An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase.

In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay to identify more AEP-type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP-type peptide ligase activity in the crude extract of all tested bamboo leaves. From a popular bamboo species, Bambusa multiplex, we identified a full-length AEP-type peptide ligase candidate (BmAEP1) via transcriptomic sequencing. After its zymogen was overexpressed in Escherichia coli and self-activated in vitro, BmAEP1 displayed high peptide ligase activity, but with considerable hydrolytic activity. After site-directed mutagenesis of its ligase activity determinants, the mutant zymogen of [G238V]BmAEP1 was normally overexpressed in E. coli, but failed to activate itself. To resolve this problem, we developed a novel protease-assisted activation approach in which trypsin was used to cleave the mutant zymogen and was then conveniently removed via ion-exchange chromatography. After the noncovalently bound cap domain was dissociated from the catalytic core domain under acidic conditions, the recombinant [G238V]BmAEP1 displayed high peptide ligase activity with much lower hydrolytic activity and could efficiently catalyze inter-molecular protein ligation and intramolecular peptide cyclization. Thus, the engineered bamboo-derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis.

Inhibitory interaction of flavonoids with organic anion transporter 3 and their structure-activity relationships for predicting nephroprotective effects.

The organic anion transporter 3 (OAT3), an important renal uptake transporter, is associated with drug-induced acute kidney injury (AKI). Screening and identifying potent OAT3 inhibitors with little toxicity in natural products, especially flavonoids, in reducing OAT3-mediated AKI is of great value. The five strongest OAT3 inhibitors from the 97 flavonoids markedly decreased aristolochic acid I-induced cytotoxicity and alleviated methotrexate-induced nephrotoxicity. The pharmacophore model clarified hydrogen bond acceptors and hydrophobic groups are the critical pharmacophores. These findings would provide valuable information in predicting the potential risks of flavonoid-containing food/herb-drug interactions and optimizing flavonoid structure to alleviate OAT3-related AKI.

A multicenter clinical study: personalized medication for advanced gastrointestinal carcinomas with the guidance of patient-derived tumor xenograft (PDTX).

BACKGROUND: Establish patient-derived tumor xenograft (PDTX) from advanced GICs and assess the clinical value and applicability of PDTX for the treatment of advanced gastrointestinal cancers. METHODS: Patients with advanced GICs were enrolled in a registered multi-center clinical study (ChiCTR-OOC-17012731). The performance of PDTX was evaluated by analyzing factors that affect the engraftment rate, comparing the histological consistency between primary tumors and tumorgrafts, examining the concordance between the drug effectiveness in PDTXs and clinical responses, and identifying genetic variants and other factors associated with prognosis. RESULTS: Thirty-three patients were enrolled in the study with the engraftment rate of 75.8% (25/33). The success of engraftment was independent of age, cancer types, pathological stages of tumors, and particularly sampling methods. Tumorgrafts retained the same histopathological characteristics as primary tumors. Forty-nine regimens involving 28 drugs were tested in seventeen tumorgrafts. The median time for drug testing was 134.5 days. Follow-up information was obtained about 10 regimens from 9 patients. The concordance of drug effectiveness between PDTXs and clinical responses was 100%. The tumor mutation burden (TMB) was correlated with the effectiveness of single drug regimens, while the outgrowth time of tumorgrafts was associated with the effectiveness of combined regimens. CONCLUSION: The engraftment rate in advanced GICs was higher than that of other cancers and meets the acceptable standard for applying personalized therapeutic strategies. Tumorgrafts from PDTX kept attributes of the primary tumor. Predictions from PDTX modeling closely agreed with clinical drug responses. PDTX may already be clinically applicable for personalized medication in advanced GICs.

Nobiletin (NOB) suppresses autophagic degradation via over-expressing AKT pathway and enhances apoptosis in multidrug-resistant SKOV3/TAX ovarian cancer cells.

Chemotherapy could be used as an effective therapeutic treatment for ovarian cancer and subsequent peritoneal metastasis. However, the occurrence of drug resistance reduced the treatment effect originated from cancer chemotherapy. Accumulating evidences indicated the significant role of autophagy in tumor cell resistance to chemotherapy. Thus, inhibition of autophagy using natural compounds could be a promising candidate to overcome multidrug resistance in human ovarian cancer cells. Nobiletin (NOB), a polymethoxyflavonoid found in citrus fruits such as Citrus depressa and Citrus reticulate, exhibits a number of bioactivities. In the present study, NOB selectively suppressed the growth and proliferation of human SKOV3/TAX cells, inducing G0/G1 phase arrest and reducing G2/M phase, along with the increase of p53 and p21. In addition, NOB induced significant apoptosis in SKOV3/TAX cells through the intrinsic apoptosis pathway, as evidenced by the up-regulation of cleaved Caspase-9/-3 and PARP. Further, NOB impaired the autophagic degradation in SKOV3/TAX cells, resulting in autophagic flux inhibition. Moreover, the impaired autophagic flux enhanced NOB-induced apoptosis in SKOV3/TAX cells. Importantly, AKT signaling was activated by NOB, which was involved in autophagic degradation and apoptotic cell death. In conclusion, the findings here supplied the illustration that NOB could overcome multidrug resistance in human ovarian cancer cells through AKT-regulated suppression of autophagic degradation.

Elevated circulating Th17 and follicular helper CD4(+) T cells in patients with rheumatoid arthritis.

It remains not fully elucidated the potential functions of Th17 cells and follicular helper T (Tfh) cells and secreting cytokines in the pathogenesis of rheumatoid arthritis (RA) and their association with disease activity. In this study, the frequencies of Th17 and Tfh cells were determined by flow cytometry, and the levels of interleukin (IL)-17, IL-21, and IL-22 were measured by ELISA in RA patients with different disease activities. The dynamic changes of cell subsets were also detected in response to disease-modify antirheumatic drugs (DMARDs) therapy. The percentages of CD3(+) CD4(+) IL-17A(+) (Th17) cells and CD3(+) CD4(+) CXCR5(+) ICOS(high) (Tfh) cells, as well as the concentrations of IL-17, IL-21, and IL-22 were significantly elevated in RA patients than those in healthy individuals. Furthermore, Tfh cells, IL-21, and IL-22 in the serum was positively correlated with the values of disease activity score. Concentrations of IL-21 and IL-22 in the serum were remarkably reduced following the DMARDs therapies. Our data suggested that Th17 cells, Tfh cells as well as the secreting cytokines may be involved in the pathogenesis of RA. The frequency of circulating Tfh cells and the productions of IL-21 and IL-22 were associated with the disease activity of RA patients, and might be potential therapeutic targets for treatment of RA.

MG132, a proteasome inhibitor, enhances LDL uptake in HepG2 cells in vitro by regulating LDLR and PCSK9 expression.

AIM: Expression of liver low-density lipoprotein receptor (LDLR), a determinant regulator in cholesterol homeostasis, is tightly controlled at multiple levels. The aim of this study was to examine whether proteasome inhibition could affect LDLR expression and LDL uptake in liver cells in vitro. METHODS: HepG2 cells were examined. Real-time PCR and Western blot analysis were used to determine the mRNA and protein levels, respectively. DiI-LDL uptake assay was used to quantify the LDLR function. Luciferase assay system was used to detect the activity of proprotein convertase subtilisin/kexin type 9 (PCSK9, a major protein mediating LDLR degradation) promoter. Specific siRNAs were used to verify the involvement of PCSK9. RESULTS: Treatment of HepG2 cells with the specific proteasome inhibitor MG132 (0.03-3 µmol/L) dose-dependently increased LDLR mRNA and protein levels, as well as LDL uptake. Short-term treatment with MG132 (0.3 µmol/L, up to 8 h) significantly increased both LDLR mRNA and protein levels in HepG2 cells, which was blocked by the specific PKC inhibitors GF 109203X, Gö 6983 or staurosporine. In contrast, a longer treatment with MG132 (0.3 µmol/L, 24 h) did not change LDLR mRNA, but markedly increased LDLR protein by reducing PCSK9-mediated lysosome LDLR degradation. Furthermore, MG132 time-dependently suppressed PCSK9 expression in the HepG2 cells through a SREBP-1c related pathway. Combined treatment with MG132 (0.3 µmol/L) and pravastatin (5 µmol/L) strongly promoted LDLR expression and LDL uptake in HepG2 cells, and blocked the upregulation of PCSK9 caused by pravastatin alone. CONCLUSION: Inhibition of proteasome by MG132 in HepG2 cells plays dual roles in LDLR and PCSK9 expression, and exerts a beneficial effect on cholesterol homeostasis.

Effects of biliary drainage on the intestinal barrier function in obstructive jaundice.

BACKGROUND/AIMS: Despite advances in preoperative evaluation and postoperative care, intervention, especially surgery, for relief of obstructive jaundice still carries high morbidity and mortality rates. In obstructive jaundice, intestinal barrier dysfunction has been postulated to be a key factor contributing to high postoperative morbidity and mortality rates. Since surgery in patients with jaundice is thought to increase the risk of postoperative complications, preoperative biliary drainage (PBD) was introduced to improve the postoperative outcome. To date, whether biliary drainage should be routinely performed in patients with jaundice undergoing a pancreatoduodenectomy remains controversial, and the effect of biliary drainage on the intestinal barrier function in obstructive jaundice remains unknown. RESULTS: Biliary drainage is almost exclusively associated with beneficial results, such as improved intestinal barrier function in experimental models. However, clinical data in this field are limited, indirect and remain controversial. Most importantly, routine PBD will result in a highly procedure-related complication rate and an increase in operative infectious complications. CONCLUSIONS: PBD should not be performed routinely, unless further improved PBD techniques would become available in clinical studies. Future studies should focus on PBD techniques, and then on the effects of biliary drainage on intestinal mucosa in obstructive jaundice in clinical.

Sodium tanshinone IIA silate inhibits oxygen-glucose deprivation/recovery-induced cardiomyocyte apoptosis via suppression of the NF-κB/TNF-α pathway.

BACKGROUND AND PURPOSE: Inhibition of apoptosis may attenuate the irreversible injury associated with reperfusion. In the current study, we focused on the cytoprotective effects and the underlying mechanism of sodium tanshinone IIA silate (STS) against damage induced by oxygen-glucose deprivation/recovery (OGD/R). in H9c2 cardiomyocytes and the underlying mechanisms. EXPERIMENTAL APPROACH: We used a model of cardiac ischaemia/reperfusion, OGD/R in H9c2 cardiomyocytes, to assess the cardioprotective effects of STS. Apoptosis of cells was measured with Hoechst 33342-based fluorescence microscopy, and annexin V-FITC-based flow cytometry. Caspase-3 and caspase-8 activities and mitochondrial membrane potential were also measured using commercial kits. TNF-α in the cell culture supernatant fractions were measured with sandwich elisa, and protein levels assayed using Western blot. KEY RESULTS: STS inhibited OGD/R-induced apoptosis by suppressing JNK-mediated activation of NF-κB, TNF-α expression, activation of caspase-3 and caspase-8 and the Bax/Bcl-2 ratio. Additionally, positive feedback between NF-κB and TNF-α and amplification of TNF-α were inhibited, suggesting that STS plays a protective role against apoptosis in cardiomyocytes, even upon activation of pro-inflammatory cytokines. Interestingly, the cytoprotective effects of STS on OGD/R-induced apoptosis and promotion of cell survival were attenuated after inhibition of PI3K. CONCLUSION AND IMPLICATIONS: The inhibitory effects of STS on TNF-α and positive feedback signalling of the NF-κB/TNF-α pathways may play important roles in myocardial protection against ischaemia/reperfusion. These protective effects of STS are mediated by suppressing JNK activity through activation of the PI3K-Akt pathway.

Clinical characteristics of non-perianal fistulating Crohn's disease in China: a single-center experience of 184 cases.

BACKGROUND: There is little information of non-perianal fistulating Crohn's disease in the consensus published by the European Crohn's and Colitis Organization in 2006 and 2010. This study was designed to demonstrate the clinical characteristics of non-perianal fistulating Crohn's disease among homogenous Chinese population. METHODS: One-hundred-and-eighty-four patients were retrospectively collected. All of these patients were diagnosed of Crohn's disease between February 2001 and April 2011. RESULTS: The male-to-female ratio was 2.7:1. The most common symptoms at onset were abdominal pain (88.0%), diarrhea (34.7%), and fever (28.3%). The most common disease location and behavior at diagnosis were small bowel (56.0%) and penetrating (51.6%). Among 324 non-perianal fistulae, the most common types were ileocolonic anastomotic (30.9%), terminal ileocutaneous (19.7%), and enteroenteric anastomotic (11.4%). One-hundred-and-thirty- eight (75.0%) patients received antibiotics, and ß-lactam (85.5%) and metronidazole (67.4%) are most frequently used. One-hundred-and-seventy-eight (96.7%) patients suffered 514 surgical operations, and the cumulative surgical rates after 1, 3, and 5 years were 38.0%, 52.2%, and 58.7% respectively. Nine patients died during the follow-up period, and the cumulative survival rates after 1, 3, and 5 years were 97.8%, 96.7%, and 96.2% respectively. CONCLUSIONS: This study displayed the clinical characteristics of non-perianal fistulating Crohn's disease in our center. Large population-based studies are required for further investigation in China.

[Experimental study of liver injury in rats of abdominal infection with abdominal compartment syndrome].

OBJECTIVE: To evaluate the liver injury in rats of abdominal infection complicated with abdominal compartment syndrome(ACS). METHODS: SD rats were divided into four groups, including the sham group, the abdominal infection group, the ACS group, and the abdominal infection plus ACS group (combination group). Rats were sacrificed at 1 h, 6 h, 12 h, 24 h after operation with 6 rats at each time point. Blood specimens were collected for liver function testing. Liver tissues were assessed by pathologically examination with hepatic injury severity scoring(HISS). The expressions of Toll-like receptor 4 (TLR4),TNF-α and IL-6 were examined by reverse transcription- polymerase chain reaction. RESULTS: At 24 h after operation, as compared to the sham group(18.2±1.3) U/L and (105.6±25.5) U/L, ALT and AST increased obviously in the abdominal infection group(68.2±17.5) U/L and (184.6±36.1) U/L, the ACS group (305.2±128.2) U/L and (638.0±104.8) U/L and the combination group (409.2±67.1) U/L and (743.2±250.2) U/L, while the combination group had a higher level as compared to the infection group and the ACS group(all P<0.05). HISS scores were significantly higher in the abdominal infection group(5.0), the ACS group(5.5) and the combination group(7.0) as compared to the sham group(1.5), but no significant differences were found among the three groups at 24 h after operation. Expressions of TLR4, TNF-α and IL-6 were significantly higher in combination group than those in the other three groups. CONCLUSIONS: Liver function can be affected by abdominal infection and ACS. Abdominal infection plus ACS results in more severe liver injury.

[Open abdomen management treatment of liver injury in rats with abdominal compartment syndrome and sepsis].

OBJECTIVE: To evaluate the open and closed management treatment of liver injury in rats with sepsis and abdominal compartment syndrome (ACS). METHODS: The sepsis and ACS rats (n = 72) were randomized divided into two groups. One group used closed management (n = 36), the other accepted the open abdomen management (n = 36). The rats were killed at 1, 6 h, 1, 3, 5, 7 d after operation. Blood was collected for liver function tests. Liver sections assessed pathologically and the expressions of Toll-like receptor 4 (TLR4), tumor necrosis factor (TNF)-α, interleukin (IL)-6, signal transducers actuators of transcription (STAT3) and suppressor of cytokine signaling 3 (SOCS3) of rat livers were examined by RT-PCR. RESULTS: The early stage after operation, TNF-α and IL-6 concentrations, STAT3 expressions in rat liver were higher in open abdomen rats than the closed management ones (P < 0.05). TLR4 and SOCS3 expressions were lower in open abdomen rats than the closed management ones (P < 0.05). Aspartate aminotransferase, alanine aminotransferase levels also was lower in open abdomen ones (P < 0.05). CONCLUSIONS: The randomized study demonstrates that open abdomen management could improve liver regeneration in the early stage after operation. Also open abdomen could reduce inflammatory response by reducing TLR4 expressions.

[Maintenance effect of polyglycosides of Tripterygium wilfordii on remission in postoperative Crohn disease].

OBJECTIVE: To observe the maintenance effect of polyglycosides of Tripterygium wilfordii (GTW) on remission in postoperative Crohn disease (CD). METHODS: From 2005 to 2007, 45 adult cases of postoperative Crohn disease were randomly divided into two groups, GTW group and mesalazine group, which received GTW and mesalazine treatment respectively. CD activity index (CDAI) and clinical markers were collected at 0, 3, 6, 12 months or at the onset of symptoms. Ileocolonoscopy was performed at the end of the trial (1 year after operation) or at the onset of symptoms, and recurrence score were recorded. RESULTS: No clinical recurrence was ascertained in both groups at 3 months. Four patients (18.2%) in GTW group relapsed and 5 (21.7%) in mesalazine group relapsed at 6 months (P=0.530). Seven patients (31.8%) in GTW group and 9 (39.1%) in mesalazine group relapsed at one year (P=0.421). Ten patients (45.5%) in GTW group had endoscopic recurrence compared with 14 (60.9%) in mesalazine group at one year(P=0.231). There were no significant differences between two groups. CONCLUSION: GTW is similar to mesalazine in maintenance of remission of postoperative Crohn disease.

[Clinical characteristics and surgical treatment of Crohn disease complicated with intra-abdominal abscess].

OBJECTIVE: To elucidate the clinical characteristics and surgical treatment of intra-abdominal abscess in patients with Crohn disease(CD). METHODS: Clinical data of 39 patients with CD complicated with intra-abdominal abscess from 2000 to 2005 were analyzed retrospectively. RESULTS: The cumulative incidence of intra-abdominal abscesses was 27.5%. Of the 39 CD patients with abscess, 61.5% had surgery the time of present study. The mean age of the patients with abscesses was (34.7+/- 12.3) years, and the duration of illness from the onset of CD until development of an abscess was (0-22) years,with 5 years of the average duration. In terms of location of abscess, it occurred most often on the right side (76.9%), especially near the site of anastomosis (48.7%). Most patients (34 cases, 94.4%) were treated with surgical drainage and intestinal resection. CONCLUSIONS: Abscess formation was noted in 27.5% of patients with CD, with nearly half of abscesses occurring near the anastomotic site. The mean age of patients with abscess was 35 years, with 5 years of the duration of illness. Most abscesses were treated with operative drainage and intestinal resection.

[Management of tertiary peritonitis in the patients complicated with intestinal fistula].

OBJECTIVE: To investigate the etiology and management of tertiary peritonitis in the patients with intestinal fistula. METHODS: One hundred and fifty-three cases of intestinal fistula complicated with tertiary peritonitis were reviewed. The microbiological characteristics, treatment Methods and outcomes were analyzed. RESULTS: There were 114 males and 39 females with a mean age of (42+/- 19) years. The main causes of intestinal fistula included gastrointestinal surgery (40.5%), trauma (31.4%) and severe pancreatitis (14.4%), etc. The most common cultured bacteria of 157 specimens from 79 patients with tertiary peritonitis were Escherichia coli (24.2%), Pseudomonas aeruginosa (12.1%), Staphylococcus aureus (10.8%), Enterobacter cloacae (10.2%), Klebsiella pneumoniae (8.3%). Debridement of the necrotic tissues, drainage of the abscess, continuous rinsing plus negative pressure drainage and antibiotics treatment were performed in 52 cases. Nineteen patients only changed from simple tube drainage to continuous rinsing plus negative pressure drainage. Twenty- eight patients changed to continuous rinsing plus negative pressure drainage and received antibiotics as well. Thirty- six patients received antibiotics and ecoimmune nutrition, while 18 patients only received ecoimmun nutrition. CONCLUSIONS: Intestinal fistula complicated with tertiary peritonitis was mainly caused by residual infectious focus and inappropriate drainage. The rational treatments include reoperation for debridement of the necrotic and infectious tissues, changing drainage to continuous rinsing plus negative pressure drainage, appropriate usage of antibiotics, and ecoimmune nutrition.

[Diagnosis and management of Crohn disease complicated with gastrointestinal fistulae].

OBJECTIVE: To investigate the diagnosis and treatment of patients with Crohn disease (CD) complicated with gastrointestinal fistulae. METHODS: Clinical data of sixty-two cases with CD complicated with gastrointestinal fistula e from 1978 to 2004 were analyzed. RESULTS: These were 68 external fistulae in 6 2 patients including recurrent fistulae in 6 cases, internal fistulae in 8 cases . Twenty- seven fistulae were located in the terminal ileum and 21 fistulae wer e located in ileocolic anastomosis site. The main surgery included 14 ileocecal resections with primary anastomosis and 26 resections of original ileocolic anastomosis with fistula and re-anastomosis. The incidence of recurrence was lower (15.4% ) in patients with postoperative medication including sulfasalazine and immunomodulator than that (34.8% ) in patients without postoperative immunomodulator,but the recurrence time was longer [(40+/- 17) months] in patients with postoperative medication than that [(8+/- 3)months] in the patients without postoperative specific medication. CONCLUSIONS: Most CD fistulae are external fistulae,most of the external fistulae are treated by resection of the fistula and anastomosis. Specific medication including sulfasalazine,mesalamine and immunomodulators should be used to prevent postoperative complications and CD recurrence.