Prediction Induced by Drug Reactive Metabolites Using Human Liver Microsome and Online 2D-Nano-LC-MS Analysis.

Hepatotoxicity is a leading cause of drug withdrawal from the market; thus, the assessment of potential drug induced liver injury (DILI) in preclinical trials is necessary. More and more research has shown that the covalent modification of drug reactive metabolites (RMs) for cellular proteins is a possible reason for DILI. Unfortunately, so far no appropriate method can be employed to evaluate this kind of DILI due to the low abundance of RM-protein adducts in complex biological samples. In this study, we proposed a mechanism-based strategy to solve this problem using human liver microsomes (HLMs) and online 2D nano-LC-MS analysis. First, RM modification patterns and potential modified AA residues are determined using HLM and model amino acids (AAs) by UHPLC-Q-TOF-MS. Then, a new online 2D-nano-LC-Q-TOF-MS method is established and applied to separate the digested modified microsomal peptides from high abundance peptides followed by identification of RM-modified proteins using Mascot, in which RM modification patterns on specific AA residues are added. Finally, the functions and relationship with hepatotoxicity of the RM-modified proteins are investigated using ingenuity pathway analysis (IPA) to predict the possible DILI. Using this strategy, 21 proteins were found to be modified by RMs of toosendanin, a hepatotoxic drug with complex structure, and some of them have been reported to be associated with hepatotoxicity. This strategy emphasizes the identification of drug RM-modified proteins in complex biological samples, and no pretreatment is required for the drugs. Consequently, it may serve as a valuable method to predict potential DILI, especially for complex compounds.