Metabolomic analyses of plasma and liver of mice fed with immature Citrus tumida peel.

In this study, we investigated the effects of dietary supplementation of Citrus tumida hort. ex Tanaka on food intake, body and fat tissue weights, and metabolic profiles of plasma and liver in mice. Supplementation with 5% (w/w) of peels of immature C. tumida (PIC) for 4 weeks significantly suppressed body weight gain and decreased adipose tissue weight in epididymal, perirenal, and subcutaneous fats. Metabolome analyses showed that 2-hydroxyvaleric acid levels were reduced in the blood plasma of mice fed with PIC. PIC supplementation significantly elevated dipeptide (Thr-Asp, Ser-Glu, and Ala-Ala), glucuronic acid, and S-methylglutathione levels, and significantly reduced betaine aldehyde levels in the liver. In conclusion, PIC supplementation affects the metabolism of fatty acids, pectin, glutathione, and choline, showing potential beneficial effects for metabolic syndrome and obesity. PIC may be developed as a functional food and used in the treatment of these diseases.

Rational Design of Peptide-Functionalized Surface Plasmon Resonance Sensor for Specific Detection of TNT Explosive.

In this study, a rationally-designed 2,4,6-trinitrotoluene (TNT) binding peptide derived from an amino acid sequence of the complementarity-determining region (CDR) of an anti-TNT monoclonal antibody was used for TNT detection based on a maleimide-functionalized surface plasmon resonance (SPR) sensor. By antigen-docking simulation and screening, the TNT binding candidate peptides were obtained as TNTHCDR1 derived from the heavy chain of CDR1, TNTHCDR2 derived from CDR2, and TNTHCDR3 from CDR3 of an anti-TNT antibody. The binding events between candidate peptides and TNT were evaluated using the SPR sensor by direct determination based on the 3-aminopropyltriethoxysilane (APTES) surface. The TNT binding peptide was directly immobilized on the maleimide-functionalized sensor chip surface from N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). The results demonstrated that peptide TNTHCDR3 was identified and selected as a TNT binding peptide among the other two candidate peptides. Five kinds of TNT analogues were also investigated to testify the selectivity of TNT binding peptide TNTHCDR3. Furthermore, the results indicated that the APTES-GMBS-based SPR sensor chip procedure featured a great potential application for the direct detection of TNT.

Array-Based Rational Design of Short Peptide Probe-Derived from an Anti-TNT Monoclonal Antibody.

Complementarity-determining regions (CDRs) are sites on the variable chains of antibodies responsible for binding to specific antigens. In this study, a short peptide probe for recognition of 2,4,6-trinitrotoluene (TNT), was identified by testing sequences derived from the CDRs of an anti-TNT monoclonal antibody. The major TNT-binding site in this antibody was identified in the heavy chain CDR3 by antigen docking simulation and confirmed by an immunoassay using a spot-synthesis based peptide array comprising amino acid sequences of six CDRs in the variable region. A peptide derived from heavy chain CDR3 (RGYSSFIYWF) bound to TNT with a dissociation constant of 1.3 µM measured by surface plasmon resonance. Substitution of selected amino acids with basic residues increased TNT binding while substitution with acidic amino acids decreased affinity, an isoleucine to arginine change showed the greatest improvement of 1.8-fold. The ability to create simple peptide binders of volatile organic compounds from sequence information provided by the immune system in the creation of an immune response will be beneficial for sensor developments in the future.

Enhancement of Biomass and Lipid Productivities of Water Surface-Floating Microalgae by Chemical Mutagenesis.

Water surface-floating microalgae have great potential for biofuel applications due to the ease of the harvesting process, which is one of the most problematic steps in conventional microalgal biofuel production. We have collected promising water surface-floating microalgae and characterized their capacity for biomass and lipid production. In this study, we performed chemical mutagenesis of two water surface-floating microalgae to elevate productivity. Floating microalgal strains AVFF007 and FFG039 (tentatively identified as Botryosphaerella sp. and Chlorococcum sp., respectively) were exposed to ethyl methane sulfonate (EMS) or 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), and pale green mutants (PMs) were obtained. The most promising FFG039 PM formed robust biofilms on the surface of the culture medium, similar to those formed by wild type strains, and it exhibited 1.7-fold and 1.9-fold higher biomass and lipid productivities than those of the wild type. This study indicates that the chemical mutation strategy improves the lipid productivity of water surface-floating microalgae without inhibiting biofilm formation and floating ability.

Array-based functional peptide screening and characterization of gold nanoparticle synthesis.

Based on inorganic material production through biomineralization in organisms, the use of biological molecules in nanomaterial production has received increasing attention as a vehicle to synthesize inorganic materials with selected properties in ambient conditions. Among various biological molecules that interact with metallic surfaces, short peptides are putative ligand molecules as they exhibit potential to control the synthesis of nanoscale materials with tailored functions. Herein, using a spot synthesis-based peptide array, the gold nanoparticle (AuNP) binding activities of approximately 1800 peptides were evaluated and revealed various activities ranging from positive (high-affinity binding peptides) to negative (weak- or null-affinity binding peptides). Among 50 peptides showing the highest AuNP binding activity, 46 sequences showed the presence of tryptophan-based motifs including W[Xn]W, H[Xn]W, and W[Xn]H (W: tryptophan, X: any amino acid, n: 1-8 amino acid residues), whereas none of these motifs was found in the WORST50 peptides. Notably, three peptides showing the highest binding affinities possessed bi-functionality in AuNP binding and Au(III) reduction in solution and on solid surfaces. In addition, the characterization of truncated peptide derivatives revealed unique peptide motifs for their function expressions that also supported the importance of tryptophan-based motifs for peptide-AuNP binding. These findings open the door for peptide-mediated precise regulation of AuNP synthesis in ambient condition and for site dependent controlled AuNP integration onto nanotechnological devices. STATEMENT OF SIGNIFICANCE: The development of a technique for functionally regulated nanosized material production in ambient condition is broadly required according to the expansion of nanomaterial based applications. Short peptides, which bind to metallic surfaces, have great potential for the technique development, but the realization remains a difficult challenge due to the lack of metal binding peptide varieties. Herein, approximately 1800 peptides with the gold nanoparticle (AuNP) binding activity are reported and characterized. Furthermore, by three highest binding peptides, the expression of bi-functionality in AuNP binding and Au(III) reduction was serendipitously discovered in solution and on solid surfaces. These findings will be attributed to new technique development of functional nanoparticle synthesis in mild condition, and for site-dependent AuNP integration in various nanotechnological devices.

Potential of water surface-floating microalgae for biodiesel production: Floating-biomass and lipid productivities.

Microalgae have been accepted as a promising feedstock for biodiesel production owing to their capability of converting solar energy into lipids through photosynthesis. However, the high capital and operating costs, and high energy consumption, are hampering commercialization of microalgal biodiesel. In this study, the surface-floating microalga, strain AVFF007 (tentatively identified as Botryosphaerella sudetica), which naturally forms a biofilm on surfaces, was characterized for use in biodiesel production. The biofilm could be conveniently harvested from the surface of the water by adsorbing onto a polyethylene film. The lipid productivity of strain AVFF007 was 46.3 mg/L/day, allowing direct comparison to lipid productivities of other microalgal species. The moisture content of the surface-floating biomass was 86.0 ± 1.2%, which was much lower than that of the biomass harvested using centrifugation. These results reveal the potential of this surface-floating microalgal species as a biodiesel producer, employing a novel biomass harvesting and dewatering strategy.

The Effect of Preoperative Subcutaneous Fat Thickness on Surgical Site Infection Risk in Patients Undergoing Colorectal Surgery: Results of a Multisite, Prospective Cohort Study.

Surgical site infection (SSI) is one of the most frequent postoperative complications among patients undergoing elective colorectal surgery. A multisite, prospective cohort study was conducted to investigate whether the thickness of subcutaneous fat (TSF) influences the occurrence of SSI in patients undergoing colorectal surgery. Participants included patients scheduled to receive colorectal laparotomy for colorectal cancer and who were under the care of a wound ostomy continence nurse at 17 participating general hospitals in Japan. Patients were not eligible to participate if they had undergone emergency surgery, reoperation, or laparoscopic surgery. Demographic, wound, and surgical data and American Society of Anesthesiologists (ASA) scale scores were collected and assessed, along with nutritional status, TSF, body mass index, and risk factors for SSI (ie, length of surgery and wound classification). The incidence of SSI and nutritional conditions was assessed weekly for 30 days after surgery. Of the 155 participants (mean age 68.9 ± 10.8 years, 53 [34.2%] of whom were women), 90 (58.1%) underwent rectal surgery, and the remaining 65 underwent colon surgery. Seventy-two (72, 46.5%) of the 155 patients underwent colostomy surgery; 24 (15.5%) developed a SSI. The mean onset of SSI was 7.3 ± 2.9 days after surgery and commonly observed in the stoma group when the ASA score was 3 or higher (P = 0.02). Patients who developed SSI resumed oral dietary intake later than those without SSI (7.4 days versus 4.6 days, P = 0.02). Multivariate analysis indicated TSF >15 mm (P = 0.01), Alb level <3.5 g/dL at postoperative days 14 (P = 0.03) and 21 (P = 0.02), and total protein level <6.8 g/dL at postoperative day 7 (P = 0.02) were statistically significantly correlated with SSI occurrence. These results suggest preoperative TSF and preoperative and postoperative serum albumin levels are independent risk factors for SSI in patients undergoing colorectal surgery, confirming current recommendations to maximize the preoperative nutritional status of patients in order to prevent major complications. The results also confirm obesity/TSF should be assessed preoperatively to ascertain the risk of SSI in patients undergoing colorectal surgery.

Peptide-mediated microalgae harvesting method for efficient biofuel production.

BACKGROUND: Production of biofuels from microalgae has been recognized to be a promising route for a sustainable energy supply. However, the microalgae harvesting process is a bottleneck for industrialization because it is energy intensive. Thus, by displaying interactive protein factors on the cell wall, oleaginous microalgae can acquire the auto- and controllable-flocculation function, yielding smarter and energy-efficient harvesting. RESULTS: Towards this goal, we established a cell-surface display system using the oleaginous diatom Fistulifera solaris JPCC DA0580. Putative cell wall proteins, termed frustulins, were identified from the genome information using a homology search. A selected frustulin was subsequently fused with green fluorescent protein (GFP) and a diatom cell-surface display was successfully demonstrated. The antibody-binding assay further confirmed that the displayed GFP could interact with the antibody at the outermost surface of the cells. Moreover, a cell harvesting experiment was carried out using silica-affinity peptide-displaying diatom cells and silica particles where engineered cells attached to the silica particles resulting in immediate sedimentation. CONCLUSION: This is the first report to demonstrate the engineered peptide-mediated harvesting of oleaginous microalgae using a cell-surface display system. Flocculation efficiency based on the silica-affinity peptide-mediated cell harvesting method demonstrated a comparable performance to other flocculation strategies which use either harsh pH conditions or expensive chemical/biological flocculation agents. We propose that our peptide-mediated cell harvest method will be useful for the efficient biofuel production in the future.

Enhancement of glycerol metabolism in the oleaginous marine diatom Fistulifera solaris JPCC DA0580 to improve triacylglycerol productivity.

BACKGROUND: Microalgal oil is a promising alternative feedstock for biodiesel fuel (BDF). Mixotrophic cultivation with glycerol, the primary byproduct of BDF production, may be used to optimize BDF production. This strategy would reduce costs through glycerol recycling and improve lipid productivity and biomass productivity by overcoming the growth retardation caused by decreased light penetration in high-density culture. RESULTS: Overexpression of the endogenous glycerol kinase (GK) gene in an oleaginous marine diatom, Fistulifera solaris JPCC DA0580, accelerates glycerol metabolism and improves lipid and biomass productivities. Two candidates were selected from a collection of 90 G418-resistant clones, based on growth and confirmation of genome integration. GK gene expression was higher in the selected clones (GK1_7 and GK2_16) than in the wild-type culture. The GK2_16 clone achieved a 12% increase in lipid productivity. CONCLUSION: We have demonstrated the potential of metabolic engineering in oleaginous microalgae to improve lipid productivity. Metabolic engineering techniques can be used to optimize BDF production.

Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome.

Oleaginous photosynthetic organisms such as microalgae are promising sources for biofuel production through the generation of carbon-neutral sustainable energy. However, the metabolic mechanisms driving high-rate lipid production in these oleaginous organisms remain unclear, thus impeding efforts to improve productivity through genetic modifications. We analyzed the genome and transcriptome of the oleaginous diatom Fistulifera solaris JPCC DA0580. Next-generation sequencing technology provided evidence of an allodiploid genome structure, suggesting unorthodox molecular evolutionary and genetic regulatory systems for reinforcing metabolic efficiencies. Although major metabolic pathways were shared with nonoleaginous diatoms, transcriptome analysis revealed unique expression patterns, such as concomitant upregulation of fatty acid/triacylglycerol biosynthesis and fatty acid degradation (ß-oxidation) in concert with ATP production. This peculiar pattern of gene expression may account for the simultaneous growth and oil accumulation phenotype and may inspire novel biofuel production technology based on this oleaginous microalga.

Chloroplast-targeting protein expression in the oleaginous diatom Fistulifera solaris JPCC DA0580 toward metabolic engineering.

The chloroplast plays critical roles in lipid metabolism of microalgae, thus it is recognized as an attractive target of metabolic engineering to enhance biofuel production. It has been well known that recombinant protein expression in microalgal chloroplasts needs specific signal sequence which governs the transition manner of nuclear-encoded polypeptides within the subcellular compartments. However certain microalgae, including diatoms, have complex membrane systems surrounding the chloroplast, and thus chloroplast-targeting protein expression with the signal sequence has rarely been demonstrated except for a few model non-oleaginous diatoms. In this study, we performed recombinant green fluorescence protein (GFP) expression and transportation into the chloroplast of the oleaginous marine diatom, Fistulifera solaris JPCC DA0580. The signal sequence of ATP synthetase gamma subunit, which was predicted to localize in the chloroplast according to a bioinformatics analysis pipeline, was employed as a key factor of this technique. As a result, specific localization of GFP in the chloroplast was observed. It would be useful to engineer the lipid synthesis pathways existing in the chloroplast. Furthermore, intensive gathering of GFP in the rod-like structure was also detected, which has not been observed in model diatom studies. As comparing with electron microscopic observation, the structure was estimated to be a pyrenoid.

Alkane production by the marine cyanobacterium Synechococcus sp. NKBG15041c possessing the α-olefin biosynthesis pathway.

The production of alkanes in a marine cyanobacterium possessing the α-olefin biosynthesis pathway was achieved by introducing an exogenous alkane biosynthesis pathway. Cyanobacterial hydrocarbons are synthesized via two separate pathways: the acyl-acyl carrier protein (ACP) reductase/aldehyde-deformylating oxygenase (AAR/ADO) pathway for the alkane biosynthesis and the α-olefin synthase (OLS) pathway for the α-olefin biosynthesis. Coexistence of these pathways has not yet been reported. In this study, the marine cyanobacterium Synechococcus sp. NKBG15041c was shown to produce α-olefins similar to those of Synechococcus sp. PCC7002 via the α-olefin biosynthesis pathway. The production of heptadecane in Synechococcus sp. NKBG15041c was achieved by expressing the AAR/ADO pathway genes from Synechococcus elongatus PCC 7942. The production yields of heptadecane in Synechococcus sp. NKBG15041c varied with the expression level of the aar and ado genes. The maximal yield of heptadecane was 4.2 ± 1.2 µg/g of dried cell weight in the transformant carrying a homologous promoter. Our results also suggested that the effective activation of ADO may be more important for the enhancement of alkane production by cyanobacteria.

Identification of a frustule-associated protein of the marine pennate diatom Fistulifera sp. strain JPCC DA0580.

Among the proteins localized on the cell wall (frustule) of diatoms (frustule-associated proteins), several proteins tightly associated with the cell wall have been implicated in frustule formation. These proteins include diatom-specific unique serine- and lysine-rich sequences represented by silaffins. Taking advantage of available genome information, we used a recently described bioinformatics approach to screen silaffin-like proteins rich in serine and lysine from the genome of the marine pennate diatom Fistulifera sp. strain JPCC DA0580 and identified 7 proteins. All of the proteins shared a sequence motif called the XGXG domain, which was also confirmed in a silaffin-like protein identified in other diatoms. In vivo localization analysis revealed that one of the identified proteins, G7408, occurs throughout the frustule with a slightly uneven distribution. This novel frustule-associated protein could be a useful tool to elucidate the mechanism of biosilica formation in diatoms and to functionalize this strain for future biotechnological applications.

Biosynthesis of polyunsaturated fatty acids in the oleaginous marine diatom Fistulifera sp. strain JPCC DA0580.

Studies of polyunsaturated fatty acid (PUFA) biosynthesis in microalgae are of great importance for many reasons, including the production of biofuel and variable omega 3-long chain PUFAs. The elucidation of the PUFA biosynthesis pathway is necessary for bioengineering to increase or decrease PUFA content in certain microalgae. In this study, we identified the PUFA synthesis pathway in the oleaginous marine diatom, Fistulifera sp. strain JPCC DA0580, a promising candidate for biodiesel production. The data revealed not only the presence of the desaturases and elongases involved in eicosapentaenoic acid (EPA) synthesis, but also the unexpected localization of ω3-desaturase expression in the chloroplast. This suggests that this microalga might perform the final step of EPA synthesis in the chloroplast and not in the endoplasmic reticulum (ER) like other diatoms. The detailed fatty acid profile suggests that the EPA was synthesized only through the ω6-pathway in this strain, which was also different from other diatoms. Finally, the transcriptome analysis demonstrated an overall down-regulation of desaturases and elongases over incubation time. These genetic features might explain the decrease of PUFA percentage over incubation time in this strain. The important insights into metabolite synthesis acquired here will be useful for future metabolic engineering to control PUFA content in this diatom.

Identification and functional analysis of delta-9 desaturase, a key enzyme in PUFA Synthesis, isolated from the oleaginous diatom Fistulifera.

Oleaginous microalgae are one of the promising resource of nonedible biodiesel fuel (BDF) feed stock alternatives. Now a challenge task is the decrease of the long-chain polyunsaturated fatty acids (PUFAs) content affecting on the BDF oxidative stability by using gene manipulation techniques. However, only the limited knowledge has been available concerning the fatty acid and PUFA synthesis pathways in microalgae. Especially, the function of Δ9 desaturase, which is a key enzyme in PUFA synthesis pathway, has not been determined in diatom. In this study, 4 Δ(9) desaturase genes (fD9desA, fD9desB, fD9desC and fD9desD) from the oleaginous diatom Fistulifera were newly isolated and functionally characterized. The putative Δ(9) acyl-CoA desaturases in the endoplasmic reticulum (ER) showed 3 histidine clusters that are well-conserved motifs in the typical Δ(9) desaturase. Furthermore, the function of these Δ(9) desaturases was confirmed in the Saccharomyces cerevisiae ole1 gene deletion mutant (Δole1). All the putative Δ(9) acyl-CoA desaturases showed Δ(9) desaturation activity for C16∶0 fatty acids; fD9desA and fD9desB also showed desaturation activity for C18∶0 fatty acids. This study represents the first functional analysis of Δ(9) desaturases from oleaginous microalgae and from diatoms as the first enzyme to introduce a double bond in saturated fatty acids during PUFA synthesis. The findings will provide beneficial insights into applying metabolic engineering processes to suppressing PUFA synthesis in this oleaginous microalgal strain.

Establishment of a genetic transformation system for the marine pennate diatom Fistulifera sp. strain JPCC DA0580--a high triglyceride producer.

A genetic transformation system for the marine pennate diatom, Fistulifera sp. JPCC DA0580, was established using microparticle bombardment methods. Strain JPCC DA0580 has been recently identified as the highest triglyceride (60 % w/w) producer from a culture collection of 1,393 strains of marine microalgae, and it is expected to be a feasible source of biodiesel fuel. The transformation conditions for strain JPCC DA0580 were optimised using the green fluorescent protein gene (gfp) and the gene encoding neomycin phosphotransferase II (nptII). The most efficient rate of transformation was attained when tungsten particles (0.6 µm in diameter) were used for microparticle bombardment. The effect of endogenous and exogenous promoters on the expression of nptII was examined. Endogenous promoters were more efficient for obtaining transformants compared with exogenous promoters. Southern hybridisation analysis suggested that nptII integrated into the nuclear genome. This genetic manipulation technique should allow us to understand the mechanisms of high triglyceride accumulation in this strain, thereby contributing to improving BDF production.

Integrated care through disease-oriented clinical care pathways: experience from Japan's regional health planning initiatives.

INTRODUCTION: In April 2008, Japan launched a radical reform in regional health planning that emphasized the development of disease-oriented clinical care pathways. These 'inter-provider critical paths' have sought to ensure effective integration of various providers ranging among primary care practitioners, acute care hospitals, rehabilitation hospitals, long-term care facilities and home care. DESCRIPTION OF POLICY PRACTICE: All 47 prefectures in Japan developed their Regional Health Plans pursuant to the guideline requiring that these should include at least four diseases: diabetes, acute myocardial infarction, cerebrovascular accident and cancer. To illustrate the care pathways developed, this paper describes the guideline referring to strokes and provides examples of the new Regional Health Plans as well as examples of disease-oriented inter-provider clinical paths. In particular, the paper examines the development of information sharing through electronic health records (EHR) to enhance effective integration among providers is discussed. DISCUSSION AND CONCLUSION: Japan's reform in 2008 is unique in that the concept of 'disease-oriented regional inter-provider critical paths' was adopted as a national policy and all 47 prefectures developed their Regional Health Plans simultaneously. How much the new regional health planning policy has improved the quality and outcome of care remains to be seen and will be evaluated in 2013 after the five-year planned period of implementation has concluded. Whilst electronic health records appear to be a useful tool in supporting care integration they do not guarantee success in the application of an inter-provider critical path.

High-throughput pyrosequencing of the chloroplast genome of a highly neutral-lipid-producing marine pennate diatom, Fistulifera sp. strain JPCC DA0580.

The chloroplast genome of the highly neutral-lipid-producing marine pennate diatom Fistulifera sp. strain JPCC DA0580 was fully sequenced using high-throughput pyrosequencing. The general features and gene content were compared with three other complete diatom chloroplast genomes. The chloroplast genome is 134,918 bp with an inverted repeat of 13,330 bp and is slightly larger than the other diatom chloroplast genomes due to several low gene-density regions lacking similarity to the other diatom chloroplast genomes. Protein-coding genes were nearly identical to those from Phaeodactylum tricornutum. On the other hand, we found unique sequence variations in genes of photosystem II which differ from the consensus in other diatom chloroplasts. Furthermore, five functional unknown ORFs and a putative serine recombinase gene, serC2, are located in the low gene-density regions. SerC2 was also identified in the plasmids of another pennate diatom, Cylindrotheca fusiformis, and in the plastid genome of the diatom endosymbiont of Kryptoperidinium foliaceum. Exogenous plasmids might have been incorporated into the chloroplast genome of Fistulifera sp. by lateral gene transfer. Chloroplast genome sequencing analysis of this novel diatom provides many important insights into diatom evolution.

[District health care network and pharmacist].

A new district health plan started in April, 2008. Under this new plan, district referral system has been largely changed and become to be formed in following four diseases of cancer, stroke, diabetes and acute myocardial infarction. A special point in the new district health plan is that district pharmacies are defined as "health care facilities". By this definition, the district pharmacy is expected to play a role as providing center of the drugs and the medical devices corresponding to the four diseases. Moreover, pharmacy of the future has to contribute to play a role of the community health coordination activities such as participation to district referral critical-pathway, home health care and pre-discharge conference in acute care hospital.

[Cancer regional alliances in critical path].

In the time of a marked increase in cancer occurrences, a regional referral system for cancer patient is called for more than ever. Regional alliances among the various health care facilities in accordance with its disease progress are essential because there are many kinds of cancer and they follow chronic processes. The regional referral critical path was developed as a tool for connecting different facilities in the region. From now on, the regional referral critical path of cancer will serve as a tool indispensable to a seamless care of cancer patients in the regional health care system.