Every 3 months the aggregated data would be stored in the kumban (through the TSC), to support selleck chemical the bi-annual analyses and discussions between district, kumban and villages. Once a year the results of these discussions would be made official and forwarded

to the provincial level. Looking for sustainability: integrating resource monitoring into the “Participatory Land Use Planning” national process Once the monitoring system, including results and activities, is embedded into the local administrative structure it requires political support to power the system and provide sustainability. During the project’s life we only proposed ways to embed the monitoring tools into existing administrative structures. However, we have not received information as to whether the villagers and kumban authorities have adopted the system or not. The Government of Laos has, in the past, introduced different LUP policies to alleviate poverty, with some success (Lestrelin

et al. 2011). The most recent one, the PLUP, is intended to give villagers a stronger role in the negotiation process of village boundaries, land zoning and land management (MAF and NLMA 2010). It also recognises the key role of the kumban in the LUP process, instead buy LY3023414 of the district as in previous LUP exercises. With the new role given to local communities, in association with the kumban, there is more likelihood of the proposed participatory monitoring system being sustained. PLUP follows 9 steps (MAF and NLMA 2010): (1) preparation; (2) socio-economic, land and

forest data collection; (3) delineation of village and village cluster boundaries; (4) village and village cluster forest and agricultural land use zoning; (5) village and village cluster land management plans; (6) land data record keeping and digital mapping; (7) land registration and titling in rural villages; (8) village and village cluster networks and networking; and (9) monitoring and evaluation. The villages of a kumban and the district authorities together designate the various zones as part of PLUP (step 4). The zones are the areas devoted to protection, conservation, economic activities (plantation and agriculture), infrastructure Edoxaban (village development) etc. They then produce a 5-year management plan for each zone. PLUP in Muangmuay Kumban had not reached the monitoring step (step 9) by the end of the project (December 2010); it had only been implemented up to step 6 (more about the PLUP process in Thiazovivin mouse Bourgoin and Castella 2011; Bourgoin et al. 2012; Lestrelin et al. 2011). However, we were still able to discuss how PLUP could utilize the proposed monitoring system (Table 4). The system can be used as a tool to assess the impact of management decisions on local livelihoods (poverty) and natural habitats (biodiversity), based on the zones proposed within PLUP (e.g. residential areas, conservation forest, sacred forest, agriculture zone).

05). The sera of all non-symptomatic individuals (non-exposed individuals and claw trimmers) that were used as AZD8931 mouse negative controls showed no specific IgE antibodies against cattle

allergen with the Hycor test or the Phadia test. Detection of cattle-related sensitizations using immunoblotting This is the first study presenting the results of a self-prepared cattle allergen mix that was designed to represent the full spectrum of cattle allergens present in a typical agricultural workplace. The self-prepared AG-014699 supplier cattle allergen mix encompasses the spectrum of proteins in a molecular range from lower than 6.5 kDa up to 66 kDa and greater (at approximately 11, 20, 22, 25, 55, 62 and 66 kDa as well as between 25 to 30 and lower than 6.5 kDa), that was obtained by SDS-PAGE-separation Inflammation related inhibitor of extracts from the hair of various cattle races (Fig. 1). The allergenic potential of the extracts concerning the different bands was verified using the sera of various confirmed cattle-allergic patients as previously described (Heutelbeck et al. 2009). Fig. 1 SDS-PAGE of the self-prepared cattle allergen mix: prepared extracts were separated using SDS-PAGE. The following marker and samples were applied: lane 1 molecular weight marker (molecular weights given in kDa), lane 2 self-prepared cattle allergen mix In this study, immunoblot investigations with a self-prepared cattle allergen mix were performed

on 37 claw trimmers of whom 27 reported work-related symptoms and 20 showed a cattle sensitization with at least one commercial test. Positive specific reactions were detected in 94.6% of the samples (n = 35). Typical results with special attention to different sensitization status, given in from the amount of specific IgE (kU/l) antibodies against cattle with the commercial cattle allergen tests of Hycor and Phadia, are shown in Fig. 2a–d.

In most of our immunoblot experiments, we observed distinct bands at a molecular weight of about 16 kDa and rarely in the range of about 20 kDa, reflecting the major component bos d 2. Sporadically, specific reactions were seen at a molecular weight of about 6 kDa, about 29 kDa and in the range between 14.3 and 21 kDa, between 21 and 29 kDa, as well as in the range greater than 45 kDa, The negative controls of all sera of non-symptomatic non-exposed individuals and non-sensitized, non-symptomatic claw trimmers showed unspecific staining in the molecular range between 45 and 67 kDa (examples are shown in Fig. 2e, f). Fig. 2 Immunoblot of the self-prepared cattle allergen mix: proteins were separated by SDS-PAGE and transferred to PVDF membranes. These were developed with serum of symptomatic claw trimmers with different sensitization status, given in the amount of specific IgE (kU/l) against cattle allergen using the commercial tests of Hycor and Phadia (a–d); a 0.11 kU/l (Hycor) and 0.05 kU/l (Phadia), b 0.

Additionally, there were two nucleotide differences between the two types of ITS1 sequences at positions 2112 and 2188 (in reference to KF110799). The Staurosporine insertion/deletion was further confirmed by PCR using primers flanking the region and subsequent BAY 11-7082 supplier sequencing of PCR amplicons. Figure 4 Comparison of the

Oxyspirura petrowi type 1 and type 2 ITS1 sequences at the insertion/deletion site. Their sequences are available at GenBank database with accession numbers [GenBank:KF110799] and [GenBank:KF110800]. While the 5.8S rRNA gene shared significant sequence homology to those of other nematodes available the NCBI databases (data not shown), both the ITS1 and ITS2 regions displayed high sequence diversity (i.e., no significant hits in BLASTN searches of the NCBI nucleotide databases). Because of the insertion/deletion at the ITS1 region, we initially designed two pairs of primers based on the ITS2 sequence for the potential of using nested PCR-based molecular detection of O. petrowi: an external primer pair QEW_2373F (5’-AAG AAT GTA ATG TTG TGG AGC-3’) and QEW_2681R MNK inhibitor (5’-GTA ATC ACA TTT GAG TTG AGG-3’), and an internal primer pair QEW_2417F and QEW_2578R (as described in the Methods section) that would give 309 bp and 162 bp

products, respectively. However, after our pilot experiments indicated that nested PCR was unnecessary, we used regular qPCR reactions with the QEW_2417F and QEW_2578R primers

in subsequent detection of O. petrowi DNA from wild bobwhite fecal specimens collected in Texas in February – March, 2013. The specificity of the QEW_2417F and QEW_2578R primer pair for O. petrowi was also confirmed by its inability to produce products from DNA isolated from the cecal worm A. pennula that is commonly present in wild quail (Figure 5). Figure 5 Agarose gel (1.5%) electrophoresis illustrating PCR-based detection of Oxyspirura petrowi DNA from quail fecal samples. Lane M: 100-bp molecular marker; Lanes EW and CW: regular PCR using DNA isolated from adult eye worm (EW, O. petrowi) and cecal worm (CW, A. pennula) isolated from wild quail as positive and negative controls (Ctl); Lanes S1 – S7: results of real-time qPCR detection from selected positive and negative stool DNA samples. On the other hand, due to the lack of molecular sequences 3-mercaptopyruvate sulfurtransferase at the ITS regions from very closely related species, we could not firmly conclude that the relatively short primers were absolutely specific to O. petrowi. In fact, only six ITS1 sequences were present in the GenBank database for the superfamily Thelazioidea, including five from Thelazia species and one from O. conjuctivalis (accession: EF417873). However, these ITS1 sequences were highly divergent from each other and from those of O. petrowi (i.e., 47.5% – 48.5% identities between the O. conjuctivalis and O. petrowi and 26.1% – 53.

Figure 2 Replacement of dhfr-ts gene with a MS/GW construct pDEST/dhfr-ts_1F8Hyg. A) Schematic of the expected genomic loci of dhfr-ts and 1f8Hyg in dhfr-ts +/-/Hyg parasites. B) PCR analysis with gDNA from cloned drug resistant parasites and WT Tulahuen parasites confirm the expected gene deletion of one allele of the dhfr-ts gene and correct insertion of 1f8Hyg. Primer H1 plus the R1, R2 or R3 downstream primers, yield the expected products of 1.8, 2.0 and 2.3 kb, respectively and the combination of H5 plus upstream primers F3, F2 and F1 give the

predicted bands of 2.1, 2.4 and 2.8 kb for respectively. See Additional file 3: Table S5 for nucleotide sequences selleck chemical of primers. C) Genomic DNA Southern blot analysis of a dhfr-ts +/-/Hyg Tulahuen clone. gDNA digested with BsrGI and hybridized with labeled Hyg CDS probe. Diagram not to scale. Numbers are sizes (bp) of expected products. Consecutive ech1 and ech2 genes are simultaneously replaced by constructs generated based on MS/GW system T. cruzi ech1 and ech2 are tandemly arranged genes (Figure 3A) with a nucleotide find more sequence

identity of 67%. Both genes encode putative enoyl-CoA hydratase/isomerase (ECH) family proteins, which catalyze the second step in the beta-oxidation pathway of fatty acid metabolism. Analysis of the T. cruzi proteome suggested that enzymes in the fatty acid oxidation pathway, including ECH, are preferentially expressed in amastigotes [28]. Therefore, we hypothesized that we would be able to knockout both ech1 and ech2 genes in epimastigotes.

The ech locus also provides an opportunity to test whether or not the MS/GW approach can be ADAMTS5 used to produce knockouts of multiple genes that are physically linked in the www.selleckchem.com/products/VX-680(MK-0457).html genome. Figure 3 Simultaneous replacement of consecutive ech1 and ech2 genes by a MS/GW construct pDEST/ ech -Hyg-GAPDH. A) Diagram of ech1, ech2 and Hyg-GAPDH-IR genomic loci in WT and ech +/-/Hyg parasites. B) PCR genotyping analysis of: no template control (water); ech +/-/Hyg (ech +/-) and WT CL (WT). See Additional file 3: Table S5 for nucleotide sequences of primers. C) Southern blot analysis of two clones (2 and 4) of ech +/-/Hyg. Left panel, gDNA digested with BanI and hybridized with Hyg CDS; right panel, gDNA digested with EcoRI and hybridized with labeled ech1 CDS. Diagrams not to scale. Numbers are sizes (bp) of expected products. In T. cruzi, transcript stability and protein translation is largely controlled by 3′UTR and intergenic regions [29, 30]. The intergenic region of a constitutively expressed gene, gapdh, gives consistently high levels of stable RNA in different constructs and in different life cycle stages [31]. Hence, we included the 3′ UTR of gapdh in our constructs, to ensure the expression of the inserted drug resistant genes in the epimastigote stage.

The MIA PaCa-2, HPAC and Capan-2 cells were transfected with pcDNA3.1 mammalian expression vector containing full-length cDNA encoding human mesothelin, or with the empty pcDNA3.1 vector. After 2 weeks of selection with G418, mesothelin-expressing cells and vector control cells were obtained for each of the three pancreatic BMS202 in vitro cancer cell lines. Mesothelin protein expression were measured by Western blot analysis (Figure 2C). All three mesothelin -expressing cells expressed high levels of mesothelin protein, whereas none of the three vector control cell lines expressed detectably increased levels of mesothelin protein Gilteritinib purchase (Figure 2C). Overexpression of mesothelin

increases cell proliferation in pancreatic cancer cells with wt-p53 by p53-dependent pathway To elucidate the role of mesothelin overexpression in pancreatic cancer cell proliferation, we used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, comparing the cell growth rate among the mesothelin -overexpressing MIA PaCa-2 stable cell line, the empty vector MIA PaCa-2 stable cell line, and AG-881 ic50 the unrelated MIA PaCa-2 cell

line. The MTT assay showed that Mesothelin transfected cells proliferated almost 3.1 times faster than the control cells at day 3 (P < 0.05; Figure 3A), and almost 2.6 times faster at day 6 (P < 0.05; Figure 3A). To confirm the role of mesothelin in cell proliferation, we did the above assay with another stably mesothelin -overexpressing pancreatic cancer cell line, Capan-2. The similarity of the results provides further evidence for the role of mesothelin in inducing cell proliferation (Figure 3B). The similarity of the results was also found in HPAC cells (data not shown). Figure 3 Overexpression of mesothelin promotes pancreatic cancer cell survival and proliferation. A, Cell proliferation of MIA PaCa-2 and Capan-2 cells according to MTT assay. Stable mesothelin PTK6 transfected MIA PaCa-2 and Capan-2 cells

and control cells were seeded in 96-well plates (2 × 103 cells/well), serum-starved (0% fetal bovine serum, FBS) for 24 h before changing to 2% FBS growth medium, and cultured for 6 day. Viability was measured with MTT. Relative increase in viability was measured by dividing viability at one time point by viability of the same cell at day 0 (day of addition of growth medium after initial serum starvation) and is plotted along the Y-axis. Points, mean of triplicate wells. B, cells grown in soft agar were counted. bars, SD. *, P < 0.05, relative to control or mock(at 14 days). C and D , Mesothelin increases bcl-2 and decrease Bax via p53-dependent pathway. Whole cell extract from cells were probed for western blot. E , Mesothelin increases bcl-2 and decrease Bax by p53-independent pathway. Whole cell extract from cells were detected for western blot.

As such, further research would be useful to investigate whether CMR can provide an ergogenic benefit during a field test that replicates field-based team games. Furthermore, as previous research suggests an increased perception of exercise intensity may hinder performance during field-based team games [13], investigation of the influence of CMR on subjective experiences during multiple P505-15 ic50 sprint exercise is required. The primary aim of our current study was to examine the effect of CMR on multiple sprint performance during a field-based exercise protocol. Secondary and tertiary aims included assessments regarding CMR on subjective experiences during multiple sprint

exercise. Methods Participants Eight physically active males (Age; 22 ± 1 y; 75.0 ± 8.8 kg; estimated VO2max 52.0 ± 3.0 ml/kg/min) volunteered to take part in the study. Seven of the participants habitually participated in field-based multiple MG-132 in vivo sprint sport such as football (i.e., soccer) and rugby, while the other was a recreationally active runner. After participants were briefed about the nature of the study, they provided written informed consent. The exclusion criteria included usage of Elafibranor clinical trial creatine supplements in the 12 weeks prior to the study, due to its influence on multiple sprint performance [14]. The ethics committee for the Department of Health at the University

of Bath approved, which was according to the Declaration of Helsinki. We have presented a schematic representation of the experimental conditions is presented in Figure 1. Figure 1 Schematic representation of the time line of study procedures. Preliminary measures and test familiarization Five days prior to

the first experimental trial, participants reported to an indoor sprints track for preliminary measurements including the participant’s height and body mass. During this visit each participant completed a progressive multistage shuttle run Chlormezanone test, which estimated maximal oxygen uptake [15]. Following this, each participant completed one 15 min section of the Loughborough Intermittent Shuttle Test (LIST) and one repeated sprint ability (RSA) test in order to familiarize themselves with the experimental tests. At the completion of this visit, participants were familiarized with the psychological scales used in this study. Experimental trials During each experimental condition, participants completed two trials consisting of a CMR and placebo (PLA) supplement administered in a randomized, counterbalanced order. To maintain blinding to the investigators and participants, all treatments were pre-labelled and subsequently dispersed by a non-affiliated researcher not participating in this trial. Experimental trials were conducted 7-9 days apart and at the same time of day. In the 24 h preceding the first experimental trial, participants were asked to record their diet and then replicate it before the second trial.

Regardless, analysis of store bought vegetables more truly represents what microorganisms are likely to be consumed by the typical consumer.

A recent study examining store bought lettuce found that 38 out of 100 leaves had internalized bacteria; although this conclusion was based solely on culture-dependent methods [39]. A few other studies have used pyrosequencing to analyse the phyllosphere bacterial #Selleckchem PXD101 randurls[1|1|,|CHEM1|]# community on lettuce and spinach [19, 25, 26], although those studies retrieved the phyllosphere community from washes from leaves and thus exclude endophytes, as well as any bacteria that adhere tightly to the leaf surface. We used a different approach, in which we surface-sterilized the surface, killing the bacterial populations associated with the leaf surface. Thus our non-sterilized samples include all leaf-associated populations (endophytes and surface-associated), while our surface sterilized samples represent just the endophytes. To our knowledge, the study presented here is the first report of pyrosequencing analysis of the endophytic bacterial community associated with SYN-117 datasheet store bought, ready-to-eat produce. Conclusions Commercial ready-to-eat salad leaf vegetables

harbor an array of endophytic and surface associated bacteria. Culture-independent analysis using pyrosequencing indicated that the majority of leaf vegetable-associated bacteria were members of the Proteobacteria and Bacteroidetes. Dominant bacterial taxa identified by pyrosequencing were also identified as culturable isolates. However, the use of pyrosequencing also allowed for the identification of numerous low abundance bacteria that would not have been identified otherwise

by culture dependent methods. Whether vegetables were cultivated under conventional or organic agricultural systems appeared to have little consistent impact on the microbial community composition. While surface sterilization significantly decreased the number of bacteria, surface sterilized salad vegetables still contained at least 2.2 × 103 to 5.8 × 105 culturable endophytic cells per gram of leaf material. Even the most extreme washing would not remove these cells, so that consumers are constantly exposed to appreciable levels of plant-associated microorganisms. Succinyl-CoA Methods Sample collection and processing Packages of ready-to-eat leaf vegetables were purchased from a grocery store in Oxford, Mississippi, USA, during September and October 2010. Leaf vegetables consisted of romaine lettuce and baby spinach (both purchased September 15th 2010), and green leaf lettuce, iceberg lettuce, and red leaf lettuce (all purchased October 11th 2010). Both organic and conventionally grown varieties of each produce type were obtained (ten samples total). Samples were in modified atmosphere packaging, stored in the chilled produce section.

Figure 5 Binding characteristics of Lsa33 and Lsa25 proteins to ECM components. (A) Wells were coated with 1 μg of laminin, collagen type I, collagen type IV, cellular fibronectin, plasma fibronectin and the control

proteins gelatin and fetuin. One μg of the recombinant proteins Lsa33 and Lsa25 was added per well and the binding was measured by ELISA. In (A) the protein binding was detected by polyclonal antibodies against each protein, while in (B) protein binding was evaluated with monoclonal anti – polyhistidine serum. Data LY2874455 molecular weight represent the mean ± the standard deviation from three independent experiments. For statistical analyses, the attachment of recombinant this website proteins to the ECM components was compared to its binding to gelatin by the two – tailed t test (*P < 0.05). (C) Dose - dependent binding experiments of recombinant proteins with laminin was performed by polyclonal antibodies against each protein; each point was performed Ro 61-8048 order in triplicate and expressed as the mean absorbance value at 492 nm ± standard error for each point. Gelatin was included

as a negative control. The dissociation constants (KD) are depicted and were calculated based on ELISA data for the recombinant proteins that reached equilibrium. (D) Immobilized laminin was treated with sodium metaperiodate (5 to 100 mM) for 15 min at 4°C in the dark. Mean absorbance values at 492 nm (± the standard deviations of three independent experiments) were compared to those obtained with untreated laminin (0 mM). Interaction of recombinant proteins to serum components Our group has recently reported that leptospires interact with PLG and that several proteins could act as PLG – receptors [17–19, 21]. Protein binding to complement regulators factor H and C4bp have also been shown [31, 32]. Therefore, we set out to evaluate whether the recombinant proteins Lsa33 and Lsa25 were capable of binding human PLG, factor H and C4bp in vitro. The components,

human PLG, factor H and C4bp and the control proteins, gelatin and fetuin, were individually immobilized onto 96 – wells plates followed by incubation with the recombinant leptospiral proteins. The results obtained using polyclonal antibodies against each protein to probe the reactions showed that Lsa33 and Lsa25 Bay 11-7085 interact with C4bp while only Lsa33 appears to bind to PLG (Figure 6A). No reaction was observed with factor H and the control proteins (Figure 6A). Similar results were achieved when binding was performed using monoclonal anti – his tag antibodies (Figure 6B). Both data show that while Lsa33 protein depicted a statistically significant absorption value for the interaction with PLG, the Lsa25 appears to have only a weak or no adherence to this component. These data were further confirmed when the reaction between the recombinant proteins and PLG were assessed on a quantitative basis as illustrated in Figure 6C. Dose – dependent and saturable binding was observed when increasing concentrations (0 to 1.

anguillarum, protein samples prepared from various subcellular fractions were separated by SDS-PAGE and analyzed by western blot analysis using polyclonal rabbit anti-Plp antiserum. An immuno-reactive band of ~45 kDa was selleck compound detected only in

the supernatant of M93Sm, but was absent in the supernatant of plp mutant (Figure 5C). Taken together with the phospholipase A2 activity data, these data indicate that Plp is a secreted protein in V. anguillarum. rPlp has a specific activity against phosphatidylcholine Various fluorescently-labeled phospholipid substrates (described in Methods) were used to determine the specificity of the rPlp protein. rPlp exhibited high activity against phosphatidylcholine, cleaving BPC to yield BLPC and free fatty acid (Figures 3 Alvocidib order and 6A). However, rPlp had almost no activity against both NBD-phosphatidylethanolamine (NBD-PE) (Figure 6B) and NBD-phosphatidylserine (NBD-PS) (Figure 6C), showing only 2% and 5%, respectively, of the activity of the standard PLA2 protein against each of the substrates. The data indicated that the rPlp protein does not efficiently cleave either phosphatidylethanolamine or phosphatidylserine. Additionally, unlike the standard sphingomyelinase, rPlp was not able to cleave the NBD-sphingomyelin into the NBD-ceramide

Idasanutlin concentration and phosphocholine (Figure 6D), indicating that rPlp had no sphingomyelinase activity. Taken together, the data demonstrated that Plp is a phosphatidylcholine-specific PLA2 enzyme. Figure 6 rPlp activity determined by TLC analysis. BPC (A), NBD-PE (B), NBD-PS (C), and NBD-Sm (D) were used as phospholipid substrates to examine the specificity of rPlp. Phosphate-buffered saline (PBS) was used as a negative control, and PLA2 enzyme from porcine pancreas as a positive control. BPC: BODIPY-labeled phosphatidylcholine; BLPC: BODIPY-labeled lysophosphatidylcholine; NBD-PE: NBD-labeled

phosphatidylethanolamine; NBD-LPE: NBD-labeled lysophosphatidylethanolamine; NBD-PS: NBD-labeled phosphatidylserine; NBD-FFA: NBD-labeled free fatty acid; NBD-SM: NBD-labeled sphingomyelin; NBD-CE: NBD-labeled ceramide. rPlp is able to lyse the fish erythrocytes directly Membrane phospholipid compositions are quite varied among the animal species, especially for phosphatidylcholine. It is known that phosphatidylcholine makes up 58% of the total phospholipid in fish erythrocytes [19]; however, no phosphatidylcholine MYO10 is found in sheep erythrocytes [20]. In order to determine whether the differential hemolysis observed for plp mutants of V. anguillarum (Figure 2) is due to the activity of Plp against PC, we tested the ability of purified rPlp to lyse Atlantic salmon erythrocytes. Addition of recombinant Plp resulted in the lysis of Atlantic salmon erythrocytes, with the amount of lysis directly related to the amount of rPlp added to the blood suspension (Figure 7). In contrast, addition of rPlp to a suspension of sheep erythrocytes resulted in no lysis of those cells (Figure 7).

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