Identification and iterative combinatorial mutagenesis of a new naringinase-producing strain, Aspergillus tubingensis MN589840.

Naringinase was mainly obtained by microbial fermentation, and mutagenesis was a major way for obtaining excellent mutants. The aim of this study was to screen out a high naringinase yielding mutant to enhance the potential application value of its industrialization and compare the effects of different mutagenic methods on the enzyme activity of the strain. A novel producing naringinase strain, Aspergillus tubingensis MN589840, was isolated from mildewed pomelo peel, later subjected to mutagenesis including UV, ARTP and UV-ARTP. After five rounds iterative mutagenesis, the mutants U1, A6 and UA13 were screened out with 1448·49, 1848·71, 2475·16 U mg-1 enzyme activity, the naringinase productivity raised by 79·08, 123·56 and 206%, respectively. In addition, the naringinase activity of three mutants rose after each round of iterative mutagenesis. These results indicated that the mutagenesis efficiency of UV-ARTP was higher than that of single ARTP, and both are better than UV. In summary, the iterative UV-ARTP mutagenesis is an effective strategy for screening high naringinase-producing strains.

Recovery of brain structural abnormalities in morbidly obese patients after bariatric surgery.

BACKGROUND/OBJECTIVES: Obesity-related brain structural abnormalities have been reported extensively, and bariatric surgery (BS) is currently the most effective intervention to produce sustained weight reduction in overtly obese (OB) people. It is unknown whether BS can repair the brain circuitry abnormalities concomitantly with long-term weight loss. SUBJECTS/METHODS: In order to investigate whether BS promotes neuroplastic structural recovery in morbidly OB patients, we quantified fractional anisotropy (FA), mean diffusivity (MD) and gray (GM) and white (WM) matter densities in 15 morbidly OB patients and in 18 normal weight (NW) individuals. OB patients were studied at baseline and also 1 month after laparoscopic sleeve gastrectomy surgery. RESULTS: Two-sample t-test between OB (baseline) and NW groups showed decreased FA values, GM/WM densities and increased MD value in brain regions associated with food intake control (that is, caudate, orbitofrontal cortex, body and genu of corpus callosum) and cognitive-emotion regulation (that is, inferior frontal gyrus, hippocampus, insula, external capsule) (P<0.05, family-wise error correction). Paired t-test in the OB group between before and after surgery showed that BS generated partial neuroplastic structural recovery in the OB group, but the differences had relative less strength and smaller volume (P<0.001). CONCLUSIONS: This study provides the first anatomical evidence for BS-induced acute neuroplastic recovery that might in part mediate the long-term benefit of BS in weight reduction. It also highlights the importance of this line of gut-brain axis research employing the combined BS and neuroimaging model for identifying longitudinal changes in brain structure that correlated with obesity status.

High c-Cbl expression in gliomas is associated with tumor progression and poor prognosis.

Casitas B-lineage lymphoma (c-Cbl) expression has been linked to the development of several types of cancer. However, no studies on the association of c-Cbl and glioma have been published thus far. The present study examined glioma samples obtained from 136 patients treated at The First Hospital of China Medical University (Shenyang, China) from January 2007 to December 2009, and the expression levels of c-Cbl in the samples were evaluated by reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and western blotting. Kaplan-Meier survival curves were generated and subjected to Cox regression analysis. The messenger RNA and protein levels of c-Cbl were observed to be upregulated in high-grade glioma, compared with low-grade glioma. A multivariate analysis revealed that the protein levels of c-Cbl were independently associated with overall survival [hazard ratio (HR)=4.923, 95% confidence interval (CI)=3.163-7.662; P<0.001]. Furthermore, the grade of the glioma (according to the World Health Organization criteria) was observed to be independent prognostic factors for progression-free survival and overall survival time (HR=8.842, 95% CI=7.827-9.989; P<0.001, and HR=10.247, 95% CI=9.009-11.655; P<0.001, respectively). Kaplan-Meier analysis and log-rank test indicated that high protein expression levels of c-Cbl were significantly associated with overall and progression-free survival (P<0.001). To the best of our knowledge, these results provide the first evidence that the overexpression of c-Cbl is correlated with advanced clinicopathological features and poor prognosis in patients with glioma.

Bortezomib-based chemotherapy to treat refractory angioimmunoblastic T-cell lymphoma: A case report and review of the literature.

The peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of aggressive neoplasms that account for <15% of all non-Hodgkin's lymphoma cases in adults. Angioimmunoblastic T-cell lymphoma (AITL) is a specific subtype of PTCL. The tumor is frequently aggressive and there is currently no general consensus regarding an effective treatment strategy. The present study reports a case in which bortezomib combined with dexamethasone was used to treat refractory AITL. A 63-year-old woman was admitted to Sir Run Run Shaw Hospital, Zhejiang University School of Medicine (Zhejiang, China) on August 17, 2013. The patient had been diagnosed with AITL for 4 months and had experienced a relapse of symptoms for the 4 days prior to admission. The patient demonstrated fever and dyspnea, accompanied by severe edema in the face and lower limbs, which later spread to the right upper limb. The patient was treated with bortezomib plus dexamethasone, which rapidly relieved the symptoms. The patient was subsequently administered an additional 2 cycles of bortezomib-based chemotherapy and survived for an additional 4 months, prior to succumbing to the disease. Only a small number of studies have reported the use of bortezomib in the treatment of T-cell lymphoma. The present study suggested that bortezomib-based treatment may be a reliable, safe and effective alternative for the treatment of relapsed/refractory PTCL. The efficacy of bortezomib as a treatment for PTCL requires additional evaluation in future studies.

The involvement of central beta-endorphin in the cardioprotective effects of remote preconditioning mediated by the intracerebroventricular administration of morphine.

BACKGROUND: Opioids can mimic the effects of remote cardiac preconditioning and mediate a subsequent reduction in myocardial infarct size. AIM: This study investigated the role of beta-endorphin (ß-EP) in intracerebroventricular morphine cardioprotection. METHODS: Anesthetized, open-chest, male Sprague-Dawley rats were assigned to 1 of 9 treatment groups 3 days after intracerebroventricular catheter placement. Remote preconditioning was induced with 3 µg/kg of morphine. The ß-EP antagonist was administered via intracerebroventricular or intravenous routes either 10 min before or immediately after morphine or saline administration. Ischemia-reperfusion injury was caused by 30 min of left coronary artery occlusion followed by 120 min of reperfusion. The infarct size, as a percentage of the area at risk, was determined by 2,3,5-triphenyltetrazolium staining. Radioimmunoassay and immunoreactivity were used to determine the ß-EP levels in the serum and brain. RESULTS: Intracerebroventricular administration of ß-EP antiserum (AEP) after morphine administration attenuated the cardioprotective effects of remote preconditioning. The addition of intravenous AEP either before or after morphine did not affect infarct size. After morphine preconditioning, the ß-EP level decreased in the hypothalamic arcuate nucleus and increased significantly in the serum, pituitary gland, ventrolateral periaqueductal gray and rostral ventrolateral medulla. CONCLUSION: Central but not peripheral ß-EP is involved in morphine remote preconditioning and plays a role in the ongoing mediation of cardioprotective effects.

Integrative genomics identifies YY1AP1 as an oncogenic driver in EpCAM(+) AFP(+) hepatocellular carcinoma.

Identification of key drivers and new therapeutic targets is important given the poor prognosis for hepatocellular carcinoma (HCC) patients, particularly those ineligible for surgical resection or liver transplant. However, the approach to identify such driver genes is facing significant challenges due to the genomically heterogenous nature of HCC. Here we tested whether the integrative genomic profiling of a well-defined HCC subset that is classified by an extreme EpCAM(+) AFP(+) gene expression signature and associated with poor prognosis, all attributes of a stem cell-like phenotype, could uncover survival-related driver genes in HCC. Following transcriptomic analysis of the well-defined HCC cases, a Gene Set Enrichment Analysis coupled with genomic copy number alteration assessment revealed that YY1-associated protein 1 (YY1AP1) is a critical oncoprotein specifically activated in EpCAM(+) AFP(+) HCC. YY1AP1 silencing eliminates oncogene addiction by altering the chromatin landscape and triggering massive apoptosis in vitro and tumor suppression in vivo. YY1AP1 expression promotes HCC proliferation and is required for the maintenance of stem cell features. We revealed that YY1AP1 cooperates with YY1 to alter the chromatin landscape and activate transcription of stemness regulators. Thus YY1AP1 may serve as a key molecular target for EpCAM(+) AFP(+) HCC subtype. Our results demonstrate the feasibility and power of a new strategy by utilizing well-defined patient samples and integrative genomics to uncover critical pathways linked to HCC subtypes with prognostic impact.

BRAF(V600E) protein expression and outcome from BRAF inhibitor treatment in BRAF(V600E) metastatic melanoma.

BACKGROUND: To examine the association between level and patterns of baseline intra-tumoural BRAF(V600E) protein expression and clinical outcome of BRAF(V600E) melanoma patients treated with selective BRAF inhibitors. METHODS: Fifty-eight BRAF(V600E) metastatic melanoma patients treated with dabrafenib or vemurafenib on clinical trials had pre-treatment tumour BRAF(V600E) protein expression immunohistochemically (IHC) assessed using the BRAF V600E mutant-specific antibody VE1. Sections were examined for staining intensity (score 1-3) and percentage of immunoreactive tumour cells, and from this an immunoreactive score (IRS) was derived (intensity × per cent positive/10). The presence of intra-tumoural heterogeneity for BRAF(V600E) protein expression was also assessed. BRAF(V600E) expression was correlated with RECIST response, time to best response (TTBR), progression-free survival (PFS) and overall survival (OS). RESULTS: Expression was generally high (median IRS 28 (range 5-30)) and homogeneous (78%). Expression of mutated protein BRAF(V600E) as measured by intensity, per cent immunoreactive cells, or IRS did not correlate with RECIST response, TTBR, PFS or OS, including on multivariate analysis. Heterogeneity of staining was seen in 22% of cases and did not correlate with outcome. CONCLUSION: In the current study population, IHC-measured pre-treatment BRAF(V600E) protein expression does not predict response or outcome to BRAF inhibitor therapy in BRAF(V600E) metastatic melanoma patients.

Effect of processing methods and heat treatment on the formation of wax matrix tablets for sustained drug release.

The objective of this study was to evaluate the effects of processing methods and heat treatment on matrix formation and subsequent drug release from wax matrix tablets for controlled release. Phenylpropanolamine hydrochloride (PPA) and Compritol were processed with appropriate diluent(s) using either dry blending (DB), wet granulation (WG), partial melt granulation (PMG), or melt granulation (MG). Then the tablets were heat-treated at 80 degrees C. Particle size distribution and compressibility, along with drug release, tablet micro-morphology, wettability, porosity, and tortuosity were investigated. The drug release was different for the four processing methods even though the tablet formulation was identical. Heat treatment further retarded drug release and its effect was related to the previous manufacturing processes. Scanning Electron Microscopy (SEM) showed that heat treatment redistributed the wax and formed a film-like structure covering drug and excipients. The contact angle of tablets made from DB, WG, and PMG methods increased after heat treatment, while that of tablets made from MG remained constant. Tablet tortuosity calculated from drug release rate constants increased dramatically after heat treatment. Drug release from the wax tablets with or without heat treatment was best described by the Higuchi equation. Different processing methods produced different matrix structures that resulted in different drug release rates. Heat treatment retarded drug release mainly by increasing tortuosity of the matrix. Contact angle measurement and SEM analysis indicated that heat treatment caused the wax to melt, redistribute, coat the drug and diluents, and form a network structure.

Crosslinking studies in gelatin capsules treated with formaldehyde and in capsules exposed to elevated temperature and humidity.

Incomplete in vitro capsule shell dissolution and subsequent drug release problems have recently received attention. A modified USP dissolution method was used to follow capsule shell dissolution, and a 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay was used to follow loss of epsilon-amino groups to study this shell dissolution problem postulated to be due to gelatin crosslinking. The dissolution problems were simulated using hard gelatin capsule (HGC) shells previously treated with formaldehyde to crosslink the gelatin. These methods were also used to study the effect of uncrosslinked HGC stored under stressed conditions (37 degrees C and 81% RH) with or without the presence of soft gelatin capsule shells (SGC). A 120 ppm formaldehyde treatment reduced gelatin shell dissolution to 8% within 45 min in water at 37 degrees C. A 200 ppm treatment reduced gelatin epsilon-amino groups to 83% of the original uncrosslinked value. The results also support earlier reports of non-amino group crosslinking by formaldehyde in gelatin. Under stressed conditions, HGC stored alone showed little change over 21 weeks. However, by 12 to 14 weeks, the HGC exposed to SGC showed a 23% decrease in shell dissolution and an 8% decrease in the number of epsilon-amino groups. These effects on the stressed HGC are ascribed to a volatile agent from SGC shells, most likely formaldehyde, that crosslinked nearby HGC shells. This report also includes a summary of the literature on agents that reduce gelatin and capsule shell dissolution and the possible mechanisms of this not-so-simple problem.

Effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells.

AIM: To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells (HSC). METHODS: The expressions of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1 (MT1-MMP) in cultured rat HSC were detected by immunocytochemistry (ICC) and in situ hybridization (ISH). The contents of MMP-2 and TIMP-2 in culture supernatant were detected with ELISA and the activity of MMP-2 in supernatant was revealed by zymography. RESULTS: In the situation of hypoxia for 12h, the expression of MMP-2 protein was enhanced (hypoxia group positive indexes: 5.7 +/- 2.0, n=10; control: 3.2 +/- 1.0, n = 7; P<0.05), while TIMP-2 protein was decreased in HSC (hypoxia group positive indexes: 2.5 +/- 0.7, n = 10; control: 3.6 +/- 1.0, n = 7; P < 0.05), and the activity (total A) of MMP-2 in supernatant declined obviously (hypoxia group: 7.334 +/- 1.922, n = 9; control: 17.277 +/- 7.424, n = 11; P < 0.01). Compared the varied duration of hypoxia, the changes of expressions including mRNA and protein level as well as activity of MMP-2 were most notable in 6h group. The highest value(A(hypoxia)-A(control)) of the protein and the most intense signal of mRNA were in the period of hypoxia for 6h, along with the lowest activity of MMP-2. In the situation of hyperoxia for 12h, the contents (A(450)) of MMP-2 and TIMP-2 in supernatant were both higher than those in the control, especially the TIMP-2 (hyperoxia group: 0.0499 +/- 0.0144, n = 16; control: 0.0219 +/- 0.0098, n = 14; P < 0.01), and so was the activity of MMP-2 (hyperoxia group: 5.252 +/- 0.771, n = 14; control: 4.304 +/- 1.083, n = 12; P < 0.05), and the expression of MT1-MMP was increased. CONCLUSION: HSC is sensitive to the oxygen, hypoxia enhances the expression of MMP-2 and the effect is more marked at the early stage; hyperoxia mainly raises the activity of MMP-2.

Fibronectin and malignant disease-associated DNA-binding protein 2 in hepatocarcinogenesis in rats.

Changes in the plasma concentration of malignant disease-associated DNA-binding protein 2 (MAD2) and in the distribution of fibronectin and MAD2 in liver tissue were studied in Fisher-344 rats with diethylnitrosamine-induced hepatocarcinogenesis. The concentration of plasma MAD2 significantly increased as pre-cancerous lesions developed into hepatocellular carcinoma. We believe that the increased plasma concentration of MAD2 is caused by an increase in the degradation of fibronectin within hepatocellular carcinoma tumors. Therefore MAD2 may be a useful marker for the early detection of hepatocellular carcinoma.

Effect of diluents on tablet integrity and controlled drug release.

The objective of this study was to evaluate the effect of diluents and wax level on tablet integrity during heat treatment and dissolution for sustained-release formulations and the resultant effect on drug release. Dibasic calcium phosphate dihydrate (DCPD), microcrystalline cellulose (MCC), and lactose were evaluated for their effect on tablet integrity during drug dissolution and heat treatment in wax matrix formulations. A newly developed direct compression diluent, dibasic calcium phosphate anhydrous (DCPA), was also evaluated. Compritol 888 ATO was used as the wax matrix material, with phenylpropanolamine hydrochloride (PPA) as a model drug. Tablets were made by direct compression and then subjected to heat treatment at 80 degrees C for 30 min. The results showed that MCC, lactose, and DCPA could maintain tablets intact during heat treatment above the melting point of wax (70 degrees C-75 degrees C). However, DCPD tablets showed wax egress during the treatment. MCC tablets swelled and cracked during drug dissolution and resulted in quick release. DCPD and lactose tablets remained intact during dissolution and gave slower release than MCC tablets. DCPA tablets without heat treatment disintegrated very quickly and showed immediate release. In contrast, heat-treated DCPA tablets remained intact through the 24-hr dissolution test and only released about 80% PPA at 6 hr. In the investigation of wax level, DCPD was used as the diluent. The drug release rate decreased as the wax content increased from 15% to 81.25%. The dissolution data were best described by the Higuchi square-root-of-time model. Diluents showed various effects during heat treatment and drug dissolution. The integrity of the tablets was related to the drug release rate. Heat treatment retarded drug release if there was no wax egress.

In situ hybridization of hepatitis B DNA in hepatitis B-associated glomerulonephritis.

Renal tissues from 43 of 49 children with hepatitis B virus-associated glomerulonephritis (HBV-GN) were examined for HBV DNA by in situ hybridization (ISH) assay within the last 10 years. HBV DNA was identified in 41 of the 43 cases (95.3%). HBV DNA was distributed generally in the nucleus and cytoplasm of epithelial cells and mesangial cells of glomeruli, and epithelial cells of renal tubules. HBV DNA also existed simultaneously in renal interstitial tissues in some of these cases. The positive results from HBV DNA ISH correlated well with HBV antigen assays. The analyses implied that the more extensive the existence of HBV DNA in the nephron unit and interstitial tissue, the more severe the clinical manifestation. The duration of proteinuria in cases with HBV DNA in renal tubules was much longer than in those with no HBV DNA in renal tubules. The persistence of the HBV genome or genes in the kidney could lead to the expression of viral antigens in renal tissues and might cause cellular pathological alteration. This would support utilization of antiviral therapy, such as cytokines, in the treatment of HBV-GN.

[Dynamic changes in extracellular matrix and related interstitial cells in experimental organ sclerosis].

The dynamic changes of extracellular matrix (ECM) and effected interstitial cells in experimental sclerosis (fibrosis) of the liver, lung and kidney glomeruli of rats were studied by immunohistochemical methods with monospecific antibodies against fibronectin, laminin, collagen type I, III, IV, V, desmin, vimentin and alpha-smooth muscle actin. The results suggest that there exists an organ (tissue) sclerosis effective cell system involved in ECM synthesis. The activation usually initiates from the Ito cell (liver), primitive mesenchymal call (lung), and mesangial cell (glomeruli), sometimes followed by fibroblast and myofibroblast differentiation with the transformation of cytoskeleton expression. Desmin is the most sensitive marker to reflect the functional state of the organ sclerosis effective cell system from the resting to the activating state.

[Studies on the antifertility constituents of Marsdenia oreophila].

A new pregnane glycoside, marsdeoreophiside B was isolated from the stems of Marsdenia oreophila (Asclepiadeaece). The structure was elucidated by means of chemical and spectrometric analysis as 12-O-cinnamyldihydrosarcostin-3-O-beta-D-glucopyranosyl (1-->4)-O-3-O-methyl-6-deoxy-beta-D-allopyranosyl(1-->4)-O-beta-D- oleandropyranosyl (1-->4)-O-beta-D-cymaropyranoside. It showed significant antifertility activity in rats.

[Further study on the immunopathology of hepatitis B virus associated glomerulonephritis].

By using immunohistochemical techniques the deposition of HBV associated immune complexes was studied in 845 consecutive cases of renal biopsy. In 665 cases of primary glomerulonephritis the frequencies of HBsAg, HBeAg and HBcAg detection in glomeruli were 11.9%, 8.3% and 3.2% respectively with a total HBV antigen positive frequency of 12.2%. High positive rates were found in membranous glomerulonephritis (MGN, 37.1%), mesangioproliferative GN (MPGN, 26%) and IgA nephropathy (IgA-NP, 18.9%). The detection of HBV infection markers in serum were simultaneously performed in 213 cases; 31.7% of the patients with primary GN were found to be positive. In patients with positive HBV infectious markers in the serum, deposits of HBV antigens in glomeruli were found in 49.1% of the cases. The incidence was significantly different in the serum negative group (10.6%). Meanwhile, about 68.3% of the cases with HBV antigen deposits in the kidney was found to have positive HBV markers in the serum. Also the incidence was significantly different in the group without HBV antigen deposits in the kidney (20.9%). It was again confirmed that the pathogenesis of hepatitis B virus associated glomerulonephritis (HBV-GN) was related to the deposition of HBV immune complexes in kidney tissue. It was noticed that the deposition of three different types of HBV antigens was somewhat associated with the development of specific forms of HBV GN. The diagnostic criteria of HBV-GN were discussed in detail.