Effects of S-adenosylmethionine on AfsKRS regulation in pristinamycin biosynthesis in Streptomyces pristinaespiralis.

In Streptomyces pristinaespiralis, AfsKRS system has differential regulation for PI and PII component biosynthesis of pristinamycin, but it is unknown whether S-adenosylmethionine (SAM) plays an important role in the AfsK-AfsR-AfsS signal transduction cascade during pristinamycin production. The possible target of exogenous SAM in the AfsKRS system and the biological role of SAM during the production of PI and PII were investigated using three mutantsΔafsK,ΔafsR andΔafsS defective in signal cascade pathway of AfsKRS. It was found that external SAM had a significant activation of PI production (1.85-fold increase) but had no obvious effect on PII production in the original strain F618 with the normal response of AfsKRS regulation. Addition of SAM resulted in a similar increase in pristinamycin yield in the mutant with defective afsK or afsR, but induced more crucial activation of PI biosynthesis than PII biosynthesis both in ΔafsK (1.65-fold and 1.15-fold increase respectively) and ΔafsR (1.27-fold and 1.09-fold increase respectively). Exogenous SAM only significantly enhanced PII production in ΔafsS (1.1-fold increase). These results could provide valuable insights into the regulatory function of the AfsKRS system in S. pristinaespiralis.

Characterization of PA12/HA composite scaffolds based on selective laser sintering.

Composite scaffolds have been extensively studied in bone tissue engineering, which can achieve excellent properties that cannot be obtained by a single material. In this study, the effect of hydroxyapatite (HA) on the reliability of polyamide 12 (PA12) scaffold for bone graft was explored in terms of mechanical and biological properties. Thermal properties testing showed that no physical or chemical reaction occurs in the prepared PA12/HA composite powders. Further, compression experiments showed that adding a small amount of HA promoted the mechanical properties of the scaffold, while excessive HA results in agglomeration and impairs the PA12/HA scaffold. For the scaffolds with the porosity of 65%, the 96% PA12/4% HA scaffold has a 7.3% higher yield strength and a 13.5% higher compressive modulus than the pure PA12 scaffold while the strength of the 88% PA12/12% HA scaffold decreases by 35.6%. Furthermore, contact angle and CCK-8 tests confirmed that 96% PA12/4% HA scaffold effectively improved the hydrophilicity and biocompatibility of the scaffold. Its OD value on the 7th day is 0.949, which is significantly higher than that of other groups. In summary, PA12/HA composites have good mechanical properties and biocompatibility, which can be used as an effective strategy in bone tissue engineering.

Mechanical properties and failure mechanisms of polyamide 12 gradient scaffolds developed with selective laser sintering.

Developing a functional gradient scaffold compatible with the fantastic biological and mechanical properties of natural bone tissue is imperative in bone tissue engineering. In this work, the stretch-dominated (cubical and circular) and bending-dominant (diamond and gyroid) pore styles were employed to design custom-graded scaffolds based on the curve interference method and then were fabricated by selective laser sintering (SLS) using polyamide 12 (PA12) powder. Subsequently, the mechanical behavior, failure mechanism, and energy absorption performance of porous structures were investigated via compression experiments and finite element (FE) simulation. The results indicated that the stretch-dominated radial gradient structures entire exhibited transverse shear failure and the bending-dominant radial gradient structures whole exhibited progressive destruction, while all of the axial gradient scaffolds suffered a predictable layer-by-layer fracture. Among them, the bending-dominated radial gradient structure of gyroid had been proven to sustain stronger deformability and energy absorption capacity. Meanwhile, the FE method powerfully predicted the mechanical behavior of the scaffold, and this research thereby possessed significant implications for the development of bone tissue engineering.

Development of the degradation bacteria in household food waste and analysis of the microbial community in aerobic composting.

By screening the strains and testing different combinations of diverse bacteria, we developed a compound bacterial agent composed of 5 g Bacillus amyloliquefaciens (B2), 10 g Pseudomonas aeruginosa (F4), 5 g Paenibacillus lautus (303), and 10 ml composite strains (DOD) for the degradation of household food waste (HFW). The final mass loss of HFW in aerobic composting with the compound bacteria agent B2+F4+303+DOD (group C) was 84.52%, increased by 20.83% over that loss in natural composting (group A). Analysis of 16S rRNA high-throughput sequencing showed that the phyla in group A and group C mainly included Firmicutes, Proteobacteria, and Cyanobacteria. At the genus level, Pediococcus was the dominant genus in group A, of which the microbial community performed better in maintaining a stable microbial system in the later stage of composting, while Weissella accounted for a larger proportion of group C, which acted well in reducing the final mass of composting. Ochrobactrum was closely related to the removal of odors in the early stage of group C. The relative abundance of compound bacterial agents was always at a rather low level, suggesting that it affected the composting process by changing the proportion of dominant bacteria in the compost.

Functional investigation of AfsKRS regulatory system for pristinamycin biosynthesis in Streptomyces pristinaespiralis.

Three genes encoding AfsK, AfsR, AfsS homologues in Streptomyces pristinaespiralis were studied, respectively, to investigate regulatory role of AfsKRS system for pristinamycin biosynthesis. Transcription change and gene inactivation analysis indicated that these genes had active transcription and positive regulation for the improvement of pristinamycin production in S. pristinaespiralis. The analysis of AfsKRS-defective mutagenesis indicated that there might be a positive correlation between the product of afsK and pristinamycin I biosynthesis, and a negative correlation to pristinamycin II biosynthesis. However, both afsR and afsS might have negative correlation to pristinamycin I production and positive correlation to pristinamycin II production. The effects on pristinamycin production of AfsKRS disruptants by protein kinase inhibitor K252a indicated that AfsR, both not AfsK and AfsS, was the inhibition target of K252a in S. pristinaespiralis, and AfsR should serve as a pleiotropic regulator to have differential regulation on biosynthesis of pristinamycin I and II components. Based on above study, it might be deduced that different signal transduction patterns via AfsK, AfsR, AfsS of AfsKRS system should be involved in respective regulation for biosynthesis of pristinamycin I and II in S. pristinaespiralis. In conclusion, the investigation could give some valuable clues for exploring furtherly regulatory function of AfsKRS system in S. pristinaespiralis.

A novel carbonyl reductase with anti-Prelog stereospecificity for the production of t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate.

A novel gene (crc1) from Candida boidinii was cloned and then overexpressed in a recombinant strain BL21(DE3)/pET30a-crc1 of Escherichia coli. The resulting carbonyl reductase was prepared through fermentations using the recombinant strain. The purified enzyme showed an NADPH-dependent activity and specific activity was 4.65 U/mg using t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (ATS-6) as substrate. The enzyme was optimally active at 35 °C and pH 7, respectively. The apparent K m and V max of the enzyme for ATS-6 are 1.5 mM and 21.1 µmol/min mg, respectively, indicating excellent anti-Prelog stereospecificity. Under the optimum condition, t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate (ATS-7) was prepared with the enzyme with high d.e. value (99.9%) and good conversion (94%) in 4 h, indicating high stereoselectivity and conversion efficiency in biotransformation of ATS-6 to ATS-7.

Curcumin inhibits the proliferation and induces apoptosis in HT-29 cell lines through a reactive oxygen species (ROS)-dependent mechanism.

Curcumin, a natural pigment extracted from Curcuma longa, has anti-carcinogenic activities in many cancer cell lines. The molecular mechanism of apoptosis induced by curcumin are still unknown. In the current study, we investigated the roles of reactive oxygen species in curcumin stimulated apoptosis in HT-29 cells. Curcumin significantly reduced cell viability, induced apoptosis, activated caspase-3 activity and stimulated concentration-dependent release of ROS. Inhibition of ROS generation by scavengers suppressed apoptosis and Bcl-2 expression induced by curcumin, indicating the critical roles of ROS in the apoptotic process. However, caspase-3 inhibitor (z-VAD-FMK) couldn't completely inhibit the curcumin induced apoptosis, indicating ROS mediated apoptosis may be caspase-independent. Together, our findings showed that ROS played significant roles in the apoptosis induced by curcumin in HT-29 cells.

Nup62, associated with spindle microtubule rather than spindle matrix, is involved in chromosome alignment and spindle assembly during mitosis.

An increasing number of the active mitotic functions of nucleoporins in the distinct steps of mitosis have been assigned over the past few years. As one of FG-repeats containing nucleoporins, Nup62 has been found to be involved in nuclear transport, cell migration, virus infection, and cell cycle regulation. However, the role and mechanism of Nup62 in mitotic regulation have not been fully revealed. In this paper, it was revealed that a fraction of Nup62 was associated with mitotic spindle microtubule instead of spindle matrix, and the localization of Nup62 in the mitotic spindle depended on its three coiled-coil domains rather than Crm1, although Nup62 strongly interacted with Crm1 during mitosis. Moreover, depletion of Nup62 by small interference of RNA seriously induced the defects of chromosome alignment and spindle assembly although the bipolar spindle was observed in most of the Nup62 knock-down cells. Notably, congression of polar chromosome defect was observed in more than 30% of Nup62 knock-down cells. These findings revealed that Nup62 was a novel mitotic spindle associated nucleoporin and involved in chromosome alignment and spindle assembly.

C. butyricum lipoteichoic acid inhibits the inflammatory response and apoptosis in HT-29 cells induced by S. aureus lipoteichoic acid.

Lipoteichoic acid (LTA) is one of microbe-associated molecular pattern (MAMP) molecules of gram-positive bacteria. In this study, we demonstrated that Clostridium butyricum LTA (bLTA) significantly inhibited the inflammatory response and apoptosis induced by Staphylococcus aureus LTA (aLTA) in HT-29 cells. aLTA stimulated the inflammatory responses by activating a strong signal transduction cascade through NF-κB and ERK, but bLTA did not activate the signaling pathway. bLTA pretreatment inhibited the activation of the NF-κB and ERK signaling pathway induced by aLTA. The expression and release of cytokines such as IL-8 and TNF-α were also suppressed by bLTA pretreatment. aLTA treatment induced apoptosis in HT-29 cells, but bLTA did not affect the viability of the cells. Further study indicated that bLTA inhibited apoptosis in HT-29 cells induced by aLTA. These results suggest that bLTA may act as an aLTA antagonist and that an antagonistic bLTA may be a useful agent for suppressing the pro-inflammatory activities of gram-positive pathogenic bacteria.

Different effects of lipoteichoic acid from C. butyricum and S. aureus on inflammatory responses of HT-29 cells.

Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria and represents one of the most critical microbe-associated molecular pattern (MAMP) molecules. In this study, we isolated and purified LTA from Clostridium butyricum (bLTA) and compared its effects on the inflammatory responses of HT-29 cells with those of LTA from Staphylococcus aureus (aLTA). We also compared the effects of bLTA and aLTA on cell growth, proliferation, and apoptosis. The results showed that the length and saturation degree of the acyl chains in the two LTA molecules were obviously different. aLTA stimulated the phosphorylation of p65 and activated the NF-κB signaling pathway, inducing the expression and secretion of cytokines. Moreover, aLTA also inhibited the growth and proliferation of HT-29 cells and induced cell apoptosis. However, bLTA had no significant effects on the NF-κB signaling pathway in HT-29 cells and did not stimulate cellular inflammatory responses or induce apoptosis. These differences in activity may result from the different lengths and saturation degrees of the acyl fatty acid chains of the two LTA molecules. These differences may also account for the distinct effects elicited by probiotic bacteria and pathogenic bacteria on host cells.

Spatial Compartmentalization Specializes the Function of Aurora A and Aurora B.

Aurora kinase A and B share great similarity in sequences, structures, and phosphorylation motif, yet they show different localizations and play distinct crucial roles. The factors that determine such differences are largely unknown. Here we targeted Aurora A to the localization of Aurora B and found that Aurora A phosphorylates the substrate of Aurora B and substitutes its function in spindle checkpoint. In return, the centrosome targeting of Aurora B substitutes the function of Aurora A in the mitotic entry. Expressing the chimera proteins of the Auroras with exchanged N termini in cells indicates that the divergent N termini are also important for their spatiotemporal localizations and functions. Collectively, we demonstrate that functional divergence of Aurora kinases is determined by spatial compartmentalization, and their divergent N termini also contribute to their spatial and functional differentiation.

Phosphorylation of Crm1 by CDK1-cyclin-B promotes Ran-dependent mitotic spindle assembly.

Mitotic spindle assembly in animal cells is orchestrated by a chromosome-dependent pathway that directs microtubule stabilization. RanGTP generated at chromosomes releases spindle assembly factors from inhibitory complexes with importins, the nuclear transport factors that facilitate protein import into the nucleus during interphase. In addition, the nuclear export factor Crm1 has been proposed to act as a mitotic effector of RanGTP through the localized assembly of protein complexes on the mitotic spindle, notably at centrosomes and kinetochores. It has been unclear, however, how the functions of nuclear transport factors are controlled during mitosis. Here, we report that human Crm1 is phosphorylated at serine 391 in mitosis by CDK1-cyclin-B (i.e. the CDK1 and cyclin B complex). Expression of Crm1 with serine 391 mutated to either non-phosphorylated or phosphorylation-mimicking residues indicates that phosphorylation directs the localization of Crm1 to the mitotic spindle and facilitates spindle assembly, microtubule stabilization and chromosome alignment. We find that phosphorylation of Crm1 at serine 391 enhances its RanGTP-dependent interaction with RanGAP1-RanBP2 and promotes their recruitment to the mitotic spindle. These results show that phosphorylation of Crm1 controls its molecular interactions, localization and function during mitosis, uncovering a new mechanism for the control of mitotic spindle assembly by CDK1-cyclin-B. We propose that nuclear transport factors are controlled during mitosis through the selection of specific molecular interactions by protein phosphorylation.