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1.
Anal Chem ; 92(1): 567-572, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31846294

RESUMO

Protein N-termini and their modifications not only represent different protein isoforms but also relate to the functional annotation and proteolytic activities. Currently, negative selection methods, such as terminal amine isotopic labeling of substrates (TAILS), are the most popular strategy to analyze the protein N-terminome, in which dimethylation or acetylation modification is commonly used to block the free amines of proteome samples. However, after tryptic digestion, the generated long peptides, caused by the missing cleavage of blocked lysine, could hardly be identified by MS, which hindered the deep-coverage analysis of N-terminome. Herein, to solve this problem, we developed an approach, named terminal amine guanidination of substrates (TAGS). 1H-Pyrazole-1-carboxamidine was used to effectively guanidinate lysine ε-amines and N-terminal α-amines, followed by tryptic digestion to generate N-terminal peptides without free amines and internal peptides with free amines. Then, the internal peptides with free amines were removed by hyperbranched polyglycerol-aldehyde polymers (HPG-ALDs) to achieve the negative enrichment of N-terminome. By TAGS, not only the cleavage rate of blocked lysine could be improved, but also the ionization efficiency of tryptic peptides was increased. In comparison, 1814 and 1620 protein N-termini were, respectively, identified by TAGS and TAILS in Saccharomyces cerevisiae (S. cerevisiae). Among them, 1012 N-termini were uniquely identified in TAGS. Furthermore, by the combination of TAGS and the stable isotope labeling with amino acids in cell culture (SILAC)/label-free quantitative method, we not only identified the known N-terminal cleavage fragment of gasdermin D but also identified some new cleavage sites during Val-boroPro-induced pyroptosis. All these results demonstrated that our developed approach, TAGS, might be of great promise for the comprehensive analysis of N-terminome and beneficial for promoting the identification of protein isoforms and studying in-depth the proteolytic activity of proteins.

2.
Anal Chem ; 2019 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-31814401

RESUMO

Chemical cross-linking combined with mass spectrometry (CXMS) has emerged as a powerful tool to study protein structure, conformation and protein-protein interactions (PPIs). Until now, most cross-linked peptides have generated by using commercial cross-linkers, such as DSS, BS3 and DSSO, which react with the primary amino groups of the lysine residues of proteins. However, trypsin, the most commonly used proteolytic enzyme, cannot cleave the C-terminus of a linked lysine, making the obtained cross-linked peptides longer than common peptides and unfavorable for MS identification and data searching. Herein, we proposed an in situ sequential digestion strategy using enzymes with distinct cleavage specificity, named as smart cutter, to generate cross-linked peptides with suitable length so that the identification coverage could improve. Through the application of such a strategy to DSS cross-linked E. coli lysates, additional cross-linked sites (1.3-fold increase) obtained in comparison with those obtained by trypsin-trypsin digestion (2879 vs. 1255). Among the different digestion combinations, AspN-trypsin performed the best, with 64% (673/1059) of the cross-linked sites complementary to trypsin-trypsin digestion, which is beneficial to ensure the depth for studying protein structure and PPIs. Taking the 60 kDa chaperonin protein as an example, more than twice cross-linked sites (30 vs. 14) identified to enrich the protein structure information. In addition, compared to the published protein interaction network for E. coli (http://www.bacteriome.org), 91 potential PPIs discovered with our strategy, of which 65 have not covered by trypsin-trypsin digestion. Therefore, these results illustrate the great significance of smart cutter based CXMS for the revelation of protein structure as well as finding new PPIs.

3.
An Bras Dermatol ; 2019 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-31789251

RESUMO

BACKGROUND: Reports regarding the causative drugs of drug-induced cutaneous adverse reactions in China are indistinct, such that different regions have reported the spectrum of drugs differs substantially in different clinical conditions. OBJECTIVE: To explore the causative drugs that led to cutaneous reactions. METHODS: Adverse drug reaction reports from central China were collected and divided into cutaneous adverse reactions and severe cutaneous adverse reactions groups. Cases were reviewed retrospectively for causative drugs. RESULTS: The male:female ratio was equal in both cutaneous adverse reactions and severe cutaneous adverse reactions. In cutaneous adverse reactions (n=482), the highest incidence happened between 51 and 60 years of age and the top three causative drugs were antibiotics (48%), Chinese medicine (16%), and allopurinol (9%). In severe cutaneous adverse reactions (n=126), the highest incidence happened between 41 and 50 years of age and the top three causative drugs were sedative-hypnotics and antiepileptics (39%), antibiotics (22%), and allopurinol (15%). Carbamazepine was the most frequently used single-drug (16/18) in sedative-hypnotics and antiepileptics. ß-lactams were the most frequently used antibiotic that induced both cutaneous adverse reactions and severe cutaneous adverse reactions. STUDY LIMITATIONS: The small sample size, retrospective design, collection of cutaneous adverse reactions and severe cutaneous adverse reactions at different time frames and locations, and exclusion of patients taking more than five medications are limitations of the study. CONCLUSIONS: Gender does not affect cutaneous adverse reactions and severe cutaneous adverse reactions. The top three drugs to induce cutaneous adverse reactions are antibiotics, Chinese medicine, and allopurinol, while those that triggered severe cutaneous adverse reactions are sedative-hypnotics and antiepileptics, antibiotics, and allopurinol. Carbamazepine is the most frequent single drug that induces severe cutaneous adverse reactions. ß-lactams are the most frequently used antibiotic that induce both cutaneous adverse reactions and severe cutaneous adverse reactions.

4.
J Mol Histol ; 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31858326

RESUMO

Deer antlers are the only mammalian organs capable of complete renewal. Antler renewal is a stem cell-based [antler stem cells (ASCs)] process. Maintenance and activation of the ASCs require them to be located in a specialized microenvironment (niche), and to interact with the cells resident in the niche. Based on previous experiments we found that niche of the ASCs is provided by the closely associated enveloping skin, which currently was known includes dermal papilla cells (DPCs) and epidermal cells. Antler generation/regeneration are triggered by the interactions between ASCs and the niche. In the present study, we established an in vitro co-culture system in which ASCs and DPCs, were cultured together to mimic the in vivo state. A MLEFF strategy was adopted to identify the interactive molecules from the co-culture system. In total, 128 molecules were identified and over 60% belonged to exosomes. Important biological processes that were activated by these molecules included osteoblast differentiation, angiogenesis, and the PI3K-AKT signaling pathway. In so doing, we have significantly simplified the process for identifying interactive molecules, which may be the key signals for triggering antler formation/renewal. Further study of these molecules will help us to gain insights into the mechanism of mammalian organ regeneration.

5.
BMC Infect Dis ; 19(1): 961, 2019 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-31711425

RESUMO

BACKGROUND: Clostridioides difficile is considered the main pathogen responsible for hospital-acquired infections. This prospective study determined the prevalence, molecular epidemiological characteristics, and risk factors for C. difficile infection (CDI) and C. difficile colonization (CDC) among patients in the intensive care unit (ICU) of a large-scale tertiary hospital in China, with the aim of providing strategies for efficient CDI and CDC prevention and control. METHODS: Stool samples were collected and anaerobically cultured for C. difficile detection. The identified isolates were examined for toxin genes and subjected to multilocus sequence typing. Patients were classified into CDI, CDC, and control groups, and their medical records were analyzed to determine the risk factors for CDI and CDC. RESULTS: Of the 800 patients included in the study, 33 (4.12%) and 25 (3.12%) were identified to have CDI and CDC, respectively. Associations with CDI were found for fever (OR = 13.993), metabolic disorder (OR = 7.972), and treatment with fluoroquinolone (OR = 42.696) or combined antibiotics (OR = 2.856). CDC patients were characterized by prolonged hospital stay (OR = 1.137), increased number of comorbidities (OR = 36.509), respiratory diseases (OR = 0.043), and treatment with vancomycin (OR = 18.168). Notably, treatment with metronidazole was found to be a protective factor in both groups (CDI: OR = 0.042; CDC: OR = 0.013). Eighteen sequence types (STs) were identified. In the CDI group, the isolated strains were predominantly toxin A and toxin B positive (A + B+) and the epidemic clone was genotype ST2. In the CDC group, the dominant strains were A + B+ and the epidemic clone was ST81. CONCLUSIONS: The prevalences of CDC and CDI in our ICU were relatively high, suggesting the importance of routine screening for acquisition of C. difficile. Future prevention and treatment strategies for CDC and CDI should consider hospital stay, enteral nutrition, underlying comorbidities, and use of combined antibiotics. Moreover, metronidazole may be a protective factor for both CDI and CDC, and could be used empirically.

6.
J Biol Eng ; 13: 81, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31737090

RESUMO

Background: Inulinase can hydrolyze polyfructan into high-fructose syrups and fructoligosaccharides, which are widely used in food, the medical industry and the biorefinery of Jerusalem artichoke. In the present study, a recombinant exo-inulinase (rKcINU1), derived from Kluyveromyces cicerisporus CBS4857, was proven as an N-linked glycoprotein, and the removal of N-linked glycan chains led to reduced activity. Results: Five N-glycosylation sites with variable high mannose-type oligosaccharides (Man3-9GlcNAc2) were confirmed in the rKcINU1. The structural modeling showed that all five glycosylation sites (Asn-362, Asn-370, Asn-399, Asn-467 and Asn-526) were located at the C-terminus ß-sandwich domain, which has been proven to be more conducive to the occurrence of glycosylation modification than the N-terminus domain. Single-site N-glycosylation mutants with Asn substituted by Gln were obtained, and the Mut with all five N-glycosylation sites removed was constructed, which resulted in the loss of all enzyme activity. Interestingly, the N362Q led to an 18% increase in the specific activity against inulin, while a significant decrease in thermostability (2.91 °C decrease in T m ) occurred, and other single mutations resulted in the decrease in the specific activity to various extents, among which N467Q demonstrated the lowest enzyme activity. Conclusion: The increased enzyme activity in N362Q, combined with thermostability testing, 3D modeling, kinetics data and secondary structure analysis, implied that the N-linked glycan chains at the Asn-362 position functioned negatively, mainly as a type of steric hindrance toward its adjacent N-glycans to bring rigidity. Meanwhile, the N-glycosylation at the other four sites positively regulated enzyme activity caused by altered substrate affinity by means of fine-tuning the ß-sandwich domain configuration. This may have facilitated the capture and transfer of substrates to the enzyme active cavity, in a manner quite similar to that of carbohydrate binding modules (CBMs), i.e. the chains endowed the ß-sandwich domain with the functions of CBM. This study discovered a unique C-terminal sequence which is more favorable to glycosylation, thereby casting a novel view for glycoengineering of enzymes from fungi via redesigning the amino acid sequence at the C-terminal domain, so as to optimize the enzymatic properties.

7.
Viruses ; 11(11)2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31752437

RESUMO

The coastal sediments were considered to contain diverse phages playing important roles in driving biogeochemical cycles based on genetic analysis. However, till now, benthic phages in coastal sediments were very rarely isolated, which largely limits our understanding of their biological characteristics. Here, we describe a novel lytic phage (named Shewanella phage S0112) isolated from the coastal sediments of the Yellow Sea infecting a sediment bacterium of the genus Shewanella. The phage has a very high replication capability, with the burst size of ca. 1170 phage particles per infected cell, which is 5-10 times higher than that of most phages isolated before. Meanwhile, the latent period of this phage is relatively longer, which might ensure adequate time for phage replication. The phage has a double-stranded DNA genome comprising 62,286 bp with 102 ORFs, ca. 60% of which are functionally unknown. The expression products of 16 ORF genes, mainly structural proteins, were identified by LC-MS/MS analysis. Besides the general DNA metabolism and structure assembly genes in the phage genome, there is a cluster of auxiliary metabolic genes that may be involved in 7-cyano-7-deazaguanine (preQ0) biosynthesis. Meanwhile, a pyrophosphohydrolase (MazG) gene being considered as a regulator of programmed cell death or involving in host stringer responses is inserted in this gene cluster. Comparative genomic and phylogenetic analysis both revealed a great novelty of phage S0112. This study represents the first report of a benthic phage infecting Shewanella, which also sheds light on the phage-host interactions in coastal sediments.

8.
Neurotherapeutics ; 2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31758411

RESUMO

The repair and regeneration of transected peripheral nerves is an important area of clinical research, and the adhesion of anastomosis sites to surrounding tissues is a vital factor affecting the quality of nerve recovery after nerve anastomosis. This study involves the generation of a novel nerve repair membrane derived from decellularized porcine nerves using a unique, innovative technique. The decellularized nerve matrix was verified to be effective in eliminating cellular components, and it still retained some neural extracellular matrix components and bioactive molecules (collagens, glycosaminoglycans, laminin, fibronectin, TGF-ß, etc.), which were mainly determined by proteomic analysis, histochemistry, immunohistochemistry, and enzyme-linked immunosorbent assay. Cytotoxicity, intracutaneous reactivity, hemolysis, and cell affinity analyses were conducted to confirm the biosecurity of the nerve repair membrane. The in vivo functionality was assessed in a rat sciatic nerve transection model, and indices of functional nerve recovery, including the measurement of the claw-spread reflex, nerve anastomosis site adhesion, electrophysiological properties, and the number of regenerated nerve fibers, were evaluated. The results indicated that the nerve repair membrane could effectively prevent adhesion between the nerve anastomosis sites and the surrounding tissues and enhance nerve regeneration, which could be attributed to its various bioactive components. In conclusion, the novel nerve repair membrane derived from xenogeneic decellularized nerves described in this study shows great potential auxiliary clinical treatment for peripheral nerve injuries.

9.
Pest Manag Sci ; 2019 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-31762173

RESUMO

BACKGROUD: Volatile terpenes can act as ecological signals to affect insect behavior. It has been proposed that the manipulation of terpenes in plants can help to control herbivore pests. In order to investigate the potential pest management function of (E)-ß-caryophyllene in cotton plants, the (E)-ß-caryophyllene synthase gene (GhTPS1) was inserted into Gossypium hirsutum variety R15 to generate overexpression lines. RESULTS: Four GhTPS1-transgenic lines were generated, and GhTPS1 expression in transgenic L18 and L46 lines was 3-5-fold higher than in R15 plants. The transgenic L18 and L46 lines also emitted significantly more (E)-ß-caryophyllene than R15. In laboratory bioassays, L18 and L46 plants reduced pests Apolygus lucorum, Aphis gossypii and Helicoverpa armigera, and attracted parasitoids Peristenus spretus and Aphidius gifuensis, but not Microplitis mediator. In open-field trials, L18 and L46 plants reduced A. lucorum, Adelphocoris suturalis and H. armigera, but had no significant effects on predators. CONCLUSION: Our findings suggest that L18 and L46 plants reduce several major hemipteran and lepidopteran cotton pests, whereas, two parasitoids P. spretus and A. gifuensis, were attracted by L18 and L46 plants. This study shows that overexpressing GhTPS1 in cotton may help to improve pest management in cotton fields. © 2019 Society of Chemical Industry.

10.
Thorac Cancer ; 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31692283

RESUMO

BACKGROUND: Distinction in the mutational profile between the common histological types, lung adenocarcinoma (LUAD) and squamous cell lung carcinoma (LUSC) has been well-established. However, comprehensive mutation profiles of the predominant histological subtypes within LUAD and LUSC remains elusive. METHODS: We analyzed the mutational profile of 318 Chinese NSCLC patients of adenocarcinoma and squamous cell carcinoma predominant subtypes from seven hospitals using capture-based ultra-deep sequencing of 68 lung cancer-related genes. RESULTS: Of the 318 NSCLC patients, 215 were diagnosed with LUAD and 103 with LUSC. Adenocarcinoma in situ and acinar adenocarcinoma were the most predominant subtypes of LUAD. On the other hand, keratinizing squamous cell carcinoma was the most predominant subtype of LUSC. Among the LUAD subtypes, EGFR sensitizing mutations were most prevalent in the invasive lepidic subtype. More than half of the patients with preinvasive adenocarcinoma in situ, minimally invasive, acinar, micropapillary and papillary subtypes were also EGFR-mutants. Patients with colloidal, invasive mucinous, and fetal subtypes had the least number of EGFR mutations. Moreover, KRAS mutations were prevalent in patients with invasive mucinous, colloid, enteric and solid subtypes. A total of 90% of the LUSC patients harbor mutations in TP53, wherein all patients except five with nonkeratinizing were TP53 mutants. PIK3CA amplifications were most prevalent in keratinizing, followed by basaloid and nonkeratinizing subtypes. CONCLUSION: These data suggest that the mutational profiles among the predominant histological subtypes were very distinct, which provided a reliable tool to improve treatment decisions.

11.
Anal Chem ; 91(23): 14860-14864, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31668058

RESUMO

Protein persulfidation is one of the most important oxidative translational modifications and plays vital roles in various important biological processes. However, the proteome-wide identification of persulfidation sites is a great challenge because of the difficulties in accurately differentiating persulfide groups with disulfide and thiol groups in proteins as well as the extremely low abundance of persulfidated peptides. By current approaches, the persulfidated peptides were often identified by the cleavage of their persulfide groups by reductants prior to MS analysis; therefore, it would bring about a false positive identification and was unable to identify persulfidation sites accurately for a single peptide with multiple cysteine residues. In this study, a novel strategy for the site-specific quantification of persulfidome (SSQPer) was developed. By this strategy, the persulfidated proteins were first labeled with cleavable isotope-coded affinity tag (c-ICAT) reagents. After digestion, the labeled persulfidated peptides were selectively enriched with streptavidin beads and fractionated by strong cation exchange chromatography, followed by LC-MS/MS identification. To evaluate the performance of SSQPer, the persulfidated BSA digests with 20 persulfidation sites identified were used to spike HeLa cell digests with mass ratios of 1:100 and 1:1000, and 16 and 13 persulfidated sites were respectively identified. We applied SSQPer to the site-specific quantification of persulfidome in the epithelial-mesenchymal transition (EMT) process, and 226 endogenous persulfidation sites were identified, of which 74.3% were newly discovered. All of these results demonstrated that the SSQPer strategy would provide a promising tool to profile the site-specific persulfidome and pave the way for future investigation to expand our knowledge of persulfidation.

12.
Mol Metab ; 29: 145-157, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31668386

RESUMO

OBJECTIVES: High fructose feeding changes fibroblast growth factor 21 (FGF21) regulation. Lactobacillus rhamnosus GG (LGG) supplementation reduces fructose-induced non-alcoholic fatty liver disease (NAFLD). The aim of this study was to determine the role of FGF21 and underlying mechanisms in the protective effects of LGG. METHODS: FGF21 knockout (KO) mice and C57BL/6 wild type (WT) mice were fed 30% fructose for 12 weeks. LGG was administered to the mice in the last 4 weeks during fructose feeding. FGF21-adiponectin (ADPN)-mediated hepatic lipogenesis and inflammation were investigated. RESULTS: FGF21 expression was robustly increased after 5-weeks of feeding and significantly decreased after 12-weeks of feeding in fructose-induced NAFLD mice. LGG administration reversed the depressed FGF21 expression, increased adipose production of ADPN, and reduced hepatic fat accumulation and inflammation in the WT mice but not in the KO mice. Hepatic nuclear carbohydrate responsive-element binding protein (ChREBP) was increased by fructose and reduced by LGG, resulting in a reduction in the expression of lipogenic genes. The methylated form of protein phosphatase 2A (PP2A) C, which dephosphorylates and activates ChREBP, was upregulated by fructose and normalized by LGG. Leucine carboxyl methyltransferase-1, which methylates PP2AC, was also increased by fructose and decreased by LGG. However, those beneficial effects of LGG were blunted in the KO mice. Hepatic dihydrosphingosine-1-phosphate, which inhibits PP2A, was markedly increased by LGG in the WT mice but attenuated in the KO mice. LGG decreased adipose hypertrophy and increased serum levels of ADPN, which regulates sphingosine metabolism. This beneficial effect was decreased in the KO mice. CONCLUSION: LGG administration increases hepatic FGF21 expression and serum ADPN concentration, resulting in a reduced ChREBP activation through dihydrosphingosine-1-phosphate-mediated PP2A deactivation, and subsequently reversed fructose-induced NAFLD. Thus, our data suggest that FGF21 is required for the beneficial effects of LGG in reversal of fructose-induced NAFLD.

13.
Chem Phys Lipids ; 226: 104848, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31705861

RESUMO

More than 200 molecular species of glycerophospholipids (GP) including glycerophosphocholine (GPC), glycerophosphoethanolamine (GPE), glycerophosphoserine (GPS), lysoglycerophosphocholine (LGPC), lysoglycerophosphoethanolamine (LGPE) and lysoglycerophosphoserine (LGPS), as well as 18 kinds of sphingomyelin (SM) were characterized by using a direct infusion-tandem mass (MS/MS) spectrometry method for lipids from the muscles of cephalopods Sepiella maindroni, Octopus ocellatus and Loligo chinensis for the first time. The majority of the GP molecular species contained long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Therefore, cephalopods can be a good possible source of dietary GP carrying n-3 LC-PUFA. The total lipids were composed of phospholipid (PL, 72.29-83.32 wt% of total lipids), cholesterol (12.70-23.60 wt% of total lipids), triacylglycerol (1.86-2.93 wt% of total lipids), diacylglycerol (0.15-1.09 wt% of total lipids), monoacylglycerol (0.06-0.18 wt% of total lipids) and free fatty acid (0.72-1.86 wt% of total lipids). For PL, phosphatidylcholine (44.47-62.30 mol%), phosphatidylethanolamine (22.57-39.08 mol%), phosphatidylserine (6.15-10.18 mol%), phosphatidylglycerol (0.68-3.11 mol%), phosphatidylinositol (2.41-7.15 mol%) and lysophosphatidylcholine (1.84-5.24 mol%) were detected. Furthermore, the total lipids from the muscles of cephalopods Sepiella maindroni, Octopus ocellatus and Loligo chinensis contained 41.80-50.02 mol% of saturated fatty acids, 11.53-21.54 mol% of monounsaturated fatty acids and 36.67-40.82 mol% of PUFA, whilst DHA (15.25-26.71 mol%) and EPA (6.29-16.57 mol%) were found to account for the majority of the PUFA. With these data presented, cephalopod muscle can be considered as a healthy food for humans.

14.
J Endocr Soc ; 3(11): 2123-2134, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31687639

RESUMO

Müllerian-inhibiting substance (MIS), also known as anti-Müllerian hormone, is thought to be a negative regulator of primordial follicle activation. We have previously reported that treatment with exogenous MIS can induce complete ovarian suppression within 5 weeks of treatment in mice. To investigate the kinetics of the return of folliculogenesis following the reversal of suppression, we treated animals with recombinant human MIS (rhMIS) protein for 40 days in adult female Nu/Nu mice and monitored the recovery of each follicle type over time. Following cessation of MIS therapy, secondary, and antral follicles returned within 30 days, along with the normalization of reproductive hormones, including LH, FSH, MIS, and Inhibin B. Furthermore, 30 days following MIS pretreatment, the number of antral follicles were significantly higher than controls, and superovulation with timed pregnant mare serum gonadotropin and human chorionic gonadotropin stimulation at this time point resulted in an approximately threefold increased yield of eggs. Use of the combined rhMIS-gonadotropin superovulation regimen in a diminished ovarian reserve (DOR) mouse model, created by 4-vinylcyclohexene dioxide treatment, also resulted in a twofold improvement in the yield of eggs. In conclusion, treatment with rhMIS can induce a reversible ovarian suppression, following which a rapid and synchronized large initial wave of growing follicles can be harnessed to enhance the response to superovulation. Therapies modulating MIS signaling may therefore augment the response to current ovarian stimulation protocols and could be particularly useful to women with DOR or poor responders to controlled ovarian hyperstimulation during in vitro fertilization.

15.
Hepatology ; 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31571251

RESUMO

Cholestatic liver disease is characterized by gut dysbiosis and excessive toxic hepatic bile acids (BAs). Modification of gut microbiota and repression of BA synthesis are potential strategies for the treatment of cholestatic liver disease. The purpose of this study was to examine the effects and to understand the mechanisms of the probiotic, Lactobacillus rhamnosus GG (LGG), on hepatic bile acid synthesis, liver injury and fibrosis in bile-duct ligation (BDL) and Mdr2-/- mice. Global and intestinal specific FXR inhibitors were used to dissect the role of FXR. LGG treatment significantly attenuated liver inflammation, injury and fibrosis with a significant reduction of hepatic BAs in BDL mice. Hepatic concentration of T-ßMCA, an FXR antagonist, was markedly increased in BDL mice and reduced in LGG-treated mice, while chenodeoxycholic acid (CDCA), an FXR agonist, was decreased in BDL mice and normalized in LGG-treated mice. LGG treatment significantly increased the expression of serum and ileum FGF15 and subsequently reduced hepatic CYP7A1 and BA synthesis in BDL and Mdr2-/- mice. At the molecular level, these changes were reversed by global and intestinal specific FXR inhibitors in BDL mice. In addition, LGG treatment altered gut microbiota, which was associated with increased BA de-conjugation and increased fecal and urine BA excretion both in BDL and Mdr2-/- mice. In vitro studies showed that LGG suppressed the inhibitory effect of T-ßMCA on FXR and FGF19 expression in Caco-2 cells. Conclusion: LGG supplementation decreases hepatic BA by increasing intestinal FXR/FGF15 signaling pathway-mediated suppression of BA de novo synthesis and enhances BA excretion, which prevents excessive BA-induced liver injury and fibrosis in mice.

16.
Biochem Biophys Res Commun ; 520(1): 67-72, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31575408

RESUMO

Intrahepatic cholestasis of pregnancy (ICP) is gestation-specific liver disease associated with liver injury and increased serum and hepatic bile acids. Although the mechanism of ICP is still not fully understood, the reproductive hormones seem to play an important role. Recent studies show that a progesterone metabolite, epiallopregnanolone sulfate (PM5S), is supraphysiologically elevated in the serum of ICP patients, indicating it may play an etiology role in ICP. Bile acid homeostasis is controlled by multiple mechanisms including farnesoid X receptor (FXR)-mediated bile acid export and synthesis. It is known that cholic acid (CA), a primary bile acid, can activate FXR, which is inhibited by PM5S, an FXR antagonist. Here we employed a mouse model of concurrent exposure of CA and PM5S-induced liver injury and determined the effects of probiotic Lactobacillus rhamnosus GG (LGG) in the prevention of the bile acid disorders and liver injury. Mice challenged with CA + PM5S had significantly increased levels of serum and hepatic bile acids and bilirubin and liver enzyme. Pretreatment with LGG significantly reduced bile acid and bilirubin levels associated with reduced liver enzyme level and mRNA expression levels of pro-inflammatory cytokines. We also showed that the beneficial effects of LGG is likely mediated by hepatic FXR activation and bile salt export pump (BSEP) upregulation. In conclusion, our results provide a rationale for the application of probiotics in the management of ICP through gut microbiota-mediated FXR activation.

17.
Se Pu ; 37(8): 836-844, 2019 Aug 08.
Artigo em Chinês | MEDLINE | ID: mdl-31642254

RESUMO

Protein persulfidation is an important oxidative translational modification which plays vital roles in many important processes including cellular senescence, endoplasmic reticulum stress, vasorelaxation, and apoptosis. The proteome-wide analysis of persulfidation is of great importance; therefore, this study combines filter-aided sample preparation with an iodoacetic acid functionalized polyamidoamine dendrimer to enrich persulfidated peptides (denoted as filter-aided dendrimer enrichment strategy, FADE). To evaluate the performance of this strategy, the synthetic persulfidated standard peptide was spiked into bovine serum albumin (BSA) digests at a mass ratio of 1:100, and was successfully identified by FADE. Moreover, in combination with stable isotope labelling by amino acids in cell culture technology, the FADE strategy was applied to enrich persulfidated peptides from NaHS-stimulated SHSY5Y cells over a concentration gradient, resulting in the identification of 163 persulfidated peptides. Bioinformatic analysis indicated that persulfidation might play important roles in the central nervous system.


Assuntos
Dendrímeros , Ácido Iodoacético/química , Peptídeos/química , Animais , Bovinos , Proteoma , Soroalbumina Bovina
18.
Biotechnol Bioeng ; 2019 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-31654413

RESUMO

Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time-consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR-Cas9 cassette or a transient CRISPR-Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis. Based on this system, we present a platform for the biosynthesis of ß-carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.

19.
Front Oncol ; 9: 921, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31649870

RESUMO

Identification of effective biomarkers is crucial for monitoring the treatment and remission of colorectal cancer (CRC) and improving survival. It is particularly important to diagnose CRC before the tumor metastasizes (stage I-II disease) where possible, to provide the greatest opportunity for patient recovery. Here, we evaluated the clinical value of serum chemokine (C-X-C) ligand 7 (CXCL7) concentration as a biomarker for CRC diagnosis. An enzyme-linked immunosorbent assay was used to measure CXCL7 concentration in 560 serum samples from patients with CRC and controls. Logistic regression and receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic efficacy and build mathematical diagnostic models. The concentration of CXCL7 in the CRC group was significantly higher than that in the control group (P < 0.001), with an area under the ROC curve (AUC) value of 0.862 [95% confidence interval (CI): 0.831-0.890]. Further, the AUC of a regression model including the markers carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and carbohydrate antigen 125 (CA125), along with CXCL7, was 0.933 (95% CI: 0.909-0.952). For stage I-II tumors, CXCL7 had the highest AUC (0.823, 95% CI: 0.783-0.858) among the four individual biomarkers. The AUC value for combination model analysis of samples from patients with stage I-II tumors was 0.904 (95% CI: 0.872-0.930), with a sensitivity of 82.76% and a specificity of 87.14%, and an optimal cut-off value of 2.66. AUC values for application of the regression model in subgroup analysis were 0.947 (0.917-0.968) and 0.919 (0.874-0.951) for males and females, respectively. These results suggest that CXCL7 has potential as a serum diagnostic biomarker for detection of CRC. Importantly, the combination of CXCL7, CEA, CA125, and CA19-9 may facilitate diagnosis of CRC with relatively high sensitivity and specificity. Clinical Trial Registration Number: LS2017001.

20.
World J Gastroenterol ; 25(38): 5814-5825, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31636474

RESUMO

BACKGROUND: Wnt1-inducible signaling pathway protein 1 (WISP1) is upregulated in several types of human cancer, and has been implicated in cancer progression. However, its clinical implications in gastric cancer (GC) remain unclear. AIM: To explore the expression pattern and clinical significance of WISP1 in GC. METHODS: Public data portals, including Oncomine, The Cancer Genome Atlas database, Coexpedia, and Kaplan-Meier plotter, were analyzed for the expression and clinical significance of WISP1 mRNA levels in GC. One hundred and fifty patients who underwent surgery for GC between February 2010 and October 2012 at the Affiliated Hospital of Jiangnan University were selected for validation study. WISP1 levels were measured at both the mRNA and protein levels by RT-qPCR, Western blot analysis, and immunohistochemistry (IHC). In addition, the in situ expression of WISP1 in the GC tissues was determined by IHC, and the patients were accordingly classified into high- and low-expression groups. The correlation of WISP1 expression status with patient prognosis was then determined by univariate and multivariate Cox regression analyses. WISP1 was knocked down by RNA interference. The 50% inhibitory concentration of oxaliplatin was detected by CellTiter-Blue assay. RESULTS: WISP1 levels at both the mRNA and protein levels were remarkably upregulated in GC tissues compared to normal tissues. Moreover, IHC revealed that WISP1 expression was associated with T stage and chemotherapy outcome, but not with lymph node metastasis, age, gender, histological grade, or histological type. GC patients with high WISP1 expression showed a poor overall survival. Multivariate survival analysis indicated that WISP1 was an important prognostic factor for GC patients. Mechanistically, knock-down of WISP1 expression enhanced sensitivity to oxaliplatin by reducing DNA repair and enhancing DNA damage. CONCLUSION: Significantly upregulated WISP1 expression is associated with cancer progression, chemotherapy outcome, and prognosis in GC. Mechanistically, knock-down of WISP1 expression enhances oxaliplatin sensitivity by reducing DNA repair and enhancing DNA damage. WISP1 may be a potential therapeutic target for GC treatment or a potential biomarker for diagnosis and prognosis.

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