Editing of a Specific Strain of Escherichia coli in the Mouse Gut Using Native Phages.

There is a lack of methodological investigation of the in situ functions of bacterial species in microecosystems. Here, we used native phages as a microbial editing tool for eliminating Escherichia coli strain MG1655 labeled with green fluorescent protein (GFP) in the mouse gut. The virulent phages (W1 and W3) possessed host specificity at both the genus and species levels, resulting in an 8.8-log10 difference in the titer of viable bacteria after 12 h of phage treatment compared with that in the phage-free control in an in vitro test. In vivo, they reduced strain MG1655 colonizing the mouse gut at concentrations of 106 to 108 CFU g-1 to a 102 CFU g-1 level, which is almost undetectable by the plate colony-counting method. Moreover, the impact of phage treatment on the microbial community structure of the mouse gut was not significant (P > 0.05), indicating that native phages can effectively edit a target bacterium, with limited perturbation of microbial diversity and relative abundance. Therefore, we developed an engineering technique for investigation of the functions of a specific bacterium by depleting its abundance in microecosystems. IMPORTANCE This report describes a gut engineering technique for investigation of the functions of a specific bacterium. Native phages with host specificity can knock down the corresponding E. coli strain in the mouse gut with limited perturbation of microbial diversity and relative abundance, indicating that they, as a microbial editing tool, can effectively edit the abundance of a target bacterium. Such an approach is undoubtedly of interest in the context of lack of knowledge of how to methodologically study the in situ function of a specific species in a complex microecosystem.

Whole-cell biosynthesis of cytarabine by an unnecessary protein-reduced Escherichia coli that coexpresses purine and uracil phosphorylase.

Currently, whole-cell catalysts face challenges due to the complexity of reaction systems, although they have a cost advantage over pure enzymes. In this study, cytarabine was synthesized by purified purine phosphorylase 1 (PNP1) and uracil phosphorylase (UP), and the conversion of cytarabine from adenine arabinoside reached 72.3 ± 4.3%. However, the synthesis was unsuccessful by whole-cell catalysis due to interference from unnecessary proteins (UNPs) in cells. Thus, we carried out a large-scale gene editing involving 377 genes in the genome of Escherichia coli to reduce the negative effect of UNPs on substrate conversion and cytarabine production. Finally, the PNP1 and UP activities of the obtained mutant were increased significantly compared with the parental strain, and more importantly, the conversion rate of cytarabine by whole-cell catalysis reached 67.4 ± 2.5%. The lack of 148 proteins and downregulation of 783 proteins caused by gene editing were equivalent to partial purification of the enzymes within cells, and thus, we provided inspiration to solve the problem caused by UNP interference, which is ubiquitous in the field of whole-cell catalysis.

Impact of the fermentation broth of Ganoderma lucidum on the quality of Chinese steamed bread.

The potential of fermentation broth of Ganoderma lucidum (FBG) in improving the quality of Chinese steamed bread (CSB) was firstly evaluated. The sensory quality scores of CSB treated by FBG are significantly higher than that of CSB in the control, and texture profile analysis also indicates the increase of CSB hardness and chewiness caused by FBG. Observation on micro-structure of CSB shows that formation of larger pores and expansion of starch granules are the important reasons for the improvement of CSB specific volume (volS), and granule expansion is due to that gluten network distributed in CSB is destroyed as a result of cross-linkage of flour proteins catalyzed by laccase, which makes starch granules releasing from the network easily contact with steam or other enzymes during the proofing and steaming of dough. Moreover, FBG contains amylases which not only convert amylopectin to amylose, but also degrade starch to glucose, maltose and polysaccharides, correspondingly resulting in changes of amylose/amylopectin (Ae/An) ratio of flour and CSB volS, and the latter is because more CO2 produced by the yeast during CSB making leads to the larger pore area in crumb. Both hardness and chewiness are determined by the comprehensive effect of protein cross-linkage, Ae/An ratio and volS change, and this viewpoint gives a logical explanation for the effects of 0.025-0.10 ml/g of FBG on hardness and chewiness of CSB.

Metagenomic insight into the bioaugmentation mechanism of Phanerochaete chrysosporium in an activated sludge system treating coking wastewater.

Phanerochaete chrysosporium was seeded to a sequencing batch reactor treating phenol wastewater. Compared to the contrast reactor (R1), the bioaugmented reactor (R2) exhibits better performance in sludge settling ability, as well as biomass and phenol removal, even though the added fungus is not persistently surviving in the reactor. Bioaugmentation improved bacterial population, growing up to 10,000 times higher than that of eukaryotes. Metagenomic sequencing results show the bioaugmentation finally increases bacterial and eukaryotic richness, but reduces their community diversity. In contrast to R1, bacterial distribution in R2 is more concentrated in Proteobacteria. The relative abundances of filamentous fungi, yeast and microalgae in R2 are all higher than those in R1 at different treatment phases, and two reactors are finally dominated by different protozoan and metazoan. In conclusion, P. chrysosporium improves reactor performances by influencing microbial community structure, and this phenomenon might be attributed to the ecological competition in sludge and toxicity reduction of phenol wastewater. The novelty of this study emphasizes why a species which is not persistently active in bioreactor still plays a crucial role in enhancing reactor performance. Results obtained here impact the conventional criteria for selection of bioaugmentation microbes used in activated sludge systems.

Removal of phenol in phenolic resin wastewater by a novel biomaterial: the Phanerochaete chrysosporium pellet containing chlamydospore-like cells.

A novel biomaterial, the Phanerochaete chrysosporium pellet (CP) composed of chlamydospore-like cells (CLCs), was prepared and its potential in treating phenolic resin wastewater was evaluated. CP possesses higher phenol removal ability in contrast with mycelial pellets of P. chrysosporium, and CLC can be seen as the naturally immobilized enzymes. At shake-flask level, the ideal pH value, temperature, and inoculation quantity of CP for treatment of 1430 mg/l phenol wastewater were pH 4-6, 30 °C, and 5.0 g/l, respectively, and the maximum specific removal rate, 41.1 mg phenol/g CP/h, was obtained in fixed bed reactor (FBR) when the flow rate of wastewater was 3.4 l/h. During the treatment, FBR harbored amounts of bacteria (135 genera) and eukaryotes, as analyzed by metagenomic sequencing. Bacterial pollution not only decreased reactor performance but also had a negative impact on reusability of CP. Hot water treatment (80-85 °C) is effective to inhibit bacterial pollution, and heat resistance of CLC makes the repeated regrowing of CP be feasible. This work presents an innovative and low-cost biomaterial for phenol removal and will be helpful for the practical application of P. chrysosporium in wastewater treatment.

Neuron-like differentiation of bone marrow mesenchymal stem cells from childrenin vitro / 中国组织工程研究

BACKGROUND:In the past, the culture and differentiation of bone marrow mesenchymal stem celsin vitrowere mostly reported in the adult or animal rather than in children. OBJECTIVE: To explore the ability of bone marrow mesenchymal stem cels from children differentiating into neural stem cels and nerve cels. METHODS: Bone marrow mesenchymal stem cels from children were isolated and cultured, and passage 12 cels were cultured in the pre-induction medium (DMEM culture medium containing 10% fetal bovine serum and 1 mmol/L β-mercapto ethanol) and induction medium (DMEM containing 2% dimethyl sulfoxide and 150 μmol/L butylated hydroxyanisole). Expression of nestin and β-tublin III was detected using immunocytochemistry method at 30 minutes and 7 days after induction, while RT-PCR was used to detect nestin mRNA expression at 0, 5.5, 6 days after induction. RESULTS AND CONCLUSION: After combined induction, the cels shrank from round shape to tapered, polygonal or oval shape, and cel processes extended gradualy and became filament-like shape. Interconnected cels formed a network at 6 days after combined induction. The expression of nestin antigen was positive at 30 minutes after induction, while the expression of β-tublin was positive at 7 days. RT-PCR findings showed that positive expression of nestin mRNA was detected at 5.5 hours of induction, and then disappeared at 6 days. These findings show that the combined use of dimethyl sulfoxide and butylated hydroxyanisole can induce bone marrow mesenchymal stem cels from children to differentiate into neural stem cels and nerve cels in vitro.

Overproduction of laccase from a newly isolated Ganoderma lucidum using the municipal food waste as main carbon and nitrogen supplement.

A strain of Ganoderma lucidum was separated and identified according to its morphological characteristics and phylogenetic data. The fungus is a laccase producer and it can secrete laccase using the municipal food waste (FW) as carbon and nitrogen supplement. After the statistic optimization, a laccase activity of 42,000 ± 600 U/l was obtained at 500 ml flask level and the activity is 12,000 U/l higher than that obtained by fermenting glucose and peptone, indicating that the use of FW to produce laccase not only reduces production cost, but also improves laccase activity. In 15 l bioreactor, FW is also suitable for laccase production and the maximum laccase activity reached 54,000 U/l. Moreover, some details of laccase overproduction using FW were investigated. The G. lucidum consumes FW by secreting a series of hydrolases and proteases and the improvement of laccase activity is because FW induces over-expression of three isoenzymes by polyacrylamide gel electrophoresis analysis.

Simulation of wastewater treatment by aerobic granules in a sequencing batch reactor based on cellular automata.

In the present paper, aerobic granules were developed in a sequencing batch reactor (SBR) using synthetic wastewater, and 81 % of granular rate was obtained after 15-day cultivation. Aerobic granules have a 96 % BOD removal to the wastewater, and the reactor harbors a mount of biomass including bacteria, fungi and protozoa. In view of the complexity of kinetic behaviors of sludge and biological mechanisms of the granular SBR, a cellular automata model was established to simulate the process of wastewater treatment. The results indicate that the model not only visualized the complex adsorption and degradation process of aerobic granules, but also well described the BOD removal of wastewater and microbial growth in the reactor. Thus, CA model is suitable for simulation of synthetic wastewater treatment. This is the first report about dynamical and visual simulation of treatment process of synthetic wastewater in a granular SBR.

Specific aerobic granules can be developed in a completely mixed tank reactor by bioaugmentation using micro-mycelial pellets of Phanerochaete chrysosporium.

Aerobic granules were firstly developed in a completely mixed tank reactor (CMTR) by seeding micro-mycelial pellets (MMPs) of Phanerochaete chrysosporium. During phenol wastewater treatment, sludge granulation rate reached 67 % after 15-day operation. The granules in CMTR are different from aerobic granules described in literature in morphology, and a majority of them are rod-shaped or rodlike sludge besides spherical granules. The polymorphic granules, having no essential difference with aerobic granules previously reported, achieve advantages over conventional activated sludge in settling ability, biomass concentration, density, integrity coefficient and removal ability to phenol wastewater. The optimized parameters for sludge granulation in CMTR including temperature, inoculum quantity, rotary speed and superficial air upflow velocity are 30 °C, 5­7 g/l, 150 rpm, and 0.5 cm/s, respectively. Analysis on sludge granulation mechanism indicates that MMPs not only result in the formation of aerobic granules containing MMPs as nuclei, but also induce the formation of biogranules which do not have MMP at their cores. The work challenges the general belief that the homogenous circular flow pattern of microbial aggregates is necessary for aerobic sludge granulation.

Abnormal expression of connexin 36 plays a role in the pathogenesis of levodopa induced dyskinesia in rat model of Parkinson' s disease / 中华神经科杂志

Objective To explore whether gap junction disturbances are involved in the pathogenesis of levodopa-induced dyskinesia ( LID ). Methods The hemi-parkinsonian ( PD ) rat was treated intraperitoneally with L-dopa methylester (20 mg/kg) and benserazid (10 mg/kg) for 21 days and abnormal involuntary movement was evaluated to establish LID rat model. The experimental animals were divided into three groups: LID group, PD group and normal control group, respectively. The behavior responses of intraperitoneal injection of different doses of carbenoxolon and intracerebroventricular injection of quinine were observed to estimate the effects of gap junctional blockade on the abnormal involuntary movement ( AIM ) in the rat model of LID. Double immunofluorescence labeling was used to analyze the expression of connexin 36 ( Cx36 ) in enkephalin positive medium spiny neurons and parvalbumin ( PV ) positive interneurons in the striatum. Western blottings was used to observe the expression of Cx36 in the striatum and moter cortex. Results Behavioral characteristics indicated that high dose of carbenoxolone ( >60 mg/kg) intraperitoneal injection and intracerebroventricular injection of quinine ( 0.5, 1.0, 2.0 μmol/L, > 2.5 μmol/L ) could decrease the AIM score of LID rats. Western blotting indicated that expression of Cx36 in lesioned striatum and motor cortex of LID rat model was 219.56% ±18.12% and 226.03% ±16.33%, respectively, which induced a significant upregulation in comparison with the normal control group (104.05% ±3.82%, t=15.389, P<0.01;105.27% ±2.82%,t=8.074, P<0.01) and untreated PD group (119.31% ±8.92%, t=13.356, P<0.01; 138.20% ±17.88%, t=5.872, P<0.01). Double immunofluorescence labeling staining revealed that Cx36 expression was increased in Enk-positive striatum neurons in LID model ( 57.59% ±5.36%) compared with that in normal control group (32.67% ±4.22%) and PD group (37.24% ±0.86%, F=78.060, P<0.01). The expression of Cx36 in PV-positive interneurons was also elevated in LID group (68.49% ±11.60%) in comparison with normal control group ( 40.43% ± 2.30%) and PD group ( 31.92% ± 5.68%, F = 39.567, P < 0.01 ).Conclusions The Cx36 expression is generally increased in lesioned striatum and motor cortex of LID rat model. In the striatum, the up-regulation of Cx36 is specifically observed in Enk-positive striatum neurons and in PV-positive interneurons. The dyskinesia behavior of LID rats can be significantly reduced by treatment with gap junction blockade. All these results suggest that gap junction dysfunction may play an important role in the pathogenesis of LID.

The glucose metabolic frame of conditions induce the overproduction of a co-culture pigment: induction mechanism of the pigment and development of a specific eutrophic-oligotrophic transition cultivation system.

Induction mechanism of a potential red pigment (RPc) was investigated in the present paper. A typical competition relationship exists between Penicillium sp. HSD07B and Candida tropicalis during co-culture, and C. tropicalis converts glucose into glycerol, organic acids and other substances, resulting in a stricter glucose limitation and the secretion of RPc. Moreover, a novel eutrophic-oligotrophic transition cultivation system (E-OTCS) was developed to produce red pigment during monoculture of Penicillium sp. HSD07B. However, the monoculture pigment (RPm) is different from RPc in components, and RP3 and RP4 only occur in RPm when glycerol is supplied. In addition, the additions of glycerol and organic acids to glucose exhaustion medium can significantly improve the pigment yield. These facts not only prove the feasibility of producing RPm using E-OTCS, but also reveal that, besides glucose exhaustion, the accumulation of metabolites of glucose including glycerol and organic acids is also an important factor influencing the production of RPc.

Overproduction of a potential red pigment by a specific self-immobilization biomembrane-surface liquid culture of Penicillium novae-zeelandiae.

A specific self-immobilization biomembrane-surface liquid culture (SIBSLC) was developed to overproduce a potential penicillium red pigment. Statistic analysis shows that both glucose concentration and membrane diameter are important factors influencing the yield of red pigment. After the optimization using central composite experimental design, the maximum yield of red pigment in shake flask reaches 4.25 g/l. The growth of strain HSD07B consists of three phases, and the pigment secreted in the decelerated phase, is originated from the interior of biomembrane where glucose exhaustion occurs. In addition, the batch and continuous SIBSLC were conducted for production of the pigment, and the latter was more competitive in consideration of the fact that it not only increased 61.5 % of pigment productivity, but also simplified the production process. Moreover, the pigment produced by SIBSLC is potentially acceptable for food applications although it is distinguished from the co-cultured red pigment we reported previously in components.

The acceleration of sludge granulation using the chlamydospores of Phanerochaete sp. HSD.

In the present paper, a novel method to accelerate sludge granulation is presented. Inoculation with chlamydospores of Phanerochaete sp. HSD accelerated sludge granulation during the treatment process of phenol wastewater, and the sludge granulation rate reached 66 ± 2% on day 7, 32 days earlier than that of the control inoculated with activated sludge only. Aerobic granule in R1 (AG(R1)) showed an annual ring-like multilayer structure and a primary core also existed in the nuclear area of the granule. The mechanism of rapid granulation revealed that the chlamydospore could survive in phenol wastewater and form the primary matrix on which aerobic granule was developed layer by layer. In addition, AG(R1) developed in a phenol uptake system to counteract the adverse effects of phenol inhibition. Higher tolerance toward wastewater with high phenol strength was exhibited, and the maximum specific phenol degradation rate reached 1.54 g phenol g(-1) VSS day(-1).

Improvement of the production of a red pigment in Penicillium sp. HSD07B synthesized during co-culture with Candida tropicalis.

Co-culture of Penicillium sp. HSD07B and Candida tropicalis resulted in the production of a red pigment consisting of six components as determined by TLC and HPLC. The pigment showed no acute toxicity in mice and was mot mutagenic in the Ames test. The pigment was stable between pH 2 and 10 and temperatures of 10-100°C and exhibited good photo-stability and resistance to oxidization by hydrogen peroxide and reduction by Na(2)SO(3). Glucose and ratio of C. tropicalis to strain HSD07B (w/w) in the inoculum were the important factors influencing production of the pigment. Under optimized conditions, a pigment yield of 2.75 and 7.7 g/l was obtained in a shake-flask and a 15 l bioreactor, respectively. Thus, co-culture of strain HSD07B and C. tropicalis is a promising way to produce a red pigment potentially useful for coloring applications.

The effect of bioaugmentation on the performance of sequencing batch reactor and sludge characteristics in the treatment process of papermaking wastewater.

In this paper, the differences between reinforced sequencing batch reactor, which was inoculated with superior mixed flora, and conventional sequencing batch reactor were compared in the process of treating papermaking wastewater under similar conditions. The results showed that the addition of superior mixed flora could not only shorten the sludge acclimation time, but also improve the treatment efficiency of reactor as well as make the reactor have higher ability to withstand high volume loading rate; the phenomenon of aerobic granulation only occurred in reinforced sequencing batch reactor, and superior mixed flora were the key reason that aerobic granular sludge could shape; aerobic granular sludge had many advantages over conventional activated sludge such as it possessed compacter microbial structure, better settling performance, and lower water content.