[Spatio-temporal Variation of PM2.5 Related Relationships in China from the Perspective of Air Pollution Regional Linkage Control and Prevention].

Identification of spatio-temporal variation of PM2.5 related relationships under joint management zones is of great significance for scientifically conducting joint control of air pollution in China. Based on the PM2.5 concentration data of 334 prefecture-level cities in China from 2000 to 2016, from the perspective of air pollution regional linkage control and prevention, this paper systematically analyzes the spatio-temporal variation of PM2.5 related relationships in China using a spatial unit aggregation strategy and geographically and temporally weighted regression. The results show that:① With PM2.5 as the primary pollutant, ten air pollution joint management areas are obtained by considering the degree of pollution, geographical location, meteorology, topography, and economy. ② Geographically and temporally weighted regression can effectively reveal the spatio-temporal non-stationarity of the relationships between PM2.5 concentration and related factors. Meanwhile, population size, secondary industry gross domestic product, SO2 emissions, annual average temperature, annual precipitation, and annual relative humidity are identified as having a significant effect on changes in PM2.5 concentration. ③ The population impacts on PM2.5 concentration in the Beijing-Tianjin-Yunmeng region are the largest of all regions during the period. The influence of the secondary industry's gross domestic product on the PM2.5 concentration in the Sichuan-Yunnan District is the most variable. Apart from these values in the northeast of China, the regression coefficient values of SO2 emissions first decrease with time, then increase, and then decrease again. The time variability of the average annual temperature of each treatment area to PM2.5 is small. The influences of annual precipitation and annual average relative humidity on PM2.5 present different variability characteristics in each region.

Efficient degradation of Azo dyes by a newly isolated fungus Trichoderma tomentosum under non-sterile conditions.

A fast-growing fungus with remarkable ability to degrade several azo dyes under non-sterile conditions was isolated and identified. This fungus was identified as Trichoderma tomentosum. Textile effluent of ten-fold dilution could be decolorized by 94.9% within 72h before optimization. Acid Red 3R model wastewater with a concentration of 85.5mgL-1 could be decolorized by 99.2% within the same time after optimization. High-level of manganese peroxidase and low-level of lignin peroxidase activities were detected during the process of decolorization from the culture supernatant, indicating the possible involvement of two enzymes in azo dye decolorization. No aromatic amine products were detected from the degradation products of Acid Red 3R by gas chromatography-mass spectrometry (GC/MS) analysis, indicating the possible involvement of a special symmetrical oxidative degradation pathway. Phytotoxicity assay confirmed the lower toxicity toward the test plant seeds of the degradation products when compared to the original dye.

Identification of insecticidal constituents of the essential oils of Dahlia pinnata Cav. against Sitophilus zeamais and Sitophilus oryzae.

The aim of this research was to determine the chemical composition of the essential oils of Dahlia pinnata, their insecticidal activity against Sitophilus zeamais and Sitophilusoryzae and to isolate insecticidal constituents. Based on bioactivity-guided fractionation, active constituents were isolated and identified as D-limonene, 4-terpineol and α-terpineol. Essential oils and active compounds tested exhibited contact toxicity, with LD50 values ranging from 132.48 to 828.79 µg/cm(2) against S. zeamais and S. oryzae. Essential oils possessed fumigant toxicity against S. zeamais and S. oryzae with LC50 from 14.10 to 73.46 mg/L. d-Limonene (LC50 = 4.55 and 7.92 mg/L) showed stronger fumigant toxicity against target insects. 4-Terpineol (88 ± 8%) and d-limonene (87 ± 5%) showed the strongest repellency against S. zeamais and S. oryzae, respectively. The results indicate that essential oils and insecticidal constituents have potential for development into natural fumigants, insecticides or repellents for control of the stored-product insect pests.

A novel cleaning process for industrial production of xylose in pilot scale from corncob by using screw-steam-explosive extruder.

Steam explosion is the most promising technology to replace conventional acid hydrolysis of lignocellulose for biomass pretreatment. In this paper, a new screw-steam-explosive extruder was designed and explored for xylose production and lignocellulose biorefinery at the pilot scale. We investigated the effect of different chemicals on xylose yield in the screw-steam-explosive extrusion process, and the xylose production process was optimized as followings: After pre-impregnation with sulfuric acid at 80 °C for 3 h, corncob was treated at 1.55 MPa with 9 mg sulfuric acid/g dry corncob (DC) for 5.5 min, followed by countercurrent extraction (3 recycles), decoloration (activated carbon dosage 0.07 g/g sugar, 75 °C for 40 min), and ion exchange (2 batches). Using this process, 3.575 kg of crystal xylose was produced from 22 kg corncob, almost 90 % of hemicellulose was released as monomeric sugar, and only a small amount of by-products was released (formic acid, acetic acid, fural, 5-hydroxymethylfurfural, and phenolic compounds were 0.17, 1.14, 0.53, 0.19, and 1.75 g/100 g DC, respectively). All results indicated that the screw-steam-explosive extrusion provides a more effective way to convert hemicellulose into xylose and could be an alternative method to traditional sulfuric acid hydrolysis process for lignocellulose biorefinery.

Candida albicans ferric reductase FRP1 is regulated by direct interaction with Rim101p transcription factor.

The opportunistic pathogen Candida albicans develops different systems to acquire essential nutrients such as iron. The Rim101 pathway controls gene expression to adapt to different environments through transcription factor Rim101p. Ferric reductase genes are important for iron acquisition, but further work should be carried out to show about how ferric reductase genes are regulated. In this study we demonstrate that ferric reductase FRP1 expression is upregulated by alkaline pH and iron-limited conditions. Moreover, promoter deletion identified that the upregulated elements lie in -822 to 437 and 263 to -124 and the downregulated elements in -437 to -263 using a beta-galactosidase reporter system. Using electrophoretic mobility shift assay we also found that Rim101p can bind directly with two putative 5'-CCAAGAA-3' sites at the -157 and -629 sites. Interestingly, site-specific mutations show that only the -629 site, not the -157 site, plays an important role in FRP1 expression under iron-limited conditions.

[Characterization of pcr2 gene of Pseudomonas aeruginosa].

Type III secretion system (TTSS) is an important virulence factor encoding by pseudomonas aeruginosa. About 40 genes are involved and they function as structure proteins, chaperons, regulators, and effectors proteins, respectively. Although some genes have been studied previously, functions of many genes remained unknown. Pcr2 gene is the third gene of popN operon that is one of the five operons of the TTSS gene clusters. Its functions were investigated in this study. First, by characterization of the phenotypes of pcr(2-) mutant, we found that the abilities of secreting or translocating effectors proteins were significantly damaged in the absence of Pcr2 protein, suggesting that Pcr2 protein involved in both the secretion and translocation processes of TSS. Second, evidences were provided that no PopN protein was detectable in supernatant of pcr(2-) mutant culture. Combined with the data from the bacterial two-hybrid system, we can conclude that Pcr2 protein might function as part of a chaperone complex for the PopN protein. Third, Pcr2 protein was found secreted in a TTSS-dependent manner, suggesting that secreted Pcr2 may play a role in the TTSS needle biogenesis.

[Type III secretion study of popN- mutant of Pseudomonas aeruginosa and proteases degradation].

Pseudomonas aeruginosa is an important opportunistic human pathogen. It encodes many virulence factors and one of them is type III secretion system (TTSS). Effectors proteins can be delivered into host cells directly by this system, causing necrosis or apoptosis. popN gene is the first gene in the popN operon of TTSS gene cluster. To investigate its function, popN gene deletion mutant was generated in this study, and we found this mutant can secrete effectors proteins constitutively under non-inducting condition in DMEM medium containing serum. The results indicated that PopN is a negative regulator of the TTSS expression. However, no secreted effector proteins were detectable when the popN- mutant was grown in LB medium under non-inducting condition. To investigate the possible reasons, effects of growth status and protease (s) inhibitors on the TTSS were investigated. We present evidences that indicate protease mediated degradation of secreted effector proteins played a key role in the phenotypic inconsistency of popN- mutant.

[Protoplast transformation of Mortierella isabellina with hygromycin B resistance plasmid PD4].

A strain Mortierella isabellina M6-22-4, which was sensitive to hygromycin B, was selected by treating parental spores with N-methyl-N' -Nitro-N-nitrosoguanidine (MNNG). Protoplasts of the strain Mortierella isabellina M6-22-4 were transformed successfully to hygromycin B resistance using the PD4 plasmid, which contains the Escherichia coli hph gene under the control of Mortierella alpina his H4.1 promoter. The PD4 plasmid was introduced by PEG/CaCl2 treatment. Transformation frequencies of 1.6 - 2.8 transformants/microg of DNA were achieved. Then they were successively incubated to non-selected PDA plates for 10 generations. About 31.6% transformants only from digested plasmid were mitotically stable and showed different hygromycin B resistance when they were incubated back to selection plates. The results of PCR and Southern analysis in three transformants indicated that the plasmid PD4 had been integrated into the fungal genome with 1 - 2 copies. This is the first report of Mortierella isabellina transformation system and supplies an important tool for further research into genetic manipulation of this filamentous fungus.

[The response to environmental pH of RIM101 pathway in Candida albicans].

Abstract: Candida albicans is the most common kind of human opportunistic pathogen and gets many attentions in recent years. A lot of research has been done. The sites this organism colonizes differ in pH. C. albicans must be able to response to the environmental changes for its survival and pathogenicity. Extracellular environment, especially pH changes,is critical for C. albicans morphological differentiation and virulence. RIM101 pathway is a conserved fungal signal transduction pathway and at least partly controls the cellular pH response through the activity of the zinc finger transcription factor Rim101p. This review concisely summarized recent research on RIM101 pathway, pH response and their relationship in C. albicans.

Hyperexcitability of distal dendrites in hippocampal pyramidal cells after chronic partial deafferentation.

Traumatic injury to the CNS results in chronic partial deafferentation of subsets of surviving neurons. Such injuries are often followed by a delayed but long-lasting period of aberrant hyperexcitability. The cellular mechanisms underlying this delayed hyperexcitability are poorly understood. We developed an in vitro model of deafferentation and reactive hyperexcitability using organotypic hippocampal slice cultures to study the underlying cellular mechanisms. One week after transection of the Schaffer collateral and temporoammonic afferents to CA1 neurons, brief tetanic stimulation of the residual excitatory synapses produced abnormally prolonged depolarizations, compared with responses in normally innervated neurons. Responses to weak stimulation, in contrast, were unaffected after deafferentation. Direct stimulation of distal apical dendrites using focal photolysis of caged glutamate triggered abnormally prolonged plateau potentials in the deafferented neurons when strong stimulation was given, but responses to weak stimulation were not different from controls. An identical phenotype was produced by chronic "chemical deafferentation" with glutamate receptor antagonists. Responses to strong synaptic and photolytic stimulation were selectively prolonged by small-conductance (SK-type) calcium-activated potassium channel blockers in normally innervated cells but not after deafferentation. No significant changes in SK2 mRNA or protein levels, GABAergic inhibition, glutamate receptor function, input resistance, or action potential parameters were observed after chronic deafferentation. We suggest that a posttranslational downregulation of SK channel function in thin distal dendrites is a significant contributor to deafferentation-induced reactive hyperexcitability.

[A method using long primers for cloning the upstream sequence of delta-6 fatty acid desaturases gene of Thamnidium elegans by nested inverse PCR].

Thamnidium elegans is a kind of phycomycete that produces essential unsaturated fatty acids, particularly y-linolenic acid. In this process, delta6-Fatty acid desaturase (D6D) plays a key role due to its enzymatic properties that catalyze the delta6 site dehydrogenation of precursor linoleic acid (18:2delta(9, 12) n-6) and a-linolenic acid (18:3delta(9, 12, 15) n-3). This reaction is the first and rate-limiting step of highly unsaturated fatty acids (HUFA) synthesis pathways. After we have isolated and cloned the gene coding delta6-fatty acid desaturase from Thamnidium elegans As3.2806 (GenBank accession number DQ099380), our interest focuses on the promotion and regulation of the gene transcription. To achieve this aim, we designed long primers and used nested inverse PCR to amplify DNA flanking sequences. First, genome of Thamnidium elegans was extracted and digested with restriction enzymes EcoR I and Kpn I , respectively. Then we ligated the digested DNA with T4 ligase at low concentration which is propitious for linear DNA to joint intromolecule. According to the sequence of delta6-fatty acid desaturase gene of Thamnidium elegans, we designed a couple of 35nt long inverse primers and two couples of shorter inverse primers for inverse PCR. Three rounds of PCR reactions were performed. In the primary reaction, the ligated DNA was used as a template, and the product was used as the template of the secondary reaction, the tertiary reaction was achieved in the same way. After all the three rounds of reactions, we got a nice product about 4 kb from the EcoR I digested sample, in which a 1.3kb 5' upstream sequence (GenBank accession number DQ309425) of delta6-fatty acid desaturase gene containing several putative regulatory elements including TATA. box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans detla6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome. box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3 kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans delta6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome.

Cloning and molecular characterization of delta12-fatty acid desaturase gene from Mortierella isabellina.

AIM: To clone delta(12)-fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo. METHODS: Reverse transcriptional polymerase chain reaction (RT-PCR) was used to clone the open reading frame of delta(12)-fatty acid desaturase gene (D12D) of Mortierella isabellina. Plasmids pEMICL12 and pYMICL12 were constructed with it. pEMICL12 was transformed into Escherichia coli (E.coli) strain BL21 using CaCl(2) method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Northern blotting method was used to investigate the effect of temperature on the transcriptional level of this gene in S.cerevisiae strain INVSc1. RESULTS: Recombinant plasmids pEMICL12 and pTMICL12 were successfully constructed and transformed into E.coli and S.cerevisiae separately with appropriate method. After induction with IPTG and galactose, it was found that expression of delta(12)-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active delta(12)-fatty acid desaturase, which could convert 17.876% and 17.604% of oleic acid respectively to linoleic acid by GC-MS detection in vitro and in vivo. CONCLUSION: Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.

[Cloning and expression of delta6-desaturase gene from Thamnidium elegans in Saccharomyces cerevisiae].

A 459 bp DNA fragment was amplified from Thamnidium elegans As3.2806 with degenerate oligonucleotides primers designed based on the sequences information from the conserved histidine-rich motifs II and near III of fungal delta6-fatty acid desaturases genes by RT-PCR and sequenced. Gene specific primers designed according to this partial sequence were used for the amplification of the 3'- and 5'- end of this cDNA by RACE method, and sequence information coming from these two ends were used to design two GSPs to clone the 1504bp full-length cDNA. Sequence analysis showed this cDNA sequence had an open reading frame (ORF) of 1377bp encoding 458 amino acids of 52.4kD. The deduced amino acid sequence of the ORF showed similarity to those of the above delta6-fatty acid desaturases which comprise the characteristics of membrane-bound desaturases, including three conserved histidine-rich boxes and hydropathy profile. A cytochrome b5-like domain was observed at the N-terminus. To elucidate the function of the protein, two specific primers corresponding to the nucleotide sequences of start and stop codons were used to amplify the coding sequence. The amplified cDNA TED6 was subcloned into the expression vector pYES2.0 to generate a recombinant plasmid pYTED6, which was subsequently transformed into Saccharomyces cerevisiae strain INVScl for heterologous expression by lithium acetate method. Grown to logarithmic phase at 30 degrees C, the transformed cells were supplemented with 0.5 mmol/L Linoleic acid and induced by 2% galactose for a further 48 h of cultivation at 20 degrees C. Total fatty acids were extracted from the induced cell and subjected to methyl-esterification. The resultant fatty acid methyl esters (FAME) were analyzed by gas chromatography (GC). A novel peak corresponding to gamma-linolenic acid (GLA) methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector. The percentage of this new fatty acid to total fatty acids was 7.5%. Gas chromatography-mass spectrometry (GC-MS) analysis of this fatty acid methyl derivative demonstrated that the novel peak was GLA methyl ester. These results showed that the coding product of this sequence exhibited the activity of converting linoleic acid (LA) to gamma-linolenic (GLA), and indicated that amino-acid sequence exhibited delta6-fatty acid desaturase activity.

Kv4 potassium channel subunits control action potential repolarization and frequency-dependent broadening in rat hippocampal CA1 pyramidal neurones.

A-type potassium channels regulate neuronal firing frequency and the back-propagation of action potentials (APs) into dendrites of hippocampal CA1 pyramidal neurones. Recent molecular cloning studies have found several families of voltage-gated K(+) channel genes expressed in the mammalian brain. At present, information regarding the relationship between the protein products of these genes and the various neuronal functions performed by voltage-gated K(+) channels is lacking. Here we used a combination of molecular, electrophysiological and imaging techniques to show that one such gene, Kv4.2, controls AP half-width, frequency-dependent AP broadening and dendritic action potential propagation. Using a modified Sindbis virus, we expressed either the enhanced green fluorescence protein (EGFP)-tagged Kv4.2 or an EGFP-tagged dominant negative mutant of Kv4.2 (Kv4.2g(W362F)) in CA1 pyramidal neurones of organotypic slice cultures. Neurones expressing Kv4.2g(W362F) displayed broader action potentials with an increase in frequency-dependent AP broadening during a train compared with control neurones. In addition, Ca(2)(+) imaging of Kv4.2g(W362F) expressing dendrites revealed enhanced AP back-propagation compared to control neurones. Conversely, neurones expressing an increased A-type current through overexpression of Kv4.2 displayed narrower APs with less frequency dependent broadening and decreased dendritic propagation. These results point to Kv4.2 as the major contributor to the A-current in hippocampal CA1 neurones and suggest a prominent role for Kv4.2 in regulating AP shape and dendritic signalling. As Ca(2)(+) influx occurs primarily during AP repolarization, Kv4.2 activity can regulate cellular processes involving Ca(2)(+)-dependent second messenger cascades such as gene expression and synaptic plasticity.