Batch dark fermentation from enzymatic hydrolyzed food waste for hydrogen production.

A combination bioprocess of solid-state fermentation (SSF) and dark fermentative hydrogen production from food waste was developed. Aspergillus awamori and Aspergillus oryzae were utilized in SSF from food waste to generate glucoamylase and protease which were used to hydrolyze the food waste suspension to get the nutrients-rich (glucose and free amino nitrogen (FAN)) hydrolysate. Both glucose and FAN increased with increasing of food waste mass ratio from 4% to 10% (w/v) and the highest glucose (36.9 g/L) and FAN (361.3mg/L) were observed at food waste mass ratio of 10%. The food waste hydrolysates were then used as the feedstock for dark fermentative hydrogen production by heat pretreated sludge. The best hydrogen yield of 39.14 ml H2/g food waste (219.91 ml H2/VSadded) was achieved at food waste mass ratio of 4%. The proposed combination bioprocess could effectively accelerate the hydrolysis rate, improve raw material utilization and enhance hydrogen yield.

N-Succinyl-chitosan nanoparticles coupled with low-density lipoprotein for targeted osthole-loaded delivery to low-density lipoprotein receptor-rich tumors.

N-Succinyl-chitosan (NSC) was synthesized and NSC nanoparticles (NPs) with loaded osthole (Ost) (Ost/NSC-NPs) were prepared by emulsion solvent diffusion. Subsequently, low-density lipoprotein (LDL)-mediated NSC-NPs with loaded Ost (Ost/LDL-NSC-NPs) were obtained by coupling LDL with Ost/NSC-NPs through amide linkage. The average particle size of Ost/NSC-NPs was approximately 145 nm, the entrapment efficiency was 78.28%±2.06%, and the drug-loading amount was 18.09%±0.17%. The release of Ost from Ost/NSC-NPs in vitro showed a more evident sustained effect than the native material. The half maximal inhibitory concentration of Ost/LDL-NSC-NPs was only 16.23% that of the free Ost at 24 hours in HepG2 cells. Ost inhibited HepG2 cell proliferation by arresting cells in the synthesis phase of the cell cycle and by triggering apoptosis. Cellular uptake and subcellular localization in vitro and near-infrared fluorescence real-time imaging in vivo showed that Ost/LDL-NSC-NPs had high targeting efficacy. Therefore, LDL-NSC-NPs are a promising system for targeted Ost delivery to liver tumor.

Low-density lipoprotein-coupled N-succinyl chitosan nanoparticles co-delivering siRNA and doxorubicin for hepatocyte-targeted therapy.

Developing safe and effective carriers of small interference RNA (siRNA) is a significant demand for the systemic delivery of siRNA. In this study, low-density lipoprotein (LDL) was isolated from human plasma and loaded with cholesterol-conjugated siRNA to silence the multidrug resistant gene of tumors. Chol-siRNA/LDL-coupled N-succinyl chitosan nanoparticles loaded with doxorubicin (Dox-siRNA/LDL-SCS-NPs) were then prepared and characterised. The Dox-siRNA/LDL-SCS-NPs had average particle size of 206.4 ± 9.2 nm, entrapment efficiency of 71.06% ± 1.42%, and drug-loading amount of 12.35% ± 0.87%. In vitro antitumor activity revealed that cell growth was significantly inhibited. The accumulation of Dox by fluorescence microscopy and flow cytometry showed that LDL-coupled nanoparticles were more easily taken up than Dox-SCS-NPs. Results of confocal microscopy and reverse transcription-PCR revealed the highly efficient uptake of siRNA and the decrease in mdr1 mRNA expression. LDL-coupled nanoparticles protected siRNA from macrophage phagocytosis by dynamic observation using live cell station. In vivo tumor-targeting suggested that Cy7-labelled Dox-LDL-SCS-NPs were markedly accumulated in an analyzed in situ liver tumor model. Results indicated that LDL-SCS-NPs were effective tumor-targeting vectors and that the preparation form may provide a new strategy for co-delivering siRNA and antitumor drugs.

Synthesis and characterization of low-toxicity N-caprinoyl-N-trimethyl chitosan as self-assembled micelles carriers for osthole.

Novel amphiphilic chitosan derivatives (N-caprinoyl-N-trimethyl chitosan [CA-TMC]) were synthesized by grafting the hydrophobic moiety caprinoyl (CA) and hydrophilic moiety trimethyl chitosan to prepare carriers with good compatibility for poorly soluble drugs. Based on self-assembly, CA-TMC can form micelles with sizes ranging from 136 nm to 212 nm. The critical aggregation concentration increased from 0.6 mg • L(-1) to 88 mg • L(-1) with decrease in the degree of CA substitution. Osthole (OST) could be easily encapsulated into the CA-TMC micelles. The highest entrapment efficiency and drug loading of OST-loaded CA-TMC micelles(OST/CA-TMC) were 79.1% and 19.1%, respectively. The antitumor efficacy results show that OST/CA-TMC micelles have significant antitumor activity on Hela and MCF-7 cells, with a 50% of cell growth inhibition (IC50) of 35.8 and 46.7 µg. mL(-1), respectively. Cell apoptosis was the main effect on cell death of Hela and MCF-7 cells after OST administration. The blank micelles did not affect apoptosis or cell death of Hela and MCF-7 cells. The fluorescence imaging results indicated that OST/CA-TMC micelles could be easily uptaken by Hela and MCF-7 cells and could localize in the cell nuclei. These findings suggest that CA-TMC micelles are promising carriers for OST delivery in cancer therapy.

[Effects of exogenous nitric oxide on physiological characteristics of longan (Dimocarpus longana) seedlings under acid rain stress].

This paper studied the effects of exogenous nitric oxide donor sodium nitroprusside (SNP) on the chlorophyll content, antioxidant enzyme activities, and osmotic regulation substances of longan (Dimocarpus longana 'Fuyan') seedlings under acid rain (pH 3.0) stress. Under the acid rain stress, the seedling leaf superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities and chlorophyll, soluble protein and soluble sugar contents decreased obviously, while the leaf malondialdedyde content had a remarkable increase, suggesting the toxic effect of the acid rain on the seedlings. Exogenous nitric oxide had dual nature on the physiological characteristics of longan seedlings under acid rain stress. Applying 0.1-0.5 mmol x L(-1) of SNP improved the SOD, POD and CAT activities and the chlorophyll, soluble protein and soluble sugar contents significantly, and decreased the malondialdedyde content. Low concentrations SNP reduced the oxidative damage caused by the acid rain stress, and 0.5 mmol x L(-1) of SNP had the best effect. Under the application of 0.5 mmol x L(-1) of SNP, the total chlorophyll, soluble protein, and soluble sugar contents and the SOD, POD and CAT activities increased by 76.0%, 107.0%, 216.1%, 150. 0%, 350.9% and 97.1%, respectively, and the malondialdedyde content decreased by 46.4%. It was suggested that low concentration (0.1-0.5 mmol x L(-1)) SNP could alleviate the toxic effect of acid rain stress on longan seedlings via activating the leaf antioxidant enzyme activities and reducing oxidative stress, while high concentration SNP (1.0 mmol x L(-1)) lowered the mitigation effect.

Pharmacokinetics, tissue distribution, and metabolites of a polyvinylpyrrolidone-coated norcantharidin chitosan nanoparticle formulation in rats and mice, using LC-MS/MS.

A novel formulation containing polyvinylpyrrolidone (PVP) K(30)-coated norcantharidin (NCTD) chitosan nanoparticles (PVP-NCTD-NPs) was prepared by ionic gelation between chitosan and sodium tripolyphosphate. The average particle size of the PVP-NCTD-NPs produced was 140.03 ± 6.23 nm; entrapment efficiency was 56.33% ± 1.41%; and drug-loading efficiency was 8.38% ± 0.56%. The surface morphology of NCTD nanoparticles (NPs) coated with PVP K(30) was characterized using various analytical techniques, including X-ray diffraction and atomic force microscopy. NCTD and its metabolites were analyzed using a sensitive and specific liquid chromatography-tandem mass spectrometry method with samples from mice and rats. The results indicated the importance of the PVP coating in controlling the shape and improving the entrapment efficiency of the NPs. Pharmacokinetic profiles of the NCTD group and PVP-NCTD-NP group, after oral and intravenous administration in rats, revealed that relative bioavailabilities were 173.3% and 325.5%, respectively. The elimination half-life increased, and there was an obvious decrease in clearance. The tissue distribution of NCTD in mice after the intravenous administration of both formulations was investigated. The drug was not quantifiable at 6 hours in all tissues except for the liver and kidneys. The distribution of the drug in the liver and bile was notably improved in the PVP-NCTD-NP group. The metabolites and excretion properties of NCTD were investigated by analyzing rat feces and urine samples, collected after oral administration. A prototype drug and two metabolites were found in the feces, and seven metabolites in the urine. The primary elimination route of NCTD was via the urine. The quantity of the parent drug eliminated in the feces of the PVP-NCTD-NP group, was 32 times greater than that of the NCTD group, indicating that the NPs dramatically increased the reduction quantity from liver to bile. We conclude that PVP-NCTD-NPs are an adequate formulation for enhancing the absorption of NCTD, and significantly improving therapeutic effects targeting the hepatic system. Decarboxylation and hydroxylation were the dominant metabolic pathways for NCTD. Metabolites were mainly excreted into rat kidney and finally into urine.

Novel norcantharidin-loaded liver targeting chitosan nanoparticles to enhance intestinal absorption.

In this paper, two novel liver-targeting nanoparticles, norcantharidin-loaded chitosan nanoparticles (NCTD-CS-NPs) and norcantharidin-associated galactosylated chitosan nanoparticles (NCTD-GC-NPs), were prepared using ionic cross-linkage. The physical properties, particle size, encapsulation efficiency, and drug release characteristics of the nanoparticles were investigated in vitro. To investigate the intestinal absorption mechanisms of the two preparations, a series of experiments was carried out, including in situ circulation method, in vitro everted gut sacs, and Ussing chamber perfusion technique. The absorption rate constants (Ka) of NCTD at different segments were found to be duodenum > jejunum > ileum > colon. The concentration had no distinctive effect on absorption kinetics, suggesting that drug absorption is not dose-dependent. The transport of NCTD was found to be inhibited by P-glycoprotein (P-gp) inhibitor, indicating that NCTD might be the substrate of P-gp. The order of the absorption enhancer effects were as follows: low molecular weight chitosan (CS-8kDa) > high molecular weight chitosan (CS-30kDa) > Poloxamer > sodium dodecyl sulfate (SDS) > sodium deoxycholate (SDCh). The results indicate that the chitosan nanoparticles can improve intestinal absorption of NCTD.

[Design and application of a cake-shaping apparatus for drug-separated moxibustion].

Acupuncturist makes herbal cakes with traditional manual way, with such disadvantages as slow in making, varying in thickness and size of the cake. When the patients are treated with medical cake-separated moxibustion, they will be unevenly affected by the heat and the patient easily suffers from burning. These hinder clinically wide application of cake-separated moxibustion. With practice of many years, the authors design and make a kind of manual cake-shaping apparatus which can rapidly and conveniently make uniform medical cake, with simple technique, ingenious structure and normal material.

Expression of tumor necrosis factor and its receptor in gallstone and gallbladder carcinoma tissue.

BACKGROUND: Some reports have confirmed that occurrence and development of tumor are related with lots of oncogene, anti-oncogene and cytokine. This study was to detect the expression of TNFmRNA, tumor necrosis factor (TNF) and TNF receptor (TNFR) in the gallbladder mucosa which developed from hyperplasia, dysplasia to carcinoma, and to further discuss the pathogenecity of gallbladder carcinoma. METHODS: The expression of TNFmRNA, TNF and TNFR was detected by in situ hybridization and immunohistochemistry on the surgical specimens of hyperplasia, dysplasia and carcinoma of the gallbladder. RESULTS: The percentage of positive cells expressed TNFmRNA in the gallbladder mucosa was 0%, 20%, and 90%, respectively in hyperplasia, dysplasia and carcinoma (P<0.05). The percentage of positive mononuclear cells (MNCs) expressed TNFmRNA in hyperplasia, dysplasia and carcinoma of gallbladder tissues was 15%, 85%, and 90%, respectively (P<0.05). The number of positive MNC high-power field (Hpf) was 4.85+/-1.50, 6.00+/-2.71, and 9.33+/-3.07, respectively (P<0.05). The number of carcinoma cells and MNCs expressed by TNFmRNA per high-power field was increased with the increase of tumor stage. The number of carcinoma cells/Hpf in stages I-III and IV-V was 9.13+/-4.39 and 7.13+/-2.53 (P<0.05), and that of MNC/Hpf was 14.80+/-4.02 and 11.10+/-2.23 (P<0.01). The number of carcinoma cells and MNCs expressed by TNFmRNA per Hpf was increased with the increase of tumor size. In tumors of more than 2 cm or less than 2 cm in diameter, the number of positive carcinoma cells/Hpf was 14.00+/-4.20 and 8.83+/-4.96, respectively (P<0.05), but that of MNC/Hpf was 10.50+/-2.54 and 7.00+/-2.83 (P<0.05). The pattern of TNF protein expression was similar to that of TNFmRNA, whereas TNF protein expression was more frequent and extensive than TNFmRNA expression. TNFR was expressed in endothelial cells and MNCs of carcinoma, and was negative in mucosal epithelial cells and tumor cells. A positive linear correlation in TNFmRNA expression was observed between tumor cells and MNCs (r=0.687, P<0.01), a correlation in TNFmRNA and TNF protein expression of tumor cells (r=0.847, P<0.001), and a correlation in TNFmRNA and TNF protein expression of MNC in tumor tissue (r=0.643, P<0.01). CONCLUSIONS: TNFmRNA and TNF protein expression is increased during the development of gallbladder mucosa from hyperplasia, dysplasia to carcinoma, and is increased with tumor stage. This finding suggests that TNF is involved in the pathogenesis of gallbladder carcinoma induced by gallstones and the TNF expression in cancer cells may serve as a marker for tumor stage.

Organ failure associated with severe acute pancreatitis.

AIM: To investigate the relationship between severe acute pancreatitis (SAP) and organ failure. METHODS: Clinical data of 74 cases of SAP from Jan. 1993 to Dec. 2002 were retrospectively reviewed, and the relationship between organ failure and age, gender, etiology, extent of necrosis, infection of necrosis and mortality was analyzed. RESULTS: A total of 47 patients (63.5%) showed organ failure, 20 patients (27.0%) multiple organ failure, whereas 27 patients (36.5%) with dysfunction of a single organ system. Pulmonary failure was the most common organ dysfunction (23.0%) among single organ failures. There were no significant differences in age, gender and gallstone pancreatitis among patients with or without organ failure (P>0.05). The incidence of organ failure in infected necrosis was not higher compared with sterile necrosis, and patients with increased amount of necrosis did not have an increased prevalence of organ failure (P>0.05). Patients with organ failure had a higher mortality rate compared with those without organ failure (P<0.05). The death of SAP was associated with multiple organ failure (P<0.005), pulmonary failure (P<0.005), cardiovascular dysfunction (P<0.05) and gastrointestinal dysfunction (P<0.05). CONCLUSION: Organ failure is common in patients with SAP, and patients with multiple organ failure and pulmonary failure have a higher mortality rate. Prevention and active treatment of organ failure can improve the outcome of patients with SAP.

Risk factors influencing mortality of patients with severe acute pancreatitis within 24 hours after admission.

OBJECTIVE: To study the risk factors for death of patients with severe acute pancreatitis (SAP) within 24 hours after admission. METHODS: Clinical and laboratory data of 74 patients with SAP were analyzed retrospectively. The 27 possible risk factors for death within 24 hours after admission were investigated using logistic regression (SPSS software package 10.0), and the equation of logistic regression was set up. RESULTS: Among the 27 possible risk factors, arterial pH, APACHE II scores, early shock, multiple organ failure were associated with mortality. Single logistic regression analysis of the 27 parameters showed that early shock, pleural effusion, arterial pH, complications and APACHE II scores were associated with death of SAP patients, but using multiple logistic regression analysis showed that only acidosis (pH<7.35) was associated with death. CONCLUSION: To maintain the function of organs and correct dysequilibrium of water-electrolyte and acid-base in early treatment of SAP is essential to lower the mortality.