Zhangli Hu

Study on interaction between curcumin and pepsin by spectroscopic and docking methods

The interaction between curcumin and pepsin was investigated by fluorescence, synchronous fluorescence, UV-vis absorption, circular dichroism (CD), and molecular docking. Under physiological pH value in stomach, the fluorescence of pepsin can be quenched effectively by curcumin via a combined quenching process. Binding constant (Ka) and binding site number (n) of curcumin to pepsin were obtained. According to the theory of Försters non-radiation energy transfer, the distance r between pepsin and curcumin was found to be 2.45 nm within the curcumin-pepsin complex, which implies that the energy transfer occurs between curcumin and pepsin, leading to the quenching of pepsin fluorescence. Fluorescence experiments also suggest that curcumin is located more closely to tryptophan residues than tyrosine residues. CD spectra together with UV-vis absorbance studies show that binding of curcumin to pepsin results in the extension of peptide strands of pepsin with loss of some β-sheet structures. Thermodynamic parameters calculated from the binding constants at different temperatures reveal that hydrophobic force plays a major role in stabilizing the curcumin-pepsin complex. In addition, docking results support the above experimental findings and suggest the possible hydrogen bonds of curcumin with Thr-77, Thr-218, and Glu-287 of pepsin, which help further stabilize the curcumin-pepsin complex.

Investigation on interaction between Ligupurpuroside A and pepsin by spectroscopic and docking methods

Ligupurpuroside A is one of the major glycoside in Ku-Din-Cha, a type of Chinese functional tea. In order to better understand its digestion and metabolism in humans, the interaction between Ligupurpuroside A and pepsin has been investigated by fluorescence spectra, UV-vis absorption spectra and synchronous fluorescence spectra along with molecular docking method. The fluorescence experiments indicate that Ligupurpuroside A can effectively quench the intrinsic fluorescence of pepsin through a combined quenching way at the low concentration of Ligupurpuroside A, and a static quenching procedure at the high concentration. The binding constant, binding sites of Ligupurpuroside A with pepsin have been calculated. The thermodynamic analysis suggests that non-covalent reactions, including electrostatic force, hydrophobic interaction and hydrogen bond are the main forces stabilizing the complex. According to the Försters non-radiation energy transfer theory, the binding distance between pepsin and Ligupurpuroside A was calculated to be 3.15 nm, which implies that energy transfer occurs between pepsin and Ligupurpuroside A. Conformation change of pepsin was observed from UV-vis absorption spectra and synchronous fluorescence spectra under experimental conditions. In addition, all these experimental results have been validated by the protein-ligand docking studies which show that Ligupurpuroside A is located in the cleft between the domains of pepsin.

Genome-wide long non-coding RNA screening, identification and characterization in a model microorganism Chlamydomonas reinhardtii

Microalgae are regarded as the most promising biofuel candidates and extensive metabolic engineering were conducted but very few improvements were achieved. Long non-coding RNA (lncRNA) investigation and manipulation may provide new insights for this issue. LncRNAs refer to transcripts that are longer than 200 nucleotides, do not encode proteins but play important roles in eukaryotic gene regulation. However, no information of potential lncRNAs has been reported in eukaryotic alga. Recently, we performed RNA sequencing in Chlamydomonas reinhardtii, and obtained totally 3,574 putative lncRNAs. 1440 were considered as high-confidence lncRNAs, including 936 large intergenic, 310 intronic and 194 anti-sense lncRNAs. The average transcript length, ORF length and numbers of exons for lncRNAs are much less than for genes in this green alga. In contrast with human lncRNAs of which more than 98% are spliced, the percentage in C. reinhardtii is only 48.1%. In addition, we identified 367 lncRNAs responsive to sulfur deprivation, including 36 photosynthesis-related lncRNAs. This is the first time that lncRNAs were explored in the unicellular model organism C. reinhardtii. The lncRNA data could also provide new insights into C. reinhardtii hydrogen production under sulfur deprivation.

Artificial miRNA inhibition of phosphoenolpyruvate carboxylase increases fatty acid production in a green microalga Chlamydomonas reinhardtii

BackgroundNutrient limitation, such as nitrogen depletion, is the most widely used method for improving microalgae fatty acid production; however, these harsh conditions also inhibit algal growth significantly and even kill cells at all. To avoid these problems, we used artificial microRNA (amiRNA) technology as a useful tool to manipulate metabolic pathways to increase fatty acid contents effectively in the green microalga Chlamydomonas reinhardtii. We down-regulated the expression of phosphoenolpyruvate carboxylase (PEPC), which catalyzes the formation of oxaloacetate from phosphoenolpyruvate and regulates carbon flux.ResultsamiRNAs against two CrPEPC genes were designed and transformed into Chlamydomonas cells and amiRNAs were induced by heat shock treatment. The transcription levels of amiRNAs increased 16–28 times, resulting in the remarkable decreases of the expression of CrPEPCs. In the end, inhibiting the expression of the CrPEPC genes dramatically increased the total fatty acid content in the transgenic algae by 28.7–48.6%, which mostly increased the content of C16–C22 fatty acids. Furthermore, the highest content was that of C18:3n3 with an average increase of 35.75%, while C20–C22 fatty acid content significantly increased by 85–160%.ConclusionsOverall our results suggest that heat shock treatment induced the expression of amiRNAs, which can effectively down-regulate the expression of CrPEPCs in C. reinhardtii, resulting in an increase of fatty acid synthesis with the most significant increase occurring for C16 to C22 fatty acids.

Microbial community structures and dynamics in the O3/BAC drinking water treatment process.

Effectiveness of drinking water treatment, in particular pathogen control during the water treatment process, is always a major public health concern. In this investigation, the application of PCR-DGGE technology to the analysis of microbial community structures and dynamics in the drinking water treatment process revealed several dominant microbial populations including: α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Bacteroidetes, Actinobacteria Firmicutes and Cyanobacteria. α-Proteobacteria and β-Proteobacteria were the dominant bacteria during the whole process. Bacteroidetes and Firmicutes were the dominant bacteria before and after treatment, respectively. Firmicutes showed season-dependent changes in population dynamics. Importantly, γ-Proteobacteria, which is a class of medically important bacteria, was well controlled by the O3/biological activated carbon (BAC) treatment, resulting in improved effluent water bio-safety.

Interaction mechanism of pepsin with a natural inhibitor gastrodin studied by spectroscopic methods and molecular docking

The interaction of gastrodin with pepsin has been investigated by enzyme activity assay, fluorescence, UV–Visible, circular dichroism spectra, and molecular docking. The pepsin activity results suggest that gastrodin is an inhibitor of pepsin. The fluorescence experiments show that gastrodin can quench the fluorescence of pepsin via a static quenching process. The thermodynamic analysis suggests that hydrophobic interaction is the main force between pepsin and gastrodin. UV–Visible and circular dichroism spectra studies suggest that the binding of gastrodin leads to a loosening and unfolding of pepsin backbone with partial α-helix being transformed into β-sheet. All these experimental results have been validated by docking studies, which further show that besides hydrophobic interaction, hydrogen bond also help stabilize the gastrodin–pepsin complex. The results reveal the potential to develop the natural compound gastrodin for the treatment of diseases related to the excessive activity of pepsin.

Study on the mechanism of the interaction between acteoside and pepsin using spectroscopic techniques

The interaction of acteoside with pepsin has been investigated using fluorescence spectra, UV/vis absorption spectra, three-dimensional (3D) fluorescence spectra and synchronous fluorescence spectra, along with a molecular docking method. The fluorescence experiments indicate that acteoside can quench the intrinsic fluorescence of pepsin through combined quenching at a low concentration of acteoside, and static quenching at high concentrations. Thermodynamic analysis suggests that hydrogen bonds and van der Waals forces are the main forces between pepsin and acteoside. According to the theory of Försters non-radiation energy transfer, the binding distance between pepsin and acteoside was calculated to be 2.018 nm, which implies that energy transfer occurs between acteoside and pepsin. In addition, experimental results from UV/vis absorption spectra, 3D fluorescence spectra and synchronous fluorescence spectra imply that pepsin undergoes a conformation change when it interacts with acteoside.

Fatty acid and metabolomic profiling approaches differentiate heterotrophic and mixotrophic culture conditions in a microalgal food supplement 'Euglena'

BackgroundMicroalgae have been recognized as a good food source of natural biologically active ingredients. Among them, the green microalga Euglena is a very promising food and nutritional supplements, providing high value-added poly-unsaturated fatty acids, paramylon and proteins. Different culture conditions could affect the chemical composition and food quality of microalgal cells. However, little information is available for distinguishing the different cellular changes especially the active ingredients including poly-saturated fatty acids and other metabolites under different culture conditions, such as light and dark.ResultsIn this study, together with fatty acid profiling, we applied a gas chromatography–mass spectrometry (GC-MS)-based metabolomics to differentiate hetrotrophic and mixotrophic culture conditions.ConclusionsThis study suggests metabolomics can shed light on understanding metabolomic changes under different culture conditions and provides a theoretical basis for industrial applications of microalgae, as food with better high-quality active ingredients.

Estimating Cyanobacteria Community Dynamics and its Relationship with Environmental Factors

The cyanobacteria community dynamics in two eutrophic freshwater bodies (Tiegang Reservoir and Shiyan Reservoir) was studied with both a traditional microscopic counting method and a PCR-DGGE genotyping method. Results showed that cyanobacterium Phormidium tenue was the predominant species; twenty-six cyanobacteria species were identified in water samples collected from the two reservoirs, among which fourteen were identified with the morphological method and sixteen with the PCR-DGGE method. The cyanobacteria community composition analysis showed a seasonal fluctuation from July to December. The cyanobacteria population peaked in August in both reservoirs, with cell abundances of 3.78 × 108 cells L-1 and 1.92 × 108 cells L-1 in the Tiegang and Shiyan reservoirs, respectively. Canonical Correspondence Analysis (CCA) was applied to further investigate the correlation between cyanobacteria community dynamics and environmental factors. The result indicated that the cyanobacteria community dynamics was mostly correlated with pH, temperature and total nitrogen. This study demonstrated that data obtained from PCR-DGGE combined with a traditional morphological method could reflect cyanobacteria community dynamics and its correlation with environmental factors in eutrophic freshwater bodies.

Optogenetic regulation of artificial microRNA improves H 2 production in green alga Chlamydomonas reinhardtii

BackgroundChlamydomonas reinhardtii is an ideal model organism not only for the study of basic metabolic processes in both plants and animals but also the production of biofuels including hydrogen. Transgenic analysis of C. reinhardtii is now well established and very convenient, but inducible exogenous gene expression systems remain under-studied. The most commonly used heat shock-inducible system has serious effects on algal cell growth and is difficult and costly to control in large-scale culture. Previous studies of hydrogen photoproduction in Chlamydomonas also use this heat-inducible system to activate target gene transcription and hydrogen synthesis.ResultsHere we describe a blue light-inducible system with which we achieved optogenetic regulation of target gene expression in C. reinhardtii. This light-inducible system was engineered in a photosynthetic organism for the first time. The photo-inducible heterodimerizing proteins CRY2 and CIB1 were fused to VP16 transcription activation domain and the GAL4 DNA-binding domain, respectively. This scheme allows for transcription activation of the target gene downstream of the activation sequence in response to blue light. Using this system, we successfully engineered blue light-inducible hydrogen-producing transgenic alga. The transgenic alga was cultured under red light and grew approximately normally until logarithmic phase. When illuminated with blue light, the transgenic alga expressed the artificial miRNA targeting photosynthetic system D1 protein, and altered hydrogen production was observed.ConclusionsThe light-inducible system successfully activated the artificial miRNA and, consequently, regulation of its target gene under blue light. Moreover, hydrogen production was enhanced using this system, indicating a more convenient and efficient approach for gene expression regulation in large-scale microalgae cultivation. This optogenetic gene control system is a useful tool for gene regulation and also establishes a novel way to improve hydrogen production in green algae.