Construction of an exosome-functionalized graphene oxide based composite bionic smart drug delivery system and its anticancer activity.

Graphene oxide has covalently modified by chito oligosaccharides andγ-polyglutamic acid to form GO-CO-γ-PGA, which exhibits excellent performance as a drug delivery carrier, but this carrier did not have the ability to actively target. In this study, the targeting property of breast cancer tumor cell exosomes was exploited to give GO-CO-γ-PGA the ability to target breast tumor cells (MDA-MB-231), and the drug mitoxantrone (MIT) was loaded to finally form EXO-GO-CO-γ-PGA-MIT with an encapsulation efficiency of 73.02%. The pH response of EXO-GO-CO-γ-PGA showed a maximum cumulative release rate of 56.59% (pH 5.0, 120 h) and 6.73% (pH 7.4, 120 h) for MIT at different pH conditions.In vitrocellular assays showed that EXO-GO-CO-γ-PGA-MIT was more potent in killing MDA-MB-231 cells due to its targeting ability and had a significantly higher pro-apoptotic capacity compared to GO-CO-γ-PGA-MIT. The results showed that this bionic nano-intelligent drug delivery system has good drug slow release function and it can increase the local drug concentration of tumor and enhance the pro-apoptotic ability of MIT, so this newly synthesized bionic drug delivery carriers (EXO-GO-CO-γ-PGA-MIT) has potential application in breast cancer treatment.

A Targeted Nano Drug Delivery System of AS1411 Functionalized Graphene Oxide Based Composites.

A novel method for the preparation of antitumor drug vehicles has been optimized. Biological materials of chitosan oligosaccharide (CO) and γ-polyglutamic acid (γ-PGA) have previously been employed as modifiers to covalently modify graphene oxide (GO), which in turn loaded doxorubicin (DOX) to obtain a nano drug delivery systems of graphene oxide based composites (GO-CO-γ-PGA-DOX). The system was not equipped with the ability of initiative targeting, thus resulting into toxicity and side effects on normal tissues or organs. In order to further improve the targeting property of the system, the nucleic acid aptamer NH2 -AS1411 (APT) of targeted nucleolin (C23) was used to conjugate on GO-CO-γ-PGA to yield the targeted nano drug delivery system APT-GO-CO-γ-PGA. The structure, composition, dispersion, particle size and morphology properties of the synthesized complex have been studied using multiple characterization methods. Drug loading and release profile data showed that APT-GO-CO-γ-PGA is provided with high drug loading capacity and is capable of controlled and sustained release of DOX. Cell experimental results indicated that since C23 was overexpressed on the surface of Hela cells but not on the surface of Beas-2B cells, APT-GO-CO-γ-PGA-DOX can target Hela cells and make increase toxicity to Hela cells than Beas-2B cells, and the IC50 value of APT-GO-CO-γ-PGA-DOX was 3.23±0.04 µg/mL. All results proved that APT-GO-CO-γ-PGA can deliver antitumor drugs in a targeted manner, and achieve the effect of reducing poison, which indicated that the targeted carrier exhibits a broad application prospect in the field of biomedicine.

Molecular mechanisms of Mycoredoxin-1 in resistance to oxidative stress in Corynebacterium glutamicum.

Glutaredoxins (Grxs) with Cys-Pro-Phe (Tyr)-Cys motif and a thioredoxin fold structure play an important role in the anti-oxidant system of bacteria by catalyzing a variety of thiol-disulfide exchange reactions with a 2-Cys mechanism or a 1-Cys mechanism. However, the catalytic and physiological mechanism of Corynebacterium glutamicum Mycoredoxin 1 (Mrx1) that shares a high amino acid sequence similarity to Grxs has not been fully elucidated. Here, we report that Mrx1 has a protective function against various adverse conditions, and the decrease of cell viability to various stress conditions by deletion of the Mrx1 in C. glutamicum was confirmed in the mrx1 mutant. The physiological roles of Mrx1 in defence to oxidative stress were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. As well as reducing mycothiol (MSH) mixed disulfide bonds via a 1-Cys mechanism, C. glutamicum Mrx1 catalytically reduced the disulfides in the Ib RNR, insulin and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) by exclusively linking the MSH/Mtr (mycothiol disulfide reductase)/NADPH electron pathway via a 2-Cys mechanism. Thus, we present the first evidence that the Mrx1 is able to protect against the damaging effects of various exogenous stresses by acting as a disulfide oxidoreductase, thereby giving a new insight in how C. glutamicum survives oxidative stressful conditions.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance.

MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)-uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882-ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to ß-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR-uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

Characterization of Xi-class mycothiol S-transferase from Corynebacterium glutamicum and its protective effects in oxidative stress.

BACKGROUND: Oxidative stress caused by inevitable hostile conditions during fermentative process was the most serious threat to the survival of the well-known industrial microorganism Corynebacterium glutamicum. To survive, C. glutamicum developed several antioxidant defenses including millimolar concentrations of mycothiol (MSH) and protective enzymes. Glutathione (GSH) S-transferases (GSTs) with essentially defensive role in oxidative stress have been well defined in numerous microorganisms, while their physiological and biochemical functions remained elusive in C. glutamicum thus far. RESULTS: In the present study, we described protein NCgl1216 belonging to a novel MSH S-transferase Xi class (MstX), considered as the equivalent of GST Xi class (GSTX). MstX had a characteristic conserved catalytic motif (Cys-Pro-Trp-Ala, C-P-W-A). MstX was active as thiol transferase, dehydroascorbate reductase, mycothiolyl-hydroquinone reductase and MSH peroxidase, while it showed null activity toward canonical GSTs substrate as 1-chloro-2,4-dinitrobenzene (CDNB) and GST Omega's specific substance glutathionyl-acetophenones, indicating MstX had some biochemical characteristics related with mycoredoxin (Mrx). Site-directed mutagenesis showed that, among the two cysteine residues of the molecule, only the residue at position 67 was required for the activity. Moreover, the residues adjacent to the active Cys67 were also important for activity. These results indicated that the thiol transferase of MstX operated through a monothiol mechanism. In addition, we found MstX played important role in various stress resistance. The lack of C. glutamicum mstX gene resulted in significant growth inhibition and increased sensitivity under adverse stress condition. The mstX expression was induced by stress. CONCLUSION: Corynebacterium glutamicum MstX might be critically involved in response to oxidative conditions, thereby giving new insight in how C. glutamicum survived oxidative stressful conditions.

OsmC in Corynebacterium glutamicum was a thiol-dependent organic hydroperoxide reductase.

Bacterial antioxidants play a vital role in the detoxification of exogenous peroxides. Several antioxidant defenses including low-molecular-weight thiols (LMWTs) and protective enzymes were developed to help the bacterium withstand the adverse stress. Although osmotically induced bacterial protein C (OsmC), classified as the organic hydroperoxide reductase (Ohr)/OsmC superfamily, has been demonstrated in some mycobacterial species, including M. tuberculosis and M. smegmatis, its physiological and biochemical functions in C. glutamicum remained elusive. Here we found the lack of C. glutamicum osmC gene resulted in decreased cell viability and increased intracellular reactive oxygen species accumulation under organic hydroperoxides (OHPs) stress conditions. The osmC expression was induced in the multiple antibiotic resistance regulator MarR-dependent manner by OHPs, and not by other oxidants or osmotic stress. Peroxide reductase activity showed that OsmC had a narrow range of substrates-only degrading OHPs, and detoxified OHPs mainly by linking the alkyl hydroperoxide reductase (AhpD) system (AhpD/dihydrolipoamide dehydrogenase (Lpd)/dihydrolipoamide acyltransferase (SucB)). Site-directed mutagenesis confirmed Cys48 was the peroxidatic cysteine, while Cys114 was the resolving Cys residue that formed an intramolecular disulfide bond with oxidized Cys48. Therefore, C. glutamicum OsmC was a thiol-dependent OHP reductase and played important role of protection against OHPs together with Ohr.

Structural characterization and immunomodulating activities of polysaccharides from a newly collected wild Morchella sextelata.

A purified polysaccharide was acquired from a newly collected wild Morchella. The strain identification initially showed that the strain was Morchella sextelata. This study aimed to investigate the structural features and immunomodulating activities of the polysaccharide. Polysaccharide extracted from mycelia was purified by DEAE-cellulose chromatography and Sephadex G-25 size-exclusion chromatography in sequence. The main fraction of polysaccharide (MSP-II) was obtained during purification process. High Performance Liquid Chromatography (HPLC) analysis revealed that MSP-II was composed of Glc, Ara, Gal, Man, Rha, Fuc, GalUA and GluUA in ratio of 34.95:8.7:9.55:4.55:5.0:1.45:12.7:7.65. The structure of MSP-II was furtherly analyzed by FT-IR spectrum and 1H and 13C NMR spectroscopy, the results showed that MSP contained ß-glycosidic bonds as well as α-glycosidic linkages. In vitro tests proved that MSP-II could not only promote the proliferation and phagocytosis of RAW264.7 cells, but also induce the section of nitric oxide (NO) of macrophages. Consequently, the polysaccharide has a potent immunomodulatory activity by stimulating macrophages and can be considered as a novel potential immunopotentiator in medical and food industries.

OhsR acts as an organic peroxide-sensing transcriptional activator using an S-mycothiolation mechanism in Corynebacterium glutamicum.

BACKGROUND: Corynebacterium glutamicum is a well-known producer of various L-amino acids in industry. During the fermenting process, C. glutamicum unavoidably encounters oxidative stress due to a specific reactive oxygen species (ROS) produced by consistent adverse conditions. To combat the ROS, C. glutamicum has developed many common disulfide bond-based regulatory devices to control a specific set of antioxidant genes. However, nothing is known about the mixed disulfide between the protein thiol groups and the mycothiol (MSH) (S-mycothiolation)-based sensor. In addition, no OhrR (organic hydroperoxide resistance regulator) homologs and none of the organic hydroperoxide reductase (Ohr) sensors have been described in the alkyl hydroperoxide reductase CF-missing C. glutamicum, while organic hydroperoxides (OHPs)-specific Ohr was a core detoxification system. RESULTS: In this study, we showed that the C. glutamicum OhsR acted as an OHPs sensor that activated ohr expression. OhsR conferred resistance to cumene hydroperoxide (CHP) and t-butyl hydroperoxide but not H2O2, hypochlorous acid, and diamide; this outcome was substantiated by the fact that the ohsR-deficient mutant was sensitive to OHPs but not inorganic peroxides. The DNA binding activity of OhsR was specifically activated by CHP. Mutational analysis of the two cysteines (Cys125 and Cys261) showed that Cys125 was primarily responsible for the activation of DNA binding. The oxidation of Cys125 produced a sulfenic acid (C125-SOH) that subsequently reacted with MSH to generate S-mycothiolation that was required to activate the ohr expression. Therefore, OhsR regulated the ohr expression using an S-mycothiolation mechanism in vivo. CONCLUSION: This is the first report demonstrating that the regulatory OhsR specifically sensed OHPs stress and responded to it by activating a specific ohr gene under its control using an S-mycothiolated mechanism.

[Expression of proteins in p53 (p14ARF-mdm2-p53-p21WAF/CIP1) pathway and their significance in exocrine pancreatic carcinoma].

OBJECTIVE: To investigate the expression of p14(ARF), p53, mdm2 and p21(WAF/CIP1) proteins and their relationship in exocrine pancreatic carcinoma. METHODS: Specimens of pancreatic carcinoma, adjacent non-neoplastic pancreatic tissue and pancreatic benign lesions were examined for p14(ARF), p53, mdm2 and p21(WAF/CIP1) protein expression by tissue microarray technique and immunohistochemistry. RESULTS: The expression of p14(ARF), p53, mdm2 and p21(WAF/CIP1) proteins in pancreatic carcinoma were 35.3% (59/167), 57.5% (96/101), 64.1% (107/167) and 39.5% (66/167) respectively. The expression of p53 proteins was increased in pancreatic carcinoma (P < 0.01), while the expression of p14(ARF) and p21(WAF/CIP1) proteins was reduced (P < 0.05), as compared with that in non-neoplastic pancreatic tissue. p21(WAF/CIP1) protein expression in pancreatic carcinoma significantly correlated with the age of patients and perineural invasion (P < 0.05). p53 protein expression correlated significantly with tumor differentiation, lymph node metastasis and perineural invasion (P < 0.05). Mdm2 protein expression correlated significantly with tumor differentiation (P < 0.05), while p14(ARF) protein expression correlated significantly with the age of patients and metastasis (P < 0.05). There was also statistic correlation between the expression of these four genes (P < 0.05). CONCLUSIONS: Overexpression of p53 and mdm2 and loss of p14(ARF) and p21(WAF/CIP1) expression may contribute to the pathogenesis of pancreatic carcinoma. These proteins play a critical role in cell cycle arrest and apoptosis after DNA damage through p14(ARF)-mdm2-p53-p21(WAF/CIP1) pathway. Detection of p53 and Mdm2 protein overexpression may be useful in evaluation of the aggressiveness of pancreatic carcinoma.

[Fine needle aspiration cytology of eyelid sebaceous gland carcinoma and its differential diagnosis].

OBJECTIVE: To investigate the fine needle aspiration cytology (FNAC) features and differential diagnosis of eyelid sebaceous gland carcinoma. METHODS: Four cases of eyelid sebaceous gland carcinoma diagnosed by FNAC were reported and confirmed by biopsy. Three of the cases were in early stages with tumor sizes smaller than 10 mm in diameter and without metastasis. The smears were stained by routine H & E and SudanIII methods. The cytologic findings were described and compared to corresponding histological features, and moreover, compared to chalazion, pilomatrixoma and eyelid basal cell carcinoma. RESULTS: Neither hemorrhage nor infection were found after the examination. Abundant cells were observed in the sebaceous carcinoma FNAC smears. Two types of tumor cells were found: one showed tumor cells differentiating toward sebaceous gland, with large pale cells and vacuolated cytoplasm, the other demonstrated poorly-differentiated cell with dark and irregular nuclei. Numerous vacuoles with inequality of size were found in cytoplasm or in background in all four cases, and the SudanIII stain showed that these vacuoles contained lipid. Some smears demonstrated cells with basaloid, fusiform or squamous features, corresponding to various histopathological types. In contrast, smears of chalazion displayed inflammatory granuloma, containing several types of inflammatory cells without malignant cells. Smears of pilomatrixoma were cellular with three cell populations, which included bland sheets of basaloid cells, nucleated basophilic cells and anucleated keratinized "ghost cells", along with calcific debris. The smears of basal cell carcinoma were typically less cellular, more tightly cohesive and had smaller clusters of uniform hyperchromatic basaloid cells without vacuolization in cytoplasm or background. Overall, the cytological features of eyelid sebaceous carcinoma were distinct from those of chalazion, pilomatricoma and basal cell carcinoma. CONCLUSIONS: FNAC is a safe and effective approach for the diagnosis of eyelid sebaceous carcinoma and lipid stain is useful in differential diagnosis. The application of FNAC may be important in reaching an early diagnosis and initial treatment of eyelid nodule.

[Ziehl-Neelsen-Victoria blue-iodine green compound staining method showing leprotic bacillus].

OBJECTIVE: To compare compound staining methods showing leprotic bacillus, elastic fibre and cell nucleus etc. in skin biopsy tissue. METHODS: A compound staining dye composed of Ziehl-Neelsen Phenol Fuchsin, Victoria blue, iodine green and Martius yellow were used in paraffin-embedding skin biopsy tissue. RESULTS: Leprotic bacilli showed red; cell nuclei, light green; cell cytoplasm, colorless; elastic fibres around tuberculoid nodules, blue; and essential substances, light yellow in skin biopsy tissue. CONCLUSION: Because the color was single in Ziehl-Neelsen staining method, its contrast was poor. The ZN-VB-IG compound staining method may show leprotic bacilli, elastic fibres and cell nuclei etc. distinctly. Therefore, this is a reliable and nice color contrast compound staining method.

[Expression of TTF-1 in thyroid tumors originating from follicular epithelium and its correlation with expression of RET, galectin-3 and mucin-1 genes].

OBJECTIVE: To explore the expression of thyroid transcription factor-1 (TTF-1) in thyroid tumors and in different parts of follicular epithelium and the correlation of expression of TTF-1 with the expression of RET, galectin-3 (Gal-3) and mucin-1 (MUC1) genes. METHODS: One hundred thirty-three samples of resected thyroid tumors were examined by streptavidin/peroxidase (S-P) immunohistochemical technique to detect the expression of TTF-1 and RET, Gal-3, and MUC1 genes. RESULTS: (1) TTF-1 was expressed in the nuclei and cytoplasm of different kinds of tumor cells and normal cells around the tumors. There was no significant difference between the expression of TTF-1 in nuclei of malignant thyroid tumor cells and that in nuclei of benign thyroid tumor cells (P > 0.05). The positive expression rate of TTF-1 was 27.40% in cytoplasm of benign tumor and was 66.10% in cytoplasm of malignant tumor was 66.10% (P < 0.05). (2) The expression rate of RET gene was 38.9% in thyroid adenoma, 88.4% in papillary carcinoma, and 87.5% in follicular carcinoma. The difference of expression rate of RET gene between adenoma and both of papillary and follicular carcinomas was statistically significant (P < 0.05). (3) The expression rate of MUC1 was 23.3% in benign tumor and 50.9% in malignant tumor. The expression rate of MUC1 was lower in benign thyroid diseases (nodular goiter and adenoma) than in thyroid papillary carcinoma and follicular carcinoma (P < 0.05). (4) The expression rate of Gal-3 gene was 38.4% in benign tumor and 88.1% in malignant tumor. (5) The expression rates of Gal-3 and MUC1 in benign lesions (nodular goiter and adenoma) were lower than those in thyroid papillary and follicular carcinomas (P < 0.05). The expression of Gal-3 and that of MUC1 in thyroid carcinomas were fundamentally parallel. However, the sensitivity of Gal-3 was higher than that of MUC1. In part of adenomas Gal-1 and MUC1 were positive locally simultaneously. The expression of TTF-1 in cytoplasm was associated with that of Gal-3 and MUC1. CONCLUSION: The expression of TTF-1 in nucleus is a phenomenon common to both normal and pathological thyroid follicles. Combined tests of RET, Gal-3 and MUC1 may act as the diagnostic markers to distinguish benign follicular tumor from malignant ones. Expression of TTF-1 in cytoplasm is probably a characteristic of malignant phenotypes of thyroid tumors.