Cloning,Expression, and Biochemical Characterization of Two New Oligoalginate Lyases with Synergistic Degradation Capability

Alginate, the most abundant carbohydrate presents in brown macroalgae, has recently gained increasing attention as an alternative biomass for the production of biofuel. Oligoalginate lyases catalyze the degradation of alginate oligomers into monomers, a prerequisite for bioethanol production. In this study, two new oligoalginate lyase genes, oalC6 and oalC17, were cloned from Cellulophaga sp. SY116, and expressed them in Escherichia coli. The deduced oligoalginate lyases, OalC6 and OalC17, belonged to the polysaccharide lyase (PL) family 6 and 17, respectively. Both showed less than 50% amino acid identity with all of the characterized oligoalginate lyases. Moreover, OalC6 and OalC17 could degrade both alginate polymers and oligomers into monomers in an exolytic mode. Substrate specificity studies demonstrated that OalC6 preferred α-L-guluronate (polyG) blocks, while OalC17 preferred poly β-D-mannuronate (polyM) blocks. The combination of OalC6 and OalC17 showed synergistic degradation ability toward both alginate polymers and oligomers. Finally, an efficient process for the production of alginate monomers was established by combining the new-isolated exotype alginate lyases (i.e., OalC6 and OalC17) and the endotype alginate lyase AlySY08. Overall, our work provides new insights for the development of novel biotechnologies for biofuel production from seaweed.     

Bone morphogenetic protein 6–mediated crosstalk between endothelial cells and hepatocytes recapitulates the iron-sensing pathway in vitro

Liver sinusoidal endothelial cell–derived bone morphogenetic protein 6 (BMP6) and the BMP6–small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin in vitro in contrast to in vivo conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using in vitro coculture models that mimic hepcidin signaling in vivo. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron in vivo. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte–endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.     

Biosynthesis of <Emphasis Type="Italic">Veratrum californicum</Emphasis> specialty chemicals in <Emphasis Type="Italic">Camelina sativa</Emphasis> seed

Economically feasible systems for heterologous production of complex secondary metabolites originating from difficult to cultivate species are in demand since Escherichia coli and Saccharomyces cerevisiae are not always suitable for expression of plant and animal genes. An emerging oilseed crop, Camelina sativa, has recently been engineered to produce novel oil profiles, jet fuel precursors, and small molecules of industrial interest. To establish C. sativa as a system for the production of medicinally relevant compounds, we introduced four genes from Veratrum californicum involved in steroid alkaloid biosynthesis. Together, these four genes produce verazine, the hypothesized precursor to cyclopamine, a medicinally relevant steroid alkaloid whose analogs are currently being tested for cancer therapy in clinical trials. The future supply of this potential cancer treatment is uncertain as V. californicum is slow-growing and not amendable to cultivation. Moreover, the complex stereochemistry of cyclopamine results in low-yield syntheses. Herein, we successfully engineered C. sativa to synthesize verazine, as well as other V. californicum secondary metabolites, in seed. In addition, we have clarified the stereochemistry of verazine and related V. californicum metabolites.     

Comparison of Hypoglycemic Activity of Trace Elements Absorbed in Fermented Mushroom of <Emphasis Type="Italic">Coprinus comatus</Emphasis>

The effect of fermented mushroom of Coprinus comatus rich in trace elements, including vanadium, chromium, zinc, magnesium, copper, iron, and nickel, on glycemic metabolism was studied in this paper. Alloxan-induced hyperglycemic mice were used in the study. The blood glucose, glycohemoglobin, and glycogen synthesis of the mice were analyzed, respectively. At the same time, the gluconeogenesis of the normal mice was also determined. After the mice were administered (ig) with C. comatus rich in vanadium (CCRV), the blood glucose and the glycohemoglobin of alloxan-induced hyperglycemic mice decreased (p < 0.05, p < 0.01), glycogen synthesis of alloxan-induced hyperglycemic mice elevated (p < 0.01), the gluconeogenesis of the normal mice was inhibited (p < 0.01), and the sugar tolerance of the normal mice was improved. However, the same result did not occur in other groups. Vanadium at lower doses in combination with C. comatus induced significant effect on glycemic metabolism in mice.     

MiR-133a-3p inhibits scar formation in scalded mice and suppresses the proliferation and migration of scar derived-fibroblasts by targeting connective tissue growth factor

Excessive scar formation post burn injury can cause great pain to the patients. MiR-133a-3p has been demonstrated to be anti-fibrotic in some fibrosis-related diseases. However, its possible role in scar formation has not been elucidated yet. In present study, the effect of miR-133a-3p on scar formation was investigated in a scalded model of mice. Moreover, the function of miR-133a-3p on proliferation and migration of scar-derived fibroblasts (SFs) was studied in vitro. It was found that miR-133a-3p was dramatically downregulated in scar tissue of scalded mice. Upregulation of miR-133a-3p by miR-133a-3p agomir obviously inhibited the scar formation in scalded mice. Histological staining showed that upregulation of miR-133a-3p attenuated the excessive deposition of collagen in scar tissue of scalded mice. In vitro study showed that upregulation of miR-133a-3p effectively suppressed the proliferation and migration of SFs. Besides, upregulation of miR-133a-3p attenuated the protein levels of α-smooth muscle actin (α-SMA) and collagen I, indicating that miR-133a-3p could suppress the activation of SFs. The expression of connective tissue growth factor (CTGF), a critical mediator in cell proliferation, migration and extracellular matrix (ECM) synthesis, was also downregulated by the upregulation of miR-133a-3p. Luciferase reporter assay validated that CTGF was directly targeted by miR-133a-3p. In addition, overexpression of CTGF abolished the effect of miR-133a-3p on inhibiting the proliferation, migration and activation of SFs, indicating that miR-133a-3p functioned by targeting CTGF. Therefore, miR-133a-3p might be a promising target for treating pathological scars.     

Construction of an effective screening system for detection of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> quorum sensing inhibitors and its application in bioautographic thin-layer chromatography

In Pseudomonas aeruginosa, quorum sensing (QS) regulates dozens of genes and proteins, many of which contribute to the virulence of this pathogen. QS inhibitory (QSI) compounds have been proposed as potential agents for treatment of bacterial infections. To search for Ps. aeruginosa QS inhibitors, we constructed an effective screening system, QSIS-lasI selector, based on the PlasI-sacB reporter, in which QS could be induced with 20 nM 3-oxo-N-[(3S)-tetrahydro-2-oxo-3-furanyl]-dodecanamide (3-oxo-C₁₂-HSL). During screening of the crude extracts from 65 marine fungi, an isolate of Penicillium atramentosum was found to have QSI activity. Thin-layer chromatography assay of the fungal extracts for bioautographic identification of QSIS-lasI indicated that this fungus produced several QSI compounds, including QS inhibitors other than penicillic acid or patulin.     

Multiple Resistances and Complex Mechanisms of Anopheles sinensis Mosquito: A Major Obstacle to Mosquito-Borne Diseases Control and Elimination in China

Malaria, dengue fever, and filariasis are three of the most common mosquito-borne diseases worldwide. Malaria and lymphatic filariasis can occur as concomitant human infections while also sharing common mosquito vectors. The overall prevalence and health significance of malaria and filariasis have made them top priorities for global elimination and control programmes. Pyrethroid resistance in anopheline mosquito vectors represents a highly significant problem to malaria control worldwide. Several methods have been proposed to mitigate insecticide resistance, including rotational use of insecticides with different modes of action. Anopheles sinensis, an important malaria and filariasis vector in Southeast Asia, represents an interesting mosquito species for examining the consequences of long-term insecticide rotation use on resistance. We examined insecticide resistance in two An. Sinensis populations from central and southern China against pyrethroids, organochlorines, organophosphates, and carbamates, which are the major classes of insecticides recommended for indoor residual spray. We found that the mosquito populations were highly resistant to the four classes of insecticides. High frequency of kdr mutation was revealed in the central population, whereas no kdr mutation was detected in the southern population. The frequency of G119S mutation in the ace-1 gene was moderate in both populations. The classification and regression trees (CART) statistical analysis found that metabolic detoxification was the most important resistance mechanism, whereas target site insensitivity of L1014 kdr mutation played a less important role. Our results indicate that metabolic detoxification was the dominant mechanism of resistance compared to target site insensitivity, and suggests that long-term rotational use of various insecticides has led An. sinensis to evolve a high insecticide resistance. This study highlights the complex network of mechanisms conferring multiple resistances to chemical insecticides in mosquito vectors and it has important implication for designing and implementing vector resistance management strategies.     

溶藻菌株NP23的紫外诱变选育

应用紫外诱变技术对溶藻菌株NP23进行紫外诱变处理。经过粗筛后,从8株诱变株中选出2株对绿藻中小球藻和蓝藻中惠氏微囊藻的去除效果明显优于原始菌株的突变株NP23-1和NP23-4,其溶藻率(叶绿素a的去除率)比原始菌株提高30%-35%。连续6代测试,2株诱变菌株NP23-1和NP23-4溶藻率都很稳定,表明所得突变株是比原始菌株更优秀的溶藻菌株。