Cai-Yun Sun

Antimicrobial peptaibols, novel suppressors of tumor cells, targeted calcium-mediated apoptosis and autophagy in human hepatocellular carcinoma cells

BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers in the world which is highly chemoresistant to currently available chemotherapeutic agents. Thus, novel therapeutic targets are needed to be sought for the successful treatment of HCC. Peptaibols, a family of peptides synthesized non-ribosomally by the Trichoderma species and other fungi, exhibit antibiotic activities against bacteria and fungi. Few studies recently showed that peptaibols exerted cytotoxicity toward human lung epithelial and breast carcinoma cells. However, the mechanism involved in peptaibol-induced cell death remains poorly understood.ResultsHere, we showed that Trichokonin VI (TK VI), a peptaibol from Trichoderma pseudokoningii SMF2, induced growth inhibition of HCC cells in a dose-dependent manner. It did not obviously impair the viability of normal liver cells at lower concentration. Moreover, the suppression of cell viability resulted from the programmed cell death (PCD) with characteristics of apoptosis and autophagy. An influx of Ca2+ triggered the activation of μ-calpain and proceeded to the translocation of Bax to mitochondria and subsequent promotion of apoptosis. On the other hand, typically morphological characteristics consistent with autophagy were also observed by punctate distribution of MDC staining and the induction of LC3-II, including extensive autophagic vacuolization and enclosure of cell organelles by these autophagosomes. More significantly, specific depletion of Bak expression by small RNA interfering (siRNA) could partly attenuate TK VI-induced autophagy. However, siRNA against Bax led to increased autophagy.ConclusionTaken together, these findings showed for the first time that peptaibols were novel regulators involved in both apoptosis and autophagy, suggesting that the class of peptaibols might serve as potential suppressors of tumor cells.

Antimicrobial peptaibols from Trichoderma pseudokoningii induce programmed cell death in plant fungal pathogens

Antibiosis is one of the widespread strategies used by Trichoderma spp. against plant fungal pathogens, the mechanism of which, however, remains poorly understood. Peptaibols are a large family of antimicrobial peptides produced by Trichoderma spp. Our previous study showed that trichokonins, a type of peptaibol from Trichoderma pseudokoningii SMF2, exhibited antibiotic activities against plant fungal pathogens. In this study, we first demonstrated that trichokonin VI (TK VI) induced extensive apoptotic programmed cell death in plant fungal pathogens. For a deeper insight into the apoptotic mechanism involved in the action of TK VI, Fusarium oxysporum was used as a model. Cells of F. oxysporum treated with TK VI showed apoptotic hallmarks, such as exposure of phosphatidylserine, the appearance of reactive oxygen species and fragmentation of nuclear DNA. Moreover, TK VI-treated cells exhibited an accumulation of cytoplasmic vacuoles with loss of the mitochondrial transmembrane potential, and this process was independent of metacaspases. Therefore, TK VI induces metacaspase-independent apoptotic cell death in F. oxysporum. This represents what is believed to be the first report to reveal the antibiotic mechanism of peptaibols against plant fungal pathogens.

Production of novel angiotensin I-converting enzyme inhibitory peptides by fermentation of marine shrimp Acetes chinensis with Lactobacillus fermentum SM 605

Acetes chinensis is an underutilized shrimp species thriving in Bo Hai Gulf of China. Its hydrolysate digested with protease SM98011 has been previously shown to have high angiotensin I-converting enzyme (ACE) inhibitory activity (He et al., J Pept Sci 12:726–733, 2006). In this article, A. chinensis were fermented by Lactobacillus fermentum SM 605 and the fermented sauce presented high ACE inhibitory activity. The minimum IC50 value (3.37 ± 0.04 mg/mL) was achieved by response surface methodology with optimized process parameters such as fermentation time of 24.19 h, incubation temperature at 38.10°C, and pH 6.12. Three ACE inhibitory peptides are purified by ultrafiltration, gel filtration, and reverse-phase high performance liquid chromatography. Identified by mass spectrometry, their amino acid sequences are Asp-Pro, Gly-Thr-Gly, and Ser-Thr, with IC50 values of 2.15 ± 0.02, 5.54 ± 0.09, and 4.03 ± 0.10 μM, respectively. Also, they are all novel ACE inhibitory peptides. Compared with protease digestion, fermentation is a simpler and cheaper method to produce ACE inhibitory peptides from shrimp A. chinensis.

Purification and enzymatic characterization of two β-endoxylanases from Trichoderma sp. K9301 and their actions in xylooligosaccharide production

Trichoderma sp. K9301 secreting endoxylanases with an activity of 2836 U/g (dry weight) was screened for XOs production. Two acidic beta-endoxylanases EX1 (30.1 kDa) and EX2 (20.1 kDa) were purified from crude extract of the strain K9301 in solid fermentation. Action modes of EX1 and EX2 towards XOs showed similar hydrolysis characters to endoxylanases belonging to glycosyl hydrolase family 10 and 11, respectively. EX1 exhibited better affinity but lower hydrolytic efficiency than EX2 to xylans from beechwood, birchwood, and oat-spelt. They had synergistic action on xylan hydrolysis. The optimum condition to prepare XOs from corncobs was obtained as 10 mg/ml corncob xylan incubated with 10 U/mg crude enzymes at 50 degrees C for 3 h. The yield of XOs reached 43.3%, and only a little amount of xylose (3.1%) was simultaneously produced, suggesting the good potential of strain K9301 in XOs production.

Gene Cloning, Expression and Characterization of a Novel Xylanase from the Marine Bacterium, Glaciecola mesophila KMM241

Marine xylanases are rather less studied compared to terrestrial xylanases. In this study, a new xylanase gene, xynB, was cloned from the marine bacterium, Glaciecola mesophila KMM241, and expressed in Escherichia coli. xynB encodes a multi-domain xylanase XynB of glycoside hydrolase (GH) family 8. The recombinant XynB comprises an N-terminal domain (NTD) with unknown function and a catalytic domain, which is structurally novel among the characterized xylanases of GH family 8. XynB has the highest identity (38%) to rXyn8 among the characterized xylanases. The recombinant XynB showed maximal activity at pH 6–7 and 35 °C. It is thermolabile and salt-tolerant. XynB is an endo-xylanase that demands at least five sugar moieties for effective cleavage and to hydrolyze xylohexaose and xylopentaose into xylotetraose, xylotriose and xylobiose. NTD was expressed in Escherichia coli to analyze its function. The recombinant NTD exhibited a high binding ability to insoluble xylan and avicel and little binding ability to chitosan and chitin. Since the NTD shows no obvious homology to any known carbohydrate-binding module (CBM) sequence in public databases, XynB may contain a new type of CBM.

Deep RNA sequencing reveals a high frequency of alternative splicing events in the fungus Trichoderma longibrachiatum

BackgroundAlternative splicing is crucial for proteome diversity and functional complexity in higher organisms. However, the alternative splicing landscape in fungi is still elusive.ResultsThe transcriptome of the filamentous fungus Trichoderma longibrachiatum was deep sequenced using Illumina Solexa technology. A total of 14305 splice junctions were discovered. Analyses of alternative splicing events revealed that the number of all alternative splicing events (10034), intron retentions (IR, 9369), alternative 5’ splice sites (A5SS, 167), and alternative 3’ splice sites (A3SS, 302) is 7.3, 7.4, 5.1, and 5.9-fold higher, respectively, than those observed in the fungus Aspergillus oryzae using Illumina Solexa technology. This unexpectedly high ratio of alternative splicing suggests that alternative splicing is important to the transcriptome diversity of T. longibrachiatum. Alternatively spliced introns had longer lengths, higher GC contents, and lower splice site scores than constitutive introns. Further analysis demonstrated that the isoform relative frequencies were correlated with the splice site scores of the isoforms. Moreover, comparative transcriptomics determined that most enzymes related to glycolysis and the citrate cycle and glyoxylate cycle as well as a few carbohydrate-active enzymes are transcriptionally regulated.ConclusionsThis study, consisting of a comprehensive analysis of the alternative splicing landscape in the filamentous fungus T. longibrachiatum, revealed an unexpectedly high ratio of alternative splicing events and provided new insights into transcriptome diversity in fungi.

Effects of Different Buffers on the Thermostability and Autolysis of a Cold-Adapted Protease MCP-01

A cold-adapted protease MCP-01 was obtained from deep-sea psychrotrophic bacterium Pseudoaltermonas sp. SM9913. The effects of four different buffers, all at 50 mmol/l concentration, on its thermostability and autolysis were studied. The autolysis process of MCP-01 was studied by capillary electrophoresis. The thermostability of MCP-01 increased successively in the following order: carbonate < Tris < phosphate < borate. The optimum temperature for casein hydrolysis also increased in the same order. This suggested that the conformation of MCP-01 was flexible and its autolytic susceptibility was affected by some factors in the buffers such as charge and ionic species. The results also showed that different buffers, in addition to affecting the autolysis speed, gave different patterns of autolysis products. In carbonate buffer, Tris buffer, phosphate buffer and borate buffer, the autolysis patterns of MCP-01 were different. These results suggested that protease MCP-01 probably have different conformations in different buffers, thus exposing different autolysis sites on the enzyme surface. In addition, the loss of activity correlated with the speed of autolysis in the four different buffers, showing that autolysis may be a reason for the low thermostability of the enzyme.

High-performance mesoporous LiFePO4 from Baker's yeast

Based on the biomineralization assembly concept, a simple and inexpensive biomimetic sol-gel method is found to synthesize high-performance mesoporous LiFePO(4) (HPM-LFP). The key step of this approach is to apply Bakers yeast cells as both a structural template and a biocarbon source. The formation mechanism of ordered hierarchical mesoporous network structure is revealed by characterizing its morphology and microstructure. The HPM-LFP exhibits outstanding electrochemical performances. The HPM-LFP has a high discharge capacity (about 153 mAh g(-1) at a 0.1 C rate), only 2% capacity loss from the initial value after 100 cycles at a current density of 0.1 C. This simple and potentially universal design strategy is currently being pursued in the synthesis of an ideal cathode-active material for high power applications.

A Novel Approach to Estimate In Vitro Antibacterial Potency of Chinese Medicine Using a Concentration-Killing Curve Method

The antibacterial pharmacodynamics against E. coli of Chinese medicine (CM) Rhizoma coptidis (Coptis Root) and its formula Sanhuang, and the control antibiotics enoxacin, were analyzed by a concentration-killing curve (CKC) approach, and the novel parameters BC50 and r for antibacterial potency were proposed. Using the agar plate method, about 400 cells of E. coli were evenly inoculated into LB agar plates containing a series of different concentrations of CM or antibiotic, and after a 24 hour incubation at 37 degrees C, all the viable colonies were enumerated. This resulted in a sigmoid concentration-killing curve , in which No, that could be closely fitted (R2 > 0.9) with the function: N = 1 + e(r(x-BC50))/N0 in which N0, BC50 and r represent meaningfully inoculums size, median bactericidal concentration, and bactericidal intensity, respectively. N modeled the survival of colony-forming units on each plate (CFU/plate) in a concentration series x of the drug. The CKC was symmetrical about its single inflexion (BC50, N0/2). Therefore theoretically, 2BC50 can replace MBC (minimum bactericidal concentration). BC1 = BC50 + r/ln(N0-1), the drug concentration at r which only one colony survived, was the least critical value of MBC in CKC. The parameters 2BC50 and BC1 agreed more closely with the definition of MBC, and were little affected by either the biochemical basis of the antibacterial or the inoculums size (200-400 CFU/plate), and were determined by a multi-point curve. As a result, these were more accurate, reproducible and practical as metrics than was the endpoint of MBC. The two-dimensional CKC, involving BC50 and r, captures the intrinsic dynamics of the antibacterial effect of CM/strain versus concentration, and it is consistent with the Logistic equation of the bacterial growth curve in the format. This verified approach has considerable value as a tool for the accurate and proper administration of CM. The CKC of CM, different from that of antibiotics, is likely to be the resultant force of each ingredient in certain CM, which provides a clue to solve the problem of antibiotic resistance.

Functional outcome of en bloc resection of a giant cell tumour of the distal radius and arthrodesis of the wrist and distal ulna using an ipsilateral double barrel segmental ulna bone graft combined with a modified Sauve-Kapandji procedure:

Giant cell tumour of the distal radius is a locally aggressive lesion. In this study, we performed a wrist arthrodesis reconstruction with an ipsilateral double barrel segmental ulnar bone graft combined with a modified Sauve-Kapandji procedure for a giant cell tumour of the distal radius. From January 2007 to September 2013, we followed eight patients for a mean duration of 36 months. One patient developed a recurrence and was treated by amputation; the other seven patients achieved radiological union in about 8 months. There was no wrist instability, deformation or dislocation; the mean range of motion of the forearm achieved 75° of supination and 70° of pronation. The patients could recover reasonable grip strength. This new operative procedure can excise the tumour with a low rate of recurrence, fewer functional deficits and fewer complications than reported for other procedures. Level of evidence: IV, therapeutic